FR2734266A1 - Polypeptide(s) able to bind to GAP protein SH3 domain - Google Patents

Abstract

A polypeptide (PP) capable of interacting with the SH3 domain of GAP (GTPase Activating Protein) and comprising one of the following sequences is new. (a) sequence (I) of 466 amino acids (AAs) given in the specification; (b) sequence (II) of 68 AAs (corresp. to residues 319-386 of (I)); or (c) one of the sequences (III)-(VI): Val-Met-Glu-Lys-Pro-Ser-Pro-Leu-leu-Val-Gly-Arg-Glu-Phe-Val-Arg-Gln-Ty r-Ile (III) Xaa<1>-Xaa<2>-Glu-Gly-Asp-Asp-Arg-Asp-Asn-Arg-Leu-Leu-Gly-Pro (IV) Leu-Pro-Asn-Phe-Gly-Phe-Val-Val-Phe-Asp-Asp-Ser-Glu-Pro-Val-Gln-Lys (V) Ser-Ala-Thr-Pro-Ala-Pro-Ala-Asp-Val-Ala-Pro-Ala-Gln-Glu-Asp-Leu-Arg-Xa a<3>-Phe (VI); Xaa<1> = Ala or Thr; Xa<2> = any AA; Xaa<3> = not defined.

Description

The present invention relates to novel peptide and nucleotide sequences, and their pharmaceutical use. More particularly, the invention relates to peptides capable of binding to the SH3 domain of the GAP protein.

The products of the ras genes, generally referred to as p21 proteins, play a key role in the control of cell division in all eukaryotic organisms where they have been sought. Certain specific modifications of these proteins cause them to lose their normal control and lead them to become oncogenic. Thus, a large number of human tumors have been associated with the presence of modified ras genes. Likewise, an overexpression of these p21 proteins can lead to a deregulation of cell proliferation. Overall, the p21 proteins are involved in 30% of human cancers.

Understanding the exact role of these p21 proteins therefore constitutes one of the target objectives of research in the field of oncology.

The model currently available to explain the functioning of the p21 proteins is based on analogies that they share with the transducing G proteins. There is a balance in cells between active p21 proteins, bound to GTP and inactive forms, having bound GDP. In a quiescent cell, where the p21 are not solicited, the majority of them are in GDP form.

When the cell is stimulated, the nucleotide exchange factor, GEF, becomes more active and facilitates the removal of GDP and its replacement by GTP. The protein then adopts an active conformation which allows it to recognize and stimulate its effector, the GAP protein "GTPase activating protein", probably associated with other proteins. The p21-GTP-GAP complex in turn probably interacts with one or other protein (s) thus allowing the transmission of the signal which causes a biological response of the cell. The association of p21-GTP with GAP simultaneously triggers the hydrolysis of GTP and the return of p2 I to the inactive form.

In the case of oncogenic p21 proteins, the mutation they carry prevents a return to the inactive state. The equilibrium is in the latter case shifted to the active form of p2i.

This complex balance between the active and inactive forms of p21 is controlled both by factors inherent in the biochemical properties of p21 proteins (relative affinity for GDP and GTP, rate of nucleotide exchange, etc.) and external factors. modulating their activity such as in particular the GAP protein.

The GAP protein is a cytosolic protein present in all eukaryotic organisms which therefore has the ability to greatly accelerate the hydrolysis of
GTP, bound to the normal p21 protein (Trahey and Mc Cormick 1987). It has two areas providing distinct functions. Its carboxy-terminal end carries the catalytic activity which binds the p21 proteins and increases their GTPase activity. On its other end, downstream of the amino-terminal part, is a juxtaposition of SH2 and SH3 domains which are capable of participating in interactions with other proteins.

At present, we know two proteins interacting with the protein
GAP. These are the proteins called p62 and psi 90, respectively 62kDa and 190kDa. These two proteins being immunoprecipitated by antibodies directed against different epitopes of GAP, obviously form a specific complex with GAP.

It is known in particular that it is at the level of the SH2 region that the interaction of the p62 protein with the GAP protein takes place.

As regards more particularly the SH3 domain, its presence in various proteins such as phospholipases Cy (P LC -y), the p85 subunit of phophatidyllinositol-3 kinase and the grb-2 protein, all involved in transduction of the p2 1 -Ras signal, suggests that this domain is very particularly important for directing protein-protein interactions and therefore necessary for the functioning of the corresponding protein and / or for its cellular localization. In the particular case of the GAP protein, this SH3 domain could therefore also participate in the transduction of the Ras signal. It is clear that understanding the exact role of this SH3 domain would be particularly valuable therapeutically.

The object of the present invention is precisely to contribute to the elucidation of the contribution of the SH3 domain to the transduction of the ras signal.

The Applicant has thus demonstrated the existence of proteins capable of associating with the GAP protein via a direct binding to its SH3 domain.

More precisely, the present invention results from the identification, isolation and characterization of proteins capable of interacting with the SH3 domain of the protein.
GAP. It also results from the structural characterization of these proteins by identification of corresponding peptide sequences.

More particularly, the polypeptide of the invention comprises all or part of a peptide sequence chosen from the sequences SEQ ID N "1, SEQ ID N" 2,
SEQ ID N "3, SEQ ID N" 4 and derivatives thereof.

