WO2004072277A2 - Elk1 phosphorylation related genes - Google Patents

Elk1 phosphorylation related genes Download PDF

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Publication number
WO2004072277A2
WO2004072277A2 PCT/JP2004/001587 JP2004001587W WO2004072277A2 WO 2004072277 A2 WO2004072277 A2 WO 2004072277A2 JP 2004001587 W JP2004001587 W JP 2004001587W WO 2004072277 A2 WO2004072277 A2 WO 2004072277A2
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Prior art keywords
elkl
action
protein
kinase
phosphorylates
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PCT/JP2004/001587
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French (fr)
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WO2004072277A3 (en
Inventor
Osamu Matsuzaki
Akio Matsuda
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Asahi Kasei Pharma Corporation
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Priority claimed from JP2003034875A external-priority patent/JP2006340601A/en
Application filed by Asahi Kasei Pharma Corporation filed Critical Asahi Kasei Pharma Corporation
Publication of WO2004072277A2 publication Critical patent/WO2004072277A2/en
Publication of WO2004072277A3 publication Critical patent/WO2004072277A3/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Definitions

  • the present invention relates to a protein capable of phosphorylating Elkl and/or activating a kinase which phosphorylates Elkl, a DNA sequence encoding the protein, a method for obtaining the DNA, a recombinant vector containing the DNA, a transformant containing the recombinant vector, and an antibody which specifically reacts with the protein.
  • the present invention also relates to use of the protein, DNA, RNA or antibody of the invention in the diagnosis, treatment or prevention of diseases associated with the excessive activation or inhibition of Elkl phosphorylation action or a kinase which phosphorylates Elkl.
  • the present invention also relates to a method for screening a substance capable of inhibiting or promoting the Elkl phosphorylation action and/or the activation of kinase which phosphorylates Elkl by using the protein, DNA, recombinant vector and transformant.
  • MAP (mitogen-activated protein) kinase was first identified in the second half of the 1980s. In 1990s, it has been elucidated that the signal transduction pathway leading to MAP kinase activation starts from cell growth factor receptors having tyrosine kinase activity, through adaptor molecules composed of SH2 (src homology 2) and SH3 (src homology 3), Ras (an oncogene product which is a GTP-binding protein), and Raf-1 (an oncogene product which is serine/threonine kinase) and leads to MAP kinase.
  • SH2 src homology 2
  • SH3 src homology 3
  • Ras an oncogene product which is a GTP-binding protein
  • Raf-1 an oncogene product which is serine/threonine kinase
  • MAP kinase signal transduction pathway is understood to be a central pathway for dete ⁇ r ⁇ iing cell proliferation, differentiation and development of higher eukaryotic organisms (Nishida, E et al., Trends Biochem. Sci., 18: 128 - 131 (1993), Marshall, C.J., Curr. Opin. Genet. Dev., 4: 82 - 89 (1994), Cobb. M. H. et al., J. Biol. Chem., 270: 14843 - 14846 (1995)).
  • MAPKK phosphorylation of Thr and Tyr within Thr-Glu-Tyr sequence between kinase sub-domains VII and V-H.
  • MAPK kinase MAPK kinase
  • MEK MAPK/ERK kinase
  • This enzyme is therefore a dual- specificity kinase which can phosphorylate serine/threon-ne and tyrosine.
  • MAPKK for its activation, requires phosphorylation of two serine (or threonine) residues between kinase domains VII and VIII.
  • MAPKK kinase The serine/threonine kinase responsible for this phosphorylation is called MAPKK kinase (MAPKKK).
  • Raf-1 is one example of MAPKKK
  • This chain of kinases consisting of three molecules, RafMAPKK/MAPK is called the classical MAP kinase cascade.
  • SAPK/JNK and p38/CSBPMPK2 are revealed as MAP kinase-like kinases, and these cascade systems are generally called MAP kinase cascade.
  • SAPK Stress-Activated Protein Kinase
  • p38 was identified and cloned as a protein which is subject to tyrosine phosphorylation in an early stage after stimulating a lymphocyte (Han, J., et al., Science, 265: 808-811(1994)).
  • CSAJD binding protein CSBP
  • CSAJD cytokine-suppressive anti-inflammatory drug
  • this has been clarified to be a human form of ⁇ 38.
  • MPK2 was independently isolated as a kinase which is activated by stress stimulation.
  • this kinase is called p38/CSBP/MPK2.
  • MAP kinase There is mutual homology between the sequences of classical MAP kinase, SAPK JNK, and p38/CSBP MPK2, and they constitute a superfamily.
  • the TEY sequence necessary for activation in classical MAP kinase is TPY (Thr-Pro-Tyr) in SAPK/JNK, and it is TGY sequence in p38/CSBP/MPK2.
  • Thr and Tyr are phosphorylated and activated by the upstream kinase MAPKK.
  • SAPK/ERK kinase- 1 SEKl
  • MKK4 mitogen-activated protein kinase kinase 4
  • MAPKKs mitogen activated protein kinase kinase 7
  • MKK7 mitogen activated protein kinase kinase 7
  • MAPKKs mitogen activated protein kinase kinase 7
  • examples of MAPKKs which specifically activates p38 include MKK3 and MKK6 (Moriguchi, T., et al., J. Biol. Chem., 271: 26981-26988 (1996), Cuenda, A., et al,. EMBO J., 15: 4156-4164 (1996)).
  • MAPKKKs which exist in the uptream of the pathway leading to classical MAP kinase, SAPK JNK, p38/CSBP/MPK2
  • MAPKKK activity such as Raf, TAK1, MEKK, MLK3, Askl, Mos, Cot, etc.
  • MAPKKK activity such as Raf, TAK1, MEKK, MLK3, Askl, Mos, Cot, etc.
  • MEKK1 is known as MAPKKK which exists in the upsteam of the pathway leading to SAPK/JNK, but it is highly possible that other kinases which work as MAPKKK exist.
  • SAPK/JNK and p38/CSBP MPK2 are not activated by growth factors which activate classical MAP kinase, and are each known to be activated by stresses such as osmotic shock or heat shock, or by inflammatory cytokines such as TNF- a or IL-1, etc. (Kyriakis., J. M., et al., J. Biol. Chem., 271: 24313-24316 (1996), Davis., R. J., Trends Biochem. Sci., 19: 470-473 (1994)). Further, they are activated under a condition which induces cell death, such as UV radiation and depletion of serum and/or growth factor(Kyriakis., J.
  • MAP kinase cascades particularly the S APK/JNK and p38/CSBP/MPK2 systems, are activated by a variety of stimuli.
  • SAPK/JNK or p38/CSBP/MPK2 there exist many MAPKK kinases and MAPK kinases, or groups of adaptor molecular which connect these, and these molecules have not yet been folly clarified. It is desired to clarify these kinases and molecules, and utilize them in the fields of medicaments, diagnostics and therapy.
  • the object of the present invention is to identify a novel gene and protein such as described above acting to activate useful MAP kinase cascade, and to provide a method of use of them in medicaments, diagnostics and therapy.
  • the aforementioned 3 kinds of MAP kinases phosphorylate Elkl as a substrate. Therefore, the object of the present invention is to provide a novel protein capable of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl, a DNA sequence encoding the protein, a recombinant vector containing the DNA, a transformant containing the recombinant vector, a process for producing the protein, an antibody directed against the protein or a peptide fragment thereof, and a process for producing the antibody.
  • Another object of the present invention is to provide a method for screening a substance capable of inhibiting or activating Elkl phosphorylation action and/or kinase which phosphorylates Elkl, a kit for the screening, a substance capable of inhibiting or activating Elkl phosphorylation action and or kinase which phosphorylates Elkl obtainable by the screening method or the screening kit, a process for producing the substance, a pharmaceutical composition containing a substance capable of inhibiting or activating Elkl phosphorylation action and/or kinase which phosphorylates Elkl, etc.
  • SAPK JNK and ⁇ 38/CSBP/MPK2 are rapidly activated by stimulations of various extracellular inflammatory cytokines, and have intensively studied on the action of Elkl phosphorylation and/or the action of activating kinase which phosphorylates Elkl.
  • the present inventors have succeeded in constructing a full-length cDNA hbrary by using the oligo-capping method; establishing a gene function assay system employing an expression cloning method using HEK293EBNA cells; and isolating new DNAs (cDNAs) encoding a protein having a function of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl by using the assay system.
  • These new DNA molecules induced Elkl phosphorylation action and/or activation of kinase which phosphorylates Elkl by its expression in HEK293EBNA cells.
  • This result shows that these new DNAs are signal transduction molecules involved in Elkl phosphorylation action and/or activating pathway of kinase which phosphorylates Elkl .
  • the present invention has been completed.
  • the present invention provides the folio wings:
  • a purified protein selected from the group consisting of:
  • a protein which consists of an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108; and
  • a purified protein that acts to phosphorylate Elkl and/or activate kinase which phosphorylates Elkl and comprises an amino acid sequence having at least 95% identity to the protein according to above item (1) over the entire length thereof.
  • a protein which comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108; and
  • a protein that acts to phosphorylate Elkl and/or activate a kinase which phosphorylates Elkl and consists of an amino acid sequence having at least one amino acid deletion, substitution or addition in an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108.
  • polynucleotide sequence which encodes a protein that acts to phosphorylate Elkl and/or activate kinase which phosphorylates Elkl, and which hybridizes under stringent conditions with a polynulceotide having a polynulceotide sequence complementary to the polynucleotide sequence of (a);
  • a polynucleotide sequence encoding a protein that acts to phosphorylate Elkl and/or activate a kinase which phosphorylates Elkl, and consists of a nucleotide sequence having at least one nucleotide deletion, substitution or addition in a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107.
  • polynucleotide sequence which encodes a protein that acts to phosphorylate Elkl and/or activate kinase which phosphorylates Elkl, and which hybridizes under stringent conditions with a polynulceotide having a polynulceotide sequence complementary to the polynucleotide sequence of (a);
  • a polynucleotide sequence encoding a protein that acts to phosphorylate Elkl and/or activate a kinase which phosphorylates Elkl, and consists of a nucleotide sequence having at least one nucleotide deletion, substitution or addition in a nucleotide sequence of protein coding region of any one selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107.
  • An isolated polynucleotide comprising a polynucleotide sequence which encodes a protein that acts to phosphorylate Elkl and/or activate a kinase which phosphorylates Elkl, and has at least 95% identity to the polynucleotide sequence according to above item (3) over the entire length thereof.
  • An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that acts to phosphorylate Elkl and/or activate a kinase which phosphorylates Elkl, and has at least 95% identity to the polynucleotide sequence according to above item (4) or (5) over the entire length thereof.
  • a recombinant vector which comprises a polynucleotide according to any one of above items (3) to (7).
  • a process for diagnosing a disease or susceptibihty to a disease related to expression or activity of the protein of above item (1), (2) or (8) in a subject comprising the steps of:
  • a method for screening compounds which inhibit or promote an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl which comprises the steps of:
  • a method for screening compounds which inhibit or promote an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl which comprises the steps of:
  • a compound which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl which is selected by the method for screening according to above item (15) or (16).
  • a process for producing a pharmaceutical composition which comprises the steps of:
  • a process for producing a pharmaceutical composition which comprises the steps of: (a) preparing a transformant by introducing a gene encoding a protein according to above item (1), (2) or (8) that has an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl into a host cell;
  • a kit for screening a compound for inhibiting or promoting an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl which comprises: (a) a transformant comprising a gene encoding a protein according to above item (1), (2) or (8) which promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl and a gene encoding a protein which provides a signal which can detect an action of Elkl phosphorylation; and
  • a ribozyme or deoxyribozyme capable of inhibiting an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl which has an action of cleavage of RNA that encodes the protein according to above item (1), (2) or (8) or an action of cleavage of RNA that encodes a protein which is involved in a pathway leading to Elkl phosphorylation and/or activation of kinase which phosphorylates Elkl.
  • a method for treating a disease associated with an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl which comprises administering to a subject a compound screened by the process according to above item (15) or (16), and/or a monoclonal or polyclonal antibody or a fragment thereof according to above item (21) or (22), and/or an antisense oligonucleotide according to above item (24), and/or a ribozyme or deoxyribozyme according to above item (25), and/or a double strand RNA according to above item (26) in an effective amount to treat a disease selected from the group consisting of inflammation, autoimmune diseases, cancers, infection diseases, bone diseases, AIDS, neurodegenerative diseases, ischemic injury, GVHD, skin diseases, IgA nephritis, purpuric nephritis, proliferative nephritis, and fulminant hepatit
  • composition according to above item (28) for the treatment and or prevention of inflammation, autoimmune diseases, cancers, infection diseases, bone diseases, AIDS, neurodegenerative diseases, ischemic injury, GVHD, skin diseases, IgA nephritis, purpuric nephritis, proliferative nephritis, and fuhninant hepatitis.
  • a method of treating inflammation, autoimmune diseases, cancers, infection diseases, bone diseases, AIDS, neurodegenerative diseases, ischemic injury, GVHD, skin diseases, IgA nephritis, purpuric nephritis, proliferative nephritis, and inlminant hepatitis which comprising administering a pharmaceutical composition produced by the process according to above item (18) or (19) to a patient suffering from a disease relating to abnormalities in an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl.
  • a pharmaceutical composition which comprises a monoclonal or polyclonal antibody or a fragment thereof according to above item (21) or (22) as an active ingredient.
  • a pharmaceutical composition which comprises an antisense oligonucleotide according to above item (24) as an active ingredient.
  • a pharmaceutical composition which comprises a ribozyme or deoxyribozyme according to above item (25) as an active ingredient.
  • a pharmaceutical composition or a gene therapy agent which comprises a double strand RNA according to above item (26) or a vector capable of expressing said double strand RNA, an active ingredient.
  • composition according to any one of (31) to (34) for the treatment and/or prevention of a disease which is selected from the group consisting of inflammation, autoimmune diseases, cancers, infection diseases, bone diseases, ADDS, neurodegenerative diseases, ischemic injury, GVHD, skin diseases, IgA nephritis, purpuric nephritis, proliferative nephritis, and ftilminant hepatitis.
  • a computer-readable medium on which a sequence data set has been stored said sequence data set comprising at least one of nucleotide sequence or that of coding region which is selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107, and/or at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76
  • a method for calculating identity to other nucleotide sequences and/or amino acid sequences which comprises comparing data on a medium according to above item (36) with data of said other nucleotide sequences and/or amino acid sequences.
  • polypeptides comprising all or a part of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108, are fixed.
  • Fig. 1 shows a results of measurement of reporter activity using p38 inhibitor B203580 which is one type of MAP kinases.
  • Fig. 2 indicates the structural formula of staurosporine.
  • SEQ ID NO: 109 and SEQ ID NO: 110 are primers.
  • a full-length cDNA was produced from mRNA prepared from human peripheral blood monocytes, and a full-length cDNA library was constructed in which the cDNA was inserted into the vector pME18S-FL3 (GenBank Accession AB009864).
  • the cDNA library was introduced into E. coli cells, and plasmid preparation was carried out per clone.
  • animal cell expression plasmid ⁇ cDNA3.1 comprising DNA encoding human JNK gene
  • animal cell expression plasmid pcDNA3.1 comprising DNA encoding human p38 gene
  • plasmid pFA2-Elkl comprising DNA encoding a fusion protein of part of Elkl being a substrate of these and GAL4DNA binding region
  • reporter plasmid pFR-Luc having a Gal4 recognition site upstream of a gene encoding luciferase were cotransfected with the above full-length cDNA plasmid into HEK293EBNA cells (INVITROGEN).
  • luciferase activity was measured, and the plasmid with significantly increased luciferase activity compared to that of a control experiment (vector pME18S-FL3 is introduced into a cell in place of a full-length cDNA )was selected (the selected plasmid showed a 3-fold or more increase in luciferase activity compared to that of the control experiment), and the entire nucleotide sequence of the cDNA cloned into the plasmid was dete ⁇ riined.
  • the protein encoded by the cDNA thus obtained is a signal molecule involved in the action of Elkl phosphorylation and/or the activation of a kinase which phosphorylates Elkl, and at the same time, can be expected to be a signal transduction molecule involved in the activation of the MAP kinase cascades.
  • activation of kinase which phosphorylates Elkl includes both direct modification and activation of a kinase which phosphorylates Elkl, and indirect activation of the kinase by activating upstream signal(s). The present invention is described in detail below.
  • the words "acts to phosphorylate Elkl” or “acts to activate a kinase which phosphorylates Elkl” refers to having an action which phosphorylates Elkl directly or indirectly when a gene is introduced into a suitable host and the protein encoded by the gene is excessively expressed.
  • Phosphorylation of Elkl can be measured, for example, by a reporter gene assay comprising cotransfecting into a cell together with a gene resulting from fusion of Elkl and GAL4DNA binding region, a reporter gene wherein a luciferase gene is linked downstream of a GAL4 recognition sequence.
  • Action of phosphorylating Elkl or action of activating a kinase which phosphorylates Elkl can be confirmed by an increase in reporter activity in cells into which the gene was introduced, compared to control cells (cells into which a null vector only was introduced).
  • Increase in reporter activity is preferably by a factor of 1.5 or more, more preferably by a factor of 2 or more, and still more preferably by a factor of 3 or more.
  • Reporter activity can be measured by cloning a polynucleotide (e.g. cDNA) encoding the protein to be expressed into a suitable expression vector, co-transfecting the thus prepared expression vector into a suitable host together with a gene resulting from fusion of Elkl and GAL4 DNA binding region, and a reporter gene wherein a luciferase gene is linked downstream of a GAL4 recognition sequence, and after culturing for a certain period, then measuring the reporter activity.
  • Suitable expression vectors are well known to those skilled in the art, and examples include pME18S-FL3, pcDNA3.1 (Invitrogen).
  • the reporter gene can be one which enables a person skilled in the art to easily detect the expression thereof, and examples include a gene encoding luciferase, chloramphenicol acetyl transferase, or ⁇ -galactosidase.
  • Use of a gene encoding luciferase is most preferable, and examples of a gene resulting from fusion of Elkl and GAL4 DNA binding region include pFA2-Elkl (STRATAGENE), and examples of a reporter gene wherein a luciferase gene is linked downstream of a GAL4 recognition sequence include pFR-Luc (STRATAGENE).
  • Suitable hosts include those which exhibit MAPK cascades activation response to stimulation by IL-1, TNF- or the like. Examples include 293-EBNA cells. Cell culture and introduction of genes into cells (transfection) can be performed and optimized by a person skilled in the art by known techniques.
  • 293-EBNA cells are inoculated on 5% FBS (Fetal Bovine Serum)-containing DMEM (Dulbecco's Modified Eagle Medium) medium in a 96-well cell culture plate to a final cell density of 1 x 10 4 cells/well, and cultured for 24 hours at 37°C, in the presence of 5% CO 2 .
  • FBS Fetal Bovine Serum
  • DMEM Dulbecco's Modified Eagle Medium
  • luciferase activity can be measured using PerkinElmer's Wallac ARVOTMST 1420 MULTILABEL COUNTER.
  • the method for gene introduction by FuGENE6, and measurement of luciferase activity by Picagene LT2.0 can be performed respectively according to the attached protocols.
  • the amount of FuGENE ⁇ per 1 well is suitably 0.3 to 0.5 ⁇ 1, preferably 0.5 ⁇ 1.
  • the respective amounts per 1 well of the plasmid genes to be introduced are: pFR-Luc plasmid amount, 50 to lOOng, preferably 60ng; pFA2-Elkl plasmid amount, 0.1 to 0.5ng, preferably 0.25ng; amount of pcDNA3.1 (+) plasmid into which human p38 gene has been incorporated, 5ng; and amount of pcDNA3.1(+) plasmid into which human JNJ1 ⁇ 1 gene has been incorporated, 30ng.
  • An ability to phosphorylate Elkl or to activate a kinase that phosphorylates Elkl can be confirmed by using, as an index, an ability to increase the reporter activity (luciferase activity) relative to the control experiment (using cells into which a null vector only was introduced).
  • Increase in reporter activity as an index is preferably by a factor of 1.5 or more, more preferably by a factor of 2 or more, and still more preferably by a factor of 3 or more.
  • the present invention provides the folio wings: (a) proteins which comprises any of the above amino acid sequences;
  • Identity as known in the art, is a relationship between two or more protein sequence or two or more polynucleotide sequences, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between protein or polynucleotide sequences, as determined by the match between protein or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
  • Identity and similarity can be readily calculated by known methods. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs.
  • Identity can be determined by using, for example, the BLAST (Basic Local Alignment Search Tool) program (for example, Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410(1990), Altschul SF, Madden TL, Schaffer AA, Zhang Z, Miller W, Lipman DJ,. Nucleic Acids Res. 25: p3389-3402 (1997)), however methods of determining identity are not hmited to this. Where software such as BLAST is used, it is preferable to use default values.
  • the main initial conditions generally used in a BLAST search are as follows, but are not limited to these.
  • An amino acid substitution matrix is a matrix numerically representing the degree of analogy of each pairing of each of the 20 types of amino acid, and normally the default matrix, BLOSUM62, is used.
  • the theory of this amino acids substitution matrix is shown in Altschul S.F., J. Mol. Biol. 219: 555-565 (1991), and its applicability to DNA sequence comparison is shown in States D. J., Gish W., Altschul S.F., Methods, 3: 66-70 (1991).
  • optimal gap cost is deterrnined empirically and in the case of BLOSUM62, preferably parameters, Existence 11, Extension 1 are used.
  • the expected value (EXPECT) is the threshold value concerning statistical significance for a match with a database sequence, and the default value is 10.
  • the present invention further provides the following isolated polynucleotides:
  • polynucleotides comprising a nucleotide sequence which has at least 95% identity, preferably at least 97-99% identity to any one of the above sequences, and encodes a protein which phosphorylate Elkl and/or activate kinase which phosphorylates Elkl;
  • Polynucleotides which are identical or almost identical to a nucleotide sequences contained in the above nucleotide sequence may be used as hybridization probes or as primers for a nucleic acid amplification reaction to isolate full-length cDNAs and genomic clones encoding proteins of the present invention or cDNA and genomic clones of other genes that have a high sequence homology to the above sequences.
  • these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical to the above sequences.
  • the probes or primers will generally comprises at least 15 nucleotides, preferably 30 nucleotides and may have 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred primers have between 20 and 25 nucleotides.
  • the polynucleotide of the present invention may be either in the form of a DNA such as cDNA ,a genomic DNA obtained by cloning or synthetically produced, or may be in the form of RNA such as mRNA.
  • the polynucleotide may be single-stranded or double-stranded.
  • the double- stranded polynucleotides may be double-stranded DNA, double-stranded RNA or DNA:RNA hybrid.
  • the single- stranded polynucleotide may be sense strand also known as coding strand or antisense strand also known as non-coding strand.
  • a protein having the same action of phosphorylating Elkl and/or the same action of activating kinase which phosphorylates Elkl as the protein having an amino acid sequence of any one of SEQ ID NOS 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108, by means of appropriate substitution of an amino acid in the protein using known methods.
