Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Reexamination Certificate
1999-05-14
2001-05-29
Prouty, Rebecca (Department: 1653)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
C435S252300, C435S254110, C435S325000, C435S320100, C536S023100, C536S023200
Reexamination Certificate
active
06238903
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a novel SH2-containing inositol-phosphatase, truncations, analogs, homologs and isoforms thereof; nucleic acid molecules encoding the protein and truncations, analogs, and homologs of the protein; and, uses of the protein and nucleic acid molecules.
BACKGROUND OF THE INVENTION
Many growth factors regulate the proliferative, differentiative and metabolic activities of their target cells by binding to, and activating cell surface receptors that have tyrosine kinase activity (Cantley, L. C., et al. 1991, Cell 64:281-302; and Ullrich, A., and J. Schlessinger. 1990, Cell 61:203-212). The activated receptors become tyrosine phosphorylated through intermolecular autophosphorylation events, and then stimulate intracellular signalling pathways by binding to, and phosphorylating cytoplasmic signalling proteins (Cantley, L. C., et al. 1991, Cell 64:281-302; and, Ullrich, A., and J. Schlessinger, 1990, Cell 61:203-212). Many cytoplasmic signalling proteins share a common structural motif, known as the src homology 2 (SH2) domain, that mediates their association with specific phosphotyrosine-containing sites on activated receptors (Heldin, C. H. 1991, Trends Biochem. Sci. 16:450-452; Koch, C. A., et al., 1991, Science 252:669-674; Margolis, B. 1992, Cell Growth Differ. 3:73-80; McGlade, C. J., et al, 1992, Mol. Cell. Biol. 12: 991-997; Moran, M. F., et al., 1990, Proc. Natl. Acad. Sci. USA 87:8622-8626; and Reedijk, M., et al., 1992, EMBO J. 11:1365-1372).
Two SH2-containing proteins, Grb2 and Shc, have been implicated in the Ras signalling pathway (Lowenstein, E. J.,et al.,1992, Cell 70:431-442, and, Pelicci, G., et al., 1992, Cell 70 93-104.). Grb2 and Shc act upstream of Ras and bind directly to activated receptors (Buday, L., and J. Downward, 1993, Cell 73:611-620; Matuoka, K. et al., 1993, EMBO J. 12:3467-3473, Oakley, B. R. et al., 1980, Anal. Biochem. 105:361-363., Reedijk, M., et al., 1992, EMBO J. 11:1365-1372; Rozakis-Adcock, M.,et al., 1992 Nature 360: 689-692; and, Songyang, Z.,et al., 1993, Cell 72:767-778), or to designated SH2 docking proteins, such as the insulin receptor substrate 1 (IRS-1), which is tyrosine phosphorylated in response to insulin (Baltensperger, K., et al., Science 260:1950-1952; Pelicci, G., et al., 1992, Cell 70:93-104; Skoinik, E. Y., 1993, EMBO J. 12:1929-1936; Skolnik, E. Y., et al., 1993, Science 260:1953-1955; and Suen, K-L., et al., 1993 Mol. Cell. Biol. 13: 5500-5512).
Grb2 is a 25 kDa adapter protein with two SH3 domains flanking one SH2 domain. It has been shown in fibroblasts to shuttle its constitutively bound Ras guanine nucleotide exchange factor, Sos1, to activated receptors (or to IRS-1 (Skolnik, E. Y., 1993, EMBO J. 12:1929-1936; and Skolnik, E. Y., et al., 1993, Science 260:1953-1955), (Baltensperger, K., et al., Science 260:1950-1952; Buday, L., and J. Downward, 1993, Cell 73:611-620; Egan, S. E. et al., 1993, Nature (London) 367:87-90; Gale, N. W., et al., 1993, Nature (London) 363:88-92; Li, N., et al., 1993, Nature (London) 363-85-88; Olivier, J. P. et al., 1993, Cell 73:179-191; and Rozakis-Adcock, M., et al., 1993 Nature (London) 363:83-85). Binding of the SH2 domain of Grb2 to tyrosine phosphorylated proteins activates Sos1 which then catalyzes the activation of Ras by exchanging GDP for GTP (Buday, L., and J. Downward. 1993. Cell 73:611-620 12,,20; Egan, S. E. Et al, 1993, Nature 363:45-51; Gale, N. W et al., 1993 Nature 363:88-92; Li, N., et al., 1993 Nature 363:85-88).
