WO2002077276A2 - Novel signalling polypeptide - Google Patents

Abstract

The present invention provides a novel signalling polypeptide that represents the first example of a signalling intermediate directly linking the MAP kinase signalling pathway to its downstream transcription factor targets. The invention further provides a binding polypeptide that is capable of binding to the polypeptide, methods of diagnosis and treatment of diseases and pharmaceutical compositions. Methods of identifying compounds that modulate the activity of the polypeptide are also provided.

Description

NOVEL SIGNALLING POLYPEPTIDE

The present invention is concerned with a novel signalling polypeptide and in particular with a polypeptide that represents the first example of a signalling intermediate directly linking the MAP kinase signalling pathway to its downstream transcription factor targets.

Transcriptional factors play a central role in regulating an organism's development as the majority of gene regulation in developmental processes occurs at the transcriptional level. Transcriptional regulators are divided into families which are usually based on the conserved structure of their DNA binding domains. The evolutionarily conserved Ras Mitogen-activated-protein-kinase (MAPK) cascade is an integral part of the processes of cell division, differentiation, movement and death. Signals received at the cell surface are relayed into the nucleus, where a MAPK phosphorylates and thereby modulates the activities of a subset of transcription factors. For example, EGFR (epidermal growth factor receptor, a tyrosine kinase, which is an essential component of many cell types), is stimulated by its ligand and activates the Ras/MAPK pathway to modulate transcription factors controlling growth, survival and differentiation of the cell.

The MAP Kinases are activated in response to a wide variety of external stimuli, including stimulation of cell growth and differentiation and the like, and they play a central role in controlling changes in cell phenotype. The name of the MAP kinases reflects their identification as mitogen-activated kinases; they are also called ERKs for extracellular signal-regulated. They are serine/threonine kinases and have several targets, including other kinases. Although they are cytosolic, they can move into the nucleus when activated, which extends the range of substrates even further to include the nuclear transcription factors. MAPK also mediates phosphorylation of substrates other than transcription factors, such as FDD (Head Involution Defective). HID exhibits proapoptotic activity and is involved in the regulation of apoptosis. MAPK phosphorylation sites in HID have been shown to be critical in the Ras/MAPK mediated apoptotic response (Bergmann et al, Cell, Vol. 95, 331-341 and Kurada et al, Cell, Vol.95, 319-329).

Phosphorylation of MAPK substrates, such as by Erk kinase, is a key link between cell signalling and the control of gene expression. The Ets family of transcription factors is one subset of nuclear transcription factors which function to regulate cell growth and differentiation, and the activities of many of its 35 members are modulated through phosphorylation by MAPK. The ETS DNA-binding domain is conserved at the structural level and is a divergent member of the hinged helix-turn-helix superfamily of DNA binding proteins. ETS domain proteins function as either transcriptional activators or repressors and regulate a diverse array of biological functions including mammalian haematopoiesis and Drosophila eye development.

Until now, it was thought that MAPK mediates the activity of its substrates, such as the transcription factors, by directly phosphorylating them.

It has now been surprisingly discovered by the present inventors that a new component of the signal transduction pathway, which functions as an intermediate adapter/coupler protein, is required to mediate phosphorylation of MAPK substrates, such as nuclear transcription factors, by MAPK. This represents the first example of a signalling intermediate that directly links the MAP kinase signalling pathway to its downstream targets. Therefore, in a first aspect the invention provides an isolated or recombinant intermediate adapter/coupler polypeptide capable of mediating modulation or phosphorylation of a MAPK substrate.

The identification of the adapter/coupler protein is a significant development and, advantageously, represents a particularly convenient location for analysing or modulating cell signalling pathways. It also provides an excellent target for intervention with specific cellular processes. Intervention in such specific cellular processes at such a specific target level is particularly beneficial because it avoids or minimises toxic side effects in the cell. For example, once a transcription factor and its role in the cellular process is known, it is possible to selectively control or modulate the cellular process without substantially affecting other cellular processes.

The inventors have also, advantageously, cloned and characterised one example of this class of intermediate adapter/coupler proteins from Drosophila melanogaster, designated herein as Mae (for modulator of the activity of ets). Accordingly, in a second aspect the invention provides an isolated or recombinant polypeptide which:

a) comprises the amino acid sequence shown in Figure lb; b) has one or more amino acid substitutions, deletions or insertions relative to the amino acid sequence given in a) above, which polypeptide is capable of mediating modulation or phosphorylation of a MAPK substrate; or c) is a fragment of a polypeptide as defined in a) or b) above, wherein the fragment is capable of mediating modulation or phosphorylation of a MAPK substrate.

Figure lb shows the a ino acid sequence of the Mae polypeptide from Drosophila. The entire genome of Drosophila melanogaster has been sequenced and published, and attempts have been made to annotate the genome. However, the present inventors are the first to characterise the gene coding for the Mae polypeptide. They are also the first to provide the correct sequence of the isolated Mae polypeptide, together with its function.

The term "polypeptide" is used herein in a broad sense to indicate that a particular molecule comprises a plurality of amino acids joined together by peptide bonds. It therefore includes within its scope substances, which may sometimes be referred to in the literature as peptides, polypeptides or proteins ( whether or not they are covalently bound to other moieties - e.g. to form fusion proteins).

The protein according to the invention may be recombinant, synthetic or naturally occurring, but is preferably recombinant or isolated. As used herein with respect to polypeptides, "isolated" means separated from its native environment and present in sufficient quantity to permit its identification or use. Isolated, when referring to a protein or polypeptide, means, for example: (i) selectively produced by expression cloning or (ii) purified as by chromatography or electrophoresis. Isolated proteins or polypeptides may, but need not be, substantially pure. The term "substantially pure" means that the proteins or polypeptides are essentially free of other substances with which they may be found in nature or in vivo systems to an extent practical and appropriate for their intended use.

The polypeptide of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the expressed polypeptide may lack the initiating methionine residue as a result of post-translational cleavage. Proteins or polypeptides which have been modified in this way are also included within the scope of the invention. Alternatively, chemical synthesis techniques may be used to produce polypeptides of the present invention. Such techniques generally utilise solid phase synthesis. Chemical synthesis techniques that allow polypeptides having particular sequences to be produced have now been automated. Apparatus capable of chemically synthesising polypeptides is available, for example, from Applied Biosystems. If necessary, short polypeptides can be synthesised initially and can then be ligated to produce longer polypeptides.

The polypeptides of the present invention are generally capable of mediating MAPK modulation and/or phosphorylation of MAPK substrates. Preferably the MAPK substrates are transcription factors, preferably Ets DNA binding transcription factors.

Functional homologues or equivalents of the polypeptide of the invention can be prepared according to methods known in the art, and which comprise, amongst others, altering the polypeptide sequence as set out in Molecular Cloning, A Laboratory Manual, Sambrook et al. Conservative amino acid substitutions can be performed by altering the nucleic acid encoding the polypeptide, using, for example, PCR or site directed mutagenesis or by chemical synthesis of the nucleic acid molecule. Computer algorithms can also be utilised which predict the amino acid sequences that may be altered or substituted to prepare said functional equivalents.

In order to appreciate the present invention more fully, polypeptides within the scope of a), b) or c) above will now be discussed in greater detail.

Polypeptides within the scope of a)

A polypeptide within the scope of a) may consist of the particular amino acid sequence shown in Figure lb, or may have an additional N-terminal and/or an additional C-terminal amino acid sequence. Additional N-terminal or C-terminal sequences may be provided for various reasons. Techniques for providing such additional sequences are well known in the art. These include using gene-cloning techniques to ligate together nucleic acid molecules encoding polypeptides or parts thereof, followed by expressing a polypeptide encoded by the nucleic acid molecule produced by ligation.

Additional sequences may be provided in order to alter the characteristics of a particular polypeptide. This can be useful in improving expression or regulation of expression in particular expression systems. For example, an additional sequence may provide some protection against proteolytic cleavage. This has been done for the hormone somatostatin by fusing it at its N-terminus to part of the β galactosidase enzyme (Itakwa et al., Science 198: 105-63 (1977)).

Additional sequences can also be useful in altering the properties of a polypeptide to aid in identification or purification.

For example, a signal sequence may be present to direct the transport of the polypeptide to a particular location within a cell or to export the polypeptide from the cell. Different signal sequences can be used for different expression systems. Another example of the provision of an additional sequence is where a polypeptide is linked to a moiety capable of being isolated by affinity chromatography. The moiety may be an antigen or an epitope and the affinity column may comprise immobilised antibodies or immobilised antibody fragments that bind to said antigen or epitope (desirably with a high degree of specificity). The fusion protein can usually be eluted from the column by addition of an appropriate buffer. Additional N-terminal or C-terminal sequences may, however, be present simply as a result of a particular technique used to obtain a substance of the present invention and need not provide any particular advantageous characteristic.

Polypeptides within the scope ofb)

Turning now to the polypeptides defined in b) above, it will be appreciated by the person skilled in the art that these are variants of the polypeptides given in a) above.

The skilled person will appreciate that various changes can sometimes be made to the amino acid sequence of a polypeptide which has a particular activity to produce variants (often known as "muteins") which still have said activity. Such variants of the polypeptides described in a) above are within the scope of the present invention and are discussed in greater detail below in sections (i) to (ϋi). They include allelic and non-allelic variants.

(i) Substitutions

An example of a variant of the present invention is a polypeptide as defined in a) above, apart from the substitution of one or more amino acids with one or more other amino acids.

The skilled person is aware that various amino acids have similar properties. One or more such amino acids of a polypeptide can often be substituted by one or more other such amino acids without eliminating a desired activity of that polypeptide.

For example, the amino acids glycine, alanine, valine, leucine and isoleucine can often be substituted for one another (amino acids having aliphatic side chains). Of these possible substitutions it is preferred that glycine and alanine are used to substitute for one another (since they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (since they have larger aliphatic side chains which are hydrophobic). Other amino acids that can often be substituted for one another include:

phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and cysteine and methionine (amino acids having sulphur containing side chains).

Substitutions of this nature are often referred to as "conservative" or "semi-conservative" amino acid substitutions.

(ii) Deletions

Amino acid deletions can be advantageous since the overall length and the molecular weight of a polypeptide can be reduced whilst still retaining activity. This can enable the amount of polypeptide required for a particular purpose to be reduced. For example if the polypeptide is to be used in medicine, dosage levels can be reduced.

(in) Insertions Amino acid insertions relative to a polypeptide as defined in a) above can also be made. This may be done to alter the properties of the polypeptide (e.g. to assist in identification, purification or expression, as explained above in relation to fusion proteins).

Polypeptides incorporating amino acid changes (whether substitutions, deletions or insertions) relative to the sequence of a polypeptide as defined in a) above can be provided using any suitable techniques. For example, a nucleic acid sequence incorporating a desired sequence change can be provided by site directed mutagenesis. This can then be used to allow the expression of a polypeptide having a corresponding change in its amino acid sequence.