For the purposes of the present invention, the term derivative denotes any molecule obtained by modification of a genetic and / or chemical nature of the polypeptide according to the invention and retaining the desired activity. By modification of a genetic and / or chemical nature, is meant any mutation, substitution, deletion, addition and / or modification of one or more residues. Such derivatives can be generated for different purposes, such as in particular that of increasing the affinity of the peptide for its site of interaction, that of improving its production levels, that of increasing its resistance to proteases, that of increasing its resistance to proteases. to increase its therapeutic efficacy or to reduce its side effects, or that of giving it new pharmacokinetic and / or biological properties.

They may also be fragments of the aforementioned sequences of derivatives thereof. Such fragments can be generated in different ways. In particular, they can be synthesized chemically, on the basis of the sequence given in FIG. 1, using peptide synthesizers known to those skilled in the art. They can also be synthesized genetically, by expression in a cellular host of a nucleotide sequence encoding the desired peptide. In this case, the nucleotide sequence can be prepared chemically using an oligonucleotide synthesizer, on the basis of the peptide sequence given in the present application and the genetic code. The nucleotide sequence can also be prepared from the sequence given in the present application, by enzymatic cleavage, ligation, cloning, etc., according to techniques known to those skilled in the art, or by screening DNA libraries with probes produced from the
SEQ ID N "1.

It is more particularly a polypeptide comprising SEQ ID N "1,
SEQ ID N "2, SEQ ID N" 3 and / or SEQ ID N "4.

Preferably, the polypeptide according to the invention has a molecular weight of the order of 68 kDa.

According to a particular embodiment of the invention, it is a polypeptide of human origin comprising all or part of SEQ ID N "5, SEQ ID N" 9 or of one of their derivatives as defined. previously. It is more particularly a polypeptide comprising SEQ ID No. 5.

More preferably, it is a polypeptide represented by SEQ ID No. 9.

Unexpectedly, this protein does not have homology with another p68 protein already identified as binding to the SH3 domain of Src. It is not phosphorylated at its tyrosine units in growing cells or in a state of mitosis.

Another subject of the invention resides in antibodies or fragments of polyclonal or monoclonal antibodies directed against a polypeptide as defined above. Such antibodies can be generated by methods known to those skilled in the art. In particular, these antibodies can be prepared by immunization of an animal against a polypeptide whose sequence is chosen from SEQ 1DN0 1, SEQ
ID N ″ 2, SEQ ID N ″ 3, SEQ ID N ″ 4, SEQ ID N ″ 5 and SEQ ID N ″ 9 or any fragment or derivative thereof, blood sample and antibody isolation. These antibodies can also be generated by preparing hybridomas according to techniques known to those skilled in the art.

The antibodies or antibody fragments of the invention can thus be used to regulate the state of activation of the product of the ras genes. Advantageously, they have the ability to inhibit the interaction between the GAP protein and a polypeptide according to the invention. Moreover, these antibodies can also be used to detect and / or assay a peptide according to the invention in biological samples, and therefore to provide information on the state of activation of the product of the ras genes.

The invention also extends to antagonists, namely any peptide capable of blocking the interaction of a polypeptide according to the invention with the SH3 domain of the GAP protein. Such peptides can be demonstrated in competition tests (see example 2-3) or in inhibition of ras activity.

The present invention therefore makes it possible to generate polypeptides derived from the sequences identified above as well as antibodies directed against these polypeptides or corresponding proteins, exhibiting interesting biological properties with a view to pharmaceutical use.

The invention also provides non-peptide or non-exclusively peptide compounds which can be used pharmaceutically. It is in fact possible, from the active protein motifs described in the present application, to produce molecules which inhibit the signaling pathway dependent on P21 proteins which are not exclusively peptide and which are compatible with pharmaceutical use. In this regard, the invention relates to the use of a polypeptide of the invention as described above for the preparation of non-peptide molecules, or not exclusively peptide molecules, which are pharmacologically active, by determining the structural elements of this polypeptide which are important for its activity and reproduction of these elements by non-peptide or non-exclusively peptide structures.

A subject of the invention is also pharmaceutical compositions comprising one or more molecules thus prepared.

A subject of the present invention is also any nucleic acid sequence encoding a polypeptide capable of binding to the SH3 domain of the GAP protein. More preferably, it is a sequence comprising
(a) all or part of SEQ ID N "6 and / or of SEQ ID N" 9 or of their complementary strand,
(b) any sequence hybridizing with a sequence (a) and encoding a polypeptide capable of binding to the SH3 domain of the GAP protein, and
(c) sequences derived from sequences (a) and (b) due to degeneration of the genetic code.

Preferably, it comprises the sequence SEQ ID N "6 and more preferably, it is represented by SEQ ID N" 9.

The various nucleotide sequences of the invention may or may not be of artificial origin. They can be genomic sequences, cDNA, RNA, hybrid sequences or synthetic or semi-synthetic sequences. These sequences can be obtained for example by screening DNA libraries (cDNA library, genomic DNA library) by means of probes produced on the basis of sequences presented above. Such libraries can be prepared from cells of different origins by standard molecular biology techniques known to those skilled in the art. The nucleotide sequences of the invention can also be prepared by chemical synthesis, in particular according to the phosphoramidite method, or alternatively by mixed methods including the chemical or enzymatic modification of sequences obtained by screening of libraries.

These nucleotide sequences according to the invention can be used in the pharmaceutical field, either for the production of the polypeptides of the invention, or for the production of antisense sequences which can be used in the context of gene therapy, or even for detection and - diagnosis, by hybridization experiments, of GAP protein activity in biological samples or for the isolation of homologous sequences from other cellular sources.

