The inositol 5-phosphatase SHIP2 regulates endocytic clathrin-coated pit dynamics

Phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P₂) and its phosphorylated product PI 3,4,5-triphosphate (PI(3,4,5)P₃) are two major phosphoinositides concentrated at the plasma membrane. Their levels, which are tightly controlled by kinases, phospholipases, and phosphatases, regulate a variety of cellular functions, including clathrin-mediated endocytosis and receptor signaling. In this study, we show that the inositol 5-phosphatase SHIP2, a negative regulator of PI(3,4,5)P₃-dependent signaling, also negatively regulates PI(4,5)P₂ levels and is concentrated at endocytic clathrin-coated pits (CCPs) via interactions with the scaffold protein intersectin. SHIP2 is recruited early at the pits and dissociates before fission. Both knockdown of SHIP2 expression and acute production of PI(3,4,5)P₃ shorten CCP lifetime by enhancing the rate of pit maturation, which is consistent with a positive role of both SHIP2 substrates, PI(4,5)P₂ and PI(3,4,5)P₃, on coat assembly. Because SHIP2 is a negative regulator of insulin signaling, our findings suggest the importance of the phosphoinositide metabolism at CCPs in the regulation of insulin signal output.     

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 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.     

Comparative mechanistic and substrate specificity study of inositol polyphosphate 5-phosphatase Schizosaccharomyces pombe Synaptojanin and SHIP2

Inositol-5-phosphatases are important enzymes involved in the regulation of diverse cellular processes from synaptic vesicle recycling to insulin signaling. We describe a comparative study of two representative inositol-5-phosphatases, Schizosaccharomyces pombe synaptojanin (SPsynaptojanin) and human SH2 domain-containing inositol-5-phosphatase SHIP2. We show that in addition to Mg2+, transition metals such as Mn2+, Co2+, and Ni2+ are also effective activators of SPsynaptojanin. In contrast, Ca2+ and Cu2+ are inhibitory. We provide evidence that Mg2+ binds the same site occupied by Ca2+ observed in the crystal structure of SPsynaptojanin complexed with inositol 1,4-bisphosphate (Ins(1,4)P2). Ionizations important for substrate binding and catalysis are defined for the SPsynaptojanin-catalyzed Ins(1,4,5)P3 reaction. Kinetic analysis with four phosphatidylinositol lipids bearing a 5-phosphate and 54 water-soluble inositol phosphates reveals that SP-synaptojanin and SHIP2 possess much broader substrate specificity than previously appreciated. The rank order for SPsynaptojanin is Ins(2,4,5)P3 > phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) approximately Ins(4,5)P2 approximately Ins(1,4,5)P3 approximately Ins(4,5,6)P3 > PtdIns(3,5)P2 approximately PtdIns(3,4,5)P3 approximately Ins(1,2,4,5)P4 approximately Ins(1,3,4,5)P4 approximately Ins-(2,4,5,6)P4 approximately Ins(1,2,4,5,6)P5. The rank order for SHIP2 is Ins(1,2,3,4,5)P5 > Ins(1,3,4,5)P4 > PtdIns(3,4,5)P4 approximately PtdIns(3,5)P2 approximately Ins(1,4,5,6)P4 approximately Ins(2,4,5,6)P4. Because inositol phosphate isomers elicit different biological activities, the extended substrate specificity for SPsynaptojanin and SHIP2 suggest that these enzymes likely have multiple roles in cell signaling and may regulate distinct pathways. The unique substrate specificity profiles and the importance of 2-position phosphate in binding also have important implications for the design of potent and selective SPsynaptojanin and SHIP2 inhibitors for pharmacological investigation.     

The SH2 domain containing inositol 5-phosphatase SHIP2 controls phosphatidylinositol 3,4,5-trisphosphate levels in CHO-IR cells stimulated by insulin

The lipid phosphatase SHIP2 (SH2 domain containing inositol 5-phosphatase 2) has recently been shown to be a potent negative regulator of insulin signaling and insulin sensitivity in vivo. We show here that SHIP2 is expressed in Chinese hamster ovary cells overexpressing the insulin receptor (CHO-IR cells) and tyrosine phosphorylated upon insulin stimulation. We show that SHIP2, which is recruited in anti-phosphotyrosine immunoprecipitates in insulin-stimulated cells, accounts for the insulin sensitivity or apparent increase in activity reported by Guilherme et al. (J. Biol. Chem. 271, 29533-29536, 1996). Overexpression of SHIP2 led to a decrease of the insulin-dependent PIP3 production as well as Akt/PKB activation and MAPK stimulation.     

The RhoA effector mDia is induced during T cell activation and regulates actin polymerization and cell migration in T lymphocytes

Regulation of actin polymerization is critical for many different functions of T lymphocytes, including cell migration. Here we show that the RhoA effector mDia is induced in vitro in activated PBL and is highly expressed in vivo in diseased tissue-infiltrating activated lymphocytes. mDia localizes at the leading edge of polarized T lymphoblasts in an area immediately posterior to the leading lamella, in which its effector protein profilin is also concentrated. Overexpression of an activated mutant of mDia results in an inhibition of both spontaneous and chemokine-directed T cell motility. mDia does not regulate the shape of the cell, which involves another RhoA effector, p160 Rho-coiled coil kinase, and is not involved in integrin-mediated cell adhesion. However, mDia activation blocked CD3- and PMA-mediated cell spreading. mDia activation increased polymerized actin levels, which resulted in the blockade of chemokine-induced actin polymerization by depletion of monomeric actin. Moreover, mDia was shown to regulate the function of the small GTPase Rac1 through the control of actin availability. Together, our data demonstrate that RhoA is involved in the control of the filamentous actin/monomeric actin balance through mDia, and that this balance is critical for T cell responses.     

The SH2 inositol 5-phosphatase Ship1 is recruited in an SH2-dependent manner to the erythropoietin receptor

Ship1 (SH2 inositol 5-phosphatase 1) has been shown to be a target of tyrosine phosphorylation downstream of cytokine and immunoregulatory receptors. In addition to its catalytic activity on phosphatidylinositol substrates, it can serve as an adaptor protein in binding Shc and Grb2. Erythropoietin (EPO), the primary regulator of erythropoiesis, has been shown to activate the tyrosine phosphorylation of Shc, resulting in recruitment of Grb2. However, the mechanism by which the erythropoietin receptor (EPO-R) recruits Shc remains unknown. EPO activates the tyrosine phosphorylation of Ship1, resulting in the interdependent recruitment of Shc and Grb2. Ship1 is recruited to the EPO-R in an SH2-dependent manner. Utilizing a panel of EPO-R deletion and tyrosine mutants, we have discovered remarkable redundancy in Ship1 recruitment. EPO-R Tyr(401) appears to be a major site of Ship1 binding; however, Tyr(429) and Tyr(431) can also serve to recruit Ship1. In addition, we have shown that EPO stimulates the formation of a ternary complex consisting of Ship1, Shc, and Grb2. Ship1 may modulate several discrete signal transduction pathways. EPO-dependent activation of ERK1/2 and protein kinase B (PKB)/Akt was examined utilizing a panel of EPO-R deletion mutants. Activation of ERK1/2 was observed in EPO-RDelta99, which retains only the most proximal tyrosine, Tyr(343). In contrast, EPO-dependent PKB activation was observed in EPO-RDelta43, but not in EPO-RDelta99. It appears that EPO-dependent PKB activation is downstream of a region that indirectly couples to phosphatidylinositol 3-kinase.     

Regulation of the Src homology 2-containing inositol 5-phosphatase SHIP1 in HIP1/PDGFbeta R-transformed cells

It has been shown previously that the Huntingtin interacting protein 1 gene (HIP1) was fused to the platelet-derived growth factor beta receptor gene (PDGFbetaR) in leukemic cells of a patient with chronic myelomonocytic leukemia. This resulted in the expression of the chimeric HIP1/PDGFbetaR protein, which oligomerizes, is constitutively tyrosine-phosphorylated, and transforms the Ba/F3 murine hematopoietic cell line to interleukin-3-independent growth. Tyrosine phosphorylation of a 130-kDa protein (p130) correlates with transformation by HIP1/PDGFbetaR and related transforming mutants. We report here that the p130 band is immunologically related to the 125-kDa isoform of the Src homology 2-containing inositol 5-phosphatase, SHIP1. We have found that SHIP1 associates and colocalizes with the HIP1/PDGFbetaR fusion protein and related transforming mutants. These mutants include a mutant that has eight Src homology 2-binding phosphotyrosines mutated to phenylalanine. In contrast, SHIP1 does not associate with H/P(KI), the kinase-dead form of HIP1/PDGFbetaR. We also report that phosphorylation of SHIP1 by HIP1/PDGFbetaR does not change its 5-phosphatase-specific activity. This suggests that phosphorylation and possible PDGFbetaR-mediated sequestration of SHIP1 from its substrates (PtdIns(3,4,5)P(3) and Ins(1,3,4,5)P(4)) might alter the levels of these inositol-containing signal transduction molecules, resulting in activation of downstream effectors of cellular proliferation and/or survival.     

Sac2/INPP5F is an inositol 4-phosphatase that functions in the endocytic pathway

The recruitment of inositol phosphatases to endocytic membranes mediates dephosphorylation of PI(4,5)P₂, a phosphoinositide concentrated in the plasma membrane, and prevents its accumulation on endosomes. The importance of the conversion of PI(4,5)P₂ to PtdIns during endocytosis is demonstrated by the presence of both a 5-phosphatase and a 4-phosphatase (Sac domain) module in the synaptojanins, endocytic PI(4,5)P₂ phosphatases conserved from yeast to humans and the only PI(4,5)P₂ phosphatases in yeast. OCRL, another 5-phosphatase that couples endocytosis to PI(4,5)P₂ dephosphorylation, lacks a Sac domain. Here we show that Sac2/INPP5F is a PI4P phosphatase that colocalizes with OCRL on endocytic membranes, including vesicles formed by clathrin-mediated endocytosis, macropinosomes, and Rab5 endosomes. An OCRL–Sac2/INPP5F interaction could be demonstrated by coimmunoprecipitation and was potentiated by Rab5, whose activity is required to recruit Sac2/INPP5F to endosomes. Sac2/INPP5F and OCRL may cooperate in the sequential dephosphorylation of PI(4,5)P₂ at the 5 and 4 position of inositol in a partnership that mimics that of the two phosphatase modules of synaptojanin.     

The inositol 5-phosphatase INPP5B regulates B cell receptor clustering and signaling

Upon antigen binding, the B cell receptor (BCR) undergoes clustering to form a signalosome that propagates downstream signaling required for normal B cell development and physiology. BCR clustering is dependent on remodeling of the cortical actin network, but the mechanisms that regulate actin remodeling in this context remain poorly defined. In this study, we identify the inositol 5-phosphatase INPP5B as a key regulator of actin remodeling, BCR clustering, and downstream signaling in antigen-stimulated B cells. INPP5B acts via dephosphorylation of the inositol lipid PI(4,5)P₂ that in turn is necessary for actin disassembly, BCR mobilization, and cell spreading on immobilized surface antigen. These effects can be explained by increased actin severing by cofilin and loss of actin linking to the plasma membrane by ezrin, both of which are sensitive to INPP5B-dependent PI(4,5)P₂ hydrolysis. INPP5B is therefore a new player in BCR signaling and may represent an attractive target for treatment of B cell malignancies caused by aberrant BCR signaling.     

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.     

The host phosphoinositide 5-phosphatase SHIP2 regulates dissemination of vaccinia virus

After fusing with the plasma membrane, enveloped poxvirus virions form actin-filled membranous protrusions, called tails, beneath themselves and move toward adjacent uninfected cells. While much is known about the host and viral proteins that mediate formation of actin tails, much less is known about the factors controlling release. We found that the phosphoinositide 5-phosphatase SHIP2 localizes to actin tails. Localization requires phosphotyrosine, Abl and Src family tyrosine kinases, and neural Wiskott-Aldrich syndrome protein (N-WASP) but not the Arp2/Arp3 complex or actin. Cells lacking SHIP2 have normal actin tails but release more virus. Moreover, cells infected with viral strains with mutations in the release inhibitor A34 release more virus but recruit less SHIP2 to tails. Thus, the inhibitory effects of A34 on virus release are mediated by SHIP2. Together, these data suggest that SHIP2 and A34 may act as gatekeepers to regulate dissemination of poxviruses when environmental conditions are conducive.     

Sertoli cell vacuolization and abnormal germ cell adhesion in mice deficient in an inositol polyphosphate 5-phosphatase

The dynamic nature of cellular interactions during differentiation of germ cells and their translocation from the basement membrane to the lumen of the seminiferous tubules requires the existence of complex and well-regulated cellular adhesion mechanisms in the testis. Successful migration of the developing germ cells is characterized by dynamic breakage and reformation of cadherin-containing adherens junctions between the germ cells and Sertoli cells, the polarized somatic cells of the testis that support and nourish the developing gametes. Here, we demonstrate the accumulation of abnormally swollen, actin-coated, endosome-like structures that contain intact adherens junctions and stain positive for N-cadherin and beta-catenin in the Sertoli cell cytosol of mice deficient in Inpp5b, an inositol polyphosphate 5-phosphatase. Simultaneous to the formation of these abnormal structures, developing germ cells are prematurely released from the seminiferous epithelium and sloughed into the epididymis. Our results demonstrate a role for Inpp5b in the regulation of cell adhesion in the testis and in the formation of junctional complexes with neighboring cells, and they emphasize the important and essential role of phosphoinositides in spermatogenesis.     

The control of phosphatidylinositol 3,4-bisphosphate concentrations by activation of the Src homology 2 domain containing inositol polyphosphate 5-phosphatase 2, SHIP2

Activation of class Ia PI3K (phosphoinositide 3-kinase) produces PtdInsP3, a vital intracellular mediator whose degradation generates additional lipid signals. In the present study vanadate analogues that inhibit PTPs (protein tyrosine phosphatases) were used to probe the mechanisms which regulate the concentrations of these molecules allowing their independent or integrated function. In 1321N1 cells, which lack PtdInsP3 3-phosphatase activity, sodium vanadate or a cell permeable derivative, bpV(phen) [potassium bisperoxo(1,10-phenanthroline)oxovanadate (V)], increased the recruitment into anti-phosphotyrosine immunoprecipitates of PI3K activity and of the p85 and p110a subunits of class Ia PI3K and enhanced the recruitment of PI3K activity stimulated by PDGF (platelet-derived growth factor). However, neither inhibitor much increased cellular PtdInsP3 concentrations, but both diminished dramatically the accumulation of PtdInsP3 stimulated by PDGF or insulin and markedly increased the control and stimulated concentrations of PtdIns(3,4)P2. These actions were accounted for by the ability of PTP inhibitors to stimulate the activity of endogenous PtdInsP3 5-phosphatase(s), particularly SHIP2 (Src homology 2 domain containing inositol polyphosphate 5-phosphatase 2) and to inhibit types I and II PtdIns(3,4)P2 4-phosphatases. Thus bpV(phen) promoted the translocation of SHIP2 from the cytosol to a Triton X-100-insoluble fraction and induced a marked (5-10-fold) increase in SHIP2 specific activity mediated by enhanced tyrosine phosphorylation. The net effect of these inhibitors was, therefore, to switch the signal output of class I PI3K from PtdInsP3 to PtdIns(3,4)P2. A key component controlling this shift in the balance of lipid signals is the activation of SHIP2 by increased tyrosine phosphorylation, an effect observed in HeLa cells in response to both PTP inhibitors and epidermal growth factor.     

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.     

The two SH2-domain-containing inositol 5-phosphatases SHIP1 and SHIP2 are coexpressed in human T lymphocytes.

The activation of many hematopoietic cells via cytokine receptors, as well as B and T cell receptors, leads to the tyrosine phosphorylation of Shc and its association with both Grb2-Sos1 complexes and with a 145 kDa protein referred to as the SH2 containing inositol 5-phosphatase (SHIP1). In a search of putative 5-phosphatase isoenzymes, we have isolated a second SH2 domain containing inositol 5-phosphatase, referred to as (SHIP2). Both SHIP1 and SHIP2 are coexpressed in human T lymphocytes. This was shown at the protein level by Western blot analysis in transformed T cell lines and in peripheral blood T lymphocytes either unstimulated or after in vitro activation through TCR-CD3 complex. SHIP1 protein level was not modulated after activation of T lymphocytes, in contrast to SHIP2, which was increased after long-term stimulation. SHIP1 was tyrosine phosphorylated in resting naive T cells. This was not observed in the transformed T cell lines. T lymphocyte is therefore a model of coexpression of the two SH2-containing inositol 5-phosphatases SHIP1 and SHIP2.     

Src homology 2 domain-containing inositol 5-phosphatase 1 mediates cell cycle arrest by FcgammaRIIB

We previously found that low affinity receptors for the Fc portion of IgG, FcgammaRIIB, which are widely expressed by hematopoietic cells, can negatively regulate receptor tyrosine kinase-dependent cell proliferation. We investigated here the mechanisms of this inhibition. We used as experimental models wild-type mast cells, which constitutively express the stem cell factor receptor Kit and FcgammaRIIB, FcgammaRIIB-deficient mast cells reconstituted with wild-type or mutated FcgammaRIIB, and Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1)-deficient mast cells. We found that, upon coaggregation with Kit, FcgammaRIIB are tyrosyl-phosphorylated, recruit SHIP1, but not SHIP2, SH2 domain-containing protein tyrosine phosphatase-1 or -2, abrogate Akt phosphorylation, shorten the duration of the activation of mitogen-activated protein kinases of the Ras and Rac pathways, abrogate cyclin induction, prevent cells from entering the cell cycle, and block thymidine incorporation. FcgammaRIIB-mediated inhibition of Kit-dependent cell proliferation was reduced in SHIP1-deficient mast cells, whereas inhibition of IgE-induced responses was abrogated. Cell proliferation was, however, inhibited by coaggregating Kit with FcgammaRIIB whose intracytoplasmic domain was replaced with the catalytic domain of SHIP1. These results demonstrate that FcgammaRIIB use SHIP1 to inhibit pathways shared by receptor tyrosine kinases and immunoreceptors to trigger cell proliferation and cell activation, respectively, but that, in the absence of SHIP1, FcgammaRIIB can use other effectors that specifically inhibit cell proliferation.     

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.