WO1995021923A1 - Production and use of map kinase phosphatases and encoding nucleic acid therefor - Google Patents
Production and use of map kinase phosphatases and encoding nucleic acid therefor Download PDFInfo
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- WO1995021923A1 WO1995021923A1 PCT/GB1995/000272 GB9500272W WO9521923A1 WO 1995021923 A1 WO1995021923 A1 WO 1995021923A1 GB 9500272 W GB9500272 W GB 9500272W WO 9521923 A1 WO9521923 A1 WO 9521923A1
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- nucleic acid
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- map kinase
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
- C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
Definitions
- the present invention relates to phosphatases.
- 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.
- 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.
- MAP kinases 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 (1) .
- MAP kinase has been shown to phosphorylate and activate effector substrates such as the transcription factors c-jun and elk-1. For a summary of MAP kinases and pathways in which they are known to be involved, see a review by Roger Davis (50) .
- 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 (2) .
- this gene product When expressed in vitro (6) 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 (7) . Furthermore, it has been shown recently that this phosphatase gene can also block cellular DNA 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.
- MAP 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 SMKl, HOGl, MPKl, 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.
- MAP kinase phosphatases are capable of hydrolysing either or preferably both phosphothreonine and phosphotyrosine residues on a MAP kinase.
- PTPs protein tyrosine phosphatases
- Tyrosine phosphorylation plays a central role in these events [8,9] , and is regulated by opposing activities of kinases and phosphatases.
- 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.
- PTK protein tyrosine kinase
- a key element in signal transduction from an activated receptor tyrosine kinase to an intracellular response is now recognised to involve the family of MAP- kinases, pathways implicated in many diverse cell types [1] .
- Two forms of MAP kinase have been purified from human fibroblasts with molecular weights P42 mapk and P44 raapk , (ERK-2 and ERK-1 respectively) , [24] .
- Activation requires an ordered phosphorylation of a threonine and tyrosine located within the conserved kinase subdomain 8, (T183, Y185) , [25,26] .
- Raf-1 oncogenic forms of Raf-1 have shown it to act as a putative MKKK [33-35] , along with MEKK [38] , and c-Mos [39,40] , which results in sequential activation of Scr/Thr kinases MEK [24,41] , which ultimately phosphorylate the MAP kinases, see Figure 1.
- JNK is activated by stress, Tumour Necrosis Factor (TNF) , Interleukin-1 (IL-1) and ultra-violet (UV) light.
- TNF Tumour Necrosis Factor
- IL-1 Interleukin-1
- UV ultra-violet
- p38 is activated by lipopolysaccharide (LPS) .
- Both kinases are related to the erk-type MAP kinases in that 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 [25] 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 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.
- 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, C 100, 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-proliterative 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".
- the phosphatases may have novel substrates. These substrates may also be key regulators of cell proliferation and potential targets for intervention by drug inhibitors.
- MAP kinase phosphatases and encoding nucleic acid therefor enables the production of 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.
- 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 wild- type 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 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.
- 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.
- 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.
- 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.
- 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 vi tro 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 (e.g. encoding another polypeptide) .
- nucleic acid according to the present invention may be wholly or partially synthetic.
- the present invention also provides a 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. Amino acid sequences encoded by the sequences of Figure 2 appear in Figure 3.
- Variants, mutants or derivatives of these polypeptides 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.
- 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 (8) . 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.
- a polypeptide comprising an amino acid sequence which comprises an allele, derivative or mutant, by way of addition, insertion, deletion or substitution of one or more amino acids, of an amino acid sequence encoded by 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 linkage 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.
- 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 encoding sequences shown in Figure 2, and which has MAP kinase phosphatase activity but is not CL100, or an orthologue thereof.
- 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.
- the homologous polypeptide is an orthologue of a polypeptide comprising an amino acid sequence encoded by a nucleotide sequence shown in Figure 2.
- 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.
- the present invention also encompasses a method of making a polypeptide which has MAP kinase phosphatase 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 vi tro systems, such as reticulocyte lysate. Alleles, derivatives, variants and mutants may be expressed likewise.
- 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.
- appropriate regulatory sequences including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate.
- 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.
- 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 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) .
- 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 WO92/01047.
- 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 antigen or other binding partner are the Fab fragment consisting of the VL, VH, CI 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.
- 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 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 in a test sample comprising cells or cell lysate. Similar proposals have been made for the known tumour suppressor gene retinoblastoma (e.g. in W094/01467 and AU-A-52461/90) .
- 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.
- antibodies may be used to determine the presence of wild- type 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.
- Antibody binding to a sample may conveniently be determined by employing a suitable labelling system for the antibody.
- 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.
- 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
- 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 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.
