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• W2106000003 abstract "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 full-length enzyme was expressed in COS-7 cells and stained with anti-His6 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. 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 full-length enzyme was expressed in COS-7 cells and stained with anti-His6 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. epidermal growth factor EGF receptor phosphoinositide 3-kinase 4-P2, phosphatidylinositol 3,4-bisphosphate phosphatidylinositol 3,4,5-trisphosphate protein kinase B Src homology truncated SHIP2 polymerase chain reaction Tris-buffered saline SH2 domain containing protein tyrosine phosphatase Protein tyrosine phosphorylation plays a central role in the regulation of protein-protein interactions and modulation of enzyme activities (1Dixon J.E. Recent Prog. Horm. Res. 1996; 51: 405-414PubMed Google Scholar). 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) (2Vanhaesebroeck B. Waterfield M.D. Exp. Cell. Res. 1999; 253: 239-254Crossref PubMed Scopus (763) Google Scholar). 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 (3Wymann M.P. Pirola L. Biochim. Biophys. Acta. 1998; 1436: 127-150Crossref PubMed Scopus (579) Google Scholar, 4Vanhaesebroeck B. Leevers S.J. Panayotou G. Waterfield M. Trends Biochem. Sci. 1997; 22: 267-272Abstract Full Text PDF PubMed Scopus (797) Google Scholar). PI 3-kinase class IA substrates in vitroare phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate, whereas the preferred substratein vivo is phosphatidylinositol 4,5-bisphosphate (2Vanhaesebroeck B. Waterfield M.D. Exp. Cell. Res. 1999; 253: 239-254Crossref PubMed Scopus (763) Google Scholar). Phosphatidylinositol 3,4,5-trisphosphate (PIP3) and/or phosphatidylinositol 3,4-bisphosphate (PI 3,4-P2) could activate protein kinase B (PKB) through the binding to the PH domain of 3′-phosphoinositide-dependent kinase 1 phosphorylating protein kinase B (5Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (675) Google Scholar, 6Rameh L.E. Cantley L.C. J. Biol. Chem. 1999; 274: 8347-8350Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar, 7Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (969) Google Scholar). 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) (8Maehama T. Dixon J.E. Trends Cell Biol. 1999; 9: 125-128Abstract Full Text Full Text PDF PubMed Scopus (505) Google Scholar). SHIP1 and SHIP2 (SH2 domain containing inositol 5-phosphatase 1 and 2) belong to the inositol and phosphatidylinositol 5-phosphatase family (9Mitchell C.A. Brown S. Campbell J.K. Munday A.D. Speed C.J. Biochem. Soc. Trans. 1996; 24: 994-1000Crossref PubMed Scopus (54) Google Scholar, 10Erneux C. Govaerts C. Communi D. Pesesse X. Biochim. Biophys. Acta. 1998; 1436: 185-199Crossref PubMed Scopus (126) Google Scholar, 11Majerus P.W. Kisseleva M.V. Norris F.A. J. Biol. Chem. 1999; 274: 10669-10672Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Both phosphatases contain different protein-protein interaction domains, such as an N-terminal-specific SH2 domain, a potential phosphotyrosine binding (PTB) domain binding site (NPAY), and C-terminal proline-rich sequences allowing binding of the SH3 domain. SHIP2 contains also a sterile α motif at the C-terminal end that was not present in SHIP1 (12Pesesse X. Deleu S. De Smedt F. Drayer L. Erneux C. Biochem. Biophys. Res. Commun. 1997; 239: 697-700Crossref PubMed Scopus (199) Google Scholar). cDNAs encoding SHIP2 have been reported in human, mouse, and rat (12Pesesse X. Deleu S. De Smedt F. Drayer L. Erneux C. Biochem. Biophys. Res. Commun. 1997; 239: 697-700Crossref PubMed Scopus (199) Google Scholar, 13Ishihara H. Sasaoka T. Hori H. Wada T. Hirai H. Haruta T. Langlois W.J. Kobayashi M. Biochem. Biophys. Res. Commun. 