Phosphoinositide lipid phosphatase SHIP1 and PTEN coordinate to regulate cell migration and adhesion

The second messenger phosphatidylinositol(3,4,5)P(3) (PtdIns(3,4,5)P(3)) is formed by stimulation of various receptors, including G protein-coupled receptors and integrins. The lipid phosphatases PTEN and SHIP1 are critical in regulating the level of PtdIns(3,4,5)P(3) during chemotaxis. Observations that loss of PTEN had minor and loss of SHIP1 resulted in a severe chemotaxis defect in neutrophils led to the belief that SHIP1 rather than PTEN acts as a predominant phospholipid phosphatase in establishing a PtdIns(3,4,5)P(3) compass. In this study, we show that SHIP1 regulates PtdIns(3,4,5)P(3) production in response to cell adhesion and plays a limited role when cells are in suspension. SHIP1((-)/(-)) neutrophils lose their polarity upon cell adhesion and are extremely adherent, which impairs chemotaxis. However, chemo-taxis can be restored by reducing adhesion. Loss of SHIP1 elevates Akt activation following cell adhesion due to increased PtdIns(3,4,5)P(3) production. From our observations, we conclude that SHIP1 prevents formation of top-down PtdIns(3,4,5)P(3) polarity to facilitate proper cell attachment and detachment during chemotaxis.     

SHIP2 interaction with the cytoskeletal protein Vinexin

The src homology 2 (SH2) domain-containing inositol 5-phosphatase 2 (SHIP2) catalyses the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] to phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P2]. We report the identification of the cytoskeletal protein Vinexin as a protein interacting with SHIP2. This was achieved by yeast two-hybrid screening using the C-terminal region of SHIP2 as bait. Vinexin has previously been identified as a vinculin-binding protein that plays a key role in cell spreading and cytoskeletal organization. The interaction between SHIP2 and Vinexin was confirmed in lysates of both COS-7 cells and mouse embryonic fibroblasts (MEF). The C-terminus was involved in the interaction, as shown by the transfection of a truncated C-terminus mutant of SHIP2. In addition, we showed the colocalization between Vinexin alpha and SHIP2 at the periphery of transfected COS-7 cells. When added in vitro to SHIP2, Vinexin did not affect the PtdIns(3,4,5)P3 5-phosphatase activity of SHIP2. Enhanced cell adhesion to collagen-I-coated dishes was shown upon transfection of either SHIP2 or Vinexin to COS-7 cells. This effect was no longer observed with either a catalytic mutant or the C-terminus mutant of SHIP2. It also appears SHIP2 specific; this was not seen with SHIP1. Adhesion to the same matrix was decreased in SHIP2-/- MEF cells compared with MEF+/+ cells. Our data suggest that SHIP2 interaction with Vinexin promotes the localization of SHIP2 at the periphery of the cells leaving its catalytic site intact. The complex formation between Vinexin and SHIP2 may increase cellular adhesion. The data reinforce the concept that SHIP2 is active both as a PtdIns(3,4,5)P3 5-phosphatase and as a modulator of focal contact formation.     

The association between the SH2-containing inositol polyphosphate 5-Phosphatase 2 (SHIP2) and the adaptor protein APS has an impact on biochemical properties of both partners

SHIP2 (SH2-containing inositol polyphosphate 5-phosphatase 2) is a phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase containing various motifs susceptible to mediate protein-protein interaction. In cell models, SHIP2 negatively regulates insulin signalling through its catalytic PtdIns(3,4,5)P(3) 5-phosphatase activity. We have previously reported that SHIP2 interacts with the c-Cbl associated protein (CAP) and c-Cbl, proteins implicated in the insulin cellular response regulating the small G protein TC10. The first steps of the TC10 pathway are the recruitment and tyrosine phosphorylation by the insulin receptor of the adaptor protein with Pleckstrin Homology and Src Homology 2 domains (APS). Herein, we show that SHIP2 can directly interact with APS in 3T3-L1 adipocytes and in transfected CHO-IR cells (Chinese hamster ovary cells stably transfected with the insulin receptor). Upon insulin stimulation, APS and SHIP2 are recruited to cell membranes as seen by immunofluorescence studies, which is consistent with their interaction. We also observed that SHIP2 negatively regulates APS insulin-induced tyrosine phosphorylation and consequently inhibits APS association with c-Cbl. APS, which specifically interacts with SHIP2, but not PTEN, in turn, increases the PtdIns(3,4,5)P(3) 5-phosphatase activity of SHIP2 in an inositol phosphatase assay. Co-transfection of SHIP2 and APS in CHO-IR cells further increases the inhibitory effect of SHIP2 on Akt insulin-induced phosphorylation. Therefore, the interaction between APS and SHIP2 provides to both proteins potential negative regulatory mechanisms to act on the insulin cascade.     