  • One such method involves conventional mutagenesis procedures using mutagens for the DNA encoding the protein.
  • Another method is, for example, site-directed mutagenesis (e.g., Mutan-Super Express Km Kit from Takara Shuzo Co., Ltd.). Mutations of amino acids in proteins may also occur in nature.
  • the present invention also includes a mutated protein which is capable of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl and which has at least one amino acid deletion, substitution or addition compared to the corresponding amino acid sequence of SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 or 108.
  • the number of mutations is preferably 1 to 10, more preferably 1 to 5, most preferably 1 to 3.
  • amino acid substitutions are preferably conservative substitutions, and include substitutions within the following groups: (glycine, alanine), (valine, isoleucine, leucine), (aspartic acid, glutamic acid), (asparagine, glutamine), (serine, threonine), (lysine, arginine) and (phenylalanine, tyrosine).
  • nucleotide sequence e.g., SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 107) encoding a protein consisting of an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102,
  • the present invention also includes a protein that phosphorylates Elkl and/or activates kinase which phosphorylates Elkl and comprises an amino acid sequence having a high identity to the amino acid sequence of above SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 or 108.
  • a protein that phosphorylates Elkl and/or activates kinase which phosphorylates Elkl and comprises an amino acid sequence having a high identity to the amino acid sequence of above SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,
  • High identity refers to an amino acid sequence having an identity of at least 90%, preferably at least 97-99% over the entire length of any one of the above SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108.
  • the proteins of the present invention may be natural proteins derived from any human or animal cells or tissues, chemically synthesized proteins, or proteins obtained by genetic recombination techniques.
  • the protein may or may not be subjected to post-translational modifications such as sugar chain addition or phosphorylation.
  • Examples of the protein encoded by the gene of the present invention includes secretory proteins (growth factors, cytokines, hormones, etc.), protein modifying enzymes (protein kinases, protein phosphatases, proteases, etc), signal transduction molecules (adaptor molecules between proteins, etc.), nuclear proteins (nuclear receptors, transcription factors) and membrane proteins.
  • Membrane proteins include receptors, cell adhesion molecules, ion channels, transporters, etc. Where the protein is a membrane protein, the protein is more useful as a research tool of medical compound since a compound selected by the below-described screening is expected to easily migrate into a cell or give signal transduction into a cell.
  • the present invention also includes a polynucleotide encoding the above protein of the present invention.
  • nucleotide sequences encoding a protein consisting of an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 or 108 include a nucleotide sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79,
  • the DNA includes cDNA, genomic DNA, and chemicaUy synthesized DNA.
  • the DNA of the present invention includes a DNA which encodes a protein capable of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl and hybridizes under stringent conditions with the DNA sequence of any one of the above nucleotide sequences of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107 or a polynucleotide sequence complementary to said nucleotide sequence.
  • Stringent conditions are apparent to those skilled in the art, and can be easily attained in accordance with various laboratory manuals such as T. Maniatis et al., Molecular Cloning
  • stringent conditions refer to overnight incubation at 37°C in a hybridization solution containing 30% formamide, 5 x SSC (0.75 M NaCl, 75mM trisodium citrate),5 x Denhardt's solution, 0.5% SDS, lOO ⁇ g/ml denatured, sheared salmon sperm DNA) followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 minutes at room temperature, then followed by washing (two times) in 1 x SSC, 0.1% SDS for 10 minutes at 37°C(low stringency).
  • Preferred stringent conditions are overnight incubation at 42 °C in a hybridization solution containing 40% formamide, followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 minutes at room temperature, then followed by washing (two times) in 0.2 x SSC, 1% SDS for 10 minutes at 42°C(moderate stringency). More preferred stringent conditions are overnight incubation at 42 °C in a hybridization solution containing 50% formamide, followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 minutes at room temperature, followed by washing (two times) in 0.2 x SSC, 0.1% SDS for 10 minutes at 50°C (high stringency).
  • the DNA sequence thus obtained must encode a protein capable of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl.
  • the present invention also includes a polynucleotide comprising a nucleotide sequence which encodes a protein capable of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl and has a high sequence similarity to the nucleotide sequence of the polynucleotide according to above item (3), (4) or (5).
  • these nucleotide sequence are 95% identical, preferably 97% identical, most preferably at least 99% identical to the nucleotide sequence of the polynucleotide according to above item (3), (4) or (5) over the entire length thereof.
  • the above DNA of the present invention can be used to produce the protein of above item (1), (2) or (8) using recombinant DNA techniques.
  • the DNA and peptide of the present invention can be obtained by:
  • Techniques for cloning the DNA encoding the protein of the above step (A) include, in addition to the methods described in the specification of the present application, PCR amplification using synthetic DNA containing a part of the nucleotide sequence of the present invention (e.g., SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 107) as a primer, and selection of the DNA inserted into a suitable vector by hybridization with a labeled DNA fragment encoding a partial or full coding region of the protein of the present invention or a labeled synthetic DNA.
  • Another technique involves direct amplification of DNA from total RNA
  • a DNA inserted into a suitable vector for example, a commercially available library (e.g., from CLONTECH and STRATAGENE) can be used. Techniques for hybridization are normally used in the art, and can be easily carried out in accordance with various laboratory manuals such as T. Maniatis et al., supra. Depending on the intended purpose, the cloned DNA encoding the protein of the present invention can be used as such or if desired after digestion with a restriction enzyme or addition of a linker.
  • the DNA thus obtained may have a nucleotide sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 107, or a polynucleotide of above items (3) to (7).
  • the DNA sequence to be inserted into an expression vector in the above step (B) may be a full-length cDNA or a DNA fragment encoding the above full-length protein, or a DNA fragment constructed so that it expresses a part thereof.
  • the present invention also includes a recombinant vector, which comprises the above DNA sequence.
  • the expression vector for the protein of the present invention can be produced, for example, by excising the desired DNA fragment from the DNA encoding the protein of the present invention, and ligating the DNA fragment downstream of a promoter in a suitable expression vector.
  • Expression vectors for use in the present invention may be any vectors derived from prokaryotes (e.g., E. coli), yeast, fungi, insect viruses and vertebrate viruses. However, the vectors should be selected to be compatible with the hosts. Suitable combinations of host cell - expression vector systems are selected depending on the desired expression product.
  • Plasmid vectors compatible with these bacteria are generally used as replicable expression vectors for recombinant DNA molecules.
  • the plasmids pBR322 and pBR327 can be used to transform E. coli.
  • Plasmid vectors normally contain an origin of replication, a promoter, and a marker gene conferring upon a recombinant DNA a phenotype useful for selecting the cells transformed with the recombinant DNA.
  • promoters include a ⁇ -lactamase promoter, lactose promoter and tryptophan promoter.
  • marker genes include an ampicillin resistance gene, and a tetracycline resistance gene.
  • suitable expression vectors include the plasmids pUC18 and pUC19 in addition to ⁇ BR322, pBR327.
  • YEp24 can be used as a replicable vector.
  • the plasmid YEp24 contains the URA3 gene, which can be employed as a marker gene.
  • promoters in expression vectors for yeast cells include promoters derived from genes for 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase.
  • promoters and terminators for use in expression vectors to express the DNA of the present invention in fungal cells include promoters and terminators derived from genes for 3-phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPD) and actin.
  • suitable expression vectors include the plasmids pPGACY2 and pBSFAHY83.
  • promoters for use in expression vectors to express the DNA of the present invention in insect cells include a polyhedrin promoter and P10 promoter.
  • expression vector suitable for insect cells include baculovirus.
  • Recombinant vectors used to express the DNA of the present invention in animal cells normally contain functional sequences to regulate genes, such as a promoter to be placed upstream of the DNA of the present invention, a polyadenylation site and a transcription termination sequence.
  • Such functional sequences which can be used to express the DNA of the present invention in eukaryotic cells, can be obtained from viruses or and viral substances.
  • Examples of such functional sequences include an SR promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter and HSV-TK promoter. Among them, a CMV promoter and SR ct promoter can be preferably used. Promoters to be placed inherently upstream of the gene encoding the protein of the present invention, can be used so long as they are suitable for use in the above host-vector systems.
  • Examples of origins of replication include foreign origins of replication, for example, those derived from viruses such as adenovirus, polyoma virus and SV40 virus. When vectors capable of integration into host chromosomes are used as expression vectors, origins of replication of the host chromosomes may be employed. Examples of suitable expression vectors include the plasmids pSV-dhfr (ATCC 37146), pBPV-l(9-l) (ATCC 37111), pcDNA3.1 (INVITROGEN) andpME18S-FL3.
  • the present invention also includes a transformed cell, which comprises the above recombinant vector.
  • Microorganisms or cells transformed with the replicable recombinant vector of the present invention can be selected from remaining untransformed parent cells based on at least one phenotype conferred by the recombinant vector. Phenotypes can be conferred by inserting at least one marker gene into the recombinant vector. Marker genes naturally contained in replicable vectors can be employed. Examples of marker genes include drug resistance genes such as neomycin resistance genes, and genes encoding dihydrofolate reductase.
  • any of prokaryotes e.g., E. coli
  • microorganisms e.g., yeast and fungi
  • insect and animal cells can be used so long as such hosts are compatible with the expression vectors used.
  • microorganisms include Escherichia coli strains such as E. coli K12 strain 294 (ATCC 31446), E. coli X1776 (ATCC 31537), E. coli C600, E. coli JM109 and E. coli B strain; bacterial strains belonging to the genus Bacillus such as Bacillus subtilis; intestinal bacteria other than E.
  • yeast such as Salmonella typhimurium or Serratia marcescens
  • various strains belonging to the genus Pseudomonas include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris.
  • yeast include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris.
  • fungi include Aspergillus nidulans, and Acremonium chrysogenum (ATCC 11550).
  • insect cells for example, Spodoptera frugiperda (Sf cells), High Five TM cells derived from eggs of Trichoplusiani, etc.
  • insect cells for example, Spodoptera frugiperda (Sf cells), High Five TM cells derived from eggs of Trichoplusiani, etc.
  • animal cells include HEK 293 cells, COS-1 cells, COS-7 cells, Hela cells, and Chinese hamster ovary (CHO) cells.
  • CHO cells and HEK 293 cells are preferred.
  • combinations of expression vectors and host cells to be used vary with experimental objects. According to such combinations, two types of expression (i.e. transient expression and constitutive expression) can be included.
  • Transformation of microorganisms and cells in the above step (C) refers to introducing DNA into microorganisms or cells by forcible methods or phagocytosis of cells and then transiently or constitutively expressing the trait of the DNA in a plasmid or an intra-chromosome integrated form.
  • Those skilled in the art can carry out transformation by known methods [see e.g., "Idenshi Kougaku Handbook (Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.].
  • DNA can be introduced into cells by known methods such as DEAE-dextran method, calcium-phosphate-mediated transfection, electroporation, lipofection, etc.
  • selection can be carried out by clonal selection of the animal cells containing the chromosomes into which the introduced expression vectors have been integrated.
  • transformants can be selected using the above selectable marker as an indication of successful transformation.
  • the animal cells thus obtained using the selectable marker can be subjected to repeated clonal selection to obtain stable animal cell strains highly capable of expressing the protein of the present invention.
  • DHFR dihydrofolate reductase
  • MTX methotrexate
  • the above transformed cells can be cultured under conditions which permit the expression of the DNA encoding the protein of the present invention to produce and accumulate the protein of the present invention. In this manner, the protein of the present invention can be produced.
  • the present invention also includes a process for producing a protein, which comprises culturing a transformed cell comprising the isolated polynucleotide according to above item (3) to (7) under conditions providing expression of the encoded protein and recovering the protein from the culture (i.e., host itself or media).
  • animal cells can be cultured by methods known to those skilled in the art (see e.g., "Bio Manual Series 4", YODOSHA CO., LTD.).
  • animal cells can be cultured by various known animal cell culture methods including attachment culture such as Petri dish culture, multitray type culture and module culture, attachment culture in which cells are attached to cell culture carriers (microcarriers), suspension culture in which productive cells themselves are suspended.
  • attachment culture such as Petri dish culture, multitray type culture and module culture
  • suspension culture in which productive cells themselves are suspended.
  • media for use in the culture include media commonly used for animal cell culture, such as D-MEM, I-MDM and RPMI 1640.
  • suitable combinations of per se known separation and purification methods can be used.
  • methods include methods based on solubility, such as salting-out and solvent precipitation; methods based on the difference in charges, such as ion-exchange chromatography; methods mainly based on the difference in molecular weights, such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis; methods based on specific affinity, such as affinity chromatography; methods based on the difference in hydrophobicity, such as reverse phase high performance hquid chromatography; and methods based on the difference in isoelectric points, such as isoelectric focusing.
  • a protein of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography.
  • high performance liquid chromatography is employed for purification.
  • Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation or purification.
  • the protein of the present invention can also be produced as a fusion protein with another protein. These fusion proteins are also included within the present invention.
  • any vectors can be used so long as the DNA encoding the protein can be inserted into the vectors and the vectors can express the fusion protein.
  • proteins to which a polypeptide of the present invention can be fused include glutathione S-transferase (GST) and a hexa-histidine sequence (6 x His).
  • GST glutathione S-transferase
  • 6 x His hexa-histidine sequence
  • membrane protein When the protein of the present invention is a membrane protein, a transformant into which DNA encoding the protein of the present invention has been introduced can express the protein on its membrane.
  • the membrane which is prepared from such transformants and contains the protein of the present invention is also included within the present invention.
  • membrane of a cell includes cell membrane, and membrane of cell organelle.
  • the membrane of a cell can be prepared by a method known to those skilled in the art. For example, cells are collected from the culture where transformants are cultured, and suspended in a suitable buffer. Then, the cells are lysed by a homogenizer or by vortex after addition of glassbeads.
  • the obtained solution is centrifuged to remove uncrushed cells and the like, and the supernatant is ultracentrifuged under a sutable condition, and the obtained precipitate is suspended in a buffer to prepare. a membrabe fraction.
  • the condition for ulfracentrifugation can be suitably selected depending on the type of membrane and the like.
  • the present invention also provides a protein capable of inhibiting the activity of the protein of the present invention.
  • proteins include antibodies, or other proteins that bind to active sites of the protein of the present invention, thereby inhibiting the expression of their activity.
  • the present invention also relates to an antibody that reacts with the protein of the present invention or a fragment thereof, and to production of such an antibody. More preferably, the present invention relates to an antibody that specifically react with the protein of the present invention or a fragment thereof, and to production of such an antibody.
  • “specifically” means that closs-reactivity is low, more preferably closs-reactivity is not present.
  • the antibody of the present invention is not specifically limited so long as it can recognize the protein of the present invention.
  • examples of such antibodies include polyclonal antibodies, monoclonal antibodies and their fragments, single chain antibodies and humanized antibodies.
  • Antibody fragments can be produced by known techniques. Examples of such antibody fragments include, but not limited to, F(ab') 2 fragments, Fab' fragments, Fab fragments and Fv fragments.
  • a monoclonal or polyclonal antibody can be produced by administering the protein according to above item (1) or (2) or epitope-bearing fragments as an antigen to a non-human animal.
  • the antibody against the protein of the present invention can be produced by using the protein of the present invention or a peptide thereof as an immunogen according to per se known process for producing antibodies or antisera.
  • Such methods are described, for example, in "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition, an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.
  • polyclonal antibodies for example, the protein of the present invention or a peptide thereof can be injected to animals such as rabbits to produce antibodies directed against the protein or peptide, and then their blood can be collected.
  • the polyclonal antibodies can be purified from the blood, for example, by ammonium sulfate precipitation or ion-exchange chromatography, or by using the affinity column on which the antigen protein has been immobilized.
  • monoclonal antibodies for example, animals such as mice are immunized with the protein of the present invention, their spleen is removed and homogenized to obtain spleen cells, which are then fused with mouse myeloma cells by using a reagent such as polyethylene glycol. From the resulting fused cells (i.e. hybridoma), the clone producing the antibody directed against the protein of the present invention can be selected. Then, the resulting clonal hybridoma can be implanted intraperitoneally into mice, and the ascitic fluid was recovered from the mice. The resulting monoclonal antibody can be purified, for example, by ammonium sulfate precipitation or ion-exchange chromatography, or by using the affinity column on which the antigen protein has been immobilized.
  • humanized antibody or human antibody When the resulting antibody is used to administer to humans, it is preferable to use a humanized antibody or human antibody in order to reduce its immunogenicity.
  • humanized antibodies or human antibodies can be produced using transgenic mice or other mammals.
  • Humanized chimeric antibodies can be produced by hnking a V region of a mouse antibody to a C region of a human antibody. Humanized antibodies can be produced by substituting a sequence derived from a human antibody for a region other man a complementarity-determining region (CDR) from a mouse monoclonal antibody.
  • CDR complementarity-determining region
  • human antibodies can be directly produced in the same manner as the production of conventional monoclonal antibodies by immunizing the mice whose immune systems have been replaced with human immune systems. These antibodies can be used to isolate or to identify clones expressing the protein.
  • these antibodies can be used to purify the protein of the present invention from a cell extract or transformant producing the protein of the present invention.
  • These proteins can also be used to construct ELISA, RIA (radioimmunoassay) and western blotting systems.
  • ELISA electrospray
  • RIA radioimmunoassay
  • western blotting systems can be used for diagnostic purposes for detecting an amount of the protein of the present invention present in a body sample in a tissue or a fluid in the blood of an animal, preferably human.
  • these antibodies can be used for diagnosis of a disease characterized by undesirable activation of MAP kinase cascade resulting from (expression) abnormality of the protein of the present invention, such as inflammation, autoimmune diseases, infection diseases (for example, HTV infection), and cancer.
  • a standard value In order to provide a basis for diagnosis of a disease, a standard value must be estabhshed. This is a well-known technique to those skilled in the art.
  • a method of calculating the standard value comprises binding a body fluid or a cell extract of normal individual of a human or an animal to an antibody against the protein of the present invention under a suitable condition for the complex formation, detecting the amount of the antibody-protein complex by chemical or physical means and then calculating the standard value for the normal sample using a standard curve prepared from a standard solution containing a known amount of an antigen (the protein of the present invention).
  • the presence of a disease can be confirmed by deviation from the standard value obtained by comparison of the standard value with the value obtained from a sample of an individual latently suffering from a disease associated with the protein of the present invention.
  • These antibodies can also be used as reagents for studying functions of the protein of the present invention.
  • the antibody of the present invention can be used as a medicament as mentioned below.
  • an antibody capable of inhibiting the function of phosphorylation Elkl and/or the function of activating kinase which phosphorylates Elkl which is possessed by the protein of present invention that is, neutralizing antibody.
  • the present invention is a pharmaceutical composition which comprises the above antibody as an active ingredient, and therapy and/or prevention using the antibody of the present invention, -h such pharmaceutical compositions, the active ingredient may be combined with other therapeutically active ingredients or inactive ingredients (e.g., conventional pharmaceutically acceptable carriers or diluents such as immunogenic adjuvants) and physiologically non-toxic stabilizers and/or excipients.
  • the resulting combinations can be sterilized by filtration, and formulated into vials after lyophilization or into various dosage forms in stabilized and preservable aqueous preparations.
  • Administration to a patient can be intra-arterial administration, intravenous administration and subcutaneous administration, which are well known to those skilled in the art.
  • the dosage range depends upon the weight and age of the patient, route of administration and the like. Suitable dosages can be determined by those skilled in the art.
  • These antibodies exhibit therapeutic activity by inhibiting the MAP kinase activation mediated by the protein of the present invention.
  • the antibody of the present invention can be useful as a medicament for treatment or prevention of diseases characterized by abnormal activation of MAP kinase cascade, such as inflammation, autoimmune diseases, infection diseases (for example, HIV infection), and cancer.
  • the DNA of the present invention can also be used to isolate, identify and clone other proteins involved in intracellular signal transduction processes.
  • the DNA sequence encoding the protein of the present invention can be used as a "bait" in yeast two-hybrid systems (see e.g., Nature 340:245-246 (1989)) to isolate and clone the sequence encoding a protein ("prey") which can associate with the protein of the present invention.
  • prey a protein which can associate with the protein of the present invention.
  • proteins which can associate with the protein of the present invention can be isolated from cell extracts by immunoprecipitation [see e.g., "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.] using antibodies directed against the protein of the present invention.
  • the protein of the present invention can be expressed as a fusion protein with another protein as described above, and immunoprecipitated with an antibody directed against the fusion protein in order to isolate a protein which can associate with the protein of the present invention.
  • the present invention provides a process for diagnosing a disease or susceptibility to a disease related to expression or activity of the protein of present invention in a subject comprising the steps of:
  • the diagnostic assays offer a. process for diagnosing diseases or determining a susceptibihty to the diseases through detection of mutation in a gene for the protein of the present invention which is involved in the function of phosphorylating Elkl and/or the function of activating kinase which phosphorylates Elkl.
  • diseases may be diagnosised by analyzing expression level of the gene in a sample derived from a subject at protein or mRNA level, and detecting an abnormally decreased or increased level of the expression.
  • Determination of the presence or absence of a mutation in the gene encoding the protein of the present invention which is involved in the function of phosphorylating Elkl and/or the function of activating kinase which phosphorylates Elkl may involve RT-PCR using a part of the nucleotide sequences of genes as a primer, followed by conventional DNA sequencing to detect the presence or absence of the mutation.
  • PCR-SSCP Genetics 5:874-879 (1989); "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.] can also be used to determine the presence or absence of the mutation.
  • Decreased or increased expression of a gene in a sample can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, for example, nucleic acid amplification methods such as RT-PCR, and methods such as RNase protection assay, Northern blotting and other hybridization methods.
  • Assay techniques that can be used to determine levels of a protein in a sample derived from a host are well-known to those skilled in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western blot analysis and ELISA assays.
  • the antibody of the present invention mentioned above can be used.
  • the degree of abnormality of expression level of gene in a sample is not particularly limited.
  • the level of the expressed protein is 2 or more times, or 1/2 or less, as compared with normal case, the subject may be diagnosed to be a disease.
  • the level of the expressed protein is 3 or more times, or 1/3 or less, as compared with normal case, the subject may be diagnosed to be a disease.
  • the mutation may cause a disease associated with the expression and/or activity of said protein which is involved in phosphorylation of Elkl.
  • the abnormal expression of the protein of the present invention which is involved in phosphorylation of Elkl may be responsible for diseases associated with the expression and/or activity of said protein which is involved in phosphorylation of Elkl.
  • the present invention also includes a method for screening compounds which inhibit or promote an action of activating MAP kinase cascade mediated by the protein of the present invention (an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl), which comprises the steps of:
  • a compound that increases the detectable signal 2-fold or higher than normal is preferably isolated or identified as an activator compound, and a compound that decreases the detectable signal 50% or less than normal is preferably isolated or identified as an inhibitor compound.
