AU696939B2 - Production and use of map kinase phosphatases and encoding nucleic acid therefor - Google Patents

Description

WO 95/21923 PCT/GB95/00272 1 PRODUCTION AND USE OF MAP KINASE PHOSPHATASES AND ENCODING NUCLEIC ACID THEREFOR The present invention relates to phosphatases. In particular, it relates to polypeptides having MAP kinase phosphatase activity, encoding nucleic acid therefor, antibodies thereto, and methods of production and use of the phosphatases, encoding nucleic acid and antibodies.

It also relates to screens for substances which have an effect on phosphatase activity and screens for MAP kinase phosphatase polypeptides and genes. Additionally, it relates to methods of diagnosis and treatment for proliferative diseases involving loss of MAP kinase phosphatase function.

The mechanism by which extracellular signals for growth and differentiation are transmitted to the nucleus to alter gene expression is the subject of much current investigation. In many cases, the transduction of these signals requires the activities of key enzymes known generally as "Mitogen activated protein (MAP) kinases".

MAP kinase pathways have been implicated in a large number of signal transduction pathways. For instance, activation of MAP kinases has been observed during growth factor stimulation of DNA synthesis and during differentiation, secretion and stimulation of glycogen synthesis MAP kinase has been shown to phosphorylate and activate effector substrates such as the transcription factors c-jun and elk-1. For a summary r IR ~LBBB;~dBBprrmuaraaa~p WO 95/21923 PCT/GB95/00272 2 of MAP kinases and pathways in which they are known to be involved, see a review by Roger Davis MAP kinase is activated by phosphorylation on threonine and tyrosine by a dual specificity kinase, "MAP kinase kinase". This kinase kinase is in turn activated by phosphorylation by "MAP kinase kinase kinase", one form of which is the proto-oncogene c-raf. The activation of c-raf is not fully understood at present but apparently there is a requirement for an interaction with GTP-bound p21 ras protein The full picture of how MAP kinase pathways are switched off is as yet unclear. Down-regulation of MAP kinase activity by de-phosphorylation is likely to be of key importance. The human gene CL100 and its murine homologue 3CH134 (Charles et al, 1992) were originally discovered as genes whose transcription was stimulated by growth factors, oxidative stress and heat shock.

Subsequently, they were shown to encode polypeptides that have both serine/threonine and tyrosine phosphatase activity (5 This removal of phosphate from both threonine and tyrosine on MAP kinase is unusual. When expressed in vitro this gene product has been shown to be very specific for MAP kinase and leads to its inactivation. Co-expression of the murine gene 3CH134 and the erk2 MAP kinase isoform in mammalian cells leads to the dephosphorylation and inactivation of the MAP kinase Furthermore, it has been shown recently that this phosphatase gene can also block cellular DNA I- WO 95/21923 PCT/GB95/00272 3 synthesis induced by an activated version of the ras oncogene in rat embryo fibroblasts (51).

The present invention has resulted from the surprising discovery-of several new genes, each encoding a polypeptide implicated in MAP kinase regulatory systems.

For present purposes, the terms "Mitogen-activated protein kinase", "MAP kinase" and "MAPK" apply to protein kinases that are activated by dual phosphorylation on threonine and tyrosine. This may be in reponse to a wide array of stimuli. Different MAP kinases are activated in repsonse to different extracellular stimuli, including (depending on the MAPK) stress, osmotic stress, mating pheromone (in yeast), growth factors, TNF, IL-1 and LPS.

MAP kinases include SMK1, HOG1, MPK1, FUS3/KSS1, spkl, ERK1/ERK2, JNK/SAPK, p38. "MAP kinase phosphatase" activity or function is the ability to dephosphorylate one or preferably both of the threonine and tyrosine residues on a MAP kinase, which residues are phosphorylated in the activation of the MAP kinase. Put another way, MAP kinase phosphatases are capable of hydrolysing either or preferably both phosphothreonine and phosphotyrosine residues on a MAP kinase.

Signalling by protein tyrosine phosphatases (PTPs).

The mechanism by which extracellular signals for growth and differentiation are transmitted to the nucleus to alter gene expression is currently the subject of much I WO 95/21923 PCT/GB95/00272 4 investigation. Tyrosine phosphorylation plays a central role in these events and is regulated by opposing activities of kinases and phosphatases. Although phosphatases may act directly by dephosphorylation of protein tyrosine kinase (PTK) receptors, it can be envisaged that they dephosphorylate the signalling molecules downstream, since PTK receptors are regulated at least in part through internalisation.

Increasing attention has been focused on the expanding family of protein tyrosine phosphatases which can be categorised as receptor-like and nonreceptor molecules [10 12]. Deregulated expression of some nontransmembrane tyrosine phosphatases has been shown to affect cell growth and increase the proportion of multinucleated cells [13,14]. Evidence suggests that PTPs may function as negative regulators of cell proliferation [15,18].

This is supported by the observations that PTPase inhibitors are able transiently to substitute for growth factors and induce mitogenic response [19,20].

Furthermore, it has been demonstrated that overexpression of PTPs can revert the transformed phenotype of v-src and suppress subsequent transformation by both the oncogenes neu and v-erbB [21,22]. However, it is apparent that PTPs are able to promote growth stimulatory effects [23], so a critical balance must exist in the cell between these activities to ensure proper growth control.

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WO 95/21923 PCT/GB95/00272 Signal transduction pathways regulating MAP kinases A key element in signal transduction from an activated receptor tyrosine kinase to an intracellular response is now recognised to involve the family of MAPkinases, pathways implicated in many diverse cell types Two forms of MAP kinase have been purified from human fibroblasts with molecular weights P 42 "pk and p 44 mpk (ERK-2 and ERK-1 respectively), [241. Activation requires an ordered phosphorylation of a threonine and tyrosine located within the conserved kinase subdomain 8, (T183, Y185), [25,26].

The use of dominant-negative mutations and homology experiments in yeast have proved to be invaluable tools in the elucidation of this signal cascade. Evidence suggests that in response to growth factor activated PTKs, a pre-existing Grb2-SOS 1 complex binds to tyrosyl phosphorylated She through SH2 domains, thus recruiting Ras activator molecules to the plasma membrane [28,29]. Alternatively, Grb2 may directly interact with autophosphorylated receptors. Extensive studies support hypotheses that signals converge through Ras [30,32], and continue through the Ser/Thr kinase Raf [33,35]. The activation of Raf-1 is not fully understood at present but apparently there is a requirement for an interaction with GTP-bound p21 ras protein [36,37]. The use of oncogenic forms of Raf-1 have shown it to act as a putative MKKK [33-35], along with MEKK and c-Mos [39,40], which results in sequential activation of ~ss~B-a WI

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WO 95/21923 PCT/GB95/00272 6 Scr/Thr kinases MEK [24,41], which ultimately phosphorylate the MAP kinases, see Figure 1.

Recently several new members of the MAP kinase gene family have been discovered These kinases, called JNK and p38 are involved in a variety of cellular responses. JNK is activated by stress, Tumour Necrosis Factor (TNF), Interleukin-1 (IL-l) and ultra-violet (UV) light. p38 is activated by lipopolysaccharide (LPS).

Both kinases are related to the erk-type MAP kinases in chat they.are activated by phosphorylation on both tyrosine and threonine. Deactivation by phosphatases is indicated.

A novel subfamily of Protein Phosphatases Pathways have been defined involving cascades of protein phosphorylation capable of inducing a complex set of immediate early genes, functionally significant in cell cycle regulation and oncogenic transformation. An essential feature of these phosphorylation events is their reversibility, and indeed tyrosine phosphorylation is often transient. It is known that removal of phosphate from either threonine by PP2A or from tyrosine by CD45 results in loss of MAP kinase activity though how exactly this pathway is switched off in vivo is yet to be identified.