Polypeptides within the scope ofc)

As discussed supra, it is often advantageous to reduce the length of a polypeptide. Feature c) of the present invention therefore covers fragments of the polypeptides a) or b) above which are at least 10 amino acids long. The fragments of the polypeptides of the present invention are capable of mediating modulation or phosphorylation of a MAPK substrate. Desirably these fragments are at least 20, at least 50 or at least 100 amino acids long.

A polypeptide according to the invention includes all possible amino acid variants encoded by its corresponding nucleic acid molecule, including a polypeptide encoded by said molecule and having conservative amino acid changes. Proteins or polypeptides according to the invention further include variants of such sequences, including naturally occurring allelic variants which are substantially homologous to said proteins or polypeptides.

Preferably the polypeptide of the present invention has substantial homology with the polypeptide having the amino acid sequence shown in Figure lb, or comprises an amino acid sequence which differs from that shown in Figure lb only in conservative amino acid changes.

The term "homologous" describes the relationship between different nucleic acid molecules or amino acid sequences wherein said sequences or molecules are related by partial identity or similarity at one or more blocks or regions within said molecules or sequences. Homology may be determined by means of computer programs known in the art. Substantial homology preferably carries with it that the amino acid sequences of the polypeptide of the invention comprise an amino acid sequence fragment corresponding and displaying a certain degree of sequence homology to the amino acid sequence of Figure lb. Preferably they share a homology of at least: 30 %, 40 %, 50 %, 60 %, 70%, 80 %, more than 90 %, or more than 95 % with respect to the amino acid sequence depicted in Figure lb.

One way of determining amino acid sequence homology is to align a given amino acid sequence with the amino acid sequence shown in Figure lb in a manner which achieves the maximum number of matches of amino acids over the full length of the amino acid sequence given in Figure 1. The percentage sequence homology will then be (m/t) x 100 , where m is the number of matches between the two aligned sequences over the length of the amino acid sequence shown in Figure lb and t is the total number of amino acids present in the amino acid sequence shown in Figure lb.

A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using, for example, the Blast program described in Altschul, S.T., et al., (1990) Basic Local Alignment Search Tool, J. Mol. Biol., 215, 403-410. In accordance with the present invention the algorithm and the parameters utilised in the Blast search were as follows: Blosum 62 (matrix), Cut Off: default, threshold: default, statistics: sumt, both strands no filter. In a sequence alignment the query and subject sequences are both DNA sequences. An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity.

Another way of determining sequence homology allows for the introduction of gaps when matching two amino acid sequences (0,1,2,3 or even more gaps may be allowed for in each sequence). This can be done, for example, by using the "Gap" program, which is available from Genetics Computer Group as part of "The Wisconsin Package". This program is based upon an algorithm provided by Smith and Waterman (Advances in Applied Mathematics, 482-489 (1981))

All of these ways of determining sequence homology may be used in respect of the present invention, although it is preferred to allow for one or more gaps.

The sequences that are homologous to the sequences described above are, for example, variations of said sequences which represent modifications having the same biological function, in particular encoding proteins with the same or substantially the same specificity, e.g. binding specificity. They may be naturally occurring variations, such as sequences from other mammals, or mutations. These mutations may occur naturally or may be obtained by mutagenesis techniques. The allelic variations may be naturally occurring allelic variants as well as synthetically produced or genetically engineered variants. In a preferred embodiment the sequences are derived from human.

According to a third aspect, the present invention provides an isolated or recombinant nucleic acid molecule which encodes a polypeptide according to the first or the second aspect.

According to the fourth aspect, the present invention provides an isolated or recombinant nucleic acid molecule which comprises or consists of:

a) the nucleic acid sequence set forth in Figure la; b) a nucleic acid sequence having a codon sequence which differs from that of the codon sequence of a) due to the degeneracy of the genetic code; c) a nucleic acid sequence which is homologous to the sequence of a) or b); or d) a complement of a), b) or c). The nucleic acid sequence of Figure la codes for the Mae polypeptide as shown in Figure lb. The polypeptide of Figure lb can be coded for by a large variety of nucleic acid molecules, taking into account the well-known degeneracy of the genetic code. All of these nucleic acid molecules are within the scope of the present invention. Thus, in accordance with the present invention, a defined nucleic acid includes not only the identical nucleic acid but also any minor base variations including in particular, substitutions in cases which result in a synonymous codon (a different codon specifying the same amino acid residue) due to the degenerate code in conservative amino acid substitutions. The term "nucleic acid sequence" also includes the complementary sequence to any single stranded sequence given regarding base variations. Thus, for example, both strands of a double stranded nucleic acid molecule are included within the scope of the present invention (whether or not they are associated with one another).. Also included are mRNA molecules and complementary DNA molecules (e.g. cDNA molecules).

As used herein with respect to nucleic acids "isolated or recombinanf'means any of a) amplified in vitro by, for example, polymerase chain reaction (PCR), b) recombinantly produced by cloning, c) purified by, for example, gel separation, or d) synthesised, such as by chemical synthesis.

The term "homologous" describes the relationship between different nucleic acid molecules or amino acid sequences wherein said sequences or molecules are related by partial identity or similarity at one or more blocks or regions within said molecules or sequences. The discussion of homology above in relation to the first aspect of the present invention applies mutatis mutandis to the nucleic acid molecules of the present invention. Nucleic acid molecules having substantial homology to the nucleic acid sequence set forth in Figure la is preferred. Preferably a nucleic acid molecule shares a homology of at least: 30 %, 40 %, 50 %, 60 %, 70%, 80 %, more than 90 %, or more than 95 % with respect to the nucleic acid sequence depicted in Figure la.

The coding strand of the nucleic acid molecules of this aspect of the present invention encodes an intermediate adapter/coupler polypeptide capable of mediating modulation or phosphorylation of MAPK substrates designated Mae, or a functional equivalent or derivative thereof. Preferably, the polypeptide is also capable of functioning as an adapter/coupler in MAPK mediated phosphorylation of MAPK substrates, such as, nuclear and preferably ets transcription factors. The nucleic acid molecule is preferably a DNA molecule, and more preferably a cDNA molecule. Preferably, the nucleic acid sequence of the invention is of mammalian, and more preferably human origin.

In view of the foregoing description, the skilled person will appreciate that a large number of nucleic acids are within the scope of the present invention. Therefore, unless the context indicates otherwise, nucleic acid molecules of the present invention may have one or more of the following characteristics:

1 ) they may be DNA or RNA;

2) they may be single or double stranded; 3) they may be provided in recombinant form i.e. covalently linked to a heterologous 5' and/or a 3' flanking sequence to provide a chimaeric molecule (e.g. a vector) which does not occur in nature; 4) they may be provided without 5' and/or 3' flanking sequences which normally occur in nature; 5) they may be provided in substantially pure form, e.g. by using probes to isolate cloned molecules having a desired target sequence or by using chemical synthesis techniques (thus they may be provided in a form which is substantially free from contaminating proteins and/or from other nucleic acids); 6) they may be provided with introns (e.g. as a full-length gene) if appropriate, or without introns (e.g. as cDNA).

The present invention also provides an isolated or recombinant nucleic acid molecule which is capable of hybridising to a nucleic acid molecule according to the third or fourth aspect.

The appropriate conditions of hybridisation may be chosen by the skilled person. Stringency of hybridisation as used herein refers to conditions under which polynucleic acids are stable. The stability of hybrids is reflected in the melting temperature (Tm) of the hybrids. Tm can be approximated by the formula:

81.5^oC+16.6(log₁₀[Na⁺]+⁰-⁴l (%G-&C)-600/l

wherein 1 is the length of the hybrids in nucleotides. Tm decreases approximately by 1 - 1.5°C with every 1% decrease in sequence homology.

The term "stringency" refers to the hybridisation conditions wherein a single-stranded nucleic acid joins with a complementary strand when the purine or pyrimidine bases therein pair with their corresponding base by hydrogen bonding. High stringency conditions favour homologous base pairing whereas low stringency conditions favour non-homologous base pairing.

"Low stringency" conditions comprise, for example, a temperature of about 37°C or less, a formamide concentration of less than about 50%, and a moderate to low salt (SSC) concentration; or, alternatively, a temperature of about 50°C or less, and a moderate to high salt (SSPE) concentration, for example 1M NaCl. "High stringency" conditions comprise, for example, a temperature of about 42°C or less, a formamide concentration of less than about 20%, and a low salt (SSC) concentration; or, alternatively, a temperature of about 65°C, or less, and a low salt (SSPE) concentration. For example, high stringency conditions comprise hybridization in 0.5 M NaHPO , 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at 65°C (Ausubel, F.M. et al. Current Protocols in Molecular Biology. Vol. I, 1989; Green Inc. New York, at 2.10.3).

"SSC" comprises a hybridization and wash solution. A stock 20X SSC solution contains 3M sodium chloride, 0.3M sodium citrate, pH 7.0.

"SSPE" comprises a hybridization and wash solution. A IX SSPE solution contains 180 mM NaCl, 9mM Na₂HPO₄ and 1 mM EDTA, pH 7.4.

There are other conditions, reagents and so forth which can be used, which result in stringent hybridisation and the skilled practitioner is familiar with such conditions.

The nucleic acid capable of hybridising to nucleic acid molecules according to the invention will generally be at least 70%, preferably at least 80, 85 or 90% and more preferably at least 95% and even more preferably at least 97% homologous to the nucleotide sequences according to the invention.

Most preferably, hybridising nucleic acid molecules of the present invention hybridise to a DNA molecule having the sequence shown in Figure la, to an RNA equivalent thereof, or to a complementary sequence to said DNA molecule or to said RNA equivalent thereof. The hybridising nucleic acid molecule or its complement may encode an intermediate adapter/coupler polypeptide capable of mediating modulation or phosphorylation of MAPK substrates. The hybridising nucleic acid molecule may be of mammalian, preferably human, origin.

Hybridising nucleic acid molecules can be useful as probes or primers, for example. Desirably such hybridising molecules are at least 10 nucleotides in length and preferably are at least 25 or at least 50 nucleotides in length.

In addition to being used as probes, hybridising nucleic acid molecules of the present invention can be used as antisense molecules to alter the expression of polypeptides of the present invention by binding to complementary nucleic acid molecules (generally this can be achieved by using RNA molecules to bind to transcribed RNA molecules, thereby preventing translation of such transcribed RNA molecules due to the formation of RNA- RNA duplexes). This technique can be used in antisense therapy. Thus, an antisense molecule may be used as a medicament or may be included in a pharmaceutical composition with a pharmaceutically acceptable carrier, diluent or excipient therefor.

Hybridising molecules may also be provided as ribozymes. Ribozymes can be used to regulate expression by binding to and cleaving RNA molecules which include particular target sequences.