1999; 260: 265-272Crossref PubMed Scopus (117) Google Scholar, 14Schurmans S. Carrio R. Behrends J. Pouillon V. Merino J. Clement S. Genomics. 1999; 62: 260-271Crossref PubMed Scopus (29) Google Scholar, 15Kudo M. Saito S. Owada Y. Suzuki H. Kondo H. Mol. Brain Res. 2000; 75: 172-177Crossref PubMed Scopus (5) Google Scholar). In humans, the protein contains 1258 amino acids, whereas a splice variant that lacks exon 28 and the C-terminal sterile α motif has been isolated in rat skeletal muscle (13Ishihara H. Sasaoka T. Hori H. Wada T. Hirai H. Haruta T. Langlois W.J. Kobayashi M. Biochem. Biophys. Res. Commun. 1999; 260: 265-272Crossref PubMed Scopus (117) Google Scholar). SHIP2 has been shown to be expressed in nonhemopoietic and hemopoietic cells, as shown by Western blotting (16Muraille E. Pesesse X. Kuntz C. Erneux C. Biochem. J. 1999; 342: 697-705Crossref PubMed Scopus (89) Google Scholar, 17Bruyns C. Pesesse X. Moreau C. Blero D. Erneux C. Biol. Chem. 1999; 380: 969-974Crossref PubMed Scopus (23) Google Scholar). When tested in vitro on recombinant SHIP2 produced in COS-7 cells, we could demonstrate PIP3 5-phosphatase activity. In another study, PIP3 5-phosphatase activity was also demonstrated in K562 cells after immunoprecipitation with anti-SHIP2 antibodies (18Wisniewski D. Strife A. Swendeman S. Erdjument-Bromage H. Geromanos S. Kavanaugh W.M. Tempst P. Clarkson B. Blood. 1999; 93: 2707-2720Crossref PubMed Google Scholar). Growth factors (EGF and platelet-derived growth factor) and insulin stimulate tyrosine phosphorylation of SHIP2 in various cell models, such as SH-SY5Y cells (19Habib T. Hejna J.A. Moses R.E. Decker S.J. J. Biol. Chem. 1998; 273: 18605-18609Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). SHIP2 was also constitutively tyrosine-phosphorylated in chronic myelogenous leukemia progenitor cells (18Wisniewski D. Strife A. Swendeman S. Erdjument-Bromage H. Geromanos S. Kavanaugh W.M. Tempst P. Clarkson B. Blood. 1999; 93: 2707-2720Crossref PubMed Google Scholar). In B cells, SHIP2 was also maximally tyrosine-phosphorylated and associated to Shc after BCR and FcγRIIB cross-linking but not after stimulation of BCR alone (16Muraille E. Pesesse X. Kuntz C. Erneux C. Biochem. J. 1999; 342: 697-705Crossref PubMed Scopus (89) Google Scholar, 20Muraille E. Bruhns P. Pesesse X. Daëron M. Erneux C. J. Immunology Lett. 2000; 72: 7-15Crossref PubMed Scopus (52) Google Scholar). SHIP2 has been shown to control insulin sensitivity in a model of SHIP2 deficient mice (21Clément S. Krause U. De Smedt F. Tanti J.-F. Behrends J. Pesesse X. Sasaki T. Penninger J. Doherty M. Malaisse W. Dumont J.E. Le Marchand-Brustel Y. Erneux C. Hue L. Schurmans S. Nature. 2000; 409: 92-97Crossref Scopus (317) Google Scholar). A role of SHIP2 in cellular adhesion and spreading has also been recently proposed (22Prasad N. Topping R.S. Decker S.J. Mol. Cell. Biol. 2001; 21: 1416-1428Crossref PubMed Scopus (120) Google Scholar). Previous data obtained in B cells have suggested that PIP3 initiates a PLCγ2-dependent inositol trisphosphate production through its ability to activate Tec kinases. Moreover FcγRIIB1, an inhibitory receptor that recruits SHIP1 eliminates BCR-induced PIP3 accumulation (23Scharenberg A.M. El-Hillal O. Fruman D.A. Beitz L.O. Li Z. Lin S. Gout I. Cantley L.C. Rawlings D.J. Kinet J.P. EMBO J. 1998; 17: 1961-1972Crossref PubMed Scopus (386) Google Scholar, 24Brauweiler A.M. Tamir I. Cambier J.C. Immunol. Rev. 2000; 176: 69-74Crossref PubMed Scopus (55) Google Scholar, 25Rohrschneider L.R. Fuller J.F. Wolf I. Liu Y. Lucas D.M. Genes Dev. 2000; 14: 505-520PubMed Google Scholar). The data implicate PIP3 as a crucial regulator of calcium signaling through its ability to initiate Tec kinase activation. The data also stressed