- nucleotide sequence complementary to any one of the sequences shown in Figure 2 (v) 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 2, or a nucleotide sequence complementary thereto; and (vii) a nucleotide sequence which is a fragment of any one of (v) , (vi) and (vii) ; may equally be used in a method of screening cells for the presence of nucleic acid encoding a polypeptide 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 nucle
- 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 (e.g. 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 (e.g. DNA) may be measured using any of a variety of techniques at the disposal of those skilled in the art.
- target nucleic acid e.g. DNA
- 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. 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.
- individuals may be screened for the presence of one or more copies of a MAP kinase phosphatase gene to assist in assessing likelihood of developing a disorder involving uncontrolled cell proliferation. The screening may be prenatal.
- 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) .
- 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.
- 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 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.
- a smaller molecule representing part of the full molecule such as those shown in Figure 2
- 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 co- transfection 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.
- the MAP kinase of the reporter plasmid may be tagged, eg with a myc-epitope (32) , which allows detection using an anti-tag antibody.
- a suitable anti- myc antibody is 9E10 (43) .
- anti-MAP kinase antibodies may be used, needing no label to be added to the protein.
- COS cells co- transfected 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.
- EGF extracellular protein kinase growth factor
- 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.
- 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.
- recombinant production and purification of a MAP kinase phosphates for mixing with MAP kinase may be by any technique known in the art.
- 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.
- effector molecules activators or inhibitors
- 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.
- 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 /S-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 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 /3-galactosidase) and will grow in the absence of histidine (and the presence of 3- aminotriazole) 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 (i.e. non erk-type) MAP kinases or novel molecules involved in signal transduction.
- 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 WO94/23039) .
- mammalian c-raf and MAP kinase have been introduced into the yeast S. po be 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. 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 ⁇ -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. po ⁇ be strain, may be used.
- MAP kinase phosphatase may be incorporated into this screening system.
- 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
- a chosen phosphatase (attenuating the blue colour) a chosen phosphatase.
- Such compounds may be useful therapeutic agents, for example antiproliferative or anti-inflammatory drugs. 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 time- consuming, so that a method for assisting in the screening process can have considerable commercial importance. Of course, the marker used may be a simple
- 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.
- 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 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.
- FISH fluorescence in situ hybridisation
- FISH fluorescence in situ hybridisation
- 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 MAP kinase phosphatase polypeptides, antibodies thereto or encoding nucleic acid therefor.
- 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.
- disorders will generally be cell- 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 (e.g. in WO91/15580 and WO94/06910) . 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.
- 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 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.
- the nucleic acid forms part of a gene construct enabling expression within cells of the individual.
- 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. Preventative 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 treated.
- 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.
- 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.
- 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.
- a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
- isotonic vehicles such as Sodium Chloride Injection, 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.
- disease sites such as tumours.
- suitable imaging devices may be employed to guide an injecting needle to the desired site.
- proliferative diseases such as those of the bone marrow, leukaemias etc.
- 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 Wang et al., PNAS USA, 85: 3014, Gilboa (1982) -J. Virology 44: 845 and Hocke (1986) Nature 320:275
- vaccinia viruses Chokrabarty 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 D ⁇ A replication for integration of their genome and expression of polypeptides encoded therein.
- retrovirus vectors include those described by Chen et al ( Science 250: 1576-80, 1990) and Miller et al (BioTechniques 7: 980-990, 1989).
- a pharmaceutical composition 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.
- 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.
- rodents such as mice, rats, hamsters etc.
- 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.
- FIG 1 is a schematic diagram of the mammalian MAP kinase signal transduction pathway (from ref 2) .
- 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.
- FIG. 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 5 and STY6 were isolated by screening a hamster liver cDNA library with a mixture of STY2 and STY3 probes shown in part (a) and (b) .
- 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 (a) and (b) . All sequences apart from STY7 and STY10 show 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. (a) - STY2; (b) - STY3; (c) - STY4;
- Figure 3 shows deduced amino acid sequences of phosphatase clones aligned with the amino acid sequence of CL100: (a) - STY2, STY3 , STY 4 AND STY5; (b) STY6; (c) STY 9; (d) STY 8.
- For parts (a) - (c) spaces indicate residues that are identical with CL100 and dots indicate residues which have not yet been determined.
- amino acid sequences shown correspond to residues 177-255 for part (a) , 231-302 for part (b) . 223- 267 for part (c) and 1-367 for part (d) .
- FIG. 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 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 1) .
- EGF stimulatory ligand
- A431 cells were grown in Dulbecco's modification of Eagle's minimal essential medium (DMEM) supplemented with 10% fetal calf serum.
- Total cellular RNA was prepared with RNAzolB (Promega) and poly(A) +RNA isolated with Dynabeads oligo(dT) 25 (Dynal) .
- 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 (A)- RNA (l ⁇ g) 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°C for 30sec, 50°C, 30sec, 72°C 1 min.
- a 270bp band was purified by agarose gel electrophoresis and subcloned into pBluescript.
