[go: up one dir, main page]

WO1997010252A1 - Adn codant une proteine sh2-inositol phosphatase (ship), proteine se liant a shc - Google Patents

Adn codant une proteine sh2-inositol phosphatase (ship), proteine se liant a shc Download PDF

Info

Publication number
WO1997010252A1
WO1997010252A1 PCT/US1996/014754 US9614754W WO9710252A1 WO 1997010252 A1 WO1997010252 A1 WO 1997010252A1 US 9614754 W US9614754 W US 9614754W WO 9710252 A1 WO9710252 A1 WO 9710252A1
Authority
WO
WIPO (PCT)
Prior art keywords
ship
seq
mammalian
cells
isolated
Prior art date
Application number
PCT/US1996/014754
Other languages
English (en)
Inventor
Larry R. Rohrschneider
Mario N. Lioubin
Original Assignee
Fred Hutchinson Cancer Research Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fred Hutchinson Cancer Research Center filed Critical Fred Hutchinson Cancer Research Center
Priority to AU69780/96A priority Critical patent/AU6978096A/en
Publication of WO1997010252A1 publication Critical patent/WO1997010252A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes

Definitions

  • Fms is a tyrosine kinase growth factor receptor closely related to the c-Kit, Flt3 and PDGF receptors. While the PDGF receptor is primarily expressed in fibroblasts and endothelial cells, Fms, Kit and Flt3 are expressed in a lineage dependent fashion in hematopoietic cells. Flt3 is expressed in very early progenitor cells, Kit in intermediate developing cells as well as some mature lineages, and Fms is expressed in later stages of hematopoiesis when monocyte/macrophage development occurs. These receptors control signals for growth, survival, differentiation, and expression of mature cell functions.
  • Fms and its signal transduction pathways appear to be integral to the overall process of hematopoietic cell development.
  • the initial stages after Fms activation in the Fms signal transduction pathways have been characterized.
  • M-CSF macrophage colony-stimulating factor
  • CSF-1 homodimeric macrophage colony-stimulating factor
  • the receptor is dimerized, transphosphorylated on specific cytoplasmic tyrosines and is down-modulated by internalization and degradation.
  • Receptor autophosphorylation sites serve as docking sites for binding of signal transduction molecules which transmit and amplify signals downstream, and additional cytoplasmic proteins are tyrosine phosphorylated, although it is not clear what kinases are involved in this process.
  • a tyrosine phosphorylated protein of 145-150 kDa has been identified in a number of hematopoietic cell lines after stimulation with a variety of lymphokines.
  • Antigen receptor cross-linking in B-cells, IL-3, Steel factor and erythropoietin stimulation of erythroid cells and megakaryocytes or M-CSF stimulation of monocytes and erythroid cells induces the tyrosine phosphorylation of a 145 kDa protein that is found in a complex with She, Grb2 and Sos.
  • She and pl45 is not well understood, due either to different pl45-150 molecules or to different phosphorylation patterns from stimulation of different cells with different lymphokines.
  • Fms induces the tyrosine phosphorylation of pl50 and its association with She and Grb2/Sos in hematopoietic cells
  • this tyrosine phosphorylated protein has not been observed in Fms-expressing fibroblasts after M-CSF stimulation.
  • PDGF stimulation of fibroblasts induces the phosphorylation of a 145 kDa protein that associates with the She-PTB domain. Whether these two molecules are different proteins or whether an intermediate molecule required in Fms signaling for pl50 activation is not present in fibroblasts is unknown.
  • the present invention provides isolated and substantially pure preparations of mammalian SHIP and biologically active fragments thereof.
  • the invention also provides antibodies to SHIP, in the form of antisera and/or monoclonal antibodies.
  • the invention provides the ability to produce SHIP and polypeptides or fragments thereof by recombinant means, preferably in cultured eukaryotic cells.
  • the expressed SHIP or fragments thereof may or may not have the biological activity of corresponding native SHIP.
  • polynucleotides which code for SHIP and fragments thereof, where the polynucleotides may be in the form of DNA, such as cDNA, or
  • probes may be used to hybridize and identify these and related genes which encode SHIP.
  • the probes may also be used to identify mutations in SHIP for diagnostic purposes.
  • the probes may be full length cDNA or as small as from 14 to 25 nucleotides, more often though from about 40 to about 50 or more nucleotides.
  • the invention concerns DNA constructs which comprise a transcriptional promoter, a DNA sequence which encodes SHIP or a fragment thereof, and a transcriptional terminator, each operably linked for expression of the SHIP.
  • the polynucleotide molecules encoding SHIP and antibodies to SHIP may be used to identify mutations in SHIP that are associated with certain diseases, such as cancer.
  • the invention relates to methods for diagnosis, where the polynucleotides molecules and antibodies are used to detect the presence of SHIP mutations in a biological sample.
  • an antibody which specifically binds SHIP or a SHIP mutant is incubated with the sample under conditions conducive to immune complex formation, which complexes are then detected, typically by means of a label such as an enzyme, fluorophore, radionuclide, chemiluminescer, particle, or a second labeled antibody.
  • a label such as an enzyme, fluorophore, radionuclide, chemiluminescer, particle, or a second labeled antibody.
  • SH2-Inositol Phosphatase is a protein capable of binding to She, a transforming protein with an SH2 domain that is implicated in mitogenic signal transduction. Activation of tyrosine kinase receptors, such as Fms, the EGF receptor, insulin receptor and PDGF receptor results in She phosphorylation and Ras activation. Ras activation to its GTP-bound form is facilitated by the nucleotide exchange factor Sos.
  • Grb2 a 23 kD protein composed entirely of one SH2 domain flanked by two SH3 domains, binds through both of its SH3 domains to proline-rich sequences of Sos and through its SH2 domain to other tyrosine-phosphorylated proteins such as activated EGF receptor or tyrosine kinase substrates such as She. Interactions of Grb2 with any of these tyrosine- phosphorylated proteins results in the translocation of Sos to the plasma membrane, where it stimulates Ras to exchange GDP for GTP.
  • She proteins phosphorylated on tyrosine and participate in a complex formation with Fms, Grb2, and SHIP by directly interacting with Grb2 and SHIP.
  • SHIP inositol phosphate 5-phosphatase
  • IP5P inositol phosphate 5-phosphatase
  • Purified SHIP has an apparent molecular weight of 150 kD as determined by SDS PAGE, and a calculated molecular weight of 133,438, as determined from an open reading frame translating for an 1190 amino acid protein. For consistency, the molecular weight of SHIP is referred to herein as 150 kD.
  • the present invention provides representative polynucleotide sequences encoding SHIP.
  • SHIP contains an SH2 (src homology-2) domain at its N- terminus, a C-terminal region with two NPXY [SEQ ID NO:14] motifs that are known to bind phosphotyrosine binding (PTB) domains and numerous proline rich clusters with motifs that are known to bind SH3 (src homology-3) domains.
  • the present invention provides the advantage that SHIP encodes a protein involved in signal transduction and the control of cell proliferation.
  • the nucleotide sequences encoding the protein appear related to sequences encoding the human 51C gene product associated with Fanconi's anemia, the X-linked Lowe's oculocerebrorenal syndrome (OCRL) gene, and inositol polyphosphate 5-phosphatases.
  • OCRL Lowe's oculocerebrorenal syndrome
  • the symptoms associated with these genes are considered to be related to errors in maintaining DNA fidelity at replication.
  • SHIP may thus function in cell cycle checkpoint control, in that the activation of SHIP following M-CSF stimulation of Fms may positively regulate a checkpoint pathway for the transit of cells from Gl to the S phase, although this is offered by way of possible explanation and not limitation.
  • SHIP was found in large complexes containing at least She, Grb2/Sos and PI3 kinase. Mutations which abolish Fms' interactions with PI3 kinase
  • the phosphorylated protein was purified and amino acid sequence information obtained for Lys C-generated peptides. Degenerate oligonucleotides based on these peptides were designed for use in PCR technology to screen cDNA libraries, but were not successful in generating cDNA fragments corresponding to SHIP.
  • a modified yeast two-hybrid system was combined with the high affinity binding of the Shc-PTB domain for phosphorylated SHIP to clone a cDNA encoding for SHIP.
  • the yeast two hybrid system includes a LexA fusion vector pBTMll ⁇ which permits the in-frame fusion of LexA to a protein of interest ("bait") and a VP16 fusion vector, pVP16, into which cDNA fragments are ligated to create a library.
  • the LexA:bait can bind to the LexA operator but is unable to activate transcription because it cannot associate with RNA polymerase.
  • VP16 can associate with RNA polymerase but does not recognize the LexA operator. Accordingly, cotransformation of pBTM116:bait and pVP16 does not activate HIS3 transcription.
  • a bait binding partner fused to VP16 provides a bridge between LexA and VP16 and consequently activates HIS3 expression.
