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.     

The c-Cbl-associated protein and c-Cbl are two new partners of the SH2-containing inositol polyphosphate 5-phosphatase SHIP2

SHIP2 is a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) 5-phosphatase which contains motifs susceptible to mediate protein-protein interaction. Using yeast two-hybrid, GST-pulldown, and coimmunoprecipitation studies, we isolated the CAP cDNA as a specific partner of SHIP2 proline-rich domain and showed by GST-pulldown experiments that the interaction took place with the SH3C of CAP. The interaction was not modulated in COS-7 cells stimulated by EGF neither in CHO cells overexpressing the insulin receptor in the presence or absence of insulin stimulation. We also showed that SHIP2 was able to coimmunoprecipitate with endogenous c-Cbl protein in the absence of CAP and with the insulin receptor in CHO-IR cell extracts. The presence of SHIP2 in a complex around the insulin receptor could account for the very specific increase in insulin sensitivity of SHIP2 knock-out mice.     

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.     

Regulation of PDGF-stimulated SHIP2 tyrosine phosphorylation and association with Shc in 3T3-L1 preadipocytes

In 3T3-L1 and human preadipocytes, insulin results in the isolated rise in phosphatidylinositol (PI)-3,4,5-P3, whereas PDGF produces PI(3,4)P2 in addition to PI(3,4,5)P3. SH2 domain-containing inositol 5-phosphatase 2 (SHIP2) converts PI(3,4,5)P3 into PI(3,4)P2. PDGF, but not insulin, stimulates SHIP2 tyrosine phosphorylation and its association with Shc in human and 3T3-L1 preadipocytes. We now demonstrate that SHIP2 tyrosine phosphorylation and association with Shc in PDGF-treated 3T3-L1 preadipocytes was reduced by bisindolylmaleimide I (BisI), an inhibitor of conventional/novel protein kinase C (PKC). However, the production of PI(3,4)P2 and PI(3,4,5)P3 by PDGF was unaffected by BisI. Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate (TPA) was not sufficient to induce SHIP2 tyrosine phosphorylation. Furthermore, we identified threonine 958 (T958) as a novel PDGF-responsive SHIP2 phosphorylation site. Mutation of T958 to alanine reduced PDGF-stimulated SHIP2 tyrosine phosphorylation and association with Shc, but did not alter its anti-proliferative effect on preadipocytes. This study demonstrates that SHIP2 tyrosine phosphorylation and Shc association can be regulated by serine/threonine signaling pathways, either indirectly (via PKC), or directly (via T958). Interestingly, the anti-proliferative effect of SHIP2 T958A, as well as another SHIP2 mutant (Y986F, Y987F) that also displays defective tyrosine phosphorylation and Shc association, does not depend on these molecular events.     

SIP/SHIP inhibits Xenopus oocyte maturation induced by insulin and phosphatidylinositol 3-kinase.

SIP (signaling inositol phosphatase) or SHIP (SH2-containing inositol phosphatase) is a recently identified SH2 domain-containing protein which has been implicated as an important signaling molecule. SIP/SHIP becomes tyrosine phosphorylated and binds the phosphotyrosine-binding domain of SHC in response to activation of hematopoietic cells. The signaling pathways and biological responses that may be regulated by SIP have not been demonstrated. SIP is a phosphatidylinositol- and inositol-polyphosphate 5-phosphatase with specificity in vitro for substrates phosphorylated at the 3' position. Phosphatidylinositol 3'-kinase (PI 3-kinase) is an enzyme which is involved in mitogenic signaling and whose phosphorylated lipid products are predicted to be substrates for SIP. We tested the hypothesis that SIP can modulate signaling by PI 3-kinase in vivo by injecting SIP cRNAs into Xenopus oocytes. SIP inhibited germinal vesicle breakdown (GVBD) induced by expression of a constitutively activated form of PI 3-kinase (p110*) and blocked GVBD induced by insulin. SIP had no effect on progesterone-induced GVBD. Catalytically inactive SIP had little effect on insulin- or PI 3-kinase-induced GVBD. Expression of SIP, but not catalytically inactive SIP, also blocked insulin-induced mitogen-activated protein kinase phosphorylation in oocytes. SIP specifically and markedly reduced the level of phosphatidylinositol (3,4,5) triphosphate [PtdIns(3,4,5)P3] generated in oocytes in response to insulin. These results demonstrate that a member of the phosphatidylinositol polyphosphate 5-phosphatase family can inhibit signaling in vivo. Further, our data suggest that the generation of PtdIns(3,4,5)P3 by PI 3-kinase is necessary for insulin-induced GVBD in Xenopus oocytes.     

