SHIP2 multiple functions: a balance between a negative control of PtdIns(3,4,5)P3 level, a positive control of PtdIns(3,4)P2 production, and intrinsic docking properties.

The SH2 domain containing inositol 5-phosphatase 2 (SHIP2) belongs to the family of the mammalian inositol polyphosphate 5-phosphatases. The two closely related isoenzymes SHIP1 (or SHIP) and SHIP2 contain a N-terminal SH2 domain, a catalytic domain, potential PTB domain-binding sites (NPXY), and C-terminal proline-rich regions with consensus sites for SH3 domain interactions. In addition, SHIP2 contains a unique sterile alpha motif (SAM) domain that could be involved in SAM-SAM domain interactions with other proteins or receptors. SHIP2 also shows the presence of an ubiquitin interacting motif at the C-terminal end. SHIP2 is essentially a PI(3,4,5)P(3) 5-phosphatase that negatively controls PI(3,4,5)P(3) levels in intact cells and produce PI(3,4)P(2) . Depending on the cells and stimuli, PI(3,4)P(2) could accumulate at important levels and be a "second messenger" by its own. It could interact with a very large number of target proteins such as PKB or TAPP1 and 2 that control insulin sensitivity. In addition to its catalytic activity, SHIP2 is also a docking protein for a large number of proteins: Cytoskeletal, focal adhesion proteins, scaffold proteins, adaptors, protein phosphatases, and tyrosine kinase associated receptors. These interactions could play a role in the control of cell adhesion, migration, or endocytosis of some receptors. SHIP2 could be acting independently of its phosphatase activity being part of a protein network of some receptors, e.g., the EGF receptor or BCR/ABL. These non-catalytic properties associated to a PI phosphatase have also been reported for other enzymes of the metabolism of myo-inositol such as Ins(1,4,5)P(3) 3-kinases, inositol phosphate multikinase (IPMK), or PTEN.

SHIP2 controls PtdIns(3,4,5)P(3) levels and PKB activity in response to oxidative stress.

Reactive oxygen species (ROS) are known to be involved in redox signalling pathways that may contribute to normal cell function as well as disease progression. The tumour suppressor PTEN and the inositol 5-phosphatase SHIP2 are critical enzymes in the control of PtdIns(3,4,5)P(3) level. It has been reported that oxidants, including those produced in cells such as macrophages, can activate downstream signalling via the inactivation of PTEN. The present study evaluates the potential impact of SHIP2 on phosphoinositides in cells exposed to sodium peroxide. We used a model of SHIP2 deficient mouse embryonic fibroblasts (MEFs) stimulated by H(2)O(2): at 15 min, PtdIns(3,4,5)P(3) was markedly increased in SHIP2 -/- cells as compared to +/+ cells. In contrast, no significant increase in PtdIns(3,4)P(2) could be detected at 15 or 120 min incubation of the cells with H(2)O(2) (0.6 mM). PKB activity was also upregulated in SHIP2 -/- cells as compared to +/+ cells in response to H(2)O(2). SHIP2 add back experiments in SHIP2 -/- cells confirm its critical role as a lipid phosphatase in the control of PtdIns(3,4,5)P(3) level in response to H(2)O(2). We conclude that SHIP2 lipid phosphatase activity plays an important role in the metabolism PtdIns(3,4,5)P(3) which is demonstrated in oxygen stressed cells.

The PI3K effector Arap3 interacts with the PI(3,4,5)P3 phosphatase SHIP2 in a SAM domain-dependent manner.

Arap3 is a phosphoinositide (PI) 3 kinase effector that serves as a GTPase activating protein (GAP) for both Arf and Rho G-proteins. The protein has multiple pleckstrin homology (PH) domains that bind preferentially phosphatidyl-inositol-3,4,5-trisphosphate (PI(3,4,5,)P3) to induce translocation of Arap3 to the plasma membrane upon PI3K activation. Arap3 also contains a Ras association (RA) domain that interacts with the small G-protein Rap1 and a sterile alpha motif (SAM) domain of unknown function. In a yeast two-hybrid screen for new interaction partners of Arap3, we identified the PI 5'-phosphatase SHIP2 as an interaction partner of Arap3. The interaction between Arap3 and SHIP2 was observed with endogenous proteins and shown to be mediated by the SAM domain of Arap3 and SHIP2. In vitro, these two domains show specificity for a heterodimeric interaction. Since it was shown previously that Arap3 has a higher affinity for PI(3,4,5,)P3 than for PI(3,4)P2, we propose that the SAM domain of Arap3 can function to recruit a negative regulator of PI3K signaling into the effector complex.