  • Yeast cotransfected with pBTM116:bait and pVP16 : ait-binding partner grows when plated on selective medium lacking histidine.
  • this system can be utilized to identify protei :protein interactions, it is limited in identifying protein:phosphotyrosine interactions because yeast do not express active tyrosine kinases.
  • the pBTM116 vector was modified to express a tyrosine kinase, in addition to the LexA:bait fusion protein.
  • the PTB domain of She was fused to the LexA protein (LexA-PTB) as bait, and the EGF receptor C-terminal tail was fused to the VP16 protein as a target for testing the ability of kinases to phosphorylate the known PTB-binding motif (NPXY) in the EGF receptor cytoplasmic tail (EGFRT) domain and bind the Shc-PTB domain.
  • NPXY PTB-binding motif
  • EGFRT EGF receptor cytoplasmic tail
  • Shc-PTB domain Shc-PTB domain.
  • the PDGF receptor cytoplasmic domain was the most effective in phosphorylating the target EGF cytoplasmic domain with resultant VP16-EGFRT binding, with growth of ⁇ -Gal positive cells.
  • the two hybrid screen used the pBTM116/PDGFR vector with the Shc-PTB domain (aa 48-209) as the bait, and a VP16-EML library. Transformants that were positive for transcriptional activation were "cured" to allow loss of the pBTMll ⁇ vector and mated with a panel of new lexA:baits to ensure specificity.
  • the same cDNA insert (EML-11) was used as a probe to screen a polyA cDNA library constructed from FDC-P1 clonel9 and Macll cells.
  • Two of the 10 clones identified contained inserts of approximately 5.0 kb and one of these clones (clone pBK-CMV-150.8) was chosen for DNA sequencing in both orientations.
  • the clone pBK-CMV-150.8 contained a 4863 nucleotide insert with an open reading frame (ORF) translating for 1190 amino acid protein with a calculated molecular weight of 133,438.
  • N-terminus of SHIP is occupied by an SH2 domain and the C-terminal region contains two NPXY motifs (NPNY).
  • NPNY amino acids 914-917 of SEQ ID NO:13
  • NPLY amino acids 1017-1020 of SEQ ID NO: 13
  • the C-terminal one-fourth of the molecule contains numerous proline rich clusters of which at least three appear to be motifs that are known to bind SH3 domains.
  • ATP or GTP binding proteins share a conserved motif or P loop within a glycine rich region that is believed to interact with one of the phosphate groups of the nucleotide.
  • SHIP contains such a motif (GQPLHGKS) [SEQ ID NO:15] only seven amino acids after the NPNY [SEQ ID NO:13] sequence.
  • the SHIP cDNA insert from clone pBK-CMV-150.8 was used to identify and clone a partial human cDNA encoding human SHIP.
  • the missing 5' coding sequence of human SHIP were obtained by PCR amplification of SHIP sequences from a human HL-60 library.
  • the human SHIP contains a 3566 bp coding seuqence and a deduced amino acid sequence of 1189 amino acids [SEQ ID NOS:26 and 27] .
  • the central portion of SHIP exhibits 55% identity and 73% similarity to the human 51C gene product.
  • the 51C cDNA was cloned by its ability to complement the Fanconi anemia group C complementation (FACC) gene for hypersensitivity to DNA-damaging agents.
  • the FACC gene encodes a 60 kDa protein and antibodies to this protein also recognize a related 150 kDa protein.
  • the similarity between SHIP and 51C also extends to the nucleotide level.
  • SHIP also shares homology with the X-linked Lowe's oculocerebrorenal syndrome gene (OCRL) .
  • SHIP, 51C, and the OCRL protein all share amino acid sequence homology with proteins involved in inositol metabolism which remove the 5' phosphate of inositol (1,4,5) and/or inositol (1,3,4,5) .
  • SHIP'S homology with these proteins is not as extensive as with 51C, numerous smaller domains are conserved among SHIP,
  • M-CSF activation of Fms leads to the downstream tyrosine phosphorylation within at least the NPNY motif of a 150 kDa inositol polyphosphate-5' -phosphatase-like protein, SHIP, and its association at this site with She through the amino-terminal PTB domain.
  • SHIP inositol polyphosphate-5' -phosphatase-like protein
  • the SH2 domain of SHIP permits additional interactions with tyrosine phosphorylated proteins, and proline-rich sequences in the carboxyl domain can couple to proteins containing SH3 domains.
  • SHIP is tyrosine phosphorylated and associates with She after stimulation of responsive erythroid cells with either Epo or M-CSF.
  • SHIP or a related tyrosine phosphorylated protein is detected after M-CSF stimulation of Fms-expressing myeloid cells after activation of the EpoR and IL-3R. Also, SHIP participates in signaling by IL-3 and M-CSF in FD(Fms) cells (and weakly by GM-CSF stimulation) . In contrast, the pl45 tyrosine phosphorylated and She-PTB-binding protein in PDGF- stimulated fibroblasts differs from SHIP because no protein with these properties was tyrosine phosphorylated after M-CSF stimulation of Rat2 (Fms) fibroblast cells.
  • inositol polyphosphate-5' - phosphatase in a Fms signaling pathway indicates a function in Ca 2+ signaling. While offered by way of possible explanation and not limitation, as inositol-1,4, 5-triphosphate increases the intracellular concentration of Ca 2+ , SHIP may terminate that signal. Additionally, 5' -phosphorylated phosphoinositides can bind to pleckstrin homology domains and activate such molecules as the Akt/PKB kinase, and again SHIP may regulate this process.
  • the genes to which SHIP shows homology are linked to several disease states in humans.
  • the 51C gene was isolated by its complementation of the DNA repair defect in cells from a Fanconi anemia patient. This defect also predisposes individuals to an increased incidence of myeloid leukemia.
  • the mutated OCRL protein linked to the oculocerebrorenal syndrome of Lowe, is characterized by congenital cataracts, mental retardation, and defective renal tubular function. The broad spectrum of symptoms in each condition indicates an error in maintaining DNA fidelity at replication.
  • a number of other hereditary syndromes also fall into this same category: ataxia telangiectasia (AT) , Bloom's, Werner's (premature aging) , and xeroderma pigmentosum.
  • SHIP fits into a group of proteins and their associated diseases that indicates a relationship to Ca 2+ metabolism and/or events that prevent the propagation of DNA damage by controlling transition at the Gl/S boundary of the cell cycle.
  • the present invention provides representative polynucleotide molecules and amino acids sequences encoding SHIP.
  • Sequences encoding SHIP include those sequences that result in minor variations in amino acid sequence, such as those due to genetic polymorphisms, allelic variations, differences between species and those in which blocks of amino acids have been added, altered or replaced without substantially altering the biological activity of the proteins.
  • polynucleotide molecules encoding SHIP are capable of hybridizing under stringent conditions to an oligonucleotide of 14 or more contiguous nucleotides of SEQ ID NO:12 or SEQ ID NO:26, and the complementary strand of SEQ ID NO:12 or SEQ ID NO:26.
  • the polynucleotide molecules encoding Ship include sequences substantially identical to the sequences of SEQ ID NO: 12 or SEQ ID NO:26
  • the terms “substantially corresponds to”, “substantially homologous”, or “substantial identity” as used herein denotes a characteristic of a nucleic acid sequence, wherein a nucleic acid sequence has at least about 70 percent sequence identity as compared to a reference sequence, typically at least about 85 percent sequence identity, and preferably at least about 95 percent sequence identity as compared to a reference sequence. The percentage of sequence identity is calculated excluding small deletions or additions which total less than 25 percent of the reference sequence.
  • the reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome. However, the reference sequence is at least 18 nucleotides long, typically at least about 30 nucleotides long, and preferably at least about 50 to 100 nucleotides long. "Substantially complementary” as used herein refers to a sequence that is complementary to a sequence that substantially corresponds to a reference sequence.
  • the disclosed sequences may be used to identify and isolate SHIP polynucleotide molecules from suitable mammalian hosts such as canine, ovine, bovine, equine, caprine, or lagomorph, or avian hosts.
  • Nucleotide sequences 5' or 3 ' of sequence domain encoding the inositol phosphate 5-phosphatase (IP-5P) may be particularly useful in identifying polynucleotide molecules encoding SHIP from other species.
  • Complementary DNA molecules encoding SHIP may be obtained by constructing a cDNA library mRNA from, for example, myeloid cells, monocytes, and macrophages.
  • DNA molecules encoding SHIP may be isolated from such a library using the disclosed sequences in standard hybridization techniques (e.g., Sambrook et al . , Molecular Cloning: A Laboratorv Manual, Second Edition, Cold Spring Harbor, NY, 1989 and Bothwell, Yancopoulos and Alt, Methods for Cloning and Analysis of Eukaryotic Genes, Jones and Bartlett Publishers, Boston, MA 1990) or by amplification of sequences using polymerase chain reaction (PCR) amplification (e.g, Loh et al.
  • PCR polymerase chain reaction
  • genomic DNA encoding SHIP may be obtained using probes designed from the sequences disclosed herein. Upstream regulatory regions of SHIP may be obtained using the same methods. Suitable PCR primers are between 7-50 nucleotides in length, more preferably between 14 and 25 nucleotides in length. Polynucleotide molecules encoding SHIP may be isolated using standard hybridization using probes of at least about 7 nucleotides in length and up to and including the full coding sequence.