the importance of SHIP1 as a PIP3 5-phosphatase in an intact cell model (26Scharenberg A.M. Kinet J.P. Cell. 1998; 94: 5-8Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Given the potential role of SHIP2 in regulation of PI 3-kinase signaling by growth factors and insulin (19Habib T. Hejna J.A. Moses R.E. Decker S.J. J. Biol. Chem. 1998; 273: 18605-18609Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), we aimed at measuring PIP3 levels in intact cells. In the course of these studies, we observed that SHIP2 was coimmunoprecipitated with the EGFR and also with the adaptor protein Shc. The EGFR was also present in SHIP2 immunoprecipitates. We have observed a colocalization of SHIP2 and the EGFR in COS-7 cells stimulated by EGF. Our data could be interpreted as the recruitment of a complex of at least three proteins (SHIP2, Shc, and the EGFR) in EGF-stimulated cells. Vector pcDNA3-His and Hyperfilm-MP were from Amersham Pharmacia Biotech. Superfect was from Qiagen. Protein A-Sepharose CL4B was obtained from Amersham Pharmacia Biotech.Anti-His6 monoclonal antibody was fromCLONTECH. Anti-phosphotyrosine monoclonal antibody 4G10, anti-Shc, and anti-PKB antibodies were purchased from Upstate Biotechnology; goat polyclonal anti-EGFR antibodies for Western blotting, immunofluorescence and monoclonal antibody to EGFR for immunoprecipitation were from Santa Cruz Biotechnology. Rabbit polyclonal antibodies against Shc were obtained from Affinity. Fluorescein-labeled mouse secondary antibodies were from Jackson ImmunoResearch Laboratories. SlowFade kit was purchased from Molecular Probes. Phosphatidylinositol 4,5-bisphosphate, phosphatidylserine, and Triton X-100 were from Sigma. TLC plates (20 × 20 cm) were from Merck. Recombinant bovine brain PI 3-kinase was prepared as described (28Vanhaesebroeck B. Higashi K. Raven C. Welham M. Anderson S. Brennan P. Ward S.G. Waterfield M.D. EMBO J. 1999; 18: 1292-1302Crossref PubMed Google Scholar) and kindly provided by Dr. Bart Vanhaesebroeck (Ludwig Institute for Cancer Research, London, United Kingdom). Protease inhibitors mixture was from Roche Molecular Biochemicals. The Quickchange site-directed mutagenesis kit was from Stratagene. Phosphocellulose P81 paper was obtained from Whatman. The peptide RPRAATF was synthesized at the Laboratoire de Chimie Biologique et de la Nutrition (Université Libre de Bruxelles). The peptide GGDGpYYDLSPL was kindly provided by Drs. Rüdiger Woscholski and Peter Parker (Imperial Cancer Research Fund). It was coupled to Actigel ALD (Sterogene) as described by the manufacturer. A truncated form of SHIP2 (tSHIP2) that lacks 366 amino acids at the C terminus (12Pesesse X. Deleu S. De Smedt F. Drayer L. Erneux C. Biochem. Biophys. Res. Commun. 1997; 239: 697-700Crossref PubMed Scopus (199) Google Scholar, 27Pesesse X. Moreau C. Drayer A.L. Woscholski R. Parker P. Erneux C. FEBS Lett. 1998; 437: 301-303Crossref PubMed Scopus (98) Google Scholar) was partially digested for 5 min by NcoI to obtain a 2.5-kilobase pair fragment. This was further digested with BamHI and resulted in an insert of 2.4 kilobase pairs. The partial SHIP2 clone (Clone 7 in Ref. 12Pesesse X. Deleu S. De Smedt F. Drayer L. Erneux C. Biochem. Biophys. Res. Commun. 