Insulin is the primary hormone involved in glucose homeostasis, and impairment of insulin action and/or secretion has a critical role in the pathogenesis of diabetes mellitus. Type-II SH2-domain-containing inositol 5-phosphatase, or 'SHIP2', is a member of the inositol polyphosphate 5-phosphatase family. In vitro studies have shown that SHIP2, in response to stimulation by numerous growth factors and insulin, is closely linked to signalling events mediated by both phosphoinositide-3-OH kinase and Ras/mitogen-activated protein kinase. Here we report the generation of mice lacking the SHIP2 gene. Loss of SHIP2 leads to increased sensitivity to insulin, which is characterized by severe neonatal hypoglycaemia, deregulated expression of the genes involved in gluconeogenesis, and perinatal death. Adult mice that are heterozygous for the SHIP2 mutation have increased glucose tolerance and insulin sensitivity associated with an increased recruitment of the GLUT4 glucose transporter and increased glycogen synthesis in skeletal muscles. Our results show that SHIP2 is a potent negative regulator of insulin signalling and insulin sensitivity in vivo.

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Distinct forms of inositol and phosphatidylinositol polyphosphate 5-phosphatases selectively remove the phosphate from the 5-position of the inositol ring from both soluble and lipid substrates. SHIP1 is the 145-kDa SH2 domain-containing inositol 5-phosphatase expressed in haematopoietic cells. SHIP2 is a related but distinct gene product. We report here that SHIP2 can be expressed in an active form both in Escherichia coli and in COS-7 cells. A truncated 103-kDa recombinant protein could be purified from bacteria that display both inositol 1,3,4,5-tetrakisphosphate (InsP 4 ) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) phosphatase activities. COS-7 cell lysates transfected with SHIP2 had increased PtdIns(3,4,5)P 3 phosphatase activity as compared to the vector alone.z 1998 Federation of European Biochemical Societies.

apoptosis ͉ inositol phosphate ͉ lymphocyte ͉ Rasa3 C ross-linking of the antigen receptor on T and B lymphocytes by antigen is followed by the rapid activation of phospholipase C␥, resulting in the hydrolysis of phosphatidylinositol 4,5-bisphosphate and the production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ] and diacylglycerol. Ins(1,4,5)P 3 causes release of calcium from the endoplasmic reticulum and is the substrate of two main metabolic pathways. Dephosphorylation by type I Ins(1,4,5)P 3 5-phosphatase generates the inactive metabolite inositol 1,4-bisphosphate (1, 2). Phosphorylation by isoforms A, B, and C of Ins(1,4,5)P 3 3-kinase (Itpkb) or inositol polyphosphate multikinase results in the production of inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P 4 ] (3). In various cellular systems, it has been demonstrated that Ins(1,3,4,5)P 4 is a modulator of Ins(1,4,5)P 3 levels and calcium mobilization (3-5). However, Ins(1,4,5)P 3 and calcium concentrations in response to T cell antigen receptor stimulation were found normal in double-positive thymocytes from Itpkb-deficient mice, despite an important decrease in Ins(1,3,4,5)P 4 production resulting in profound defects in the final maturation of these cells (6, 7). Alternative pathways to mediate the cellular effects of Ins(1,3,4,5)P 4 include the binding to pleckstrin homology (PH) domains of specific proteins acting as Ins(1,3,4,5)P 4 receptor, and the synthesis of higher inositol phosphates (8).To assess the potential contribution of the Ins(1,4,5)P 3 -ItpkbIns(1,3,4,5)P 4 signaling pathway to B cell function and development, we have analyzed the B cell lineage of Itpkb Ϫ/Ϫ mice. Our results indicate that Itpkb and Ins(1,3,4,5)P 4 mediate a survival signal in B cells by regulating Erk signaling pathway and proapoptotic Bim gene expression.

The lipid phosphatase SHIP2 (Src homology 2 domain containing inositol 5-phosphatase 2) has been shown to be expressed in nonhemopoietic and hemopoietic cells. It has been implicated in signaling events initiated by several extracellular signals, such as epidermal growth factor (EGF) and insulin. In COS-7 cells, SHIP2 was tyrosine-phosphorylated at least at two separated tyrosine phosphorylation sites in response to EGF. SHIP2 was coimmunoprecipitated with the EGF receptor (EGFR) and also with the adaptor protein Shc. A C-terminal truncated form of SHIP2 that lacks the 366 last amino acids, referred to as tSHIP2, was also precipitated with the EGFR when transfected in COS-7 cells. The Src homology 2 domain of SHIP2 was unable to precipitate the EGFR in EGF-stimulated cells. Moreover, when transfected in COS-7 cells, it could not be detected in immunoprecipitates of the EGFR. When the His-tagged fulllength enzyme was expressed in COS-7 cells and stained with anti-His 6 monoclonal antibody, a signal was observed at plasma membranes in EGF-stimulated cells that colocalize with the EGFR by double staining. Upon stimulation by EGF, phosphatidylinositol 3,4,5-trisphosphate and protein kinase B activity were decreased in SHIP2-transfected COS-7 cells as compared with the vector alone. SHIP2 appears therefore in a tyrosine-phosphorylated complex with at least two other proteins, the EGFR and Shc.Protein tyrosine phosphorylation plays a central role in the regulation of protein-protein interactions and modulation of enzyme activities (1). Key events in receptor signaling are the interactions of proteins such as the adaptor protein Shc with other phosphorylated proteins. In epidermal growth factor (EGF) 1 signaling, the activation of the EGF receptor (EGFR)could mediate the phosphorylation of a series of proteins, such as the p85 subunit of the phosphoinositide 3-kinase (PI 3-kinase) (2). This phosphorylation has been linked to the activation of PI 3-kinase, which plays an important role in signaling. All mammalian cell types express at least class IA PI 3-kinase isoform, and stimulation of almost every receptor that induces Tyr kinase activity also leads to class IA PI 3-kinase activation (3, 4). PI 3-kinase class IA substrates in vitro are phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate, whereas the preferred substrate in vivo is phosphatidylinositol 4,5-bisphosphate (2). Phosphatidylinositol 3,4,5-trisphosphate (PIP3) and/or phosphatidylinositol 3,4-bisphosphate (PI 3,4-P 2 ) could activate protein kinase B (PKB) through the binding to the PH domain of 3Ј-phosphoinositide-dependent kinase 1 phosphorylating protein kinase B (5-7). The control of the levels of the second messenger PIP3 depends on the activity of both PI 3-kinase and PIP3 phosphatases (which are members of inositol and phosphatidylinositol 5-phosphatase family) and 3-phosphatases, such as PTEN (phosphatase and tensin deleted from chromosome 10) (8). SHIP1 and SHIP2 (SH2 domain containing inositol 5-phospha...

The termination of activation signals is a critical step in the control of the immune response; perturbation of inhibitory feedback pathways results in profound immune defects culminating in autoimmunity and overwhelming inflammation. FcgammaRIIB receptor is a well described inhibitory receptor. The ligation of B-cell receptor (BCR) and FcgammaRIIB leads to the inhibition of B-cell activation. Numerous studies have demonstrated that the SH2-domain-containing inositol 5-phosphatase SHIP (referred hereto as SHIP-1) is essential in this process. The cDNA encoding a second SH2-domain-containing inositol 5-phosphatase, SHIP-2, has been cloned [Pesesse, Deleu, De Smedt, Drayer and Erneux (1997) Biochem. Biophys. Res. Commun. 239, 697-700]. Here we report the distribution of SHIP-2 in mouse tissues: a Western blot analysis of mouse tissues reveals that SHIP-2 is expressed in both haemopoietic and non-haemopoietic cells. In addition to T-cell and B-cell lines, spleen, thymus and lung are shown to coexpress SHIP-1 and SHIP-2. Moreover, SHIP-2 is detected in fibroblasts, heart and different brain areas. SHIP-2 shows a maximal tyrosine phosphorylation and association to Shc after ligation of BCR to FcgammaRIIB but not after stimulation of BCR alone. Our results therefore suggest a possible role for SHIP-2 in the negative regulation of immunocompetent cells.

In brain, type I inositol-1,4,5-trisphosphate 5-phosphatase (InsP 3 5-phosphatase) is the major isoenzyme hydrolyzing the calcium-mobilizing second messenger InsP 3 . Activity of this enzyme could be measured in both soluble and particulate fractions of tissue homogenates. The protein sequence showed a putative C-terminal isoprenylation site (CVVQ). In this study, two mutants have been generated. The first mutant (C409S) has a serine replacing a cysteine at position 409 of the wild-type enzyme. The second mutant (K407D1) is a deletion mutant that lacks the last five C-terminal amino acids. These constructs were individually expressed by transfection in COS-7 cells. Western blot analysis of wild-type transfected cells indicated that both soluble and particulate fractions had a 43-kDa immunoreactive band, with a higher proportion of the original homogenate associated with the particulate part. On the contrary, when the two mutated constructs were transfected in COS-7 cells, the phosphatase was predominantly soluble. Confocal immunofluorescence studies showed the wild-type enzyme to be present on the cell surface of transfected COS-7 cells and in subcellular compartments around the nucleus. This was not observed for the two mutants, where uniform immunofluorescence labeling was observed throughout the cytosol. Recombinant type I InsP 3 5-phosphatase expressed in Escherichia coli was a substrate of purified farnesyltransferase. Altogether, the data therefore suggest a direct participation of Cys-409 in a C-terminally anchored InsP 3 5-phosphatase by farnesylation.