  • hybridization conditions will be evident to one skilled in the art and will generally be guided by the purpose of the hybridization, the type of hybridization (DNA-DNA or DNA-RNA) , and the level of relatedness between the sequences.
  • Methods for hybridization are well established in the literature; See, for example: Sambrook, ibid.; Hames and Higgins, eds, Nucleic Acid Hybridization A Practical Approach, IRL Press, Washington DC, 1985; Berger and Kimmel, eds, Methods in Enzymology, Vol. 52. Guide to Molecular Cloning Techniques, Academic Press Inc., New York, NY, 1987; and Bothwell, Yancopoulos and Alt, eds, Methods for Cloning and Analysis of Eukaryotic Genes. Jones and Bartlett Publishers,
  • Hybridization stringency can be altered by: adjusting the temperature of hybridization; adjusting the percentage of helix-destabilizing agents, such as formamide, in the hybridization mix; and adjusting the temperature and salt concentration of the wash solutions. In general, the stringency of hybridization is adjusted during the post-hybridization washes by varying the salt concentration and/or the temperature.
  • Stringency of hybridization may be reduced by reducing the percentage of formamide in the hybridization solution or by decreasing the temperature of the wash solution.
  • High stringency conditions may involve high temperature hybridization (e.g., 65-68°C in aqueous solution containing 4-6X SSC, or 42°C in 50% formamide) combined with high temperature (e.g., 5-25°C below the T m ) and a low salt concentration (e.g., 0.IX SSC) .
  • Reduced stringency conditions may involve lower hybridization temperatures (e.g., 35-42°C in 20-50% formamide) with intermediate temperature (e.g., 40-60°C) and washes in a higher salt concentration (e.g., 2-6X SSC) .
  • Moderate stringency conditions which may involve hybridization at a temperature between 50°C and 55°C and washes in 0. lx SSC, 0.1% SDS at between 50°C and 55°C, may be used to identify clones encoding polynucleotide molecules encoding SHIP from other species.
  • Recombinant SHIP may be produced by inserting an isolated DNA molecule encoding SHIP into a suitable expression vector, which is in turn used to transfect or transform a suitable host cell.
  • Suitable expression vectors for use in carrying out the present invention will comprise a promoter capable of directing the transcription of a polynucleotide molecule of interest in a host cell.
  • Representative expression vectors may include both plasmid and/or viral vector sequences.
  • Suitable vectors include retroviral vectors, vaccinia viral vectors, CMV viral vectors, BLUESCRIPT (Stratagene Cloning Systems) , baculovirus vectors, and the like.
  • Promoters capable of directing the transcription of a cloned gene or cDNA may be inducible or constitutive promoters and include viral and cellular promoters.
  • suitable viral promoters include the immediate early cytomegalovirus promoter (Boshart et al., Cell 41: 521-530, 1985) and the SV40 promoter (Subramani et al . , Mol. Cell. Biol. 1: 854-864, 1981) .
  • Suitable cellular promoters for expression of proteins in mammalian host cells include the mouse metallothionien-1 promoter (Palmiter et al . , U.S. Patent No.
  • transcription termination signal located downstream of the coding sequence of interest. Suitable transcription termination signals include the early or late polyadenylation signals from SV40 (Kaufman and Sharp, Mol . Cell. Biol. 2:1304-1319, 1982) , the polyadenylation signal from the Adenovirus 5 elB region and the human growth hormone gene terminator (DeNoto et al . , Nucleic Acid. Res. 9: 3719- 3730, 1981) . Mammalian cells for example may be transfected by a number of methods including calcium phosphate precipitation (Wigler et al .
  • Mammalian cells can be transduced with virus vectors such as SV40, CMV and the like.
  • virus vectors such as SV40, CMV and the like.
  • cloned DNA molecules may be introduced by infection of susceptible cells with viral particles.
  • Retroviral vectors may be preferred for use in expressing SHIP in mammalian cells particularly if SHIP is used for gene therapy (for review, see, Miller et al. , Meth. Enzymol.
  • selectable markers may be used to identify cells that contain the cloned DNA.
  • Selectable markers are generally introduced into the cells along with the cloned DNA molecules and include genes that confer resistance to drugs, such as neomycin, hygromycin and methotrexate. Selectable markers may also complement auxotrophies in the host cell . Yet other selectable markers provide detectable signals, such as beta- galactosidase to identify cells containing the cloned DNA molecules.
  • Selectable markers may be amplifiable. Such amplifiable selectable markers may be used to amplify the number of sequences integrated into the host genome.
  • the polynucleotide molecules of the present invention may be expressed Saccharomyces cerevisiae, filamentous fungi, and E. coli.
  • Methods for expressing cloned genes in Saccharomyces cerevisiae are generally known in the art (see, "Gene Expression Technology,” Meth. Enzymol. , vol. 185, Goeddel (ed.), Academic Press, San Diego, CA, 1990 and "Guide to Yeast Genetics and Molecular Biology, " Meth. Enzymol. , Guthrie and Fink (eds.), Academic Press, San Diego, CA, 1991; which are incorporated herein by reference).
  • Filamentous fungi may also be used to express the proteins of the present invention.
  • Methods for expressing genes and cDNAs in cultured mammalian cells and in E. coli is discussed in detail in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Second Ed.. Cold Spring Harbor, NY, 1989; which is incorporated herein by reference) .
  • one could express the protein of the instant invention in other host cells such as avian, insect and plant cells using regulatory sequences, vectors and methods well established in the literature.
  • the SHIP polynucleotides of the present invention may be useful in gene therapy protocols in which it is desirable to overexpress SHIP, such as in protocols designed to re-establish checkpoint control in cancer or other diseases associated with loss of checkpoint control.
  • SHIP proteins produced according to the present invention may be purified using a number of established methods such as affinity chromatography using anti-SHIP antibodies coupled to a solid support. Additional purification may be achieved using conventional purification means such as liquid chromatography, gradient centrifugation and gel electrophoresis among others. Methods of protein purification are known in the art (see generally, Scopes, R., Protein Purification, Springer-Verlag, NY, 1982, which is incorporated herein by reference) and may be applied to the purification of recombinant SHIP described herein. Thus, SHIP is provided isolated from its natural cellular environment, and substantially free of other cellular proteins.
  • Purified SHIP is also provided, where substantially pure SHIP of at least about 50% is preferred, at least about 70-80% is more preferred, and 95-99% or more homogeneity most preferred.
  • the recombinant SHIP or native SHIP may be used to generate antibodies, diagnostically in assay procedures, etc.
  • Antisense SHIP nucleotide sequences may be used to block overexpression of SHIP or expression of mutant forms of SHIP.
  • Suitable antisense oligonucleotides are at least 11 nucleotide in length and up to and including the upstream untranslated and associated coding sequences.
  • the optimal length of antisense oligonucleotides is dependent on the strength of the interaction between the antisense oligonucleotides and their complementary sequence on the mRNA, the temperature and ionic environment in which translation takes place, the base sequence of the antisense oligonucleotide, and the presence of secondary and tertiary structure in the mRNA and/or in the antisense oligonucleotide.
  • Suitable target sequences for antisense oligonucleotides include intron-exon junctions (to prevent proper splicing) , regions in which DNA/RNA hybrids will prevent transport of mRNA from the nucleus to the cytoplasm, initiation factor binding sites, ribosome binding sites, and sites that interfere with ribosome progression.
  • a particularly preferred target region for antisense oligonucleotide is the 5' untranslated region of the gene of interest.
  • Antisense oligonucleotides may be prepared by the insertion of a DNA molecule containing the target DNA sequence into a suitable expression vector such that the DNA molecule is inserted downstream of a promoter in a reverse orientation as compared to the gene itself.
  • antisense oligonucleotides may be synthesized using standard manual or automated synthesis techniques. Synthesized oligonucleotides may be introduced into suitable cells by a variety of means including electroporation, calcium phosphate precipitation microinjection. The selection of a suitable antisense oligonucleotide administration method will be evident to one skilled in the art. With respect to synthesized oligonucleotides, the stability of antisense oligonucleotides- mRNA hybrids may be increased by the addition of stabilizing agents to the oligonucleotide.
  • Stabilizing agents include intercalating agents that are covalently attached to either or both ends of the oligonucleotide.