1997; 239: 697-700Crossref PubMed Scopus (199) Google Scholar) was partially digested by NcoI and XhoI to obtain a 2-kilobase pair fragment. Both fragments were subcloned into pcDNA3-His vector digested with BamHI andXhoI. A construct corresponding to the SH2 domain of SHIP2 was obtained by PCR using the tSHIP2 (27Pesesse X. Moreau C. Drayer A.L. Woscholski R. Parker P. Erneux C. FEBS Lett. 1998; 437: 301-303Crossref PubMed Scopus (98) Google Scholar) as template and a 5′-primer containing aBamHI restriction site (underlined), 5′-GTGCGGATCCATGGCCCCCTCCTGGTA-3′, and a 3′-primer containing an XhoI restriction site (underlined), 5′-CCGCTCGAGTCACTCTACAGGAAGAAGC-3′. The PCR product was subcloned into pcDNA3-His C vector. The same construct was also subcloned in pTrc-His vector to produce the SH2 domain in bacteria as His-tagged construct. Production and purification on Probond resin was performed as reported before (27Pesesse X. Moreau C. Drayer A.L. Woscholski R. Parker P. Erneux C. FEBS Lett. 1998; 437: 301-303Crossref PubMed Scopus (98) Google Scholar). The catalytic domain of SHIP2 was obtained by PCR using tSHIP2 as template and a 5′-primer containing aBamHI restriction site (underlined), 5′-CGCGGATCCATGAAGGACCGGACTCAGCGCAA-3′, and a 3′-primer containing an XhoI restriction site (underlined) 5′-CGCTCTCGAGTCACGTGCTGCCGATCATGGAT-3′. The PCR product was subcloned into pcDNA3-His C vector. A SHIP2 construct that does not have SHIP2 SH2 domain (ΔSH2-SHIP2) was prepared with SHIP2 as template and a 5′-primer containing an EcoRI restriction site (underlined), 5′-CGGAATTCATGTCAGATGGGGAGGATGAG-3′, and a 3′-primer containing a XhoI site (underlined), 5′-CCGCTCGAGTCACTTGCTGAGCTGC-3′. The PCR product was subcloned in pcDNA3-HisC vector. The catalytic mutant of SHIP2 in which cysteine 689 was replaced by a serine was generated by PCR-based mutagenesis using SHIP2 subcloned into pBlueScript as a template according to the manufacturer's instructions. COS-7 cells (platted at 1.5 × 106 cells/dish the previous day) were transfected in 10-cm dishes using the Superfect method of transfection according to the manufacturer's instructions. Cells were stimulated with 50 ng/ml EGF at 37 °C for different times. The cells were washed with sterile phosphate-buffered saline and recovered in 1 ml of Buffer A containing 50 mm Tris/HCl, pH 7.5, 100 mm NaCl, 5 mm EDTA, 1% Brij, 2 mmNa3VO4, 2 mm phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin. After shaking the lysates for 20 min at 4 °C and centrifugation at 10,000 ×g, the supernatants were recovered and immediately subjected to immunoprecipitation. The following antibodies were used for immunoprecipitations: anti-SHIP2 (12Pesesse X. Deleu S. De Smedt F. Drayer L. Erneux C. Biochem. Biophys. Res. Commun. 1997; 239: 697-700Crossref PubMed Scopus (199) Google Scholar, 16Muraille E. Pesesse X. Kuntz C. Erneux C. Biochem. J. 1999; 342: 697-705Crossref PubMed Scopus (89) Google Scholar), anti-Shc (Upstate Biotechnology), anti-EGFR (Santa Cruz Biotechnology), anti-His6 (CLONTECH), and anti-phosphotyrosine 4G10 (Upstate Biotechnology). The supernatants were precleared for 5 min at 4 °C with 150 µl of 10% (w/v) protein A-Sepharose CL4B. This was centrifuged at 12000 ×g for 20 min at 4 °C. The soluble fraction was collected and incubated with the adequate antibodies and protein A-Sepharose for 2 h at 4 °C (10 µl of serum for anti-SHIP2, 10 µl for anti-Shc, 5 µl for anti-His6, and 25 µl for anti-EGFR). The immune complexes were recovered by centrifugation and washed four times in lysis buffer. The last wash was made without protease and phosphatase inhibitors. The immunoprecipitates were applied on SDS gels, followed by Western blotting. The blots were analyzed by enhanced chemiluminescence detection. Affinity adsorption of SHIP2 and tSHIP2 was performed using coupled GGDGpYYDLSPL peptide to Actigel ALD. After addition of 30 µl of peptide conjugated resin to 1 ml of COS-7 lysate, the complex was recovered by centrifugation and washed as described above. The [3-32P]PIP3 was prepared as described (27Pesesse X. Moreau C. Drayer A.L. Woscholski R. Parker P. Erneux C. FEBS Lett. 1998; 437: 301-303Crossref PubMed Scopus (98) Google Scholar) using recombinant PI 3-kinase. TLC-purified [32P]PIP3 was evaporated under nitrogen and resuspended with 100 µg of phosphatidylserine into