The present invention is founded on the discovery and isolation of several nucleic acid molecules encoding proteins which are related to the known MAP kinase -su le, I_ I WO 95/21923 PCT/GB95/00272 7 phosphatases. Using insight gained from specialist knowledge in the field, the inventors were able to design an investigative procedure which resulted in the obtention of the new genes. The actual procedure used is described in detail below.

The sequences of the polypeptides encoded by the novel nucleic acid sequences share a degree of homology with the sequence of the known MAP kinase phosphatase, CL100, which is sufficient for indication as phosphatases, particularly MAP kinase phosphatases. This is interesting and useful: MAP kinase phosphatases are likely to act as off switches for cell proliferation. The fact that there are multiple MAP kinase phosphatases suggests that there may be some specificity to the off switches. Activators of the MAP kinase phosphatases either general or for specific family members may be anti-proliferative agents.

Provision of nucleic acid encoding phosphatases enables screening for such activators. Loss of MAP kinase phosphatase activity by, for example, mutation may lead to uncontrolled cell proliferation. Hence, some of these genes may prove to be "tumour suppressor genes".

In addition to MAP kinase, the phosphatases may have novel substrates. These substrates may also be key regulators of cell proliferation and potential targets for intervention by drug inhibitors.

The provision of various MAP kinase phosphatases and encoding nucleic acid therefor enables the production of I WO 95/21923 PCT/GB95/00272 8 antibodies able to bind, or specific for, the phosphatase polypeptides. Such antibodies are useful in the determination of the presence of a phosphatase in a test sample, e.g. containing tissue or cellular material, for example to determine some abnormality in the level or nature of the polypeptide. Antibodies able to discriminate between normal and abnormal molecules may be used in a diagnostic or screening context, e.g. in the determination of the underlying cause of a proliferative disorder such as a tumour.

Similarly, nucleic acid probes may be used in screening nucleic acid from cells of an individual, for example to determine whether those cells contain the wild-type gene encoding a particular phosphatase, and if they do whether they are homozygous or heterozygous.

Since MAP kinase phosphatases are involved in deactivation of MAP kinases, they are likely to have tumour suppressor function such that the absence of wildtype may have adverse effects on control of cell proliferation and heterozygosity may predispose an individual to a proliferative disorder. Thus important clinical information may be obtained, enabling appropriate therapeutic action to be taken.

Nucleic acid encoding a MAP kinase phosphatase may be used in a therapeutic context to counter the effect of loss of normal MAP kinase phosphatase activity in cells.

Loss of such activity, which may be total or partial, may lead to a proliferative disorder wherein normal I-I. -eI WO 95/21923 PCT/GB95/00272 9 regulation of cell growth is disrupted. Uncontrolled cell growth, i.e. cell growth which is not properly controlled, is involved in numerous disorders, both malignant (cancer) and benign. Gene therapy using one or more MAP kinase phosphatase-encoding nucleic acid molecules may be used in amelioration of disorders resulting from a loss of normal MAP kinase phosphatase activity.

Sequence information is presented in the accompanying figures, discussed below, along with experimental protocols and results demonstrating MAP kinase phosphatase activity. As of 9 February 1995, none of the sequences are present in the EMBL/GENBANK database.

According to one aspect of the present invention there is provided a nucleic acid molecule comprising a sequence of nucleotides encoding a polypeptide which comprises a sequence of amino acids encoded by nucleic acid with any one of the encoding sequences shown in Figure 2. The nucleic acid molecule may comprise any of the sequences shown in Figure 2 or may comprise a sequence which is a mutant, derivative or allele of the sequences shown. The sequence may differ from any of those shown by a change which is addition, insertion, deletion or substitution of one or more nucleotides of any of the sequences shown. Changes to a nucleotide sequence may result in an amino acid change at the protein level, or not, as determined by the genetic code.

WO 95/21923 PCT/GB95/00272 Thus, nucleic acid according to the present invention may comprise a sequence different from any of the sequences shown in Figure 2, yet encode a polypeptide with the same amino acid sequence as any of those shown sequences. On the other hand the encoded polypeptide may comprise an amino acid sequence which differs by one or more amino acid residues from any of those encoded by the encoding sequences shown in Figure 2.

Also provided by the present invention are a vector comprising nucleic acid as set out above, particularly any expression vector from which the encoded polypeptide can be expressed under appropriate conditions, and a host cell containing any such vector or nucleic acid. An expression vector in this context is a nucleic acid molecule comprising nucleic acid encoding a polypeptide of interest and appropriate regulatory sequences for expression of the polypeptide, either in an in vitro expression system, e.g. reticulocyte lysate, or in vivo, e.g. in eukaryotic cells such as COS or CHO cells or in prokaryotic cells such as E. coli.

Nucleic acid according to the present invention may be isolated (an "isolate") in the sense of being removed from its natural environment, or free from other nucleic acid obtainable from the same species encoding another polypeptide). Of course, nucleic acid according to the present invention may be wholly or partially synthetic.

The present invention also provides a polypeptide 9 P WO 95/21923 PCT/GB95/00272 11 having MAP kinase phosphatase activity and comprising a sequence of amino acids encoded by nucleic acid with any one of the encoding sequences shown in Figure 2. Amino acid sequences encoded by the sequences of Figure 2 appear in Figure 3.

Variants, mutants or derivatives of these polypeptides, especially but not necessarily those which retain MAP kinase phosphatase activity, (eg variants resulting from insertion, deletion or substitution of one or more amino acids) are also encompassed by the present invention. Variant, mutant or derivative polypeptides lacking MAP kinase phosphatase activity may be useful, particularly if they retain ability to interact or bind with MAP kinase. For example, tyrosine and dual specificity phosphatases have a cysteine residue located at the active site. Alteration of this cysteine to a serine in CL100 and its murine homolog 3CH134 leads to the abolition of catalytic activity However, expression of this catalytically dead form leads to an increase in the phosphorylation of MAP kinase (ERK2).

The mutant/derivative forms a specific complex with ERK2 MAP kinase. Presumably this association blocks dephosphorylation of ERK2 by endogenous CL100/3CH134.

Thus, also provided by the present invention is a polypeptide comprising an amino acid sequence which comprises an allele, derivative or mutant, by way of addition, insertion, deletiol or substitution of one or more amino acids, of an amino acid sequence encoded by WO 95/21923 PCT/GB95/00272 12 nucleic acid with any one of the encoding sequences shown in Figure 2.

A derivative is a substance derivable from a polypeptide. The derivative may differ from a polypeptide from which it may be derived by the addition, deletion, substitution or insertion of one or more amino acids, or the lnkage or fusion of other molecules to the polypeptide. Changes such as addition, deletion, substitution or insertion may be made at the nucleotide or protein level.

The provision of amino acid and nucleic acid sequence information for various polypeptides with MAP kinase phosphatase activity, the first demonstration that a family of such polypeptides exists, enables the obtention of other polypeptides and encoding nucleic acid therefor having a significant degree homology to the sequences given herein. Such homology might be greater than about 70%, preferably greater than about 80%, more preferably greater than about 85% or about 90% and most preferably greater than about 95%. Homology between orthologues, that the equivalent sequence in different species, is very high, while homology between sequences of a given species varies somewhat, as illustrated herein.

