SHIP2 controls plasma membrane PI(4,5)P2 thereby participating in the control of cell migration in 1321 N1 glioblastoma cells.

Phosphoinositides, particularly phosphatidylinositol (3,4,5)-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2], are recognized by SHIP2 (also known as INPPL1) a member of the inositol polyphosphate 5-phosphatase family. SHIP2 dephosphorylates PI(3,4,5)P3 to form PI(3,4)P2; the latter interacts with specific target proteins (e.g. lamellipodin). Although the preferred SHIP2 substrate is PI(3,4,5)P3, PI(4,5)P2 can also be dephosphorylated by this enzyme to phosphatidylinositol 4-phosphate (PI4P). Through depletion of SHIP2 in the glioblastoma cell line 1321 N1, we show that SHIP2 inhibits cell migration. In different glioblastoma cell lines and primary cultures, SHIP2 staining at the plasma membrane partly overlaps with PI(4,5)P2 immunoreactivity. PI(4,5)P2 was upregulated in SHIP2-deficient N1 cells as compared to control cells; in contrast, PI4P was very much decreased in SHIP2-deficient cells. Therefore, SHIP2 controls both PI(3,4,5)P3 and PI(4,5)P2 levels in intact cells. In 1321 N1 cells, the PI(4,5)P2-binding protein myosin-1c was identified as a new interactor of SHIP2. Regulation of PI(4,5)P2 and PI4P content by SHIP2 controls 1321 N1 cell migration through the organization of focal adhesions. Thus, our results reveal a new role of SHIP2 in the control of PI(4,5)P2, PI4P and cell migration in PTEN-deficient glioblastoma 1321 N1 cells.

When worlds collide: inositol pyrophosphates and phosphoinositides intersect at the plasma membrane.

Highly phosphorylated inositol pyrophosphates are present in the cells of many organisms such as yeast, Dictyostelium and mammals. They can act as signal molecules in growth factor and insulin signalling both in cultured cells and in intact mice. Their action involves protein pyrophosphorylation or binding to multiple protein interactors such as PH (pleckstrin homology)-domain-containing proteins. One key enzyme in their synthesis, PPIP5K (diphosphoinositol pentakisphosphate kinase) 1/2, can phosphorylate InsP6 and 5-InsP7 to 1-InsP7 and InsP8 respectively. Stephen Shears's laboratory reported in this issue of the Biochemical Journal that PPIP5K1's unexpectedly high affinity for PtdIns(3,4,5)P3, which is synthesized at the plasma membrane, provides a recruitment mechanism for this enzyme in response to growth factor receptor activation. In competition experiments, they observed that PtdIns(3,4,5)P3 binding to PPIP5K1 could be displaced by inositol pyrophosphates and that PPIP5K1 substrates were more potent inhibitors than PPIP5K1 products. Those findings reveal a mechanism for localized depletion of InsP6 and 5-InsP7 at the plasma membrane and further translocation of PtdIns(3,4,5)P3-binding PH-domain-containing proteins.

Evidence of SHIP2 Ser132 phosphorylation, its nuclear localization and stability.

PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are major signalling molecules in mammalian cell biology. PtdIns(3,4)P2 can be produced by PI3Ks [PI (phosphoinositide) 3-kinases], but also by PI 5-phosphatases including SHIP2 [SH2 (Src homology 2)-domain-containing inositol phosphatase 2]. Proteomic studies in human cells revealed that SHIP2 can be phosphorylated at more than 20 sites, but their individual function is unknown. In a model of PTEN (phosphatase and tensin homologue deleted on chromosome 10)-null astrocytoma cells, lowering SHIP2 expression leads to increased PtdIns(3,4,5)P3 levels and Akt phosphorylation. MS analysis identified SHIP2 phosphosites on Ser132, Thr1254 and Ser1258; phosphotyrosine-containing sites were undetectable. By immunostaining, total SHIP2 concentrated in the perinuclear area and in the nucleus, whereas SHIP2 phosphorylated on Ser132 was in the cytoplasm, the nucleus and nuclear speckles, depending on the cell cycle stage. SHIP2 phosphorylated on Ser132 demonstrated PtdIns(4,5)P2 phosphatase activity. Endogenous phospho-SHIP2 (Ser132) showed an overlap with PtdIns(4,5)P2 staining in nuclear speckles. SHIP2 S132A was less sensitive to C-terminal degradation and more resistant to calpain as compared with wild-type enzyme. We have identified nuclear lamin A/C as a novel SHIP2 interactor. We suggest that the function of SHIP2 is different at the plasma membrane where it recognizes PtdIns(3,4,5)P3, and in the nucleus where it may interact with PtdIns(4,5)P2, particularly in speckles.