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.     

Therapeutic potential of SH2 domain-containing inositol-5'-phosphatase 1 (SHIP1) and SHIP2 inhibition in cancer

Many tumors present with increased activation of the phosphatidylinositol 3-kinase (PI3K)-PtdIns(3,4,5)P(3)-protein kinase B (PKB/Akt) signaling pathway. It has long been thought that the lipid phosphatases SH2 domain-containing inositol-5'-phosphatase 1 (SHIP1) and SHIP2 act as tumor suppressors by counteracting with the survival signal induced by this pathway through hydrolysis or PtdIns(3,4,5)P(3) to PtdIns(3,4)P(2). However, a growing body of evidence suggests that PtdInd(3,4)P(2) is capable of, and essential for, Akt activation, thus suggesting a potential role for SHIP1/2 enzymes as proto-oncogenes. We recently described a novel SHIP1-selective chemical inhibitor (3α-aminocholestane [3AC]) that is capable of killing malignant hematologic cells. In this study, we further investigate the biochemical consequences of 3AC treatment in multiple myeloma (MM) and demonstrate that SHIP1 inhibition arrests MM cell lines in either G0/G1 or G2/M stages of the cell cycle, leading to caspase activation and apoptosis. In addition, we show that in vivo growth of MM cells is blocked by treatment of mice with the SHIP1 inhibitor 3AC. Furthermore, we identify three novel pan-SHIP1/2 inhibitors that efficiently kill MM cells through G2/M arrest, caspase activation and apoptosis induction. Interestingly, in SHIP2-expressing breast cancer cells that lack SHIP1 expression, pan-SHIP1/2 inhibition also reduces viable cell numbers, which can be rescued by addition of exogenous PtdIns(3,4)P(2). In conclusion, this study shows that inhibition of SHIP1 and SHIP2 may have broad clinical application in the treatment of multiple tumor types.     

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.     

Distinct phosphatidylinositol 3-kinase lipid products accumulate upon oxidative and osmotic stress and lead to different cellular responses

Signaling by phosphatidylinositol (PI) 3-kinases is mediated by 3-phosphoinositides, which bind to Pleckstrin homology (PH) domains that are present in a wide spectrum of proteins. PH domains can be classified into three groups based on their different lipid binding specificities. Distinct 3-phosphoinositides can accumulate upon PI 3-kinase activation in cells in response to different stimuli and mediate specific cellular responses. In Swiss 3T3 mouse fibroblasts, oxidative stress induced by 1 mM H(2)O(2) caused almost exclusive accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3, 4)P(2)), whereas osmotic stress increased both phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) and PtdIns(3,4)P(2) levels. The increase in PtdIns(3,4)P(2) levels, caused by oxidative stress, correlated with the activation of protein kinase B, which has a promiscuous PH domain that binds both PtdIns(3,4,5)P(3) and PtdIns(3, 4)P(2). p70 S6 kinase, another signaling component downstream of PI 3-kinase, however, was not activated by this oxidative stress-induced increase in PtdIns(3,4)P(2) levels. Increased PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) levels in response to osmotic stress did not correlate with protein kinase B activation, because of concomitant activation of an inhibitory pathway, but p70 S6 kinase was activated by osmotic stress. These results demonstrate that PtdIns(3,4)P(2) can accumulate independently of PtdIns(3,4, 5)P(3) and exerts a pattern of cellular responses that is distinct from that induced by accumulation of PtdIns(3,4,5)P(3).     

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 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.     

SHIP1 and Lyn Kinase Negatively Regulate Integrin alpha IIb beta 3 signaling in platelets

Integrin alpha(IIb)beta(3) plays a critical role in platelet function, promoting a broad range of functional responses including platelet adhesion, spreading, aggregation, clot retraction, and platelet procoagulant function. Signaling events operating downstream of this receptor (outside-in signaling) are important for these responses; however the mechanisms negatively regulating integrin alpha(IIb)beta(3) signaling remain ill-defined. We demonstrate here a major role for the Src homology 2 domain-containing inositol 5-phosphatase (SHIP1) and Src family kinase, Lyn, in this process. Our studies on murine SHIP1 knockout platelets have defined a major role for this enzyme in regulating integrin alpha(IIb)beta(3)-dependent phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) accumulation, necessary for a cytosolic calcium response and platelet spreading. SHIP1 phosphorylation and PtdIns(3,4,5)P(3) metabolism is partially regulated through Lyn kinase, resulting in an enhanced calcium flux and spreading response in Lyn-deficient mouse platelets. Analysis of platelet adhesion dynamics under physiological blood flow conditions revealed an important role for SHIP1 in regulating platelet adhesion on fibrinogen. Specifically, SHIP1-dependent PtdIns(3,4,5)P(3) metabolism down-regulates the stability of integrin alpha(IIb)beta(3)-fibrinogen adhesive bonds, leading to a decrease in the proportion of platelets forming shear-resistant adhesion contacts. These studies define a major role for SHIP1 and Lyn as negative regulators of integrin alpha(IIb)beta(3) adhesive and signaling function.     

Regulation of phosphoinositide signaling by the inositol polyphosphate 5-phosphatases

Phosphoinositide signaling molecules control cellular growth, proliferation and differentiation, intracellular vesicle trafficking, and cytoskeletal rearrangement. The inositol polyphosphate 5-phosphatase family remove the D-5 position phosphate from PtdIns(3,4,5)P3, PtdIns(4,5)P2 and PtdIns(3,5)P2 forming PtdIns(3,4)P2, PtdIns(4)P and PtdIns(3)P respectively. This enzyme family, comprising ten mammalian members, exhibit seemingly non-redundant functions including the regulation of synaptic vesicle recycling, hematopoietic cell function and insulin signaling. Here we highlight recently established insights into the functions of two well characterized 5-phosphatases OCRL and SHIP2, which have been the subject of extensive functional studies, and the characterization of recently identified members, SKIP and PIPP, in order to highlight the diverse and complex functions of this enzyme family.     

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.     

Overexpression of SH2-containing inositol phosphatase 2 results in negative regulation of insulin-induced metabolic actions in 3T3-L1 adipocytes via its 5'-phosphatase catalytic activity

Phosphatidylinositol (PI) 3-kinase plays an important role in various metabolic actions of insulin including glucose uptake and glycogen synthesis. Although PI 3-kinase primarily functions as a lipid kinase which preferentially phosphorylates the D-3 position of phospholipids, the effect of hydrolysis of the key PI 3-kinase product PI 3,4,5-triphosphate [PI(3,4,5)P3] on these biological responses is unknown. We recently cloned rat SH2-containing inositol phosphatase 2 (SHIP2) cDNA which possesses the 5'-phosphatase activity to hydrolyze PI(3,4,5)P3 to PI 3,4-bisphosphate [PI(3,4)P2] and which is mainly expressed in the target tissues of insulin. To study the role of SHIP2 in insulin signaling, wild-type SHIP2 (WT-SHIP2) and 5'-phosphatase-defective SHIP2 (Delta IP-SHIP2) were overexpressed in 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer. Early events of insulin signaling including insulin-induced tyrosine phosphorylation of the insulin receptor beta subunit and IRS-1, IRS-1 association with the p85 subunit, and PI 3-kinase activity were not affected by expression of either WT-SHIP2 or Delta IP-SHIP2. Because WT-SHIP2 possesses the 5'-phosphatase catalytic region, its overexpression marked by decreased insulin-induced PI(3,4,5)P3 production, as expected. In contrast, the amount of PI(3,4,5)P3 was increased by the expression of Delta IP-SHIP2, indicating that Delta IP-SHIP2 functions in a dominant-negative manner in 3T3-L1 adipocytes. Both PI(3,4,5)P3 and PI(3,4)P2 were known to possibly activate downstream targets Akt and protein kinase C lambda in vitro. Importantly, expression of WT-SHIP2 inhibited insulin-induced activation of Akt and protein kinase C lambda, whereas these activations were increased by expression of Delta IP-SHIP2 in vivo. Consistent with the regulation of downstream molecules of PI 3-kinase, insulin-induced 2-deoxyglucose uptake and Glut4 translocation were decreased by expression of WT-SHIP2 and increased by expression of Delta IP-SHIP2. In addition, insulin-induced phosphorylation of GSK-3beta and activation of PP1 followed by activation of glycogen synthase and glycogen synthesis were decreased by expression of WT-SHIP2 and increased by the expression of Delta IP-SHIP2. These results indicate that SHIP2 negatively regulates metabolic signaling of insulin via the 5'-phosphatase activity and that PI(3,4,5)P3 rather than PI(3,4)P2 is important for in vivo regulation of insulin-induced activation of downstream molecules of PI 3-kinase leading to glucose uptake and glycogen synthesis.     

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.     

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.     

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.     

Pathway for the formation of D-3 phosphate containing inositol phospholipids in intact human platelets

We have identified the structure of phosphatidylinositol 3-phosphate (PtdIns(3)P), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in human platelets. These lipids accounted for less than 2% of the total 32P incorporated into inositol phospholipids in the platelets. All three lipids were labeled in unstimulated platelets, but incorporation of 32P changed rapidly by 15 s after thrombin stimulation, suggesting that they are important in platelet activation. Specific inositol polyphosphate phosphatases were used to both identify the lipid structures and to determine the route of synthesis of these lipids. During 32P labeling and after thrombin stimulation of human platelets, as much as 60% of the total radioactivity present in PtdIns(3,4)P2 was found in the D-4 phosphate and only 35% in the D-3 phosphate indicating that PtdIns(3)P is the precursor of PtdIns(3,4)P2. In addition, the D-5 and D-4 phosphates of PtdIns(3,4,5)P3 each contained 35-40% of the total radioactivity in the molecule compared with only 18-28% in the D-3 position, suggesting that PtdIns(3,4)P2 and not PtdIns(4,5)P2 is the major precursor of this lipid. These results define the predominant pathway for synthesis of these lipids in platelets as PtdIns----PtdIns(3)P----PtdIns(3,4)P2----PtdIns(3,4,5)P3.     

Direct activation of the epithelial Na(+) channel by phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate produced by phosphoinositide 3-OH kinase

The phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) is accepted to be a direct modulator of ion channel activity. The products of phosphoinositide 3-OH kinase (PI3K), PtdIns(3,4)P(2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), in contrast, are not. We report here activation of the epithelial Na(+) channel (ENaC) reconstituted in Chinese hamster ovary cells by PI3K. Insulin-like growth factor-I also activated reconstituted ENaC and increased Na(+) reabsorption across renal A6 epithelial cell monolayers via PI3K. Neither IGF-I nor PI3K affected the levels of ENaC in the plasma membrane. The effects of PI3K and IGF-I on ENaC activity paralleled changes in the plasma membrane levels of the PI3K product phospholipids, PtdIns(3,4)P(2)/PtdIns(3,4,5)P(3), as measured by evanescent field fluorescence microscopy. Both PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) activated ENaC in excised patches. Activation of ENaC by PI3K and its phospholipid products corresponded to changes in channel open probability. We conclude that PI3K directly modulates ENaC activity via PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3). This represents a novel transduction pathway whereby growth factors, such as IGF-I, rapidly modulate target proteins independent of signaling elicited by kinases downstream of PI3K.