For the production of the polypeptides of the invention, the nucleic acid sequences defined above are generally placed under the control of signals allowing their expression in a cellular host. The choice of these signals (promoters, terminators, secretion "leader" sequence, etc.) can vary depending on the cellular host used. Preferably, these nucleotide sequences of the invention form part of a vector, which can be autonomously replicating or integrative. More particularly, autonomously replicating vectors can be prepared using sequences which replicate autonomously in the chosen host. As regards the integrative vectors, these can be prepared for example by using sequences homologous to certain regions of the genome of the host, allowing, by homologous recombination, the integration of the vector.

The cellular hosts which can be used for the production of the polypeptides of the invention are both eukaryotic and prokaryotic hosts. Among the eukaryotic hosts which are suitable, mention may be made of animal cells, yeasts or fungi. In particular, as regards yeasts, mention may be made of yeasts of the genus
Saccharomyces, Kluyveromyces, Pichia, ScErwanniomyces, or Hwiseiiula. As regards animal cells, mention may be made of COS, CHO, C127 cells, etc. Among the fungi, there may be mentioned more particularly Aspergillus ssp. or Trichoderma ssp.

As prokaryotic hosts, it is preferred to use the following bacteria E.coli, Bacillus, or Sfreptomyces.

The nucleic acid sequences according to the invention can also be used for the production of ante-sense nucleic acids which can be used as pharmaceutical agents.

The inhibition of the expression of certain oncogenes by antisense nucleic acids has been shown to be a useful strategy in understanding the role of these oncogenes and a particularly promising pathway in carrying out an anticancer treatment. The ante sense oligonucleotides are small oligonucleotides, complementary to the coding strand of a given gene, and therefore capable of specifically hybridizing with the transcribed mRNA, inhibiting its translation into protein. Such oligonucleotides may consist of all or part of the nucleic acid sequences defined above. They are generally sequences or fragments of sequences complementary to sequences coding for peptides according to the invention. Such oligonicleotides can be obtained from the aforementioned sequences, by fragmentation, etc., or by chemical synthesis.

The invention also relates, as nucleotide sequences, to nucleotide probes, synthetic or not, capable of hydriding with the nucleotide sequences defined above which encode a polypeptide of the invention, or with the corresponding mRNA. Such probes can be used in vitro as a diagnostic tool. Such probes must be labeled beforehand, and for this various techniques are known to those skilled in the art. The hybridization conditions under which these probes can be used are the normal conditions of stringency. These probes can also be used for the demonstration and isolation of homologous nucleic acid sequences encoding a polypeptide of the invention, from other cellular sources. As an illustration of these probes, it is more particularly possible to mention the probes represented by the SEQs
ID N "7 and SEQ ID N" 8, which are used in Example 3 below.

A subject of the invention is also any pharmaceutical composition comprising as active principle at least one polypeptide as defined above.

It also relates to any pharmaceutical composition comprising as active principle at least one antibody and / or an antibody fragment as defined above, as well as any pharmaceutical composition comprising as active principle at least one antisense oligonucleotide as defined above.

Moreover, a subject of the invention is also pharmaceutical compositions in which the antisense polypeptides, antibodies and oligonucleotides defined above are combined with one another or with other active principles.

The pharmaceutical compositions according to the invention can be used to modulate the activation of p2 1 proteins and therefore to modulate the proliferation of certain cell types. More particularly, these pharmaceutical compositions are intended for the treatment of cancers. Many cancers have in fact been associated with the presence of oncogenic ras proteins. Among the cancers most often containing mutated ras genes, mention may be made in particular of adenocarcinomas of the pancreas, 90% of which have a Ki-ras oncogene mutated on the twelfth codon (Almoguera et al., Cell 53 (1988) 549), colon adenocarcinomas and thyroid cancers (50%), or lung carcinomas and myeloid leukemias (30%, Bos,
JL Cancer Res. 49 (1989) 4682).

A subject of the invention is also the use of the molecules described above for modulating the activity of the p2 proteins. In particular, the invention relates to the use of these molecules for at least partially inhibiting the activation of the p2 proteins. 1.

The invention also provides a method of detecting the expression and / or overexpression of the p68 protein in a biological sample. Such a method comprises, for example, bringing such a sample into contact with an antibody or antibody fragment according to the invention, revealing the antigen-antibody complexes, and comparing the results obtained with a standard sample. In such a method, the antibody can be in suspension or immobilized beforehand on a support. This method can also comprise bringing the sample into contact with a nucleotide probe according to the invention, the identification of the hybrids obtained, and the comparison with those obtained in the case of a standard sample.

The present invention can be used for several reasons in the therapeutic field: the polypeptides, antibodies and nucleotide sequences of the invention being capable of modulating the activity of the ras genes, they in fact make it possible to intervene in the process of cancer development. . Due to their strong expression in striated skeletal muscle cells, these peptides are also probably involved in pathologies linked to a signaling defect, such as diabetes, for example. Another aspect of the invention consists in using DNA or RNA nucleotide sequences capable of interacting with the claimed polypeptides.

These sequences can be prepared according to the method described in international application WO 91/19813.

The invention can also be used in the field of diagnosis and typing of cancers.

Other advantages of the present invention will emerge on reading the examples which follow, which should be considered as illustrative and not limiting.