The present invention also advantageously provides oligonucleotides comprising at least 10 consecutive nucleotides of a nucleic acid according to the invention and preferably from 10 to 40 consecutive nucleotides of a nucleic acid according to the invention. As would be appreciated by one of skill in the art, it is also possible to use as primers those untranslated regions (UTR's) of the gene encoding the polypeptide of the invention. For example, 3' and 5' UTR's can be used to identify homologues of the polypeptide of the invention. The oligonucleotides of the invention may, advantageously be used as probes or primers to initiate replication, or the like. Oligonucleotides having a defined sequence may be produced according to techniques well known in the art, such as by recombinant or synthetic means. They may also be used in diagnostic kits or the like for detecting the presence of a nucleic acid according to the invention. These tests generally comprise contacting the probe with the sample under hybridising conditions and detecting for the presence of any duplex or triplex formation between the probe and any nucleic acid in the sample.

According to the present invention these probes may be anchored to a solid support. Preferably, they are present on an array so that multiple probes can simultaneously hybridize to a single biological sample. The probes can be spotted onto the array or synthesised in situ on the array. (See Lockhart et al., Nature Biotechnology, vol. 14, December 1996 "Expression monitoring by hybridisation to high density oligonucleotide arrays".

The nucleic acid sequences according to the invention may be produced using recombinant or synthetic techniques, such as for example using PCR which generally involves making a pair of primers, which may be from approximately 10 to 50 nucleotides to a region of the gene which is desired to be cloned, bringing the primers into contact with cDNA, or genomic DNA from a human cell, performing a polymerase chain reaction under conditions which bring about amplification of the desired region, isolating the amplified region or f agment and recovering the amplified DNA. Generally, such techniques are well known in the art, such as described in Sambrook et al. (Molecular Cloning: a Laboratory Manual, 1989).

The nucleic acids or oligonucleotides according to the invention may carry a revealing label. Suitable labels include radioisotopes such as ³ P or ³⁵S, enzyme labels or other protein labels such as biotin or fluorescent markers. Such labels may be added to the nucleic acids or oligonucleotides of the invention and may be detected using known techniques per se.

Advantageously, human allelic variants or polymorphisms of the nucleic acid according to the invention may be identified by, for example, probing cDNA or genomic libraries from a range of individuals, for example, from different populations. Furthermore, nucleic acids and probes according to the invention may be used to sequence genomic DNA from patients using techniques well known in the art, such as the S anger Dideoxy chain termination method, which may, advantageously, ascertain any predisposition of a patient to disorders associated with variants of the Mae polypeptide of the invention.

In the very least, the nucleotide sequences can be used as molecular weight markers on Southern gels, as diagnostic probes for the presence of a specific mRNA in a particular cell type, as a probe to "subtract-out" known sequences in the process of discovering novel nucleotide sequences, for selecting and making oligomers for attachment to a "gene chip" or other support, to raise anti-DNA antibodies using DNA immunization techniques, and as an antigen to elicit an immune response.

The nucleic acid molecules according to the invention may, advantageously, be included in a suitable expression vector to express the proteins encoded therefrom in a suitable host. Accordingly the present invention provides a vector comprising a nucleic acid molecule of the present invention. Incorporation of cloned DNA into a suitable expression vector for subsequent transformation of said cell and subsequent selection of the transformed cells is well known to those skilled in the art as provided in Sambrook et al. (1989), Molecular cloning: A Laboratory Manual, Cold Spring Harbour Laboratory. An expression vector, according to the invention, includes a vector having a nucleic acid according to the invention operably linked to regulatory sequences, such as promoter regions, that are capable of effecting expression of said DNA fragments. A vector can include a large number of nucleic acids which can have a desired sequence inserted therein by, for example, using an appropriate restriction enzyme and ligating the sequence in the vector, for transport between cells of different genetic composition. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. Such vectors may be transformed into a suitable host cell to provide for expression of a protein according to the invention. The vectors are usually capable of replicating within a host environment and they also comprise one of a number of restriction sites for endonucleases which permits them to be cut in a selective manner at a particular location for insertion of a new nucleic acid molecule or sequence therein. Thus, in a further aspect, the invention provides a process for preparing polypeptides according to the invention, which comprises cultivating a host cell, transformed or transfected with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the protein, and recovering the expressed protein.

In this regard, the nucleic acid molecule may encode a mature protein or a protein having a prosequence, including encoding a leader sequence on the preprotein which is cleaved by the host cell to form a mature protein.

The vectors may be, for example, plasmid, virus or phagemid vectors provided with an origin of replication, and optionally a promoter for the expression of said nucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable markers, such as, for example, an antibiotic resistance. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and to direct an appropriate level of transcription initiation and also translation initiation sequences for ribosome binding. For example, a bacterial expression vector may include a promoter such as the lac promoter and for translation initiation the Shine-Dalgarno sequence and the start codon AUG. Similarly, a eukaryotic expression vector may include a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome. However, the precise regulatory elements required for expression of a gene of interest may vary between different cell types but generally include 5' non-transcribing and non-translating regions which are required for initiation of translation and transcription. Such vectors may be obtained commercially or be assembled from the sequences described by methods well known in the art.

Transcription of DNA encoding the polypeptides of the present invention by higher eukaryotes is optimised by including an enhancer sequence in the vector. Enhancers are cis-acting elements of DNA that act on a promoter to increase the level of transcription. Vectors will also generally include origins of replication in addition to the selectable markers.

Nucleic acid molecules according to the invention may be inserted into the vectors described in an antisense orientation in order to provide for the production of antisense RNA. Antisense RNA or other antisense nucleic acids, including antisense peptide nucleic acid (PNA), may be produced by synthetic means.

The present invention is further directed to inhibiting expression or activity of the polypeptides of the invention in vivo by, for example, inhibiting Mae transcription or Mae mRNA by, for example, the use of antisense technology. However, as would be appreciated by the skilled practitioner any other suitable method may be utilised. As discussed in more detail below, other methods of inhibiting Mae expression may utilise antibodies or binding polypeptides or other small molecules which, for example, bind or block the binding region of the polypeptides of the invention. As used herein, the term "antisense nucleotide" or "antisense" describes an oligonucleotide that is an oligoribonucleotide, oligodeoxyribonucleotide, modified oligoribonucleotide, or modified oligodeoxyribonucleotide which hybridises under physiological conditions to DNA encoding Mae polypeptide or to an mRNA transcript of the gene and, thereby, inhibits the transcription of that gene and/or translation of mRNA. Antisense technology can be used to control gene expression through triple-helix formation of antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion or the mature protein sequence, which encodes for the protein of the present invention, is used to design an antisense RNA oligonucleotide of from 10 to 40 base pairs in length. The antisense RNA oligonucleotide hybridises to the mRNA in vivo and blocks translation of an mRNA molecule into the protein (antisense - Okano, J. Neurochem., 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple-helix - see Lee et al. Nucl. Acids Res., 6:3073 (1979); Cooney et al., Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991), thereby preventing transcription and the production of the polypeptide.

The term "modified oligonucleotide" as used herein describes an oligonucleotide in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the oligonucleotide. Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, peptides, and carboxymethyl esters.

The term "modified oligonucleotide" also encompasses oligonucleotides with a covalently modified base and/or sugar. For example, modified oligonucleotides include oligonucleotides having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Thus, modified oligonucleotides may include a 2-O-alkylated ribose group. In addition, modified oligonucleotides may include sugars such as arabinose instead of ribose. Modified oligonucleotides also can include base analogs such as C-5 propyne modified bases (Wagner et al., Nature Biotechnology 14:840-844, 1996). The present invention, thus, contemplates pharmaceutical preparations containing modified antisense molecules that are complementary to and hybridizable with, under physiological conditions, nucleic acids encoding Mae polypeptide together with pharmaceutically acceptable carriers.

The antisense oligonucleotide described above can be delivered to cells by procedures in the art such that the anti-sense RNA and DNA may be expressed in vivo to inhibit production of the protein in the manner described above.

Encompassed within the scope of the invention are hybrid and modified forms of the polypeptide according to the invention including fusion proteins and fragments. The hybrid and modified forms include, for example, when certain amino acids have been subjected to some modification or replacement, such as for example, by point mutation and yet which results in a polypeptide or protein which possesses the same function as the polypeptides of the invention. A further aspect of the invention provides a host cell or organism, transformed or transfected with an expression vector according to the invention. The cell or organism may be transformed or transfected using techniques that are well known in the art, such as, electroporation or liposomes. The host cell or organism may advantageously be used in a method of producing Mae polypeptide, which comprises recovering any expressed polypeptide from the host or organism transformed or transfected with the expression vector.

According to a further aspect of the invention there is also provided a transgenic cell, tissue or organism comprising a transgene capable of expressing a polypeptide according to the invention. The term "transgene capable of expressing" as used herein encompasses any suitable nucleic acid sequence which leads to expression of polypeptides having the same function and/or activity. The transgene, may include, for example, genomic nucleic acid isolated from human cells or synthetic nucleic acid, including DNA integrated into the genome or in an extrachromosomal state. Preferably, the transgene comprises the nucleic acid sequence encoding the polypeptide according to the invention as described herein, or a functional fragment of said nucleic acid. A functional fragment of said nucleic acid should be taken to mean a fragment of the gene comprising said nucleic acid coding for the polypeptides according to the invention or a functional equivalent, derivative or a non-functional derivative such as a dominant negative mutant of said polypeptides.

Transgenic non-human organisms are being utilised as model systems for studying both normal and disease cell processes. In general, to create such transgenic animals an exogenous gene with or without a mutation is transferred to the animal host system and the phenotype resulting from the transferred gene is observed. Other genetic manipulations can be undertaken in the vector or host system to improve the gene expression leading to the observed phenotype (phenotypic expression). The gene may be transferred on a vector under the control of different inducible or constitutive promoters, may be overexpressed or the endogenous homologous gene may be rendered unexpressible, and the like (WO 92/11358). The vector may be introduced by transfection or other suitable techniques such as electroporation, for example, in embryonic stem cells. The cells that have the exogenous DNA incorporated into their genome, for example, by homologous recombination, may subsequently be injected into blastocytes for generation of the transgenic animals with the desired phenotype. Successfully transformed cells containing the vector may be identified by well known techniques such as lysing the cells and examining the DNA, by, for example, Southern blotting or using the polymerase chain reaction.

Knock-out organisms may be generated to further investigate the role of the polypeptide of the invention in vivo. By "knock-out" it is meant an animal which has its endogenous gene knocked out or inactivated. Typically, homologous recombination is used to insert a selectable gene into an essential exon of the gene of interest. Furthermore, the gene of interest can be knocked out in favour of a homologous exogenous gene (Robbins, J., GENE TARGETING. The Precise Manipulation of the Mammalian Genome Circ. Res. 1993, J.W.; 73; 3-9). Transgenic animals, such as mice or Drosophila or the like, may therefore be used to overexpress the Mae protein according to the invention to further investigate its role in vivo.