Phosphoinositide 5-phosphatase activities control cell motility in glioblastoma: Two phosphoinositides PI(4,5)P2 and PI(3,4)P2 are involved.

Inositol polyphosphate 5-phosphatases or phosphoinositide 5-phosphatases (PI 5-phosphatases) are enzymes that can act on soluble inositol phosphates and/or phosphoinositides (PIs). Several PI 5-phosphatases have been linked to human genetic diseases, in particular the Lowe protein or OCRL which is mutated in the Lowe syndrome. There are 10 different members of this family and 9 of them can use PIs as substrate. One of these substrates, PI(3,4,5)P3 binds to specific PH domains and recruits as effectors specific proteins to signaling complexes. Protein kinase B is one target protein and activation of the kinase will have a major impact on cell proliferation, survival and cell metabolism. Two other PIs, PI(4,5)P2 and PI(3,4)P2, are produced or used as substrates of PI 5-phosphatases (OCRL, INPP5B, SHIP1/2, SYNJ1/2, INPP5K, INPP5J, INPP5E). The inositol lipids may influence many aspects of cytoskeletal organization, lamellipodia formation and F-actin polymerization. PI 5-phosphatases have been reported to control cell migration, adhesion, polarity and cell invasion particularly in cancer cells. In glioblastoma, reducing SHIP2 expression can positively or negatively affect the speed of cell migration depending on the glioblastoma cell type. The two PI 5-phosphatases SHIP2 or SKIP could be localized at the plasma membrane and can reduce either PI(3,4,5)P3 or PI(4,5)P2 abundance. In the glioblastoma 1321 N1 cells, SHIP2 controls plasma membrane PI(4,5)P2 thereby participating in the control of cell migration.

SHIP2 signaling in normal and pathological situations: Its impact on cell proliferation.

Phosphoinositide 5-phosphatases are critical enzymes in modulating the concentrations of PI(3,4,5)P3, PI(4,5)P2 and PI(3,5)P2. The SH2 domain containing inositol 5-phosphatases SHIP1 and SHIP2 belong to this family of enzymes that dephosphorylate the 5 position of PI(3,4,5)P3 to produce PI(3,4)P2. Data obtained in zebrafish and in mice have shown that SHIP2 is critical in development and growth. Exome sequencing identifies mutations in the coding region of SHIP2 as a cause of opsismodysplasia, a severe but rare chondrodysplasia in human. SHIP2 has been reported to have both protumorigenic and tumor suppressor function in human cancer very much depending on the cell model. This could be linked to the relative importance of PI(3,4)P2 (a product of SHIP2 phosphatase activity) which is also controlled by the PI 4-phosphatase and tumor suppressor INPP4B. In the glioblastoma cell line 1321 N1, that do not express PTEN, lowering SHIP2 expression has an impact on the levels of PI(3,4,5)P3, cell morphology and cell proliferation. It positively stimulates cell proliferation by decreasing the expression of key regulatory proteins of the cell cycle such as p27. Together the data point out to a role of SHIP2 in development in normal cells and at least in cell proliferation in some cancer derived cells.

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.

SHIP2 signalling at the plasma membrane, in the nucleus and at focal contacts.

Phosphoinositide 5-phosphatases are critical enzymes in modulating the concentrations of PI(3,4,5)P(3), PI(4,5)P(2) and PI(3,5)P(2). The SH2 domain containing inositol 5-phosphatases SHIP1 and SHIP2 belong to this family of enzymes very much involved in physiopathology and development. Therefore activity and localization of the enzymes are particularly important taking into account both catalytic and non-catalytic mechanisms of the SHIP phosphatases. Several different mechanisms have been reported for SHIP2 targeting that often result from specific protein:protein interactions. In unstimulated astrocytoma cells, SHIP2 has a perinuclear and cytoplasmic localization. In serum-stimulated cells, SHIP2 can be localized at the plasma membrane and at focal contacts in polarized cells. A phosphorylated form of SHIP2 on S132 can be found in the nucleus and nuclear speckles. When present at the plasma membrane, SHIP2 may control the intracellular level of PI(3,4,5)P(3) thereby producing PI(3,4)P(2). When present in the nucleus, SHIP2 probably associates to other nuclear proteins such as lamin A/C and could potentially control nuclear PI(4,5)P(2). Finally, its presence at focal adhesions and lamellipodia could suggest a role in cell adhesion and migration. It is proposed that the complex phenotype observed in SHIP2 mutant mice in tissue development and growth could result from the addition of plasma membrane and nuclear effects consecutive to SHIP2 alteration.