MATERIAL AND METHODS
General cloning techniques
The methods conventionally used in molecular biology such as preparative extractions of plasmid DNA, centrifugation of plasmid DNA in cesium chloride gradient, electrophoresis on agarose or acrylamide gels, purification of DNA fragments by electroelution, the extractions of proteins with phenol or phenol-chloroform, the precipitation of DNA in a saline medium with ethanol or isopropanol, the transformation into Escherichia coli, etc., are well known to the man of profession and are abundantly described in the literature [Maniatis
T. et al., "Molecular Cloning, a Laboratory Manual", Cold Spring Harbor Laboratory,
Cold Spring Harbor, NY, 1982; Ausubel FM et al. (eds), "Current Protocols in
Molecular Biology ", John Wiley & Sons, New York, 1987].

Restriction enzymes were supplied by New England Biolabs (Biolabs), Bethesda Research Laboratories (BRL) or Amersham and are used as recommended by the suppliers.

The pGEX 2T plasmid is of commercial origin.

Enzymatic amplification of DNA fragments by the so-called PCR technique [Polymerase-catalyzed Chain Reaction, Saiki R.K. et al., Science 230 (1985) 13501354; Mullis K.B. and Faloona F.A., Meth. Enzym. 155 (1987) 335-350] is performed using a "DNA thermal cycler" (Perkin Elmer Cetus) according to the manufacturer's specifications.

Verification of the nucleotide sequences is carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kit distributed by Amersham.

For hybridization experiments, the normal stringency conditions are generally as follows. 3 x SCC hybridization in the presence of 5 x Denhart's at 65 "C; wash: 0.5 x SSC at 65" C.

EXAMPLE 1
Preparation of Glutathione-S-transferase SH3 fusion proteins
The DNA sequences encoding the SH3 domains of the GAP (residues 275 to 350) and c-Src (residues 84 to 148) proteins are amplified by the PCR technique and cloned into an expression vector pGEX2T between the restriction sites Bam HI and ECORI.

The transformed ansi bacteria are cultured, induced with IPTG (I-thio-B-D galactopyranoside) and lysed by sonication. The GST SH3 fusion proteins are purified by affinity chromatography on glutathione agarose beads (Pharmacia LKB biotech) then eluted with 10 mM of reduced glutathione.

EXAMPLE 2 1) Preparation of cell lysates
ER22 cells (hamster fibroblasts overexpressing the human EGF receptor) or NIH 3T3 cells expressing pp60 C-Src (F-527) are cultured on DMEM medium (Dubelcco's Modified Eagle Medium) enriched with 10% fetal calf serum containing the antibiotic G41 8 (200 mg / ml) and 2 mM / l of glutamine (5GBICO-BRL), at 37 ° C. under 5% CO 2.

In each test, the ER22 cells are cultured in 100 mm dishes until confluence and then without serum for 18 hours. Sodium orthovanadate is added to a final concentration of 100M and the incubation continued for 30 minutes.

EGF is then added directly to the medium for a final concentration of 80nM in 10 minutes and at 370C.

For certain tests, the mitotic cells are recovered by treatment with 0.4> g of nocodazole (SIGMA) per ml for 18 hours. They are quickly washed with a phosphate-based saline buffer, cooled in ice, then solubilized over 30 minutes at 4 ° C. in 1 ml of lysis buffer, HNTG (50 mM Hepès, pH 7.5, 150 mM of NaCl, 1% Triton x 100, 10% Glycerol, 1mM MgCl2, 1mM EGTA, in the presence of phosphatase inhibitors (1mM Na3 VO4, 10mM Na4P2Ov, 10mM NaF) and protease inhibitors (1 pg / ml leupeptin, 1 zg / ml trypsin inhibitor, 1 zg / ml pepstatin A, 2 pg / ml aprotinin, 10 Hg / ml benzamidine, 1 mM phenylmethanesulfonyl fluoride, I llg / mlssg / ml of antipain, 1 Zg / ml of chymostatin).

The lysates are decanted by centrifugation at 15,000 revolutions / min for 10 min. The protein concentration is then determined (Bio-rad micro-test).

2) Direct link test
All the cell lysates (200 μg) and the immunoprecipitated proteins are separated on 7.5% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) then transferred to a polyvinylidene difluoride membrane (PVDF Millipore
Corpo.). The nonspecific binding at the filters is blocked by 2% skimmed milk in PBS containing 0.05% Tween 20, for 2 hours, at room temperature. The filters are incubated with the GST protein and GST-SH3 proteins in the blocking buffer for 12 hours at 40 C. After having been washed with PBS0.05% Tween 20, the bound proteins are detected by successive incubation with an anti- monoclonal antibody. GST (0.25 µg / ml) (Hybridolab Pasteur Inst.), An anti-mouse antibody conjugated to an alkaline phosphatase and 5-bromo-4-chloro-3-indolyphosphate toluidinium nitroblue tetrazolium salt (PROMEGA).

3) Competition test
Synthetic peptides corresponding to the sequences (275-305), (299-326), (317326), (320-350) of GAP SH3 or to the putative sequence of dynamin (RRAPAVPPARPGS) binding to the SH3 domain are synthesized on a apparatus
Applied Biosystems 431 A, using FMOC chemistry.

The purified p68 protein is isolated by electrophoresis on sodium polyacrylamidedodecyl sulfate gel and transferred by electrophoresis on a PVDF membrane. The membranes are incubated with increasing amounts of peptides. Poly peptide
L-proline (SIGMA) is used at 500 zM in a control reaction. After 1 hour of incubation, the GST-GAP-SH3 protein (2 pg / ml) is added to each reaction medium. The filtrates are probed with an anti-GST monoclonal antibody as described above.