  • genes encoding a signal which can detect the action of Elkl phosphorylation include reporter genes. Reporter genes are used to detect the activity of trasnscription factor instead of directly detecting the activation of transcription factors of interest to be tested. The transcriptional activity of a promoter of a gene is analyzed by linking the promoter to a reporter gene and measuring the activity of the product of the reporter gene ("Bio Manual Series 4" (1994), YODOSHA CO., LTD.).
  • Any genes which code the peptide or protein can be used as the reporter genes so long as those skilled in the art can measure the activity or amount of the expression product (including the amount of the produced mRNA). For example, enzymatic activity of chloramphenicol acetyltransferase, j3 -galactosidase, luciferase, etc., can be measured.
  • Any reporter plasmids can be used to evaluate activation of Elkl phosphorylation.
  • One example is the reporter plasmids that have a yeast transcription factor Gal4 recognition sequence inserted upstream of the reporter gene.
  • pFR-Luc can be used by introduction into a host cell together with various fusion proteins of genes being substrates of MAP kinase such as Elkl and ATF2 connected to yeast Gal4 protein DNA binding region gene: e.g. pFA2-Elkl orpFA2-ATF2 (STRATAGENE).
  • Any host cells can he used so long as action of Elkl phosphorylation and/or action of activation of kinase which phosphorylates Elkl can be detected in the host cells.
  • Preferred host cells are mammalian cells such as HEK293-EBNA cells. Transformation and culture of the cells can be carried out as described above.
  • the method for screening a compound which inhibits or promotes action of Elkl phosphorylation and/or action of activation of kinase which phosphorylates Elkl comprises culturing the transformed cell for a certain period of time, adding a certain amount of a test compound, measuring the reporter activity expressed by the cell after a certain period of time, and comparing the activity with that of a cell to which the test compound has not been added.
  • the reporter activity can be measured by methods known in the art (see e.g., "Bio Manual Series 4" (1994), YODOSHA CO., LTD.).
  • test compounds for the screening include, but not limited to, low molecular weight compounds, high molecular weight compounds and peptides.
  • Test compounds may be artificially synthesized compounds or naturally occurring compounds.
  • Test compounds may be a single compound or mixtures.
  • Usable examples includes a library of low molecular weight compounds, a compound hbrary which was synthesized by combinatorial chemistry, a narurally occurring product containing cells, plants, animals or a part thereof, or an extracred product of such narurally occurring product.
  • test substance which shows an activity of inhibiting or promoting action of Elkl phosphorylation and/or action of activation of kinase which phosphorylates Elkl can be further screened to isolate a single substance having the activity.
  • Isolation and purification of a desired compound from a mixture can be carried out by using any known method such as filteration, extraction, washing, drying, concentration, crystallization or various chromatography in combination.
  • the method for screening according to the present invention can be carried out by the following steps:
  • examples of the method of measuring an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl include a method of obtaining cell extracts and detecting the activation of various MAP kinases using an antibody against various phosphorylated MAP kinases.
  • the antibodies against various phosphorylated MAP kinases are available from NEB (New England Biolabs).
  • the examples of the method of detection using the antibody include ELISA, Western blotting, dot hybridyzation and the like.
  • the activity of various MAP kinases can be directly measured by an immunoprecipitation method using an antibody against various MAP kinases.
  • the antibody against various MAP kinases, and substrates thereto are available from Upstate Biotechnology.
  • the present invention further provides a method of producing a pharmaceutical composition, which comprises the following steps (a) to (e):
  • a pharmaceutical composition may also be produced by the following steps (a) to (e):
  • step (d) of the method of producing a pharmaceutical composition it is preferable to isolate or identify as an activator compound, a compound that increases said detectable signal 2-fold or higher than normal, and to isolate or identify as an inhibitor compound, a compound that decreases said detectable signal 50% or less than normal.
  • the protein of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the protein, by:
  • the present invention also provides a compound which is selected by the above screening method.
  • This compound has an activity of inhibiting or promoting the activation of MAP kinase cascade mediated by the protein of the present invention. More specifically, this compound has an activity of inhibiting or promoting an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl which are mediated by the protein of the present invention.
  • the compounds obtained by the above screening methods have an activity of inhibiting or promoting an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, they are useful as therapeutic or preventive pharmaceuticals for the diseases resulting from unfavorable activation or inactivation of Elkl phosphorylation and/or kinase which phosphorylates Elk.
  • a compound which is obtained in the form of a salt can be purified as it is.
  • a compound which is obtained in the free form can be converted into a salt by isolating and purifying a salt obtained by dissolving or suspending the compound into a suitable solvent by conventional methods and then adding a desired acid or base.
  • Examples of a step to optimize the compounds or salts thereof obtained by the method of the present invention as a pharmceutical composition include methods of formulating according to ordinary processes such as the following.
  • the above compounds or their pharmaceutically acceptable salts in an amount effective as an active ingredient, and pharmaceutically acceptable carriers can be mixed.
  • a form of formulation suitable for the mode of administration is selected.
  • a composition suitable for oral administration includes a solid form such as tablet, granule, capsule, pill and powder, and solution form such as solution, syrup, elixir and dispersion.
  • a form useful for parenteral administration includes sterile solution, emulsion and suspension.
  • the above carriers include, for example, sugars such as gelatin, lactose and glucose, starches such as corn, wheat, rice and maize, fatty acids such as stearic acid, salts of fatty acids such as calcium stearate, magnesium stearate, talc, vegetable oil, alcohol such as stearyl alcohol and benzyl alcohol, gum, and polyalkylene glycol.
  • examples of such liquid carriers include generally water, saline, sugar solution of dextrose and the like, glycols such as ethylene glycol, propylene glycol and polyethylene glycol.
  • the present invention also includes a kit for screening a compound for activity as an inhibitor or activator of Elkl phosphorylation and/or activation of a kinase which phosphorylates Elkl.
  • the kit comprises reagents, etc. necessary for screening compounds for activity as an inhibitor or activator of Elkl phosphorylation and or activation of a kinase which phosphorylates Elkl, including:
  • a transformant comprising a gene encoding a protein that phosphorylates Elkl and/or activates kinase which phosphorylates Elkl according to the present invention and a gene encoding a protein which provides a signal which can detect an action of Elkl phosphorylation; and (b) reagents for measuring the signal.
  • the present invention relates to a diagnostic kit which comprises:
  • a polynucleotide of the present invention having a nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 107;
  • a protein of the present invention having an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 or 108 or a fragment thereof; or
  • a kit which comprises at least one of (a), (b), (c) or (d), is useful for diagnosing a disease or susceptibility to a disease such as inflammation, autoimmune diseases, infectious diseases (e.g., HIV infection) and cancers.
  • a disease or susceptibility to a disease such as inflammation, autoimmune diseases, infectious diseases (e.g., HIV infection) and cancers.
  • MAP kinase cascade is involved in a wide variety of pathological conditions such as inflammation, autoimmune diseases, cancers and viral infections, it is an attractive target for drug design and therapeutic intervention.
  • Many experiments show the possibility that the inhibition of MAP kinase cascade activity has significant physiological effects [e.g., J Cell Biochem 2001 Apr 3-27; 82(1): 68-77, Pharmacol Res 2001 Mar; 43(3): 275-83, Diabetes 2001 Jun; 50(6): 1495-504, Diabetes 2001 Jun; 50(6): 1464-71, Atherosclerosis 2001 May; 156 (1): 81-90].
  • the present invention also relates to use of a compound which inhibits the function of the protein capable of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl described above, for inhibiting MAP kinase cascade activation.
  • the present invention also relates to use of a compound which promotes the function of the protein capable of phosphorylating Elkl and or activating kinase which phosphorylates Elkl described above, for promoting MAP kinase cascade activation.
  • the compound obtained by the above screening method which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, is useful as a medicament to treat or prevent diseases characterized by undesirable activation of MAP kinase cascade, such as inflammation, autoimmune diseases, infectious diseases (e.g., HIV infection) and cancers.
  • diseases characterized by undesirable activation of MAP kinase cascade such as inflammation, autoimmune diseases, infectious diseases (e.g., HIV infection) and cancers.
  • GVHD skin diseases such as toxic epidermal necrolysis (TEN), prohferative nephritis (e.g., IgA nephritis, purpuric nephritis and lupus nephritis) and fulminant hepatitis, hi contrast
  • examples of patients for whom the induction of apoptosis may be exploited in treatment include tumor patients.
  • the compound obtained by the above screening method which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, is useful as a medicament to treat or prevent these diseases.
  • the gene encoding the protein of the present invention is useful for gene therapy to treat various diseases such as cancers, autoimmune diseases, allergy diseases and inflammatory response.
  • Gene therapy refers to administering into the human body a gene or a cell into which a gene has been introduced.
  • the protein of the present invention and the DNA encoding the protein can also be used for diagnostic purposes.
  • the present invention provides an agent for gene therapy which comprises a gene encoding the protein of the present invention.
  • RNAi RNA interference
  • the form of the agent for gene therapy is not particularly limited, but includes a pharmaceutical composition which comprises a expression vector containing a gene of the present invention in a pharmaceutical carrier of physiological buffer.
  • the pharmaceutical carrier may contain suitable stabilizer (for example, nuclease inhibitor), chelate agent (for example, EDTA), and/or other auxiliary agent.
  • the agent for gene therapy of the present invention may be provided as a complex of an expression vector containing a gene of the present invention and a liposome.
  • the agent for gene therapy may be applied using a catheter.
  • the agent for gene therapy of the present invention can be directly injected into a blood vessel of patient and the like.
  • the dosage of the agent for gene therapy of the present invention should be selected depending on the conditions such as age, sex, body weight and symptom of patient, and administration route, and is generally about 1 ⁇ g/kg to about 1000 mg/kg, more preferably about 10 g kg to about 100 mg/kg, as an amount of DNA (which is an effective ingredient) per one administration for adult.
  • the number of administration is not particularly limited.
  • the compound obtained by the screening method of the present invention or a salt thereof can be formulated into the above pharmaceutical compositions (e.g., tablets, capsules, elixirs, microcapsules, sterile solutions and suspensions) according to conventional procedures.
  • the formulations thus obtained are safe and of low toxicity, and can he administered, for example, to humans and mammals (e.g., rats, rabbits, sheep, pigs, cattle, cats, dogs and monkeys).
  • Administration to patients can be carried out by methods known in the art, such as intra-arterial injection, intravenous injection and subcutaneous injection.
  • the dosage and the administration route may vary with the weight and age of the patient, but those skilled in the art can appropriately select the administration route, and can appropriately select suitable dosage which is suitable for the administration route.
  • the compound can be encoded by DNA
  • the DNA can be inserted into a vector for gene therapy, and gene therapy can be carried out.
  • the present invention relates to a medicament which comprises a compound which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl.
  • the above compound is useful as a medicament to treat or prevent diseases characterized by undesirable activation of MAP kinase cascade, such as inflammation, autoimmune diseases, viral diseases, infectious diseases and cancers.
  • diseases characterized by undesirable activation of MAP kinase cascade such as inflammation, autoimmune diseases, viral diseases, infectious diseases and cancers.
  • the present invention also relates to a pharmaceutical composition for inflammation, autoimmune diseases, viral diseases, cancers, etc., which comprises the compound which promotes or inhibits the activation of MAP kinase cascade.
  • the pharmaceutical composition is useful as a therapeutic and prophylactic drug against, for example, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, diabetes, sepsis, asthma, allergic rhinitis, ischemic heart diseases, inflammatory intestinal diseases, subarachnoid hemorrhage, viral hepatitis, AIDS, GVHD, skin conditions such as toxic epidermal necrolysis (TEN), hyperplastic nephritis (IgA nephritis, purpuric nephritis, lupus nephritis), and fulminant hepatitis.
  • TEN toxic epidermal necrolysis
  • IgA nephritis IgA nephritis, purpuric nephritis, lupus nephritis
  • fulminant hepatitis for example, rheumatoid arthritis, osteo
  • the present invention also relates to the use of the aforementioned compound for manufacturing a medicament for therapy and prevention of inflammation, autoimmune diseases, viral diseases, cancers, etc.
  • the present invention also provides an antisense oligonucleotide against the polynucleotide of any one of above items (3) to (7).
  • An antisense oligonucleotide refers to an oligonucleotide complementary to the target gene sequence.
  • the antisense oligonucleotide can inhibit the expression of the target gene by inhibiting RNA functions such as translation to proteins, transport to the cytoplasm and other activity necessary for overall biological functions.
  • the antisense oligonucleotide may be RNA or DNA.
  • the DNA sequence of the present invention can be used to produce an antisense oligonucleotide capable of hybridizing with the mRNA transcribed from the gene encoding the protein of the present invention.
  • an antisense oligonucleotide generally has an inhibitory effect on the expression of the corresponding gene (see e.g., Saibou Kougaku Vol.13, No .4 (1994)).
  • the oligonucleotide containing an antisense coding sequence against a gene encoding the protein ofthe present invention can be introduced into a cell by standard methods.
  • the oligonucleotide effectively blocks the translation of mRNA of the gene encoding the protein of the present invention, thereby blocking its expression and inhibiting undesirable activity.
  • the antisense oligonucleotide of the present invention may be a naturally occurring oligonucleotide or its modified form [see e.g., Murakami & Makino, Saibou Kougaku Vol.13, No.4, p.259-266 (1994); Akira Murakami, Tanpakushitsu Kakusan Kouso (PROTEIN, NUCLEIC ACID AND ENZYME) Vol.40, No.lO, p.1364-1370 (1995),Tunenari Takeuchi et al., Jikken Igaku (Experimental Medicine) Vol. 14, No. 4 p85-95(1996)].
  • the oligonucleotide may have modified sugar moieties or inter-sugar moieties.
  • modified forms include phosphothioates and other sulfur-containing species used in the art.
  • at least one phosphodiester bond in the oligonucleotide is substituted with the structure which can enhance the ability of the composition to permeate cellular regions where RNA with the activity to be regulated is located.
  • Such substitution preferably involves a phosphorothioate bond, a phosphoramidate bond, methylphosphonate bond, or a short-chain alkyl or cycloalkyl structure.
  • the oligonucleotide may also contain at least some modified base forms. Thus, it may contain purine and pyrimidine derivatives other than naturally occurring purine and pyrimidine.
  • the furanosyl moieties of the nucleotide subunits can be modified so long as the essential purpose of the present invention is attained. Examples of such modifications include 2'-O-aIkyl and 2'-haIogen substituted nucleotides.
  • sugar moieties at their 2- ⁇ osition examples include OH, SH, SCH 3 , OCH 3 , OCN or O(CH 2 ) n CH 3 , wherein n is 1 to about 10, and other substituents having similar properties. All the analogues are included in the scope of the present invention so long as they can hybridize with the mRNA of the gene of the present invention to inhibit functions of the mRNA.
  • the antisense oligonucleotide of the present invention contains about 3 to about 50 nucleotides, preferably about 8 to about 30 nucleotides, more preferably about 12 to about 25 nucleotides.
  • the antisense oligonucleotide of the present invention can be produced by the well-known sohd phase synthesis technique. Devices for such synthesis are commercially available from some manufactures including Applied Biosystems. Other oligonucleotides such as phosphothioates can also be produced by methods known in the art.
  • the antisense oligonucleotide of the present invention is designed to hybridize with the mRNA transcribed from the gene of the present invention.
  • Those skilled in the art can easily design an antisense ohgonucleotides based on a given gene sequence (For example, Murakami and Makino: Saibou Kougaku Vol. 13 No.4 p259-266 (1994), Akira Murakami: Tanpakushitsu Kakusan Kouso (PROTEIN, NUCLEIC ACID AND ENZYME) Vol. 40 No.lO pl364-1370 (1995), Tunenari Takeuchi et al., Jikken Igaku (Experimental Medicine) Vol. 14 No. 4 p85-95 (1996)).
  • antisense oligonucleotides which are designed in a region containing 5' region of mRNA, preferably, the translation initiation site, are most effective for the inhibition of the expression of a gene.
  • the length of the antisense oligonucleotides is preferably 15 to 30 nucleotides and more preferably 20 to 25 nucleotides. It is important to confirm no interaction with other mRNA and no formation of secondary structure in the oligonucleotide sequence by homology search.
  • the evaluation of whether the designed antisense oUgonucleotide is functional or not can be determined by introducing the antisence oligonucleotide into a suitable cell and measuring the amount of the target mRNA, for example by northern blotting or RT-PCR, or the amount of the target protein, for example by western blotting or fluorescent antibody technique, to confirm the effect of expression inhibition.
  • Another method includes the triple helix technique.
  • This technique involves forming a triple helix on the targeted intra-nuclear DNA sequence, thereby regulating its gene expression, mainly at the transcription stage.
  • the oligonucleotide is designed mainly in the gene region involved in the transcription and inhibits the transcription and the production of the protein of the present invention.
  • Such RNA, DNA and oUgonucleotide can be produced using known synthesizers.
  • the antisense oligonucleotide may be introduced into the ceUs containing the target nucleic acid sequence by any of DNA transfection methods such as calcium phosphate method, lipofection, electroporation, microinjection, or gene transfer methods including the use of gene transfer vectors such as viruses.
  • An antisense oUgonucleotide expression vector can be prepared using a suitable retrovirus vector, then the expression vector can be introduced into the cells containing the target nucleic acid sequence by contacting the vector with the cells in vivo or ex vivo.
  • the DNA of the present invention can be used in the antisense RNA/DNA technique or the triple helix technique for the purpose of inhibiting MAP kinase cascade activation mediated by the protein of the present invention.
  • the antisense oUgonucleotide against the gene encoding the protein of the present invention is useful as a medicament to treat or prevent diseases characterized by undesirable activation of MAP kinase cascade, such as inflammation, autoimmune diseases, infectious diseases (e.g., HIV infection) and cancers.
  • the present invention also includes a pharmaceutical composition which comprises the above antisense oUgonucleotide as an active ingredient.
  • the antisense oUgonucleotide can also be used to detect such diseases using northern hybridization or PCR.
  • the present invention also provides a ribozyme or deoxyribozyme which inhibits an action of Elkl phosphorylation and/or activation of kinase which phosphorylates Elkl.
  • a ribozyme and deoxyribozyme is an RNA capable of recognizing a nucleotide sequence of a nucleic acid and cleaving the nucleic acid (see e.g., Hiroshi Yanagawa, "Jikken Igaku (Experimental Medicine) Bioscience 12: New Age of RNA).
  • the ribozyme or deoxyribozyme can be produced so that it cleaves the selected target RNA (e.g., mRNA encoding the protein of the present invention).
  • the ribozyme or deoxyribozyme specifically cleaving the mRNA of the protein of the present invention can be designed.
  • Such ribozyme or deoxyribozyme has a complementary sequence to the mRNA for the protein of the present invention, complementarily associates with the mRNA and then cleaves the mRNA, which results in reduction or entire loss of the expression of the protein of the present invention.
  • the level of the reduction of the expression is dependent on the level of the ribozyme or deoxyribozyme expression in the target cells.
  • ribozyme or deoxyribozyme commonly used: a hammerhead ribozyme and a hairpin ribozyme.
  • hammerhead ribozymes or deoxyribozymes have been weU studied regarding their primary and secondary structure necessary for their cleavage activity, and those skilled in the art can easily design the ribozymes nucleotided solely on the nucleotide sequence information for the DNA encoding the protein of the present invention [see e.g., Ma et al., Saibou Kougaku Vol.16, No.3, p.438-445 (1997); Ohkawa & Taira, Jikken Igaku (Experimental Medicine) Vol.12, No.12, p.83-88 (1994)].
  • the hammerhead ribozymes or deoxyribozymes have a structure consisting of two recognition sites (recognition site I and recognition site II fo ⁇ ning a chain complementary to target RNA) and an active site, and cleave the target RNA at the 3 'end of its sequence NUX (wherein N is A or G or C or U, and X is A or C or U) after the formation of a complementary pair with the target RNA in the recognition sites.
  • the sequence GUC (or GUA) has been found to have the highest activity [see e.g., Koizumi, M. et al., Nucl. Acids Res.
  • a ribozyme is designed to form several,up to 10 to 20 complementary base pairs around that sequence.
  • the suitability of the designed ribozyme can be evaluated by checking whether the prepared ribozyme can cleave the target mRNA in vitro according to the method described for example in Ohkawa & Taira, Jikken Igaku (Experimental Medicine) Vol.12, No.12, p.83-88 (1994).
  • the ribozyme can be prepared by methods known in the art to synthesize RNA molecules.
  • the sequence of the ribozyme can be synthesized on a DNA synthesizer and inserted into various vectors containing a suitable RNA polymerase promoter (e.g., T7 or SP6) to enzymaticaUy synthesize an RNA molecule in vitro.
  • a suitable RNA polymerase promoter e.g., T7 or SP6
  • Such ribozymes can be introduced into ceUs by gene transfer methods such as microinjection.
  • Another method involves inserting DNA encoding a ribozyme into a suitable expression vector and introducing the vector into cell strains, ceUs or tissues. Suitable vectors can be used to introduce the ribozyme into a selected cell.
  • vectors commonly used for such purpose include plasmid vectors and animal virus vectors (e.g., retrovirus, adenovirus, herpes or vaccinia virus vectors).
  • animal virus vectors e.g., retrovirus, adenovirus, herpes or vaccinia virus vectors.
  • ribozymes are capable of inhibiting the Elkl phosphorylation action and/or kinase which phosphorylates Elkl mediated by the protein of the present invention.
  • RNA which inhibits an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl.
  • RNA interference RNA interference
  • siRNA small interfering RNA
  • RNAi RNA sequence complementary to the sense RNA
  • siRNA Expression Vector available from Ambion can be used (Morita Takashi et al., Protein, Nucleic Acid and Enzyme, Vol.4 No.14 p. 1939-p. 1945 (2001); Sugimoto Asako, Kagaku to Seibutu (Chemistry and Biology), Vol.40 No.ll pp.713-718).
  • fuU-length cDNA is used in the present invention. That means 5' end sequence of the cDNA in the present invention is the transcription initiation site of the corresponding mRNA. Therefore the cDNA sequence can be used to identify the promoter region of the gene by comparing the cDNA with the genomic nucleotide sequence. Genomic nucleotide sequences are available from various databases when the sequences have been deposited in the databases. Alternatively, the cDNA can also be used to clone the desired sequence from a genomic library, for example, by hybridization, and determine its nucleotide sequence.
  • the promoter region of the gene located upstream the cDNA can be identified, hi addition, the promoter fragment thus identified can be used to construct a reporter plasmid for evaluating the expression of the gene, hi general, the DNA fragment spanning 2kb (preferably lkb) upstream from the transcription initiation site can be inserted upstream of the reporter gene to produce the reporter plasmid.
  • the reporter plasmid can be used to screen for a compound which enhances or reduces the expression of the gene.
  • such screening can be carried out by fransforming a suitable cell with the reporter plasmid, culturing the transformed cell for a certain period of time, adding a certain amount of a test compound, measuring the reporter activity expressed by the ceU after a certain period of time, and comparing the activity with that of a cell to which the test compound has not been added.