The polypeptide expressed by said transgenic cell, tissue or organism or a functional equivalent thereof also forms part of the present invention. Recombinant proteins or polypeptides may be recovered and purified from host cell cultures by methods known in the art, 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.

In a still further aspect the invention provides a binding polypeptide which is capable of binding to the polypeptide of the invention or an epitope thereof. In one embodiment, the binding polypeptide comprises an antibody, for example, or a polypeptide exhibiting regions of homology with the polypeptide of the invention and capable of binding thereto. Such an antibody may be polyclonal, for example, and may be raised according to standard techniques well known to those skilled in the art by using the polypeptide of the invention or a fragment or single epitope thereof as the challenging antigen. Alternatively, the antibody may be monoclonal in nature and may be produced according to the techniques described by Kohler & Milstein (Nature (1975) 256, 495- 497). Techniques for producing monoclonal and polyclonal antibodies which bind to a particular polypeptide are now well developed in the art. They are discussed in standard immunology textbooks, for example in Roitt etal, Immunology second edition (1989), Churchill Livingstone, London.

When the binding protein or polypeptide is an antibody the present invention includes not only complete antibody molecules but fragments thereof. Antibody fragments which contain the idiotype of the molecule can be generated by known techniques, for example, such fragments include but are not limited to the F(ab')₂ fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragments and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent. Chimeric, humanized and fully humanized monoclonal antibodies can now be made by recombinant engineering. By addition of the human constant chain to F(ab )₂ fragments it is possible to create a humanized monoclonal antibody which is useful in immunotherapy applications where patients making antibodies against the mouse Ig would otherwise be at a disadvantage. Breedveld F.C. Therapeutic Monoclonal Antibodies. Lancet 2000 Feb 26; 335, P735-40.

Other synthetic constructs which can be used include CDR peptides. These are synthetic peptides comprising antigen-binding determinants. Peptide mimetics may also be used. These molecules are usually conformationally restricted organic rings which mimic the structure of a CDR loop and which include antigen-interactive side chains. Synthetic constructs include chimaeric molecules. Thus, for example, humanised (or primatised) antibodies or derivatives thereof are within the scope of the present invention. An example of a humanised antibody is an antibody having human framework regions, but rodent hypervariable regions. Synthetic constructs also include molecules comprising an additional moiety which provides the molecule with some desirable property in addition to antigen binding. For example the moiety may be a label (e.g. a fluorescent or radioactive label). Alternatively, it may be a pharmaceutically active agent.

Furthermore, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epiptope (see, in general, Clark, W.R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The pFc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')₂ fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation.

Polypeptides that bind to the polypeptide of the invention may be identified by Phage Display. In this technique a phage library is prepared, displaying inserts from between about 4 to 80 amino acid residues using techniques which are well known in the art It is then possible to select those Phage bearing inserts that bind to the polypeptide of the invention. DNA sequence analysis is then performed to identify the nucleic acid sequences encoding the expressed polypeptides.

Antibody fragments of predetermined binding specificity may also be constructed using Phage Display technology, which obviates the need for hybridoma technology and immunization. These antibody fragments are created from repertoires of antibody V genes which are harvested from populations of lymphocytes, or assembled in vitro, and cloned for display of associated heavy and light chain variable domains on the surface of filamentous bacteriophage. The process mimics immune selection. Antibodies with many different binding specificities have been isolated from the same Phage repertoire. (Winter et al, Annu. Rev. Immunol. 1994; 12 : 433-55). Such antibodies are also embraced within the scope of the binding polypeptides of the present invention.

The antibodies or derivatives thereof of the present invention have a wide variety of uses. They can be used in purification and or identification of polypeptides of the present invention. Thus they may be used in diagnosis. They can be provided in the form of a kit for screening for the polypeptides of the present invention.

Other types of binding polypeptides that may be utilised in accordance with the invention are termed immunoadhesins. Immunoadhesins are a class of fusion proteins, which combine the target-binding region of a receptor, an adhesion molecule, a ligand or an enzyme, with the Fc portion or an immunoglobulin. Production of immunoadhesins is described in Byrn et al (1990) Nature 344, pp 667-670.

A further aspect of the present invention also provides a method of identifying a polypeptide of the invention in a sample, which method comprises contacting said sample with a binding polypeptide as described herein and monitoring for any specific binding of any polypeptides to said binding polypeptide. A kit for identifying the presence of such polypeptides in a sample is also provided comprising a binding polypeptide as described above and means for contacting said binding polypeptide with said sample.

In a further aspect the invention provides an in vitro method of detecting expression of a polypeptide of the invention which method comprises contacting a sample of tissue, cells or cell lysates from a subject with a binding protein as previously described and detecting any binding of said binding polypeptide to a protein in the sample.

Preferably the method of the invention is performed on cells or tissues removed from a human subject. However, it is also within the scope of the invention to perform the method on cells or tissues removed from non-human mammals such as mouse or monkey by using an antibody which is cross-reactive against a homologous protein expressed in the non-human mammalian species.

Other intermediate adapter/coupler polypeptides may be identified using the yeast-two hybrid vector system first proposed by Chien et al. (1991), Proc. Natl. Acad. Sci. USA 88; 9578-9582. As would be apparent to those skilled in the art, once it has been appreciated that an intermediate adapter/coupler polypeptide is involved in the modulation of MAPK substrates, such as nuclear transcription factors, it would be a matter of routine experimentation using techniques that are known in the art, and which are described in more detail in the example below, to identify other such adapter/coupler polypeptides. More specifically, the procedure will involve identifying critical amino acids, particularly serine or threonine residues in known MAPK substrates by mutating the sites involved in phosphorylation, to identify those residues which cannot be phosphorylated in the presence of MAPK alone. For example, the consensus site PxS/TP can be used as a guide (where P is proline, S/T is serine or threonine and x is any amino acid). Mutations may then be performed on the serine or threonine residues of these sites which are then assayed using the in vitro kinase assay of MAPK phosphorylation described in the example provided. This will identify those critical sites that cannot be phosphorylated by MAPK alone.

Yeast two hybrid experiments may then be performed using the sequence of the MAPK substrate as bait. This technique is based on functional reconstitution in vivo of a transcription factor which activates a reporter gene. More particularly the technique comprises providing an appropriate host cell with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA binding domain and an activating domain, expressing in the host cell a first hybrid DNA sequence encoding a first fusion of a fragment or all of a nucleic acid sequence according to the invention and either said DNA binding domain or said activating domain or the transcription factor, expressing in the host at least one second hybrid DNA sequence, such as, a library or the like, encoding putative binding proteins to be investigated together with the DNA binding or activating domain of the transcription factor which is not incorporated in the first fusion; detecting any binding of the proteins to be investigated with a protein according to the invention by detecting for the presence of any reporter gene product in the host cell, optionally isolating second hybrid DNA sequences encoding the binding protein. The above methods can also be used to isolate proteins or polypeptides interacting with Mae, the polypeptide of the invention may be used in a yeast two hybrid assay as bait. In this regard, the nucleic acid molecule of the invention may be fused in frame with a DNA binding domain and co-transfected into yeast along with a 24 hour Drosophila embryo library. Proteins or polypeptides that interact with Mae will be identified on the basis of a change in the nutritional requirements of yeast cells that express Mae and a gene which encodes a polypeptide that associates with the expressed Mae polypeptide.

Any clones identified can tfien be assayed using the in vitro kinase assay of MAPK phosphorylation as set out in the example provided. The assay will be performed with the mutant substrate that expresses the serine or threonine residue that cannot be phosphorylated with MAPK alone, and subsequently incubating it in the presence of the clones identified from the yeast-two hybrid screen. In this manner it is possible to identify other adapter/coupler polypeptides that are required to mediate phosphorylation of the critical serine or threonine residue of the substrate. Therefore, the present invention also encompasses those adapter/coupler polypeptides that may be identified by the aforementioned procedure which would be embraced within the general knowledge of the skilled practitioner.

Furthermore, according to a further aspect of the invention there is provided a method of identifying an intermediate adapter/coupler of MAPK substrates, which method comprises a) identifying critical amino residues on MAPK substrates which cannot be phosphorylated in the presence of MAPK alone, b) identifying proteins or polypeptides that bind to said MAPK substrate, c) incubating any identified proteins or polypeptides from step b) in the presence of MAPK and the substrate of MAPK, wherein phosphorylation of any of said critical residues in the MAPK substrates is an indication that said protein or polypeptide is an intermediate adapter/coupler of MAPK substrates. The nucleic acid molecules and the amino acid or polypeptide sequences of the invention, may advantageously be used in the treatment of the human or animal body.

The present invention also provides a method for controlling Ras/MAPK mediated signalling in a cell, comprising altering the levels of activity or expression of an intermediate adapter/coupler protein which binds to a substrate of MAPK, such as an ets DNA binding transcription factor, in said cell and which adapter/coupler polypeptide mediates phosphorylation of MAPK substrates by MAPK upon binding of said adapter/coupler polypeptide to said substrate.

MAPK, as aforementioned, is a generally ubiquitous protein, that is involved in phosphorylating nuclear transcription factors to regulate cellular processes and development. Aberrant levels of MAPK in a cell have been found to be associated with a number of disease states. Elevated levels of MAPK, for example, have been identified in cancerous cells. In view of the fact that MAPK is involved in the regulation of many cellular processes, it is not desirable to target MAPK in methods for alleviating any specific disease or condition. However, the findings by the present inventors now present the possibility of treating or alleviating the symptoms of specific diseases associated with aberrant expression of MAPK. For example, by inhibiting the expression or activity of the adapter/coupler polypeptide that has been found to mediate the MAPK phosphorylation of the MAPK substrates, such as nuclear transcription factor(s), that are themselves found to be responsible for mediating the diseased condition in those conditions where MAPK has been found to elevated, this should selectively target those cellular pathways that are involved and thus aid in treating the disease or condition. When, for example, a particular transcription factor is identified that is phosphorylated by or otherwise modulated by MAPK and which plays a role in the disease condition, preventing or inhibiting its phosphorylation may now be performed by selectively targeting the adapter polypeptide. It is likely that a whole family or group of such adapter proteins exist which are specific for individual MAPK substrates, including transcription factors. This, therefore, represents a highly selective target for developing therapies for controlling those conditions involving elevated or aberrant levels of MAPK. For example, the polypeptide according to the present invention, designated Mae is a component of the EGFR signalling pathway as EGFR, when stimulated, activates the MAPK pathway. Mutated/overexpressed EGFR receptor Erk has been implicated in cancer, particularly breast cancer. Accordingly, it is possible to specifically target the adapter/coupler protein that is selectively involved in mediating the phosphorylation by MAPK, to develop therapies for treating breast cancer. Other conditions which have been linked to aberrant levels of MAPK include, among others, cancer, such as prostate, pancreas, colon and lung cancer, myoproliferative disorders, leukemia, arthritis, allergies, diabetes, and host rejection of grafted tissue.

