SH2-containing inositol 5'-phosphatase SHIP2 associates with the p130(Cas) adapter protein and regulates cellular adhesion and spreading.

Products of phosphatidylinositol (PI) metabolism are important second messengers in cellular signaling pathways (1, 11, 70, 76). Activation of PI 3′-kinase, which phosphorylates the 3′ position of the inositol ring of PI, is a critical event in growth factor, insulin, and G protein-mediated signal transduction (14, 25, 49). In addition, PI 3′-kinase plays an important role in the regulation of adhesion and migration (68). PI 3′-kinase activation localizes to cell-cell and cell-matrix adhesion sites in epithelial cells, as well as to membrane ruffles in fibroblasts (78). Inhibition of PI 3′-kinase attenuated integrin-mediated adhesion and migration in several cell types, while expression of the catalytic p110 subunit of PI 3′-kinase enhanced cell adhesion (18, 22, 29, 31, 33, 34, 52, 80). Moreover, p85, the regulatory subunit of PI 3′-kinase, interacted with proteins regulating adhesion and migration such as focal adhesion kinase (FAK) and p130Crk-associated substrate (p130Cas) (3, 8, 43). In vivo, the major substrate for PI 3′-kinase is phosphatidylinositol-4,5-bisphosphate [PI-(4,5)-P2] leading to the formation of phosphatidylinositol-3,4,5-trisphosphate [PI-(3,4,5)-P3] (63). Significant pools of phosphatidylinositol-3,4-bisphosphate [PI-(3,4)-P2] are also generated following PI 3′-kinase activation primarily through dephosphorylation of PI-(3,4,5)-P3 by 5′ inositol phosphatases (27). PI-(3,4,5)-P3 and PI-(3,4)-P2 specifically interact with pleckstrin homology (PH) domains of proteins, regulating activity or intracellular localization of cellular enzymes such as Akt/PKB and its upstream kinase phosphoinositide-dependent kinase 1 (PDK1) (75). In addition to Akt and PDK1, phospholipid products of PI 3′-kinase regulate the activity of a number of other cellular proteins containing PH domains. These include the Btk family tyrosine kinases, as well as guanine nucleotide exchange factors such as Vav, Dbl, and general receptor for phosphoinositides 1 (Grp1) (54). Vav, Dbl, and Grp1 are important regulators of cytoskeletal organization, adhesion, invasion, vesicle budding, membrane trafficking, and cell spreading (7, 39, 77). Inositol phosphatases are important in regulating the cellular levels of lipid second messengers. Inactivation of the tumor suppressor gene PTEN/MMAC1, which hydrolyzes the 3′-phosphate of PI-(3,4,5)-P3, is frequently observed in tumor cells (2), leading to increased basal levels of PI-(3,4,5)-P3 and activation of downstream targets of PI 3′-kinase (23). PTEN also regulates integrin-mediated activation of extracellular-signal-regulated kinase (ERK), interacts with FAK, and inhibits adhesion, migration, and invasion processes (19, 44, 71, 72). Thus, PTEN has been implicated in the regulation of adhesion and/or integrin-mediated survival signaling and detachment-induced cell death or “anoikis” (5, 69). Several inositol phosphatases that dephosphorylate the 5′ position of PI-(3,4,5)-P3 have been cloned (79). Among the known 5′ inositol phosphatases, SH2-containing inositol 5′-phosphatases 1 and 2 (SHIP1 and SHIP2) are specific for PI-(3,4,5)-P3 and inositol-(1,3,4,5)-tetrakiphosphate (12). SHIP1 is expressed primarily in hematopoietic tissues, while SHIP2 expression appears to be more ubiquitous (15, 24, 48, 59, 67). Studies using SHIP1 knockout mice revealed a negative regulatory role for SHIP1 in myeloid cell proliferation and immune system function (46, 47). Negative regulation of growth factor and antigen receptor-mediated signaling by SHIP1 is well documented (28, 45). On the other hand, the role of SHIP2 in cellular functions remains largely unknown, although some studies suggest a negative role for SHIP2 in insulin and FcγRIIB receptor signaling (30, 53). Besides an amino-terminal SH2 domain, both SHIP1 and SHIP2 possess a proline-rich region and NPXY motifs that could possibly provide interaction sites with proteins. Indeed, SHIP1 forms complexes with adapter proteins, Shc and Grb2, as well as tyrosine phosphatase SHP-2 (6, 26, 42, 61). We have previously reported that SHIP2 was tyrosine phosphorylated and associated with adapter protein Shc in response to growth factors and insulin (24). In this study, we report a novel function for SHIP2 in cell adhesion and spreading. We have identified p130Cas, an important regulator of adhesion and migration processes, as an SHIP2-interacting protein. In HeLa cells, SHIP2 localized to focal contacts during attachment and to leading edges of membranes, lamellipodia, in spreading cells. Wild-type SHIP2 promoted adhesion, while catalytically inactive SHIP2 inhibited the spreading of HeLa cells.