  • Oligonucleotides may be made resistant to nucleases by, for example, modifications to the phosphodiester backbone by the introduction of phosphotriesters, phosphonates, phosphorothioates, phosphoroselenoates, phosphoramidates or phosphorodithioates.
  • Oligonucleotides may also be made nuclease resistant by the synthesis of the oligonucleotides with alpha-anomers of the deoxyribonucleotides.
  • the invention also provides SHIP protein fragments and peptides.
  • the SHIP peptides and fragments of the invention are desirably biologically active.
  • the peptides can be prepared via chemical synthesis, as described hereinbelow, or by recombinant DNA technology, or as fusion proteins, and the like. Desirably, the peptide will be as small as possible while still maintaining substantially all of the reactivity of a larger peptide.
  • the subject peptides have an amino acid sequence encoded by a nucleic acid which hybridizes under stringent conditions with an oligonucleotide of 15 or more contiguous nucleotides of SEQ ID NO:12 or SEQ ID NO:27.
  • the peptides and fragments of the present invention or analogs thereof which have She binding or other reactivity may be modified from a native SHIP sequence as necessary to provide other desired attributes, e.g., improved binding or inhibitory activity (e.g., increased competition with native protein) , improved adsorption to a solid phase, etc.
  • the peptides may be subject to various changes, such as insertions, deletions, and substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use.
  • conservative substitutions is meant replacing an amino acid residue with another which is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another.
  • substitutions include combinations such as Gly, Ala; Val, lie, Leu; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • sequence of the peptide will not differ by more than about 20% from the native SHIP sequence, except where additional amino acids may be added at either terminus for the purpose of modifying the physical or chemical properties of the peptide for, e.g., ease of linking or coupling, and the like. Having identified different peptides of the invention, in some instances it may be desirable to join two or more peptides in a composition or admixture.
  • the peptides in the composition can be identical or different, and together they should provide equivalent or greater reactivity than the parent peptide (s) .
  • the subject peptides find a variety of uses including preparation of specific antibodies. Because of their relatively short size, the peptides can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis. 2d. ed. , Pierce Chemical Co. (1984) ; Tarn et al . , J. Am. Chem. Soc.
  • Short peptide sequences, or libraries of overlapping peptides usually from about 6 up to about 35 to 50 amino acids, which correspond to the selected regions described herein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides.
  • recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • the invention provides antibodies which bind to and are specific for SHIP.
  • non-human antisera or monoclonal antibodies e.g., murine, lagomorpha, porcine, equine
  • monoclonal antibodies e.g., murine, lagomorpha, porcine, equine
  • antibody producing cells are obtained from immunized animals, immortalized and screened, or screened first for the production of the antibody that binds to the SHIP protein or peptides and then immortalized.
  • a human monoclonal antibody or portions thereof may be identified by first screening a human B-cell cDNA library for DNA molecules that encode antibodies that specifically bind to SHIP according to the method generally set forth by Huse et al. (Science 246: 1275-1281, 1989, which is incorporated by reference herein in its entirety) . The DNA molecule may then be cloned and amplified to obtain sequences that encode the antibody (or binding domain) of the desired specificity.
  • polynucleotide molecules, proteins and antibodies of the present invention provide methods for detecting genetic abnormalities at the SHIP locus. Such methods may be useful for genetic counseling, prognosis and staging of disease.
  • diagnostic methods and compositions are disclosed. Given the disclosed isolated polynucleotide molecules of the present invention and antibodies to SHIP, a variety of diagnostic assays are provided.
  • the present invention provides a number of reagents that find use in assays to detect and/or quantitate levels of SHIP.
  • Such reagents may be labeled with compounds that provide a detectable signal using conventional methods.
  • Such labels which include, for example, chemiluminescers, paramagnetic particles, fluorophores, radionuclides, enzymes, enzyme substrates, and the like may be used in such assay methods to facilitate detection and/or quantitation of SHIP.
  • Antibodies against SHIP proteins may be used as reagents to detect wild-type and/or mutant SHIP in biological samples such as tumor biopsy samples, tissue and organ sections, peripheral blood cells and the like.
  • antibodies of the present invention may be used in immunoassays to detect and/or quantitate SHIP.
  • Immunoassays suitable for use in the present invention include, but are not limited to, enzyme-linked immunosorbant assays, immunoblots, inhibition or competition reactions, sandwich assays, radioimmunoprecipitation, and the like, as generally described in, e.g., U.S. Pat. Nos.
  • Isolated DNA probes and PCR primers are designed as reagents for diagnostic assays for detecting the presence of SHIP or SHIP mutant sequences.
  • the nature of the specific assay may depend on the type of mutational analysis to be carried out and the type of biological sample to be assayed. High molecular weight DNA may be obtained from suitable sources using commercially available kits.
  • kits include, the Genomic Isolation Kit A.S.A.P. (Boehringer Mannheim, Indianapolis, IN) , Genomic DNA Isolation System (GIBCO BRL, Gaithersburg, MD) , Elu-Quik DNA
  • PCR primers find used in the amplification of SHIP sequences from normal and diseased tissue. Such sequences may be analyzed by direct sequence analysis or by a variety of means to distinguish normal from mutant sequence. A comparison of fragment size and or comparison of sequences may be used to diagnose a number of diseases such as cancer.
  • SHIP-specific DNA probes are used in restriction fragment length polymorphism (RFLP) assays on DNA samples isolated from normal and diseased tissues to detect rearrangements and/or deletions of the SHIP gene. More subtle mutations may be detected by a variety of methods which include but are not restricted to single strand conformation polymorphism (SSCP) (Orita et al . , Proc. Natl. Acad. Sci. USA 86: 2766-2770, 1989; which is incorporated by reference herein) ; dideoxy fingerprinting
  • SSCP single strand conformation polymorphism
  • the diagnostic and screening methods of the invention find use for individuals suspected of being at risk for developing a SHIP-associated or related disease such as cancer (e.g., family history of disease) or for patients in which such a screening is used to diagnose or eliminate SHIP- associated disease as a causative agent behind a patient's symptoms.
  • methods for screening involve biological samples from the patient is provided (e.g., tissue biopsy and amniotic fluid samples) and the sample is screened for the presence of mutations in SHIP.
  • a patient's SHIP gene is compared to normal SHIP DNA (i.e., wild-type SHIP) using a variety of methods, including RFLP analyses, SSCP, and the like, and mutations in SHIP are detected.
  • An aberrant SHIP DNA size pattern such as for RFLP analysis or SSCP analysis, aberrant SHIP protein and/or aberrant SHIP protein levels as determined by antibody assays would indicate that the patient has developed or is at risk to develop a SHIP-associated disease.
  • Prenatal diagnosis can be performed when desired, using a variety of methods to obtain fetal cells. These methods include, but are not limited to amniocentesis, chorionic villous sampling and fetoscopy. Prenatal analysis of the SHIP gene is carried out using SSCP, RFLP, DDF and the like.
  • kits and multicontainer units comprising reagents and components for practicing the assay methods of the present invention.
  • Kits of the present invention may, in addition to reagents for detecting SHIP, contain enzymatic reagents such as reverse transcriptase or polymerase; suitable buffers; nucleoside triphosphates; suitable labels for labeling the reagents for detecting SHIP and developing reagents for detecting the signal from the label.
  • kits of the present invention contain sequence-specific oligonucleotide primers for detecting polynucleotide molecules encoding SHIP. Such primers may be provided in separate containers or may be provided in combinations of one or more primer pairs in a series of containers.
  • kits containing SHIP sequence-specific probes contain antibodies useful for detecting SHIP (or mutants thereof) in a sample.
  • the kits may also contain instructions for carrying out the assay and/or additional containers suitable for carrying out the reactions of the assay.
  • assays are designed for the screening of therapeutic compounds capable of replacing SHIP function or suppressing mutant SHIP function in cells containing SHIP mutants that result in abnormal cell proliferation.
  • cells demonstrating abnormal cell proliferation due to a mutation in SHIP are exposed to test compounds.