Ets transcription factors have been strongly implicated in tumorigenic growth. Therefore strategies that can specifically inhibit their function are of potential therapeutic value. Of particular interest are cancers, such as, for example, prostate cancer. In prostate cancer, prostate epithelial cells express a prostate specific Ets transcription factor that expresses a pointed domain (Foos G, Hauser CA Oncogene 2000 Nov 16;19(48):5507-16) and might therefore be a target of a human homologue of Mae. Therefore, advantageously it is possible to inhibit the growth of the cancerous prostate cells specifically by targeting the activity of the prostate specific Ets transcription factor by interfering with the activity of the Mae homologue. In this regard it has been shown that Mae protein physically associates with an isolated Drosophilia Ets transcription factor that is homologous to human prostate specific Ets.

Many cancers have mutations of the ras gene, such as cancers of the pancreas, lung and colon. This leads to expression of a constitutively activated form of Ras and which therefore must constitutively activate the activity of MAPK. These cancers therefore represent very good examples where treatments designed to specifically inhibit the activity of MAPK substrates such as the Ets transcription factors would be of great therapeutic value (Minamoto T, Mai M, Ronai Z Cancer Detect Prev 2000; 24(1): 1- 12).

The nucleic acid molecules or the polypeptides of the invention may also be included in a pharmaceutical composition together with any suitable pharmaceutically acceptable carrier diluent or excipient therefor. The nucleic acid molecule or polypeptides may be encapsulated and/or combined with suitable carriers in solid dosage forms for oral administration which would be well known to those of skill in the art or alternatively with suitable carriers for administration in an aerosol spray.

In the pharmaceutical composition of the invention, preferred compositions include pharmaceutically acceptable carriers including, for example, non-toxic salts, sterile water or the like. A suitable buffer may also be present allowing the compositions to be lyophilized and stored in sterile conditions prior to reconstitution by the addition of sterile water for subsequent administration. The carrier can also contain other pharmaceutically acceptable excipients for modifying other conditions such as pH, osmolarity, viscosity, sterility, lipophilicity, somobility or the like. Pharmaceutical compositions which permit sustained or delayed release following administration may also be used.

Furthermore, as would be appreciated by the skilled practitioner, the specific dosage regime may be calculated according to the body surface area of the patient or the volume of body space to be occupied, dependent on the particular route of administration to be used. The amount of the composition actually administered will, however, be determined by a medical practitioner based on the circumstances pertaining to the disorder to be treated, such as the severity of the symptoms, the age, weight and response of the individual.

The invention also contemplates gene therapy. This involves introduction in vitro of a functional copy of a gene into a cell(s) of a subject which contains a defective copy of the gene and returning the genetically engineered cell(s) to the subject. The functional copy of the gene is under operable control of regulatory elements which permit expression of the gene in the genetically engineered cells. Numerous transfection and transduction techniques as well as appropriate expression vectors for carrying out such procedures are well known in the art. In vivo gene therapy using plasmids or viral vectors, such as adenovirus, vaccina virus and the like, is also contemplated according to the invention.

In an even further aspect, the invention comprises a method of modulating activity/function of MAPK mediated ets DNA binding transcription factors, which method comprises inhibiting or enhancing expression or activity in a cell of a Mae polypeptide according to the invention. As described herein, numerous methods and techniques are available in the art for inhibiting expression or function of the polypeptide of the invention which would be known to the skilled practitioner. For example, increased expression of Mae may be achieved by transformation of a suitable expression vector incorporating the nucleic acid sequence of Mae, whereas inhibiting its function or expression may be accomplished using antisense techniques described herein or by using a blocking or binding protein. Furthermore, as would be well known to the skilled practitioner, other small molecules, such as binding peptides or polypeptides or other compounds may be synthesised or produced which can inhibit function or activity of the polypeptides of the invention. A further aspect of the invention comprises a method of treating a disease or condition in an individual which is associated with under expression or activity of a polypeptide according to the invention which method comprises administering to said individual a polypeptide as described herein or an expression vector according to the invention.

A further aspect comprises a method of diagnosing the medical significance of a polypeptide according to the invention in a disease condition, which comprises monitoring expression or activity levels of said polypeptide and/or the level of mRNA encoding said polypeptide, and comparing said levels to those which are found in a non-disease state.

In an even further aspect, the invention comprises a method for diagnosing or monitoring the progression of a disorder that is characterised by expression of a polypeptide of the invention, comprising contacting a biological sample isolated from a subject with an agent specific for the polypeptide and/or for mRNA coding for said polypeptide to detect the presence of the polypeptide or mRNA thereof in the biological sample.

An even further aspect comprises a method of identifying compounds capable of modulating activity of a polypeptide according to the invention comprising, i) contacting said polypeptide with said compound in the presence of MAPK and a substrate of MAPK which is capable of being phosphorylated in the presence of said polypeptide and MAPK, and ii) monitoring for any phosphorylation of said compound, wherein an increase or decrease in the phosphorylation profile of said substrate is indicative of said compound being capable of modulating activity/function of said polypeptide. Also provided is a method of identifying compounds capable of modulating activity of a polypeptide according to the invention comprising administering said compound to a transgenic cell, tissue or organism according to the invention, and monitoring the effect of said compound on said transgenic cell, tissue or organism compared to a cell tissue or organism that has not been contacted with said compound. An even further aspect of the invention comprises a method of producing a compound that modulates the activity or function of a polypeptide according to the invention, comprising i) synthesising the compound obtained or identified in the invention, or a physiologically acceptable analogue or derivative thereof, in an amount sufficient to provide said modulators in a therapeutically effective amount to a patient, and/or ii) combining the compound obtained or identified according to the invention or an analogue or derivative thereof, with a pharmaceutically acceptable carrier.

The compounds isolated by the above methods also form part of the invention and may be used in treating the human or animal body or in the manufacture of a medicament for treating any of cancer, such as prostate, lung, pancreas and colon cancer, or myoproliferative disorders, diabetes, arthritis, allergies, leukemia and host rejection of graft tissue.

The compounds identified may also, as would be appreciated by those of skill in the art, serve as lead compounds for the development of analogue compounds. The analogues should have a stabilized electronic configuration and molecular conformation that allows key functional groups to be presented to the polypeptides of the invention in substantially the same way as the lead compound. In particular, the analogue compounds have spatial electronic properties which are comparable to the binding region, but can be smaller molecules than the lead compound, frequently having a molecular weight below about 2 kD and preferably below about 1 kD. Identification of analogue compounds can be performed through use of techniques such as self- consistent field (SCF) analysis, configuration interaction (CI) analysis, and normal mode dynamics analysis. Computer programs for implementing these techniques are available; e.g., Rein, Computer-Assisted Modeling of Receptor-Ligand Interactions (Alan Liss, New York, 1989). Methods for the preparation of chemical derivatives and analogues are well known to those skilled in the art and are described in, for example, Beilstein, Handbook of Organic Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, New York, USA. Furthermore, said derivatives and analogues can be tested for their effects according to methods known in the art; see also supra. Furthermore, peptidomimetics and/or computer aided design of appropriate derivatives and analogues can be used.

The invention may be more clearly understood with reference to the following experimental details together with the accompanying Figures and which are purely exemplary, wherein:

Figure 1 is an illustration of Mae sequence and gene organisation, a) the full-length nucleotide sequence and b) corresponding translation of Mae. A cDNA clone of Mae was sequenced and shown to encompass the entire coding region and 400 bp of 3'UTR including the poly A tail. Mae is encoded by a single exon and contains a 4.3 kb intron in the 5'UTR. It is characterised by the presence of a pointed (Pnt) domain (underlined) and absence of a DNA binding ets domain. Comparison with known genomic sequences shows that Mae derives from the gene corresponding to the predicted ORF CGI 5085 (Berkeley Drosophila Genome Project) located at 55 E6 on chromosome 2R. c) Mae shares significant homology with Pnt- domain-containing Ets transcription factors. The Pnt-domains of representative family members are compared with Mae. Exact matches are shown in bold; similar amino acids are highlighted in grey. No significant matches were found outside the Pnt-domain. d) Schematic representation of Yan and Mae. Yan is a Drosophila Ets transcription factor. Yan functions downstream of several receptor tyrosine kinase (RTK) pathways where it regulates the differentiation of a wide variety of precursor cells^5"8. The Pnt-domain is marked as a hatched box at the N-terminus of Yan and at the C-terminus of Mae. The ets DNA-binding domain of Yan is shown as a black box, and positions of the 9 putative sites of phosphorylation by Erk {PX(S/T)P}, are indicated.

Figure 2. Physical association of Yan and Mae is mediated by their Pnt-domains and this inhibits Yan from binding to DNA. a,b) GST pulldown assays, a) 50 ng of the indicated GST- Yan fusions were incubated with either ³⁵S-labelled Mae (lanes 2-4) or ³⁵S-labelled Mae ^(G139>P) (lane 5). The GST-Yan fusions and bound Mae were eluted and run on a 15% denaturing polyacrylamide gel. Mae was detected by autoradiography and Yan by Coomassie blue staining, b) Similarly, GST-Pnt-P2 fusions (lanes 2 & 3) were incubated with either ³⁵S-labelled Mae (lane 2) or Mae ^(Gi ₃ ^9>p) fø^ ^ _c ^ Gei _mobility shift assays. An 80 bp probe containing a high affinity ets binding site ²⁷ at its centre was incubated for 20 minutes with each of the indicated purified GST-fusion proteins and the DNA-protein complexes resolved on a 5% acrylamide gel. c) In all cases 10 ng each of the indicated purified proteins were incubated immediately with the DNA probe, except in lane 7 where Yan and Mae were preincubated for 20 minutes before addition of the DNA probe. In lane 8, Yan was first mixed with the DNA probe for 20 minutes followed by the addition of Mae for a further 20 minutes, d) 10 ng of GST Yan was mixed with the indicated amounts of Mae protein, immediately incubated with the DNA probe and resolved on a gel as described above. In GST Yan, the full length Yan protein is fused in-frame with Glutathione-S-transferase. GST Yan ^(G84>P) is identical to GST Yan except that an invariant glycine residue at position 84 and in the Pnt-domain has been mutated to proline. In GST Yan ^(Δ46-¹⁰⁷⁾ the entire Pnt-domain of Yan is deleted. In Yan ^*ets2 invariant arginine residues R455 and R458 in the ets domain of Yan that are indispensable for binding to DNA , are mutated to glycine. In Yan all of the consensus MAPK phosphorylation sites have been mutated to alanine ⁶. In Mae^(G139>P), glycine 139 in the Pnt-domain of Mae, and in an equivalent position to glycine 84 of Yan, is mutated to proline. Figure 3. Mae allows Erk kinase to phosphorylate critical serine or threonine residues of Yan and Pnt-P2 and by this means regulates Ets transcriptional activity, a-d) Luciferase reporter assays of the activity of the Yan and Pnt transcription factors, a) Yan is an active repressor of transcription. The indicated constructs were cotransfected with a reporter construct that has 3 high affinity ets DNA binding sites cloned upstream of a thymidine kinase (TK) promoter that controls transcriptional activation of a luciferase reporter. In Yan ^Δets, the entire DNA binding ets domain has been deleted. b,c) Mae is required for Ras-dependent modulation of the transcriptional activities of Yan and Pnt-P2. b) The transcriptional activity of the indicated constructs were tested in combination with co-transfected Mae and constitutively activated ras (¹²H ras). c) Cells transfected with 400 ng of yan and the indicated amounts of Mae with or without ras. Experiments 3(a-c) were repeated at least 3 times with similar results, d) All of the constructs were fused-in-frame with the flag epitope, and equivalent expression in Cos-7 cells was shown by Western blotting with an ?-flag antibody. The presence of the flag tag had no effect upon the activity of any of the constructs in the assay (data not shown), e-f) Mae mediates the phosphorylation by Erk of Ser- 127 of Yan. e) 100 ng of the indicated GST-purified proteins were incubated with 100 ng of activated Erk (Calbiochem), ³²P γ ATP, with or without Mae. Erk alone phosphorylates wild type Yan, but not Yan^ACT or Yan^S127. The signal for Yan^S127 (lane 7) is weaker than that for wild type (lane 1), because the former reflects phosphorylation at a single site, f) Luciferase reporter assay of the Yan constructs used in Fig 3d. In Yan 4(S/T), a common Bsm I site has been utilised to construct a chimera in which the N-terminus of Yan is fused to the C-terminus of Yan^ΛCT. This construct retains the 4 most N-terminal consensus sites of Erk phosphorylation. Yan 3(S/T) contains the 3 most N-terminal consensus phosphorylation sites. Yan SI 27, retains only the most N-terminus consensus phosphorylation site. Yan S127^(G84>P) is identical to Yan SI 27 except that glycine 84 in the Pnt-domain of Yan, has been mutated to a proline (see Fig 2a). g, h) GST pulldown assays, g) Erk physically associated with Yan, Pnt-P2 and Mae. 50 ng of either GST Yan (lane 2), GST Pnt P2 (lane 3), GST Mae (lane 4) or GST alone (lane 5) were incubated with ³⁵S-labelled Erk. Bound Erk was eluted and run on a 12% denaturing polyacrylamide gel. h) Erk binding to Yan is weak by comparison with Mae binding to Yan. 50 ng of GST Yan (lanes 4-6) or GST alone (lane 3) was incubated with the indicated ³⁵S-labelled proteins. Bound Mae and Erk were eluted and run on a 15% denaturing polyacrylamide gel. ³⁵S-labelled Mae was prepared using Mae with a flat epitope. Two Mae protein species are translated as a result of alternate usage of the flag and Mae initiation codon. i) Mae but not Erk coimmunoprecipitates with Yan. Yan was fused in-frame with the flag epitope and Mae and Erk were each fused in-frame with the myc epitope. Yan was immunoprecipitated with α-flag antibody from lysates prepared form Cos 7 cells that were transfected with the indicated constructs (lanes 3-8). Any Yan-bound Mae and Erk were detected through Western blotting with an α-Myc antibody. In lanes (1 & 2) lysates prepared from cells transfected with either erk (lane 1) or Mae were Western blotted with an α-Myc antibody.

Figure 4. Evidence that Mae regulates EGFR signalling in the Drosophila embryonic neurectoderm. a-c) Ventral views of wild type embryos at a) stage 6/7, b) stage 9 and c) stage 11, showing Mae expression along the midline. d) Undetectable Mae expression in I(2)k06602 embryos (stage 9). e,f) l(2)k06602 embryos at e) stage 9 and f) stage 11, showing that the pattern of β-galactosidase expression resembles that of Mae. g, h) argos expression is inhibited in h) I(2)k06602/Df(2R)PC4 embryos but not g) I(2)k06602/+ embryos (or DF(2R)PC4X). i) rho expression is normal in l(2)k06602/Df(2R)PC4 embryos. The ventral midline is indicated by arrows with anterior to the left. The same results were obtained using the I(2)kl2907 Mae mutant fly line (data not shown), j) Ventral cuticle of I(2)kl 2907/Df(2R)PC4 larvae showing disrupted denticle patterning (arrowheads) around the midline in segments Al-4. Anterior is to the left.

To identify novel signalling intermediates, the present inventors isolated proteins that interact with the Drosophila Ets transcription factor Yan. Yan functions downstream of several receptor tyrosine kinase (RTK) pathways where it regulates the differentiation of a wide variety of precursor cells^5"8. Experiments in tissue culture cells suggest that Yan functions as a transcriptional repressor whose activity is rapidly attenuated by MAPK phosphorylation⁹.

Using full-length yan cDNA as bait, we conducted a yeast two-hybrid screen of an 18 hour Drosophila embryo library and isolated a 1 kb 3 '-terminal fragment of Mae which allowed us to recover the full length cDNA clone shown in Fig. la (see Methods). The only region of homology between Yan and the predicted Mae protein is a Pnt-domain located at the C-terminus (see Fig. 1). The Pnt-domain (also called the SAM domain) defines a sub-family of Ets proteins, including Ets-1, Ets-2, GABP, and TEL from vertebrates, and Drosophila Yan and Pnt-P2¹⁰. It is also found in Polycomb-group proteins where it has been shown to mediate protein-protein interactions, and in MEK kinases that are components of the MAP kinase cascade¹¹. We confirmed the biochemical interaction between Yan and Mae using a fusion of Yan to glutathione-S- transferase (GST) Fig. 2a shows that Mae binds to Yan (lane 2) and that the Pnt- domains of Yan and Mae are essential for this interaction. Thus, mutations that disrupt this domain (Yan ^G84>p, lane 4; Yan ^(46"107), lane 3; Mae^G139>p, lane 5) abrogate the binding of Mae to Yan. This is the first direct evidence that the Pnt-domain of Ets transcription factors mediates a heterotypic protein interaction.

As Yan represses transcription by binding to consensus Ets DNA-binding sites, the inventors tested whether Mae interferes with Yan binding in a gel mobility shift assay. Fig. 2c shows that Mae does not bind to the consensus Ets DNA binding site (lane 2) but it prevents Yan from doing so (compare lanes 3 and 7-9; see Fig 2d). This inhibition is mediated through the Pnt-domain dependent binding of Mae to Yan (compare lanes 7 & 10). DNA binding by Yan requires a functional Ets DNA binding domain (Fig 2c; lanes 3 and 5) but not intact phosphorylation sites or a functional Pnt- domain, because Yan^ACT (in which all 9 MAPK consensus sites are mutated to Ala⁶) binds the DNA site normally (Fig 2c; lane 4), as does Yan^G84>p in which an amino acid conserved in all Pnt-domains is mutated (Fig 2c; lane 6).

To determine whether Mae modulates transcriptional repression by Yan, the inventors first established a transcription assay in which Yan represses constitutive expression of a luciferase reporter gene. Because Drosophila S2 cells express Mae endogenously (data not shown), the inventors expressed Yan and Mae in Cos-7 cells. Fig 3 a shows that Yan represses the activity of a TK-luciferase reporter 10-fold. Repression depends on the Ets DNA binding-site (data not shown), and on the Ets DNA-binding domain in Yan (Fig 3 a). As expected, Mae alone does not affect reporter activity because it lacks an Ets DNA binding domain (Fig. 3 a).

RTK signalling downregulates Yan activity via phosphorylation mediated by Ras and the Erk/Rolled MAPK^6,9. Expression of a constitutively active form of Ras (¹²H-Ras) completely abrogates transcriptional repression by Yan but only in the presence of Mae (Fig 3b & 3c). Consequently, Mae is required for Ras to inactivate Yan in Cos-7 cells. An excess of Mae alone can reduce the repressive effect of Yan {~3 fold; (Fig 3b), presumably via formation of Mae/Yan heterodimers. However, low quantities of Mae that alone are ineffective at inhibiting the repressive effect of Yan (-30%), are sufficient to mediate the complete, Ras-dependent inactivation of Yan (Fig 3 c). Furthermore, the non-phosphorylatable Yan^ACT mutation is insensitive to Ras/Mae induced inhibition (Fig. 3b), indicating that Mae inhibits Yan by promoting its MAPK phosphorylation. Indeed Yan mutants that cannot bind Mae (Yan^G84>p, Yan ^(46"107); Fig. 3b), are resistant to inactivation by Ras/Mae.

Although Yan has nine sites of phosphorylation by MAPK, only one is vital for regulating its activity. Rebay & Rubin (1995) have shown that Ser- 127 is the critical regulatory site of Yan because mutation to Ala creates a constitutive repressor that is refractory to downregulation by Ras signalling. To test if Mae mediates phosphorylation of Ser-127 of Yan, the inventors examined in vitro phosphorylation of Yan by activated Erk (see Methods). Fig 3e, lanes 1 & 2 shows that phosphorylates wild-type Yan independently of Mae and as expected is unable to phosphorylate the constitutive repressor, Yan^' (Fig 3e; lane 2) which is mutated for all nine MAPK phosphorylation sites. Yan^sm in which all consensus phosphorylation sites except Ser- 127 are mutated to Ala, is resistant to phosphorylation by Erk alone (Fig. 3e; lane 4), even with very high amounts (up to 1 mig) of the kinase (data not shown). However, Yan^S127 is efficiently phosphorylated by Erk and Mae together (lane 7), indicating that Mae is indeed needed for phosphorylation of this Ser-127 residue. This phosphorylation depends on the Pnt-domain of Yan (Fig 3e; compare lanes 4 & 7 with lanes 6 & 8), indicating that the ability of Mae to promote Erk-directed phosphorylation of Ser-127 is dependent on binding of Mae to Yan.

Figure 3f shows that Yan^S127 represses transcription of the luciferase reporter as efficiently as the wild-type protein, and is also completely susceptible to inactivation by ¹²H-Ras/Mae. Therefore, Mae is required for MAPK to abolish transcriptional repression by Yan by mediating phosphorylation of the critical regulatory residue Ser- 127.

Inhibition of Yan repression by RTK signalling in Drosophila is accompanied by activation of a transcription factor encoded by the pointed (pnt) gene pnt encodes two alternative Ets transcription factors ; PI, a constitutively active transcriptional activator whose expression is induced by Ras/MAPK signalling, and P2, a transcriptional activator that like Yan, contains an N-terminal Pnt-domain and whose activity is stimulated through phosphorylation by the Ras/MAPK pathway¹³. Activation of Pnt-P2 by Erk also appears to depend on Mae; the two proteins physically associate and this interaction is dependent on the Pnt-domain (Fig. 2b; compare lanes 2 & 3). Pnt-P2 is a weak activator in our transcription assay (Fig. 3a) and is not stimulated by Ras alone (Fig. 3b). However, Ras and Mae together augment its transcriptional activity 4-fold (Fig. 3b). Thus, Mae also regulates Pnt-P2 activity, presumably by promoting its phosphorylation by MAPK.