- STY2 STY5 with CL100 being STY1.
- STY6-STY10 were isolated by screening cDNA libraries with a 32P- labelled probe made from the inserts of plasmids containing STY2 and STY3 sequence shown in Figure 2a. STY6 was isolated from a human brain library. 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 [46] . 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.
- 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 [7,48] .
- 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.
- Recombinant activated MAP kinase kinase (MEK/EE) (50ng) was incubated with recombinant ERK2 (3/xg) or recombinant ERK2 (3 ⁇ g) and recombinant STY8 (0.75 ⁇ g) in the presence of 32 P-ATP (2.5nCi) in 50mM Tris- Cl (pH 7.5)/0.1mM EGTA/lOmM MgAcetate/O .125mM ATP for the times indicated (in minutes) in Figure 5.
- STY8 has also been cloned into a baculovirus transfer vector, resulting, after recombination, in production of a Glutathione-S-transferase (GST) - STY8 fusion protein.
- GST Glutathione-S-transferase
- the vector was co-transfected into 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) (52) .
- the sequence is related to CL100 and STY8 throughout, particularly in the catalytic domain.
- nucleic acid encoding MAP kinase phosphatases enables the following to be performed.
- 3CH134 is expressed predominantly in the lung of the adult mouse [4] .
- STY8/9 and CL100 are expressed ubiquitously across a range of tissues.
- the expression of 3CH134 corresponds to post-mitotic cells [48] , which suggests the phosphatase may play a role in cellular differentiation, acting as a negative effector of cell growth.
- 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] .
- MAP kinase pathway promotes cell proliferation and tumorigenesis, and so MAP kinase phosphatases may function as tumour suppressor genes.
Abstract
Description
Claims
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AU15861/95A AU696939B2 (en) | 1994-02-10 | 1995-02-10 | Production and use of map kinase phosphatases and encoding nucleic acid therefor |
JP7521061A JPH09508795A (en) | 1994-02-10 | 1995-02-10 | Production and use of MAP kinase phosphatases and nucleic acids encoding them |
EP95907771A EP0742827A1 (en) | 1994-02-10 | 1995-02-10 | Production and use of map kinase phosphatases and encoding nucleic acid therefor |
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GB9402573.1 | 1994-02-10 | ||
GB9402573A GB9402573D0 (en) | 1994-02-10 | 1994-02-10 | Phosphatases |
GBPCT/GB94/00694 | 1994-03-31 | ||
PCT/GB1994/000694 WO1994023039A1 (en) | 1993-04-07 | 1994-03-31 | Methods for screening of substances for therapeutic activity and yeast for use therein |
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JP (1) | JPH09508795A (en) |
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US5948885A (en) * | 1996-05-20 | 1999-09-07 | Signal Pharmaceuticals, Inc. | Mitogen-activated protein kinase p38-2 and methods of use therefor |
US5998188A (en) * | 1995-06-16 | 1999-12-07 | Oregon Health Sciences University | Mitogen activated protein kinase phosphatase cDNAS and their biologically active expression products |
US6074862A (en) * | 1995-12-20 | 2000-06-13 | Signal Pharmaceuticals Inc. | Mitogen-activated protein kinase kinase MEK6 and variants thereof |
WO2002062979A2 (en) * | 2001-02-06 | 2002-08-15 | Oxford Biomedica (Uk) Limited | Enzyme |
US6551810B1 (en) | 1999-04-23 | 2003-04-22 | Ceptyr, Inc. | DSP-10 dual-specificity phosphatase |
US6649391B1 (en) | 1999-07-20 | 2003-11-18 | Ceptyr, Inc. | DSP-11 dual-specificity phosphatase |
US6677130B1 (en) | 1996-05-20 | 2004-01-13 | Signal Pharmaceuticals, Inc. | Mitogen-activated protein kinase p38-2 and methods of use therefor |
US6897019B1 (en) | 1998-04-17 | 2005-05-24 | Tufts College | Methods for treating and preventing insulin resistance and related disorders |
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WO1994013796A1 (en) * | 1992-12-14 | 1994-06-23 | United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Expression cloning of a human phosphatase |
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1995
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- 1995-02-10 EP EP95907771A patent/EP0742827A1/en not_active Withdrawn
- 1995-02-10 JP JP7521061A patent/JPH09508795A/en active Pending
- 1995-02-10 CA CA002182967A patent/CA2182967A1/en not_active Abandoned
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WO1994013796A1 (en) * | 1992-12-14 | 1994-06-23 | United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Expression cloning of a human phosphatase |
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Also Published As
Publication number | Publication date |
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CA2182967A1 (en) | 1995-08-17 |
JPH09508795A (en) | 1997-09-09 |
EP0742827A1 (en) | 1996-11-20 |
AU1586195A (en) | 1995-08-29 |
AU696939B2 (en) | 1998-09-24 |
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