  • the cell proliferation of the treated cells are compared with the cell proliferation of untreated cells.
  • Test compounds capable of replacing SHIP function or suppressing mutant SHIP function are those compounds that cause a reduction in the abnormal cell proliferation of the mutant cell.
  • the polynucleotide molecules encoding SHIP are used in screen for compounds that enhance or inhibit the interaction between SHIP and its binding partners.
  • a yeast two hybrid screen essentially as described in detail herein, is established in which the bait portion of the LexA-bait hybrid protein is a SHIP-binding domain such as the PTB domain of She or the SH3 domain of Grb2 and the bait-binding portion of the bait-binding-VP16 hybrid protein contains at least the complementary binding domain of SHIP.
  • the bait portion of the LexA-bait hybrid protein is at least one binding domain of SHIP and the bait-binding portion of the bait-binding-VP16 hybrid protein is a SHIP-binding domain such as the SHIP-binding domains present in the She or Grb2.
  • Binding domains of SHIP include the SH2 domain, the PTB domain and the SH3 domain.
  • Test compounds that inhibit the interaction of the bait and bait-binding domains result in a decrease in reporter gene expression relative to the expression level in the absence of the test compound.
  • Test compounds that enhance the interaction between the bait and bait-binding domains may be assessed by detecting or measuring an increase in the expression of the reporter gene. Such compounds may find use, for example, in re-establishing checkpoint control in cancer and other diseases associated with the loss of checkpoint control.
  • polynucleotide molecules of the present invention may be used within the yeast two hybrid system described herein to screen for polynucleotide molecules capable of binding to other domains within SHIP.
  • discrete binding domains of SHIP are used as bait in the LexA-bait hybrid and a VP16 fusion library is screened for clones capable of binding to the SHIP binding domain in the bait hybrid by screening for clones capable of expressing the reporter gene(s) .
  • animals such as mice, and cell lines may be constructed that are heterozygous or homozygous for deletions of the SHIP genes.
  • Such "knock-out" animals and cell lines may be useful as disease models and as test systems for therapeutics capable of overcoming SHIP deletion or mutations.
  • the SHIP gene may be deleted by homologous recombination using the method essentially set forth by Buerstedde and Takeda (Cell 67: 179- 188, 1991; which is incorporated by reference herein) .
  • homologous recombination may be used to delete the SHIP gene in pluripotent mouse embryonic-derived stem (ES) cells (Thomas and Capecchi, Cell 51: 503-512, 1987 and Doetschman et al. , Nature 330: 576-578, 1987; which are incorporated by reference herein) .
  • ES pluripotent mouse embryonic-derived stem
  • murine 32D WT cells (Greenberger et al., Proc. Natl. Acad. Sci. USA 80: 2931-2935 (1983)) that had been infected with a retroviral vector encoding wild-type fms were grown at 37°C, 5% C0 2 and 95% humidity in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and WEHI conditioned medium which provided the IL-3 (see Lioubin et al., Mol. Cell. Biol. 14: 5682-5691 (1994); which is incorporated by reference herein in its entirety) .
  • Approximately 4-6 x 10 11 cells were stimulated with a total of 8 x IO 6 Units of M-CSF for one minute at 37°C to stimulate tyrosine phosphorylation.
  • the cells were lysed by incubation in lysis buffer (20 mM Tris (pH 7.5), 10 mM EDTA, 100 mM NaCl, 1 mM ZnCl 2 , 0.05% NaN 3 , 2% polyoxylethylene 9 lauryl ether (referred to hereinafter as C 12 E 9 ) , 2 mM orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 20 mM iodoacetamide and 5 ⁇ g/ml leupeptin) for 15 minutes on ice.
  • lysis buffer (20 mM Tris (pH 7.5), 10 mM EDTA, 100 mM NaCl, 1 mM ZnCl 2 , 0.05% NaN 3 , 2% poly
  • lysates were centrifuged at 25,000 x g for thirty minutes. The pellet was discarded, and the lysate was loaded onto an anti- phosphotyrosine column prepared essentially as described by Schneider et al. , J. Biol. Chem. 257: 10766-10769 (1982) ; which is incorporated by reference herein in its entirety) in load buffer (50 mM Tris (pH 7.5) , 0.05% NaN 3 , 150 mM NaCl, 2% C 12 E 9 ) .
  • load buffer 50 mM Tris (pH 7.5) , 0.05% NaN 3 , 150 mM NaCl, 2% C 12 E 9
  • wash buffer 50 mM Tris (pH 7.5) , 0.05% NaN 3 , 300 mM NaCl, 0.1% SDS, 0.1% C 12 E 9 .
  • the tyrosine phosphorylated proteins were eluted from the column by the addition of 100 mM phenyl phosphate in a buffer containing 50 mM Tris (pH 7.5) , 0.05% NaN 3 , 150 mM NaCl, and fractions were collected.
  • the 150 kD tyrosine phosphorylated protein was cleaved using the method essentially described by Patterson et al . (Anal. Biochem. 202: 193-203 (1992) ; which is incorporated by reference herein in its entirety) .
  • the column fractions were concentrated by 10% trifluoroacetic acid (TFA) precipitation followed by washes with 90% ethanol.
  • TFA trifluoroacetic acid
  • the proteins were transferred to an IMMOBILON-CD transfer membrane (a cationic, PVDF membrane; Millipore Corporation, Bedford, MA) according to the manufacturer's instructions, and the membrane was stained using an IMMOBILON CD staining kit (Millipore) according the manufacturer's instructions.
  • IMMOBILON-CD transfer membrane a cationic, PVDF membrane; Millipore Corporation, Bedford, MA
  • the protein band at 150 kD was excised.
  • the excised membrane was washed five times with MILLI-Q H 2 0 (Millipore) before the membrane was immersed in Digestion buffer (50 mM Tris-HCl (pH 8.5) , 2 mM DTT) .
  • Digestion buffer 50 mM Tris-HCl (pH 8.5) , 2 mM DTT
  • Endopeptidase Lys-C digestion was initiated by the addition of 1 ⁇ g of sequencing grade Endopeptidase Lys-C (Promega) .
  • the reaction mixture was sonicated for 5-10 seconds followed by an overnight incubation at 37°C. After overnight incubation, an additional 0.5 ⁇ g of Lys-C was added to the reaction mixture, and the mixture was incubated at 37°C for an additional eight hours.
  • the combined supernatant was diluted with 0.1% TFA in H 2 0 to achieve a final CH 3 CN concentration of no more than 3.5%.
  • the sample was applied by multiple injections on an Ultrafast Microprotein Analyzer (Michrom BioResources, Inc.) fitted with a 1 x 150 mm Reliasil C18 column.
  • a stem cell factor-dependent lympho-hematopoietic progenitor cell line (EML-Cl; deposited under accession number
  • EML-Cl cDNA library CRL 11691 on July 25, 1994, at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland, 20852 was used as a cell source for the preparation of an EML-Cl cDNA library.
  • the EML-Cl cell line was derived from bone marrow cells of male BDF X mice that had been infected with the retroviral vector containing the truncated retinoic acid receptor-alpha (RAR ⁇ ) cDNA and has been described in PCT application WO 95/04142 (which is incorporated by reference herein) .
  • PolyA enriched RNA from EML-Cl cells was prepared. Following second-strand synthesis the ds-cDNAs were size- selected for cDNAs greater than 350 bp.
  • the cDNAs were ligated to Not I linkers, and the linkered cDNAs were ligated into the Not I cloning site in the 2 ⁇ yeast expression vector pVP16 (Vojtek et al. , Cell 74: 205-214 (1993) and Hollenberg et al., Mol. Cell. Biol. 15: 3812-3822 (1995) ; which are incorporated by reference herein in their entirety) .
  • the pVPl ⁇ vector contains the VP16 activation domain of Herpes simplex virus (HSV) under the control of the Saccharomyces cerevisiae alcohol dehydrogenase promoter; and both the LEU2 and Ampicillin-resistance selectable markers.
  • HSV Herpes simplex virus
  • VP16-EML library Insertion of a DNA molecule of interest into the Not I site of the pVP16 vector resulted in expression of a VP16 hybrid protein having the protein of interest joined in-frame with VP16.
  • the resultant cDNA library was termed the "VP16-EML library”.
  • the two-hybrid screen relies on the interaction between two hybrid proteins (the LexA-bait protein and bait- binding protein-VP16 hybrids) to activate the expression of a reporter gene it is of limited use for proteins that require post-translational processing before binding can occur.
  • the interaction between SHIP and She requires the phosphorylation of SHIP.
  • She the bait portion of the LexA- bait hybrid in a screen for binding with a SHIP-VP16 hybrid (the bait-binding-VP16 hybrid)
  • the host cells must be capable of phosphorylating SHIP in the yeast host cell.
  • the yeast two hybrid screen was modified to include an additional expression unit that directed the expression of a tyrosine kinase.
  • tyrosine kinases were tested for the ability to promote phosphorylation of a hybrid protein containing the full-length She joined to VP16 in a two hybrid system containing a LexA-bait hybrid containing the Grb2 SH2 domain as the bait.
  • the /3-PDGF receptor cytoplasmic domain (amino acids 570-1106) was shown to provide the most effective tyrosine phosphorylation of target proteins fused to VP16.
  • the LexA-bait containing the phosphotyrosine-binding domain of She (Shc-PTB) was also tested for its ability to bind the EGFR tail with PDGFR as the tyrosine kinase.
  • the Shc-PTB domain was used as the bait portion of the LexA-bait hybrid.
  • the LexA-Shc-PTB hybrid was capable of binding DNA but not activating transcription of a downstream reporter gene.
