5′ Phospholipid Phosphatase SHIP-2 Causes Protein Kinase B Inactivation and Cell Cycle Arrest in Glioblastoma Cells

Disruption of signaling pathways crucial for the regulation of cellular proliferation and differentiation plays a major role in the pathogenesis of human cancer. Phosphoinositide 3-kinase (PI3K) is a key component of multiple signaling pathways, including those which regulate cell survival (12). This is mediated through one of the downstream targets of PI3K, protein kinase B (PKB, also known as c-akt) (19). One isoform of PKB, PKBβ, is amplified in some human tumors (10, 14, 57). PKB is activated by many growth factors through a process that involves translocation to the plasma membrane (6), presumably by binding of the transiently produced lipid products of PI3K activity, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] and phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2], to the pleckstrin homology (PH) domain of PKB (30). Phospholipid binding also serves to allow phosphorylation of PKB on Thr308 by phosphoinositide-dependent kinase 1 (PDK-1) (1, 2, 61), which causes PKB activation. Full activation of PKB requires phosphorylation at a second site, Ser473, which is also regulated in a PI3K-dependent manner. The kinase responsible for this phosphorylation has not been conclusively identified; however, integrin-linked kinase (17), PDK-1 (8), and PKB itself (63) have all been proposed. The tumor suppressor protein PTEN dephosphorylates the 3′ phosphate of PtdIns(3,4,5)P3 and PtdIns(3,4)P2, thus antagonizing PI3K activation (11). PTEN is mutated or deleted at high frequency in a wide variety of human cancers (39, 58) and several familial cancer predisposition disorders (18). PTEN functions both as a dual specificity protein phosphatase (48) and an inositol phospholipid phosphatase (44, 47), although it is the lipid phosphatase activity of PTEN which has been shown to be critical for its tumor suppressor function (47). PTEN is frequently mutated in advanced glioblastomas, and mutation of PTEN in these tumors results in deregulated signaling through the PI3K-PKB pathway (11). Reintroduction of a functional PTEN into glioblastoma cells results in decreased PKB activity (24, 37, 47) and a decrease in cell growth due to an arrest of the cell cycle in G1 (23, 37, 54, 62). Although these cells lack a functional PTEN, the addition of the PI3K inhibitor, LY294002, nevertheless decreased PKB activity in all glioblastoma cell lines examined (24). This suggests that once basal activity of PI3K is inhibited, mechanisms in addition to PTEN exist for disposing of cellular PtdIns(3,4,5)P3. At present it is not known which other pathways in glioblastoma cells can act on PtdIns(3,4,5)P3 to abolish signaling through the PI3K-PKB pathway. One possibility is that phosphoinositide 5′ phosphatases may be involved. SH2 domain-containing inositol phosphatase (SHIP) was recently identified through its association with the adaptor proteins Shc (33, 40) and Grb2 (16, 33) and was defined as a 5-phosphatase by the ability to dephosphorylate PtdIns(3,4,5)P3 (16, 33, 40). SHIP is expressed predominantly in hematopoietic cells (16, 40), whereas a closely related homologue, SHIP-2, is more ubiquitously expressed (51). SHIP-2 and SHIP both contain an N-terminal SH2 domain, multiple proline-rich sites representing possible SH3 domain binding sites, and, respectively, one and two NPXY phosphorylation motifs (31, 41, 50, 51). SHIP has been implicated as a crucial negative regulator of B-cell activation (42, 49), immunoglobulin E-mediated mast-cell degranulation (27, 50) and cytokine signaling in myeloid cells (43). In both B cells and myeloid cells, this negative regulatory role of SHIP has been linked to its ability to inhibit PKB activity (3, 43). Relatively little is known about the function of SHIP-2, although initial studies suggest that it may play a role in the regulation of PI3K signaling by growth factors and insulin (25, 28). Our studies on the role of SHIP-2 in glioblastoma cells were prompted by two observations. Firstly, inhibition of PI3K activity in glioblastoma cells expressing mutant PTEN results in a rapid loss of PtdIns(3,4,5)P3 and inhibition of PKB activity, suggesting that additional mechanisms for the disposal of PtdIns(3,4,5)P3 are present and active. Secondly, despite the ability of PtdIns(3,4)P2 to bind to the PH domain of PKB and allow phosphorylation and activation by PDK-1 to the same extent as PtdIns(3,4,5)P3 in vitro (1, 59), SHIP appears to act as a negative regulator of PKB activity in B cells (3, 29, 43) and upon overexpression in 3T3L1 adipocytes (64). We report here that overexpression of SHIP-2 in U87-MG cells reduces PtdIns(3,4,5)P3 levels but not PtdIns(3,4)P2 levels and that this is sufficient to abolish PKB phosphorylation and activation, as well as displace PKB from cell membranes. We show that phosphorylation of the C-terminal tyrosine of SHIP-2 does not significantly alter its catalytic activity or inhibitory effect on PKB. In addition, as previously reported with PTEN, we observed that expression of SHIP-2 in U87-MG cells causes a potent arrest of the cell cycle in G1, associated with increased stability of the cell cycle inhibitor p27KIP1. Finally, reduction in SHIP-2 levels in HeLa cells using antisense oligonucleotides causes an increase in PKB activity, implicating SHIP-2 as a biologically relevant regulator of PKB.