According to a further aspect of the present invention there is provided a polypeptide which has an amino acid sequence which is homologous to a sequence of amino acids encoded by nucleic acid with any one of the

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WO 95/21923 PCT/GB95/00272 13 encoding sequences shown in Figure 2, and which has MAP kinase phosphatase activity but is not CL100, or an orthologue thereof. As discussed, the present invention provides the first demonstration that a multitute of MAP kinase phosphates exist. Previously, only CL100 and its mouse orthologue 3CH134 were known as MAP kinase phosphatases obtainable from mammals. Prior to the making of the present invention PAC-1 (42) had been identified as a T-cell specific protein. Since then, this has been found to have MAP kinase phosphatase activity. Accordingly, PAC-1, nucleic acid encoding it, and so on may also be excluded from the present invention. In one embodiment of the present invention the homologous polypeptide is an orthologue of a polypeptide comprising an amino acid sequence encoded by a nucleotide sequence shown in Figure 2.

Of course, the present invention extends to variant polypeptides of those naturally occuring polypeptides homologous to those encoded by sequences shown in Figure 2, for example alleles, derivatives or mutants, wherein there is addition, insertion, deletion or substitution of one or more amino acids. These may or may not have MAP kinase phosphatase activity, but preferably are at least able to interact with or bind to a MAP kinase.

A convenient way of producing a polypeptide according to the present invention is to express nucleic acid encoding it.

Accordingly, the present invention also encompasses WO 95/21923 PCT/GB95/00272 14 a method of making a polypeptide which has MAP kinase phoaphatase activity, the method comprising expression from a vector which comprises nucleic acid encoding the polypeptide, the nucleic acid comprising nucleic acid encoding a polypeptide comprising an amino acid sequence encoded by an encoding nucleotide sequence shown in Figure 2. This may conveniently be achieved by growing a host cell, containing such a vector, under conditions which cause or allow expression of the polypeptide.

Polypeptides may also be expressed in in vitro systems, such as reticulocyte lysate. Alleles, derivatives, variants and mutants may be expressed likewise.

Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.

Suitable host cells include bacteria, eukaryotic cells such as mammalian cells and yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others. A common, preferred bacterial host is E. coli.

Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor WO 95/21923 PCT/GB95/00272 Laboratory Press. Transformation procedures depend on the host used, but are well known.

The proteins provided by the present invention may be purified from natural sources, or being produced recombinantly. Such purified proteins and methods of their purification, from natural sources or recombinantly produced, are encompassed by the present invention.

The provision of novel polypeptides enables for the first time the production of antibodies able to bind them. Accordingly, a further aspect of the present invention provides an antibody able to bind a polypeptide disclosed herein. Such an antibody may be specific for one or more of the polypeptides, in the sense of being able to distinguish between a polypeptide it is able to bind and other MAP kinase phosphatases which it is either not able to bind or which it binds more weakly. Other antibodies according to the present invention are able to bind MAP kinase phosphatases generally.

Preferred antibodies according to the invention are isolated, in the sense of being free from contaminants such as antibodies able to bind other polypeptides and/or free of serum components. Monoclonal antibodies are preferred for some purposes, though polyclonal antibodies are within the scope of the present invention.

Antibodies may be obtained using techniques which are standard in the art. Methods of producing antibodies include immunising a mammal (eg mouse, rat, rabbit, horse, goat, sheep or monkey) with the protein or a 9 IP WO 95/21923 PCT/GB95/00272 16 fragment thereof. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to antigen of interest. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al, 1992, Nature 357: 80-82).

As an alternative or supplement to immunising a mammal with a peptide, an antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, eg using lambda bacteriophage or filamentous bacteriophage which display functional immunoglobulin binding domains on their surfaces; for instance see W092/01047. The library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with any of the proteins (or fragments), or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest.

Antibodies according to the present invention may be modified in a number of ways. Indeed the term "antibody" should be construed as covering any binding substance having a binding domain with the required specificity.

Thus the invention covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, including synthetic molecules and molecules whose shape mimicks that of an antibody enabling it to bind an antigen or epitope.

Example antibody fragments, capable of binding an WO 95/21923 PCT/GB95/00272 17 antigen or other binding partner are the Fab fragment consisting of the VL, VH, Cl and CHI domains; the Fd fragment consisting of the VH and CHI domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; the dAb fragment which consists of a VH domain; isolated CDR regions and F(ab')2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulphide bridge at the hinge region. Single chain Fv fragments are also included.

A hybridoma producing a monoclonal antibody according to the present invention may be subject to genetic mutation or other changes. It will further be understood by those skilled in the art that a monoclonal antibody can be subjected to the techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the complementarity determining regions (CDRs), of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP184187A, GB 2188638A or EP-A-0239400. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP- A-0125023.

Hybridomas capable of producing antibody with desired binding characteristics are within the scope of the present invention, as are host cells, eukaryotic or WO 95/21923 PCT/GB95/00272 18 prokaryotic, containing nucleic acid encoding antibodies (including antibody fragments) and capable of their expression. The invention also provides methods of production of the antibodies comprising growing a cell capable of producing the antibody under conditions in which the antibody is produced, and preferably secreted.

Antibodies according to the present invention may be used in screening for the presence of a polypeptide, for example 4.i a test sample comprising.cells or cell lysate.

Similar proposals have been made for the known tumour suppressor gene retinoblastoma in W094/01467 and AU-A-52461/90).

For instance, a particular antibody may be able to distinguish between a wild-type polypeptide and a corresponding polypeptide with some difference in one or more epitopes as a result in a variation in amino acid sequence. The presence of a particular epitope may be indicative of a loss of MAP kinase phosphatase activity, or otherwise predictive of susceptibility or predisposition to a proliferative disorder. Similarly, antibodies may be used to determine the presence of wildtype polypeptide in cases wherein loss of expression of the polypeptide is predictive of poor patient prognosis or susceptibility to a proliferative disorder.

Antibodies may be used to determine whether cells of a tumour lack or have aberrerant MAP kinase phosphatase activity, e.g. because of mutation of the polypeptide or loss of expression of the polypeptide.

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WO 95/21923 PCT/GB95/00272 19 Antibody binding to a sample may conveniently be determined by employing a suitable labelling system for the antibody. Numerous approaches and labels are well known in the art, especially for immunoassays. Labelling may be direct or indirect. Detectable labels may be any substance having a physical or chemical property which may be detected, including enzymatic groups such as alkaline phosphatase and peroxidases, fluorescers, chromophores, luminescers and radioisotopes. Biotin and avidin/streptavidin systems may be employed.

Particularly suitable is the avidin-biotin compleximmunoperoxidase technique described by Cordon-Cardo et al (Amer. J. Pathol. 126: 269-284, 1987).

Antibodies according to the present invention may additionally be used in isolating or purifying any polypeptide as disclosed herein, including mutants, alleles, derivatives etc, according to standard techniques.

The new genes were isolated using a combination of PCR and low stringency hybridisation analysis. The primers used in the PCR had the sequences

TA(T,C)GA(T,C)CA(A,G)GG(A,G,T)GG(T,C,G,A)CC(A,T)GT(A,G,T)

GA and AT(G,C,T) CC(A,T)GC(T,C)TG(A,G) CA(A,G)TG(T,C,G,A)AC, and were designed based on amino acid sequences, YDQGGPVE and VHCQAGI conserved between human and mouse CL100 and the human PAC-1 gene (At the time of making the present invention, PAC-1 was not known to have MAP kinase ~0 1111~ WO 95/2'1923 PCT/GB95/00272 phosphatase activity.) A further aspect of the present invention provides an oligonucleotide with one of these sequences, a sequence complementary to one of these, for use in a method of obtaining nucleic acid encoding a protein with phosphatase activity, particularly MAP kinase phosphatase activity, comprising hybridisation of two primers to target nucleic acid. The hybridisation may be as part of a PCR procedure, or as part of a probing procedure not involving PCR. An example procedure would be a combination of PCR and low stringency hybridisation comparable to that used by the present inventors. A screening procedure, chosen from the many available to those skilled in the art, is used to identify successful hybridisation events and isolated hybridised nucleic acid.