vesicles. The [32P]PIP3 5-phosphatase activity was measured as described (28Vanhaesebroeck B. Higashi K. Raven C. Welham M. Anderson S. Brennan P. Ward S.G. Waterfield M.D. EMBO J. 1999; 18: 1292-1302Crossref PubMed Google Scholar, 29Giuriato S. Payrastre B. Drayer A.L. Plantavid M. Woscholski R. Parker P. Erneux C. Chap H. J. Biol. Chem. 1997; 272: 26857-26863Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Briefly, a total of 20,000 cpm/sample of [32P]PIP3 was resuspended in 50 mm Tris/HCl, pH 7.5, and 100 µg of phosphatidylserine. After sonication, the reaction was started by adding immunoprecipitated SHIP2 from transfected COS-7 cells and 5 mm MgCl2. The assay was stopped, and lipids were extracted. PIP3 and PI 3,4-P2 were separated by thin layer chromatography in chloroform/acetone/methanol/acetic acid/water (50:30:26:24:14, v/v/v/v/v). The corresponding spots were analyzed by PhosphorImager and autoradiography. Two days after the transfection of 1.2 × 106 cells in 10-cm-diameter dishes (vector alone, tSHIP2, or full-length SHIP2 cDNAs), COS-7 cells were labeled with [32P]orthophosphate for 5 h at 37 °C. Cells were treated with 50 ng/ml EGF for different time, and reactions were stopped by addition of chloroform/methanol (1:1, v/v). After lipid extraction, phospholipid were separated by TLC using chloroform/acetone/methanol/acetic acid/water (50:30:26:24:14, v/v/v/v/v), and PIP3-PI 3,4-P2 spots were scraped and deacylated. Lipids were resolved by high performance liquid chromatography (strong anion-exchange column partisphere SAX) and eluted by a linear gradient (0–1 m(NH4)2HPO4) as described in Ref.30Gratacap M.-P. Payrastre B. Viala C. Mauco G. Plantavid M. Chap H. J. Biol. Chem. 1998; 273: 12321-24314Abstract Full Text Full Text PDF Scopus (159) Google Scholar. PIP3 and PI 3,4-P2 were identified with internal standards. Immunofluorescence using anti-His6 and anti-EGFR antibodies was performed on transfected COS-7 cells. 1.2 × 105 transfected COS-7 cells were grown on uncoated glass coverslips in 3-cm-diameter dishes. Cells were stimulated or not with EGF, rinsed in Tris-buffered saline (TBS), and then fixed in 4% of paraformaldehyde solution in 0.1m phosphate-buffered saline, pH 7.4, for 10 min. The cells were washed three times for 10 min in TBS, permeabilized with 0.15% Triton X-100 in TBS for 10 min, and washed again with TBS. The fixed cells were incubated for 1 h at room temperature with 1:20 normal serum in TBS (goat or horse serum depending on the origin of the secondary antiserum). Incubation with immune serum was performed overnight at room temperature in the presence of blocking serum diluted 1:20 in TBS. The anti-His6 antibody was used at a 1:1000 dilution, and the anti-EGFR antibody was used at a 1:250 dilution. After being rinsed with TBS, cells were incubated for 60 min in the dark with a fluorescein-labeled goat anti-mouse secondary antibody (direct immunofluorescence). For the colocalization experiments, a Texas Red-labeled donkey anti-mouse secondary antibody at a 1:200 dilution and a fluorescein donkey anti-goat secondary antibody at a 1:250 dilution were used. The cells were then washed three times with TBS for 10 min and mounted with the SlowFade light antifade kit following the manufacturer's instructions. Cells were observed under a Nikon Optiphot fluorescence microscope, and images were obtained using a laser-scanning confocal microscope (MRC 1000, Bio-Rad) equipped with argon-krypton laser and COSMOS software (Bio-Rad). After transfection and stimulation of 1.2 × 106 COS-7 cells in 10-cm-diameter dishes, cells were lysed in 800 µl of ice-cold lysis buffer (80 mm Tris/HCl, pH 7.5, 20 mm EDTA, 200 mm NaCl, 0.75% Triton X-100, 80 mm sodium pyrophosphate, 4 mm sodium orthovanadate, 200 mm NaF, protease inhibitors mixture). After 20 min of agitation at 4 °C, the different cell supernatants were immunoprecipitated with 4 µg of antibody to PKB coupled to 25 µl of protein A-Sepharose in a total volume of 400 µl of Buffer H (80 mm Tris/HCl, pH 7.5, 20 mm EDTA, 1 mm EGTA, 200 mm NaCl, 0.2% Triton X-100, 0.1% β-mercaptoethanol, protease inhibitors mixture). PKB activity was determined as described previously (31Blero D. De Smedt F. Pesesse X. Paternotte N. Moreau C. Patrastre B. Erneux C. Biochem. Biophys. Res. Commun. 