  • the VP16-EML library (Example 2) provided a library of VP16 hybrid proteins containing candidate PTB- binding domains.
  • the yeast two hybrid screen was used to identify members of the VP16-EML library capable of binding to the LexA-Shc-PTB hybrid and therefore activating transcription of reporter genes.
  • the S_. cerevisiae host strain L40 contained multimerized LexA binding sites inserted upstream of two reporter genes, the HIS3 gene and the S-galactosidase gene.
  • An expression unit comprising the ADH promoter joined to the PDGF receptor cytoplasmic domain (the tyrosine kinase) was inserted into the vector pBTM116 at the Pvu II site.
  • the Shc-PTB domain (from amino acid 48 to 209 of She) was subcloned into the Sal I cloning site of the same vector such that the Shc-PTB and LexA coding sequences were in-frame.
  • the resulting plasmid pBTM116/PDGFR/Shc-PTB was co-transformed with Quiagen-purified (Quiagen, Chatsworth, CA) VP16-EML-CDNA library.
  • Transformants were grown on medium lacking histidine, leucine, tryptophan, uracil, lysine and containing 50 mM 3-amino triazol to select for the presence of both plasmids (the LexA-Shc-PTB plasmid and the VP16-EML-CDNA library plasmid) .
  • High levels of 3-amino triazol were essential to reduce false positives by reducing non-specific interactions between the LexA and VP16 domains.
  • Colonies capable of growing on the selective medium contained VP16-EML library clones that were capable of interacting with the LexA- ShcPTB hybrid after tyrosine phosphorylation.
  • pBTM116/PDGFR/Shc-PTB LexA-Shc-PTB and the PDGFR expression unit
  • diploids were then selected on medium lacking histidine, leucine, tryptophan, uracil, lysine and containing 50 mM 3-amino triazol.
  • the colonies were scored for ⁇ - galactosidase activity. Only those clones that required both the presence of the tyrosine kinase and the Shc-PTB domain for growth under selection and that demonstrated / S-galactosidase activity were selected for further analysis.
  • Plasmid DNA prepared from thirty-six positive transformants were subjected to Hae III restriction enzyme analysis.
  • the plasmid DNA was also subjected to PCR amplification (35 cycles of (94°C for 15 seconds, 55°C for 15 seconds, 72°C for 2 minutes)) using primers derived from flanking sequences in the pVP16 vector.
  • the PCR products from fourteen of the clones were subjected to DNA sequence analysis. All fourteen clones contained an NPXY motif [SEQ ID NO:14] known to bind the Shc-PTB domain. Ten of the fourteen clones appeared to be derived from the same gene, which appeared to be unique.
  • the deduced amino acid sequence of the overlapping cDNAs was shown to contain two of the peptides [SEQ ID Nos:7 and 11] determined by Lys-C digestion. This confirmed that the ten clones contained sequences encoding SHIP.
  • the cDNA insert from clone EML-11 was random-primed and used as a probe to screen a combined polyA cDNA library constructed from murine FDC-P1 clone 19 (FDC-P1 cells that were infected with the Fms provirus produced by ⁇ 2 cells; Lioubin et al . , Mol. Cell. Biol. 14: 5682-5691 (1994)) and Macll cells (Gliniak and Rohrschneider; Cell 63: 1073-1083 (1990)) using the Stratagene UNIZAP EXPRESS kit (Stratagene Cloning Systems; La Jolla, CA) according to the manufacturer's instructions.
  • Positive clones were subjected to secondary screens, tertiary screens and PCR amplification using primers designed from internal SHIP sequences. Ten positive clones were subjected to further analysis. Of the ten clones, two contained inserts of approximately 5.0 kb. One of the two clones, designated pBK-CMV-150.8, was sequenced in both orientations. Plasmid pBK-CMV-150.8 was deposited as an E. coli transformant at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, on September 12, 1995, under accession number ATCC 69898.
  • the insert present in pBK-CMV-150.8 contained a 4863 nucleotide insert with an open reading frame translating for a 1190 amino acid protein with a calculated molecular weight of 133,438 [SEQ ID Nos :12 and 13] .
  • the methionine at position 1 is believed to be the translational start given the presence of a SHIP peptide fragment [SEQ ID NO:6] corresponding to amino acids 2 through 9 that contained the methionine at position 4.
  • Lys-C peptides [SEQ ID NOs:1-11] derived from the native SHIP were distributed throughout the open reading frame (amino acids 2-9; amino acids 18-27; amino acids 66-73; amino acids of 307-322; amino acids 346-351; amino acids 355-358; amino acids 359-364; amino acids 514-523; amino acids 789-799; amino acids 866-871; and amino acids 1162-1171 of SEQ ID NO:13) .
  • the amino-terminus of SHIP contains an SH2 domain (residues 8 to 105 of SEQ ID NO:13) , and the carboxyl-terminus region contains two NPXY [SEQ ID NO:14] motifs (at amino acids 914-917 and 1017-1020 of SEQ ID NO:13) that are known to bind PTB domains. From the 10 clones identified using the two hybrid screen, three contained only the NPNY (amino acids 914- 917, of SEQ ID NO:13) motif and seven clones contained both the NPXY motifs.
  • the carboxy-terminal one-fourth of the molecule contains numerous proline rich clusters, of which at least three show homology to motifs known to bind SH3 domains.
  • the deduced amino acid sequence also contained a GQPLHGKS [SEQ ID NO:15] motif seven amino acids C-terminal to the NPNY (amino acids 914-917, of SEQ ID NO:13) that shows homology with the conserved motif shown for ATP or GTP binding proteins.
  • Clone pBK-CMV-150.8 showed 55% identity from amino acids 188 to 856 of SEQ ID NO:12 with the human 51C gene product (Genbank Accession No. L36818) at both the amino acid and nucleotide level. Protein sequence comparisons with other proteins showed SHIP to have an IP-5P domain (amino acids 430- 805 of SEQ ID NO:13) that shared homology with both type I and type II inositol phosphate 5-phosphatases. An alignment of the conserved amino acid sequences among inositol phosphate 5- phosphatases and SHIP (subdomains of the IP-5P domain) showed that the SHIP IP-5P domain contained signature sequences for inositol phosphate 5-phosphatases. TABLE I. Alignment of conserved amino acid sequences among inositol phosphate 5-phosphatases and SHIP.
  • IT5P2 270 EGELTFQPTYKY 301 PGVIG.FLWK 326 TSDHKPV
  • the numbers at the left of each column denote amino acid position within each molecule [SEQ ID NO:13] .
  • b 51C human 51C gene product [SEQ ID NO:18] .
  • c OCRL human oculocerebrorenal gene product [SEQ ID NO:19] .
  • d IT5P human inositol triphosphate 5-phosphatase gene product [SEQ ID NO:20] .
  • the cDNA insert from clone EML-11 was random-primed and used as a probe to screen a polyA cDNA library constructed from human HL-60 cells (Nature 270: 347-349 (1977)) ; available from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852 under accession number CCL-240) using the Stratagene UNIZAP kit (Stratagene Cloning Systems; La Jolla, CA) according to the manufacturer's instructions.
  • the library was plated, and plaques were lifted onto maximum strength NYTRAN PLUS (Schleicher & Schuell, Keene, NH) membranes.
  • plaque lifts were then autoclaved for one minute and UV crosslinked with a Stratalinker (Stratagene Cloning Systems) UV Irradiator.
  • the lifts were prehybridized for a minimum of three hours in prehybridization solution (50% deionized formamide, 4X SSC, 5X Denhardt's, 50 mM NaPi (pH)
  • the murine cDNA insert from clone EML-11 was random-primed using the READY-TO-GO (Pharmacia Biotech, Piscataway, NJ) kit according to the manufacturer's instructions. The lifts were removed from the prehybridization solution and hybridized for a minimum of 18 hours with the 32 P-labeled SHIP cDNA in hybridization solution (50% deionized formamide, 4X SSC, IX Denhardt's, 50 mM NaPi (pH 7.0), 0.5 mg/ml NaPPi, 0.1 mg/ml sheared salmon DNA, 1%SDS) .
  • clone h9.1 contained an insert of approximately 4.2 kb.
  • the insert was sequenced using DYEDEOXY TERMINATORS (Applied Biosystems, Foster City, CA) . Sequence analysis confirmed that the clone contained sequences with high homology to the EML-11 clone, and comparison of the 5' end of this clone revealed that it did not contain the full-length sequence.
  • the HL-60 library was rescreened for a full-length cDNA using a PCR-amplified probe.
  • the HL-60 library was subjected to PCR amplification (one cycle of 95°C for 1 minute, 35 cycles of (94°C for 30 seconds, 55°C for 30 seconds, 72°C for 90 seconds) , cooled to 4°C) using a sense primer designed to correspond to nucleotides 181 to 198 of SEQ ID NO:12 (from the murine SHIP), and an antisense primer (CGTTACCCATGTTCCAGG; SEQ ID NO:21) designed to complement to nucleotides 1320 to 1337 of the human SHIP nucleotide sequence [SEQ ID NO:26] to generate a radiolabeled probe.
  • the library was probed essentially as described above with the exception that the hybridization was carried out at room temperature. Positive clones were subjected to secondary screens, and tertiary screens. The longest clone, designated h4.2-A2, contained an approximately 4.7 kb insert. Sequence analysis indicated that this clone contained more 5 ' sequence than the initial clone, but did not contain a full-length open reading frame.
  • the HL-60 library was amplified (one cycle of 94°C for 1 minute, 35 cycles of (94°C for 30 seconds, 57°C for 30 seconds, 72°C for 60 seconds) , cooled to 4°C) using a sense T3 primer (CGAAATTAACCCTCACTAAAGGG; SEQ ID NO:22) and an antisense primer (GATGAGCTGGTCCAGCTTG; SEQ ID NO:23) complimentary to nucleotides 322 to 340 of the human SHIP sequence [SEQ ID NO:26] .
