WO1999042833A1 - Protein kinase c - Google Patents
Protein kinase c Download PDFInfo
- Publication number
- WO1999042833A1 WO1999042833A1 PCT/GB1999/000510 GB9900510W WO9942833A1 WO 1999042833 A1 WO1999042833 A1 WO 1999042833A1 GB 9900510 W GB9900510 W GB 9900510W WO 9942833 A1 WO9942833 A1 WO 9942833A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- pkc
- protein kinase
- site
- activation
- phosphorylatable
- Prior art date
Links
Classifications
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Definitions
- the present invention relates to protein kinase C (PKC), and in particular to a method of determining whether PKC has been activated and reagents for use in such a method.
- PKC protein kinase C
- PKC protein kinase C
- DAG second messenger diacylglycerol
- a first aspect of the invention provides a method of dete ⁇ niiiing whether a protein kinase C (PKC) has been activated, the method comprising dete ⁇ nining whether or to what extent a phosphorylatable site on the said protein kinase C, which site is associated with activation of said protein kinase C, has been phosphorylated.
- PLC protein kinase C
- Protein kinase C is defined as a polypeptide encoding a CIA and/or B regulatory domain (defined in Hurley 1997) and a protein kinase catalytic domain (see Hanks 1995). Most PKC isotypes also encode a C2 or Cllike domain (see Ponting 1996), but these are not essential to the definition of a PKC.
- Activated PKC may be defined as a PKC protein where through interaction with its allosteric activators or through interaction with certain proteins such as RACKS (receptors for activated C kinases, which are well known in the art, and which bind to PKC in the active conformation and may be used to trap PKC in the active conformation), its intramolecular inhibition is relieved permitting expression of efficient phosphotransferase activity.
- RACKS receptors for activated C kinases
- Activated PKC includes a PKC bound or recently bound to its allosteric activator(s). Allosteric activators for PKC include diacylglycerol, phospholipids, some free fatty acids, Ca 2+ , certain phorbol esters and detergents. Not all PKCs are activated by all allosteric activators. Pharmacological agents other than phorbol esters also activate (eg bryostatins, mezerin).
- phosphorylatable site we mean any site on a PKC which may be phosphorylated by the action of an enzyme and a suitable phosphate 4 donor, such as ATP.
- a “phosphorylatable site” may be a serine residue or a threonine residue within the PKC polypeptide which is phosphorylated to form phosphoserine and phosphothreonine, respectively. Not all threonine residues or serine residues are phosphorylatable sites and, as will become clear below, in the context of the invention the phosphorylatable site is one which is associated with activation of a particular PKC.
- the phosphorylatable site being a "site associated with activation of protein kinase C" we mean a site which is substantially phosphorylated when the PKC is substantially activated and which is not substantially phosphorylated when the PKC is not substantially activated.
- the site associated with activation of protein kinase C is an autophosphorylation site.
- An autophosphorylation site is a site in an enzyme which is a substrate for the same enzyme and becomes phosphorylated when the enzyme is active.
- Autophosphorylation sites for PKCs can readily be determined, for example, by methods disclosed in the Examples in relation to PKC ⁇ .
- the particular isoform of PKC for which it is desired to identify the autophosphorylation site is provided in a substantially pure form (at least substantially free of other protein kinases), for example by expressing a suitable PKC cDNA in a host cell and isolating the PKC enzyme.
- the substantially pure enzyme is then incubated with a suitable phosphate donor such as ATP in the absence of any other protein kinase.
- the donor ( ⁇ ) phosphate of ATP is labelled with a radioactive phosphorus atom such as 32 P or 33 P.
- Incorporation of the donor phosphate into the PKC enzyme is monitored, and the identity of the site which has become phosphorylated may be determined using any 5 suitable method, such as by proteolytic or chemical cleavage of the PKC, followed by identification of the peptide containing the donor phosphate by FPLC analysis and radio detection, and sequencing of the identified peptide.
- autophosphorylation may require the presence of suitable cofactors.
- a suitable amount of tetradecanoyl phorbol acetate and phosphatidyl serine is present for autophosphorylation of other cPKCs or nPKCs.
- effectors are less well-defined but, typically, mixed brain phospholipids can activate to a limited extent and so it is preferred if they are present for the autophosphorylation of aPKC.
- Suitable PKC enzyme preparations for carrying out autophosphorylation may be obtained using recombinant DNA technology as is well known in the art.
- Appropriate cDNAs encoding the PKC isozymes can be obtained by methods well known in the art by reference, for example, to the above mentioned GenBank accession numbers and records associated with the sequences in the data library.
- the DNA is then expressed in a suitable host to produce a PKC.
- the DNA encoding the PKC may be used in accordance with known techniques, 8 appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the PKC.
- Such techniques include those disclosed in US Patent Nos.
- the DNA encoding the PKC may be joined to a wide variety of other DNA sequences for introduction into an appropriate host.
- the companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into me host, and whether episomal maintenance or integration is desired.
- the DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression.
- an expression vector such as a plasmid
- the DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector.
- the vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells.
- One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. 9
- the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
- Host cells that have been transformed by the recombinant DNA of the invention are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of me teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.
- bacteria for example E. coli and Bacillus subtilis
- yeasts for example Saccharom ces cerevisiae
- filamentous fungi for example Aspergillus
- plant cells animal cells and insect cells.
- the vectors include a prokaryotic replicon, such as the ColEl ori, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic, cell types.
- the vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed therewith.
- a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur.
- Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment of the present invention.
- Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA, USA) and p7/r99A and pKK223-3 available from Pharmacia, Piscataway, NJ, USA.
- a typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, NJ, USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-1 cells.
- an inducible mammalian expression vector is pMSG, also available from Pharmacia. This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
- Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
- Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRP1, LEU2 and URA3.
- Plasmids pRS413-416 are Yeast Centromere plasmids (YCps) .
- Useful insect cell vectors are the baculovirus vectors as can be obtained from Invitrogen, De Schelp 12, 9351 NV Leek, Netherlands.
- a variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen 11 bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
- Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors.
- the DNA segment generated by endonuclease restriction digestion as is well known in the art, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded terrriini with their 3'-5'-exonucleolytic activities, and fill in recessed 3 '-ends with their polymerizing activities.