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.     

Endogenous SHIP2 does not localize in lipid rafts in 3T3-L1 adipocytes

SH2 domain containing inositol polyphosphate 5-phosphatase (SHIP2) dephosphorylates phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) into phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)). SHIP2 knock-out mice demonstrated that SHIP2 acts as a negative regulator of insulin cascade in vivo. Our two-hybrid study showed that SHIP2 interacts with c-Cbl associated protein (CAP) and c-Cbl, implicated in the insulin signaling. As some proteins implicated in insulin signaling, like insulin receptor, CAP, c-Cbl or TC10, were reported to localize in lipid rafts, we addressed the same question for SHIP2. SHIP2 was detected in the non-raft fraction in CHO-IR, C2C12 myotubes and 3T3-L1 adipocytes except when it is overexpressed in CHO-IR, where we detected SHIP2 in the raft fraction.     

Phosphatidylinositol 3,4,5-trisphosphate modulation in SHIP2-deficient mouse embryonic fibroblasts

SHIP2, the ubiquitous SH2 domain containing inositol 5-phosphatase, includes a series of protein interacting domains and has the ability to dephosphorylate phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)]in vitro. The present study, which was undertaken to evaluate the impact of SHIP2 on PtdIns(3,4,5)P(3) levels, was performed in a mouse embryonic fibroblast (MEF) model using SHIP2 deficient (-/-) MEF cells derived from knockout mice. PtdIns(3,4,5)P(3) was upregulated in serum stimulated -/- MEF cells as compared to +/+ MEF cells. Although the absence of SHIP2 had no effect on basal PtdIns(3,4,5)P(3) levels, we show here that this lipid was significantly upregulated in SHIP2 -/- cells but only after short-term (i.e. 5-10 min) incubation with serum. The difference in PtdIns(3,4,5)P(3) levels in heterozygous fibroblast cells was intermediate between the +/+ and the -/- cells. In our model, insulin-like growth factor-1 stimulation did not show this upregulation. Serum stimulated phosphoinositide 3-kinase (PI 3-kinase) activity appeared to be comparable between +/+ and -/- cells. Moreover, protein kinase B, but not mitogen activated protein kinase activity, was also potentiated in SHIP2 deficient cells stimulated by serum. The upregulation of protein kinase B activity in serum stimulated cells was totally reversed in the presence of the PI 3-kinase inhibitor LY-294002, in both +/+ and -/- cells. Altogether, these data establish a link between SHIP2 and the acute control of PtdIns(3,4,5)P(3) levels in intact cells.     

The docking properties of SHIP2 influence both JIP1 tyrosine phosphorylation and JNK activity

SHIP2 (SH2-containing inositol polyphosphate 5-phosphatase 2) is an ubiquitously expressed phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase which contains various motifs susceptible to mediate protein-protein interaction. In cell models, evidence has been provided that SHIP2 plays a role in insulin and growth factor signaling, cytoskeletal organization, cell adhesion and migration. Herein we describe the c-Jun NH2-terminal kinase (JNK)-interacting protein 1 (JIP1) as a new protein partner of SHIP2. The interaction between SHIP2 and JIP1 was confirmed in both overexpression systems and native cells. Without modifying the association of JIP1 with the MAPKs in the scaffold complex and with no apparent change of Akt phosphorylation, SHIP2 positively modulated the MLK3/JIP1-mediated JNK1 activation. Moreover, SHIP2 positively regulated the tyrosine phosphorylation of JIP1. This up-regulation was prevented by inhibitors of the Src family and Abl kinases, PP2 and Glivec. The effects of SHIP2 on JNK activity and JIP1 tyrosine phosphorylation were independent of the SHIP2 phosphoinositide 5-phosphatase activity, as similar results were obtained when using a SHIP2 catalytic inactive mutant instead of wild-type SHIP2. Together, these data suggest that by its docking properties, SHIP2 can modulate JIP1-mediated JNK pathway signaling.     