2001; 282: 839-843Crossref PubMed Scopus (56) Google Scholar). The molecular mass of SHIP2 was approximately 160 kDa in B cells (16Muraille E. Pesesse X. Kuntz C. Erneux C. Biochem. J. 1999; 342: 697-705Crossref PubMed Scopus (89) Google Scholar, 20Muraille E. Bruhns P. Pesesse X. Daëron M. Erneux C. J. Immunology Lett. 2000; 72: 7-15Crossref PubMed Scopus (52) Google Scholar). A similar value was determined in COS-7 cells. EGF was particularly potent in stimulating the tyrosine phosphorylation of SHIP2 in COS-7 cells: Fig. 1Ashows a time course study. SHIP2 tyrosine phosphorylation could be seen from 0.5 min to 120 min. When the blot was stripped and reprobed with SHIP2 antibodies, SHIP2 was recovered in the presence and absence of EGF, confirming that the immunoprecipitation was efficient in both cases (Fig. 1 A, bottom panel). Similar results were obtained in SHIP2-transfected cells, although a higher basal phosphorylation of SHIP2 could be observed depending on the transfection (data not shown). In similar transfection experiments, immunoprecipitations were performed with an anti-phosphotyrosine, and the blot was probed with SHIP2 antibodies; we observed that a 160-kDa band corresponding to SHIP2 was increased when cells had been stimulated for 5 min with EGF (Fig. 1 B). The high basal level seen in unstimulated cells results either from the migration of other tyrosine-phosphorylated proteins at the same molecular weight that could recruit SHIP2 or of a basal SHIP2 phosphorylation seen in transfected cells. The association between the adaptor protein Shc and SHIP2 has been reported by others in EGF-stimulated and platelet-derived growth factor-stimulated cells and also in K562 cells or in B cells (17Bruyns C. Pesesse X. Moreau C. Blero D. Erneux C. Biol. Chem. 1999; 380: 969-974Crossref PubMed Scopus (23) Google Scholar, 19Habib T. Hejna J.A. Moses R.E. Decker S.J. J. Biol. Chem. 1998; 273: 18605-18609Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 20Muraille E. Bruhns P. Pesesse X. Daëron M. Erneux C. J. Immunology Lett. 2000; 72: 7-15Crossref PubMed Scopus (52) Google Scholar). This was also observed in our model of transfected COS-7 cells. COS-7 cells were transfected with SHIP2 and immunoprecipitated with anti-Shc antibodies. Fig. 1 C shows that when probed with SHIP2, a 160-kDa protein band was detected in EGF-stimulated but not in control cells. We also tested whether SHIP2 could associate to the EGFR. When COS-7 cells were subjected to immunoprecipitation of the EGFR, SHIP2 was immunodetected in the immunoprecipitate provided the cells had been stimulated by EGF. This result was obtained in untransfected or SHIP2-transfected cells (Fig.1 D). Fig. 1 D, bottom panel, shows the presence of the EGFR by immunodetection of the same blot. Moreover, when the cell lysates were immunoprecipitated with SHIP2 and probed with EGFR antibodies, a protein of 170 kDa was detected in EGF-stimulated cells (Fig. 1 E). This band was not detected in control cells. Fig.1 E, bottom panel, shows that the same amounts of SHIP2 were immunoprecipitated in control and EGF-stimulated cells. Immunoprecipitation of the EGFR in SHIP2-transfected cells also shows the presence of Shc in immunoprecipitates (data not shown). Our data therefore indicate the presence of two proteins that co-precipitated with the EGFR: SHIP2 and Shc in EGF-stimulated cells. The phosphorylation of SHIP2 in response to EGF prompted us to test whether it was phosphorylated at its NPAY site at the C-terminal part of SHIP2 (Fig. 2). This site was indeed proposed to account for Shc binding through its PTB domain of SHIP2 (18Wisniewski D. Strife A. Swendeman S. Erdjument-Bromage H. Geromanos S. Kavanaugh W.M. Tempst P. Clarkson B. Blood. 1999; 93: 2707-2720Crossref PubMed Google Scholar). We have tested an antibody made against a tyrosine-phosphorylated peptide, KNSFNNPApYYVLEGV, that surrounded SHIP2 NPAY site (Fig.3A). When SHIP2 was transfected in COS-7 cells, the antibody recognized SHIP2 in EGF-stimulated cells, particularly upon stimulation. It did not recognize tSHIP2, which does not have the NPAY site (Figs. 2 and3 A). Antibodies to SHIP2-phosphorylated peptide cross-reacted with the EGFR at 170 kDa that was strongly phosphorylated in response to EGF (confirmed by reprobing experiments with EGFR antibodies, data not shown). The presence of tyrosine-phosphorylated NPXY sites in autophosphorylated EGFR could perhaps explain the cross-reactivity. The data are consistent with the tyrosine phosphorylation of SHIP2 at NPAY site, i.e.Tyr-986, in response to EGF.Figure 3Western blot analysis of tyrosine-phosphorylated SHIP2 in response to EGF. A,COS-7 cells (1.2 × 106 cells/condition) were transfected with SHIP2, tSHIP2, or ΔSH2-SHIP2 encoding cDNA and stimulated or not with 50 ng/ml EGF for 5 min. After lysis, crude lysates were probed with phosphorylated SHIP2 peptide antibodies (pSHIP2). B, a sample of each lysate was analyzed by Western blotting using SHIP2 antibodies. The migration of SHIP2 (160 kDa), ΔSH2-SHIP2 (150 kDa), and tSHIP2 (105 kDa) inA and B are indicated by arrows. The data are representative of one experiment out of three.View Large Image Figure ViewerDownload (PPT) We have prepared a construct, ΔSH2-SHIP2, that does not have SHIP2 SH2 domain. This construct was much less phosphorylated in response to EGF as compared with wild type SHIP2 (Fig. 3 A). Fig.3 B shows the expression of the constructs as detected by immunoblotting with anti-SHIP2 antibodies (SHIP2, ΔSH2-SHIP2, and tSHIP2). In previous studies, it was proposed that the optimal ligand for SHIP1 SH2 domain was Y(Y/D)X(L/I/V), consistent with SHIP1 binding to immunoreceptors (32Osborne M.A. Zenner G. Lubinus M. Zhang X. Songyang Z. Cantley L.C. Majerus P. Burn P. Kochan J.P. J. Biol. Chem. 1996; 271: 29271-29278Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). SHIP2 could be isolated by the same peptides by affinity chromatography. 2Erneux, C., unpublished data.We used this procedure to isolate transfected SHIP2 and tSHIP2. Western blot analysis shows that the two constructs were tyrosine-phosphorylated, particularly in EGF-stimulated cells (Fig.4). Fig. 4, bottom panel,shows the expression of both constructs by anti-His immunoblotting. His-tagged SHIP2 was expressed in COS-7 cells, and its cellular localization was revealed by anti-His antibody and confocal analysis. Cells were stimulated or not by EGF. Cells transfected with the vector alone did not show any signal (Fig.5, A and B), in contrast to cells transfected with SHIP2 (Fig. 5, C andD). SHIP2-transfected cells showed a cytoplasmic localization in the absence of EGF (Fig. 5 C). In EGF-stimulated cells, a relocation of SHIP2 could be seen at plasma membranes, as shown in Fig. 5 D. The colocalization of SHIP2 and the EGFR in the same membranes by double staining is shown in Fig.5, E–G, arrows. Because we h" @default.
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• W2106000003 title "The 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" @default.
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