The sequences provided in Figure 2 are themselves useful for identifying nucleic acid encoding other phosphatase proteins, such as those with MAP kinase phosphatase activity. Accordingly, the present invention provides a method of obtaining nucleic acid encoding a protein with phosphatase activity, particularly a protein with MAP kinase phosphatase activity, the method comprising hybridisation of a probe having any of the sequences shown in Figure 2 or a complementary sequence, to target nucleic acid. Hybridisation is generally followed by identification of successful hybridisation and isolation of nucleic acid which has hybridised to the

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WO 95/21923 PCT/GB95/00272 21 probe. The method may involve one or more steps of PCR.

It will not always be necessary to use a probe with one of the complete sequences shown in the figures.

Shorter fragments, particularly fragments with a sequence conserved between two or more of the sequences, may be used. Nucleic acid which has some alteration, eg insertion, deletion or substitution of one or more nucleotides, in the sequence will be useful, provided the degree of homology with one of the sequence given is sufficiently high.

A nucleic acid probe with a sequence selected from:

TA(T,C)GA(T,C)CA(A,G)GG(A,G,T)GG(T,C,G,A)CC(A,T)

GT(A,G,T)GA;

(ii) AT(G,C,T) CC(A,T)GC(T,C)TG(A,G)CA(A,G)TG(T,C,G,A)AC; (iii) a sequence complementary to or (ii); (iv) any one of the nucleotide sequences shown in Figure 2; a nucleotide sequence complementary to any one of the sequences shown in Figure 2; (vi) a nucleotide sequence which comprises an allele, derivative or mutant, by way of addition, insertion, deletion or substitution of one or more nucleotides, of any one of the sequences shown in Figure 3, or a nucleot.de sequence complementary thereto; and (vii) a nucleotide sequence which is a fragment of any one of (vi) and (vii); may equally be used in a method of screening cells for the presence of nucleic acid encoding a polypeptide WO 95/21923 PCT/GB95/00272 22 comprising a sequence of amino acids encoded by nucleic acid with any one of the encoding sequences shown in Figure 2, or an allele, derivative or mutant, by way of addition, insertion, deletion or substitution of one or more nucleotides, of any one of the encoding sequences shown in Figure 2, the method comprising hybridising such a nucleic acid probe to a sample of nucleic acid of.the cells and determining binding of the probe to the sample.

Where the nucleic acid is double-stranded DNA, hybridisation will generally be preceded by denaturing to produce single-stranded DNA. Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells of interest from normal, suspect or tumour tissue) and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturationg and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined.

Binding of a probe to target nucleic acid DNA) may be measured using any of a variety of techniques at the disposal of those skilled in the art. For instance, probes may be radioactively, fluorescently or enzymatically labelled. Other methods not employing labelling of probe include examination of restriction fragment length polymorphisms, amplification using PCR, RNAase cleavage and allele specific oligonucleotide probing.

WO 95/21923 PCT/GB95/00272 23 Abnormalities in binding of the probe to target DNA from an individual's cells may indicate that the individual is susceptible to a cell proliferation disorder arising from aberrant MAP kinase phosphatase function. Thus, individuals may be screened for the presence of one or more copies of a MAP kinase phosphatase gene to assist in asseiaing likelihood of developing a disorder involving uncontrolled cell proliferation. The screening may be prenatal.

Similarly, tumours may be identified as having resulted from a loss of MAP kinase phosphatase function if probe binding to nucleic acid from cells of the tumour does not match probe binding to nucleic acid from normal cells.

This may facilitate identification of appropriate therapy, for example involving manipulation of MAP kinase phosphatase activity in tumour cells (for which see below).

As primers, oligonucleotides may be used in PCR with cDNA derived from mRNA isolated from any tissue of human or animal or plant or microbial origin to amplify related genes which are likely to act as MAP kinase phosphatases.

In addition genomic DNA may also be amplified providing a method of accessing all MAP kinase related genes in the genome of, e.g. the human, mouse etc. The clones and fragments already isolated may be used to isolate further members of the gene family by low stringency hybridisation. Preliminary experiments may be performed by hybridising under low stringency conditions various I '--JII~ WO 95/21923 PCT/GB95/00272 24 probes to Southern blots of human DNA digested with restriction enzymes. Suitable conditions would be achieved when a large number of hybridising fragments were obtained while the background hybridisation was low.

Using these conditions cDNA libraries representative of expressed sequences in various human or animal or plant tissues or libraries made from genomic DNA of human or animal or plant or microbial origin. The screening of genomic libraries by this method.may lead to the isolation of all such homologous genes from the above species.

Where a full-length phosphatase encoding nucleic acid molecule has not been obtained, a smaller molecule representing part of the full molecule, such as those shown in Figure 2, may be used to obtain full-length clones. Inserts may be prepared from partial cDNA clones and used to screen cDNA libraries made from any of various human tissues. The full-length clones isolated may be subcloned into mammalian expression vectors and MAP kinase phosphatase activity assayed by cotransfection into COS cells, or other suitable host cells, with a reporter plasmid encoding MAP kinase or other substrate, or a suitable fragment thereof. MAP kinase has a different electrophoretic mobility depending on whether it is phosphorylated or not. The shift between the phosphorylated form to the dephosphorylated form in the presence of active MAP kinase phosphatase is detectable, eg using Western blotting.

WO 95/21923 PCT/GB95/00272 For instance, the MAP kinase of the reporter plasmid may be tagged, eg with a myc-epitope which allows detection using an anti-tag antibody. A suitable antimyc antibody is 9E10 Many ways of labelling proteins for detection/visualisation are known to those skilled in the art, so details need not be given here.

Indeed, anti-MAP kinase antibodies may be used, needing no label to be added to the protein. COS cells cotransfected with a labelled MAP kinase may be stimulated with EGF, which, in the absence of phosphatase activity, leads to a mobility shift in the MAP kinase which can be detected by means of the label. The presence of MAP kinase phosphatase activity within the cell leads to abolition of the mobility shift, thus providing a convenient assay for MAP kinase phosphatase activity, and enabling identification of encoding nucleic acid.

Other assays for MAP kinase phosphatase activity of a protein encoded by cloned nucleic acid may be used.

These include expression in a bacterial host, such as E.

coli, with a suitable tag or other label (eg a histidine tag or as glutathione-S-transferase fusion protein), followed by purification of the recombinant protein and subsequent incubation with recombinant phosphorylated MAP kinase. Dephosphorylation can be assayed by scintillation counting. Of course, recombinant production and purification of a MAP kinase phosphates for mixing with MAP kinase may be by any technique known in the art.

WO 95/21923 PCT/GB95/00272 26 A reticulocyte lysate in vitro translation system containing MAP kinase may be used, again to assay phosphorylation of the MAP kinase by mobility shift.

Candidate phosphatase clones may be translated in vitro in reticulocyte lysate and abolition of the mobility shift assayed.

The mobility shift may be used in the screening of effector molecules (activators or inhibitors) for a MAP kinase phosphatase, in any of the above assay systems.

For instance, an inhibitor of the MAP kinase phosphatase chosen for study will abolish or reduce the dephosphorylating activity of the phosphatase and so restore the mobility shift of MAP kinase or other substrate, where appropriate. Such screens are provided as an aspect of the present invention. Biochemical assays may be used to screen for effector molecules.

In any assay, a suitable fragment of MAP kinase (or any other substrate of the phosphatase of interest) may be use, eg a fragment including a site of action of the phosphatase.