Regulation of SHIP2 function through plasma membrane interaction

A fundamental aspect of signalling concerns the precise cellular localization of the enzymes acting on phosphoinositides, particularly PtdIns(3,4,5)P₃. SHIP1/2 phosphatases, that dephosphorylate PtdIns(3,4,5)P₃ to PtdIns(3,4)P₂, have a series of interacting motives that favour protein:protein interactions. A large number of proteins, receptors, protein kinases, cytoskeletal and adaptor proteins have been shown to bind to SHIP1/2 thereby modulating their own function and localization. SHIP2 localization has been studied at endogenous level in a series of cell models (MEFs, COS-7 and HeLa cells). In cells maintained in serum, SHIP2 localization was shown to be perinuclear with some staining at the nuclear level as well. In response to EGF, SHIP2 has been shown to translocate to the periphery of the cells (i.e. plasma membranes). This effect was transient with a maximum at 15 min stimulation by EGF.     

The Src homology 2-containing inositol 5-phosphatase 1 (SHIP1) is involved in CD32a signaling in human neutrophils

Phosphatidylinositol(3,4,5)triphosphate (PtdIns(3,4,5)P(3)) plays important signaling roles in immune cells, particularly in the control of activating pathways and of survival. It is formed by a family of phosphatidylinositol 3'-kinases (PI3Ks) which phosphorylate PtdIns(4,5)P(2) in vivo. In human neutrophils, the levels of PtdIns(3,4,5)P(3) increase rapidly at the leading edge of locomoting cells and at the base of the phagocytic cup during FcgammaR-mediated particle ingestion. Even though these, and other, data indicate that PtdIns(3,4,5)P(3) is involved in the control of chemotaxis and phagocytosis in human neutrophils, the mechanisms that regulate its levels have yet to be fully elucidated in these cells. We evaluated the potential implication of SHIP1 and PTEN, two lipid phosphatases that utilize PtdIns(3,4,5)P(3) as substrate, in the signaling pathways called upon in response to CD32a cross-linking. We observed that the cross-linking of CD32a resulted in a transient accumulation of PtdIns(3,4,5)P(3). CD32a cross-linking also induced the tyrosine phosphorylation of SHIP1, its translocation to the plasma membrane and its co-immunoprecipitation with CD32a. CD32a cross-linking had no effect on the level of serine/threonine phosphorylation of PTEN and did not stimulate its translocation to the plasma membrane. PP2, a Src kinase inhibitor, inhibited the tyrosine phosphorylation of SHIP1 as well as its translocation to the plasma membrane. Wortmannin, a PI3K inhibitor, had no effect on either of these two indices of activation of SHIP1. Our results indicate that SHIP1 is involved, in a Src kinase-dependent manner, in the early signaling events observed upon the cross-linking of CD32a in human neutrophils.     

The SH2 domain containing inositol polyphosphate 5-phosphatase-2: SHIP2

Phosphoinositides are membrane-bound signaling molecules that recruit, activate and localize target effectors to intracellular membranes regulating apoptosis, cell proliferation, insulin signaling and membrane trafficking. The SH2 domain containing inositol polyphosphate 5-phosphatase-2 (SHIP2) hydrolyzes phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) generating phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). Overexpression of SHIP2 inhibits insulin-stimulated phosphoinositide 3-kinase (PI3K) dependent signaling events. Analysis of diabetic human subjects has revealed an association between SHIP2 gene polymorphisms and type 2 diabetes mellitus. Genetic ablation of SHIP2 in mice has generated conflicting results. SHIP2 knockout mice were originally reported to show lethal neonatal hypoglycemia resulting from insulin hypersensitivity, but in addition to inactivating the SHIP2 gene, the Phox2a gene was also inadvertently deleted. Another SHIP2 knockout mouse has now been generated which inactivates the SHIP2 gene but leaves Phox2a intact. These animals show normal insulin and glucose tolerance but are highly resistant to weight gain on high fat diets, exhibiting an obesity-resistant phenotype. Therefore, SHIP2 remains a significant therapeutic target for the treatment of both obesity and type 2 diabetes.     