  • the present invention also relates to a computer-readable medium on which a sequence data set has been stored, said sequence data set comprising at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107 and/or at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78
  • the present invention relates to a method for calculating a homology, which comprises comparing data on the above medium with data of other nucleotide sequences.
  • the gene and amino acid sequence of the present invention provide valuable information for determining their secondary and tertiary structure, e.g., information for identifying other sequence having a similar function and high homology.
  • These sequences are stored on the computer-readable medium, then a database is searched using data stored in a known macromolecule structure program and a known search tool such as GCG program package (Devereux, J. et al, Nucleic Acids Research 12(1):387 (1984)). In this manner, a sequence in a database having a certain homology can be easily found.
  • the computer-readable medium may be any composition of materials used to store information or data. Examples of such media include commerciaUy available floppy disks, tapes, chips, hard discs, compact disks and video disks.
  • the data on the medium aUows a method for calculating a homology by comparing the data with other nucleotide sequence data. This method comprises the steps of providing a first polynucleotide sequence containing the polynucleotide sequence of the present invention for the computer-readable medium, and then comparing the first polynucleotide sequence with at least one second polynucleotide or polypeptide sequence to identify the homology.
  • the present invention also relates to an insoluble substrate to which a polynucleotide comprising aU or part of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107, is fixed.
  • a polynucleotide comprising aU or part of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61
  • a plurality of the various polynucleotides which are DNA probes are fixed on a specifically processed solid substrates such as slide glass to form a DNA microarray and then a labeled target polynucleotide is hybridized with the fixed polynucleotides to detect a signal from each of the probes.
  • the data obtained is analyzed and the gene expression is determined.
  • the present invention further relates to an insoluble substrate to which a polypeptide comprising all or part of an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108 is fixed.
  • Proteins which are expected to be useful for diagnosis or development of a new drug, can be isolated and or identified by mixing the insoluble substrate and a ceU extract from organisms, and capturing the proteins onto the insoluble substrate.
  • Example 1 Construction of a fuU-length cDNA library using the oligo-capping method
  • PBMC peripheral blood mononuclear cell
  • RNA extraction source (2) One portion was cultured for further 24 hours, and the ceUs were collected as mentioned above to obtain the RNA extraction source (2).
  • the other portion was cultured for 24 hours in the medium containing PMA (Phorbol-12-myristate-13-acetate) at a final concentration of lOnM and A23187 at a final concentration of 10 M, and the cells were collected to obtain the RNA extraction source (3).
  • total RNAs were respectively obtained from these (1) to (3) (3 types of the recovered cells) by using the RNA extraction reagent ISOGEN (purchased from NIPPON GENE) according to the manufacture's protocol.
  • poly A + RNA was obtained from the total RNA by using an oligo-dT ceUulose column according to Maniatis et al., supra.
  • a full-length cDNA library was constructed from the above poly A + RNA of human PBMC by the oligo-capping method according to the method of Sugano S. et al. [e.g., Maruyama, K. & Sugano, S., Gene, 138:171-174 (1994); Suzuki, Y. et al, Gene, 200:149-156 (1997); Suzuki, Y & Sugano, S. "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition (1999), an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.].
  • the full-length cDNA library constructed as above was transfected into E. coli strain TOP 10 by electroporation, then spread on LB agar medium containing 100 ⁇ g/ml ampicillin, and incubated overnight at 37°C. Then, using QIAwell 96 Ultra Plasmid Kit (QIAGEN) according to the manufacturer's protocol, the plasmids were recovered from the E.coU colonies grown on ampicillin-containing LB agar medium.
  • Example 2 Cloning of DNA capable of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl
  • 293-EBNA cells purchased from Invitrogen
  • 293-EBNA cells were grown to 1 x 10 4 ceUs/well in a 96 well plate for cell culture for 24 hours at 37°C (in the presence of 5% CO 2 ) using 5% FBS containing DMEM medium.
  • the reporter activity reflecting activation of MAP kinase cascade was measured using long-term luciferase assay system, PicaGene LT2.0 (TOYO INK) according to the attached manufacturer's instructions.
  • the luciferase activity was measured using Wallac ARVOTMST 1420 MULTILABEL COUNTER (Perkin Elmer).
  • the sequencing was carried out using the reagent Thermo Sequenase ⁇ Dye Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech) or BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (AppUed Biosystems) and the device ABI PRISM 377 sequencer or ABI PRISM 3100 sequencer according to the manufacturer's instructions.
  • BLAST Basic local alignment search tool searching [S. F. Altschul et al., J. Mol. Biol., 215:403-410 (1990)] was carried out in GenBank for the obtained nucleotide sequences. The results showed that 54 clones represented 54 genes encoding new proteins capable of phosphorylating Elkl and or activating kinase which phosphorylates Elkl.
  • the fuU-length DNA sequences for the 54 new clones were determined (SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107).
  • amino acid sequences of the protein coding regions were deduced (SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108).
  • Example 3 Screening compounds inhibiting activation of MAP kinase cascade, in particular, p38 signal.
  • 293-EBNA cells were seeded on DMEM medium containing 5% FBS in a 96-weU cell culture plate to a final cell density of 1 x 10 4 cells/100 ⁇ 1 medium/well, and cultured for 24 hours at 37°C in the presence of 5% CO 2 .
  • Example 4 Measurement of reporter activity corrected using an internal control.
  • 293-EBNA cells were inoculated on a 5% FBS (Fetal Bovine Serum)-containing DMEM medium in a 96-well ceU culture plate to a final cell density of 1 x 10 4 ceUs/lOO ⁇ 1 medium/well, and cultured for 24 hours at 37°C (in the presence of 5% CO 2 ).
  • FBS Fetal Bovine Serum
  • reporter activity firefly luciferase acitivity
  • reporter activity which reflects MAP kinase cascade activation
  • reporter activity being the internal control (sea pansy luciferase)
  • Picagene Dual purchased from Toyo Ink Manufacturing
  • Luciferase activity was measured with Perkin Elmer's WaUac ARVOTMST 1420 MULTILABEL COUNTER.
  • reporter activity which reflects MAP kinase cascade activation corrected with internal control reporter activity, exhibited increases that were dependent on the amount of expression plasmid comprising a gene encoding a MAP kinase cascade activating protein according to SEQ ID NO: 2, 4, 6, 10, 16, 18, 24, 26, 32, 36, 44, 50, 52, 56, 60, 62, 64, 68, 70, 74, 78, 82, 86, 88, 92, 96, 100, 102, 106 or 108 obtained in Example 2 above, as the amount of plasmid introduced was changed through 25ng, 50ng, and lOOng, respectively.
  • Reporter activity where an expression plasmid comprising the above gene was introduced which reflects MAP kinase cascade activation corrected with internal control reporter activity, is shown in Table 1, which is expressed as a factor of the same reporter activity where a null vector was introduced (S B value).
  • 293-EBNA cells were inoculated on a 5% FBS (Fetal Bovine Serum)-containing DMEM medium in a 96-well ceU culture plate to a final ceU density of 1 x 10 4 ceUs/100 ⁇ 1 medium/well, and cultured for 24 hours at 37°C (in the presence of 5% CO 2 ).
  • FBS Fetal Bovine Serum
  • the method used for introduction was in accordance with the attached protocol. After culturing for 2 hours at 37°C, any one of 1000 types of compound that were held in stock, was added to a final concentration of 10 ⁇ M per well, and after further culturing for 24 hours at 37°C, reporter activity, which reflects MAP kinase cascade activition, was measured by Picagene LT2 (purchased from Toyo Ink Manufacturing) in accordance with the manufacturer's instructions. Using a weU to which no compound was added as a control, compounds were screened to identify a compound which reduced reporter activity in the well to which it was added to 50% or lower.
  • the present invention provides proteins capable of phosphorylating Elkl and or activating a kinase which phosphorylates Elkl, and genes encoding the proteins. These proteins and genes are highly likely to posses highly industrially useful MAP kinase cascade activating activity.
  • the proteins of the present invention and the genes encoding the proteins allow not only screening for compounds useful for treating and preventing diseases associated with the excessive activation or inhibition of MAP kinase cascade, but also production of diagnostics for such diseases.
  • the genes of the present invention are also useful as a gene source used for gene therapy.

Abstract

Proteins capable of phosphorylating Elk1 and/or activating a kinase which phosphorylates Elk1, which are used for diagnosing, treating or preventing diseases associated with the excessive activation or inhibition of MAP kinase cascade are provided. Using the plasmids pFR-Luc and pFA2-Elk1, the cDNA encoding a protein capable of phosphorylating Elk1 and/or activating a kinase wich phosphorylates Elk1 was cloned from the cDNA library constructed from human peripheral blood monocytes, and the cDNA sequence and the deduced amino acid sequence were determined. The protein, the DNA encoding the protein, a recombinant vector containing the DNA, and a transformant containing the recombinant vector are useful for screening a substance inhibiting or promoting an action of Elk1 phosphorylation and/or an action of activation of kinase which phosphorylates Elk1.

Description

DESCRIPTION
ELK1 PHOSPHORYLATION RELATED GENE
TECHNICAL FIELD
The present invention relates to a protein capable of phosphorylating Elkl and/or activating a kinase which phosphorylates Elkl, a DNA sequence encoding the protein, a method for obtaining the DNA, a recombinant vector containing the DNA, a transformant containing the recombinant vector, and an antibody which specifically reacts with the protein. The present invention also relates to use of the protein, DNA, RNA or antibody of the invention in the diagnosis, treatment or prevention of diseases associated with the excessive activation or inhibition of Elkl phosphorylation action or a kinase which phosphorylates Elkl.
The present invention also relates to a method for screening a substance capable of inhibiting or promoting the Elkl phosphorylation action and/or the activation of kinase which phosphorylates Elkl by using the protein, DNA, recombinant vector and transformant.
BACKGROUND ART
MAP (mitogen-activated protein) kinase (MAPK) was first identified in the second half of the 1980s. In 1990s, it has been elucidated that the signal transduction pathway leading to MAP kinase activation starts from cell growth factor receptors having tyrosine kinase activity, through adaptor molecules composed of SH2 (src homology 2) and SH3 (src homology 3), Ras (an oncogene product which is a GTP-binding protein), and Raf-1 (an oncogene product which is serine/threonine kinase) and leads to MAP kinase. Now, the MAP kinase signal transduction pathway is understood to be a central pathway for deteπrώiing cell proliferation, differentiation and development of higher eukaryotic organisms (Nishida, E et al., Trends Biochem. Sci., 18: 128 - 131 (1993), Marshall, C.J., Curr. Opin. Genet. Dev., 4: 82 - 89 (1994), Cobb. M. H. et al., J. Biol. Chem., 270: 14843 - 14846 (1995)).
For the activation of MAP kinase, phosphorylation of Thr and Tyr within Thr-Glu-Tyr (TEY) sequence between kinase sub-domains VII and V-H is required. Identified as an enzyme which catalyzes the phosphorylation of both these amino acid residues, is MAPK kinase (MAPKK) or MAPK/ERK kinase (MEK). This enzyme is therefore a dual- specificity kinase which can phosphorylate serine/threon-ne and tyrosine. Further, MAPKK, for its activation, requires phosphorylation of two serine (or threonine) residues between kinase domains VII and VIII. The serine/threonine kinase responsible for this phosphorylation is called MAPKK kinase (MAPKKK). Raf-1 is one example of MAPKKK, and the Ras- Raf-1 (=MAPKKK) -> MAPKK -> MAPK chain is one of the major signal transduction pathways. This chain of kinases consisting of three molecules, RafMAPKK/MAPK is called the classical MAP kinase cascade. Now, the presences of SAPK/JNK and p38/CSBPMPK2 are revealed as MAP kinase-like kinases, and these cascade systems are generally called MAP kinase cascade.
The Stress-Activated Protein Kinase (SAPK) was identified as a kinase which is activated when a cell is subjected to chemical stress such as protein synthesis inhibiting agents or physical stress such as heat shock or osmotic pressure changes (Kyriakis, J.M., Nature, 369: 156-160 (1994)). SAPK was later found to be identical to c-Jun N-terminal Kinase (JNK), which increases transcription activity by phosphorylating the N-terminus of transcription factor Jun (Derijard, B., Cell, 76: 1025-1037 (1994)). Therefore, this kinase is called SAPK/JNK.
On the other hand, p38 was identified and cloned as a protein which is subject to tyrosine phosphorylation in an early stage after stimulating a lymphocyte (Han, J., et al., Science, 265: 808-811(1994)). At around the same time, CSAJD binding protein (CSBP) was identified as protein which binds to cytokine-suppressive anti-inflammatory drug (CSAJD); a drug suppressing the production of inflammatory cytokines in lymphocytes (Lee, J. C, et al., Nature, 372: 739-746 (1994)). At present, this has been clarified to be a human form of ρ38. Further, this is also the same molecule as MPK2 which was independently isolated as a kinase which is activated by stress stimulation. Thus, this kinase is called p38/CSBP/MPK2.
There is mutual homology between the sequences of classical MAP kinase, SAPK JNK, and p38/CSBP MPK2, and they constitute a superfamily. For example, the TEY sequence necessary for activation in classical MAP kinase is TPY (Thr-Pro-Tyr) in SAPK/JNK, and it is TGY sequence in p38/CSBP/MPK2. In both cases, Thr and Tyr are phosphorylated and activated by the upstream kinase MAPKK.
There is mutual homology between the sequences of MAPKKs specific to each of classical MAP kinase, SAPK JNK, and ρ38/CSBP/MPK2, and they constitute a superfamily. MEK, SEKl, MKK3, MKK4, MKK6, MKK7, etc belong to this superfamily. (Kyriakis, J. M., et al., J. Biol. Chem., 271: 24313-24316 (1996), Davis, R. J., Trends Biochem. Sci., 19: 470-473 (1994)). Examples of MAPKKs whichs specifically phosphorylate SAPK JNK include SAPK/ERK kinase- 1 (SEKl), mitogen-activated protein kinase kinase 4 (MKK4) (Lin, A., et al., Science, 268: 286 - 290 (1995), Sanchez, I., Nature, 372: 794-798 (1994), Moriguchi, T, et al., J. Biol. Chem., 270: 12969-12972 (1995)), and mitogen activated protein kinase kinase 7 (MKK7) (Moriguchi, T, et al, EMBO J., 16: 7045-7053 (1997), Tournier, C, et al, P.N.A.S., 94: 7337-7342 (1997), and others). Also, examples of MAPKKs which specifically activates p38 include MKK3 and MKK6 (Moriguchi, T., et al., J. Biol. Chem., 271: 26981-26988 (1996), Cuenda, A., et al,. EMBO J., 15: 4156-4164 (1996)).
In contrast, between MAPKKKs which exist in the uptream of the pathway leading to classical MAP kinase, SAPK JNK, p38/CSBP/MPK2, mutual sequence homology is relatively low. There is only about 30% homology even in the kinase domain between kinases having MAPKKK activity such as Raf, TAK1, MEKK, MLK3, Askl, Mos, Cot, etc. This matches well with the adaptedness for purpose of a system which specifically activates a necessary MAP kinase signal transduction pathway in response to a great variety of stresses. Now, MEKK1 is known as MAPKKK which exists in the upsteam of the pathway leading to SAPK/JNK, but it is highly possible that other kinases which work as MAPKKK exist.
SAPK/JNK and p38/CSBP MPK2 are not activated by growth factors which activate classical MAP kinase, and are each known to be activated by stresses such as osmotic shock or heat shock, or by inflammatory cytokines such as TNF- a or IL-1, etc. (Kyriakis., J. M., et al., J. Biol. Chem., 271: 24313-24316 (1996), Davis., R. J., Trends Biochem. Sci., 19: 470-473 (1994)). Further, they are activated under a condition which induces cell death, such as UV radiation and depletion of serum and/or growth factor(Kyriakis., J. M., et al., J. Biol. Chem., 271: 24313-24316 (1996), Davis., R. J., Trends Biochem. Sci., 19: 470-473 (1994)). Unlike classical MAP kinase which is activated by a signal induced from a tyrosine kinase-type receptor, the systems of SAPK/JNK and p38/CSBP/MPK2 are characterized by the extreme variety of entry points thereto.
As described above, it is known that MAP kinase cascades, particularly the S APK/JNK and p38/CSBP/MPK2 systems, are activated by a variety of stimuli. Along the pathway from an extracellular stimulus finally to the activation of SAPK/JNK or p38/CSBP/MPK2, there exist many MAPKK kinases and MAPK kinases, or groups of adaptor molecular which connect these, and these molecules have not yet been folly clarified. It is desired to clarify these kinases and molecules, and utilize them in the fields of medicaments, diagnostics and therapy.
DISCLOSURE OF THE INVENTION
The object of the present invention is to identify a novel gene and protein such as described above acting to activate useful MAP kinase cascade, and to provide a method of use of them in medicaments, diagnostics and therapy. The aforementioned 3 kinds of MAP kinases phosphorylate Elkl as a substrate. Therefore, the object of the present invention is to provide a novel protein capable of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl, a DNA sequence encoding the protein, a recombinant vector containing the DNA, a transformant containing the recombinant vector, a process for producing the protein, an antibody directed against the protein or a peptide fragment thereof, and a process for producing the antibody.
Another object of the present invention is to provide a method for screening a substance capable of inhibiting or activating Elkl phosphorylation action and/or kinase which phosphorylates Elkl, a kit for the screening, a substance capable of inhibiting or activating Elkl phosphorylation action and or kinase which phosphorylates Elkl obtainable by the screening method or the screening kit, a process for producing the substance, a pharmaceutical composition containing a substance capable of inhibiting or activating Elkl phosphorylation action and/or kinase which phosphorylates Elkl, etc. In order to solve the above objects, the present inventors have attended to the fact that SAPK JNK and ρ38/CSBP/MPK2 are rapidly activated by stimulations of various extracellular inflammatory cytokines, and have intensively studied on the action of Elkl phosphorylation and/or the action of activating kinase which phosphorylates Elkl. As a result, the present inventors have succeeded in constructing a full-length cDNA hbrary by using the oligo-capping method; establishing a gene function assay system employing an expression cloning method using HEK293EBNA cells; and isolating new DNAs (cDNAs) encoding a protein having a function of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl by using the assay system. These new DNA molecules induced Elkl phosphorylation action and/or activation of kinase which phosphorylates Elkl by its expression in HEK293EBNA cells. This result shows that these new DNAs are signal transduction molecules involved in Elkl phosphorylation action and/or activating pathway of kinase which phosphorylates Elkl . Thus, the present invention has been completed.
That is, the present invention provides the folio wings:
(1) A purified protein selected from the group consisting of:
(a) a protein which consists of an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108; and
(b) a protein that acts to phosphorylate Elkl and/or activate kinase which phosphorylates Elkl and consists of an amino acid sequence having at least one amino acid deletion, substitution or addition in an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 10, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108.
(2) A purified protein that acts to phosphorylate Elkl and/or activate kinase which phosphorylates Elkl and comprises an amino acid sequence having at least 95% identity to the protein according to above item (1) over the entire length thereof.
(3) An isolated polynucleotide which comprises a polynucleotide sequence encoding a protein selected from the group consisting of:
(a) a protein which comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108; and
(b) a protein that acts to phosphorylate Elkl and/or activate a kinase which phosphorylates Elkl, and consists of an amino acid sequence having at least one amino acid deletion, substitution or addition in an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108.
(4) An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of:
(a) a polynucleotide sequence represented by SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 107;
(b) a polynucleotide sequence which encodes a protein that acts to phosphorylate Elkl and/or activate kinase which phosphorylates Elkl, and which hybridizes under stringent conditions with a polynulceotide having a polynulceotide sequence complementary to the polynucleotide sequence of (a); and,
(c) a polynucleotide sequence encoding a protein that acts to phosphorylate Elkl and/or activate a kinase which phosphorylates Elkl, and consists of a nucleotide sequence having at least one nucleotide deletion, substitution or addition in a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107.
(5) An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of:
(a) a polynucleotide sequence represented by protein coding region of any of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 107;
(b) a polynucleotide sequence which encodes a protein that acts to phosphorylate Elkl and/or activate kinase which phosphorylates Elkl, and which hybridizes under stringent conditions with a polynulceotide having a polynulceotide sequence complementary to the polynucleotide sequence of (a); and,
(c) a polynucleotide sequence encoding a protein that acts to phosphorylate Elkl and/or activate a kinase which phosphorylates Elkl, and consists of a nucleotide sequence having at least one nucleotide deletion, substitution or addition in a nucleotide sequence of protein coding region of any one selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107.
(6) An isolated polynucleotide comprising a polynucleotide sequence which encodes a protein that acts to phosphorylate Elkl and/or activate a kinase which phosphorylates Elkl, and has at least 95% identity to the polynucleotide sequence according to above item (3) over the entire length thereof.
(7) An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that acts to phosphorylate Elkl and/or activate a kinase which phosphorylates Elkl, and has at least 95% identity to the polynucleotide sequence according to above item (4) or (5) over the entire length thereof.
(8) A purified protein encoded by the polynucleotide according to any one of above items (3) to (7).
(9) A recombinant vector which comprises a polynucleotide according to any one of above items (3) to (7).
(10) A gene therapy agent which comprises the recombinant vector according to above item (9).
(11) A transformant which comprises the recombinant vector according to above item (9).
(12) A membrane of the transformant according to above item (11) having the protein according to above item (1) or (2) which is a membrane protein.
(13) A process for producing a protein according to above item (1), (2) or (8) comprising the steps of;
(a) culturing a transformant according to (11) under conditions providing expression of the protein according to above item (1), (2) or (8); and
(b) recovering the protein from the culture product.
(14) A process for diagnosing a disease or susceptibihty to a disease related to expression or activity of the protein of above item (1), (2) or (8) in a subject comprising the steps of:
(a) determining the presence or absence of a mutation in the gene encoding said protein in the genome of said subject; and/or
(b) analyzing the amount of expression of said protein in a sample derived from said subject.
(15) A method for screening compounds which inhibit or promote an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, which comprises the steps of:
(a) preparing a transformant by introducing a gene encoding a protein according to above item (1), (2) or (8) that has an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl and a gene encoding a protein which generates a signal which can detect an action of Elkl phosphorylation into a host cell;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the signal which can detect an action of Elkl phosphorylation; and
(d) selecting a candidate compound which can change the signal amount as compared with the case of the absence of candidate compounds, as a compound which inhibits or promotes an action of Elkl phosphorylation and or an action of activation of kinase which phosphorylates Elkl.
(16) A method for screening compounds which inhibit or promote an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, which comprises the steps of:
(a) preparing a transformant by introducing a gene encoding a protein according to above item (1), (2) or (8) that has an action of Elkl phosphorylation and or an action of activation of kinase which phosphorylates Elkl into a host cell;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring an action of Elkl phosphorylation and or an action of activation of kinase which phosphorylates Elkl; and
(d) selecting a candidate compound which can change the action of Elkl phosphorylation and/or the action of activation of kinase which phosphorylates Elkl as compared with the case of the absence of candidate compounds, as a compound which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl.