Shc is also an adapter protein that is widely expressed in all tissues. The protein contains an N-terminal phosphotyrosine binding (PTB) domain (Kavanaugh, V. M. Et al., 1995 Science, 268:1177-1179; Craparo, A., et al., 1995, J. Biol. Chem. 270:15639-15643; van der Geer, P., & Pawson, T., 1995, TIBS 20:277-280; Batzer, A. G., et al., Mol. Cell. Biol. 1995, 15:4403-4409; and Trub, T., et al., 1995, J. Biol. Chem. 270:18205-18208) and a C-terminal SH2 domain (Pelicci, G., et al., 1992. Cell 70:93-104) and can associate, in its tyrosine phosphorylated form, with Grb2-Sos1 complexes and may increase Grb2-Sos1 interactions following growth factor stimulation (Egan, S. E. Et al, 1993, Nature 363:45-51;Rozakis-Adcock, M., et al., 1992, Nature 360:689-692; and Ravichandran, K. S., 1995, Mol. Cell. Biol. 15:593-600). Shc appears to function as a bridge between Grb2-Sos1 complexes and tyrosine kinases where the latter are incapable, for lack of an appropriate consensus sequence, of binding Grb2-Sos1 directly (Egan, S. E. Et al, 1993, Nature 363:45-51).
Preliminary evidence suggests that Shc and Grb2 may be used by members of the hemopoietin receptor superfamily (Cutler, R. L., et al., 1993, J. Biol. Chem. 268:21463-21465, Damen, J. E.,et al., 1993, Blood 82:2296-2303). Although members of this family lack endogenous kinase activity, following ligand binding, they are apparently tyrosine phosphorylated by a closely associated JAK family member (Argetsinger, L. S., et al., 1993, Cell 74:237-244; Lutticken, C., et al., 1994, Science 263:89-92; Silvennoinen, O., et al., 1993, Proc. Natl. Acad. Sci. USA 90:8429-8433; and Witthuhn, B. A., et al., 1993, Cell 74:227-236). The hemopoietic growth factors, erythropoietin (Ep), interleukin-3 (IL-3) and steel factor (SF) (which utilizes a receptor with endogenous tyrosine kinase activity, i.e., c-kit, (Chabot, B., et al., 1988, Nature (London) 335:88-89)), have been shown to induce the tyrosine phosphorylation of Shc and its subsequent association with Grb2 (Cutler, R. L., et al., 1993, J. Biol. Chem. 268:21463-21465). Stimulation of members of the hemopoietin receptor superfamily has also been reported to result in the association of Shc with uncharacterized proteins with molecular masses of 130 kDa (Smit, L., et al., J. of Biol. Chem. 269(32):20209, 1994), 150 kDa (Lioubin, M. N., et al., Mol. Cell. Biol. 14(9):5682, 1994), and 145 kDa (Damen, J., et al., Blood 82(8):2296, 1993, and Saxton, T. M. et al.,J. Immunol. 623, 1994).
SUMMARY OF THE INVENTION
The present inventor has identified and characterized a protein that associates with Shc in response to multiple cytokines. The unique protein, herein referred to as “SH2-containing inositol-phosphatase” or “SHIP” (for SH2-containing, inositol 5-phosphatase), contains an amino terminal src homology 2 (SH2) domain, two phosphotyrosine binding (PTB) consensus sequences, a proline rich region, and two motifs highly conserved among inositol polyphosphate-5-phosphatases (phosphoIns-5-ptases). Cell lysates immunoprecipitated with antiserum to the protein exhibit phosphoIns-5-ptase activity, in particular, both phosphatidylinositol trisphosphate (PtdIns-3,4,5-P
3
) and inositol tetraphosphate (Ins-1,3,4,5-P
4
) 5-phosphatase activity. This activity implicates SHIP in the regulation of signalling pathways that control gene expression, cell proliferation, differentiation, activation, and metabolism, in particular, the Ras and phospholipid signalling pathways. This finding permits the identification of substances which affect SHIP and which may be used in the treatment of conditions involving perturbation of signalling pathways.
The present invention therefore provides a purified and isolated nucleic acid molecule comprising a sequence encoding an SH2-containing inositol-phosphatase which has a src homology 2 (SH2) domain and exhibits phosphoIns-5-ptase activity. The SH2-containing inositol-phosphatase is further characterized by it ability to associate with Shc and by having two phosphotyrosine binding (PTB) consensus sequences, a proline rich region, and motifs highly conserved among inositol polyphosphate-5-phosphatases (phosphoIns-5-ptases).
In an embodiment of the invention, the purified and isolated nucleic acid molecule comprises (i) a nucleic acid sequence encoding an SH2-containing inositol-phosphatase having the amino acid sequence as shown in SEQ ID NO:2 or
FIG. 2
(A); and, (ii) nucleic acid sequences complementary to (i). In another embodiment of the invention, the purified and isolated nucleic acid molecule comprises (i) a nucleic ac
Bereskin & Parr
Gravelle Micheline
Hutson Richard
Prouty Rebecca
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