Control of cell polarity and motility by the PtdIns(3,4,5)P3 phosphatase SHIP1

Proper neutrophil migration into inflammatory sites ensures host defense without tissue damage. Phosphoinositide 3-kinase (PI(3)K) and its lipid product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) regulate cell migration, but the role of PtdIns(3,4,5)P(3)-degrading enzymes in this process is poorly understood. Here, we show that Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1), a PtdIns(3,4,5)P(3) phosphatase, is a key regulator of neutrophil migration. Genetic inactivation of SHIP1 led to severe defects in neutrophil polarization and motility. In contrast, loss of the PtdIns(3,4,5)P(3) phosphatase PTEN had no impact on neutrophil chemotaxis. To study PtdIns(3,4,5)P(3) metabolism in living primary cells, we generated a novel transgenic mouse (AktPH-GFP Tg) expressing a bioprobe for PtdIns(3,4,5)P(3.) Time-lapse footage showed rapid, localized binding of AktPH-GFP to the leading edge membrane of chemotaxing ship1(+/+)AktPH-GFP Tg neutrophils, but only diffuse localization in ship1(-/-)AktPH-GFP Tg neutrophils. By directing where PtdIns(3,4,5)P(3) accumulates, SHIP1 governs the formation of the leading edge and polarization required for chemotaxis.     

SHIP2 overexpression strongly reduces the proliferation rate of K562 erythroleukemia cell line

SHIP2 belongs to the inositol 5-phosphatase family and is characterized by a phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) 5-phosphatase activity. Evidence based on mice lacking the SHIP2 gene has demonstrated its predominant role in the control of insulin sensitivity. However, SHIP2 expression in both hematopoietic and non-hematopoietic cells suggests additional functions. SHIP2 was previously identified in chronic myelogenous progenitor cells, in which its constitutive tyrosine phosphorylation was reported by Wisniewski et al., [Blood 93 (1999) 2707-2720]. Here, we further investigated the function of SHIP2 in this hematopoietic and malignant context. A detailed analysis of the substrate specificity of SHIP2 indicated that this phosphatase is primarily directed towards PI(3,4,5)P(3) both in vitro and in K562 chronic myeloid leukemia cells. The SHIP2-mediated decrease in PI(3,4,5)P(3) levels and increase in phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)) was accompanied by a reduction of cell proliferation, characterized by an accumulation of the cells in the G2/M phase of the cell cycle. Thus, in addition to its role in the control of insulin sensitivity, SHIP2 may also play a role in cell proliferation, at least in chronic myelogenous progenitor cells.     

The influence of anionic lipids on SHIP2 phosphatidylinositol 3,4,5-trisphosphate 5-phosphatase activity

The SH2 domain containing inositol 5-phosphatase 2 (SHIP2) catalyzes the dephosphorylation of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃) to phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P₂) and participates in the insulin signalling pathway in vivo. In a comparative study of SHIP2 and the phosphatase and tensin homologue deleted on chromosome 10 (PTEN), we found that their lipid phosphatase activity was influenced by the presence of vesicles of phosphatidylserine (PtdSer). SHIP2 PtdIns(3,4,5)P₃ 5-phosphatase activity was greatly stimulated in the presence of vesicles of PtdSer. This effect appears to be specific for di-C8 and di-C16 fatty acids of PtdIns(3,4,5)P₃ as substrate. It was not observed with inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P₄) another in vitro substrate of SHIP2, nor with Type I Ins(1,4,5)P₃/Ins(1,3,4,5)P₄ 5-phosphatase activity, an enzyme which acts on soluble inositol phosphates. Vesicles of phosphatidylcholine (PtdCho) stimulated only twofold PtdIns(3,4,5)P₃ 5-phosphatase activity of SHIP2. Both a minimal catalytic construct and the full length SHIP2 were sensitive to the stimulation by PtdSer. In contrast, PtdIns(3,4,5)P₃ 5-phosphatase activity of the Skeletal muscle and Kidney enriched Inositol Phosphatase (SKIP), another member of the mammaliam Type II phosphoinositide 5-phosphatases, was not sensitive to PtdSer. Our enzymatic data establish a specificity in the control of SHIP2 lipid phosphatase activity with PtdIns(3,4,5)P₃ as substrate which is depending on the fatty acid composition of the substrate.     