(17) A compound which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, which is selected by the method for screening according to above item (15) or (16).
(18) A process for producing a pharmaceutical composition, which comprises the steps of:
(a) preparing a transformant by introducing a gene encoding a protein according to above item (1), (2) or (8) that has an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl and a gene encoding a protein which generates a signal which can detect an action of Elkl phosphorylation into a host cell;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the signal which can detect an action of Elkl phosphorylation;
(d) selecting a candidate compound which can change the signal amount as compared with the case of the absence of candidate compounds, as a compound which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl; and
(e) producing a pharmaceutical composition which comprises a compound selected in the step of (d).
(19) A process for producing a pharmaceutical composition, which comprises the steps of: (a) preparing a transformant by introducing a gene encoding a protein according to above item (1), (2) or (8) that has an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl into a host cell;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl;
(d) selecting a candidate compound which can change the action of Elkl phosphorylation and/or the action of activation of kinase which phosphorylates Elkl as compared with the case of the absence of candidate compounds, as a compound which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl; and
(e) producing a pharmaceutical composition which comprises a compound selected in the step of (d).
(20) A kit for screening a compound for inhibiting or promoting an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, which comprises: (a) a transformant comprising a gene encoding a protein according to above item (1), (2) or (8) which promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl and a gene encoding a protein which provides a signal which can detect an action of Elkl phosphorylation; and
(b) reagents for measuring the signal.
(21) A monoclonal or polyclonal antibody or a fragment thereof, which recognizes the protein according to above item (1), (2) or (8).
(22) The monoclonal or polyclonal antibody or a fragment thereof according to above item (21), which inhibits an action of Elkl phosphorylation andor an act on of activation of kinase which phosphorylates Elkl which are possessed by the protein according to above item (l), (2) or (8).
(23) A process for producing a monoclonal or polyclonal antibody according to above item (21) or (22), which comprises administering the protein according to above item (1), (2) or (8) or epitope-bearing fragments thereof to a non-human animal as an antigen. (24) An antisense oligonucleotide having a sequence complementary to a part of the polynucleotide according to any one of above items (3) to (7), which prevents the expression of a protein which promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl.
(25) A ribozyme or deoxyribozyme capable of inhibiting an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, which has an action of cleavage of RNA that encodes the protein according to above item (1), (2) or (8) or an action of cleavage of RNA that encodes a protein which is involved in a pathway leading to Elkl phosphorylation and/or activation of kinase which phosphorylates Elkl.
(26) A double strand RNA having a sequence corresponding to a part of the nucleotide sequence according to any one of above items (3) to (7), which inhibits expression of a protein which promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl .
(27) A method for treating a disease associated with an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, which comprises administering to a subject a compound screened by the process according to above item (15) or (16), and/or a monoclonal or polyclonal antibody or a fragment thereof according to above item (21) or (22), and/or an antisense oligonucleotide according to above item (24), and/or a ribozyme or deoxyribozyme according to above item (25), and/or a double strand RNA according to above item (26) in an effective amount to treat a disease selected from the group consisting of inflammation, autoimmune diseases, cancers, infection diseases, bone diseases, AIDS, neurodegenerative diseases, ischemic injury, GVHD, skin diseases, IgA nephritis, purpuric nephritis, proliferative nephritis, and fulminant hepatitis.
(28) A pharmaceutical composition which is produced the process according to above item (18) or (19), for inhibiting or promoting an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl.
(29) The pharmaceutical composition according to above item (28) for the treatment and or prevention of inflammation, autoimmune diseases, cancers, infection diseases, bone diseases, AIDS, neurodegenerative diseases, ischemic injury, GVHD, skin diseases, IgA nephritis, purpuric nephritis, proliferative nephritis, and fuhninant hepatitis.
(30) A method of treating inflammation, autoimmune diseases, cancers, infection diseases, bone diseases, AIDS, neurodegenerative diseases, ischemic injury, GVHD, skin diseases, IgA nephritis, purpuric nephritis, proliferative nephritis, and inlminant hepatitis, which comprising administering a pharmaceutical composition produced by the process according to above item (18) or (19) to a patient suffering from a disease relating to abnormalities in an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl.
(31) A pharmaceutical composition which comprises a monoclonal or polyclonal antibody or a fragment thereof according to above item (21) or (22) as an active ingredient.
(32) A pharmaceutical composition which comprises an antisense oligonucleotide according to above item (24) as an active ingredient.
(33) A pharmaceutical composition which comprises a ribozyme or deoxyribozyme according to above item (25) as an active ingredient.
(34) A pharmaceutical composition or a gene therapy agent, which comprises a double strand RNA according to above item (26) or a vector capable of expressing said double strand RNA, an active ingredient.
(35) The pharmaceutical composition according to any one of (31) to (34) for the treatment and/or prevention of a disease which is selected from the group consisting of inflammation, autoimmune diseases, cancers, infection diseases, bone diseases, ADDS, neurodegenerative diseases, ischemic injury, GVHD, skin diseases, IgA nephritis, purpuric nephritis, proliferative nephritis, and ftilminant hepatitis.
(36) A computer-readable medium on which a sequence data set has been stored, said sequence data set comprising at least one of nucleotide sequence or that of coding region which is selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107, and/or at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108.
(37) A method for calculating identity to other nucleotide sequences and/or amino acid sequences, which comprises comparing data on a medium according to above item (36) with data of said other nucleotide sequences and/or amino acid sequences.
(38) An insoluble substrate to which polynucleotides comprising all or part of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107 are fixed.
(39) An insoluble substrate to which polypeptides comprising all or a part of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108, are fixed.
The contents of the specifications and/or drawings of Japanese Patent Apphcation Nos.2003-34875 and U.S. Provisional Applications Nos.60/447,320, which form the bases of priority of the instant application, are incorporated herein.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows a results of measurement of reporter activity using p38 inhibitor B203580 which is one type of MAP kinases.
Fig. 2 indicates the structural formula of staurosporine.
Explanation of the Sequence Listing
SEQ ID NO: 109 and SEQ ID NO: 110 are primers.
BEST MODE FOR CARRYING OUT THE INVENTION
At first, in order to further clarify the basic feature of the present invention, the present invention is explained by following how the present invention is completed. In order to obtain a new gene having a function of activating MAP kinase cascade, the following experiments were carried out as shown in the examples.
First, using the oligo-capping method, a full-length cDNA was produced from mRNA prepared from human peripheral blood monocytes, and a full-length cDNA library was constructed in which the cDNA was inserted into the vector pME18S-FL3 (GenBank Accession AB009864). Next, the cDNA library was introduced into E. coli cells, and plasmid preparation was carried out per clone. Then, animal cell expression plasmid ρcDNA3.1 (INVITROGEN) comprising DNA encoding human JNK gene, animal cell expression plasmid pcDNA3.1 (INVITROGEN) comprising DNA encoding human p38 gene, plasmid pFA2-Elkl (STRATAGENE) comprising DNA encoding a fusion protein of part of Elkl being a substrate of these and GAL4DNA binding region, and reporter plasmid pFR-Luc (STRATAGENE) having a Gal4 recognition site upstream of a gene encoding luciferase were cotransfected with the above full-length cDNA plasmid into HEK293EBNA cells (INVITROGEN). After 24 hours of culture, luciferase activity was measured, and the plasmid with significantly increased luciferase activity compared to that of a control experiment (vector pME18S-FL3 is introduced into a cell in place of a full-length cDNA )was selected (the selected plasmid showed a 3-fold or more increase in luciferase activity compared to that of the control experiment), and the entire nucleotide sequence of the cDNA cloned into the plasmid was deteπriined. As discussed above, since p38, JNK, and ERK etc, existing furthermost downstream of MAP kinase cascade, phosphorylate Elkl as a good substrate, signal transduction molecules involved in the action of Elkl phosphorylation and/or the activation of a kinase which phosphorylates Elkl, are highly likely to be signal transduction molecules which activate the MAP kinase cascades. Therefore, the protein encoded by the cDNA thus obtained is a signal molecule involved in the action of Elkl phosphorylation and/or the activation of a kinase which phosphorylates Elkl, and at the same time, can be expected to be a signal transduction molecule involved in the activation of the MAP kinase cascades.
The phrase "activation of kinase which phosphorylates Elkl" includes both direct modification and activation of a kinase which phosphorylates Elkl, and indirect activation of the kinase by activating upstream signal(s). The present invention is described in detail below.
In the present invention, the words "acts to phosphorylate Elkl" or "acts to activate a kinase which phosphorylates Elkl" refers to having an action which phosphorylates Elkl directly or indirectly when a gene is introduced into a suitable host and the protein encoded by the gene is excessively expressed. Phosphorylation of Elkl can be measured, for example, by a reporter gene assay comprising cotransfecting into a cell together with a gene resulting from fusion of Elkl and GAL4DNA binding region, a reporter gene wherein a luciferase gene is linked downstream of a GAL4 recognition sequence. Action of phosphorylating Elkl or action of activating a kinase which phosphorylates Elkl can be confirmed by an increase in reporter activity in cells into which the gene was introduced, compared to control cells (cells into which a null vector only was introduced). Increase in reporter activity is preferably by a factor of 1.5 or more, more preferably by a factor of 2 or more, and still more preferably by a factor of 3 or more.
Reporter activity can be measured by cloning a polynucleotide (e.g. cDNA) encoding the protein to be expressed into a suitable expression vector, co-transfecting the thus prepared expression vector into a suitable host together with a gene resulting from fusion of Elkl and GAL4 DNA binding region, and a reporter gene wherein a luciferase gene is linked downstream of a GAL4 recognition sequence, and after culturing for a certain period, then measuring the reporter activity. Suitable expression vectors are well known to those skilled in the art, and examples include pME18S-FL3, pcDNA3.1 (Invitrogen). The reporter gene can be one which enables a person skilled in the art to easily detect the expression thereof, and examples include a gene encoding luciferase, chloramphenicol acetyl transferase, or β -galactosidase. Use of a gene encoding luciferase is most preferable, and examples of a gene resulting from fusion of Elkl and GAL4 DNA binding region include pFA2-Elkl (STRATAGENE), and examples of a reporter gene wherein a luciferase gene is linked downstream of a GAL4 recognition sequence include pFR-Luc (STRATAGENE). Suitable hosts include those which exhibit MAPK cascades activation response to stimulation by IL-1, TNF- or the like. Examples include 293-EBNA cells. Cell culture and introduction of genes into cells (transfection) can be performed and optimized by a person skilled in the art by known techniques.
As a preferable method, 293-EBNA cells are inoculated on 5% FBS (Fetal Bovine Serum)-containing DMEM (Dulbecco's Modified Eagle Medium) medium in a 96-well cell culture plate to a final cell density of 1 x 104 cells/well, and cultured for 24 hours at 37°C, in the presence of 5% CO2. Then, pFR-Luc (STRATAGENE), pFA2-Elkl (STRATAGENE), pcDNA3.1 (+) (INVITROGEN) into which human p38 gene has been incorporated, pcDNA3.1 (+) (INVITROGEN) into which human JNK1 β 1 gene has been incorporated, and the expression vector are co-transfected into the cells in a well using FuGENE 6 (Roche). After 24 hours of culture at 37°C, phosphorylation of Elkl or activation of a kinase that phosphorylates Elkl is then measured by measuring luciferase activity using a long term luciferase assay system, Picagene LT2.0 (Toyo Ink). For example, luciferase activity can be measured using PerkinElmer's Wallac ARVOTMST 1420 MULTILABEL COUNTER. The method for gene introduction by FuGENE6, and measurement of luciferase activity by Picagene LT2.0 can be performed respectively according to the attached protocols. In a method of gene introduction with a 96-well plate using FuGENEό, the amount of FuGENEό per 1 well is suitably 0.3 to 0.5 μ 1, preferably 0.5 μ 1. The respective amounts per 1 well of the plasmid genes to be introduced are: pFR-Luc plasmid amount, 50 to lOOng, preferably 60ng; pFA2-Elkl plasmid amount, 0.1 to 0.5ng, preferably 0.25ng; amount of pcDNA3.1 (+) plasmid into which human p38 gene has been incorporated, 5ng; and amount of pcDNA3.1(+) plasmid into which human JNJ1 β 1 gene has been incorporated, 30ng. An ability to phosphorylate Elkl or to activate a kinase that phosphorylates Elkl can be confirmed by using, as an index, an ability to increase the reporter activity (luciferase activity) relative to the control experiment (using cells into which a null vector only was introduced). Increase in reporter activity as an index is preferably by a factor of 1.5 or more, more preferably by a factor of 2 or more, and still more preferably by a factor of 3 or more.
Related to the amino acid sequences of SEQ ID NOS. 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108, the present invention provides the folio wings: (a) proteins which comprises any of the above amino acid sequences;
(b) peptides having one of the above amino acid sequences;
(c) proteins which phosphorylate Elkl and/or activate kinase which phosphorylates Elkl and consist of an amino acid sequence having at least one amino acid, preferably several amino acids deletion, substitution or addition in any one of the above amino acid sequences; and
(d) proteins which phosphorylate Elkl and/or activate kinase which phosphorylates Elkl and comprise an amino acid sequence, which has at least 95% identity, preferably at least 97-99% identity, to any one of the above amino acid sequences over the entire length thereof, and .
"Identity" as known in the art, is a relationship between two or more protein sequence or two or more polynucleotide sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between protein or polynucleotide sequences, as determined by the match between protein or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "Identity" and "similarity" can be readily calculated by known methods. Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. "Identity" can be determined by using, for example, the BLAST (Basic Local Alignment Search Tool) program (for example, Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410(1990), Altschul SF, Madden TL, Schaffer AA, Zhang Z, Miller W, Lipman DJ,. Nucleic Acids Res. 25: p3389-3402 (1997)), however methods of determining identity are not hmited to this. Where software such as BLAST is used, it is preferable to use default values. The main initial conditions generally used in a BLAST search are as follows, but are not limited to these.
An amino acid substitution matrix is a matrix numerically representing the degree of analogy of each pairing of each of the 20 types of amino acid, and normally the default matrix, BLOSUM62, is used. The theory of this amino acids substitution matrix is shown in Altschul S.F., J. Mol. Biol. 219: 555-565 (1991), and its applicability to DNA sequence comparison is shown in States D. J., Gish W., Altschul S.F., Methods, 3: 66-70 (1991). In this case, optimal gap cost is deterrnined empirically and in the case of BLOSUM62, preferably parameters, Existence 11, Extension 1 are used. The expected value (EXPECT) is the threshold value concerning statistical significance for a match with a database sequence, and the default value is 10.
The Examples described below demonstrate that the protein consisting of any one of the amino acid sequences of the above SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108, is capable of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl.
Related to the polynucleotide of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 107, or the polynucleotides of the coding region (CDS) in these sequences, the present invention further provides the following isolated polynucleotides:
(a) polynucleotides having any of the above sequences;
(b) polynucleotides comprising a nucleotide sequence which has at least 95% identity, preferably at least 97-99% identity to any one of the above sequences, and encodes a protein which phosphorylate Elkl and/or activate kinase which phosphorylates Elkl;
(c) polynucleotides having a polynucleotide sequence encoding a protein which has at least 95%, preferably 97-99% identity, to any one of the amino acid sequences of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108, and phosphorylate Elkl and/or activate kinase which phosphorylates Elkl:
Polynucleotides which are identical or almost identical to a nucleotide sequences contained in the above nucleotide sequence may be used as hybridization probes or as primers for a nucleic acid amplification reaction to isolate full-length cDNAs and genomic clones encoding proteins of the present invention or cDNA and genomic clones of other genes that have a high sequence homology to the above sequences. Typically, these nucleotide sequences are 70% identical, preferably 80% identical, more preferably 90% identical, most preferably 95% identical to the above sequences. The probes or primers will generally comprises at least 15 nucleotides, preferably 30 nucleotides and may have 50 nucleotides. Particularly preferred probes will have between 30 and 50 nucleotides. Particularly preferred primers have between 20 and 25 nucleotides.
The polynucleotide of the present invention may be either in the form of a DNA such as cDNA ,a genomic DNA obtained by cloning or synthetically produced, or may be in the form of RNA such as mRNA. The polynucleotide may be single-stranded or double-stranded. The double- stranded polynucleotides may be double-stranded DNA, double-stranded RNA or DNA:RNA hybrid. The single- stranded polynucleotide may be sense strand also known as coding strand or antisense strand also known as non-coding strand.
Those skilled in the art can prepare a protein having the same action of phosphorylating Elkl and/or the same action of activating kinase which phosphorylates Elkl as the protein having an amino acid sequence of any one of SEQ ID NOS 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108, by means of appropriate substitution of an amino acid in the protein using known methods. One such method involves conventional mutagenesis procedures using mutagens for the DNA encoding the protein. Another method is, for example, site-directed mutagenesis (e.g., Mutan-Super Express Km Kit from Takara Shuzo Co., Ltd.). Mutations of amino acids in proteins may also occur in nature. Thus, the present invention also includes a mutated protein which is capable of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl and which has at least one amino acid deletion, substitution or addition compared to the corresponding amino acid sequence of SEQ ID NO 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 or 108. The number of mutations is preferably 1 to 10, more preferably 1 to 5, most preferably 1 to 3.
Examples of such amino acid substitutions are preferably conservative substitutions, and include substitutions within the following groups: (glycine, alanine), (valine, isoleucine, leucine), (aspartic acid, glutamic acid), (asparagine, glutamine), (serine, threonine), (lysine, arginine) and (phenylalanine, tyrosine). Based on a nucleotide sequence (e.g., SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 107) encoding a protein consisting of an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 or 108, or fragments thereof, those skilled in the art can routinely isolate a DNA with a high sequence similarity to any one of these nucleotide sequences by using hybridization techniques and the like, and obtain proteins having the same action of phosphorylating Elkl and/or the same action of activating kinase which phosphorylates Elkl as the protein having an amino acid sequence of SEQ ID NOS 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 or 108.
Thus, the present invention also includes a protein that phosphorylates Elkl and/or activates kinase which phosphorylates Elkl and comprises an amino acid sequence having a high identity to the amino acid sequence of above SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 or 108. "High identity" refers to an amino acid sequence having an identity of at least 90%, preferably at least 97-99% over the entire length of any one of the above SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108.
The proteins of the present invention may be natural proteins derived from any human or animal cells or tissues, chemically synthesized proteins, or proteins obtained by genetic recombination techniques. The protein may or may not be subjected to post-translational modifications such as sugar chain addition or phosphorylation.
Examples of the protein encoded by the gene of the present invention includes secretory proteins (growth factors, cytokines, hormones, etc.), protein modifying enzymes (protein kinases, protein phosphatases, proteases, etc), signal transduction molecules (adaptor molecules between proteins, etc.), nuclear proteins (nuclear receptors, transcription factors) and membrane proteins. Membrane proteins include receptors, cell adhesion molecules, ion channels, transporters, etc. Where the protein is a membrane protein, the protein is more useful as a research tool of medical compound since a compound selected by the below-described screening is expected to easily migrate into a cell or give signal transduction into a cell.
The present invention also includes a polynucleotide encoding the above protein of the present invention. Examples of nucleotide sequences encoding a protein consisting of an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 or 108 include a nucleotide sequences of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 107. The DNA includes cDNA, genomic DNA, and chemicaUy synthesized DNA. In accordance with the degeneracy of the genetic code, at least one nucleotide in the nucleotide sequence encoding a protein consisting of an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 or 108 can be substituted with other nucleotides without altering the amino acid sequence of the protein produced from the gene. Therefore the DNA sequences of the present invention also include nucleotide sequences altered by substitution based on the degeneracy of the genetic code. Such DNA sequences can be synthesized using known methods.
The DNA of the present invention includes a DNA which encodes a protein capable of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl and hybridizes under stringent conditions with the DNA sequence of any one of the above nucleotide sequences of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107 or a polynucleotide sequence complementary to said nucleotide sequence. Stringent conditions are apparent to those skilled in the art, and can be easily attained in accordance with various laboratory manuals such as T. Maniatis et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory 1982, 1989.
That is, "stringent conditions" refer to overnight incubation at 37°C in a hybridization solution containing 30% formamide, 5 x SSC (0.75 M NaCl, 75mM trisodium citrate),5 x Denhardt's solution, 0.5% SDS, lOOμg/ml denatured, sheared salmon sperm DNA) followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 minutes at room temperature, then followed by washing (two times) in 1 x SSC, 0.1% SDS for 10 minutes at 37°C(low stringency). Preferred stringent conditions are overnight incubation at 42 °C in a hybridization solution containing 40% formamide, followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 minutes at room temperature, then followed by washing (two times) in 0.2 x SSC, 1% SDS for 10 minutes at 42°C(moderate stringency). More preferred stringent conditions are overnight incubation at 42 °C in a hybridization solution containing 50% formamide, followed by washing (three times) in 2 x SSC, 0.1% SDS for 10 minutes at room temperature, followed by washing (two times) in 0.2 x SSC, 0.1% SDS for 10 minutes at 50°C (high stringency). The DNA sequence thus obtained must encode a protein capable of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl.
The present invention also includes a polynucleotide comprising a nucleotide sequence which encodes a protein capable of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl and has a high sequence similarity to the nucleotide sequence of the polynucleotide according to above item (3), (4) or (5). Typically these nucleotide sequence are 95% identical, preferably 97% identical, most preferably at least 99% identical to the nucleotide sequence of the polynucleotide according to above item (3), (4) or (5) over the entire length thereof.
The above DNA of the present invention can be used to produce the protein of above item (1), (2) or (8) using recombinant DNA techniques. In general, the DNA and peptide of the present invention can be obtained by:
(A) cloning the DNA encoding the protein of the present invention;
(B) inserting the DNA encoding the entire coding region of the protein or a part thereof into an expression vector to construct a recombinant vector;
(C) fransforming host cells with the recombinant vector thus constructed; and
(D) culturing the obtained cells to express the protein or its analogue, and then purifying it by column chromatography.
General procedures necessary to handle DNA and recombinant host cells (e.g., E. coli) in the above steps are well known to those skilled in the art, and can be easily carried out in accordance with various laboratory manuals such as T Maniatis et al., supra. All the enzymes, reagents, etc., used in these procedures are commercially available, and unless otherwise stated, such commercially available products can be used according to the conditions specified by the manufactures' instructions to attain completely its objects. The above steps (A) to (D) can be further illustrated in more details as follows.
Techniques for cloning the DNA encoding the protein of the above step (A) include, in addition to the methods described in the specification of the present application, PCR amplification using synthetic DNA containing a part of the nucleotide sequence of the present invention (e.g., SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 107) as a primer, and selection of the DNA inserted into a suitable vector by hybridization with a labeled DNA fragment encoding a partial or full coding region of the protein of the present invention or a labeled synthetic DNA. Another technique involves direct amplification of DNA from total RNAs or mRNA fractions prepared from cells or tissues, using the reverse transcriptase polymerase chain reaction (RT-PCR method).