- the combination of mese activities therefore generates blunt-ended DNA segments.
- the blunt-ended segments are men incubated with a large molar excess of linker molecules in the presence of an enzyme mat is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
- the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved widi the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces temiini compatible with those of the DNA segment.
- Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CN, USA.
- a desirable way to modify the DNA encoding the PKC so that it may be readily introduced into an expression vector is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491. 12
- the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA.
- the said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
- the host cell which is to express the PKC can be either prokaryotic or eukaryotic.
- Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1 available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No ATCC 31343).
- Preferred eukaryotic host cells include yeast and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic cell line.
- Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
- Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swiss mouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, and monkey kidney-derived COS-1 cells available from the ATCC as CRL 1650.
- Insect cells such as Sf9 and Hi5, are also useful eukaryotic host cells.
- Transformation of appropriate cell hosts with a DNA construct encoding PKC is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast 13 Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 104-109 is also useful.
- reagents useful in transfecting such cells for example calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA.
- Electroporation is also useful for transforming cells and is well known in the art for transforming yeast cells, bacterial cells and vertebrate cells.
- bacterial species may be transformed by the methods described in Luchansky et al (1988) Mol. Microbiol. 2, 637-646 incorporated herein by reference. The greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2.5X PEB using 6250V per cm at 25 ⁇ FD.
- Successfully transformed cells ie cells that contain a DNA construct of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an expression construct can be grown to produce the PKC. Cells can be harvested and lysed and their DNA content examined for me presence of the DNA using a method such as that described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208.
- phosphorylation of the phosphorylatable site associated with activation of said protein kinase C is not itself required for activation but it is a consequence of activation.
- the site associated with activation exhibits a variable extent of phosphorylation which is a reflection of the extent of activation.
- PKC phosphopeptides which exhibit a varied stoichiometry based on recovery when isolated from their natural cells under different conditions may contain (as the site at which phosphorylation has occurred) a site associated with activation of said protein kinase C. Such sites may be identified using methods herein disclosed.
- PKC isotypes that display an activation-associated transphosphorylation at a site that is dephosphorylated on deactivation
- these can also be employed as activation markers.
- the activation-associated transphosphorylation sites are conveniently defined empirically by identification following isolation of 32 P-phosphate labelled PKC from unstimulated and stimulated cells. It is also possible to identify such potential sites by, for example, random or precedent-directed mutagenesis or phospho-site antibody production. The usefulness of positively identified sites can be determined 15 in retrospect. For example, aPKC ⁇ can be shown to be phosphorylated on a well conserved activation loop site at threonine 410.
- This phosphorylation is found to vary as a function of activation state (ie go up and down), and detection of phosphorylation (or the extent of phosphorylation) at this site, for example using antibodies specific for this site, is a determination of activation.
- Threonine 410 in a PKC ⁇ is not an autophosphorylation site.
- phosphorylatable sites which serve as activation markers may be conserved between related protein kinases, specificity for any one unique gene product may be effected by use of a monospecific antibody mat captures the protein kinase (eg ELISA, immunoprecipitation) while me activation specific antibody is employed as a second layer (eg ELISA) or, for example, in a subsequent western (immunoprecipitation).
- a monospecific antibody captures the protein kinase (eg ELISA, immunoprecipitation) while me activation specific antibody is employed as a second layer (eg ELISA) or, for example, in a subsequent western (immunoprecipitation).
- the method additionally comprises distinguishing the said PKC, whose activation state is to be determined by determining whether the said phosphorylatable site has been phosphorylated, from another PKC.
- the PKC whose activation state is to be determined can be distinguished from another PKC by contacting the sample with a compound which distinguishes the PKCs.
- a compound is a PKC-selective antibody.
- PKC-selective antibodies may be readily made by the person skilled in the art, for example by making use of the amino acid sequence differences between PKCs in order to raise antibodies.
- antibodies which are PKC-selective may be raised against PKC-specific peptides using methods well known in the art. 16
- the PKC-selective antibody may be used to separate the PKC whose activation is to be determined from other PKCs such as by immunoprecipitation, or it may be used to distinguish the PKCs.
- the method of the invention may be used to determine whether a protein kinase C of any class has been activated. For example, it may be used for "a"-class (atypical) PKCs and for "c"-class ("classical)” PKCs and for "n"-class ("novel") PKCs.
- the "a" class PKCs include PKC ⁇ , PKCi and PKC ⁇ .
- PKCi and PKC ⁇ are human and mouse homologues. They are often referred to as PKQ / ⁇ .
- the "c" class PKCs include PKC ⁇ , PKC ⁇ l , PKC ⁇ 2 and PKC ⁇ .
- the "n" class PKCs include PKC ⁇ , PKC ⁇ , PKC ⁇ , PKC ⁇ and PKC ⁇ .
- the method of the present invention has significant advantages over the previously used "translocation" method for determining the activation of a PKC isoform.
- the basis of the "translocation” method was the observations that activation leads to a membrane-bound form of PKC and mat activation is a membrane-binding step.
- the previous method therefore, relied on determining the intracellular location of a particular PKC isoform.
- no cellular fractionation is required and the quality of the sample to be tested is not so important.
- the present method allows for historical or archival samples, such as frozen tissue samples or even samples stored as paraffin-fixed tissue sections, to be analysed as well as fresh tissue samples and cell lines. 17
- the memod is used to detect activation of a human PKC but it may be used in relation to any PKC.
- the method comprises dete ⁇ nining whether PKC ⁇ or PKCp, or PKCp 2 or PKC ⁇ has been activated.
- T250 threonine 250
- the phosphorylation status of T250 serves as an activation marker for PKC ⁇ .
- the determination of the phosphorylation status of T250 of PKC ⁇ provides an indication of the activation status of PKC ⁇ . If T250 has been phosphorylated, PKC ⁇ is activated; if T250 is not phosphorylated, PKC ⁇ is not activated.
- Such measurements may be achieved, for example, by combining a simple two site ELISA (eg PKC capture using, for example, a PKC-selective antibody and activation state detection, for example using an antibody which detects phosphorylation of a phosphorylatable site associated with activation of that PKC) with a parallel reading to determine the quantity of PKC captured (using a PKC antibody to a second protein epitope).