Molecular cloning of rat SH2-containing inositol phosphatase 2 (SHIP2) and its role in the regulation of insulin signaling.

SH2-containing inositol 5'-phosphatase (SHIP) plays a negative regulatory role in hematopoietic cells. We have now cloned the rat SHIP isozyme (SHIP2) cDNA from skeletal muscle, which is one of the most important target tissue of insulin action. Rat SHIP2 cDNA encodes a 1183-amino-acid protein that is 45% identical with rat SHIP. Rat SHIP2 contains an amino-terminal SH2 domain, a central 5'-phosphoinositol phosphatase activity domain, and a phosphotyrosine binding (PTB) consensus sequence and a proline-rich region at the carboxyl tail. Specific antibodies to SHIP2 were raised and the function of SHIP2 was studied by stably overexpressing rat SHIP2 in Rat1 fibroblasts expressing human insulin receptors (HIRc). Endogenous SHIP2 underwent insulin-mediated tyrosine phosphorylation and phosphorylation was markedly increased when SHIP2 was overexpressed. Although overexpression of SHIP2 did not affect insulin-induced tyrosine phosphorylation of the insulin receptor beta-subunit and Shc, subsequent association of Shc with Grb2 was inhibited, possibly by competition between the SH2 domains of SHIP2 and Grb2 for the Shc phosphotyrosine. As a result, insulin-stimulated MAP kinase activation was reduced in SHIP2-overexpressing cells. Insulin-induced tyrosine phosphorylation of IRS-1, IRS-1 association with the p85 subunit of PI3-kinase, and PI3-kinase activation were not affected by overexpression of SHIP2. Interestingly, although both PtdIns-(3,4,5)P3 and PtdIns(3,4)P2 have been implicated in the regulation of Akt activity in vitro, overexpression of SHIP2 inhibited insulin-induced Akt activation, presumably by its 5'-inositol phosphatase activity. Furthermore, insulin-induced thymidine incorporation was decreased by overexpression of SHIP2. These results indicate that SHIP2 plays a negative regulatory role in insulin-induced mitogenesis, and regulation of the Shc. Grb2 complex and of the downstream products of PI3-kinase provides possible mechanisms of SHIP2 action in insulin signaling.     

FcgammaRIIb modulation of surface immunoglobulin-induced Akt activation in murine B cells

We examined activation of the serine/threonine kinase Akt in the murine B cell line A20. Akt is activated in a phosphoinositide 3-kinase (PtdIns 3-kinase)-dependent manner upon stimulation of the antigen receptor, surface immunoglobulin (sIg). In contrast, Akt induction is reduced upon co-clustering of sIg with the B cell IgG receptor, FcgammaRIIb. Co-clustering of sIg-FcgammaRIIb transmits a dominant negative signal and is associated with reduced accumulation of the PtdIns 3-kinase product phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P3), known to be a potent activator of Akt. PtdIns 3-kinase is activated to the same extent with and without FcgammaRIIb co-ligation, indicating conditions supporting the generation of PtdIns 3,4,5-P3. We hypothesized that the decreased Akt activity arises from the consumption of PtdIns 3,4,5-P3 by the inositol-5-phosphatase Src homology 2-containing inositol 5-phosphatase (SHIP), which has been shown by us to be tyrosine-phosphorylated and associated with FcgammaRIIb when the latter is co-ligated. In direct support of this hypothesis, we report here that Akt induction is greatly reduced in fibroblasts expressing catalytically active but not inactive SHIP. Likewise, the reduction in Akt activity upon sIg-FcgammaRIIb co-clustering is absent from avian B cells lacking expression of SHIP. These findings indicate that SHIP acts as a negative regulator of Akt activation.     

Role of TAPP1 and TAPP2 adaptor binding to PtdIns(3,4)P2 in regulating insulin sensitivity defined by knock-in analysis

Insulin sensitivity is critically dependent on the activity of PI3K (phosphoinositide 3-kinase) and generation of the PtdIns(3,4,5)P(3) second messenger. PtdIns(3,4,5)P(3) can be broken down to PtdIns(3,4)P(2) through the action of the SHIPs (Src-homology-2-domain-containing inositol phosphatases). As PtdIns(3,4)P(2) levels peak after those of PtdIns(3,4,5)P(3), it has been proposed that PtdIns(3,4)P(2) controls a negative-feedback loop that down-regulates the insulin and PI3K network. Previously, we identified two related adaptor proteins termed TAPP [tandem PH (pleckstrin homology)-domain-containing protein] 1 and TAPP2 that specifically bind to PtdIns(3,4)P(2) through their C-terminal PH domain. To determine whether TAPP1 and TAPP2 play a role in regulating insulin sensitivity, we generated knock-in mice that express normal endogenous levels of mutant TAPP1 and TAPP2 that are incapable of binding PtdIns(3,4)P(2). These homozygous TAPP1(R211L/R211L) TAPP2(R218L/R218L) double knock-in mice are viable and exhibit significantly enhanced activation of Akt, a key downstream mediator of insulin signalling. Consistent with increased PI3K and Akt activity, the double knock-in mice display enhanced whole body insulin sensitivity and disposal of glucose uptake into muscle tissues. We also generated wild-type and double TAPP1(R211L/R211L) TAPP2(R218L/R218L) knock-in embryonic fibroblasts and found that insulin triggered enhanced production of PtdIns(3,4,5)P(3) and Akt activity in the double knock-in fibroblasts. These observations provide the first genetic evidence to support the notion that binding of TAPP1 and TAPP2 adap-tors to PtdIns(3,4)P(2) function as negative regulators of the insulin and PI3K signalling pathways.     

Regulation of protein kinase B activity by PTEN and SHIP2 in human prostate-derived cell lines

Protein Kinase B (PKB/Akt) is a key regulator of cell proliferation, motility and survival. The activation status of PKB is regulated by phosphatidylinositol 3-kinase (PI3K) via the synthesis of phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3, PIP3). PTEN antagonises PI3K by degrading PIP3 to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Deregulation of PKB through loss of functional PTEN has frequently been implicated in the progression of tumours, including prostate cancer, and the PTEN-negative prostate cancer cell lines LNCaP and PC3 have been widely used as models for this mechanism of constitutive PKB activation. However, other enzymes in addition to PTEN can antagonise PI3K, including SHIP2, which degrades PIP3 to phosphatidylinositol-3,4-bisphosphate (PI(3,4)P2). We investigated the role of PTEN and SHIP2 in the regulation of PKB phosphorylation in a panel of human prostate-derived epithelial cell lines. In the PTEN-positive prostate-derived cell lines PNT2, PNT1a and P4E6, PI3K inhibition by LY294002 caused rapid dephosphorylation of PKB at ser473 (T(1/2)<2 min), leading to its inactivation. In the PTEN-null line LNCaP, LY294002-induced PKB dephosphorylation was much slower (T(1/2)>20 min), but in PC3 cells (also PTEN-null) it was only slightly slower than in PTEN-positive cells (T(1/2)=3 min). PKB dephosphorylation paralleled loss of plasma membrane PIP3. PNT1a, P4E6 and PC3, but not PNT2 or LNCaP, expressed SHIP2. SiRNA-mediated knockdown of SHIP2 expression markedly slowed PKB inactivation in response to LY294002 in PC3 but not in other SHIP2-positive cells, whereas knockdown of PTEN expression in PNT2, PNT1a and P4E6 resulted in higher steady-state levels of PKB phosphorylation and slowed, but did not prevent, LY294002-induced PKB inactivation. Thus SHIP2 substitutes for PTEN in the acute regulation of PKB in PC3 cells but not other prostate cell lines, where PTEN may share this role with further PIP3-degrading mechanisms.     

SHIP family inositol phosphatases interact with and negatively regulate the Tec tyrosine kinase

The Tec family of protein-tyrosine kinases (PTKs), that includes Tec, Itk, Btk, Bmx, and Txk, plays an essential role in phospholipase Cgamma (PLCgamma) activation following antigen receptor stimulation. This function requires activation of phosphatidylinositol 3-kinase (PI 3-kinase), which promotes Tec membrane localization through phosphatidylinositol 3,4,5-trisphosphate (PtdIns 3,4,5-P(3)) generation. The mechanism of negative regulation of Tec family PTKs is poorly understood. In this study, we show that the inositol 5' phosphatases SHIP1 and SHIP2 interact preferentially with Tec, compared with other Tec family members. Four lines of evidence suggest that SHIP phosphatases are negative regulators of Tec. First, SHIP1 and SHIP2 are potent inhibitors of Tec activity. Second, inactivation of the Tec SH3 domain, which is necessary and sufficient for SHIP binding, generates a hyperactive form of Tec. Third, SHIP1 inhibits Tec membrane localization. Finally, constitutively targeting Tec to the membrane relieves SHIP1-mediated inhibition. These data suggest that SHIP phosphatases can interact with and functionally inactivate Tec by de-phosphorylation of local PtdIns 3,4,5-P(3) and inhibition of Tec membrane localization.     

Regulation of endogenous SH2 domain-containing inositol 5-phosphatase (SHIP2) in 3T3-L1 and human preadipocytes

The role of the inositol lipid 5-phosphatase (SHIP2) in preadipocyte signaling is not known. Although overexpression of SHIP2 inhibited proliferation and (3)H-thymidine incorporation in 3T3-L1 preadipocytes, there was no effect on insulin-induced adipogenesis. Insulin promoted SHIP2 tyrosine phosphorylation in differentiated 3T3-L1 adipocytes, but did not do so in preadipocytes. The absence of SHIP2 tyrosine phosphorylation suggests a potential explanation for the isolated rise in PI(3,4,5)P3, without any changes in PI(3,4)P2, previously observed following insulin treatment of these cells. Lack of SHIP2 tyrosine phosphorylation by insulin was also observed in primary cultures of human abdominal subcutaneous preadipocytes. These cells also produced PI(3,4,5)P3, but not PI(3,4)P2, in response to insulin. Comparison of insulin vs. PDGF treatment on SHIP2 tyrosine phosphorylation in 3T3-L1 and human preadipocytes revealed that only PDGF, which stimulates the accumulation of PI(3,4,5)P3 as well as PI(3,4)P2, was active in this regard, and only PDGF promoted the association of 52 kDa form of Shc with SHIP2. Nevertheless, both insulin and PDGF were equally effective in translocating SHIP2 to the plasma membrane in 3T3-L1 preadipocytes. Lack of SHIP2 tyrosine phosphorylation may account for the insulin-specific inositol phospholipid pattern of accumulation in preadipocytes.     

A selective role for phosphatidylinositol 3,4,5-trisphosphate in the Gi-dependent activation of platelet Rap1B

The small GTP-binding protein Rap1B is activated in human platelets upon stimulation of a G(i)-dependent signaling pathway. In this work, we found that inhibition of platelet adenylyl cyclase by dideoxyadenosine or SQ22536 did not cause activation of Rap1B and did not restore Rap1B activation in platelets stimulated by cross-linking of Fcgamma receptor IIA (FcgammaRIIA) in the presence of ADP scavengers. Moreover, elevation of the intracellular cAMP concentration did not impair the G(i)-dependent activation of Rap1B. Two unrelated inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, totally prevented Rap1B activation in platelets stimulated by cross-linking of FcgammaRIIA, by stimulation of the P2Y(12) receptor for ADP, or by epinephrine. However, in platelets from PI3Kgamma-deficient mice, both ADP and epinephrine were still able to normally stimulate Rap1B activation through a PI3K-dependent mechanism, suggesting the involvement of a different isoform of the enzyme. Moreover, the lack of PI3Kgamma did not prevent the ability of epinephrine to potentiate platelet aggregation through a G(i)-dependent pathway. The inhibitory effect of wortmannin on Rap1B activation was overcome by addition of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), but not PtdIns(3,4)P(2), although both lipids were found to support phosphorylation of Akt. Moreover, PtdIns(3,4,5)P(3) was able to relieve the inhibitory effect of apyrase on FcgammaRIIA-mediated platelet aggregation. We conclude that stimulation of a G(i)-dependent signaling pathway causes activation of the small GTPase Rap1B through the action of the PI3K product PtdIns(3,4,5)P(3), but not PtdIns(3,4)P(2), and that this process may contribute to potentiation of platelet aggregation.