As a DNA inserted into a suitable vector, for example, a commercially available library (e.g., from CLONTECH and STRATAGENE) can be used. Techniques for hybridization are normally used in the art, and can be easily carried out in accordance with various laboratory manuals such as T. Maniatis et al., supra. Depending on the intended purpose, the cloned DNA encoding the protein of the present invention can be used as such or if desired after digestion with a restriction enzyme or addition of a linker. The DNA thus obtained may have a nucleotide sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 107, or a polynucleotide of above items (3) to (7). The DNA sequence to be inserted into an expression vector in the above step (B) may be a full-length cDNA or a DNA fragment encoding the above full-length protein, or a DNA fragment constructed so that it expresses a part thereof.
Thus, the present invention also includes a recombinant vector, which comprises the above DNA sequence. The expression vector for the protein of the present invention can be produced, for example, by excising the desired DNA fragment from the DNA encoding the protein of the present invention, and ligating the DNA fragment downstream of a promoter in a suitable expression vector.
Expression vectors for use in the present invention may be any vectors derived from prokaryotes (e.g., E. coli), yeast, fungi, insect viruses and vertebrate viruses. However, the vectors should be selected to be compatible with the hosts. Suitable combinations of host cell - expression vector systems are selected depending on the desired expression product.
When bacteria are used as hosts, plasmid vectors compatible with these bacteria are generally used as replicable expression vectors for recombinant DNA molecules. For example, the plasmids pBR322 and pBR327 can be used to transform E. coli. Plasmid vectors normally contain an origin of replication, a promoter, and a marker gene conferring upon a recombinant DNA a phenotype useful for selecting the cells transformed with the recombinant DNA. Example of such promoters include a β -lactamase promoter, lactose promoter and tryptophan promoter. Examples of such marker genes include an ampicillin resistance gene, and a tetracycline resistance gene. Examples of suitable expression vectors include the plasmids pUC18 and pUC19 in addition to ρBR322, pBR327.
In order to express the DNA of the present invention in yeast, for example, YEp24 can be used as a replicable vector. The plasmid YEp24 contains the URA3 gene, which can be employed as a marker gene. Examples of promoters in expression vectors for yeast cells include promoters derived from genes for 3-phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase.
Examples of promoters and terminators for use in expression vectors to express the DNA of the present invention in fungal cells include promoters and terminators derived from genes for 3-phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPD) and actin. Examples of suitable expression vectors include the plasmids pPGACY2 and pBSFAHY83.
Examples of promoters for use in expression vectors to express the DNA of the present invention in insect cells include a polyhedrin promoter and P10 promoter. Examples of expression vector suitable for insect cells include baculovirus.
Recombinant vectors used to express the DNA of the present invention in animal cells normally contain functional sequences to regulate genes, such as a promoter to be placed upstream of the DNA of the present invention, a polyadenylation site and a transcription termination sequence. Such functional sequences, which can be used to express the DNA of the present invention in eukaryotic cells, can be obtained from viruses or and viral substances.
Examples of such functional sequences include an SR promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter and HSV-TK promoter. Among them, a CMV promoter and SR ct promoter can be preferably used. Promoters to be placed inherently upstream of the gene encoding the protein of the present invention, can be used so long as they are suitable for use in the above host-vector systems. Examples of origins of replication include foreign origins of replication, for example, those derived from viruses such as adenovirus, polyoma virus and SV40 virus. When vectors capable of integration into host chromosomes are used as expression vectors, origins of replication of the host chromosomes may be employed. Examples of suitable expression vectors include the plasmids pSV-dhfr (ATCC 37146), pBPV-l(9-l) (ATCC 37111), pcDNA3.1 (INVITROGEN) andpME18S-FL3.
The present invention also includes a transformed cell, which comprises the above recombinant vector. Microorganisms or cells transformed with the replicable recombinant vector of the present invention can be selected from remaining untransformed parent cells based on at least one phenotype conferred by the recombinant vector. Phenotypes can be conferred by inserting at least one marker gene into the recombinant vector. Marker genes naturally contained in replicable vectors can be employed. Examples of marker genes include drug resistance genes such as neomycin resistance genes, and genes encoding dihydrofolate reductase.
As hosts for use in the above step (C), any of prokaryotes (e.g., E. coli), microorganisms (e.g., yeast and fungi) as well as insect and animal cells can be used so long as such hosts are compatible with the expression vectors used. Examples of such microorganisms include Escherichia coli strains such as E. coli K12 strain 294 (ATCC 31446), E. coli X1776 (ATCC 31537), E. coli C600, E. coli JM109 and E. coli B strain; bacterial strains belonging to the genus Bacillus such as Bacillus subtilis; intestinal bacteria other than E. coli, such as Salmonella typhimurium or Serratia marcescens; and various strains belonging to the genus Pseudomonas. Examples of such yeast include Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris. Examples of such fungi include Aspergillus nidulans, and Acremonium chrysogenum (ATCC 11550).
As insect cells, for example, Spodoptera frugiperda (Sf cells), High Five ™ cells derived from eggs of Trichoplusiani, etc., can be used when the virus is AcNPV. Examples of such animal cells include HEK 293 cells, COS-1 cells, COS-7 cells, Hela cells, and Chinese hamster ovary (CHO) cells. Among them, CHO cells and HEK 293 cells are preferred. When cells are used as hosts, combinations of expression vectors and host cells to be used vary with experimental objects. According to such combinations, two types of expression (i.e. transient expression and constitutive expression) can be included.
"Transformation" of microorganisms and cells in the above step (C) refers to introducing DNA into microorganisms or cells by forcible methods or phagocytosis of cells and then transiently or constitutively expressing the trait of the DNA in a plasmid or an intra-chromosome integrated form. Those skilled in the art can carry out transformation by known methods [see e.g., "Idenshi Kougaku Handbook (Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.]. For example, in the case of animal cells, DNA can be introduced into cells by known methods such as DEAE-dextran method, calcium-phosphate-mediated transfection, electroporation, lipofection, etc. For stable expression of the protein of the present invention using animal cells, there is a method in which selection can be carried out by clonal selection of the animal cells containing the chromosomes into which the introduced expression vectors have been integrated. For example, transformants can be selected using the above selectable marker as an indication of successful transformation. In addition, the animal cells thus obtained using the selectable marker can be subjected to repeated clonal selection to obtain stable animal cell strains highly capable of expressing the protein of the present invention. When a dihydrofolate reductase (DHFR) gene is used as a selectable marker, one can culture animal cells while gradually increasing the concentration of methotrexate (MTX) and select the resistant strains, thereby amplifying the DNA encoding the protein of the present invention together with the DHFR gene to obtain animal cell strains having higher levels of expression.
The above transformed cells can be cultured under conditions which permit the expression of the DNA encoding the protein of the present invention to produce and accumulate the protein of the present invention. In this manner, the protein of the present invention can be produced. Thus, the present invention also includes a process for producing a protein, which comprises culturing a transformed cell comprising the isolated polynucleotide according to above item (3) to (7) under conditions providing expression of the encoded protein and recovering the protein from the culture (i.e., host itself or media).
The above transformed cells can be cultured by methods known to those skilled in the art (see e.g., "Bio Manual Series 4", YODOSHA CO., LTD.). For example, animal cells can be cultured by various known animal cell culture methods including attachment culture such as Petri dish culture, multitray type culture and module culture, attachment culture in which cells are attached to cell culture carriers (microcarriers), suspension culture in which productive cells themselves are suspended. Examples of media for use in the culture include media commonly used for animal cell culture, such as D-MEM, I-MDM and RPMI 1640.
In order to separate and purify the protein of the present invention from the above culture, suitable combinations of per se known separation and purification methods can be used. Examples such methods include methods based on solubility, such as salting-out and solvent precipitation; methods based on the difference in charges, such as ion-exchange chromatography; methods mainly based on the difference in molecular weights, such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis; methods based on specific affinity, such as affinity chromatography; methods based on the difference in hydrophobicity, such as reverse phase high performance hquid chromatography; and methods based on the difference in isoelectric points, such as isoelectric focusing. For example, a protein of the present invention can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding proteins may be employed to regenerate active conformation when the polypeptide is denatured during intracellular synthesis, isolation or purification.
The protein of the present invention can also be produced as a fusion protein with another protein. These fusion proteins are also included within the present invention. For the expression of such fusion proteins, any vectors can be used so long as the DNA encoding the protein can be inserted into the vectors and the vectors can express the fusion protein. Examples of proteins to which a polypeptide of the present invention can be fused include glutathione S-transferase (GST) and a hexa-histidine sequence (6 x His). The fusion protein of the protein of the present invention with another protein can be advantageously purified by affinity chromatography using a substance with an affinity for the fusion partner protein. For example, fusion proteins with GST can be purified by affinity chromatography using glutathione as a ligand.
When the protein of the present invention is a membrane protein, a transformant into which DNA encoding the protein of the present invention has been introduced can express the protein on its membrane. The membrane which is prepared from such transformants and contains the protein of the present invention is also included within the present invention. As used herein, "membrane of a cell" includes cell membrane, and membrane of cell organelle. The membrane of a cell can be prepared by a method known to those skilled in the art. For example, cells are collected from the culture where transformants are cultured, and suspended in a suitable buffer. Then, the cells are lysed by a homogenizer or by vortex after addition of glassbeads. The obtained solution is centrifuged to remove uncrushed cells and the like, and the supernatant is ultracentrifuged under a sutable condition, and the obtained precipitate is suspended in a buffer to prepare. a membrabe fraction. The condition for ulfracentrifugation can be suitably selected depending on the type of membrane and the like.
The present invention also provides a protein capable of inhibiting the activity of the protein of the present invention. Examples of such proteins include antibodies, or other proteins that bind to active sites of the protein of the present invention, thereby inhibiting the expression of their activity.
The present invention also relates to an antibody that reacts with the protein of the present invention or a fragment thereof, and to production of such an antibody. More preferably, the present invention relates to an antibody that specifically react with the protein of the present invention or a fragment thereof, and to production of such an antibody. As used herein, "specifically" means that closs-reactivity is low, more preferably closs-reactivity is not present.
The antibody of the present invention is not specifically limited so long as it can recognize the protein of the present invention. Examples of such antibodies include polyclonal antibodies, monoclonal antibodies and their fragments, single chain antibodies and humanized antibodies. Antibody fragments can be produced by known techniques. Examples of such antibody fragments include, but not limited to, F(ab')2 fragments, Fab' fragments, Fab fragments and Fv fragments. For example, a monoclonal or polyclonal antibody can be produced by administering the protein according to above item (1) or (2) or epitope-bearing fragments as an antigen to a non-human animal. The antibody against the protein of the present invention can be produced by using the protein of the present invention or a peptide thereof as an immunogen according to per se known process for producing antibodies or antisera. Such methods are described, for example, in "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition, an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.
In the case of polyclonal antibodies, for example, the protein of the present invention or a peptide thereof can be injected to animals such as rabbits to produce antibodies directed against the protein or peptide, and then their blood can be collected. The polyclonal antibodies can be purified from the blood, for example, by ammonium sulfate precipitation or ion-exchange chromatography, or by using the affinity column on which the antigen protein has been immobilized.
In the case of monoclonal antibodies, for example, animals such as mice are immunized with the protein of the present invention, their spleen is removed and homogenized to obtain spleen cells, which are then fused with mouse myeloma cells by using a reagent such as polyethylene glycol. From the resulting fused cells (i.e. hybridoma), the clone producing the antibody directed against the protein of the present invention can be selected. Then, the resulting clonal hybridoma can be implanted intraperitoneally into mice, and the ascitic fluid was recovered from the mice. The resulting monoclonal antibody can be purified, for example, by ammonium sulfate precipitation or ion-exchange chromatography, or by using the affinity column on which the antigen protein has been immobilized.
When the resulting antibody is used to administer to humans, it is preferable to use a humanized antibody or human antibody in order to reduce its immunogenicity. These humanized antibodies or human antibodies can be produced using transgenic mice or other mammals. For a general review of humanized antibodies or human antibodies, see, for example, Morrison, S.L. et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984); Jones, P.T. et al., Nature 321:522-525 (1986); Hiroshi Noguchi, Igaku no Ayumi (J. Clin. Exp. Med.) 167:457-462 (1993); Takashi Matsumoto, Kagaku to Seibutsu (Chemistry and Biology) 36:448-456 (1998). Humanized chimeric antibodies can be produced by hnking a V region of a mouse antibody to a C region of a human antibody. Humanized antibodies can be produced by substituting a sequence derived from a human antibody for a region other man a complementarity-determining region (CDR) from a mouse monoclonal antibody.
In addition, human antibodies can be directly produced in the same manner as the production of conventional monoclonal antibodies by immunizing the mice whose immune systems have been replaced with human immune systems. These antibodies can be used to isolate or to identify clones expressing the protein.
Also, these antibodies can be used to purify the protein of the present invention from a cell extract or transformant producing the protein of the present invention. These proteins can also be used to construct ELISA, RIA (radioimmunoassay) and western blotting systems. These assay systems can be used for diagnostic purposes for detecting an amount of the protein of the present invention present in a body sample in a tissue or a fluid in the blood of an animal, preferably human. For example, these antibodies can be used for diagnosis of a disease characterized by undesirable activation of MAP kinase cascade resulting from (expression) abnormality of the protein of the present invention, such as inflammation, autoimmune diseases, infection diseases (for example, HTV infection), and cancer.
In order to provide a basis for diagnosis of a disease, a standard value must be estabhshed. This is a well-known technique to those skilled in the art. For example, a method of calculating the standard value comprises binding a body fluid or a cell extract of normal individual of a human or an animal to an antibody against the protein of the present invention under a suitable condition for the complex formation, detecting the amount of the antibody-protein complex by chemical or physical means and then calculating the standard value for the normal sample using a standard curve prepared from a standard solution containing a known amount of an antigen (the protein of the present invention). The presence of a disease can be confirmed by deviation from the standard value obtained by comparison of the standard value with the value obtained from a sample of an individual latently suffering from a disease associated with the protein of the present invention. These antibodies can also be used as reagents for studying functions of the protein of the present invention.
The antibody of the present invention can be used as a medicament as mentioned below. When the antibody of the present invention is used as a medicament, it is preferred to use an antibody capable of inhibiting the function of phosphorylation Elkl and/or the function of activating kinase which phosphorylates Elkl which is possessed by the protein of present invention (that is, neutralizing antibody).
The antibodies of the present invention can be purified and then administered to patients characterized by undesirable activation of kinase which phosphorylates Elkl resulting from (expression) abnormality of the protein of the present invention, such as inflammation, autoimmune disease, infection (such as HIV infection), cancer and the like. Thus in another aspect, the present invention is a pharmaceutical composition which comprises the above antibody as an active ingredient, and therapy and/or prevention using the antibody of the present invention, -h such pharmaceutical compositions, the active ingredient may be combined with other therapeutically active ingredients or inactive ingredients (e.g., conventional pharmaceutically acceptable carriers or diluents such as immunogenic adjuvants) and physiologically non-toxic stabilizers and/or excipients. The resulting combinations can be sterilized by filtration, and formulated into vials after lyophilization or into various dosage forms in stabilized and preservable aqueous preparations.
Administration to a patient can be intra-arterial administration, intravenous administration and subcutaneous administration, which are well known to those skilled in the art. The dosage range depends upon the weight and age of the patient, route of administration and the like. Suitable dosages can be determined by those skilled in the art. These antibodies exhibit therapeutic activity by inhibiting the MAP kinase activation mediated by the protein of the present invention. Specifically, the antibody of the present invention can be useful as a medicament for treatment or prevention of diseases characterized by abnormal activation of MAP kinase cascade, such as inflammation, autoimmune diseases, infection diseases (for example, HIV infection), and cancer.
The DNA of the present invention can also be used to isolate, identify and clone other proteins involved in intracellular signal transduction processes. For example, the DNA sequence encoding the protein of the present invention can be used as a "bait" in yeast two-hybrid systems (see e.g., Nature 340:245-246 (1989)) to isolate and clone the sequence encoding a protein ("prey") which can associate with the protein of the present invention. In a similar manner, it can be determined whether the protein of the present invention can associate with other cellular proteins (e.g., JNK, p38). In another method, proteins which can associate with the protein of the present invention can be isolated from cell extracts by immunoprecipitation [see e.g., "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.] using antibodies directed against the protein of the present invention. In still another method, the protein of the present invention can be expressed as a fusion protein with another protein as described above, and immunoprecipitated with an antibody directed against the fusion protein in order to isolate a protein which can associate with the protein of the present invention.
The present invention provides a process for diagnosing a disease or susceptibility to a disease related to expression or activity of the protein of present invention in a subject comprising the steps of:
(a) determining the presence or absence of a mutation in the gene encoding said protein in the genome of said subject; and/or
(b) analyzing the amount of expression of said protein in a sample derived from said subject.
The diagnostic assays offer a. process for diagnosing diseases or determining a susceptibihty to the diseases through detection of mutation in a gene for the protein of the present invention which is involved in the function of phosphorylating Elkl and/or the function of activating kinase which phosphorylates Elkl. hi addition, such diseases may be diagnosised by analyzing expression level of the gene in a sample derived from a subject at protein or mRNA level, and detecting an abnormally decreased or increased level of the expression.
Determination of the presence or absence of a mutation in the gene encoding the protein of the present invention which is involved in the function of phosphorylating Elkl and/or the function of activating kinase which phosphorylates Elkl, may involve RT-PCR using a part of the nucleotide sequences of genes as a primer, followed by conventional DNA sequencing to detect the presence or absence of the mutation. PCR-SSCP [Genomics 5:874-879 (1989); "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.] can also be used to determine the presence or absence of the mutation.
Decreased or increased expression of a gene in a sample can be measured at the RNA level using any of the methods well known in the art for the quantitation of polynucleotides, for example, nucleic acid amplification methods such as RT-PCR, and methods such as RNase protection assay, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein in a sample derived from a host are well-known to those skilled in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western blot analysis and ELISA assays. When an expression level is determined at a protein level, the antibody of the present invention mentioned above can be used.
The degree of abnormality of expression level of gene in a sample is not particularly limited. For example, when the level of the expressed protein is 2 or more times, or 1/2 or less, as compared with normal case, the subject may be diagnosed to be a disease. In another example, when the level of the expressed protein is 3 or more times, or 1/3 or less, as compared with normal case, the subject may be diagnosed to be a disease.
When the nucleotide sequence encoding the protein of the present invention in a genome of an individual contains a mutation, the mutation may cause a disease associated with the expression and/or activity of said protein which is involved in phosphorylation of Elkl.
When the amount of the expression of the protein in a sample from an individual is different from the normal value, the abnormal expression of the protein of the present invention which is involved in phosphorylation of Elkl may be responsible for diseases associated with the expression and/or activity of said protein which is involved in phosphorylation of Elkl.
The present invention also includes a method for screening compounds which inhibit or promote an action of activating MAP kinase cascade mediated by the protein of the present invention (an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl), which comprises the steps of:
(a) preparing a transformant by introducing a gene encoding a protein according to the present invention that has an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl and a gene encoding a protein which generates a signal which can detect an action of Elkl phosphorylation into a host cell;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the signal which can detect an action of Elkl phosphorylation; and
(d) selecting a candidate compound which can change the signal amount as compared with the case of the absence of candidate compounds, as a compound which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl.
A compound that increases the detectable signal 2-fold or higher than normal is preferably isolated or identified as an activator compound, and a compound that decreases the detectable signal 50% or less than normal is preferably isolated or identified as an inhibitor compound.
Examples of genes encoding a signal which can detect the action of Elkl phosphorylation include reporter genes. Reporter genes are used to detect the activity of trasnscription factor instead of directly detecting the activation of transcription factors of interest to be tested. The transcriptional activity of a promoter of a gene is analyzed by linking the promoter to a reporter gene and measuring the activity of the product of the reporter gene ("Bio Manual Series 4" (1994), YODOSHA CO., LTD.).
Any genes which code the peptide or protein can be used as the reporter genes so long as those skilled in the art can measure the activity or amount of the expression product (including the amount of the produced mRNA). For example, enzymatic activity of chloramphenicol acetyltransferase, j3 -galactosidase, luciferase, etc., can be measured. Any reporter plasmids can be used to evaluate activation of Elkl phosphorylation. One example is the reporter plasmids that have a yeast transcription factor Gal4 recognition sequence inserted upstream of the reporter gene. For example, pFR-Luc (STRATAGENE) can be used by introduction into a host cell together with various fusion proteins of genes being substrates of MAP kinase such as Elkl and ATF2 connected to yeast Gal4 protein DNA binding region gene: e.g. pFA2-Elkl orpFA2-ATF2 (STRATAGENE).
Any host cells can he used so long as action of Elkl phosphorylation and/or action of activation of kinase which phosphorylates Elkl can be detected in the host cells. Preferred host cells are mammalian cells such as HEK293-EBNA cells. Transformation and culture of the cells can be carried out as described above.
In a specific embodiment, the method for screening a compound which inhibits or promotes action of Elkl phosphorylation and/or action of activation of kinase which phosphorylates Elkl comprises culturing the transformed cell for a certain period of time, adding a certain amount of a test compound, measuring the reporter activity expressed by the cell after a certain period of time, and comparing the activity with that of a cell to which the test compound has not been added. The reporter activity can be measured by methods known in the art (see e.g., "Bio Manual Series 4" (1994), YODOSHA CO., LTD.).
Examples of test compounds for the screening include, but not limited to, low molecular weight compounds, high molecular weight compounds and peptides. Test compounds may be artificially synthesized compounds or naturally occurring compounds. Test compounds may be a single compound or mixtures. Usable examples includes a library of low molecular weight compounds, a compound hbrary which was synthesized by combinatorial chemistry, a narurally occurring product containing cells, plants, animals or a part thereof, or an extracred product of such narurally occurring product. When a mixture containing several compounds is used as a test substance for screening, the test substance which shows an activity of inhibiting or promoting action of Elkl phosphorylation and/or action of activation of kinase which phosphorylates Elkl can be further screened to isolate a single substance having the activity. Isolation and purification of a desired compound from a mixture can be carried out by using any known method such as filteration, extraction, washing, drying, concentration, crystallization or various chromatography in combination.
The method for screening according to the present invention can be carried out by the following steps:
(a) preparing a transformant by introducing a gene encoding a protein according to the present invention that has an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl into a host cell;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the signal which can detect an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl; and
(d) selecting a candidate compound which can change the signal amount as compared with the case of the absence of candidate compounds, as a compound which inhibits or promotes an action of Elkl phosphorylation and or an action of activation of kinase which phosphorylates Elkl.
In the above method, examples of the method of measuring an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl include a method of obtaining cell extracts and detecting the activation of various MAP kinases using an antibody against various phosphorylated MAP kinases. The antibodies against various phosphorylated MAP kinases are available from NEB (New England Biolabs). The examples of the method of detection using the antibody include ELISA, Western blotting, dot hybridyzation and the like. The activity of various MAP kinases can be directly measured by an immunoprecipitation method using an antibody against various MAP kinases. The antibody against various MAP kinases, and substrates thereto are available from Upstate Biotechnology.