These results show that Mae is needed in cultured cells to permit regulation of Ets transcription factor activity by Erk. This requirement was not evident in previous experiments on Yan because Drosphila S2 cells ⁹ were used in which endogenous Mae is expressed at high levels (unpublished).

To study Mae function in vivo, the present inventors used in situ hybridisation to examine its expression in early Drosophila embryos. In stage 6/7 embryos mae is expressed within bilaterally-symmetric anteroposterior stripe 3-4 cells wide flanking the ventral midline (Fig 4a). These stripes of expression narrow to a width of 2 cells and then 1 cell adjacent to the midline by stage 9 (Fig 4b) and stage 11 (Fig 4c), respectively.

This expression is very similar to the expression pattern of EGFR signalling regulators such as rhomboid (rho), vein, argos, yan and/wt^14'18 (see Fig 4g & 4i), and it marks the ventral neurectodermal zone that is patterned by EGFR signalling. Mae is subsequently expressed in tracheal pits, in ventral denticle domains and in areas such as the optic lobe and the medial domains of the brain (data not shown), all of which are also sites of EGFR activity¹⁹.

To investigate the in vivo role of mae further, we analysed the enhancer trap lines l(2)k06602²⁰ and l(2)k!2907^2X, which contain single P-element insertions that have been shown to be responsible for the lethal phenotype(see Methods)²¹. It is found that these P-elements insert into the 5' UTR of the mae gene {0.8kb upstream of the mae initiation codon in l(2)k06602, and 50 bp upstream of the Mae initiation codon of l(2)kl2907 (Supplementary Information; Berkely Fly Database)}. B(-galactosidase in early l(2)k06602 and l(2)kl2907 embryos is, like mae, expressed in the ventral neurectoderm, tracheal pits and ventral denticle belts (see Fig. 4e & f). Furthermore, homozygous mae mutant embryos (approximately 25% of embryos derived from either heterozygous l(2)k06602 or 1(2) kl 2907 parents) lack detectable mae expression (see Fig. 4d). These results indicate that the P element insertions disrupt development because they disrupt mae expression.

Embryonic patterning is affected in mae mutant embryos especially towards the midline of the interior abdominal segments. Ventral denticle belts are somewhat thinner and , around the midlne, denticle bands are missing or misorientated (Fig. 4j). mae^kl2907 and mae^k06602 embryos each show a similar phenotype when hemizygous over Df(2R)PC4, a deficiency for the region, indicating that the patterning defects result from lack of mae expression. Preferential disruption of midline patterning in anterior abdominal segments is reminiscent of the phenotype of pnt mutant larvae^22,23' although the complex regulation by EGFR signalling via MAPK of both pnt¹²¹³ and yan⁶⁹, makes detailed comparison difficult.

A role for Mae in EGFR signalling is further supported by a reduction of expression of argos, a downstream target of Pnt and Yan¹⁶'²⁴'²⁵, in mae mutant embryos (Fig. 4g & h). By contrast, expression oirho, the spatially restricted limiting component of EGFR signalling²⁶ is unaffected in mae mutant embryos (Fig 4i) indicating that the upstream elements of EGFR signalling are normal. Conversely, expression of both mae and argos are inhibited in rho mutant embryos (DAB, unpublished). In summary, these data suggest that Mae is both a target of and required for normal EGFR signalling in vivo.

The role of Mae in prostate cancer has been demonstrated by the inventors by the following. A novel Drosophilia ets transcription factor has been isolated that is homologous to human prostate specific ets. This factor is expressed in Drosophilia pole cells that ultimately give rise to Drosophilia germ cells. The mouse homologue of this gene is expressed in the sertoli cells which serve to regulate differentiation of spermatocytes into spermatozoa. Mae has been demonstrated to physically associate with the Drosophilia ets transcription factor. Therefore Mae can be targeted in order to treat prostate cancer and in particular to inhibit the growth of cancerous prostate cells.

Identification of the human homologue of mae

The skilled person will be aware of the various suitable techniques for identifying a homologue polypeptide of Mae in other organisms, such as humans. For example, the Drosophila DNA sequence (Fig la) can be used to obtain cDNAs encoding other homologues of Mae by cross-species hybridization techniques. An oligonucleotide probe may be created from the nucleotide sequence of Mae (Fig la) by standard techniques, such as those described in Sambrook et al, Molecular Biology: A Laboratory Manual, 1989. The probe may then be used to screen a mammalian, such as human, cDNA library or genomic library under moderate stringency conditions.

A search of the EST databases can be performed for human sequences that might be used as probes in order to isolate a human homologue from cDNA libraries. In the first instance such probes may be used to perform Northern hybridisations of different tissues to identify mae expression and choose the appropriate cDNA library to screen.

In the absence of an appropriate human sequence, a search for EST sequences from other species can be performed as an intermediate step. Once any sequence located is identified as being a mae homologue, degenerate primers can be designed which are centred in and around the Mae pointed domain and which can be used to amplify up a human mae probe from cDNA prepared from the RNA of different human tissues. This probe can then be used to screen human cDNA libraries, as described above.

Conclusions

The above experiments show that Mae mediates inactivation of Yan by MAPK phosphorylation of the critical Ser-127 residue. Presumably, binding of Mae causes a local change in conformation of Yan which exposes Ser-127 to MAPK. Further evidence for this view is that Mae-binding to the Pnt-domain in Yan also affects the latter' s ability to bind DNA via its Ets domain(Fig. 2b). Erk can associated with Mae and Yan in GST pulldown assays (Fig. 3g). However, unlike Mae-binding to Yan this interaction is weak and is not evident in co-precipitation assays (Fig. 3h & I; Supplementary Information. It is therefore suggested that Mae-binding to Yan or Pnt- P2 allows MAPK to phosphorylate their critical residues in a transitory ternary enzyme: substrate complex.

This is the first report of a requirement for an intermediary protein linking MAPK to its substrate, and suggests a novel mechanism for achieving tissue-specific responses to the generic RTK/Ras/MAPK signalling pathways , MAPK substrate determination by tissue-specific adapter/coupling proteins. Cells will respond differently to MAPK according to the adapter/coupling proteins they express.

Materials and Methods Yeast 2-hybrid and cDNA cloning

Full length yan cDNA was cloned into pAS2-l (Clontech) in-frame with the GAL4 DNA binding domain. 5 x 10³ independent transformants from a GAL4 activation domain Drosophila embryo library were screened in Hf7c yeast cells according to the manufacturer's protocol (Clontech). Specificity of interaction was confirmed by transforming Yeast with the cDNA of positive clones either in the presence or the absence of the yan bait or an unrelated bait. By these means a partial mae cDNA was isolated, and used to screen a λGTl 11-18 hour Drosophila embryo cDNA library for a complete mae cDNA. Molecular biology and Biochemistry Mutagenesis was carried out using the Transformer site-directed mutagenesis kit (Clontech). cDNA fragments were cloned into pGEX -2TK and GST-fusion proteins purified from BL21 E. coli using glutathione sepharose beads. For pulldown assays Mae proteins were labelled with ³⁵S methionine using the TNT-coupled reticulocyte in vitro translation system (Promega), and incubated with GST- Yan fusions that were immobilised onto glutathione sepharose beads. For gel mobility shift assays, proteins were incubated with 0.1 ng of ³²P-labelled probe for 20 minutes at 4 °C in the presence of 2 μg of poly dl/dC, 110 mM KC1, 10 mM HEPES, 5.5 MgCl₂, 5 mM Nab glycerophosphate, 0.05 mM EDTA, 0.05 mM spermine and 17.5 % glycerol. In vitro kinase assays were performed in a volume of 20 ml in 'kinase' buffer (10 uCi g³²P ATP, 25mM Na b- glycerophosphate, 20mM MOPS pH 7.2, lOmM MgCl₂, lOmM MnCl₂, 2mM NaF, lmM DTT and lmM NaVO ). Reactions proceeded for 30 minutes at 30°C and were resolved using SDS-PAGE. Yan and associated proteins were immunoprecipitated from cell lysates (1% Nonidet P40, 10% glycerol, 75mM NaCl, 20mM Tris pH 7.5) on to beads coated with flag antibody (Sigma) for 2 hours at 4°C. The beads were extensively washed, and associated proteins were eluted and resolved using SDS- PAGE. Western blotting was carried out as previously described²⁹.

Luciferase reporter assays Constructs were calcium phosphate transfected into 1 x 10⁵ Cos-7 cells growing in 2.4 cm wells in Dulbecco's Modified Eagles Medium without serum. Following a single change of medium 7 hours post-transfection, cells were lysed for analysis of luciferase activity after 16 hours of culture. We used 1 mg of reporter (modified pGL 3 vector- see Fig 3), and all other constructs were used at 400 ng. To ensure that we added equivalent amounts of DNA/well, the total amount of DNA to be transfected was adjusted using control vector. Equal transfection efficiencies were determined by co- transfection of the control pRL-TK vector and assay with Renilla luciferase (Promega). In every experiment each condition was done in triplicate. Drosophila genetics, in situ hybridisation and immunohistochemistry Stocks were obtained from Bloomington, Gerald Rubin and Matt Freeman, mae stocks were maintained using balancer chromosomes expressing GFP or wg-lac Z to allow selection of homozygous mutants. Cuticle preparations of mutant larvae were made according to {Wieschaus, E. & Nusslein-Volhard, C. in Drosophila, a practical approach (eds. Roberts, D.B), pp 199-226 (IRL Press, Oxford, 1986)}. Whole mount in situ hybridisation was carried out as described previously ³⁰ using digoxygenin-labelled (Roche) anti-sense RNA probes. Probes were derived from the following cDNAs: 1) 3'-most 800 bp of mae including 250 bp of 3' UTR, 2) 1.1 kb 5' Bam HI fragment of yan that excluded the ets DNA-binding domain, 3) Entire coding region of argos, 4) 2.5 kb fragment of rho encompassing the entire 1 kb coding region.

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Claims

1. An isolated or recombinant intermediate adapter/coupler polypeptide capable of mediating modulation or phosphorylation of a MAPK substrate.

2. An isolated or recombinant polypeptide which:

a) comprises the amino acid sequence shown in Figure lb; b) has one or more amino acid substitutions, deletions or insertions relative to the amino acid sequence given in a) above, which polypeptide is capable of mediating modulation or phosphorylation of a MAPK substrate; or c) is a fragment of a polypeptide as defined in a) or b) above, wherein the fragment is capable of mediating modulation or phosphorylation of a MAPK substrate.