- PKC capture using, for example, a PKC-selective antibody and activation state detection for example using an antibody which detects phosphorylation of a phosphorylatable site associated with activation of that PKC
- Phosphorylation of T410 in PKC ⁇ , and phosphorylation of T403 in PKCi are markers of the respective PKCs.
- the amino acid sequence of human PKC ⁇ , human PKC ⁇ l , human PKC ⁇ 2 and rat PKC ⁇ is shown in Figure 10.
- a threonine residue equivalent to T250 of human PKC ⁇ is conserved in human PKC ⁇ l , human PKC ⁇ 2 and rat PKC ⁇ , and I have found that phosphorylation of the equivalent threonine residue (as shown in Figure 10) is also a marker for activation of human PKC P1 , human PKC ⁇ 2 and rat PKC ⁇ .
- the determination of whether or to what extent said phosphorylatable site has been phosphorylated may be made using any suitable method, for example, by amino acid sequencing. However, it is preferred that the determination is made using a reagent which is capable of distinguishing between the presence or absence of a phosphate moiety at said phosphorylatable site.
- the reagent is an antibody or a suitable fragment or derivative thereof which is capable of distinguishing between the presence or absence of a phosphate at the phosphorylatable site. 19
- a “suitable fragment or derivative of an antibody” we include any molecule derived from an antibody or which has an antibody-like binding site, which can distinguish between the presence or absence of a phosphate in a PKC as said.
- Suitable fragments and derivatives include F(ab') 2 fragments, Fab fragments, ScFvs, domain antibodies (dAbs) and the like.
- the antibody or suitable fragment or derivative mereof recognises and binds to the phosphorylated phosphorylatable site.
- the antibody is, therefore, preferably one which binds to a specific phosphopeptide. It is preferred that the antibody does not bind to phosphoserine or phosphothreonine in the absence of further specific determinants in the polypeptide surrounding the specific phospho-residue.
- Antibodies which cross-react with a PKC when phosphorylated at a specific site and which do not cross-react to any significant extent with the PKC when not phosphorylated at that specific site may be made using any suitable method. Antibodies may be raised against specifically phosphorylated PKC and screened for their ability to bind to said specific phosphorylated PKC and for their inability to bind to said specific non- phosphorylated PKC.
- the preferred method for making antibodies which are useful in the practice of the invention involves the use of phosphopeptides based on the primary amino acid sequence surrounding the phosphorylatable site in which the site is phosphorylated. For example, the peptide WDRT(P)TRND. amide, where T(P) denotes a phosphorylated threonine residue, is particularly useful for raising antibodies which react with human PKC ⁇ phosphorylated at T250.
- the antibodies may be monoclonal or polyclonal. 20
- Monoclonals provide an easily renewable resource, polyclonals may require extensive characterisation (for each new batch). Polyclonals are usually of a different animal origin to monoclonals and mis can be useful where two-site detection is required (obviating the need for monoclonals of distinct subclasses since secondary reagents such as antibodies can distinguish polyclonal antibodies from different animal sources (such as anti-sheep antibody antibodies, or anti-rabbit antibody antibodies)). It is preferable if there are a range of monoclonals of different IgG subclass, particularly when a two-stage system is used, since the positive binding of each subclass can be measured using a subclass-selective reagent.
- antibodies can be prepared to most antigens.
- the antigen-binding portion may be a part of an antibody (for example a Fab fragment) or a synthetic antibody fragment (for example a single chain Fv fragment [ScFv]).
- Suitable monoclonal antibodies to selected antigens may be prepared by known techniques, for example those disclosed in 'Monoclonal Antibodies: A manual of techniques ", H Zola (CRC Press, 1988) and in 'Monoclonal Hybridoma Antibodies: Techniques and Applications ", J G R Hurrell (CRC Press, 1982).
- the phosphopeptide may be prepared as glutaraldehyde coupled conjugates to keyhole limpet haemocyanin (KLH) or ovalbumin for immunisation; ELISA may be used to screen hybridomas; and western blotting may be used on activated or non-activated samples to confirm specificity of die monoclonal antibody.
- the antibodies of the invention may be used in any suitable way to detect me presence of the specifically phosphorylated, and hence activated, PKC.
- the antibodies may be used in Western blotting, or ELISA, or in in situ binding assays.
- In-situ binding assays include those involving tissue sections, fixed cells, microinjected live cells with coincident detection of the protein using FRET (fluorescence resonance energy transfer) and related analyses. Additionally dot/ slot blots, that is cell extract applied directly to a filter or appropriate support and then treated as a western, may be used. For this latter type of analysis, it is preferred if the reagents are of very high quality since no independent criteria are used, ie recognition by another antibody (ELISA) or size (western).
- FRET fluorescence resonance energy transfer
- Methods for identifying the activation marker site for any given PKC are included in the invention.
- the most direct method for identifying marker sites in any given PKC is based upon autophosphorylation in vitro.
- purified PKC is isolated and incubated in the presence of appropriate activators and Mg 2+ -[ 32 P]ATP.
- Incorporation of 32 P into the PKC protein through autophosphorylation provides a marker for the newly phosphorylated site(s).
- the protein may be fragmented with a protease (trypsin is most useful, although others will also work) or through chemical cleavage (for example, using cyanogen bromide).
- the labelled protein fragment(s) are identified by monitoring the presence of 32 P following purification; this can be achieved by electrophoresis, thin layer chromatography, HPLC and other related column chromatographic methods or any other suitable method.
- the site may be identified by use of sequential Edman degradation using a commercially available automated device (as supplied by Applied 22
- the PTH-amino acids sequentially derived may be identified by HPLC; a proportion of the sample is employed to determine 32 P content. It may be desirable to use phosphoaminoacid analysis as an independent means of confirming the nature of the residue involved (i.e. serine, threonine or tyrosine). This can be effected through partial acid hydrolysis of the peptide and subsequent electrophoresis with phosphoaminoacid standards.
- Phosphopeptide analysis can also be carried out by mass spectrometry. This can circumvent the need to purify and also the need to 32 P label, although 'before' and 'after' autophosphorylation analyses are needed to define the new site(s).