The present invention further provides a method of producing a pharmaceutical composition, which comprises the following steps (a) to (e):
(a) preparing a transformant by introducing a gene encoding a protein according to the present invention that has an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl and a gene encoding a protein which generates a signal which can detect an action of Elkl phosphorylation into a host cell;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the signal which can detect an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl;
(d) selecting a candidate compound which can change the signal amount as compared with the case of the absence of candidate compounds, as a compound which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl; and
(e) producing a pharmaceutical composition which comprises a compound selected in the step of (d).
In the present invention, a pharmaceutical composition may also be produced by the following steps (a) to (e):
(a) preparing a transformant by introducing a gene encoding a protein according to the present invention that has an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl into a host cell;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl;
(d) selecting a candidate compound which can change the action of Elkl phosphorylation and/or the action of activation of kinase which phosphorylates Elkl as compared with the case of the absence of candidate compounds, as a compound which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl; and
(e) producing a pharmaceutical composition which comprises a compound selected in the step of (d).
In the step (d) of the method of producing a pharmaceutical composition, it is preferable to isolate or identify as an activator compound, a compound that increases said detectable signal 2-fold or higher than normal, and to isolate or identify as an inhibitor compound, a compound that decreases said detectable signal 50% or less than normal.
The protein of the present invention may also be used in a method for the structure-based design of an agonist, antagonist or inhibitor of the protein, by:
(a) determining in the first instance the three-dimensional structure of the protein;
(b) deducing the three-dimensional structure for the likely reactive or binding site(s) of an agonist, antagonist or inhibitor;
(c) synthesising candidate compounds that are predicted to bind to or react with the deduced binding or reactive site; and
(d) testing whether the candidate compounds are indeed agonists, antagonists or inhibitor.
The present invention also provides a compound which is selected by the above screening method. This compound has an activity of inhibiting or promoting the activation of MAP kinase cascade mediated by the protein of the present invention. More specifically, this compound has an activity of inhibiting or promoting an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl which are mediated by the protein of the present invention.
Since the compounds obtained by the above screening methods have an activity of inhibiting or promoting an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, they are useful as therapeutic or preventive pharmaceuticals for the diseases resulting from unfavorable activation or inactivation of Elkl phosphorylation and/or kinase which phosphorylates Elk.
When obtainment of a salt of the compounds is desired, a compound which is obtained in the form of a salt can be purified as it is. A compound which is obtained in the free form can be converted into a salt by isolating and purifying a salt obtained by dissolving or suspending the compound into a suitable solvent by conventional methods and then adding a desired acid or base. Examples of a step to optimize the compounds or salts thereof obtained by the method of the present invention as a pharmceutical composition, include methods of formulating according to ordinary processes such as the following. The above compounds or their pharmaceutically acceptable salts in an amount effective as an active ingredient, and pharmaceutically acceptable carriers can be mixed. A form of formulation suitable for the mode of administration is selected. A composition suitable for oral administration includes a solid form such as tablet, granule, capsule, pill and powder, and solution form such as solution, syrup, elixir and dispersion. A form useful for parenteral administration includes sterile solution, emulsion and suspension. The above carriers include, for example, sugars such as gelatin, lactose and glucose, starches such as corn, wheat, rice and maize, fatty acids such as stearic acid, salts of fatty acids such as calcium stearate, magnesium stearate, talc, vegetable oil, alcohol such as stearyl alcohol and benzyl alcohol, gum, and polyalkylene glycol. Examples of such liquid carriers include generally water, saline, sugar solution of dextrose and the like, glycols such as ethylene glycol, propylene glycol and polyethylene glycol.
The present invention also includes a kit for screening a compound for activity as an inhibitor or activator of Elkl phosphorylation and/or activation of a kinase which phosphorylates Elkl. The kit comprises reagents, etc. necessary for screening compounds for activity as an inhibitor or activator of Elkl phosphorylation and or activation of a kinase which phosphorylates Elkl, including:
(a) a transformant comprising a gene encoding a protein that phosphorylates Elkl and/or activates kinase which phosphorylates Elkl according to the present invention and a gene encoding a protein which provides a signal which can detect an action of Elkl phosphorylation; and (b) reagents for measuring the signal.
In another aspect, the present invention relates to a diagnostic kit which comprises:
(a) a polynucleotide of the present invention having a nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 107;
(b) a polynucleotide having a nucleotide sequence complementary to the nucleotide sequence of (a);
(c) a protein of the present invention having an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 or 108 or a fragment thereof; or
(d) an antibody to the protein of the present invention of above item (c).
A kit, which comprises at least one of (a), (b), (c) or (d), is useful for diagnosing a disease or susceptibility to a disease such as inflammation, autoimmune diseases, infectious diseases (e.g., HIV infection) and cancers.
Because MAP kinase cascade is involved in a wide variety of pathological conditions such as inflammation, autoimmune diseases, cancers and viral infections, it is an attractive target for drug design and therapeutic intervention. Many experiments show the possibility that the inhibition of MAP kinase cascade activity has significant physiological effects [e.g., J Cell Biochem 2001 Apr 3-27; 82(1): 68-77, Pharmacol Res 2001 Mar; 43(3): 275-83, Diabetes 2001 Jun; 50(6): 1495-504, Diabetes 2001 Jun; 50(6): 1464-71, Atherosclerosis 2001 May; 156 (1): 81-90].
The finding of the new protein described herein capable of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl has provided a new method for inhibiting an abnormal MAP kinase cascade activation. Thus, the present invention also relates to use of a compound which inhibits the function of the protein capable of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl described above, for inhibiting MAP kinase cascade activation. The present invention also relates to use of a compound which promotes the function of the protein capable of phosphorylating Elkl and or activating kinase which phosphorylates Elkl described above, for promoting MAP kinase cascade activation. The compound obtained by the above screening method, which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, is useful as a medicament to treat or prevent diseases characterized by undesirable activation of MAP kinase cascade, such as inflammation, autoimmune diseases, infectious diseases (e.g., HIV infection) and cancers.
It is known that there are cases where MAP kinase cascade activation induces apoptosis, and it is thought that there is a possibility that an inhibitor of Elkl phosphorylation action and/or a kinase which phosphorylates Elkl will control apoptosis. Diseases which may be treated by the inhibition of apoptosis include GVHD, skin diseases such as toxic epidermal necrolysis (TEN), prohferative nephritis (e.g., IgA nephritis, purpuric nephritis and lupus nephritis) and fulminant hepatitis, hi contrast, examples of patients for whom the induction of apoptosis may be exploited in treatment include tumor patients. Thus, the compound obtained by the above screening method, which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, is useful as a medicament to treat or prevent these diseases.
In addition, the gene encoding the protein of the present invention is useful for gene therapy to treat various diseases such as cancers, autoimmune diseases, allergy diseases and inflammatory response. "Gene therapy" refers to administering into the human body a gene or a cell into which a gene has been introduced. The protein of the present invention and the DNA encoding the protein can also be used for diagnostic purposes. Thus, the present invention provides an agent for gene therapy which comprises a gene encoding the protein of the present invention.
When a gene encoding the protein of the present invention is used for a agent for gene therapy, a technique of RNA interference (RNAi) mentioned below may be applied. Thus, the present invention provides a vector for gene therapy which expresses double strand RNA having a gene sequence encoding the protein of the present invention.
The form of the agent for gene therapy is not particularly limited, but includes a pharmaceutical composition which comprises a expression vector containing a gene of the present invention in a pharmaceutical carrier of physiological buffer. The pharmaceutical carrier may contain suitable stabilizer (for example, nuclease inhibitor), chelate agent (for example, EDTA), and/or other auxiliary agent. Alternatively, the agent for gene therapy of the present invention may be provided as a complex of an expression vector containing a gene of the present invention and a liposome. The agent for gene therapy may be applied using a catheter. For example, the agent for gene therapy of the present invention can be directly injected into a blood vessel of patient and the like.
The dosage of the agent for gene therapy of the present invention should be selected depending on the conditions such as age, sex, body weight and symptom of patient, and administration route, and is generally about 1 μ g/kg to about 1000 mg/kg, more preferably about 10 g kg to about 100 mg/kg, as an amount of DNA (which is an effective ingredient) per one administration for adult. The number of administration is not particularly limited.
The compound obtained by the screening method of the present invention or a salt thereof can be formulated into the above pharmaceutical compositions (e.g., tablets, capsules, elixirs, microcapsules, sterile solutions and suspensions) according to conventional procedures. The formulations thus obtained are safe and of low toxicity, and can he administered, for example, to humans and mammals (e.g., rats, rabbits, sheep, pigs, cattle, cats, dogs and monkeys). Administration to patients can be carried out by methods known in the art, such as intra-arterial injection, intravenous injection and subcutaneous injection. The dosage and the administration route may vary with the weight and age of the patient, but those skilled in the art can appropriately select the administration route, and can appropriately select suitable dosage which is suitable for the administration route. When the compound can be encoded by DNA, the DNA can be inserted into a vector for gene therapy, and gene therapy can be carried out.
Thus, the present invention relates to a medicament which comprises a compound which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl.
In addition, the above compound is useful as a medicament to treat or prevent diseases characterized by undesirable activation of MAP kinase cascade, such as inflammation, autoimmune diseases, viral diseases, infectious diseases and cancers. Thus, the present invention also relates to a pharmaceutical composition for inflammation, autoimmune diseases, viral diseases, cancers, etc., which comprises the compound which promotes or inhibits the activation of MAP kinase cascade. Specifically, the pharmaceutical composition is useful as a therapeutic and prophylactic drug against, for example, rheumatoid arthritis, osteoarthritis, systemic lupus erythematosus, diabetes, sepsis, asthma, allergic rhinitis, ischemic heart diseases, inflammatory intestinal diseases, subarachnoid hemorrhage, viral hepatitis, AIDS, GVHD, skin conditions such as toxic epidermal necrolysis (TEN), hyperplastic nephritis (IgA nephritis, purpuric nephritis, lupus nephritis), and fulminant hepatitis.
The present invention also relates to the use of the aforementioned compound for manufacturing a medicament for therapy and prevention of inflammation, autoimmune diseases, viral diseases, cancers, etc.
The present invention also provides an antisense oligonucleotide against the polynucleotide of any one of above items (3) to (7). An antisense oligonucleotide refers to an oligonucleotide complementary to the target gene sequence. The antisense oligonucleotide can inhibit the expression of the target gene by inhibiting RNA functions such as translation to proteins, transport to the cytoplasm and other activity necessary for overall biological functions. In this case, the antisense oligonucleotide may be RNA or DNA. The DNA sequence of the present invention can be used to produce an antisense oligonucleotide capable of hybridizing with the mRNA transcribed from the gene encoding the protein of the present invention. It is known that an antisense oligonucleotide generally has an inhibitory effect on the expression of the corresponding gene (see e.g., Saibou Kougaku Vol.13, No .4 (1994)). The oligonucleotide containing an antisense coding sequence against a gene encoding the protein ofthe present invention can be introduced into a cell by standard methods. The oligonucleotide effectively blocks the translation of mRNA of the gene encoding the protein of the present invention, thereby blocking its expression and inhibiting undesirable activity.
The antisense oligonucleotide of the present invention may be a naturally occurring oligonucleotide or its modified form [see e.g., Murakami & Makino, Saibou Kougaku Vol.13, No.4, p.259-266 (1994); Akira Murakami, Tanpakushitsu Kakusan Kouso (PROTEIN, NUCLEIC ACID AND ENZYME) Vol.40, No.lO, p.1364-1370 (1995),Tunenari Takeuchi et al., Jikken Igaku (Experimental Medicine) Vol. 14, No. 4 p85-95(1996)]. Thus, the oligonucleotide may have modified sugar moieties or inter-sugar moieties. Examples of such modified forms include phosphothioates and other sulfur-containing species used in the art. According to several preferred embodiments of the present invention, at least one phosphodiester bond in the oligonucleotide is substituted with the structure which can enhance the ability of the composition to permeate cellular regions where RNA with the activity to be regulated is located.
Such substitution preferably involves a phosphorothioate bond, a phosphoramidate bond, methylphosphonate bond, or a short-chain alkyl or cycloalkyl structure. The oligonucleotide may also contain at least some modified base forms. Thus, it may contain purine and pyrimidine derivatives other than naturally occurring purine and pyrimidine. Similarly, the furanosyl moieties of the nucleotide subunits can be modified so long as the essential purpose of the present invention is attained. Examples of such modifications include 2'-O-aIkyl and 2'-haIogen substituted nucleotides. Examples of modifications in sugar moieties at their 2-ρosition include OH, SH, SCH3, OCH3, OCN or O(CH2)nCH3, wherein n is 1 to about 10, and other substituents having similar properties. All the analogues are included in the scope of the present invention so long as they can hybridize with the mRNA of the gene of the present invention to inhibit functions of the mRNA.
The antisense oligonucleotide of the present invention contains about 3 to about 50 nucleotides, preferably about 8 to about 30 nucleotides, more preferably about 12 to about 25 nucleotides. The antisense oligonucleotide of the present invention can be produced by the well-known sohd phase synthesis technique. Devices for such synthesis are commercially available from some manufactures including Applied Biosystems. Other oligonucleotides such as phosphothioates can also be produced by methods known in the art.
The antisense oligonucleotide of the present invention is designed to hybridize with the mRNA transcribed from the gene of the present invention. Those skilled in the art can easily design an antisense ohgonucleotides based on a given gene sequence (For example, Murakami and Makino: Saibou Kougaku Vol. 13 No.4 p259-266 (1994), Akira Murakami: Tanpakushitsu Kakusan Kouso (PROTEIN, NUCLEIC ACID AND ENZYME) Vol. 40 No.lO pl364-1370 (1995), Tunenari Takeuchi et al., Jikken Igaku (Experimental Medicine) Vol. 14 No. 4 p85-95 (1996)). Recent sutudy suggests that antisense oligonucleotides which are designed in a region containing 5' region of mRNA, preferably, the translation initiation site, are most effective for the inhibition of the expression of a gene. The length of the antisense oligonucleotides is preferably 15 to 30 nucleotides and more preferably 20 to 25 nucleotides. It is important to confirm no interaction with other mRNA and no formation of secondary structure in the oligonucleotide sequence by homology search. The evaluation of whether the designed antisense oUgonucleotide is functional or not can be determined by introducing the antisence oligonucleotide into a suitable cell and measuring the amount of the target mRNA, for example by northern blotting or RT-PCR, or the amount of the target protein, for example by western blotting or fluorescent antibody technique, to confirm the effect of expression inhibition.
Another method includes the triple helix technique. This technique involves forming a triple helix on the targeted intra-nuclear DNA sequence, thereby regulating its gene expression, mainly at the transcription stage. The oligonucleotide is designed mainly in the gene region involved in the transcription and inhibits the transcription and the production of the protein of the present invention. Such RNA, DNA and oUgonucleotide can be produced using known synthesizers.
The antisense oligonucleotide may be introduced into the ceUs containing the target nucleic acid sequence by any of DNA transfection methods such as calcium phosphate method, lipofection, electroporation, microinjection, or gene transfer methods including the use of gene transfer vectors such as viruses. An antisense oUgonucleotide expression vector can be prepared using a suitable retrovirus vector, then the expression vector can be introduced into the cells containing the target nucleic acid sequence by contacting the vector with the cells in vivo or ex vivo.
The DNA of the present invention can be used in the antisense RNA/DNA technique or the triple helix technique for the purpose of inhibiting MAP kinase cascade activation mediated by the protein of the present invention.
The antisense oUgonucleotide against the gene encoding the protein of the present invention is useful as a medicament to treat or prevent diseases characterized by undesirable activation of MAP kinase cascade, such as inflammation, autoimmune diseases, infectious diseases (e.g., HIV infection) and cancers. Thus, the present invention also includes a pharmaceutical composition which comprises the above antisense oUgonucleotide as an active ingredient. The antisense oUgonucleotide can also be used to detect such diseases using northern hybridization or PCR.
The present invention also provides a ribozyme or deoxyribozyme which inhibits an action of Elkl phosphorylation and/or activation of kinase which phosphorylates Elkl. A ribozyme and deoxyribozyme is an RNA capable of recognizing a nucleotide sequence of a nucleic acid and cleaving the nucleic acid (see e.g., Hiroshi Yanagawa, "Jikken Igaku (Experimental Medicine) Bioscience 12: New Age of RNA). The ribozyme or deoxyribozyme can be produced so that it cleaves the selected target RNA (e.g., mRNA encoding the protein of the present invention). Based on the nucleotide sequence of the DNA encoding the protein of the present invention, the ribozyme or deoxyribozyme specifically cleaving the mRNA of the protein of the present invention can be designed. Such ribozyme or deoxyribozyme has a complementary sequence to the mRNA for the protein of the present invention, complementarily associates with the mRNA and then cleaves the mRNA, which results in reduction or entire loss of the expression of the protein of the present invention. The level of the reduction of the expression is dependent on the level of the ribozyme or deoxyribozyme expression in the target cells.
There are two types of ribozyme or deoxyribozyme commonly used: a hammerhead ribozyme and a hairpin ribozyme. In particular, hammerhead ribozymes or deoxyribozymes have been weU studied regarding their primary and secondary structure necessary for their cleavage activity, and those skilled in the art can easily design the ribozymes nucleotided solely on the nucleotide sequence information for the DNA encoding the protein of the present invention [see e.g., Ma et al., Saibou Kougaku Vol.16, No.3, p.438-445 (1997); Ohkawa & Taira, Jikken Igaku (Experimental Medicine) Vol.12, No.12, p.83-88 (1994)]. It is known that the hammerhead ribozymes or deoxyribozymes have a structure consisting of two recognition sites (recognition site I and recognition site II foπning a chain complementary to target RNA) and an active site, and cleave the target RNA at the 3 'end of its sequence NUX (wherein N is A or G or C or U, and X is A or C or U) after the formation of a complementary pair with the target RNA in the recognition sites. In particular, the sequence GUC (or GUA) has been found to have the highest activity [see e.g., Koizumi, M. et al., Nucl. Acids Res. 17:7059-7071 (1989); Ma et al., Saibou Kougaku Vol.16, No.3, p.438-445 (1997); Ohkawa & Taira, Jikken Igaku (Experimental Medicine) Vol.12, No.12, p.83-88 (1994); Kawasaki & Taira, Jikken Igaku (Experimental Medicine) Vol.18, No.3, p.381-386 (2000)].
Therefore the sequence GTC (or GTA) is searched out, and a ribozyme is designed to form several,up to 10 to 20 complementary base pairs around that sequence. The suitability of the designed ribozyme can be evaluated by checking whether the prepared ribozyme can cleave the target mRNA in vitro according to the method described for example in Ohkawa & Taira, Jikken Igaku (Experimental Medicine) Vol.12, No.12, p.83-88 (1994). The ribozyme can be prepared by methods known in the art to synthesize RNA molecules.
Alternatively, the sequence of the ribozyme can be synthesized on a DNA synthesizer and inserted into various vectors containing a suitable RNA polymerase promoter (e.g., T7 or SP6) to enzymaticaUy synthesize an RNA molecule in vitro. Such ribozymes can be introduced into ceUs by gene transfer methods such as microinjection. Another method involves inserting DNA encoding a ribozyme into a suitable expression vector and introducing the vector into cell strains, ceUs or tissues. Suitable vectors can be used to introduce the ribozyme into a selected cell. Examples of vectors commonly used for such purpose include plasmid vectors and animal virus vectors (e.g., retrovirus, adenovirus, herpes or vaccinia virus vectors). Such ribozymes are capable of inhibiting the Elkl phosphorylation action and/or kinase which phosphorylates Elkl mediated by the protein of the present invention.
According to the present invention, a double-stranded RNA is provided, which inhibits an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl.
The introduction of the double-stranded RNA into a ceU enables the specific degradation of mRNA corresponding to the sequence of the RNA, and the degradation suppresses gene expression. Recently, this phenomenon, referred to as RNA interference (RNAi), has been revealed. One example of a method using RNAi is a method for introducing an artificiaUy synthesized small interfering RNA (siRNA) into a cell. siRNA is a double-stranded RNA of 19 to 25 base pairs which is mentioned as an important trigger to induce RNAi phenomena. With regard to the 19-to-25-nucleotide sequence in a suitable region of the sequence of a gene encoding the protein of the present invention, a sense RNA (wherein DNA sequence is substituted by RNA sequence) and an antisense RNA (having a sequence complementary to the sense RNA) are synthesized to prepare siRNA, and the siRNA is introduced into a cell by lipofection using, for example, FuGENE6, thereby enabling the use of RNAi. h addition to the introduction of synthesized siRNA, a method that is also effective has been recently and gradually unveiled, which comprises: incorporating the 19-to-25-nucleotide sequence in a suitable region of the sequence of a gene encoding the protein of the present invention and a sequence complementary thereto into a plasmid; and temporarily expressing siRNA in the cell. More specifically, for example, pSilencer siRNA Expression Vector available from Ambion can be used (Morita Takashi et al., Protein, Nucleic Acid and Enzyme, Vol.4 No.14 p. 1939-p. 1945 (2001); Sugimoto Asako, Kagaku to Seibutu (Chemistry and Biology), Vol.40 No.ll pp.713-718).
As mentioned above, fuU-length cDNA is used in the present invention. That means 5' end sequence of the cDNA in the present invention is the transcription initiation site of the corresponding mRNA. Therefore the cDNA sequence can be used to identify the promoter region of the gene by comparing the cDNA with the genomic nucleotide sequence. Genomic nucleotide sequences are available from various databases when the sequences have been deposited in the databases. Alternatively, the cDNA can also be used to clone the desired sequence from a genomic library, for example, by hybridization, and determine its nucleotide sequence. Thus, by comparing the nucleotide sequence of the cDNA of the present invention with a genomic sequence, the promoter region of the gene located upstream the cDNA can be identified, hi addition, the promoter fragment thus identified can be used to construct a reporter plasmid for evaluating the expression of the gene, hi general, the DNA fragment spanning 2kb (preferably lkb) upstream from the transcription initiation site can be inserted upstream of the reporter gene to produce the reporter plasmid. The reporter plasmid can be used to screen for a compound which enhances or reduces the expression of the gene. For example, such screening can be carried out by fransforming a suitable cell with the reporter plasmid, culturing the transformed cell for a certain period of time, adding a certain amount of a test compound, measuring the reporter activity expressed by the ceU after a certain period of time, and comparing the activity with that of a cell to which the test compound has not been added. These methods are also included in the scope of the present invention.
The present invention also relates to a computer-readable medium on which a sequence data set has been stored, said sequence data set comprising at least one nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107 and/or at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108.
In another aspect, the present invention relates to a method for calculating a homology, which comprises comparing data on the above medium with data of other nucleotide sequences. Thus, the gene and amino acid sequence of the present invention provide valuable information for determining their secondary and tertiary structure, e.g., information for identifying other sequence having a similar function and high homology. These sequences are stored on the computer-readable medium, then a database is searched using data stored in a known macromolecule structure program and a known search tool such as GCG program package (Devereux, J. et al, Nucleic Acids Research 12(1):387 (1984)). In this manner, a sequence in a database having a certain homology can be easily found.