3. A polypeptide as claimed in claim 1 or 2, which has substantial homology with the polypeptide having the amino acid sequence shown in Figure lb.

4. A polypeptide as claimed in claim 1 or 2, comprising an amino acid sequence which differs from that shown in Figure lb only in conservative amino acid changes.

5. A polypeptide as claimed in any of claims 1-4, which exhibits at least 30% homology with the amino acid sequence set forth in Figure lb.

6. A polypeptide as claimed in claim 5, which exhibits at least 50% homology with the amino acid sequence set forth in Figure lb.

7. A polypeptide as claimed in claim 6, which exhibits at least 60% homology with the amino acid sequence set forth in Figure lb.

8. A polypeptide as claimed in claim 7, which exhibits at least 70% homology with the amino acid sequence set forth in Figure lb.

9. A polypeptide as claimed in claim 8, which exhibits at least 80% homology with the amino acid sequence set forth in Figure lb.

10. A polypeptide as claimed in claim 9, which exhibits at least 90% homology with the amino acid sequence set forth in Figure lb.

11. An isolated or recombinant nucleic acid molecule which encodes a polypeptide according to any of claims 1-10.

12. An isolated or recombinant nucleic acid molecule which comprises or consists of: a) the nucleic acid sequence set forth in Figure la; b) a nucleic acid sequence having a codon sequence which differs from that of the codon sequence of a) due to the degeneracy of the genetic code; c) a nucleic acid sequence which is homologous to the sequence of a) or b); or d) a complement of a), b) or c).

13. An isolated or recombinant nucleic acid molecule which is capable of hybridising to a nucleic acid molecule as defined in claim 11 or claim 12.

14. An isolated or recombinant nucleic acid molecule which has a codon sequence which differs from that of the codon sequence of the nucleic acid molecule as defined in claim 13 due to the degeneracy of the genetic code.

15. An isolated or recombinant nucleic acid molecule which is complementary to the nucleic acid molecule claimed in claim 13 or claim 14.

16. A nucleic acid molecule as claimed in any of claims 11-15, wherein the nucleic acid molecule encodes an intermediate adapter/coupler polypeptide capable of mediating modulation or phosphorylation of a MAPK substrate.

17. A nucleic acid molecule as claimed in any of claims 11-16 which is a DNA molecule or a cDNA molecule.

18. An isolated nucleic acid molecule comprising a sequence of 10 or more consecutive nucleotides of the nucleotide sequence set forth in Figure la, or a complement thereof.

19. Use of a nucleic acid molecule as defined in any of claims 11-18 as a probe.

20. A vector comprising a nucleic acid molecule according to any of claims 11-17.

21. A host cell transformed or transfected with a vector as defined in claim 20.

22. A method for obtaining a polypeptide according to any of claims 1-10, comprising incubating a host according to claim 21 under conditions causing expression of the polypeptide and then obtaining the polypeptide.

23. A binding polypeptide which is capable of binding to the polypeptide of any one of claims 1-10.

24. A polypeptide as claimed in claim 23, wherein the polypeptide is an antibody.

25. A polypeptide according to any of claims 1-10 and 22-23 for use in the treatment of a human or non-human animal or for use in diagnosis.

26. Use of a binding polypeptide according to claim 23 or claim 24 in the manufacture of a medicament for treating a disease or condition mediated or associated with expression or function of a polypeptide according to any of claims 1-10.

27. A method of modulating activity/function of MAPK phosphorylated substrates which method comprises inhibiting or enhancing expression or activity in a cell of a polypeptide according to any of claims 1-10.

28. A method of treating a disease or condition in an individual associated with overexpression or activity of a polypeptide according to any of claims 1-10 and which polypeptide mediates MAPK phosphorylation of MAPK substrates, which method comprises administering to said individual a composition that selectively inhibits binding of said polypeptide to said MAPK substrate.

29. A method according to claim 27 or claim 28 wherein said MAPK substrate comprises a transcription factor.

30. A method according to claim 29 wherein said transcription factor is an Ets DNA binding transcription factor.

31. A method of treating a disease or condition in an individual which is associated with underexpression or activity of a polypeptide according to any of claims 1-10, which method comprises administering to said individual a polypeptide according to any of claims 1-10 or a vector according to claim 20.

32. An in vitro method of detecting expression of a polypeptide according to any of claims 1-10 or the mRNA therefor, which method comprises contacting a sample of tissue, cells or cell lysates from a subject with a binding polypeptide according to any of claims 1-10, 23 or 24 or a paratope of an antibody of claim 24 or an agent capable of identifying an mRNA of a polypeptide of claims 1-10, and detecting any binding of said binding polypeptide or paratope to a polypeptide in the sample or detecting the presence of any mRNA in the sample.

33. A kit for detecting expression of a polypeptide according to any of claims 1-10 comprising a binding polypeptide according to claim 23 or claim 24, means for contacting said binding polypeptide with a sample to be tested, and a means for detecting binding of said binding polypeptide with a polypeptide according to any of claims 1-10.

34. A method of diagnosing the medical significance of a polypeptide according to any of claims 1-10 in a disease condition, which comprises either monitoring expression or activity levels of said polypeptide or the level of mRNA encoding said polypeptide and comparing said levels to those which are found in cells from the same tissue in non- disease state.

35. A kit for diagnosing the medical significance of a polypeptide according to any of claims 1-10 in a disease condition, comprising one or more cells found in a diseased tissue and at least one control cell from the same tissue in a non-disease state, together with means for monitoring expression of said polypeptide or mRNA encoding said polypeptide in any of said cells.

36. A method of treating a disease or condition in a patient associated with overexpression of a polypeptide according to any of claims 1-10 which comprises administering to said patient a therapeutically effective amount of a molecule according to claim 13 or a binding polypeptide according to any of claims 23-24 or a blocking peptide.

37. A pharmaceutical composition comprising any of a polypeptide according to any of claims 1-10, a nucleic acid molecule according to any of claims 11-18, a binding polypeptide according to any of claims 23-24, or other small molecules capable of inhibiting function or activity of a polypeptide according to any of claims 1-10 together with a pharmaceutically acceptable carrier, diluent or excipient therefor.

38. A method for controlling Ras/MAPK mediated signalling in a cell, comprising altering the levels of activity or expression of an intermediate adapter/coupler polypeptide which binds to a substrate of MAPK in said cell and which adapter/coupler polypeptide mediates modulation or phosphorylation of MAPK substrates by MAPK upon binding of said adapter/coupler polypeptide to said substrate.

39. A method according to claim 38 wherein said adapter protein comprises a Pnt- domain.

40. A method according to claim 38 wherein said substrate comprises an Ets transcription factor.

41. A method of treating or alleviating the symptoms of cancer, myoproliferative disorders, arthritis, diabetes, allergies or host rejection of graft tissue, which method comprises inhibiting expression or activity of one or more adapter/coupler proteins capable of mediating MAPK phosphorylation of MAPK substrates, including nuclear transcription factors, involved in progression of said disorders.

42. A method according to claim 41 wherein said disorder is leukemia or prostate, pancreas, breast, lung or colon cancer.

43. A transgenic cell, tissue or non-human organism transformed or transfected with a vector according to claim 20.

44. A transgenic non-human organism comprising a transgene capable of expressing a polypeptide according to any of claims 1-10.

45. A non-human organism wherein the endogenous gene coding for a polypeptide according to any of claims 1-10 is knocked out or inactivated.

46. An organism as claimed in claim 45, which is a mouse.

47. A method of treating a disease or condition associated with elevated levels of MAPK, comprising inhibiting expression or activity of an adapter/coupler protein which mediates activation or repression of a transcription factor which is associated with progression of the disease.

48. A method according to claim 47 wherein said disease or condition is any of cancer, myoproliferative disorders, diabetes, arthritis, allergies and host rejection of graft tissue.

49. A method according to claim 47 wherein said disease or condition is any of leukemia or prostate, lung, breast, pancreas or colon cancer.

49. A method according to any of claims 47-49, wherein said adaptor/coupler is a polypeptide according to any of claims 1-10.

51. A method of identifying compounds capable of modulating activity of a polypeptide according to any of claims 1-10 comprising, i) contacting said polypeptide with said compound in the presence of MAPK and a substrate of MAPK which is capable of being phosphorylated in the presence of said polypeptide and MAPK, and ii) monitoring for any phosphorylation of said compound, wherein an increase or decrease in the phosphorylation profile of said substrate is indicative of said compound being capable of modulating activity/function of said polypeptide.

52. A method according to claim 51 wherein said polypeptide is a polypeptide as defined in any of claims 1-10.

53. A method according to claim 51 or 52 wherein said MAPK is Erk kinase.

54. A method of identifying compounds capable of modulating activity of a polypeptide according to any of claims 1-10 comprising administering said compound to a transgenic cell, tissue or organism according to claim 43-46, and monitoring the effect on said transgenic cell, tissue or organism compared to said cell tissue or organism that has not been contacted with said compound.

55. A method of producing a compound that modulates the activity or function of a polypeptide according to any of claims 1-10, comprising i) synthesising the compound obtained or identified according to any of claims 51-54, or a physiologically acceptable analogue or derivative thereof, in an amount sufficient to provide said modulators in a therapeutically effective amount to a patient, and/or ii) combining the compound obtained or identified according to any of claims 51-54 or an analogue or derivative thereof, with a pharmaceutically acceptable carrier.

56. A compound which is capable of modulating activity or function of a polypeptide according to any of claims 1-10, obtainable according to the method of any of claims 51-54.

57. A compound according to claim 56 for use in treatment of the human or animal body.

58. Use of a compound according to claim 56 in the manufacture of a medicament for treating a disease or condition including cancer, myoproliferative disorders, arthritis, diabetes, allergies and host rejection of graft tissue.

59. Use according to claim 58 wherein said disease or condition is any of leukemia, prostate, pancreas, breast, colon or lung cancer.

60. A pharmaceutical composition comprising a compound according to claim 56 with a pharmaceutically acceptable carrier, diluent or excipient therefor.

61. A method of identifying an intermediate adapter/coupler protein of MAPK substrates, which method comprises a) identifying critical amino residues on MAPK substrates which cannot be phosphorylated in the presence of MAPK alone, b) identifying proteins or polypeptides that bind to said MAPK substrate, c) incubating any identified proteins or polypeptides from step b) in the presence of MAPK and the substrate of MAPK and wherein phosphorylation of any of said critical residues in the MAPK substrates is an indication that said protein or polypeptide of MAPK substrates is an intermediate adapter/coupler.

62. A kit for detecting for the presence of a molecule according to any of claims 11 , 12, or 16-18, comprising a nucleic acid molecule according to claim 13 and means for contacting said nucleic acid molecule with any nucleic acid molecule of a sample to be tested.

63. A substance, composition, method or use, substantially as hereinbefore described, with reference to any of the accompanying examples.