4! Immunoprecipitation and immunoblotting
The soluble cell lysates (3 mg) are decanted with 50 Cll of protein A sepharose
CL-4B (Pharmacia Biotech) for 2 H at 4 "C. The decanted cell lysates are incubated with the antiphosphotyrosine monoclonal antibody (4G10 monoclonal antibody - Upstate Biotechnology Incorporated) for 4 H at 4" C. Then, 50 zl of protein A
Sepharose are added to the complex and the incubation is continued overnight at 40C.

The immunoprecipitates are washed 3 times with an HNTG buffer and solubilized in a sample of SDS buffer (100 μl). The complexes are then separated by SDS
PAGE and electrophoretically transferred to PVDF membranes.

The membranes are incubated with the phosphotyrosine monoclonal antibody in
TBS (10 mM Tris, pH 7.4, 150 mM NaCl, 3% bovine serum albumin) and further incubated with second antibodies conjugated to alkaline phosphatase. Substrates for alkaline phosphatase are then added for proper color development.

5! Purification and sequence analysis
About 5.109 ER22 cells are lysed in 200 ml of HNTG buffer. The lysate is centrifuged at 15,000 g for 15 min and diluted 5 times in HNG buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 10% Glycerol, I mM EGTA, phosphatase inhibitors and protease inhibitors). The lysate is incubated overnight with 6 ml of S-Sepharose Fast flow equilibrated in the same buffer. The complex is transferred onto a column (2.5 x 1.3 cm - IBF). The column is washed with 10 times its volume in buffer and the bound proteins are then eluted at an elution rate of 60 ml hl, with a linear gradient of 60 ml from 0 to 1 M NaCl of the same buffer. The fractions having 1 'binding activity determined under the conditions of Example 2.2 (0.150.37 M NaCl) are combined, diluted 10 times in HNG buffer and loaded onto
Heparin-Sepharose CL-6B (3ml) (Pharmacia LKB), pre-equilibrated with the same buffer at an elution rate of 24 ml hl. After washing with HNG buffer, the column is eluted with a gradient of 24 ml, from 0 to 1 M NaCl. The active fractions of the S Fast Flow chromatography are combined, diluted 4 times in a buffer.
MES (100 mM MES, ph 6.8, 1 mM MgSO4, 1 mM EGTA) and transferred to an ATP agarose column (3 ml) (Sigma N "A9264) pre-equilibrated with the buffer
MES containing 50 mM NaCl at an elution rate of 6 ml.hl The column is then washed with 20 ml of MES buffer containing 50 mM NaCl and eluted at 30 ml.hl with a linear gradient from 50 mM to 2 M NaCl in MES buffer. The p68 protein elutes between 0.3 M and 0.4 M NaCl. The active fractions are combined, dialyzed with 20 mM NH4HCO3 pH 8.3 and concentrated. The proteins, brought to dryness, are resuspended in SDS buffer, separated by electrophoresis on polyacrylamide gel. The gel is stained with coomassie blue and the band of molecular weight 68kDA, corresponding to the SH3 binding activity is collected. It is washed for 1 hour with the following solutions: water, water / methanol (90/10), water / CH3CN (80/20), water / CH3CN (50/50).

The gel band, containing the purified p68 protein, is then divided into small fragments and dried under SPEED / VAC (SAVANT). 400 µl of a solution containing 25 mM Tris pH 8.5, 1 mM EDTA, 0.05% SDS and 5 µg of Lys-c endoproteinase (Boehringer Mannheim) are added and the whole is incubated overnight at 37 "C. The hydrolyzate is injected onto a reverse phase HPLC column (Vydac C18: 2.1 x 250mm). The column is eluted at 0.2mVmin with a linear gradient from 0 to 35% B in
150 minutes. (A: H2O + TFA 0.07% and B: CH3CN + TFA 0.07%) and the elution peaks observed at tr 113.7, 117.7 and 133.7 min are collected and directly sequenced using a Applied Biosystems 477A microsequencer. The sequence of the corresponding peptides thus obtained is presented in SEQ ID N "1, SEQ ID N02, SEQ ID N" 3 and SEQ ID N "4. These sequences show no homology with proteins referenced in the bases protein data (PIR 33. Swiss-Prot 33 (intelligenetics)).

In order to determine whether the p68 protein is tyrosine phosphorylated in mitotic or non-synchronous cells, lysate proteins from ER22 cells cultured in 10% fetal calf serum or from cells treated with nocodazole are immunoprecipitated with anti-phosphotyrosine antibodies, transferred onto a membrane and either immunodetected with anti-phosphotyrosine antibodies or incubated with either GST-GAP-SH3 or GST-Src-SH3, under the conditions described in Examples 2.2 and 2.4.

As a control, the proteins of NIH 3T3 cells transformed with an activated allele of c-Src (c-Src Y527F) are tested under the same conditions as those described for the ER22 proteins. The results obtained show that proteins are phosphorylated at the tyrosine level, more particularly in the 60-70kDa region.

In ER22 cells, no binding to a phosphorylated protein p68 is detected with the GST-GAP-SH3 or GST-Src-SH3 probes. In NIH3T3 cells (c-Src
Y527F), the GST-Src-SH3 probe binds to a protein of 68 kDa molecular weight phosphorylated in tyrosine while the GST-GAP-SH3 probe does not interact with any protein.

In order to confirm the functional involvement of this protein in the ras signaling pathway, competition experiments were carried out under the conditions described in Example 2.3. The results obtained show that the peptides derived from
GAP capable of blocking ras-induced disruption of xenopus egg germinal vesicles (GVBD), GAPSH3 peptides (299-326) and (317-326) are also capable of blocking the interaction between p68 protein and GAP.

These results clearly confirm that the ability to block the activity of the protein according to the invention or to interfere with its activity constitutes a particularly promising new approach for the treatment of cancer.

EXAMPLE 3
Isolation of the human sequence SEQ ID N "6 from a human placental cDNA library.

SEQ ID N "7 and N08, established from the peptide sequences SEQ ID N" 1 and SEQ ID N "3, are used as probes. The DNA of the Clonetech HL1008B bank was prepared and used in a reaction of PCR 30 amplification cycles were carried out under the following conditions: denaturation 1 min at 94 ° C., pairing 1 min between 35 and 400 ° C. and elongation 1 min at 72 ° C. This reaction makes it possible to isolate a DNA fragment. of 1.3 kb, part of the sequence of which is given in SEQ ID N "6.

Northern blot analysis shows that the corresponding RNA (3.3 kb) is ubiquitous and expressed very strongly in adult skeletal muscle.

Example 4:
Isolation of the SEO ID N "9 sequence encoding human p68 from a human placental cDNA library.

We use the two oligonucleotides inosine GTI ATG GAI AAI CCI TCC CCI CTI
CTI GTI GG (SEQ ID N "7) and GAA TCA TCG AAI ACI ACG AAI CCG AA (SEQ
ID N "8) as primers for the amplification of a cDNA in a human placental bank. PCR was carried out using the Amplitaq Perkin Elmer at an annealing temperature of 35" C followed by lmn of extension at 72 ° C in the presence of 10% formamide. We amplified a cDNA fragment of 1164 bp. After direct cloning into a pMOSblue vector (Amersham) comprising -20 and reverse sequences, the fragment was sequenced using fluorescent probes This PCR fragment was then used as a probe to screen 106 phages from a kgtll human placental cDNA library (Clontech). The probe was synthesized by the Amersham Rediprime system and the filters were incubated 16 hours in hybridization buffer (6X
SSC, 5X Denhardt's, 100 μg / ml salmon sperm, 0.25% SDS) containing the probe at 45 ° C. The filters were then washed for 1 hour at room temperature and 20 minutes at hybridization temperature in 2X SSC, 0.05% SDS Eight positive clones were identified Two of them were purified and the cDNAs were digested with EcoRI to bring out the inserts They were subcloned into M13mp18 / EcoRI to be sequenced. was carried out on the fragments resulting from a progressive deletion at exonuclease III of the cDNA (Nested Deletion Kit, Pharmacia Biotech).

The differences in size of the fragments were analyzed by PCR amplification between the -20 and reverse primers of the M13 vector and different size populations were chosen to undergo sequencing. The assembly of the different sequences obtained made it possible to reconstitute the entire open phase of P68.

SEQUENCE LIST
(1) GENERAL INFORMATION:
(i) DEPOSITOR:
(A) NAME: RHONE-POULENC RORER SA

(B) STREET: 20, avenue Raymond ARON
(C) CITY: ANTONY
(E) COUNTRY: FRANCE
(F) POSTAL CODE: 92165
(ii) TITLE OF THE INVENTION: PEPTIDE CAPABLE OF BINDING TO
SH3 DOMAIN OF GAP PROTEIN.

(iii) NUMBER OF SEQUENCES: 9
(iv) COMPUTER READABLE FORM:
(A) MEDIA TYPE: Tape
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS / MS-DOS
(D) SOFTWARE: PatentIn Release # 1.0 Version # 1.25 (EPO)
(2) INFORMATION FOR SEQ ID NO: 1:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: Protein
(vi) ORIGIN: HAMSTER
(xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 1: VM (E) KPSPLLVGREF (V) RQYI (-) (G, L) (3) INFORMATION FOR SEQ ID NO: 2:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 15 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: Protein
(vi) ORIGIN: HAMSTER
(xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 2: (T, A) (-) EGD (D) RDNRL (L) GP (4) INFORMATION FOR SEQ ID NO: 3:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: Protein
(vi) ORIGIN: HAMSTER
(xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 3: LPNFGFVVFDDSEPVQK (5) INFORMATION FOR SEQ ID NO: 4:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: Protein
(vi) ORIGIN: HAMSTER
(xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 4: (S) A (T) PAPADVAPAQEDLR (-) F (6) INFORMATION FOR SEQ ID NO: 5:
(i) CHARACTERISTICS OF THE SEQUENCE:
(A) LENGTH: 68 amino acids
(B) TYPE: amino acid
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: Protein
(vi) ORIGIN: HUMAN
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: REAGEQGDIEPRRMVRHPDSHQLFIGNLPHEVDKSELKDFFQSYGNVVE
LRINSGPKLPNFAFVVFDD
(7) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF SEQUENCE: SEQ ID NO: 6:
(A) LENGTH: 204 base pairs
(B) TYPE: nucleic acid
(C) NUMBER OF STRANDS: double
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN:
(A) ORGANISM: Human
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: 5 'CGTGAGGCTGGTGAGCAAGGTGACATTGAACCCCGAAGAATGGTGAGACAC
CCTGACAGTCACCAACTCTTCATTGGCAACCTGCCTCATGAAGTGGACAAATCA
GAGCTTAAAGATTTCTTTCAAAGTTATGGAAACGTGGTGGAGTTGCGCATTAAC AGTGGTGGGAAATTACCCAATTTCGCCTTCGTCGTCTTCGATGAT 3 '(8) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF SEQUENCE: 7: SEQ ID
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN:
(A) ORGANISM: Human
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7:
5V GTIATGGAIAAICCITCCCCICTICTIGTIGG 3 '(9) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF SEQUENCE: SEQ ID NO: 8:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) NUMBER OF STRANDS: single
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN:
(A) ORGANISM: Human
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8:
5V GAATCATCGAAIACIACGAAICCGAA 3 '(9) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF SEQUENCE: SEQ ID NO: 9:
(A) LENGTH: 2089base pairs
(B) TYPE: nucleic acid
(C) NUMBER OF STRANDS: double
(D) CONFIGURATION: linear
(ii) TYPE OF MOLECULE: cDNA
(iii) HYPOTHETICAL: NO
(iii) ANTI-SENSE: NO
(vi) ORIGIN:
(A) ORGANISM: Human
(xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9:
gaattcgggcggggtttgtactatcctcggtgctgtggtgcagagctagttcctctccagctcagccgcgta 72
ggtttggacatatttactcttttccccccaggttgaattgaccaaagca ATG GTG ATG GAG AAG 136
MVMEK 5
CCT AGT CCC CTG CTG GTC GGG CGG GAA TTT GTG AGA CAG TAT TAC ACA CTG CTG 190
PSPLLVGREFVRQYYTLL 23
AAC CAG GCC CCA GAC ATG CTG CAT AGA TTT TAT GGA AAG AAC TCT TCT TAT GTC 244
NQAPDMLHRFYGKNSSYV 41
CAT GGG GGA TTG GAT TCA AAT GGA AAG CCA GCA GAT GCA GTC TAC GGA CAG AAA 298
HGGLD 5 NGKPAOAVYGQK 59
GAA ATC CAC AGG AAA GTG ATG TCA CAA AAC TTC ACC AAC TGC CAC ACC AAG ATT 352
EIHRKVMSQNFTNCHTKI 77
CGC CAT GTT GAT GCT CAT GCC ACG CTA AAT GAT GGT GTG GTA GTC CAG GTG ATG 406
RHVOAHATLNOGVVVQVM 95
GGG CTT CTC TCT AAC AAC AAC CAG GCT TTG AGG AGA TTC ATG CAA ACG TTT GTC 460
GLLSNNNQA t RRFMQTFV 113
CTT GCT CCT GAG GGG TCT GTT GCA AAT AAA TTC TAT GTT CAC AAT GAT ATC TTC 514
LAPEGSVANKFYVHNDIF 131
AGA TAC CAA GAT GAG GTC TTT GGT GGG TTT GTC ACT GAG CCT CAG GAG GAG TCT 568
RYQOEVFGGFVTEPQEES 149
GAA GAA GAA GTA GAG GAA CCT GAA GAA AGA CAG CAA ACA CCT GAG GTG GTA CCT 622
EEEVEEPEERQQTPEVVP 167
GAT GAT TCT GGA ACT TTC TAT GAT CAG GCA GTT GTC AGT AAT GAC ATG GAA GAA 676
OOSGTFYOQAVVSNOMEE 185
CAT TTA GAG GAG CCT GTT GCT GAA CCA GAG CCT GAT CCT GAA CCA GAA CCA GAA 730
HLEEPVAEPEPOPEPEPE 203
CAA GAA CCT GTA TCT GAA ATC CAA GAG GAA AAG CCT GAG CCA GTA TTA GAA GAA 784
QEPVSEIQEEKPEPV t EE 221
ACT GCC CCT GAG GAT GCT CAG AAG AGT TCT TCT CCA GCA CCT GCA GAC ATA GCT 838
TAPEOAQKSSSPAPADIA 239
CAG ACA GTA CAG GAA GAC TTG AGG ACA TTT TCT TGG GCA TCT GTG ACC AGT AAG 892
QTVQEO t RTFSWASVTSK 257
AAT CTT CCA CCC AGT GGA GCT GTT CCA GTT ACT GGG ATA CCA CCT CAT GTT GTT 946
NLPPSGAVPVTGIPPHVV 275
AAA GTA CCA GCT TCA CAG CCC CGT CCA GAG TCT AAG CCT GAA TCT CAG ATT CCA 1000
KVPASQPRPESKPESQIP 293
CCA CAA AGA CCT CAG CGG GAT CAA AGA GTG CGA GAA CAA CGA ATA AAT ATT CCT 1054
PQRPQROQRVREQRINIP 311
CCC CAA AGG GGA CCC AGA CCA ATC CGT GAG GCT GGT GAG CAA GGT GAC ATT GAA 1108
PQRGPRPIREAGEQGOIE 329
CCC CGA AGA ATG GTG AGA CAC CCT GAC AGT CAC CAA CTC TTC ATT GGC AAC CTG 1162
PRRMVRHPOSHQLFIGNL 347
CCT CAT GAA GTG GAC AAA TCA GAG CTT AAA GAT TTC TTT CAA AGT TAT GGA AAC 1216
PHEVOKSE t KOFFQSYGN 365
GTG GTG GAG TTG CGC ATT AAC AGT GGT GGG AAA TTA CCC AAT TTT GGT TTT GTT 1270
VVELRINSGGK t PNFGFV 383
GTG TTT GAT GAT TCT GAG CCT GTT CAG AAA GTC CTT AGC AAC AGG CCC ATC ATG 1324
VFDOSEPVQKVLSNRPIM 401
TTC AGA GGT GAG GTC CGT CTG AAT GTC GAA GAG AAG AAG ACT CGA GCT GCC AGG 1378
FRGEVR t NVEEKKTRAAR 419
GAA GGC GAC CGA CGA GAT AAT CGC CTT CGG GGA CCT GGA GGC CCT CGA GGT GGG 1432
EGORRONR t RGPGGPRGG 437
CTG GGT GGT GGA ATG AGA GGC CCT CCC CGT GGA GGC ATG GTG CAG AAA CCA GGA 1486
LGGGMRGPPRGGMVQKPG 455
TTT GGA GTG GGA AGG GGG CTT GCG CCA CGG CAG TAA tcttcatggatcttcatgcagcc 1545
FGVGRGLAPRQ * 467 atacaaaccctggttccaacagaatggtgaattttcgaoagcctttggtatcttggagtatgaccccagtct 1617 gttataaactgcttaagtttgtataattttactttttttgtgtgttaatggtgtgtgctccctctccctctc ttccctttcctgacctttagtctttcacttccaattttgtggaatgatattttaggaataacggacttttac 1689 1761 1833 ccgaattcgtaatcatggtcatagctgtttccgtgtgaaattgttatccgctcacaattccacacaacatac gagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgct cactgcccgctttccagtcgggaaacctgtcgtgccagcgcattaatgaatcggccaacgcgcggggagagg 1905 1977 2049 cggtttgcgtattgggcgccagggtggttttcttttcaccagtgagacgggcaacagctgattgcccttcac cgctggccctgagagagttgcagcaagcggtccacgctgg 2089

Claims (23)

1. Polypeptide capable of interacting with the SH3 domain of the GAP protein. 2. Polypeptide according to claim 1, characterized in that it comprises all or part of a peptide sequence chosen from the sequences SEQ ID N01, SEQ ID N "2, SEQ ID N" 3, SEQ ID N "4 and the derivatives. of these. 3. Polypeptide according to one of the preceding claims, characterized in that it comprises SEQ ID N0 i, SEQ ID N02, SEQ ID N "3 and / or SEQ ID N" 4. 4. Polypeptide according to one of the preceding claims, characterized in that it has a molecular weight of the order of 68 kDa. 5. Polypeptide according to claim I characterized in that it comprises all or part of the sequence SEQ ID N05 SEQ ID N "9 or a derivative thereof. 6. Polypeptide according to claim I or 5 characterized in that it comprises SEQ ID N "5. 7. Polypeptide according to claim 1, 5 or 6 characterized in that it is represented by SEQ ID N "9. 8. Polypeptide according to one of the preceding claims, characterized in that its tyrosine units do not phosphorylate in mitotic or growing cells. 9. Antibody or antibody fragment directed against a polypeptide according to one of claims I to 8. 10. Antibody or antibody fragment according to claim 9, characterized in that it is directed against a sequence chosen from the peptide sequences presented in SEQ ID N0l SEQ ID N 2, SEQ ID N "3, SEQ ID N" 4, SEQ ID N05 and SEQ ID N "9. I I. Antibody or antibody fragment according to claim 9 or 10, characterized in that it has the ability to inhibit the interaction between the GAP protein and a polypeptide according to one of claims 1 to 8. 12. Nucleotide sequence encoding a polypeptide as defined according to one of claims I to 8. 13. Nucleotide sequence according to claim 12 characterized in that it comprises (a) all or part of the sequence SEQ ID N "6 and / or of the sequence SEQ 1DON09 or of their complementary strand, (b) any sequence hybridizing with a sequence (a) and encoding a polypeptide capable of binding to the SH3 domain of the GAP protein, and (c) sequences derived from sequences (a) and (b) due to degeneration of the genetic code. 14. Nucleotide sequence according to claim 12 or 13, characterized in that it comprises the sequence SEQ ID N "6. 15. Nucleotide sequence according to claim 12, 13 or 14 characterized in that it is represented by SEQ ID N "9. 16. Antisense nucleic acid capable of at least partially inhibiting the production of polypeptides according to one of claims I to 8. 17. Use of a sequence according to one of claims 12 to 15 for the detection of the expression of a polypeptide according to one of claims 1 to 8 or for the demonstration of genetic abnormalities (poor splicing, polymorphism, point mutations, etc.). 18. Use of a polypeptide according to one of claims 1 to 8 for the production of a non-peptide or non-exclusively peptide compound capable of interacting with the GAP protein, by determining the structural elements of this polypeptide which are important for its activity and reproduction of these elements by non-peptide or non-exclusively peptide structures. 19. Pharmaceutical composition comprising as active principle at least one polypeptide according to one of claims 1 to 8 and / or an antibody or antibody fragment according to claim 9 to 11 and / or an antisense nucleic acid according to claim 16 and / or or a compound prepared according to claim 18. 20. A pharmaceutical composition according to claim 18 intended to modulate the activation of p21 proteins. 21. A pharmaceutical composition according to claim 19 intended for the treatment of cancers. 22. Use of an antibody or antibody fragment according to claim 9 to 11 and / or of a nucleotide sequence according to claim 12 to 15 for the detection of the expression and / or of an overexpression of a polypeptide. according to one of claims 1 to 8 amplified, mutated or rearranged in a biological sample. 23. Use of an antibody or antibody fragment according to one of claims 9 to 11 and / or of a nucleotide sequence according to one of claims 12 to 15 for the typing of cancers or of diseases linked to defects. signaling. 24. Use of an RNA or DNA nucleotide sequence capable of interacting with a polypeptide according to one of claims I to 8 for the preparation of a pharmaceutical composition intended for the treatment of cancers.