  • a sense T3 primer CGAAATTAACCCTCACTAAAGGG; SEQ ID NO:22
  • an antisense primer GATGAGCTGGTCCAGCTTG; SEQ ID NO:23
  • Amplification products greater than 400 bp in length were pooled and ligated into pT7BLUE(R) vector (Novagen, Madison, WI) .
  • the ligation products were transformed into £. coli strain XLl-BLUE (Stratagene Cloning Systems) .
  • the transformants were screened using a blue/white beta-galactosidase screen.
  • the PCR products were electrophoresed in an agarose gel and products of greater than the expected 650 bp fragment size were eluted and subjected to sequence analysis.
  • the SHIP vector and an empty LXSN vector were each transfected into Psi2 packaging cells (Mann et al. , ibid.) and selected in Dulbecco's modified Eagles medium containing 10% calf serum and 0.5 mg/ml G418 to permit packaging of the vector.
  • the resultant retrovirus was used to infect Rat2 cells (ATCC CRL 1764) .
  • Rat2 cells were grown in Dulbecco's modified Eagles medium containing 10% fetal bovine serum.
  • Western analysis of lysates of cells containing the SHIP vector demonstrated a 150 kD protein in contrast to cells containing an empty vector.
  • FDC-P1 cells FDC-P1 cells transformed with Fms
  • FDC-P1 cells were originally described by Duhrsen and Metcalf (Leukemia 2: 329- 333, (1989) ; which is incorporated herein by reference in its entirety) and a clone (clone 19) was selected for its ability to differentiate along the macrophage lineage after retroviral expression of Fms and growth in M-CSF (Bourette et al., Growth Diff. 6:631-645, (1995) ; which is incorporated herein by reference in its entirety) .
  • FDC-P1 and FD-Fms were each infected by co-culture over Psi2 cells infected with either empty vector (LXSN) or the SHIP vector.
  • LXSN empty vector
  • SHIP vector the SHIP vector
  • Factor-dependent growth was measured in 1 milliliter agar cultures with modification of the methods described by Rohrschneider and Metcalf (Mol. Cell. Biol. 9 : 5081-5092, (1989) ; which is incorporated herein by reference in its entirety) . Briefly, one volume of 2x Iscove's medium (3.54 g Iscove's powder (Sigma, St. Louis, MO) , 0.605 g sodium bicarbonate, 20 ml fetal bovine serum and 200 U/ml penicillin + 200 micrograms/milliliter streptomycin in 100 ml of water) was mixed with one volume of 0.62% agar at 42°C.
  • 2x Iscove's medium 3.54 g Iscove's powder (Sigma, St. Louis, MO) , 0.605 g sodium bicarbonate, 20 ml fetal bovine serum and 200 U/ml penicillin + 200 micrograms/milliliter streptomycin in 100 ml of water
  • the cells were added to the agar mixture in a small volume of Dulbecco's Modified Eagles (DME) containing 10% fetal bovine serum, and one milliliter of each mixture was plated onto a non-treated 33 mm plastic culture dish. Each assay was performed in quadruplicate. Appropriate growth factors were added to the bottom of the dishes prior to the addition of cells. After 7- 10 days, the colonies were counted and cultures were photographed. The average number of colonies for each culture condition (four plates) was calculated with the standard error. No differences in SHIP expression or cellular behavior were detected between cells infected with the empty vector and cell infected with the SHIP vector.
  • DME Dulbecco's Modified Eagles
  • FD-Lx cells formed large colonies and FD-Ship cells formed fewer, and in general, smaller colonies.
  • FD-Fms cells infected with empty vector produced colonies of the size and number seen previously for FD-Fms cells (RohrSchneider and Metcalf, ibid.).
  • the FD- Fms/Ship cells (FD-Fms cells infected with Ship vector) formed fewer and significantly smaller colonies than the control FD- Fms/Lx cells grown in the presence of M-CSF.
  • Fms/Ship cells were grown in IL-3 the colony size was similar to the control FD-Fms/Lx cells but a reduction in colony number was observed.
  • FD-Fms/Ship cells grown in the presence of M-CSF formed mainly small colonies in soft agar, a few were larger and compact like those formed in the presence of IL-3. These larger colonies presumably lack the retrovirally expressed SHIP yet were G418 resistant and accounted for the outgrowth of "normal" cells and lack of Ship overexpression after longer term culture.
  • the expression level of Ship was examined in both FD-Fms/Lx and FD-Fms/Ship cells after infection by 2-day growth over Psi2 cells followed by a three day selection in 0.75 milligram/milliliter G418. Immunofluorescence detection of the SHIP protein with affinity purified antibodies demonstrated that the FD-Fms/Ship cells expressed slightly higher levels of the protein than the endogenous Ship detected in the FD-Fms/Lx cells. These results contrasted sharply with the relative ease of Ship overexpression in Rat2 and Psi2 cells and indicated that expression levels are very tightly regulated in the FDC-Pl cells and that overexpression may have deleterious effects.
  • the growth inhibitory role of Ship and its inositol 5 phosphatase activity in Fms signaling is opposite that reported for the positive mitogenic effects of PI-3 Kinase activity.
  • Polyclonal rabbit antibodies to murine SHIP domains were raised to GST fusion proteins containing either amino acids 670-688 of SEQ ID NO:12 or amino acids 889-1046 of SEQ ID NO:12.
  • Antisera to the C-terminal GST fusion were affinity purified by antigen affinity chromatography.
  • the affinity purified antisera against the unique C- terminal region of SHIP (amino acids 889-1046 of SEQ ID NO:12) recognized a major 150 kD protein in hematopoietic (32D-Fms) cells that co-migrated with the tyrosine phosphorylated protein observed after M-CSF stimulation (Lioubin et al., Mol. Cell. Biol. 14: 5682-5691, (1994) ; which is incorporated herein by reference in its entirety) .
  • the 150kD SHIP band was the most prominant but often appeared as a doublet, and multiple minor bands (up to five) below the main protein were always detectable. All of the SHIP proteins contained phosphotyrosine.
  • 5-phosphatase assays were carried out on murine Ship immunoprecipitates. Briefly, radiolabeled phospholipids were first prepared in a kination reaction with phosphotidyl inositol 3-kinase (PI3 Kinase) . PI3 Kinase was prepared by immunoprecipitation from Psi2 cells (Mann et al . , Cell 33: 153-159, (1983) ; which is incorporated herein by reference in its entirety) using a rabbit polyclonal antibody (Upstate Biotechnology Inc., Lake Placid, NY) .
  • PI3 Kinase phosphotidyl inositol 3-kinase
  • the PI3 Kinase- containing immune pellets were washed six times in kinase buffer (50 mM Hepes (pH 7.5), 0.5 mM EDTA, 5 mM MgCl 2 ) , and 25 microliter portions were aliquoted into microfuge tubes.
  • Radiolabeled Ptdlns (3*,4) P 2 and Ptdlns (3*,4, 5) P 3 were prepared by first drying 100 micrograms each of Ptdlns (4) P and Ptdlns (4, 5) P 2 (Sigma, St. Louis, MO) under argon.
  • the dried material was resuspended in 100 microiiters of kinase buffer by sonication for five minutes on ice in a batch sonicator. Each lipid suspension was added to an aliquot of the PI3 Kinase immune pellet. The reaction was initiated by the addition of 3 P- ⁇ -ATP (Dupont/NEN) (at 3,000 Ci/mmol) to a final concentration of 150 nM. After an incubation for one hour at room temperature, 100 microiiters of chloroform/methanol) (1:1 v:v) was added to each reaction, and the organic phase of each reaction containing the radiolabeled phospholipids was washed twice with 200 microiiters of 2M KCI.
  • 3 P- ⁇ -ATP (Dupont/NEN) (at 3,000 Ci/mmol)
  • the radiolabeled phospholipids were used immediately or were stored under argon at -70°C.
  • reaction products were separated on Silica Gel 60 TLC plates that had been treated with 1% potassium oxalate in 50% ethanol and allowed to dry at least 24 hours at room temperature before use.
  • the TLC plates were developed as described by Traynor-Kaplan et al . (Nature 334 : 353-356, (1988); which is incorporated by reference herein in its entirety) using a solvent mixture of chloroform/acetone/methanol/glacial acetic acid/water (80:30:26:24:14 - v:v:v:v) .
  • Radiolabeled phospholipids were detected by radiography. The identity of the phospholipids was obtained by comparison of chromatographic mobility on the same TLC plate.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Immunology (AREA)
  • General Engineering & Computer Science (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention porte sur des molécules polynucléotidiques isolées codant une protéine SHIP de mammifère ou des fragments de celles-ci. On peut utiliser ces molécules polynucléotidiques isolées dans des procédés de production d'une protéine SHIP de recombinaison ou de fragments biologiquement actifs de celle-ci ou bien dans le cadre de méthodes diagnostiques. Il est possible d'utiliser des polynucléotides de la protéine SHIP selon l'invention dans des criblages pour l'identification de composés à usage thérapeutique, capables d'inhiber ou de renforcer l'expression de cette protéine. Il est également possible d'employer des anticorps dirigés contre la protéine SHIP pour identifier des molécules mutantes de cette protéine dans des méthodes diagnostiques de maladies associées à la protéine SHIP telles que le cancer.
PCT/US1996/014754 1995-09-14 1996-09-13 Adn codant une proteine sh2-inositol phosphatase (ship), proteine se liant a shc WO1997010252A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU69780/96A AU6978096A (en) 1995-09-14 1996-09-13 Dna encoding an sh2-inositol phosphatase, an shc-binding protein