Phosphorylated SHIP2 on Y1135 localizes at focal adhesions and at the mitotic spindle in cancer cell lines.

The SH2 containing inositol 5-phosphatase SHIP2 is a member of the mammalian phosphoinositide polyphosphate 5-phosphatase family. It is a multi-domain protein comprising a central catalytic domain, an SH2 domain at its N-terminus, proline rich sequences and SAM domain at its C-terminus. It can dephosphorylate both phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and can participate in multiple signaling events in response to growth factors such as EGF, FGF or PDGF. Human SHIP2 can be phosphorylated at two major tyrosine residues Tyr986 and Tyr1135. Here, we report two intracellular localizations of pSHIP2(Y1135): pSHIP2(Y1135)-ir localizes at focal adhesions in EGF-stimulated HeLa cells and at the mitotic spindle in HeLa, in GIST882 cells, a human model of gastrointestinal stromal tumors derived cells, and in human astrocytoma 1321N1 cells. pSHIP2(Y1135)-ir is maximal at metaphase. In N1 cells, evidence is provided that the SHIP2 pathway impacts the distribution of mitotic centrosomes, particularly Ò¯-tubulin. Our data reinforce the concept that SHIP2 localization in intact cells is dependent on phosphorylation mechanisms on both Ser/Thr and Tyr residues, i.e. Y1135, in three cancer cell lines.

A specific increase in inositol 1,4,5-trisphosphate 3-kinase B expression upon differentiation of human embryonic stem cells.

Human embryonic stem cells (hESCs) are of great hope for regenerative medicine due to their dual pluripotency and self-renewal properties. We report a comparison of inositol phosphate (InsP(s)) production in undifferentiated, differentiated hESCs and in two cancer cell lines, Ntera2 cells, a human embryonal carcinoma cell (hECC) line and HeLa cells. To evaluate the potential impact of InsP(s) in differentiation, hESCs were spontaneously differentiated in culture for two weeks. The distribution of the different InsP(s) was affected upon differentiation: the level of highly phosphorylated InsP(s) was decreased. In contrast, the total level of phosphoinositides (PI) was increased. Using real time quantitative PCR (qPCR), the mRNA expression of several enzymes of the metabolism of InsP(s) was determined: a specific increase in inositol 1,4,5-trisphosphate 3-kinase A and B (ITPKA and ITPKB) was observed upon hESCs spontaneous differentiation. Ins(1,4,5)P(3) 3-kinase activity, undetectable in undifferentiated hESCs, increased upon differentiation. The same observation was made by Western blotting using an antibody directed against human ITPKB. This is the first report showing the potential implication of soluble InsP(s) in hESCs and possible function of isoenzymes of the inositol trisphosphate 3-kinase family in differentiation.

Abnormal elevated PTEN expression in the mouse antrum of a model of GIST Kit(K641E/K641E).

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Approximately 85% of GISTs harbor activating mutations of the KIT or PDGFRA receptor tyrosine kinases. PTEN and SHIP2 are major phosphatases that dephosphorylate PI(3,4,5)P(3), one of the intracellular signal pathways downstream of KIT. PTEN is an important tumor suppressor, whereas the involvement of SHIP2 in cancer has been proposed based essentially on cell line studies. We have used a mouse model of GIST, i.e. Kit(K641E) knock-in mice, resulting in the substitution of a Lys by Glu at position 641 of Kit. In homozygous Kit(K641E) mice, PTEN-immunoreactivity (ir) in antrum was found in the hyperplastic Kit-ir layer. The same localization was found for SHIP2. Western blot analysis in antrum showed a large increase in PTEN expression in Kit(K641E) homozygous mice as compared to wild type. In contrast, SHIP2 expression was not affected between the two genotypes. Erk1, but not PKB, phosphorylation appears to be upregulated in Kit(K641E) homozygous mice. In the human GIST882 imatinib sensitive cell line, both PTEN and SHIP2 were expressed and showed, in part, a nuclear localization. The upregulation of PTEN in antrum in Kit(K641E) mice might serve as a feedback mechanism to limit PI 3-kinase activation downstream of Kit in a context of oncogenic mutation.