- mass spectrometry it is possible to first enrich phosphopeptides by small scale metal chelate HPLC. The whole mixture of peptides is then subjected to mass spectroscopic analysis to determine mass and irough fragmentation, sequence.
- a series of synthetic peptides may be made such that each peptide has a single serine, the others being altered to alanine. These may be tested as substrates for a protein kinase to determine which serine is phosphorylated. The phosphorylation site may also be identified using site directed mutagenesis.
- a further aspect of the invention provides a method for identifying a marker associated with activation of a protein kinase C the method comprising comparing the phosphorylation state of a phosphorylatable site in activated said protein kinase C with that of the phosphophorylatable site in non-activated said protein kinase C and determining those 23 phosphorylatable sites whose phosphorylation state varies with the activation status of the protein kinase C.
- a further aspect of the invention provides the use of a reagent which is capable of distinguishing between the presence or absence of a phosphate moiety at a phosphorylatable site in a protein kinase C, which site is associated with activation of said protein kinase C for determining whether a protein kinase C has been activated.
- the reagent is preferably an antibody as disclosed above.
- a still further aspect of the invention provides a reagent which is capable of distinguishing between the presence or absence of a phosphate moiety at a phosphorylatable site in a protein kinase C, which site is associated wim activation of said protein kinase C.
- the reagent is an antibody or a suitable fragment or derivative thereof.
- the antibodies may be made as disclosed above.
- phosphopeptides which are used for raising antibodies may be readily made using methods well known in the art.
- Peptides may be synthesised by the Fmoc-polyamide mode of solid-phase peptide synthesis as disclosed by Lu et al (1981) J. Org. Chem. 46, 3433 and references therein. Temporary N-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is effected using 20% piperidine in N,N-dimethylformamide. Side-chain functionalities may be protected as their butyl ethers (in me case of serine, threonine and tyrosine), butyl esters 24
- glutamic acid and aspartic acid in the case of glutamic acid and aspartic acid
- butyloxycarbonyl derivative in the case of lysine and histidine
- trityl derivative in the case of cysteine
- 4-memoxy-2,3,6-trimethylbenzenesulphonyl derivative in the case of arginine
- glutamine or asparagine are C-terminal residues
- the solid-phase support is based on a polydimethyl-acrylamide polymer constituted from e tiiree monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalising agent).
- the peptide-to-resin cleavable linked agent used is the acid-labile 4- hydroxymethyl-phenoxyacetic acid derivative.
- Scavengers commonly used are ethanedimiol, phenol, anisole and water, the exact choice depending on me constituent amino acids of the peptide being synfhesised. Trifluoroacetic acid is removed by evaporation in vacuo, wim subsequent trituration with diethyl edier affording me crude peptide. Any scavengers present are removed by a simple extraction procedure which on lyophilisation of the aqueous phase affords the crude peptide free of scavengers. Reagents for peptide synthesis are generally available from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK.
- Purification may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ion-exchange chromatography and 25 (principally) reverse-phase high performance liquid chromatography.
- Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis.
- FAB fast atom bombardment
- the phosphate esters are alkali labile and to some extent acid labile. They do not survive complete acid (6N HC1) hydrolysis of peptides, but can be identified on partial hydrolysis (110°C, lh 15mins). It is routine to analyse synthetic peptides using matrix assisted laser description mass spectrometry. This is a time of flight (ToF) device that gives precise mass measurements. For synthetic peptides mis provides confirmation of predicted sequence (can not distinguish leucine and isoleucine) and assessment of purity is also obtained.
- THF time of flight
- the peptides when used as immunogens, may be present as single copies or as multiples, for example tandem repeats. Such tandem or multiple repeats may be sufficiently antigenic memselves to obviate the use of a carrier. It may be advantageous for the peptide to be formed as a loop, with the N- terminal and C-temiinal ends joined together, or to add one or more Cys residues to an end to increase antigenicity and/or to allow disulphide bonds to be formed. If the peptide is covalently linked to a carrier, preferably a polypeptide, then the arrangement is preferably such that the peptide of me invention forms a loop.
- a carrier function should be present in any immunogenic formulation in order to stimulate, or enhance stimulation of, the immune system. It is thought that me best carriers embody (or, together with the antigen, create) a T-cell epitope.
- the 26 peptides may be associated, for example by cross-linking, with a separate carrier, such as serum albumins, myoglobins, bacterial toxoids and keyhole limpet haemocyanin.
- More recently developed carriers which induce T-cell help in e immune response include me hepatitis-B core antigen (also called me nucleocapsid protein), presumed T-cell epitopes such as Thr-Ala-Ser- Gly-Val-Ala-Glu-Thr-Thr-Asn-Cys, beta-galactosidase and the 163-171 peptide of interleukin-1.
- the latter compound may variously be regarded as a carrier or as an adjuvant or as both.
- several copies of the same or if appropriate different peptides may be cross-linked to one another; in this situation there is no separate carrier as such, but a carrier function may be provided by such cross-linking.
- Suitable cross-linking agents include those listed as such in me Sigma and Pierce catalogues, for example glutaraldehyde, carbodiimide and succimmidyl 4-(N-maleimidomemyl) cyclohexane-1-carboxylate, the latter agent exploiting the -SH group on any cysteine residue (if present).
- the antibodies or other reagents of the invention are detectably labelled.
- detectably labelled we mean mat the antibody or other reagent is labelled in such a manner that it can be readily detected; for example, the labelling may be direct such as radiolabelling, or fluorescent or coloured labelling; or the labelling may be indirect such as labelling with an enzyme-linked system in which the enzyme converts an uncoloured substrate into a coloured product.
- a further aspect of the invention provides a kit of parts comprising a reagent which is capable of distinguishing between the presence or absence of a phosphate moiety at a phosphorylatable site in a protein kinase C, which site is associated widi activation of said protein kinase C and, as 27 controls, a sample comprising activated said protein kinase C and a sample comprising non-activated said protein kinase C.
- the reagent of die kit is an antibody as herein described.
- the controls are provided by cell extracts comprising said protein kinase C wherein said cells have been stimulated (to produce activated said PKC) or unstimulated (to produce non-activated said PKC).
- the kit further comprises a reagent which distinguishes me said PKC whose activation state is to be deteirmned from another PKC.