A yeast two-hybrid system (43,44) may be used to identify molecules that interact with a phosphatase molecule. This system utilises a yeast containing a GAL4 responsive promoter linked to 3-galactosidase gene and to a gene (His3) that allows the yeast to grow in the absence of the amino acid histidine and to grow in the presence of the toxic compound 3-aminotriazole. The phosphatases may be cloned into yeast vectors that will WO 95/21923 PCT/GB95/00272 27 express these proteins as fusions with the DNA binding domain of GAL4. These yeast may then be transformed with cDNA libraries constructed from various human tissues in vectors designed to express proteins as GAL4 activator fusions. Yeast that have a blue colour on indicator plates (due to activation of -galactosidase) and will grow in the absence of histidine (and the presence of 3aminotriazole) may be selected and the library plasmid isolated. The library plasmid may encode a protein that can interact with the phosphatase. Such a protein may be a molecule which interacts with the phosphatase and modulates the activity. It also seems likely that substrates for the phosphatase other than MAP kinase may be isolated. These may be known non-classical non erk-type) MAP kinases or novel molecules involved in signal transduction.

The provision of MAP kinase phosphatases and encoding nucleic acid sequences therefor enables effector molecules to be screened using a novel yeast system, eg in Schizosaccharomyces pombe.

A phosphatase may be incorporated into the screening system that has been devised for the identification of molecules that can specifically inhibit components of the MAP kinase system (46 and W094/23039). In this system mammalian c-raf and MAP kinase have been introduced into the yeast S. pombe so that they can complement the sterility of yeast mutant in the Byrl or Byr2 genes.

These yeast genes are involved in the mating pathway.

WO 95/21923 PCT/GB95/00272 28 The mammalian genes are able to substitute for components of the pathway and can then be targeted. This screen may be adapted so that the activity of the mammalian enzymes (c-raf and MAP kinase) leads via activation of mammalian MAP kinase to the production of P-galactosidase enzyme so that these yeast will be blue on suitable indicator media. This system may be used to assay for substances that can specifically inhibit the mammalian signal transduction molecules by scoring for yeast that have lost their blue colour. The specificity of the screen is ensured as any non-specific kinase or other inhibitors would prevent growth of the yeast rather than simply loss of the blue colour. Other markers, such as the mating ability of the S. pombe strain, may be used.

Any MAP kinase phosphatase, including those provided herein and the known CL100, may be incorporated into this screening system. For instance, constitutive expression of a phosphatase may be manipulated by the use of suitable expression vectors to reduce partially the activity of the mammalian MAP kinase present in the yeast so that the 3-galactosidase activity is partially reduced, resulting in a diminution of the blue colour of the yeast on suitable indicator plates. This system may then be used to screen for compounds that can inhibit (leading to a stronger blue colour) or activate (attenuating the blue colour) a chosen phosphatase. Such compounds'may be useful therapeutic agents, for example antiproliferative or anti-inflammatory drugs.

a WO 95/21923 PCT/GB95/00272 29 It is well known that pharmaceutical research leading to the identification of a new drug generally involves the screening of very large numbers of candidate substances, both before, and even after, a lead compound has been found. This is one factor which makes pharmaceutical research very expensive and timeconsuming, so that a method for assisting in the screening process can have considerable commercial importance.

Of course, the marker used may be a simple "positive/negative" indicating the presence or absence of MAP kinase activity and so the respective absence or presence of MAP kinase phosphatase activity. The inhibition of the MAP kinase phosphatase by a test molecule (allowing MAP kinase activity) would then manifest as a positive result, eg blue colour, mating ability and so on, identifying the molecule as an.

inhibitor of the phosphatase.

It seems probable that the MAP kinase phosphatases act to switch off cellular proliferation. As such, loss of their enzyme activity by e.g. deletion or mutation may lead to uncontrolled cellular proliferation and cancer.

Several regions of the human genome have been described which show loss of heterozygosity, i.e. are deleted in various human tumours. Whether any of the phosphatase genes provided herein map near any of these regions of the human genome may be determined. Methods for the mapping of genes within the human genome are well known I- I- WO 95/21923 PCT/GB95/00272 and include analysis using specific PCR primers of the presence of genes in cell hybrids segregating human chromosomes as well as fluorescence in situ hybridisation (FISH). Any genes which are deleted in specific tumours, may be useful as reagents for the classification of such tumours and their diagnosis.

STY8 like sequences have been detected on human chromosomes lq and 8p using fluorescence in situ hybridisation (FISH). More detailed mapping and analysis of tumour material may be used to confirm tumour suppressor function.

As discussed, methods of diagnosis, comprising the use of any nucleic acid molecule with a sequence provided herein, or any fragment, mutant, allele or derivative thereof, are encompassed by the present invention.

Conveniently, this may involve use of specific PCR primers that recognise polymorphic regions of these genes or by using probes derived from these genes on Southern blots of DNA isolated from tumour material.

Also provided by the present invention are therapeutic methods employing MAF kinase phosphatase polypeptides, antibodies thereto or encoding nucleic acid therefor.

In principle, gene therapy using nucleic acid encoding a polypeptide with MAP kinase phosphatase activity may be used in treatment of any disorder which arises from a loss of wild-type MAP kinase phosphatase activity. Such disorders will generally be cell- -I WO 95/21923 PCT/GB95/00272 31 proliferative, involving inappropriate cell growth as a result of cellular pathways which normally regulate growth using a MAP kinase being "switched on" with the "off-switch", dephosphorylation of MAP kinase by a MAP kinase phosphatase, not being applied as normal.

Disorders of cell proliferation and growth may be benign or malignant.

Retinoblastoma is the "classic" example of a cell proliferative disorder which results from loss of function of a normally expressed gene. Individuals who are heterozygous for the Rb-1 gene, i.e. they have only one wild-type copy of the gene, are predisposed to get the disease because of the increased likelihood of a mutation leaving them with no working copy of the gene.

Gene therapy for retinoblastoma, using a nucleic acid construct comprising the Rb-1 gene, has been proposed in W091/15580 and W094/06910). Introduction of the Rb-1 gene into a tumour cell that has lost the Rb-1 gene results in suppression of growth of the tumour.

Significantly, although the tumorigenic phenotype was suppressed by the Rb-1 gene, it had no effect on normal cells.

p53 is another gene which has a "tumour suppressor function" and is an appropriate target for gene therapy.

In accordance with the present invention, gene therapy may be employed in the treatment of a disorder, especially a disorder of cell proliferation, involving loss of activity, total or partial, of a MAP kinase WO 95/21923 PCT/GB95/00272 32 phosphatase. A method of treatment practised on the human or animal body in accordance with the present invention may comprise administration of nucleic acid encoding a polypeptide which has MAP kinase phosphatase activity, as disclosed herein. Preferably, the nucleic acid forms part of a gene construct enabling expression within cells of the individual. Conveniently, the nucleic acid may be introduced into cells using a retroviral vector, preferably one which will not transform non-proliferating cells, or using liposome technology.

The treatment may be of existing disease, or it may be prophylactic. Prefventative treatment may be appropriate for individuals who have been identified as at risk of developing a disorder, e.g. because they lack two copies of a wild-type MAP kinase phosphatase gene or have mutated genes encoding a MAP kinase phosphatase with aberrant activity.

Administration is preferably in a "therapeutically effective amount", this being sufficient to show benefit to a patient. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated.

Prescription of treatment, eg decisions on dosage etc, is within the responsibility of general practioners and other medical doctors. Administration may be alone or in combination.with other treatments, either simultaneously or sequentially dependent upon the condition to be WO 95/21923 PCT/GB95/00272 33 treated.