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US384195P 1995-09-14 1995-09-14
US60/003,841 1995-09-14

Publications (1)

Publication Number Publication Date
WO1997010252A1 true WO1997010252A1 (fr) 1997-03-20

Family

ID=21707846

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1996/014754 WO1997010252A1 (fr) 1995-09-14 1996-09-13 Adn codant une proteine sh2-inositol phosphatase (ship), proteine se liant a shc

Country Status (2)

Country Link
AU (1) AU6978096A (fr)
WO (1) WO1997010252A1 (fr)

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997022690A3 (fr) * 1995-12-08 1997-09-12 Chiron Corp 5-phosphatases de polyphosphate d'inositol de signalisation
FR2792000A1 (fr) * 1999-04-08 2000-10-13 Centre Nat Rech Scient Procede de cartographie d'une molecule d'adn comprenant une etape d'amplification ad infinitum
WO2002024233A3 (fr) * 2000-09-19 2003-03-13 Univ South Florida Regulation de la fonction et de la survie de la cellule nk par modulation de l'activite ship
WO2004032880A3 (fr) * 2002-10-11 2004-08-12 Isis Pharmaceuticals Inc Methode permettant d'inhiber l'angiogenese a l'aide d'inhibiteurs ship-1
WO2003053341A3 (fr) * 2001-12-06 2005-05-26 Isis Pharmaceuticals Inc Modulation antisens de l'expression de ship-1
US7250505B1 (en) 1997-08-21 2007-07-31 Fred Hutchinson Cancer Research Center Isolation and expression of a disabled protein gene mDab1 and methods
WO2008067666A1 (fr) * 2006-12-04 2008-06-12 British Columbia Cancer Agency Branch Modulation allostérique de polypeptides ship et ses utilisations
US7691821B2 (en) 2001-09-19 2010-04-06 University Of South Florida Inhibition of SHIP to enhance stem cell harvest and transplantation
US7713945B2 (en) 2000-09-19 2010-05-11 University Of South Florida Control of NK cell function and survival by modulation of SHIP activity
US7763592B1 (en) 2003-11-20 2010-07-27 University Of South Florida SHIP-deficiency to increase megakaryocyte progenitor production
US7807646B1 (en) 2003-11-20 2010-10-05 University Of South Florida SHIP-deficiency to increase megakaryocyte progenitor production
US8956824B2 (en) 2006-12-04 2015-02-17 British Columbia Cancer Agency Branch Methods for identifying allosteric modulators of ship polypeptides

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BLOOD, October 15, 1996, Vol. 88, No. 8, WARE et al., "Cloning and Characterization of Human SHIP, the 145-kD Inositol 5-Phosphatase that Associates with SHC After Cytokine Stimulation", pages 2833-2840. *
GENES AND DEV., 01 May 1996, Vol. 10, MAJERUS, P.W., "Inositols do it All", pages 1051-1053. *
GENES AND DEV., May 1996, Vol. 10, LIOUBIN et al., "P150Ship, a Signal Transduction Molecule with Inositol Polyphosphate-5-Phosphatase Activity", pages 1084-1095. *
NATURE, 19 September 1996, Vol. 383, ONO et al., "Role of the Inositol Phosphatase SHIP in Negative Regulation of the Immune System by the Receptor Fc RIIB", pages 263-266. *
PROC. NATL. ACAD. SCI. U.S.A., February 1996, Vol. 93, DAMEN et al., "The 145-kDa Protein Induced to Associate with Shc by Multiple Cytokines is an Inositol Tetraphosphate and Phosphatidylinositol 3,4,5-Trisphosphate 5-Phosphatase", pages 1689-1693. *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997022690A3 (fr) * 1995-12-08 1997-09-12 Chiron Corp 5-phosphatases de polyphosphate d'inositol de signalisation
US7250505B1 (en) 1997-08-21 2007-07-31 Fred Hutchinson Cancer Research Center Isolation and expression of a disabled protein gene mDab1 and methods
FR2792000A1 (fr) * 1999-04-08 2000-10-13 Centre Nat Rech Scient Procede de cartographie d'une molecule d'adn comprenant une etape d'amplification ad infinitum
WO2000061798A3 (fr) * 1999-04-08 2001-03-15 Centre Nat Rech Scient Procede de cartographie d'une molecule d'adn comprenant une etape d'amplification $i(ad infinitum)
US6780593B1 (en) 1999-04-08 2004-08-24 Centre National De La Recherche Scientifique (Cnrs) Method for mapping a DNA molecule comprising an ad infinitum amplification step
WO2002024233A3 (fr) * 2000-09-19 2003-03-13 Univ South Florida Regulation de la fonction et de la survie de la cellule nk par modulation de l'activite ship
US8163710B2 (en) 2000-09-19 2012-04-24 University Of South Florida Reduction of graft-versus-host disease by modulation of SHIP activity
US7713945B2 (en) 2000-09-19 2010-05-11 University Of South Florida Control of NK cell function and survival by modulation of SHIP activity
US7691821B2 (en) 2001-09-19 2010-04-06 University Of South Florida Inhibition of SHIP to enhance stem cell harvest and transplantation
WO2003053341A3 (fr) * 2001-12-06 2005-05-26 Isis Pharmaceuticals Inc Modulation antisens de l'expression de ship-1
WO2004032880A3 (fr) * 2002-10-11 2004-08-12 Isis Pharmaceuticals Inc Methode permettant d'inhiber l'angiogenese a l'aide d'inhibiteurs ship-1
US7807646B1 (en) 2003-11-20 2010-10-05 University Of South Florida SHIP-deficiency to increase megakaryocyte progenitor production
US7763592B1 (en) 2003-11-20 2010-07-27 University Of South Florida SHIP-deficiency to increase megakaryocyte progenitor production
US8008273B2 (en) 2003-11-20 2011-08-30 University Of South Florida SHIP-deficiency to increase megakaryocyte progenitor production
WO2008067666A1 (fr) * 2006-12-04 2008-06-12 British Columbia Cancer Agency Branch Modulation allostérique de polypeptides ship et ses utilisations
US8956824B2 (en) 2006-12-04 2015-02-17 British Columbia Cancer Agency Branch Methods for identifying allosteric modulators of ship polypeptides

Also Published As

Publication number Publication date
AU6978096A (en) 1997-04-01

Similar Documents

Publication Publication Date Title
Dahl et al. A testis-specific form of the human pyruvate dehydrogenase E1α subunit is coded for by an intronless gene on chromosome 4
CA2111205C (fr) Polypeptides ayant une activite kinase, preparation et utilisation
US5686412A (en) Protein kinases
Mizuki et al. Functional modules and expression of mouse p40 phox and p67phox, SH3‐domain‐containing proteins involved in the phagocyte NADPH oxidase complex
CA2343939A1 (fr) Genes et proteines predictifs et methodes therapeutiques destinees aux attaques, a l'hypertension, aux diabetes et a l'obesite
US6210666B1 (en) Truncated α-galactosidase A to treat fabry disease
JPH08506480A (ja) 新種蛋白質分離のための相互作用を用いる補捉システム
WO1997010252A1 (fr) Adn codant une proteine sh2-inositol phosphatase (ship), proteine se liant a shc
EP0914452A2 (fr) Nouvelles proteines ptp20, pcp-2, bdp1, clk et sirp, produits et procedes connexes
AU2001263952B2 (en) Tumour suppressor and uses thereof
AU2001263952A1 (en) Tumour suppressor and uses thereof
EP0732398B1 (fr) Nouvelle tyrosine kinase
US6797501B2 (en) Protein tyrosine phosphatase PTP20 and related products and methods
JPH11123086A (ja) セリン−トレオニンプロテインキナーゼ（ｈ−ｓｇｋ２）
US7368113B2 (en) Hormonally up-regulated, neu-tumor-associated kinase
JP3907247B2 (ja) Ｔａｂ１蛋白質及びそれをコードするｄｎａ
JP2000512147A (ja) ＩＬ―１／ＴＮＦ―α活性化キナーゼ（ＩＴＡＫ）、並びに当該キナーゼを作成しそして使用する方法
US6297019B1 (en) Recombinant polynucleotides encoding CYP7 promoter-binding factors
US5627064A (en) Protein kinases
JPH10117788A (ja) ヒト・ｍｙｔ−１キナーゼクローン
US20030022311A1 (en) Human CIS protein
US5840536A (en) Growth factor receptor-binding insulin receptor
EP1017826A2 (fr) Compositions et techniques permettant d'identifier les inhibiteurs de la kinase de la pkb
US6682920B1 (en) Compositions and methods for identifying PKB kinase inhibitors
WO1997020573A1 (fr) Homologue de proteine 2 se liant au recepteur du facteur de croissance

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA