Phosphatidylinositol 3,4,5-trisphosphate modulation in SHIP2-deficient mouse embryonic fibroblasts Daniel Blero 1 , Jing Zhang 1 , Xavier Pesesse 1 , Bernard Payrastre 2 , Jacques E. Dumont 1 , Ste ´ phane Schurmans 3 and Christophe Erneux 1 1 Interdisciplinary Research Institute (IRIBHM), Universite ´ Libre de Bruxelles, Belgium 2 INSERM U563, Departement d’Oncogenese et Signalization dans les Cellules Hematopoietiques, Ho ˆ pital Purpan, Toulouse Cedex, France 3 IRIBHM, IBMM, Gosselies Belgique The SHIPs (SH2 domain containing inositol 5-phos- phatases) are members of the inositol 5-phosphatase family. Two isoenzymes, named SHIP1 and SHIP2 have been identified and characterized [1–4]. The cellu- lar and tissue distribution of SHIP2 is very wide [5], particularly in cells that do not express SHIP1 (e.g. in heart, muscle or adipocytes). Tyrosine phosphorylation of SHIP2 occurs in response to treatment of cells with various stimuli, e.g. epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin or macrophage colony-stimulating factor (M-CSF) but the biological significance of this phosphorylation is unknown [6–8]. In 3T3-L1 preadipocytes, SHIP2 translocation to the plasma membrane occurs in response to insulin or PDGF. In this model, SHIP2 translocation does not seem to require its tyrosine Keywords inositol 5-phosphatase; mouse embryonic fibroblasts; phosphatidylinositol 3,4,5-trisphosphate; SH2 domain; signal transduction. Correspondence C. Erneux, Institute of Interdisciplinary Research (IRIBHM), Campus Erasme Building C, 808 Route de Lennik, 1070 Brussels, Belgium Fax: +32 2 555 4655 Tel: +32 2 555 4162 E-mail: cerneux@ulb.ac.be Note D. Blero and J. Zhang contributed equally to this work. (Received 27 July 2004, revised 6 February 2005, accepted 21 March 2005) doi:10.1111/j.1742-4658.2005.04672.x 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. Abbreviations CHO-IR, chinese hamster ovary cells overexpressing the insulin receptor; EGF, epidermal growth factor; FBS, foetal bovine serum; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; IGF, insulin-like growth factor; MAP, mitogen activated protein; M-CSF, macrophage colony-stimulating factor; MEF, mouse embryonic fibroblast; PDGF, platelet-derived growth factor; PI 3-kinase, phosphoinositide 3-kinase; PKB, protein kinase B; PtdIns(3,4)P 2 , phosphatidylinositol 3,4-bisphosphate; PtdIns(3,4,5)P 3 , phosphatidylinositol 3,4,5-trisphosphate; PtdIns4P, phosphatidylinositol 4-phosphate; PtdIns(4,5)P 2 , phosphatidylinositol 4,5-bisphosphate; PTEN, phosphate and tension homolog deleted on chromosome 10; SHIP, SH2 domain containing inositol phosphatase. 2512 FEBS Journal 272 (2005) 2512–2522 ª 2005 FEBS phosphorylation [9]. As phosphatidylinositol 3,4,5-tris- phosphate [PtdIns(3,4,5)P 3 ] is a major intracellular sig- nal generated by insulin and insulin-like growth factor (IGF)-1, it has been suggested that SHIP2 is a physio- logically negative regulator of their signalling. Indeed, overexpression of SHIP2 inhibited insulin-induced glu- cose uptake and glycogen synthesis in 3T3-L1 adipo- cytes and L6 myotubes [10,11]. SHIP2 also appears to inhibit the insulin-induced phosphorylation of Akt2, but not Akt1, in 3T3-L1 adipocytes. Upon insulin sti- mulation, SHIP2 is translocated to the plasma mem- brane, where it inhibits the insulin-specific subcellular redistribution of Akt2 [12]. The expression of SHIP2 was enhanced in an animal model of type 2 diabetes which was accompanied by an attenuation of insulin signalling [13]. However, a role for SHIP2 has also been suggested in other pathways: in rat vascular smooth muscle cells, SHIP2 downregulates PDGF and IGF-1 mediated signalling downstream of PI 3-kinase [14]. In glioblastoma cells, SHIP2 inhibits protein kin- ase B (PKB) and provokes a potent cell cycle arrest in G 1 [15]. SHIP2 could play an essential role in cell adhesion and spreading as shown in HeLa cells [16]. A regulatory role for SHIP2 in M-CSF-induced signalling has been recently suggested [8]. SHIP2 also functions in the maintenance and dynamic remodelling of actin structures as well as in endocytosis and downregula- tion of the EGF receptor [17]. In vivo, homozygous disruption of SHIP2 by remo- ving exons 19–29 causes severe hypoglycemia and death within a few hours after birth. Heterozygous dis- ruption of this gene leads to hypersensitivity to insulin demonstrated by the increased glycogen synthesis in skeletal muscles in response to insulin. Injection of d-glucose resulted in a more rapid glucose clearance in SHIP2+ ⁄ – than in SHIP2+ ⁄ + mice. Moreover, the incidences of spontaneous or irradiated-induced tumours were not affected in SHIP2+ ⁄ – mice [18]. Removal of exons 1–18 of SHIP2 resulted in a differ- ent phenotype: the mice were viable and had no increased insulin sensitivity but they were smaller in body weight and length, and were highly resistant to weight gain when placed on a high-fat diet [19]. The reason for this discrepancy between the two pheno- types is currently not understood but several explana- tions have been proposed [19]. We and others previously reported that SHIP2 displays inositol 5-phosphatase activity when PtdIns(3,4,5)P 3 and phosphatidylinositol 4,5-bisphos- phate [PtdIns(4,5)P 2 ] were used as substrate in vitro. Inositol tetrakisphosphate was also a substrate of the enzyme expressed in bacteria [15,20,21]. Moreover, both in COS-7 cells and in chinese hamster ovary cells over- expressing the insulin receptor (CHO-IR) cells transfected with SHIP2, the levels of PtdIns(3,4,5)P 3 were decreased in both EGF and insulin stimulated cells [22,23]. This was also observed in rat vascular smooth muscle cells, where PtdIns(3,4,5)P 3 levels were decreased in SHIP2 transfected cells stimulated by PDGF or IGF-1 [14]. Both PKB and mitogen activated protein (MAP) kinase activities were also decreased in SHIP2 transfected cells suggesting that SHIP2 is a down-regulator of both arms of receptor tyrosine kinase activation [10,15,22,23]. The present study was therefore undertaken to establish the extent of PtdIns(3,4,5)P 3 regulation in SHIP2 – ⁄ – cells derived from 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 – ⁄ – MEF cells but only after short-term (i.e. 5–10 min) incubation with serum. In our model, IGF-1 stimulation did not show this upregulation and PtdIns(4,5)P 2 levels were comparable between SHIP2+ ⁄ + and – ⁄ – MEF cells. Results Status of SHIP2, PTEN, insulin and IGF-1 receptor expression in SHIP2 +/+ and –/– MEF cells SHIP2 – ⁄ – mice were obtained as reported previously [18]. As our SHIP2 – ⁄ – mice died very shortly after birth, we chose to work with MEF cells as a model to measure the 3-phosphorylated phosphoinositides. MEF cells were prepared from embryos of hetero- zygous crosses and genotyped by PCR analysis. Two series of MEF cells (1 and 2) were prepared from two independent crosses to validate the measurements of phosphoinositides (see below). Western blot analy- sis of SHIP2 was performed to confirm the absence of expression of SHIP2 in – ⁄ – MEF cells (Figs 1A and B). The expression of SHIP2 in + ⁄ – MEF cells was decreased as compared to wild type (+ ⁄ +) MEF cells (Fig. 1A) as reported previously [18]. Although we detected the presence of the IGF-1 receptor in MEF cells by western blotting, the b sub- unit of the insulin receptor was not be seen by this method suggesting that it is either not expressed or was below the detection level of the antibodies used in our immunodetection method (Fig. 1B). The expression of the PtdIns(3,4,5)P 3 3-phosphatase, phos- phatase and tension homolog deleted on chromosome 10 (PTEN) [24,25] was not significantly modified between SHIP2+ ⁄ + and – ⁄ – MEF cells (Fig. 1B). No changes in expression of the regulatory subunits of PI 3-kinase p85 were seen between the two types of cells (data not shown). D. Blero et al. Phosphatidylinositol 3,4,5-trisphosphate levels in mouse embryonic fibroblasts FEBS Journal 272 (2005) 2512–2522 ª 2005 FEBS 2513 No change in PtdIns(4,5)P 2 levels in SHIP2 +/+ and –/– MEF cells As in vitro, PtdIns(4,5)P 2 is also a substrate of SHIP2 [15], we compared the levels of [ 3 H]PtdIns(4,5)P 2 and [ 3 H] phosphatidylinositol 4-phosphate (PtdIns4P) after labelling the cells with [ 3 H]inositol in the presence of 10% FBS for 72 h: the amount of [ 3 H]PtdIns(4,5)P 2 and [ 3 H]PtdIns4P did not change significantly between + ⁄ + and – ⁄ – MEF cells (Fig. 2A). Similar results were obtained when we labelled the cells with [ 32 P]orthophosphate for more than 4 h. In our assay SHIP2 MEF SHIP2 +/+ 1 MEF SHIP2 +/+ 2 MEF SHIP2 +/ - MEF SHIP2 - / - 2 MEF SHIP2 - / - 1 CHO - IR 150 100 75 250 kDa AB SHIP2 InsR IGF-IR PTEN MEF SHIP2 + /+ 2 MEF SHIP2 - / - 1 MEF SHIP2 +/+ 1 MEF SHIP2 - / - 2 CHO - IR Fig. 1. Western blot analysis of MEF SHIP2 + ⁄ +, + ⁄ –and–⁄ – cells. Twenty micrograms of proteins from a lysate made of MEF cells or CHO-IR were applied to SDS gels. Immunodetection was performed with antibodies against SHIP2, PTEN, IGF-1 and the insulin receptor (IGF-1R and InsR). MEF cells 1 and 2 were from two independent preparations of cells. PtdIns(3,4,5)P 3 levelsLabelling with [ 3 H] inositol 0 0,2 0,4 0,6 0,8 1 1,2 010203040 TIME (minute) %(of PtdIns(4,5)P2) SHIP2+/+ SHIP2-/- 0 1 2 3 4 5 6 AB CD PtdInsP PtdIns(4,5)P2 % (of PtdIns) SHIP2+/+ SHIP2-/- PtdIns(3,4,5)P 3 levels 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 0 min 10 min %(of PtdIns(4,5)P2) SHIP2+/+ SHIP2-/- PtdIns(3,4,5)P 3 levels 0 0,2 0,4 0,6 0,8 1 1,2 1,4 0 min 10 min %(of PtdIns(4,5)P2) SHIP2+/+ SHIP2+/- SHIP2-/- Fig. 2. PtdIns(3,4,5)P 3 levels in serum stimulated SHIP2 + ⁄ +, + ⁄ –and–⁄ – MEFs. (A) MEF cells were labelled with 50 lCi [ 3 H]inositol for 72 h in the presence of FBS. [ 3 H]phosphoinositides were isolated as described. The data were normalized with respect to the total radioac- tivity present in the phosphatidylinositol fraction. The data are means of triplicates ± SD. (B) + ⁄ + and – ⁄ – MEF cells were labelled with [ 32 P]orthophosphate for 4 h and stimulated with 10% serum for various periods of time. [ 32 P]PtdIns(3,4,5)P 3 was isolated as described. The data are expressed as a percentage of total [ 32 P]PtdIns(4,5)P 2 measured in the same HPLC profile and are means of triplicates ± SD. (C) MEF cells were labelled with [ 32 P] for 4 h and stimulated by 10% FBS for 10 min. The data are expressed as a percentage of total [ 32 P] PtdIns(4,5)P 2 measured in the same HPLC profile. The data are means of three independent experiments using the two series of MEF cells (1 and 2) ± SD. (D) + ⁄ +,+ ⁄ – and – ⁄ – MEF cells were labelled with [ 32 P] and stimulated for 10 min by 10% FBS. The data are means of duplicates ± SD. The data are representative of two different experiments. Phosphatidylinositol 3,4,5-trisphosphate levels in mouse embryonic fibroblasts D. Blero et al. 2514 FEBS Journal 272 (2005) 2512–2522 ª 2005 FEBS of [ 32 P]-labelled lipids, we scraped off together the region of the TLC containing both [ 32 P]PtdIns(3,4,5)P 3 and [ 32 P]PtdIns(4,5)P 2 , the levels of [ 32 P]-labelled 3-phosphoinositides were normalized with respect to [ 32 P]PtdIns(4,5)P 2 . SHIP2 modulated PtdIns(3,4,5)P 3 levels after short-term serum stimulation The levels of PtdIns(3,4,5)P 3 were compared between the two types of cells that had been stimulated by serum. PtdIns(3,4,5)P 3 was increased following stimu- lation of both types of cells by 10% FBS. In res- ponse to the addition of serum, the production of PtdIns(3,4,5)P 3 was upregulated in SHIP2 – ⁄ – cells as compared to + ⁄ + cells. A maximal effect was seen between 5 and 10 min after stimulation by 10% serum (Figs 2B and 3A). This effect was observed in the two independently prepared MEF cells (Fig. 2C). In these experiments, the levels of phosphatidylinositol 3,4-bis- phosphate; [PtdIns(3,4)P 2 ] were not significantly differ- ent between the two types of cells (see below). This probably reflects the complex pathway of PtdIns(3,4)P 2 production ⁄ degradation in response to FBS via PI 3-kinase, PTEN, SHIP2 and several other phosphati- dylinositol 5-phosphatases [25]. The difference in PtdIns(3,4,5)P 3 levels between serum stimulated + ⁄ + and – ⁄ – cells was also observed in heterozygous MEF cells but the effect was intermediate as compared to the + ⁄ + and – ⁄ – MEF cells. Basal levels of PtdIns(3,4,5)P 3 were not different between + ⁄ + and – ⁄ – cells (Fig. 2D). In contrast to serum, IGF-1 stimulation resulted in maximal production of PtdIns(3,4,5)P 3 after 2 min and no significant differences in PtdIns(3,4,5)P 3 levels were seen between SHIP2+ ⁄ +and – ⁄ – cells (Fig. 3B). When the MEF cells were stimulated with insulin (1–100 nm), we did not see any significant increase in PtdIns(3,4,5)P 3 levels in contrast to CHO-IR cells sti- mulated with insulin that were used as positive control (data not shown). SHIP2 did not modulate PtdIns(3,4,5)P 3 levels after long-term serum stimulation Previous studies in MEF cells deficient in PTEN have shown that PtdIns(3,4,5)P 3 was upregulated about twofold in – ⁄ – cells after 4 h of incubation in the presence of 5% FBS. The data suggested that PtdIns(3,4,5)P 3 is a physiological substrate of PTEN [26]. We therefore measured PtdIns(3,4,5)P 3 levels in SHIP2 deficient cells after long-term stimulation by FBS (under the same conditions used in the study of PTEN deficient MEF cells). PtdIns(3,4,5)P 3 levels in SHIP2 – ⁄ – cells were not significantly different as com- pared to + ⁄ + cells after 30 min or 4 h of stimulation by 5% FBS (Figs 2B and 4, respectively). Therefore, no significant differences in PtdIns(3,4,5)P 3 levels between SHIP2 + ⁄ + and – ⁄ – were observed under the conditions where PtdIns(3,4,5)P 3 levels were upreg- ulated in PTEN deficient cells. PI 3-kinase activity in SHIP2 +/+ and –/– MEF cells PI 3-kinase activity in SHIP2 + ⁄ + and – ⁄ – cells was determined in both the presence and absence of the PI 3- kinase inhibitor LY-294002. Basal activity was stimula- ted in the presence of 10% FBS or IGF-1 at 10 nm. This activity was reversed when lysates were prepared in the presence of LY-294002 (Fig. 5). No differences in the 0 0,2 0,4 0,6 0,8 1 1,2 0510 TIME (minute) 0510 TIME (minute) %(of PtdIns(4,5)P 2 ) %(of PtdIns(4,5)P 2 ) SHIP2+/+ SHIP2-/- A PtdIns(3,4,5)P 3 levels PtdIns(3,4,5)P 3 levels +serum 0 0,1 0,2 0,3 0,4 0,5 0,6 SHIP2+/+ SHIP2-/- B + IGF-1 Fig. 3. PtdIns(3,4,5)P 3 levels in serum and IGF-1 stimulated MEF cells. Time course of [ 32 P] PtdIns(3,4,5)P 3 production in (A) serum and (B) IGF-1 stimulated MEF cells. + ⁄ + and – ⁄ – MEF cells were labelled with [ 32 P]orthophosphate for 4 h and stimulated with 10% serum or IGF-1 at 10 n M for the indicated times. [ 32 P] PtdIns(3,4,5)P 3 was quantified as before. D. Blero et al. Phosphatidylinositol 3,4,5-trisphosphate levels in mouse embryonic fibroblasts FEBS Journal 272 (2005) 2512–2522 ª 2005 FEBS 2515 level of activation was determined in the SHIP2 + ⁄ + and – ⁄ – MEF cells at the time of maximal production of PtdIns(3,4,5)P 3 , i.e. 5 min for FBS and 2 min for IGF-1. Therefore, changes in PI 3-kinase activity are probably not responsible for the upregulation of PtdIns(3,4,5)P 3 levels in SHIP2 – ⁄ – MEF cells. Effect of serum on PKB and MAP kinase activities In overexpression studies, SHIP2 causes PKB inactiva- tion and MAP kinase inhibition [10,15,22,23]. Activa- ted PKB was detected using phosphospecific antibodies against T308 and S473. PKB activity was upregulated in serum stimulated SHIP2 –⁄ – cells as compared to + ⁄ + cells (Fig. 6A). A similar result was obtained by using an enzymatic assay for PKB after immunopre- cipitation of PKB. The net increase in PKB activity in serum stimulated cells was approximately two times higher in SHIP2 – ⁄ – cells as compared to + ⁄ + cells (Fig. 6B). We also showed that the upregulation of PKB phosphorylation was totally reversed when cells were preincubated in the presence of the PI 3-kinase inhibitor LY-294002 (Fig. 7). In contrast, MAK kinase activities (p-Erk1 ⁄ 2) following serum stimulation were not different between the two types of cells (Fig. 6A). Stimulation of MEF with various agonists MEF cells were also stimulated for 5 min by various agonists: EGF, hepatocyte growth factor (HGF), b fibroblast growth factor (FGF), IGF-1 and PDGF (Fig. 8). HGF, b FGF, IGF-1 (1 nm), did not increase PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 production. EGF sti- mulated both PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 levels but the differences in the two lipid levels were not signifi- cant between the two types of cells (SHIP2 deficient or not). IGF-1 at 10 nm stimulated PtdIns(3,4,5)P 3 produc- tion but PtdIns(3,4,5)P 3 levels were not significantly upregulated in the – ⁄ – cells compared to the + ⁄ + cells SHIP2 MEF +/+ -/- +/+ -/- Origin PI3P Control Control with LY No lysates A SHIP2 MEF +/+ -/- +/+ -/- Origin PI3P FBS10% FBS10% +LY B +/+ -/- +/+ -/- Origin PI3P SHIP2 MEF IGF-1 10 nM IGF-1 10 nM +LY C - LY + LY Fig. 5. PI 3-kinase activity in serum or IGF-1 stimulated SHIP2 + ⁄ +and–⁄ – MEF cells PI 3-kinase activty was measured in (A) con- trol, and in stimulated cells (B) 10% serum for 5 min and (C) 10 n M IGF-1 for 2 min. The assay was performed as described. When used, LY-294002 (25 l M) was added to the preincubation for 30 min. PtdIns(3,4,5)P 3 levels 0 0.05 0.1 0.15 0.2 0.25 MEF SHIP2+/+ MEF SHIP2-/- % (of PtdIns(4,5)P2) + serum Fig. 4. PtdIns(3,4,5)P 3 levels in serum stimulated SHIP2 + ⁄ + and – ⁄ – MEF cells after long-term stimulation. + ⁄ + and – ⁄ – MEF cells were labelled with [ 32 P] and stimulated with 5% serum for 4 h. [ 32 P]PtdIns(3,4,5)P 3 was quantified as before. Phosphatidylinositol 3,4,5-trisphosphate levels in mouse embryonic fibroblasts D. Blero et al. 2516 FEBS Journal 272 (2005) 2512–2522 ª 2005 FEBS as shown before in the kinetics of the lipid production (Fig. 3B). In PDGF stimulated cells, PtdIns(3,4,5)P 3 levels increased but no significant differences were seen between +⁄ + and – ⁄ – cells although PtdIns(3,4)P 2 was lower in SHIP2 deficient cells as compared to control cells (Fig. 8). In order to verify that the agonists used stimulated MEF cells efficiently, we determined MAP kinase activity (p-Erk1 ⁄ 2) in the two types of cells: the agonists tested also stimulated phospho MAP kinase activity in both SHIP2 + ⁄ + and – ⁄ – cells (Fig. 9). Discussion SHIP2 is a typical signalling enzyme potentially involved in the biochemical cascade of many growth factors and insulin [2,4,7,10–15,23]. Its sequence shows the presence of an SH2 domain, proline rich sequences, a NPXY site that can be phosphorylated on tyrosine and a catalytic domain which is typical for a member of the inositol 5-phosphatase family. SHIP2 appears to be able to dephosphorylate at the 5-position of the inositol ring of PtdIns(4,5)P 2 , PtdIns(3,4,5)P 3 and ino- sitol tetrakisphoshate in vitro [7,15,21]. SHIP2 p-ser 473 PKB A B p-thr 308 PKB total PKB p-Erk1/2 Erk2 1 2 3 4 5 6 7 8 Western blotting PKB assay 0 1000 2000 3000 4000 5000 6000 7000 8000 9000 0min 10min FBS CPM SHIP2+/+ SHIP2-/- Fig. 6. PKB and MAP kinase activities in SHIP2 + ⁄ + and – ⁄ – MEF cells. (A) SHIP2 + ⁄ + (lane 1,2 and 5,6) and – ⁄ – (lane 3,4 and 7,8) MEF cells were stimulated by 10% FBS for 10 min (lane 1–4 unstimulated, 5–8 stimulated by FBS). Protein (20 lg) wase immuno- blotted with the indicated antibodies. Phosphorylation was assayed by using phospho-specific antibodies against p-Thr308 or p-Ser473 for PKB and p-Erk1 ⁄ 2 for MAK kinase activities. Western blot against total PKB or Erk2 is shown below. Results are representa- tive of three experiments. (B) PKB activities determined after immunoprecipitation of endogenous PKB as described. Protein cell lysate (100 lg) was used in the assay. Results are means of dupli- cates ± SD. SHIP2 MEF +/+ -/- +/+ -/- +/+ -/- +/+ -/- LY 294002 FBS 10% FBS 10% + LY294002 Control +/+ -/- +/+ -/- +/+ -/- +/+ -/- P-Ser 473 PKB Total PKB Fig. 7. Phospho PKB activity in SHIP2 + ⁄ +and–⁄ – cells MEF cells. MEF cells were stimulated in the 10% serum for 5 min in the pres- ence and absence of LY-294002 (25 l M). Protein (20 lg) was immunoblotted. Phosphorylation of PKB was assayed in the pres- ence of antibodies against p-Ser473 and total PKB is shown below. PtdIns(3,4)P 2 levels in MEF cells (5 min stimulation) 0 0,5 1 1,5 2 2,5 3 3,5 NS FBS 10% EGF 50n g /ml HGF 15n g /ml β-FGF 100n g /ml IGF-1 1nM IGF-1 10nM PDGF 30n g /ml %(of tP dI sn4(,5P)2) SHIP2+/+ SHIP2-/- 0 0,2 0,4 0,6 0,8 1 1,2 S N BF S %01 G E F 5 0 g n m / l GH F 1 n 5 g m / l β- G FF01 0 n g / m l I G -F 1 n 1 M GI F - 1 1 n0 M P D FG 3 n0 g / lm SHIP2+/+ SHIP2-/- PtdIns(3,4,5)P 3 levels ( 5 min stimulation) %(of tP dI sn4(5,P)2) Fig. 8. PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 levels in SHIP2 + ⁄ +and – ⁄ – MEF cells. + ⁄ + and – ⁄ – MEF cells were stimulated by 10% FBS, EGF 50 ngÆmL )1 , HGF 15 ngÆmL )1 , b FGF 100 ngÆmL )1 , IGF-1 1 and 10 n M or PDGF 30 ngÆmL )1 for 5 min. [ 32 P]PtdIns(3,4,5)P 3 (upper panel) and [ 32 P]PtdIns(3,4)P 2 (lower panel) are expressed as a percentage of total [ 32 P]PtdIns(4,5)P 2 and are means of dupli- cates ± SD. NS ¼ non stimulated cells. D. Blero et al. Phosphatidylinositol 3,4,5-trisphosphate levels in mouse embryonic fibroblasts FEBS Journal 272 (2005) 2512–2522 ª 2005 FEBS 2517 The ability of SHIP2 to be a physiological regulator of PtdIns(3,4,5)P 3 is often assumed based on a similar function attributed to SHIP1 [27,28]. Two SHIP2 knockout mice have now been reported. The first knockout mice showed an increased sensitivity to insu- lin although it was later recognized that another gene Phox2a was also deleted in the targeting construct together with the 19–29 exons of SHIP2 [18,29]. It is not known whether the phenotype was influenced by this second gene deletion. The second knockout mice, in which the first 18 exons were deleted, showed a dif- ferent phenotype with normal insulin and glucose tol- erances; the mice were however, resistant to dietary obesity [19]. Interestingly, an increased activation of PKB phosphorylation was observed in skeletal muscle and liver of these SHIP2 null mice on stimulation with insulin [19]. The data obtained in analysing the pheno- types of both knockout mice are consistent with SHIP2 being directly responsible for the dephosphory- lation of PtdIns(3,4,5)P 3 . This is not to say that SHIP2 is only acting as a phosphoinositide 5-phosphatase (EC 3.1.3.36). For example, the SHIP2 C-terminal region is quite specific as compared to that of SHIP1 and could interact with specific protein partners such as filamin or c-Cbl associated protein thereby provi- ding multiple molecular interactions and possible bio- chemical regulation mechanisms of its activity and localization [30,31]. The presence of SHIP2 in mem- brane ruffles has been reported and this could account for the regulation of actin rearrangement by regulating local levels of SHIP2 lipid substrate and ⁄ or interacting with cytoskeleton regulatory proteins [30]. The trans- location of SHIP2 to plasma membranes upon insulin stimulation and the requirement for the negative regu- lation of insulin signalling could account for SHIP2 specificity [32]. The role of tyrosine phosphorylation of SHIP2 is unclear in preadipocytes [9] and multiple phosphorylation sites have been identified, e.g. in Jurkat cells [33]. Thus, the biochemistry of SHIP2 in the insulin (and probably other signalling cascades) is not fully understood. We were interested to measure PtdIns(3,4,5)P 3 lev- els in SHIP2 depleted cells. As SHIP2 did not appear to be specific for a given signalling pathway in cellu- lar models, we compared the effects of a series of agonists including EGF, PDGF and IGF-1 that had been previously used in SHIP2 overexpression studies. We also compared our data with PTEN which is often presented as the principal regulator of PtdIns(3,4,5)P 3 levels [25]. This is the first report showing a direct comparison of PtdIns(3,4,5)P 3 pro- duction in SHIP2 + ⁄ + and – ⁄ – cells. PtdIns(3,4,5)P 3 levels [but not PtdIns(4,5)P 2 levels] were potentiated in serum stimulated SHIP2 – ⁄ – MEF cells as com- pared to + ⁄ + cells; this effect was not observed in unstimulated cells, or after long-term stimulation (i.e. 1 or 4 h) of the cells. PKB activity (but not MAP kinase activity) was potentiated in serum stimulated SHIP2 – ⁄ – cells with this effect being completely reversed in the presence of LY-294002. Serum stimu- lated PI 3-kinase activity appeared to be comparable between SHIP2 + ⁄ + and – ⁄ – cells and in both cases, the activity was decreased in the presence of LY-294002. Therefore, the results obtained with PtdIns(3,4,5)P 3 in our study could not be explained by an upregulation of PI 3-kinase in serum stimulated SHIP2 – ⁄ – cells. We concluded that the increase in PtdIns(3,4,5)P 3 levels and PKB activity measured in our study is a consequence of an effect on PtdIns(3,4,5)P 3 dephosphorylation. Stambolic et al. [26] reported that PtdIns(3,4,5)P 3 levels were also potentiated (about twofold) in PTEN – ⁄ – cells that had been incubated and labelled with 5% FBS for 4 h; however, no kinetics were provided for compari- son with our data. In contrast, in our study no change in PtdIns(3,4,5)P 3 levels were seen in SHIP2 depleted cells as compared to + ⁄ + cells either at the basal level or after long-term stimulation. We found that SHIP2 acted after short-term (5–10 min) serum stimulation as a modulator of PtdIns(3,4,5)P 3 levels. Previous data obtained with SHIP1 also indicated that SHIP1 does not regulate basal PtdIns(3,4,5)P 3 EGF 50ng/ml HGF 15ng/ml FGF 100ng/ml IGF - 1 1 n M IGF 1 10nM PDGF 30ng/ml FBS 10% control SHIP2 +/+ SHIP2 -/- EGF 50ng/ml HGF 15ng/ml FGF 100ng/ml IGF - 1 1 nM IGF 1 10nM PDGF 30ng/ml FBS 10% control Erk2Erk2Erk2 p-Erk1/2 Fig. 9. Phospho MAP kinase activity in SHI- P2 + ⁄ + and – ⁄ – MEF cells. + ⁄ +and–⁄ – MEF cells were stimulated as in Fig. 8. Protein (100 lg) was immunoblotted and probed with p-Erk1 ⁄ 2 for MAP kinase activity. Total MAP kinase (Erk2) is shown below. Phosphatidylinositol 3,4,5-trisphosphate levels in mouse embryonic fibroblasts D. Blero et al. 2518 FEBS Journal 272 (2005) 2512–2522 ª 2005 FEBS levels but that it may control the duration and mag- nitude of stimulated increases in this lipid [25,34]. We did not detect any upregulation of PtdIns(3,4,5)P 3 by stimulation of the cells with HGF, b-FGF, IGF-1 or EGF. We excluded any difference in regulation of PI 3-kinase activity between serum and IGF-1 stimulated cells as no differences in PI 3-kinase activity could be detected between SHIP2 + ⁄ + and – ⁄ – MEF cells (this effect was reversed in the presence of LY-294002). The reason for not observing an upreg- ulation of PtdIns(3,4,5)P 3 with every agonist is not yet understood, however, maximal production of PtdIns(3,4,5)P 3 was observed after 5 min stimulation by serum and after only 2–3 min by IGF-1; the ampli- tude of the PtdIns(3,4,5)P 3 production was also not comparable being higher for serum as compared to IGF-1 (as shown in Fig. 3). Finally, we cannot ignore the fact that our method does not allow the measure- ment of minor or local changes in PtdIns(3,4,5)P 3 levels that are observed only in the presence of serum. These observations could contribute to the specificity we have observed with serum. However, in addition to serum, we found that short-term H 2 O 2 treatment of the SHIP2 – ⁄ – MEF cells also upregulates the phos- phorylation of PKB (J. Zhang, unpublished data). This observation suggests the following model: it is possible that serum is producing some reactive oxygen species which could be responsible for the inactivation of PTEN. The production of reactive oxygen species in response to growth factors or insulin and inactivation of PTEN has been recently reported by others [35,36]. In this model, we assume that SHIP2 is less active than PTEN in terms of enzymatic activity and that SHIP2 will only be able to control the PtdIns(3,4,5)P 3 levels once PTEN is inactivated by oxidation. This mechan- ism may be dependent on both the type of agonist and the cell type. In another study, others have recently reported enhanced PKB activation in response to M-CSF in fetal liver-derived macrophages prepared from SHIP2 knockout mice [8]. Our data also suggest that one or several compo- nents of the serum allows SHIP2 to be effectively recruited near the sites of PtdIns(3,4,5)P 3 production. The localization of SHIP2 at the membrane is import- ant for its lipid phosphatase activity as shown in 3T3- L1 adipocytes where insulin provokes a redistribution of SHIP2 from the cytosol to the plasma membrane fraction following a mechanism which is in part dependent on PI 3-kinase activity [14]. Moreover, as discussed above, PTEN is also competing for SHIP2 in the regulation of PtdIns(3,4,5)P 3 and PtdIns(3,4)P 2 lev- els and this may affect the kinetics of the phosphoino- sitides in stimulated cells. The influence of PTEN activity in this complex pathway is not known but we clearly established in our study that PTEN expression is unchanged between SHIP2 wild type and SHIP2 deficient cells. In contrast to data obtained in cells transfected with SHIP2, no difference in MAP kinase activity was observed between serum stimulated SHIP2 + ⁄ + and – ⁄ – cells; this suggests that the SHIP2 pathway does not interfere with MAP kinase activity in serum stimulated cells. In conclusion, our data are consistent with SHIP2 affecting the transient control of PtdIns(3,4,5)P 3 levels which influences PKB activity, at least in cells stimula- ted by serum. The specificity of PtdIns(3,4,5)P 3 hydro- lysis by SHIP2 with regard to serum stimulation and the short-term kinetics of the SHIP2 action add an interesting twist to the sophistication of these signal- ling systems. This concept, is quite reminiscent of some of the characteristics of Ca 2+ signalling, e.g. the differ- ent aspects of neuronal differentiation encoded by the frequency of Ca 2+ transients [37]. Experimental procedures Materials SHIP1 and SHIP2 antibodies have been described previously [5]. PTEN antibody was from A.G. Scientific, Inc. (San Diego, CA, USA) Anti-PI 3-Kinase p85 and antiphosphotyr- osine were from Upstate (AH Veemendaal, the Netherlands). Anti-Insulin receptor (InsR) and anti IGF-1 antibodies were kindly provided by K. Siddle (Department of Clinical Biochemistry, Cambridge University, UK). HGF, IGF-1 and PDGF were provided by Upstate. LY-294002, PI and phos- phatidylserine were from Sigma (Bonnem, Belgium). Easi- tides [c- 32 P] ATP (3000 CiÆmmol )1 ) was from NEM. [ 32 P]Orthophosphate (10 mCiÆmL )1 ) was from Amersham (Rosendaal, the Netherlands). PtdIns(3,4,5)P 3 measurements Cells (1.5 · 10 6 ) were cultured in 10% serum overnight. Cells were washed twice in medium without serum and twice in medium without either phosphate or serum. They were labelled for at least 4 h in medium with [ 32 P]ortho- phosphate (250 lCiÆmL )1 ) but without serum. Cells were stimulated with various agents as indicated in the text. The reaction was terminated by 5 mL cold NaCl ⁄ Pi. Cells were lysed in 3.75 mL 2.4 N HCl. Lipids were extracted in 3 mL methanol and 4.5 mL CHCl 3 . After TLC and deacylation of the phosphoinositides, separation was per- formed by HPLC on Whatman SAX columns (Leuven, Belgium) [23]. Radioactivity was estimated with an online detector from Raytest (Straubenhardt, Germany). D. Blero et al. Phosphatidylinositol 3,4,5-trisphosphate levels in mouse embryonic fibroblasts FEBS Journal 272 (2005) 2512–2522 ª 2005 FEBS 2519 PtdIns(4,5)P 2 was also determined by labelling the cells with [ 3 H] inositol as reported [38]. The various 3-phos- phorylated phosphoinositides standards were prepared in insulin stimulated CHO-IR cells or in platelets as reported previously [22,39]. Preparation of MEF cells Primary MEF cells were isolated from 14-day postcoitus C57BL6 mouse embryos [18]. Embryos were surgically removed and separated from maternal tissues following pro- tocol approved by the Ethics Committee for animals. The head of each embryo was removed and kept for genotyping. After visceral organ removal, the rest of the body was minced finely by repetitive syringe aspiration, then washed twice with 1 · NaCl ⁄ Pi and incubated in 500 lL trypsin⁄ EDTA (2.5% trypsin, 1 mm EDTA) at 37 °C for 60 min. The embryo fragments were resuspended by adding 1.5 mL of complete medium (DMEM, 2% streptomycin ⁄ ampicillin, 50 lm b-mercaptoethanol) with a 2-mL glass pipette. The cells were dissociated with a 10-mL pipette by adding another 7.5 mL complete medium. The supernatant was transferred to a T75 culture flask after 2 min resting. MEF were obtained after incubation of the cells at 37 °C for 2–3 days. Genotyping of MEF cells Genotyping of SHIP2 MEF was performed by PCR using specific primers to amplify the neo gene and a specific exon deleted in the recombinant allele [18]. The same forward primer was used for each of the + ⁄ + and – ⁄ – alleles: 5¢-GGGTCTTTGGAGCTGTGGACT-3¢. While specific reverse primers were used for the + ⁄ + allele: 5¢-CCCAAGTGTCTCCCATCATCC-3¢ and for the – ⁄ – allele: 5¢-TAAGGGTTCCGGATCTGCC-3¢. The PCR reaction was performed under the following conditions: denaturation at 95 °C for 3 min, followed by 40 cycles at 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s and elonga- tion of 72 °C for 7 min. Cell lysates, PKB and MAP kinase assay MEF cells were lysed in 50 mm Tris ⁄ HCl pH 7.4, 1% NP- 40, 0.5% cholate, 0.1% Triton X-100, 1 mm EDTA, 1 mm EGTA, 50 mm NaF, 20 mm b glycerophosphate, 15 mm sodium pyrophosphate, 2 mm orthovanadate, 10 nm oka- daic acid, protease inhibitors (Roche, Vilvoorde, Belgium), 0.1 m NaCl. Activated PKB was detected using phospho- specific antibodies against T308 or S473. Activated MAP kinase was detected using phospho-p44 ⁄ 42 MAP (Erk1 ⁄ 2) kinase antibody (Cell Signalling, Leusden, the Netherlands). Antibodies against total PKB and MAP kinase (Erk2) were from Cell Signalling. For PKB assay, immunoprecipitation was performed with anti Akt1 ⁄ PKB (Upstate) following the protocol provided. PKB activities were determined in the presence of 30 lm Crosstide substrate peptide and [c- 32 P]ATP. Phosphorylated substrate was measured on P81 phosphocellulose papers. Measurement of PI 3-kinase activity Cell lysates of serum or IGF-1 stimulated MEF cells (2 · 10 6 cells per condition) were immunoprecipitated with 2 lL antiphosphotyrosine antibodies overnight at 4 °C. The immunoprecipitates were washed twice in 50 mm Tris ⁄ HCl pH 7.4, 200 mm NaCl, 0.1% Brij, protease inhibitors (Roche) and once in the kinase reaction buffer (see below). The pellet ( 30 lL) was incubated at 37 °C for 30 min in the presence of 80 lL2· kinase buffer containing 100 mm Tris ⁄ HCl pH 7.4, 200 mm NaCl, 10 mm MgCl 2 ,1mm EDTA, 200 lm ATP together with [c- 32 P]ATP (10 lCi per condition) and 50 lL sonicated vesicles of PI and phos- phatidylserine. When the effect of the PI 3-kinase inhibitor LY-294002 was tested, it was added to the kinase buffer at 25 lm. The lipids were extracted following a Bligh and Dyer modified procedure and resuspended in 30 lLof CHCl 3 ⁄ CH 3 OH (1 : 1). Separation of the reaction product was performed by TLC on a silica plate in acetone ⁄ CH 3 OH ⁄ acetic acid ⁄ H 2 O ⁄ CHCl 3 (30 : 26 : 24 : 14 : 80, v ⁄ v ⁄ v ⁄ v ⁄ v). The corresponding spots were analysed by autoradiography. Acknowledgements We would like to thank Mrs Colette Moreau, Dr Len Stephens, Louis Hue, Mark Rider, Franc¸ ois Willer- main, Fabrice Vandeput, Vale ´ rie Dewaste and Natha- lie Paternotte for many helpful discussions. This work was supported by grants of the Fonds de la Recherche Scientifique Me ´ dicale, Action de Recherche Concerte ´ e of the Communaute ´ Franc¸ aise de Belgique and INSERM-Communaute ´ Franc¸ aise de Belgique exchange contract. This work was executed in the framework of research network IAPV-O5 (Belgium Science Policy). Daniel Blero and Xavier Pesesse are Charge ´ de Recherche FNRS. References 1 Damen JE, Liu L, Rosten P, Humphries RK, Jefferson AB, Majerus PW & Krystal G (1996) The 145-kDa pro- tein induced to associate with Shc by multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5- triphosphate 5-phosphatase. Proc Natl Acad Sci USA 93, 1689–1693. 2 Ishihara H, Sasaoka T, Hori H, Wada T, Hirai H, Har- uta T, Langlois WJ & Kobayashi M (1999) Molecular Phosphatidylinositol 3,4,5-trisphosphate levels in mouse embryonic fibroblasts D. Blero et al. 2520 FEBS Journal 272 (2005) 2512–2522 ª 2005 FEBS cloning of rat SH2-containing inositol phosphatase 2 (SHIP2) and its role in the regulation of insulin signal- ing. Biochem Biophys Res Commun 260, 265–272. 3 Lioubin MN, Algate PA, Tsai S, Carlberg K, Aebersold A & Rohrschneider LR (1996) p150Ship, a signal trans- duction molecule with inositol polyphosphate-5-phos- phatase activity. Genes Dev 10, 1084–1095. 4 Pesesse X, Deleu S, De Smedt F, Drayer L & Erneux C (1997) Identification of a second SH2-domain-contain- ing protein closely related to the phosphatidylinositol polyphosphate 5-phosphatase SHIP. Biochem Biophys Res Commun 239, 697–700. 5 Muraille E, Pesesse X, Kuntz C & Erneux C (1999) Dis- tribution of the src-homology-2-domain-containing ino- sitol 5- phosphatase SHIP-2 in both non-haemopoietic and haemopoietic cells and possible involvement of SHIP-2 in negative signalling of B-cells. Biochem J 342, 697–705. 6 Habib T, Hejna JA, Moses RE & Decker SJ (1998) Growth factors and insulin stimulate tyrosine phosphory- lation of the 51C ⁄ SHIP2 protein. J Biol Chem 273, 18605–18609. 7 Wisniewski D, Strife A, Swendeman S, Erdjument-Bro- mage H, Geromanos S, Kavanaugh WM, Tempst P & Clarkson B (1999) A novel SH2-containing phosphati- dylinositol 3,4,5-trisphosphate 5-phosphatase (SHIP2) is constitutively tyrosine phosphorylated and associated with src homologous and collagen gene (SHC) in chronic myelogenous leukemia progenitor cells. Blood 93, 2707–2720. 8 Wang YJ, Keogh RJ, Hunter MG, Mitchell CA, Frey RS, Javaid K, Malik AB, Schurmans S, Tridandapani S & Marsh CB (2004) SHIP2 is recruited to the cell mem- brane upon macrophage colony-stimulating factor (M-CSF) stimulation and regulates M-CSF-induced signaling. J Immunol 173, 6820–6830. 9 Gagnon A, ArtemenkoY, Crapper T & Sorisky A (2003) Regulation of endogenous SH2 domain-containing inositol 5-phosphatase (SHIP2) in 3T3-L1 and human preadipocytes. J Cellular Physiol 197, 243–250. 10 Sasaoka T, Hori H, Wada T, Ishiki M, Haruta T, Ishi- hara H & Kobayashi M (2001) SH2-containing inositol phosphatase 2 negatively regulates insulin- induced gly- cogen synthesis in L6 myotubes. Diabetologia 44, 1258– 1267. 11 Wada T, Sasaoka T, Funaki M, Hori H, Murakami S, Ishiki M, Haruta T, Asano T, Ogawa W, Ishihara H & Kobayashi M (2001) 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. Mol Cell Biol 21, 1633–1646. 12 Sasaoka T, Wada T, Fukui K, Murakami S, Ishihara H, Suzuki R, Tobe K, Kadowaki T & Kobayashi M (2004) SH2-containing inositol phosphatase 2 predomi- nantly regulates Akt2, and not Akt1, phosphorylation at the plasma membrane in response to insulin in 3T3-L1 adipocytes. J Biol Chem 279, 14835–14843. 13 Hori H, Sasaoka T, Ishihara H, Wada T, Murakami S, Ishiki M & Kobayashi M (2002) Association of SH2- containing inositol phosphatase 2 with the insulin resis- tance of diabetic db ⁄ db mice. Diabetes 51, 2387–2394. 14 Sasaoka T, Kikuchi K, Wada T, Sato A, Hori H, Murakami S, Fukui K, Ishihara H, Aota R, Kimura I & Kobayashi M (2003) Dual role of Src homology domain 2-containing inositol phosphatase 2 in the regu- lation of platelet-derived growth factor and insulin-like growth factor I signaling in rat vascular smooth muscle cells. Endocrinology 144, 4204–4214. 15 Taylor V, Wong M, Brandts C, Reilly L, Dean NM, Cowsert LM, Moodie S & Stokoe D (2000) 5¢ phos- pholipid phosphatase SHIP-2 causes protein kinase B inactivation and cell cycle arrest in glioblastoma cells. Mol Cell Biol 20, 6860–6871. 16 Prasad N, Topping RS & Decker SJ (2001) SH2-con- taining inositol 5¢-phosphatase SHIP2 associates with the p130 (Cas) adapter protein and regulates cellular adhesion and spreading. Mol Cell Biol 21, 1416–1428. 17 Prasad N & Decker SJ (2005) SH2-containing 5¢ inositol phosphatase SHIP2 regulates cytoskeleton organization and ligand-dependent downregulation of the epidermal growth factor receptor. J Biol Chem 280, 13129–13136. 18 Clement S, Krause U, Desmedt F, Tanti JF, Behrends J, Pesesse X, Sasaki T, Penninger J, Doherty M, Mala- isse W, Dumont JE, Marchand-Brustel Y, Erneux C, Hue L & Schurmans S (2001) The lipid phosphatase SHIP2 controls insulin sensitivity. Nature 409, 92–97. 19 Sleeman MW, Wortley KEV, Lai KM, Gowen LC, Kintner J, Kline WO, Garcia K, Stitt TN, Yancopoulos GD, Wiegand SJ & Glass DJ (2005) Absence of the lipid phosphatase SHIP2 confers resistance to diary obesity. Nat Med 11, 199–205. 20 Giuriato S, Blero D, Robaye B, Bruyns C, Payrastre B & Erneux C (2002) SHIP2 overexpression strongly reduces the proliferation rate of K562 erythroleukemia cell line. Biochem Biophys Res Commun 296, 106–110. 21 Pesesse X, Moreau C, Drayer AL, Woscholski R, Par- ker P & Erneux C (1998) The SH2 domain containing inositol 5-phosphatase SHIP2 displays phosphatidylino- sitol 3,4,5-trisphosphate and inositol 1,3,4,5- tetraki- sphosphate 5-phosphatase activity. FEBS Lett 437, 301–303. 22 Blero D, De Smedt F, Pesesse X, Paternotte N, Moreau C, Payrastre B & Erneux C (2001) The SH2 domain containing inositol 5-phosphatase SHIP2 controls phos- phatidylinositol 3,4,5-trisphosphate levels in CHO-IR cells stimulated by insulin. Biochem Biophys Res Com- mun 282, 839–843. 23 Pesesse X, Dewaste V, De Smedt F, Laffargue M, Giur- iato S, Moreau C, Payrastre B & Erneux C (2001) The D. Blero et al. Phosphatidylinositol 3,4,5-trisphosphate levels in mouse embryonic fibroblasts FEBS Journal 272 (2005) 2512–2522 ª 2005 FEBS 2521 [...]... homology 2-containing inositol 5¢-phosphatase 2 via Shc association is required for the negative regulation of insulin signaling in Rat1 fibroblasts overexpressing insulin receptors Mol Endocrinol 16, 2371–2381 Salomon AR, Ficarro SB, Brill LM, Brinker A, Phung QT, Ericson C, Sauer K, Brock A, Horn DM, Schultz PG & Peters EC (2003) Profiling of tyrosine phosphorylation pathways in human cells using mass spectrometry.. .Phosphatidylinositol 3,4,5-trisphosphate levels in mouse embryonic fibroblasts 24 25 26 27 28 29 30 31 Src homology 2 domain containing inositol 5-phosphatase SHIP2 is recruited to the epidermal growth factor (EGF) receptor and dephosphorylates phosphatidylinositol 3,4,5-trisphosphate in EGF- stimulated COS-7 cells J Biol Chem 276, 28348–28355 McConnachie G, Pass I, Walker SM & Downes CP (2003) Interfacial... J & Erneux C (2003) The termination of PI3K signalling by SHIP1 and SHIP2 inositol 5-phosphatases Adv Enzyme Regul 43, 15–28 Scharenberg AM, El HillalO, Fruman DA, Beitz LO, Li ZM, Lin SQ, Gout I, Cantley LC, Rawlings DJ & Kinet JP (1998) Phosphatidylinositol- 3,4,5-trisphosphate (PtdIns-3,4,5-P.-3) Tec kinase-dependent calcium signaling pathway: a target for SHIP-mediated inhibitory signals EMBO J 17,... platelet-derived growth-factor stimulation Methods Enzymol 198, 78–87 Giuriato S, Pesesse X, Bodin S, Sasaki T, Viala C, Marion E, Penninger J, Schurmans S, Erneux C & Payrastre B (2003) SH2-containing inositol 5-phosphatases 1 and 2 in blood platelets their interactions and roles in the control of phosphatidylinositol 3,4,5-trisphosphate levels Biochem J 376, 199–207 FEBS Journal 272 (2005) 2512–2522 ª 2005... species in response to insulin stimulation is phosphatase and tensin homolog and not PI-3 kinase in the PI-3 kinase pathway Mol Biol Cell 16, 348–357 Gu XN & Spitzer NC (1995) Distinct Aspects of Neuronal Differentiation Encoded by Frequency of Spontaneous Ca2+ Transients Nature 375, 784–787 Serunian LA, Auger K & Cantley LC (1991) Identification and quantification of polyphosphoinositides produced in response... Sasaki T, Penninger J, Doherty M, Malaisse W, Dumont JE, Marchand-Brustel Y, Erneux C, Hue L & Schurmans S (2005) Corrigendum: the lipid phosphatase SHIP2 controls insulin sensitivity Nature 431, 878 Dyson JM, O’Malley CJ, Becanovic J, Munday AD, Berndt MC, Coghill ID, Nandurkar HH, Ooms LM & Mitchell CA (2001) The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates... The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin J Cell Biol 155, 1065– 1079 Vandenbroere I, Paternotte N, Dumont JE, Erneux C & Pirson I (2003) The c-Cbl-associated protein and c-Cbl are two new partners of the SH2-containing inositol 2522 32 33 34 35 36 37 38 39 D Blero et al polyphosphate 5-phosphatase SHIP2 Biochem Biophys Res Commun... Walker SM & Downes CP (2003) Interfacial kinetic analysis of the tumour suppressor phosphatase, PTEN: evidence for activation by anionic phospholipids Biochem J 371, 947–955 Leslie NR & Downes CP (2002) PTEN: The down side of PI 3-kinase signalling Cell Signal 14, 285–295 Stambolic V, Suzuki A, de la Pompa JL, Brothers GM, Mirtsos C, Sasaki T, Ruland J, Penninger JM, Siderovski DP & Mak TW (1998) Negative... Sasaki T, Kozieradzki I, Wakeham A, Itie A, Dumont DJ & Penninger JM (1999) SHIP is a negative regulator of growth factor receptor-mediated PKB ⁄ Akt activation and myeloid cell survival Genes Dev 13, 786–791 Kwon J, Lee SR, Yang KS & Ahn Y (2004) Kim, Stadtman ER & Rhee SG Reversible oxidation and inactivation of the tumor suppressor PTEN in cells stimulated with peptide growth factors Proc Natl Acad . domain containing inositol 5-phosphatase, includes a series of protein interacting domains and has the ability to dephosphorylate phosphatidylinositol 3,4,5-trisphosphate. Phosphatidylinositol 3,4,5-trisphosphate modulation in SHIP2-deficient mouse embryonic fibroblasts Daniel Blero 1 , Jing Zhang 1 , Xavier