Pharmaceutical compositions for administration in accordance with the present invention may comprise a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration, which may be in principle be oral, intranasal, topical, or by cutaneous, subcutaneous, intravenous or intramuscular injection.

Pharmaceutical compositions for oral administration may be in tablet, capsule, powder or liquid form. A tablet may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil.

Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection,

I

WO 95/21923 PCT/GB95/00272 34 Ringer's Injection, Lactated Ringer's Injection.

Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.

Injection may be used to deliver nucleic acid to disease sites, such as tumours. Internally, e.g. in internal organs, body cavities etc., suitable imaging devices may be employed to guide an injecting needle to the desired site.

In some proliferative diseases, such as those of the bone marrow, leukaemias etc., it may be desirable in certain cases to remove cells, including normal and tumour cells, from the body, treat them, then return them to the body.

Nucleic acid may be introduced locally into cells using transfection, electroporation, microinjection, lipsomes, lipofectin or as naked DNA or RNA, or using any other suitable technique. Retroviral vectors (Wilson et al., PNAS USA, 85: 3014, Gilboa (1982) J. Virology 44: 845 and Hocke (1986) Nature 320:275) and vaccinia viruses (Chakrabarty et al (1985) Mol. Cell Biol. 5: 3403) are amongst the choices available to those skilled in the art. Proliferating cells may be targeted especially using defective retroviruses lacking genes required for replication, since such retroviruses must rely on cellular DNA replication for integration of their genome and expression of polypeptides encoded therein. Such "replication incompetent" retrovirus vectors include those described by Chen et al (Science 250: 1576-80, I WO 95/21923 PCT/GB95/00272 1990) and Miller et al (BioTechniques 7: 980-990, 1989).

According to further aspects of the present invention there are provided a pharmaceutical composition, as disclosed, comprising nucleic acid encoding a polypeptide with MAP kinase phosphatase activity, as disclosed, for use in therapy or prophylaxis, especially of a cell-proliferative disorder, and the use of nucleic acid encoding a polypeptide which has MAP kinase phosphatase activity in the manufacture of a composition or medicament for use in treatment as disclosed.

Techniques of introduction of nucleic acid into mammalian cells may also be used to create transgenic animals, e.g. rodents such as mice, rats, hamsters etc., which carry one or more genes encoding defective, or at least altered, MAP kinase phosphatase. For instance, techniques involving homologous recombination may be used. The animals may have a germline or somatic mutated gene encoding a polypeptide with MAP kinase phosphatase activity. Such transgenic animals may be used in studying progression of disease involving a MAP kinase phosphatase or in screening or assessment of substances for therapeutic action, or testing of therapies, for example involving gene therapy wherein a wild-type gene may be introduced.

Aspects of the present invention will now be illustrated with reference to the accompanying figures, WO 95/21923 PCT/GB95/00272 36 by way of example and not limitation. Further aspects and embodiments will be apparent to those of ordinary skill in the art. All documents mentioned in the text are incorporated herein by reference.

In the figures: Figure 1 is a schematic diagram of the mammalian MAP kinase signal transduction pathway (from ref Ligand "interacts with a receptor tyrosine kinase at the cell surface. The receptor becomes phosphorylated on tyrosine residues which allows it to associate with GRB2 and the ras exchange factor SOS. SOS activates ras to the GTP bound form which is then able to interact with the MAP kinase kinase kinase raf. In a process which is not yet understood this activates raf so that it can phosphorylate MAP kinase kinase, which in turn phosphorylates MAP kinase on threonine and tyrosine residues. This activated form of MAP kinase can then stimulate cellular profileration.

Figure 2 shows DNA sequences of novel phosphatase molecules. STY2-STY4 are PCR products amplified from RNA produced from A431 cells as described in the text. STY and STY6 were isolated by screening a hamster liver cDNA library with a mixture of STY2 and STY3 probes shown in part and STY7-STY10 are parts of cDNA clones isolated by screening a human brain cDNA library with a mixture of STY2 and STY3 probes shown in part and All sequences apart from STY7 and STY10 show WO 95/21923 PCT/GS95/00272 37 homology to CL100. In the case of these clones the sequence shown does not show homology to CL100 but the cDNA clones hybridised strongly to the STY2/3 probe suggesting that these clones also encode novel phosphatase genes. STY2; STY3; STY4; STY5; STY6; STY7; STY8; STY9; Figure 3 shows deduced amino acid sequences of phosphatase clones aligned with the amino acid sequence of CL100: STY2, STY3, STY 4 AND STY5; STY6; STY 9; STY 8. For parts spaces indicate residues that are identical with CL100 and dots indicate residues which have not yet been determined.

For part which is a comparison of the full length clone for STY8 with CL100 dashes indicate gaps introduced into the sequences to optimise their alignment. Shaded residues correspond to residues that are identical between STY8 and CL100.

The amino acid sequences shown correspond to residues 177-255 for part 231-302 for part 223- 267 for part and 1-367 for part Figure 4 shows proof that STY8 encodes MAP kinase phosphatase activity. Protein extracts were prepared from COS cells transfected with various recombinant plasmids before or after stimulation of the cells with EGF. These extracts were electrophoresed on SDS/polyacrylamide gels and the-proteins then transferred to a nitrocellulose membrane. This membrane was then WO 95/21923 PCT/GB95/00272 38 incubated with the anti-myc antibody 9E10, treated by the ECL procedure and the resulting chemiluminescence detected on x-ray film. It can be seen that in the absence of stimulatory ligand (EGF) the anti-myc antibody 9E10 reveals only a single band of MAP kinase on western blotting (lane In the presence of EGF (lane 2) a clear doublet of bands is present indicating the partial phosphorylation of the MAP kinase. This is unaffected by expression of the parental expression vector (lanes 3 and However, expression of CL100 or STY8 in the presence of EGF (lanes 7-10) leads to abolition of the EGF induced shift indicating that both these molecules encode MAP kinase phosphatases. Lanes 5 and 6 in which the cells are transfected with Myc-tagged STY8 shows that the STY8 protein is indeed expressed. Lane 1 MAPK; Lane 2 MAPK EGF; Lane 3 MAPK pMT; Lane 4 MAPK pMT EGF; Lane 5 Myc-STY8; Lane 6 Myc-STY8 EGF; Lane 7 MAPK CL100; Lane 8 MAPK CL100 EGF; Lane 9 MAPK STY8; Lane 10 MAPK STY8 EGF.

Figure 5 shows the results of autoradiography of SDS-polyacrylamide gels on which samples containing (a) MAP kinase kinase and ERK2 or MAP kinase kinase, ERK2 and STY8 were incubated in the presence of "P-ATP for times indicated in minutes across the top of the lanes.

The positions of phosphorylated ERK2 and STY8 are indicated. The left hand lane shows molecular weight markers.

q WO 95/21923 PCITGB95/00272 39 Isolation of MAP kinase phosphatase encoding genes To identify related protein amino acids sequences human CL100 and its murine homologue 3CH134 and the human PAC-1 gene a related T cell specific gene of unknown function, were compared. It proved possible to design degenerate PCR primers, based on conserved regions of the proteins. These primers were used to amplify related sequences from cDNA made from poly(A)'RNA isolated from the human squamous cell line A431. A fragment of 270bp was purified and subcloned. Of fifty individual clones sequences six proved to be identical to CL100. A further twelve clones were found to be homologous to, but distinguishable from, CL100:- STY2 isolated six times End STY3 four times, with single isolates of STY4 and In order to identify further related genes, we screened human brain and liver cDNA libraries with a mixed probe from STf2 and-3 PCR products. Several hybridising clones were analysed in more detail by restriction endonuclease mapping and partial DNA sequencing. This resulted in the identification of several additional gene families, STY6with STY1 being CL100. In total nine new genes were identified and these are compared to amino acid sequences of CL100, see figure 3. The high degree of similarity of these genes suggested that they encode proteins with MAP kinase phosphatase activity.