- a reagent which distinguishes me said PKC whose activation state is to be deteirmned from another PKC.
- at least some antibodies which are useful in distinguishing between me presence or absence of a phosphate moiety at a phosphorylatable site in a PKC, which site is associated wim activation of said protein kinase C may also detect me presence or absence of a phosphate moiety at a phosphorylatable site in a different, but related, PKC.
- a kit might contain: an activation marker antibody; an antibody to me specific protein kinase C; a protein kinase C transfected cell extract from (i) unstimulated and (ii) stimulated cells as a control; and, optionally, a second layer detection conjugate (optional). If both antisera are from the same species, it is desirable to mark the detecting, activation marker antibody, with for example biotin. This would permit the second layer detection to be some suitable strep tavidin-X complex, where X is any convenient detection system such as alkaline phosphatase or horse radish peroxidase. 28
- the kit further comprises directions on use.
- the methods of the invention may be used on fresh material, such as cultured cells or tissue samples (once suitably prepared) or it may be used on archival or historic material (once suitably prepared).
- the methods and reagents of the invention may be used whenever it is desirable to deteirnine the activation status of a protein kinase C. This may be useful in die research laboratory for determining me effects of reagents or other conditions on PKC. It may also be useful in clinical and odier situations.
- a still further aspect of the invention provides a peptide comprising a phosphorylatable site in a protein kinase C which site is associated wim activation of said protein kinase C, or a peptide in which said phosphorylatable site is phosphorylated.
- the peptide is an immunogenic portion of the PKC (whether phosphorylated at die phosphorylatable site or not) and typically has between 5 and 50 amino acids, preferably between 6 and 40, more preferably between 7 and 30 and still more preferably between 8 and 20 amino acids. It is particularly preferred if the peptide comprises the phosphorylatable site which is found at T250 in bovine PKC ⁇ or site equivalent thereto as herein disclosed. A preferred such peptide is die phosphorylated peptide WDRT(P)TRND.
- the peptides are suitably included in an immunogenic formulation for raising antibodies.
- the immunogenic formulation comprises the 29 antibody and a suitable carrier or adjuvant and, desirably, is sterile and pyrogen-free.
- substantially no proteolysis, substantially no dephosphorylation and substantially no phosphorylation take place.
- this may be achieved by extraction into buffers with appropriate preservatives (eg phenylmethylsulfonyl fluoride, aprotinin, leupeptin, benzamidine, all of which are protease inhibitors; microcystin, okadaic acid and sodium fluoride + EDTA, all of which are phosphatase inhibitors; EDTA to chelate Mg 2+ and so inhibit kinases).
- preservatives eg phenylmethylsulfonyl fluoride, aprotinin, leupeptin, benzamidine, all of which are protease inhibitors; microcystin, okadaic acid and sodium fluoride + EDTA, all of which are phosphatase inhibitors; EDTA to chelate Mg 2+ and so inhibit kinases.
- preservatives
- these may be diluted 10-fold widi 1 % Triton X-100 and the resulting detergent mixed micelles (l %Triton X-100, 0.1 % SDS) do not interfere with PKC capture (ELISA) or direct immunoprecipitation.
- PKC activation by the methods of the invention in order to understand when PKC is triggered physiologically; or define which PKC is triggered physiologically; or determine where PKC is becoming activated (subcellular); or to follow efficacy of drug treatments directed at or through PKC; or to assess PKC action in (human) diseases and normal tissues.
- Figure 1 shows the identification of two PKC ⁇ phosphorylation sites.
- the histogram shows the 32 P radioactivity (counts per minute, cpm) released 30 from a 32 P-labelled PKC ⁇ tryptic peptide subjected to sequential Edman degradation. The counts are released at cycles 1 and 11. Based upon die specificity of trypsin (cleavage C-terminal to arginine, R and lysine, K) it can be predicted diat die peptide is me one shown.
- the underscored amino acids are threonine (T) 250 and serine (S) 260.
- FIG. 2 shows mat PKC ⁇ mreonine 250 is phosphorylated in response to activation.
- COS cells transfected with PKC ⁇ were stimulated for 0, or 30 minutes wim TPA in me absence (-) or presence (+) of a PKC inhibitor (10 ⁇ M bisindolylmaleimide I).
- the phosphorylation of PKC ⁇ is detected by Western analysis of whole cell extracts with a polyclonal antiserum specific for the phosphorylated T250 site.
- the lower panel shows that die amount of PKC ⁇ protein does not vary as evidenced by immunoreaction wim a PKC ⁇ antiserum (MC5). Recognition of PKC ⁇ by the antiserum MC5 is independent of the phosphorylation state of PKC ⁇ .
- FIG. 3 shows that threonine 250 is a PKC ⁇ autophosphorylation site.
- PKC ⁇ protein was purified from transfected COS cells and incubated for the times indicated widi Mg-ATP in die presence of TPA and phosphatidylserine (allosteric activators). Reactions were terminated by denaturation and subjected to Western analysis.
- the upper panel shows immunoreactivity wim the phosphorylated threonine 250 (T(P)250) specific antiserum.
- T(P)250 phosphorylated threonine 250 specific antiserum.
- the lower panel demonstrates diat die amount of PKC ⁇ does not alter.
- FIG. 4 shows PKC ⁇ autophosphorylation on threonine 250 in COS cells.
- A shows mat TPA induces a time-dependent increase in the phosphorylation of COS cell expressed PKC ⁇ .
- the upper panel is a 31 Western of whole cell extracts from samples treated with TPA for the times indicated.
- the lower panel shows that die amount of PKC ⁇ does not vary during the first 30 minutes. By 60 minutes there is some downregulation, consistent with bodi a loss of PKC ⁇ immunoreactivity and of T(P)250 immunoreactivity.
- B shows that the protein identified in PKC ⁇ expressing COS cell extracts is PKC ⁇ .
- the PKC ⁇ protein was immunoprecipitated from cell extracts (using a PKC ⁇ antibody, MC5) and men subjected to Western analysis. The immunoreactivity with die T(P)250 antiserum is shown.
- FIG 4A there is an increase in phosphorylation during the first 30 minutes. This is followed by a decline, reflecting downregulation of the protein.
- FIG. 5 shows that TPA induces endogenous PKC ⁇ direonine 250 phosphorylation in Swiss 3T3 cells.
- Quiescent Swiss 3T3 cells were treated with TPA (400 nM) for the times indicated and whole cell extracts subjected to Western blotting with the T(P)250-specific antiserum.
- the upper panel shows T(P)250 serum immunoreactivity; the specific (phosphopeptide competable) PKC ⁇ band is indicated by the arrow (the unmarked faster migrating band is variably observed and is not competed).
- the lower panel shows that over mis time course there is no change in total PKC ⁇ content of the cells.
- Figure 6 shows that direonine 250 phosphorylation is reversible.
- Swiss 3T3 cells were treated with PDBu for 20 minutes and then washed widi phorbol dibutyrate (PDBu)-free medium (at 4°C) and incubated further at 37°C for the times indicated. Extracts were prepared and subjected to Western blotting with the T(P)250-antiserum. Immunoreactivity was quantified following scanning of autoradiographs. There is a lag in 32 immunoreactivity loss before an exponential decline; note die immunoreactivity is shown as a log scale.
- FIG. 7 shows that mitogens stimulate PKC ⁇ autophosphorylation in Swiss 3T3 cells.
- Quiescent Swiss 3T3 cells were treated for the times indicated widi platelet derived growth factor (PDGF), bombesin or vasopressin as indicated.
- Cell extracts were prepared and subjected to Western blotting.
- the specific T(P)250 positive PKC ⁇ band is indicated by die arrows for each panel.
- FIG. 8 shows that PKC ⁇ phosphorylation on T250 is blocked by a PKC inhibitor.
- Quiescent Swiss 3T3 cells were stimulated for 10 minutes in the presence (+) or absence (-) of lO ⁇ M bisindolylmaleimide I (BIM).
- the agonists are: bombesin (Bom.), vasopressin (Vasop.) and PDGF.
- the arrow indicates die immunoreactive PKC ⁇ protein.
- FIG. 9 shows that insulin stimulates PKC ⁇ autophosphorylation in Swiss 3T3 cells.
- Quiescent Swiss 3T3 cells were treated widi insulin (10 "7 M) for the times indicated.
- Whole cell extracts were prepared and subjected to Western blotting with the T(P)250 serum or PKC ⁇ antibody as indicated.
- Figure 10 is an alignment of the amino acid sequences of human PKC ⁇ , ⁇ 2 and ⁇ l and rat PKC ⁇ .
- a "*" indicates perfect correspondence in amino acid; a " : “ indicates a conservative amino acid substitution; a " . " indicates related amino acid substitutions.
- Phosphorylation sites in PKC are marked with an arrow (T250; T497; T638; and S657).
- Activated PKC ⁇ is selectively recognised by the T(P)250 antiserum in situ.
- Swiss 3T3 cells were processed as described in the Mediods Section of Example 1. Control cells were untreated (con) or treated for 10 minutes wim TPA (400 nM) as indicated. Following treatment cells were fixed and stained for PKC ⁇ (monoclonal 9E10) or for T(P)250 (PPA245 polyclonal). In addition to activated PKC ⁇ , die latter revealed a non-PKC ⁇ nuclear reaction in bom uninjected (arrowhead) and injected cells (see text). Similar results were obtained employing an affinity purified PPA182, T(P)250 antiserum.
- Figure 12 shows a section of a breast tumour showing T(P)250 staining quite intensely me tumour and much less so the surrounding tissue.
- Figure 13 shows that a PKC ⁇ is poorly phosphorylated at direonine 410 under normal cell culture conditions, but this can be greatly enhanced by stimulation of cells with die broad specificity agonist okadaic acid.
- Example 1 Mapping a novel autophosphorylation site on protein kinase C a identifies an activation marker.
- T250 threonine 250
- S260 serine 260
- TPA tetradecanoyl phorbol acetate
- T(P)250- specific antisera have been employed to monitor PKC ⁇ activation in quiescent Swiss 3T3 cells.
- TPA, PDGF, bombesin and vasopressin were all found to induce T250 phosphorylation in a time- and dose-dependent manner, consistent wim known targets and coupling mechamsms. Further, it is shown that insulin also induces T250 phosphorylation, demonstrating control of PKC ⁇ by this agonist.
- COS-7 cells were cultured in Dulbecco's modified Eagles medium (DMEM) containing 10% foetal calf serum at 37°C and in a 10% CO 2 atmosphere. Cells were transfected by electroporation as described previously [Bornancin, 1996]. Transfected cells were stimulated as indicated in the text and figure legends, 48 hours after transfection. Where indicated bisindolylmaleimide I (Calbiochem) was added 20 minutes prior to agonist treatment. Following stimulation, cells were then harvested directly into Laemmli sample buffer [Laemmli, 1970]. For the purification of COS-7 cell expressed PKC ⁇ , die His-tagged recombinant protein was processed as described previously [Bornancin, 1996].
- DMEM Dulbecco's modified Eagles medium
- PKC ⁇ die His-tagged recombinant protein was processed as described previously [Bornancin, 1996].
- Swiss 3T3 cells were maintained at 37 °C in a 10% C0 2 incubator in medium containing 10% foetal calf serum. Three days following seeding, cells were switched to DMEM containing 31 % Weymouth's medium and 35
- Double-label immunofluorescence staining with the anti-Myc mAb 9E10 [Evan, 1985] and T(P)250 antiserum (PPA245) was performed as described [Kiley, 1997] except for the following modifications. Cells were permeabilised with 0.2% Triton- 37
- Protein concentration was determined by the metiiod of Bradford [Bradford, 1976] using bovine serum albumin (Sigma) as a standard.
- Phosphorylation was time-dependent being maximal by 5 minutes, sustained for a further 5 minutes with dephosphorylation occurring after 10 minutes. This method of assessment of PKC ⁇ activation can thus be employed to monitor me action of physiological agonists.
- other well studied agonists in Swiss 3T3 cells were also analysed. Both vasopressin and bombesin were found to induce time dependent increases in PKC ⁇ T250 phosphorylation ( Figure 7 lower panels).
- the effect of the PKC inhibitor BIM was determined. For all three agonists, BIM was found to block die T250 phosphorylation 41 of PKC ⁇ ( Figure 8).
- T(P)250 Antisera specific to the occupied site (T(P)250) are characterised and shown to provide a means of following PKC ⁇ activation directly.
- the definition of the T250 site as an autophosphorylation site is based upon direct analysis of purified PKC ⁇ .
- the COS-7 cell expressed and purified protein phosphorylates the T250 site on ineubation with Mg-ATP and lipid activators. This is evidenced by increased immunoreactivity with the T(P)250-specific antisera and parallels an increase in 32 P- orthophosphate incorporation into the protein.
- the T250 site may not be the sole site autophosphorylated in vitro, but that its analysis here establishes the principle of an activation marker. Consistent with the in vitro data, it is found diat the agonist induced phosphorylation of the T250 site in PKC ⁇ is inhibited by the PKC inhibitor BIM. Thus, although activation through ligand binding may induce a conformation (or 42 localisation) susceptible to T250 phosphorylation by an heterologous activity in vivo, the inhibition by BIM is consistent with autophosphorylation.
- T250 site as an autophosphorylation site and die development of selective antisera have provided a rationale for assessing activation in vivo.
- the efficacy of mis approach is dependent upon the turnover of phosphate at this site and die detectability of endogenous phosphorylated PKC ⁇ (as opposed to transfected protein).
- the reversibility of the induced T250 phosphorylation is clear, with an observed half-life of approx. 5 minutes following a lag period.
- T250 phosphorylation is not an irreversible process, on me contrary turnover is relatively rapid.
- nPKC and aPKC isotypes The identification of autophosphorylation site markers for nPKC and aPKC isotypes involves the use of isolated recombinant enzymes (for example expressed in bacteria, insect cells or mammalian cells). These purified proteins are incubated under die conditions required for activation of each PKC, employing [ ⁇ 32 P] -labelled ATP as substrate. Radioactivity incorporated into me kinase (autophosphorylation) is detected following SDS-PAGE separation and die labelled protein digested from the gel (or if desired following transfer to a membrane such as PVDF) with protease (trypsin is most efficient in mis respect). Peptides derived from the digest are separated by HPLC and phosphorylation sites identified by a 45 combination of mass spectrometry, Edman degradation and phosphoaminoacid analysis.
- Antibodies mat specifically recognise die defined phosphorylation sites are obtained tiirough immunisation (of for example rabbits or mice) with syndietic phospho-peptides (usually employing a seven amino acid peptide widi the phosphorylated residue in me fourth position) coupled to a carrier (usually keyhole limpet haemocyanin).
- the antibodies so derived can dien be employed to monitor PKC activation status.
- Threonine-497 is a critical site for permissive activation of protein kinase C ⁇ . Biochem. J. 301, 443-448.
- Interleukin-2 stimulates association of protein kinase C with plasma membrane. Nature 315, 233- 235.
- Protein kinases 6 The eukaryotic protein kinase super family: kinase (catalytic) domain structure and classification. Faseb J 9, 57696.
- Faciogenital dysplasia protein (FGD1) and Vav two related proteins required for normal embryonic development, are upstream regulators of Rho-GTPases. Curr. Biol. 6, 1628-1633.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Organic Chemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Cell Biology (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2000532724A JP2002504686A (en) | 1998-02-19 | 1999-02-18 | Protein kinase C |
CA002320787A CA2320787A1 (en) | 1998-02-19 | 1999-02-18 | Protein kinase c |
AU25388/99A AU2538899A (en) | 1998-02-19 | 1999-02-18 | Protein kinase c |
EP99905090A EP1057025A1 (en) | 1998-02-19 | 1999-02-18 | Protein kinase c |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9803399.6 | 1998-02-19 | ||
GBGB9803399.6A GB9803399D0 (en) | 1998-02-19 | 1998-02-19 | Protein kinase c |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1999042833A1 true WO1999042833A1 (en) | 1999-08-26 |
Family
ID=10827176
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB1999/000510 WO1999042833A1 (en) | 1998-02-19 | 1999-02-18 | Protein kinase c |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP1057025A1 (en) |
JP (1) | JP2002504686A (en) |
AU (1) | AU2538899A (en) |
CA (1) | CA2320787A1 (en) |
GB (1) | GB9803399D0 (en) |
WO (1) | WO1999042833A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000057184A2 (en) * | 1999-03-19 | 2000-09-28 | Kudos Pharmaceuticals Limited | Screening methods based on fha domains |
JP2006225396A (en) * | 2000-09-27 | 2006-08-31 | Biosource Internatl | Method for quantifying phosphokinase activity on proteins |
JP2013078330A (en) * | 2001-02-27 | 2013-05-02 | Blanchette Rockefeller Neurosciences Inst | Alzheimer's disease diagnosis based on mitogen-activated protein kinase phosphorylation |
CN110520144A (en) * | 2017-02-19 | 2019-11-29 | 国家生物技术研究所公司 | Peptide kinase inhibitor and its application method |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11624082B2 (en) * | 2018-06-21 | 2023-04-11 | Universiteit Utrecht Holding B.V. | Method for monitoring kinase activity in a sample |
-
1998
- 1998-02-19 GB GBGB9803399.6A patent/GB9803399D0/en not_active Ceased
-
1999
- 1999-02-18 CA CA002320787A patent/CA2320787A1/en not_active Abandoned
- 1999-02-18 EP EP99905090A patent/EP1057025A1/en not_active Withdrawn
- 1999-02-18 JP JP2000532724A patent/JP2002504686A/en not_active Withdrawn
- 1999-02-18 WO PCT/GB1999/000510 patent/WO1999042833A1/en not_active Application Discontinuation
- 1999-02-18 AU AU25388/99A patent/AU2538899A/en not_active Abandoned
Non-Patent Citations (8)
Title |
---|
BORNANCIN, F. ET AL.: "Phosphorylation of threonine 638 critically controls the dephosphorylation and inactivation of protein kinase C-alpha", CURRENT BIOLOGY, vol. 6, no. 9, 1 September 1996 (1996-09-01), pages 1114 - 1123, XP002109456 * |
CAZAUBON, S. ET AL.: "Threonine-497 is a critical site for permissive activation of protein kinase C-alpha", BIOCHEMICAL JOURNAL, vol. 301, no. 2, 15 July 1994 (1994-07-15), pages 443 - 448, XP002109459 * |
CAZAUBON, SYLVIE ET AL: "Effector-dependent conformational changes in protein kinase C-gamma through epitope mapping with inhibitory monoclona antibodies", EUR. J. BIOCHEM., 194(3), 799-804, October 1990 (1990-10-01), XP002109461 * |
KERANEN, L.M. ET AL.: "Protein kinase C is regulated in vivo by three functionally distinct phosphorylations", CURRENT BIOLOGY, vol. 5, no. 12, 1 October 1995 (1995-10-01), pages 1394 - 1403, XP002109458 * |
LINDBERG R A ET AL: "IDENTIFICATION OF cDNA CLONES THAT CODE FOR PROTEIN-TYROSINE KINASES BY SCREENING EXPRESSION LIBRARIES WITH ANTIBODIES AGAINST PHOSPHOTYROSINE", ONCOGENE, vol. 3, no. 6, 1 December 1988 (1988-12-01), pages 629 - 633, XP000196785, ISSN: 0950-9232 * |
LINDBERG, R.A.: "Characterization of a human protein threonine kinase isolated by screening an expression library with antibodies to phosphotyrosine", ONCOGENE, vol. 8, no. 2, February 1993 (1993-02-01), pages 351 - 359, XP002109460 * |
MITCHELL, F.E. ET AL.: "The phosphorylation of protein kinase C as a potential measure of activation", THE BIOCHEMICAL JOURNAL, vol. 261, no. 1, 1 July 1989 (1989-07-01), pages 131 - 136, XP002109457 * |
TSUTAKAWA, S. E.: "Determination of in Vivo Phosphorylation Sites in Protein Kinase C", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 270, no. 45, 10 November 1995 (1995-11-10), pages 26807 - 26812, XP002109455 * |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000057184A2 (en) * | 1999-03-19 | 2000-09-28 | Kudos Pharmaceuticals Limited | Screening methods based on fha domains |
WO2000057184A3 (en) * | 1999-03-19 | 2001-03-08 | Kudos Pharm Ltd | Screening methods based on fha domains |
JP2006225396A (en) * | 2000-09-27 | 2006-08-31 | Biosource Internatl | Method for quantifying phosphokinase activity on proteins |
US7888050B2 (en) | 2000-09-27 | 2011-02-15 | Life Technologies Corporation | Method for quantifying phosphokinase activity on proteins |
US8114617B2 (en) | 2000-09-27 | 2012-02-14 | Life Technologies Corporation | Method for quantifying phosphokinase activity on proteins |
JP2013078330A (en) * | 2001-02-27 | 2013-05-02 | Blanchette Rockefeller Neurosciences Inst | Alzheimer's disease diagnosis based on mitogen-activated protein kinase phosphorylation |
CN110520144A (en) * | 2017-02-19 | 2019-11-29 | 国家生物技术研究所公司 | Peptide kinase inhibitor and its application method |
CN110520144B (en) * | 2017-02-19 | 2024-04-05 | 本-古里安大学B.G.内盖夫技术和应用公司 | Peptide kinase inhibitors and methods of use thereof |
Also Published As
Publication number | Publication date |
---|---|
CA2320787A1 (en) | 1999-08-26 |
AU2538899A (en) | 1999-09-06 |
JP2002504686A (en) | 2002-02-12 |
EP1057025A1 (en) | 2000-12-06 |
GB9803399D0 (en) | 1998-04-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Hoffmann et al. | Activation of the phosphatase activity of human cdc25A by a cdk2‐cyclin E dependent phosphorylation at the G1/S transition. | |
Reuther et al. | Association of the protein kinases c-Bcr and Bcr-Abl with proteins of the 14-3-3 family | |
US7629132B2 (en) | Goodpasture antigen binding protein | |
US5854206A (en) | Compounds and methods for treatment and diagnosis of prostate cancer | |
US5744313A (en) | Assay employing novel protein domain which binds tyrosine phosphorylated proteins | |
US6383734B1 (en) | Method to determine inhibition of PAK3 activation of Raf-1 | |
EP2624856B1 (en) | Moesin fragments for use in the diagnosis of immune thrombocytopenia | |
CA2814026C (en) | Moesin fragments associated with aplastic anemia | |
US6472197B1 (en) | GRB2 associating polypeptides and nucleic acids encoding therefor | |
US7799538B2 (en) | Method for identifying agents which modulate GTPase activity involved in insulin-stimulated GLUT4 translocation | |
EP1057025A1 (en) | Protein kinase c | |
EP0891551B1 (en) | Protein phosphatase-1 catalytic subunit interactions | |
WO1997019101A9 (en) | Novel grb2 associating polypeptides and nucleic acids encoding therefor | |
AU2003257962A1 (en) | Mk2 interacting proteins | |
WO1997012900A2 (en) | Ring finger protein | |
JPH07203963A (en) | Method of inhibiting serinekinase activity, method of adjusting bond activity between subunits of pi3- kinase, bond antibody for subunit of pi3-kinase, hybrydoma cell, nucleic acid molecule, plasmid, agonist and antagonist for producing said antibody, subunit bonding molecule for inhibiting pi3-kinase activity, method of detecting presence of subunit, method of preparing inhibitor for pi3-kinase activity and inhibitor for pi3-kinase activity | |
US20170218430A1 (en) | Detection of protein arginine demethylase activity | |
WO1997003090A2 (en) | Nuclear localized transcription factor kinase and diagnostic assays related thereto |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
WWE | Wipo information: entry into national phase |
Ref document number: 1999905090 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 09629975 Country of ref document: US |
|
ENP | Entry into the national phase |
Ref document number: 2320787 Country of ref document: CA Ref country code: CA Ref document number: 2320787 Kind code of ref document: A Format of ref document f/p: F |
|
NENP | Non-entry into the national phase |
Ref country code: KR |
|
WWP | Wipo information: published in national office |
Ref document number: 1999905090 Country of ref document: EP |
|
REG | Reference to national code |
Ref country code: DE Ref legal event code: 8642 |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1999905090 Country of ref document: EP |