The computer-readable medium may be any composition of materials used to store information or data. Examples of such media include commerciaUy available floppy disks, tapes, chips, hard discs, compact disks and video disks. The data on the medium aUows a method for calculating a homology by comparing the data with other nucleotide sequence data. This method comprises the steps of providing a first polynucleotide sequence containing the polynucleotide sequence of the present invention for the computer-readable medium, and then comparing the first polynucleotide sequence with at least one second polynucleotide or polypeptide sequence to identify the homology.
The present invention also relates to an insoluble substrate to which a polynucleotide comprising aU or part of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107, is fixed. A plurality of the various polynucleotides which are DNA probes are fixed on a specifically processed solid substrates such as slide glass to form a DNA microarray and then a labeled target polynucleotide is hybridized with the fixed polynucleotides to detect a signal from each of the probes. The data obtained is analyzed and the gene expression is determined.
The present invention further relates to an insoluble substrate to which a polypeptide comprising all or part of an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108 is fixed. Proteins, which are expected to be useful for diagnosis or development of a new drug, can be isolated and or identified by mixing the insoluble substrate and a ceU extract from organisms, and capturing the proteins onto the insoluble substrate.
EXAMPLES
The foUowing examples further illustrate, but do not Umit the present invention.
Example 1: Construction of a fuU-length cDNA library using the oligo-capping method
(1) Preparation of RNA from human peripheral blood mononuclear ceU (PBMC)
About 2X 107 human peripheral blood mononuclear cell (PBMC) were cultured in the presence of 5% CO2 at 37°C for 7 days in a RPMI1640 medium (purchased from GIBCO) containing 10% FBS (Fetal Bovine Serum; purchased from GIBCO) and PHA (Phytohemagglutinin) at a final concentration of 10 μ g/ml. The thus obtained culture solution was centrifuged and washed once with PBS, and about 1 X 108 cells were collected to obtain the RNA extraction source (1). The remaning cells (about 1 X 108 cells) were cultured for 24 hours in the aforementioned medium lacking PHA, and were devided into 2 portions equally. One portion was cultured for further 24 hours, and the ceUs were collected as mentioned above to obtain the RNA extraction source (2). The other portion was cultured for 24 hours in the medium containing PMA (Phorbol-12-myristate-13-acetate) at a final concentration of lOnM and A23187 at a final concentration of 10 M, and the cells were collected to obtain the RNA extraction source (3). Then, total RNAs were respectively obtained from these (1) to (3) (3 types of the recovered cells) by using the RNA extraction reagent ISOGEN (purchased from NIPPON GENE) according to the manufacture's protocol. Then, poly A+ RNA was obtained from the total RNA by using an oligo-dT ceUulose column according to Maniatis et al., supra.
(2) Construction of a full-length cDNA Ubrary by the oligo-capping method
A full-length cDNA library was constructed from the above poly A+ RNA of human PBMC by the oligo-capping method according to the method of Sugano S. et al. [e.g., Maruyama, K. & Sugano, S., Gene, 138:171-174 (1994); Suzuki, Y. et al, Gene, 200:149-156 (1997); Suzuki, Y & Sugano, S. "Shin Idenshi Kougaku Handbook (New Genetic Engineering Handbook)", the third edition (1999), an extra issue of "Jikken Igaku (Experimental Medicine)", YODOSHA CO., LTD.].
(3) Preparation of plasmid DNA
The full-length cDNA library constructed as above was transfected into E. coli strain TOP 10 by electroporation, then spread on LB agar medium containing 100 μ g/ml ampicillin, and incubated overnight at 37°C. Then, using QIAwell 96 Ultra Plasmid Kit (QIAGEN) according to the manufacturer's protocol, the plasmids were recovered from the E.coU colonies grown on ampicillin-containing LB agar medium.
Example 2: Cloning of DNA capable of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl
(1) Screening of the cDNA encoding the protein capable of phosphorylating Elkl and/or activating kinase which phosphorylates Elkl
293-EBNA cells (purchased from Invitrogen) were grown to 1 x 104 ceUs/well in a 96 well plate for cell culture for 24 hours at 37°C (in the presence of 5% CO2) using 5% FBS containing DMEM medium. Then, 60ng of pFR-Luc (purchased from STRATAGENE), 0.25ng of pFA2-Elkl (purchased from STRATAGENE), 5ng of pcDNA3.1 (+) (INVITROGEN) into which human p38 had been incorporated, 30ng of pcDNA3.1(+) into which human JNJl β 1 gene had been incorporated; and 2 t 1 of the full-length cDNA prepared in above Example 1.(3) were cotransfected into the cells in a well using FuGENE 6 (purchased from Roche) according to the manufacturer's protocol. After 24 hours of culture at 37°C, the reporter activity reflecting activation of MAP kinase cascade (luciferase activity) was measured using long-term luciferase assay system, PicaGene LT2.0 (TOYO INK) according to the attached manufacturer's instructions. The luciferase activity was measured using Wallac ARVO™ST 1420 MULTILABEL COUNTER (Perkin Elmer).
(2) DNA sequencing The above screening was carried out, and plasmids showing a 3-fold or more increase in luciferase activity compared to that of the control experiment (luciferase activity of the ceU into which vacant vector pME18S-FL3 is introduced instead of fuU-length cDNA) were selected. One pass sequencing was carried out from the 5' end of the cloned cDNA (sequencing primer: 5'-CTTCTGCTCTAAAAGCTGCG-3' (SEQ ID NO: 109)) and from the 3' end (sequencing primer: 5'-CGACCTGCAGCTCGAGCACA-3* (SEQ ID NO: 110)) so that as long sequence as possible is determined. The sequencing was carried out using the reagent Thermo Sequenase π Dye Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech) or BigDye Terminator Cycle Sequencing FS Ready Reaction Kit (AppUed Biosystems) and the device ABI PRISM 377 sequencer or ABI PRISM 3100 sequencer according to the manufacturer's instructions.
(3) Database analysis of the obtained clones
BLAST (Basic local alignment search tool) searching [S. F. Altschul et al., J. Mol. Biol., 215:403-410 (1990)] was carried out in GenBank for the obtained nucleotide sequences. The results showed that 54 clones represented 54 genes encoding new proteins capable of phosphorylating Elkl and or activating kinase which phosphorylates Elkl.
(4) Full-length sequencing
The fuU-length DNA sequences for the 54 new clones were determined (SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107). The amino acid sequences of the protein coding regions (open reading frames) were deduced (SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108).
Example 3: Screening compounds inhibiting activation of MAP kinase cascade, in particular, p38 signal. 293-EBNA cells were seeded on DMEM medium containing 5% FBS in a 96-weU cell culture plate to a final cell density of 1 x 104 cells/100 μ 1 medium/well, and cultured for 24 hours at 37°C in the presence of 5% CO2. Then, lOng of the plasmid containing the gene encoding MKK3, a protein known to act to activate MAP kinase cascade, obtained by the screening in Example 2 and 60ng of pFR-Luc (purchased from STRATAGENE), 0.25ng pFA2-Elkl (purchased from STRATAGENE), 5ng of pcDNA3.1 (+) (INVITROGEN) into which human p38 had been incorporated were cotransfected into the cells in a well using FuGENE 6. Within 1 hour, SB203580 (purchased from CALBIOCHEM) which is known to be an inhibitor of p38, which is one kind of MAP kinase, was added to the culture to a final concentration of 0.082-20 μ M. After 24 hours of culture at 37°C, the reporter activity was measured using PicaGene LT2.0. The results showed that SB203580 inhibited the expression of the reporter gene to a level of IC50 is about 0.4 μ M (Fig. 1).
Example 4: Measurement of reporter activity corrected using an internal control.
293-EBNA cells were inoculated on a 5% FBS (Fetal Bovine Serum)-containing DMEM medium in a 96-well ceU culture plate to a final cell density of 1 x 104 ceUs/lOOμ 1 medium/well, and cultured for 24 hours at 37°C (in the presence of 5% CO2). Then, using FuGENE 6 (purchased from Roche), 25ng, 50ng and lOOng of each of the expression plasmids obtained in Example 2 above which comprise a gene encoding the MAP kinase cascade activating protein according to SEQ ID NO: 2, 4, 6, 10, 16, 18, 24, 26, 32, 36, 44, 50, 52, 56, 60, 62, 64, 68, 70, 74, 78, 82, 86, 88, 92, 96, 100, 102, 106 or 108 were respectively co-transfected in a well with 60ng of pFR-Luc (purchased from STRATAGENE), 0.25ng of pFA2-Elkl (STRATAGENE), 5ng of pcDNA 3.1 (+) (INVITROGEN) into which human p38 gene had been incorporated, 30ng of pcDNA 3.1 (+) (INVITROGEN) into which human JNJl β 1 gene had been incorporated, and lOng of pRL-tk (purchased from Toyo Ink) as a reporter gene for internal control use. The method used for introduction was in accordance with the attached protocol. After culturing for 24 hours at 37°C, reporter activity (firefly luciferase acitivity), which reflects MAP kinase cascade activation, and reporter activity being the internal control (sea pansy luciferase) were measured using Picagene Dual (purchased from Toyo Ink Manufacturing) in accordance with the manufacturer's instructions. Luciferase activity was measured with Perkin Elmer's WaUac ARVOTMST 1420 MULTILABEL COUNTER.
As a result, reporter activity, which reflects MAP kinase cascade activation corrected with internal control reporter activity, exhibited increases that were dependent on the amount of expression plasmid comprising a gene encoding a MAP kinase cascade activating protein according to SEQ ID NO: 2, 4, 6, 10, 16, 18, 24, 26, 32, 36, 44, 50, 52, 56, 60, 62, 64, 68, 70, 74, 78, 82, 86, 88, 92, 96, 100, 102, 106 or 108 obtained in Example 2 above, as the amount of plasmid introduced was changed through 25ng, 50ng, and lOOng, respectively.
Reporter activity where an expression plasmid comprising the above gene was introduced, which reflects MAP kinase cascade activation corrected with internal control reporter activity, is shown in Table 1, which is expressed as a factor of the same reporter activity where a null vector was introduced (S B value).
Table 1
Figure imgf000056_0001
Figure imgf000057_0001
Example 5: Screening for inhibitors
293-EBNA cells were inoculated on a 5% FBS (Fetal Bovine Serum)-containing DMEM medium in a 96-well ceU culture plate to a final ceU density of 1 x 104 ceUs/100 μ 1 medium/well, and cultured for 24 hours at 37°C (in the presence of 5% CO2). Then, using FuGENE6 (Roche), 50ng of each of the expression plasmids obtained in Example 2 above which comprise a gene encoding the MAP kinase cascade activating protein according to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 or 108 were respectively co-transfected in a well with 60ng of pFR-Luc (STRATAGENE), 0.25ng of pFA2-Elkl (STRATAGENE), 5ng of pcDNA 3.1 (+) (INVITROGEN) into which human p38 gene had been incorporated and 30ng of pcDNA 3.1 (+) (INVITROGEN) into which human JNJl β 1 gene had been incorporated. The method used for introduction was in accordance with the attached protocol. After culturing for 2 hours at 37°C, any one of 1000 types of compound that were held in stock, was added to a final concentration of 10 μ M per well, and after further culturing for 24 hours at 37°C, reporter activity, which reflects MAP kinase cascade activition, was measured by Picagene LT2 (purchased from Toyo Ink Manufacturing) in accordance with the manufacturer's instructions. Using a weU to which no compound was added as a control, compounds were screened to identify a compound which reduced reporter activity in the well to which it was added to 50% or lower. As a result, in systems to which was introduced an expression plasmid comprising a gene encoding the MAP kinase cascade activating protein according to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 or 108, staurosporine, which is known to inhibit the activity of various kinases non-specifically, was obtained as an inhibitor. The structural formula of the staurosporine obtained here is shown in Figure 2.
EFFECTS OF THE INVENTION
As described above, the present invention provides proteins capable of phosphorylating Elkl and or activating a kinase which phosphorylates Elkl, and genes encoding the proteins. These proteins and genes are highly likely to posses highly industrially useful MAP kinase cascade activating activity. The proteins of the present invention and the genes encoding the proteins allow not only screening for compounds useful for treating and preventing diseases associated with the excessive activation or inhibition of MAP kinase cascade, but also production of diagnostics for such diseases. The genes of the present invention are also useful as a gene source used for gene therapy.

Claims

1. A purified protein selected from the group consisting of:
(a) a protein which consists of an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108; and
(b) a protein that acts to phosphorylate Elkl and or activate kinase which phosphorylates Elkl and consists of an amino acid sequence having at least one amino acid deletion, substitution or addition in an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108.
2. A purified protein that acts to phosphorylate Elkl and/or activate kinase which phosphorylates Elkl and comprises an amino acid sequence having at least 95% identity to the protein according to claim 1 over the entire length thereof.
3. An isolated polynucleotide which comprises a polynucleotide sequence encoding a protein selected from the group consisting of:
(a) a protein which comprises an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108; and
(b) a protein that acts to phosphorylate Elkl and/or activate a kinase which phosphorylates Elkl, and consists of an amino acid sequence having at least one amino acid deletion, substitution or addition in an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108.
4. An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of:
(a) a polynucleotide sequence represented by SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 107;
(b) a polynucleotide sequence which encodes a protein that acts to phosphorylate Elkl and/or activate kinase which phosphorylates Elkl, and which hybridizes under stringent conditions with a polynulceotide having a polynulceotide sequence complementary to the polynucleotide sequence of (a); and,
(c) a polynucleotide sequence encoding a protein that acts to phosphorylate Elkl and/or activate a kinase which phosphorylates Elkl, and consists of a nucleotide sequence having at least one nucleotide deletion, substitution or addition in a nucleotide sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107.
5. An isolated polynucleotide comprising a polynucleotide sequence selected from the group consisting of:
(a) a polynucleotide sequence represented by protein coding region of any of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or 107;
(b) a polynucleotide sequence which encodes a protein that acts to phosphorylate Elkl and/or activate kinase which phosphorylates Elkl, and which hybridizes under stringent conditions with a polynulceotide having a polynulceotide sequence complementary to the polynucleotide sequence of (a); and,
(c) a polynucleotide sequence encoding a protein that acts to phosphorylate Elkl and/or activate a kinase which phosphorylates Elkl, and consists of a nucleotide sequence having at least one nucleotide deletion, substitution or addition in a nucleotide sequence of protein coding region of any one selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107.
6. An isolated polynucleotide comprising a polynucleotide sequence which encodes a protein that acts to phosphorylate Elkl and/or activate a kinase which phosphorylates Elkl, and has at least 95% identity to the polynucleotide sequence according to claim 3 over the entire length thereof.
7. An isolated polynucleotide comprising a nucleotide sequence which encodes a protein that acts to phosphorylate Elkl and/or activate a kinase which phosphorylates Elkl, and has at least 95% identity to the polynucleotide sequence according to claim 4 or 5 over the entire length thereof.
8. A purified protein encoded by the polynucleotide according to any one of claims 3 to 7.
9. A recombinant vector which comprises a polynucleotide according to any one of claims 3 to 7.
10. A gene therapy agent which comprises the recombinant vector according to claim 9.
11. A transformant which comprises the recombinant vector according to claim 9.
12. A membrane of the transformant according to claim 11 having the protein according to claim 1 or 2 which is a membrane protein.
13. A process for producing a protein according to claim 1, 2 or 8 comprising the steps of;
(a) culturing a transformant according to claim 11 under conditions providing expression of the protein according to claim 1, 2 or 8; and
(b) recovering the protein from the culture product.
14. A process for diagnosing a disease or susceptibihty to a disease related to expression or activity of the protein of claim 1, 2 or 8 in a subject comprising the steps of:
(a) determining the presence or absence of a mutation in the gene encoding said protein in the genome of said subject; and/or
(b) analyzing the amount of expression of said protein in a sample derived from said subject.
15. A method for screening compounds which inhibit or promote an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, which comprises the steps of:
(a) preparing a transformant by introducing a gene encoding a protein according to claim 1, 2 or 8 that has an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl and a gene encoding a protein which generates a signal which can detect an action of Elkl phosphorylation into a host ceU;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the signal which can detect an action of Elkl phosphorylation; and
(d) selecting a candidate compound which can change the signal amount as compared with the case of the absence of candidate compounds, as a compound which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl.
16. A method for screening compounds which inhibit or promote an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, which comprises the steps of:
(a) preparing a transformant by introducing a gene encoding a protein according to claim 1, 2 or 8 that has an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl into a host cell;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl; and
(d) selecting a candidate compound which can change the action of Elkl phosphorylation and or the action of activation of kinase which phosphorylates Elkl as compared with the case of the absence of candidate compounds, as a compound which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl .
17. A compound which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, which is selected by the method for screening according to claim 15 or 16.
18. A process for producing a pharmaceutical composition, which comprises the steps of:
(a) preparing a transformant by introducing a gene encoding a protein according to claiml, 2 or 8 that has an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl and a gene encoding a protein which generates a signal which can detect an action of Elkl phosphorylation into a host cell;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring the signal which can detect an action of Elkl phosphorylation;
(d) selecting a candidate compound which can change the signal amount as compared with the case of the absence of candidate compounds, as a compound which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl; and
(e) producing a pharmaceutical composition which comprises a compound selected in the step of (d).
19. A process for producing a pharmaceutical composition, which comprises the steps of:
(a) preparing a transformant by introducing a gene encoding a protein according to claim 1, 2 or 8 that has an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl into a host cell;
(b) culturing the transformant under conditions which permit the expression of the gene in the presence or absence of one or more candidate compounds;
(c) measuring an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl;
(d) selecting a candidate compound which can change the action of Elkl phosphorylation and or the action of activation of kinase which phosphorylates Elkl as compared with the case of the absence of candidate compounds, as a compound which inhibits or promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl; and
(e) producing a pharmaceutical composition which comprises a compound selected in the step of (d).
20. A kit for screening a compound for inhibiting or promoting an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, which comprises:
(a) a transformant comprising a gene encoding a protein according to claim 1, 2 or 8 which promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl and a gene encoding a protein which provides a signal which can detect an action of Elkl phosphorylation; and
(b) reagents for measuring the signal.
21. A monoclonal or polyclonal antibody or a fragment thereof, which recognizes the protein according to claim 1, 2 or 8.
22. The monoclonal or polyclonal antibody or a fragment thereof according to claim 21, which inhibits an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl which are possessed by the protein according to claim 1, 2 or 8.
23. A process for producing a monoclonal or polyclonal antibody according to claim 21 or 22, which comprises administering the protein according to claim 1, 2 or 8 or epitope-bearing fragments thereof to a non-human animal as an antigen.
24. An antisense oligonucleotide having a sequence complementary to a part of the polynucleotide according to any one of claims 3 to 7, which prevents the expression of a protein which promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl.
25. A ribozyme or deoxyribozyme capable of inhibiting an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, which has an action of cleavage of RNA that encodes the protein according to claim 1, 2 or 8 or an action of cleavage of RNA that encodes a protein which is involved in a pathway leading to Elkl phosphorylation and or activation of kinase which phosphorylates Elkl.
26. A double strand RNA having a sequence corresponding to a part of the nucleotide sequence according to any one of claims 3 to 7, which inhibits expression of a protein which promotes an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl.
27. A method for treating a disease associated with an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl, which comprises administering to a subject a compound screened by the process according to claim 15 or 16, and/or a monoclonal or polyclonal antibody or a fragment thereof according to claim 21 or 22, and/or an antisense oligonucleotide according to claim 24, and/or a ribozyme or deoxyribozyme according to claim 25, and/or a double strand RNA according to claim 26 in an effective amount to treat a disease selected from the group consisting of inflammation, autoimmune diseases, cancers, infection diseases, bone diseases, AIDS, neurodegenerative diseases, ischemic injury, GVHD, skin diseases, IgA nephritis, purpuric nephritis, proliferative nephritis, and -nlminant hepatitis.
28. A pharmaceutical composition which is produced the process according to claim 18 or 19, for inhibiting or promoting an action of Elkl phosphorylation and/or an action of activation of kinase which phosphorylates Elkl.
29. The pharmaceutical composition according to claim 28 for the treatment and/or prevention of inflammation, autoimmune diseases, cancers, infection diseases, bone diseases, AIDS, neurodegenerative diseases, ischemic injury, GVHD, skin diseases, IgA nephritis, purpuric nephritis, proliferative nephritis, and fulminant hepatitis.
30. A method of treating inflammation, autoimmune diseases, cancers, infection diseases, bone diseases, AIDS, neurodegenerative diseases, ischemic injury, GVHD, skin diseases, IgA nephritis, purpuric nephritis, proliferative nephritis, and -nlminant hepatitis, which comprising administering a pharmaceutical composition produced by the process according to claim 18 or 19 to a patient suffering from a disease relating to abnormalities in an action of Elkl phosphorylation and or an action of activation of kinase which phosphorylates Elkl.
31. A pharmaceutical composition which comprises a monoclonal or polyclonal antibody or a fragment thereof according to claim 21 or 22 as an active ingredient.
32. A pharmaceutical composition which comprises an antisense oligonucleotide according to claim 24 as an active ingredient.
33. A pharmaceutical composition which comprises a ribozyme or deoxyribozyme according to claim 25 as an active ingredient.
34. A pharmaceutical composition or a gene therapy agent, which comprises a double strand RNA according to claim 26 or a vector capable of expressing said double strand RNA, an active ingredient.
35. The pharmaceutical composition according to any one of claims 31 to 34 for the treatment and/or prevention of a disease which is selected from the group consisting of inflammation, autoimmune diseases, cancers, infection diseases, bone diseases, AIDS, neurodegenerative diseases, ischemic injury, GVHD, skin diseases, IgA nephritis, purpuric nephritis, proliferative nephritis, and fuhninant hepatitis.
36. A computer-readable medium on which a sequence data set has been stored, said sequence data set comprising at least one of nucleotide sequence or that of coding region which is selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107, and/or at least one amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62,
64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108.
37. A method for calculating identity to other nucleotide sequences and/or amino acid sequences, which comprises comparing data on a medium according to claim 36 with data of said other nucleotide sequences and or amino acid sequences.
38. An insoluble substrate to which polynucleotides comprising all or part of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 51, 59, 61, 63,
65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 and 107 are fixed.
39. An insoluble substrate to which polypeptides comprising aU or a part of the amino acid sequences selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106 and 108, are fixed.
PCT/JP2004/001587 2003-02-13 2004-02-13 Elk1 phosphorylation related genes WO2004072277A2 (en)

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WO2007096854A1 (en) * 2006-02-21 2007-08-30 The Provost, Fellows And Scholars Of The College Of The Holy And Undivided Trinity Of Queen Elizabeth, Near Dublin Compositions and methods for modulating an immune response
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