Cell Culture and RNA Preparation A431 cells were grown in Dulbecco's modification of e I" I- I-- WO 95/21923 PCT/GB95/00272 Eagle's minimal essential medium (DMEM) supplemented with fetal calf serum. Total cellular RNA was prepared with RNAzolB(Promega) and poly(A)+RNA isolated with Dynabeads Isolation of CL100-related cDNAs Two degenerate oligonucleotides

TA(T,C)GA(T,C)CA(A,G)GG(A,G,T)GG(T,C,G,A)CC(A,T)GT(A,G,T)

GA and AT(G,C,T)CC(A,T)GC(T,C)TG(A,G)CA(A,G)TG(T,C,G,A)AC were designed based on amino acid sequences, YDQGGPVE and VHCQAGI conserved between human and mouse CL100 and the human PAC-1 gene. A431 poly RNA was reverse transcribed with SuperScript reverse transcriptase (BRL- GIBCO) and subject to PCR on a Techne PHC-1 thermal cycler with these oligonucleotides (47) under the following conditions 94 0 C for 30sec, 50 0 C, 30sec, 72 0 C 1 min. A 270bp band was purified by agarose gel electrophoresis and subcloned into pBluescript.

Fifty individual subclones were sequenced and of these six proved to be CL100. Twelve others were found to be homologous to-but not identical to CL100, and these were grouped as four different potential phosphatases, designated STY2'STY5 with CL100 being STY1. STY6-STY10 were isolated by screening cDNA libraries with a 32Plabelled probe made front the inserts of plasmids containing STY2 and STY3 sequence shown in Figure 2a.

STY6 was isolated from a human brain library.

WO 95'21923 PCT/GB95/00272 41 Structural Analysis of STY cDNAs One of the cDNA clones isolated from the human brain is full length. Colinear alignments of the STY genes with CL100 show that amino acids around the highly conserved catalytic domain differ, and two conserved regions between CL100 and cdc25 are also present in STY8.

Studies on the genomic structure of 3CH134 reveal that the transcription unit is 2.8kbp long and split into four exons It will be of interest to elucidate the genomic structure of the STY genes, and determine if their promoter regions contain consensus sequences for transcription factors. Preliminary studies suggest that STY8 has a similar gene structure to 3CH134.

Functional Assays The human CL100 and its murine counterpart 3CH134 function as immediate-early genes whose transcription is rapidly and transiently induced within minutes, with protein accumulation seen in the first hour upon growth factor stimulation As observed for the expression of several immediate-early genes, the rapid increase in growth factor receptor tyrosine kinase activity and subsequent activation of signalling molecules needs to return to normal levels to avoid abnormal growth. One method for accomplishing this implicates protein phosphatases whose expression is induced by external signals, such that they are present in the cell only under certain circumstances.

WO 95/21923 PCT/GB95/00272 42 Evidence indicates that when CL100 and 3CH134 are expressed in vitro or in vivo the gene product leads to selective dephosphorylation of p 42 apk blocking its activation by serum, oncogenic Ras, or activated Raf, whilst the catalytically inactive mutant of the phosphatase augments MAP kinase phosphorylation.

We tested whether the phosphatase STY8 exhibited similar specificity in vivo using a COS cell transient expression system. We cotransfected Cos cells with the reporter plasmid pEXV3-Myc-p42m ap together with various plasmids including pMT-Myc-STY8. Figure 4 is typical of such an experiment.

It can be seen that in the absence of stimulatory ligand (EGF) the anti-myc antibody 9E10 reveals only a single band of MAP kinase on western blotting (lane 1).

In the presence of EGF (lane 2) a clear doublet of bands is present indicating the partial phosphorylation of the MAP kinase. This is unaffected by expression of the parental expression vector (lanes 3 and However expression of CL100 or STY8 in the presence of EGF (lanes 7-10) leads to abolition of the EGF induced shift indicating that both these molecules encode MAP kinase phosphatases. Lanes 5 and 5 in which the cells are transfected with Myc-tagged STY8 shows that the STY8 protein is indeed expressed.

We have demonstrated that recombinant STY8 can dephosphorylate MAP kinase (erk2) when the two proteins are incubated in vitro. Recombinant activated MAP kinase I WO 95/21923 PCT/GB95/00272 43 kinase (MEK/EE) (50ng) was incubated with recombinant ERK2 (3xg) -or recombinant ERK2 (3jg) and recombinant STY8 (0.75Ag) in the presence of 32 P-ATP (2.5nCi) in 50mM Tris- C1 (pH 7.5)/0.1mM EGTA/lOmM MgAcetate/0.125mM ATP for the times indicated (in minutes) in Figure 5. Samples were then electrophoresed on a 10% SDS/polyacrylamide gel and the gel dried and autoradiographed-. The positions of phosphorylated ERK2 and (surprisingly) STY8 are shown in the Figure.

STY8 is shown by this experiment surprisingly to be itself a substrate of the MAP kinase erk2. This can be seen in Figure 5 by the appearance of the phosphorylated ST8 during the incubation with erk2. The significance of this phosphorylation is at present unknown but several potential sites for phosphorylation are located in the sequence of STY8. It is possible that phosphorylation of STY8 by erk2 serves to modulate its activity, either increasing or reducing it. Another possibility is that phosphorylation targets the protein for degradation. A further possibility is that the presence of potential MAP kinase phosphorylation sites within STY8 serves to facilitate the interaction of erk2 and STY8 and the consequence is phosphorylation of STY8 and dephosphorylation of erk2.

STY8 has also been cloned into a baculovirus transfer vector, resulting, after recombination, in production of a Glutathione-S-transferase (GST) STY8 fusion protein. The vector was co-transfected into I WO 95/21923 PCT/GB95/00272 44 insect cells along with linear viral genomic DNA.

Recombination resulted in production of viral particles containing baculovirus DNA having incorporated the expression vector. This virus was used to infect further insect cells and the recombinant GST-STY8 protein purified by affinity chromatography on glutathione agarose.

STY2 has been fully cloned and sequenced and shown to have MAP kinase phosphatase activity, i.e. the ability the dephosphorylate.a MAP kinase (ERK2) The sequence is related to CL100 and STY8 throughout, particularly in the catalytic domain.

The provision of the nucleic acid encoding MAP kinase phosphatases enables the following to be performed.

Expression Patterns of STY cDNAs Experiments have demonstrated that 3CH134 is expressed predominantly in the lung of the adult mouse We have shown through northern blot analysis that STY8/9 and CL100 are expressed ubiquitously across a range of tissues. Interestingly, the expression of 3CH134 corresponds to post-mitotic cells which suggests the phosphatase may play a role in cellular differentiation, acting as a negative effector of cell growth. We have also shown through microinjection that Ib arsl r~ 41- PAOPIERMROX586-.95.SPE 1215198 Myc-tagged STY8 is located in the nucleus of transfected cells. This is where a MAPK phosphatase might be expected to act in the light of evidence that reports MAP kinase activity is biphasic, with the second sustained peak correlating with nuclear translocation and initiation of DNA synthesis [49].

The MAP kinase pathway promotes cell proliferation and tumorigenesis, and so MAP kinase phosphatases may function as tumour suppressor genes. We intend to determine the chromosomal location of the STY genes using a chromosomal hybrid panel of hamster cells with single human chromosomes. These will be screened by PCR using unique primers to each of the STY genes, and the exact position identified using in situ hybridisation. (STY8 like sequences have been detected using flurescence in situ hybridisation on human chromosomes 1q and 8p.) These loci may then be compared with regions of known loss of heterozygosity in the BICR cell lines or that correspond to known tumour suppressor loci.

9 *9a09 15 Throughout this specification and the claims which follow, unless the context requires 90 otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

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Claims (23)

1. An isolated or recombinant polypeptide having MAP kinase phosphatase activity and comprising a sequence of amino acids encoded by nucleic acid with any one of the encoding sequences shown in Figure 2.

2. An isolated or recombinant polypeptide which has an amino acid sequence which shows at least 80% homology to a sequence of amino acids encoded by a nucleic acid molecule which comprises any one of the encoding sequences shown in Figure 2, 10 wherein said polypeptide has MAP kinase phosphatase activity but is not CL100, PAC-1, or an orthologue thereof. *4

3. The isolated or recombinant polypeptide according to claim 2 wherein said S•polypeptide is an orthologue of a polypeptide encoded by one or more nucleotide sequences set forth in Figure 2, subject to the proviso that said orthologue is not CL100, PAC-1 or an orthologue thereof.

4. An isolated nucleic acid molecule comprising a sequence of nucleotides which encodes a polypeptide having MAP kinase phosphatase activity, wherein said polypeptide o 20 comprises a sequence of amino acids encoded by any one of the encoding nucleotide sequences shown in Figure 2. The isolated nucleic acid molecule according to claim 5 comprising any one of the encoding nucleotide sequences shown in Figure 2.

6. An isolated nucleic acid molecule comprising a sequence of nucleotides which encodes a polypeptide which: has MAP kinase phosphatase activity; (ii) comprises a allele, derivative or mutant, by way of addition, insertion, R A^ deletion or substitution of one or more amino acids, of an amino acid sequence PAOPERW1ROU586.9S.CI.M -1318/98 -51- encoded by any one of the encoding sequences shown in Figure 2; and (iii) comprises an amino acid sequence which shows at least 80% homology to a polypeptide encoded by an encoding sequence shown in Figure 2; subject to the proviso that said polypeptide is not CL100, PAC-1, or an orthologue thereof.

7. An isolated nucleic acid molecule comprising a nucleotide sequence which encodes a polypeptide with MAP kinase phosphatase activity said nucleotide sequence being complementary to a nucleotide sequence which is hybridisable with any one of the 10 sequences shown in Figure 2.

8. A vector comprising the isolated nucleic acid molecule according to any one of claims 4 to 7 and one or more regulatory sequences for expression of a polypeptide encoded by said nucleic acid molecule.

9. A host cell comprising the isolated nucleic acid molecule according to any one of claims 4 to 7.

10. A method of making a polypeptide, which comprises expression from a vector 20 according to claim 8.

11. A host cell comprising the vector according to claim 8.

12. The host cell according to claim 11, wherein said host cell is a prokaryotic cell.

13. The host cell according to claim 11, wherein said host cell is a eukaryotic cell.

14. A method of making a polypeptide, which comprises growing the host cell according to any one of claims 11 to 13 under conditions which are sufficient for expression of 6 said polypeptide to occur. rN\OPERWMROIS86995.CLM 13/8/98 -52- An oligonucleotide having a sequence: TA(T,C)GA(T,C)CA(A,G)GG(A,G,T)GG(T,C,G,A,)CC(A,T)GT(A,G,T)GA; (ii) AT(G,C,T)CC(A,T)GC(T,C)TG(A,G)CA(A,G)TG(T,C,G,A)AC; or (iii) a sequence complementary to either of these sequences.

16. A method of obhaining a nucleic acid molecule which is capable of encoding a polypeptide with MAP kinase phosphatase activity, said method comprising the step of hybridizing the oligonucleotide according to claim 15, or a nucleic acid molecule 10 comprising said oligonucleotide, to a target nucleic acid molecule.

17. A method of obtaining a nucleic acid molecule which is capable of encoding a polypeptide with MAP kinase phosphatase activity, comprising the step of hybridizing 0*0*00 a nucleic acid molecule which comprises a nucleotide sequence selected from the list comprising: any one of the nucleotide sequences shown in Figure 2; (ii) a nucleotide sequence complementary to any one of the sequences shown in S" Figure 2; (iii) a nucleotide sequence which comprises an allele, derivative or mutant, by way 20 of addition, insertion, deletion or substitution of one or more nucleotides, of any one of the encoding sequences shown in Figure 2, and which encodes a polypeptide which shows at least 80% homology to the polypeptide encoded by said encoding sequence which polypeptide has MAP kinase phosphatase activity subject to the proviso that said polypeptide is not CL100, PAC-1 or an orthologue thereof; (iv) a nucleotide sequence complementary to (iii); and a nucleotide sequence which is a fragment of any one of (iii) or (iv); to a target nucleic acid molecule. /f 8. The method according to claims 16 or 17, further comprising the steps of identifying II w -e I a~ P:\OI'Pt\MRO\I586l.95.CLM. 13/8/98 -53- the hybridisation and isolating the hybridized target nucleic acid molecule.

19. The method according to any one of claims 16 to 18 involving use of the polymerase chain reaction (PCIR) A method of screening cells for the presence of a nucleotide sequence which corresponds to the nucleotide sequence of the isolated nucleic acid molecule according to any one of claims 4 to 7, wherein said method comprises the steps of: hybridising a nucleic acid probe to a nucleic acid sample derived from said 10 cells; and (ii) determining binding of the probe to the sample; Swherein said probe comprises a nucleotide sequence selected from the list: TA(T,C)GA(T,C)CA(A,G)GG(A,G,T)GG(T,C,G,A,)CC(A,T)GT(A,G,T)GA; (ii) AT(G,C,T)CC(A,T)GC(T,C)TG(A,G)CA(A,G)TG(T,C,G,A)AC; (iii) a sequence complementary to or (ii); (iv) any one of the nucleotide sequences shown in Figure 2; a nucleotide sequence which is complementary to any one of the sequences shown in Figure 2; (vi) a nucleotide sequence which comprises an allele, derivative or mutant, by way 20 of addition, insertion, deletion or substitution of one or more nucleotides, of any one of the encoding sequences shown in Figure 2, which encodes a polypeptide which shows at least 80% homology to the polypeptide encoded by said encoding sequence; (vii) a nucleotide sequence complementary to and (viii) a nucleotide sequence which is a fragment of any one of (vi) and (vii).

21. An antibody which is capable of binding specifically to the isolated or recombinant polypeptide according to any one of claims 1 to 3. I 9 F i P:\OPER\Mro\l5S61-9SCL1 3O)R -54-

22. The antibody according to claim 21 which is monoclonal.

23. The antibody according to claim 21 which is polyclonal.

24. The antibody according to any one of claims 21 to 23 when used to screen for the presence of a polypeptide which is substantially the same as the polypeptide according to any one of claims 1 to 3. The host cell according to any one of claims 9 or 11 to 13 substantially as 10 hereinbefore described with reference to the Figures and/or Examples.

26. The method according to any one of claims 16 to 19 substantially as hereinbefore described with reference to the Figures and/or Examples. 15 27. The method according to claim 20 substantially as hereinbefore described with reference to the Figures and/or Examples. *9

28. The isolated nucleic acid molecule according to any one of claims 4 to 7 substantially as hereinbefore described with reference to the Figures and/or Examples. 20

29. The isolated or recombinant polypeptide according to any one of claims 1 to 3 substantially as hereinbefore described with reference to the Figures and/or Examples. DATED this THIRTEENTH day of AUGUST, 1998. CANCER RESEARCH CAMPAIGN TECHNOLOGY LIMITED by DAVIES COLLISON CAVE Patent Attorneys for the Applicant I D-pl I$