FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2076523960> ?p ?o ?g. }

• W2076523960 endingPage "14843" @default.
• W2076523960 startingPage "14835" @default.
• W2076523960 abstract "SH2-containing inositol phosphatase 2 (SHIP2) is a physiologically important negative regulator of insulin signaling by hydrolyzing the phosphatidylinositol (PI) 3-kinase product PI 3,4,5-trisphosphate in the target tissues of insulin. Targeted disruption of the SHIP2 gene in mice resulted in increased insulin sensitivity without affecting biological systems other than insulin signaling. Therefore, we investigated the molecular mechanisms by which SHIP2 specifically regulates insulin-induced metabolic signaling in 3T3-L1 adipocytes. Insulin-induced phosphorylation of Akt, one of the molecules downstream of PI3-kinase, was inhibited by expression of wild-type SHIP2, whereas it was increased by expression of 5′-phosphatase-defective (ΔIP) SHIP2 in whole cell lysates. The regulatory effect of SHIP2 was mainly seen in the plasma membrane (PM) and low density microsomes but not in the cytosol. In this regard, following insulin stimulation, a proportion of Akt2, and not Akt1, appeared to redistribute from the cytosol to the PM. Thus, insulin-induced phosphorylation of Akt2 at the PM was predominantly regulated by SHIP2, whereas the phosphorylation of Akt1 was only minimally affected. Interestingly, insulin also elicited a subcellular redistribution of both wild-type and ΔIP-SHIP2 from the cytosol to the PM. The degree of this redistribution was inhibited in part by pretreatment with PI3-kinase inhibitor. Although the expression of a constitutively active form of PI3-kinase myr-p110 also elicited a subcellular redistribution of SHIP2 to the PM, expression of SHIP2 appeared to affect the myr-p110-induced phosphorylation, and not the translocation, of Akt2. Furthermore, insulin-induced phosphorylation of Akt was effectively regulated by SHIP2 in embryonic fibroblasts derived from knockout mice lacking either insulin receptor substrate-1 or insulin receptor substrate-2. These results indicate that insulin specifically stimulates the redistribution of SHIP2 from the cytosol to the PM independent of 5′-phosphatase activity, thereby regulating the insulin-induced translocation and phosphorylation of Akt2 at the PM. SH2-containing inositol phosphatase 2 (SHIP2) is a physiologically important negative regulator of insulin signaling by hydrolyzing the phosphatidylinositol (PI) 3-kinase product PI 3,4,5-trisphosphate in the target tissues of insulin. Targeted disruption of the SHIP2 gene in mice resulted in increased insulin sensitivity without affecting biological systems other than insulin signaling. Therefore, we investigated the molecular mechanisms by which SHIP2 specifically regulates insulin-induced metabolic signaling in 3T3-L1 adipocytes. Insulin-induced phosphorylation of Akt, one of the molecules downstream of PI3-kinase, was inhibited by expression of wild-type SHIP2, whereas it was increased by expression of 5′-phosphatase-defective (ΔIP) SHIP2 in whole cell lysates. The regulatory effect of SHIP2 was mainly seen in the plasma membrane (PM) and low density microsomes but not in the cytosol. In this regard, following insulin stimulation, a proportion of Akt2, and not Akt1, appeared to redistribute from the cytosol to the PM. Thus, insulin-induced phosphorylation of Akt2 at the PM was predominantly regulated by SHIP2, whereas the phosphorylation of Akt1 was only minimally affected. Interestingly, insulin also elicited a subcellular redistribution of both wild-type and ΔIP-SHIP2 from the cytosol to the PM. The degree of this redistribution was inhibited in part by pretreatment with PI3-kinase inhibitor. Although the expression of a constitutively active form of PI3-kinase myr-p110 also elicited a subcellular redistribution of SHIP2 to the PM, expression of SHIP2 appeared to affect the myr-p110-induced phosphorylation, and not the translocation, of Akt2. Furthermore, insulin-induced phosphorylation of Akt was effectively regulated by SHIP2 in embryonic fibroblasts derived from knockout mice lacking either insulin receptor substrate-1 or insulin receptor substrate-2. These results indicate that insulin specifically stimulates the redistribution of SHIP2 from the cytosol to the PM independent of 5′-phosphatase activity, thereby regulating the insulin-induced translocation and phosphorylation of Akt2 at the PM. Phosphatidylinositol (PI) 1The abbreviations used are: PI, phosphatidylinositol; PI(3,4,5)P3, PI 3,4,5-trisphosphate; PKC, protein kinase C; SHIP2, SH2-containing inositol phosphatase 2; WT, wild-type; PM, plasma membrane; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; m.o.i., multiplicity of infection; pfu, plaque-forming unit; LDM, low density microsomes; IRS, insulin receptor substrate. 3-kinase plays a central role in the metabolic actions of insulin. PI(3,4,5)P3 produced by activated PI3-kinase is thought to function as a key lipid second messenger for signaling to further downstream molecules including Akt and atypical PKC (1Virkamäki A. Ueki K. Kahn C.R. J. Clin. Invest. 1999; 103: 931-943Crossref PubMed Scopus (726) Google Scholar, 2Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4678) Google Scholar, 3Rameh L.E. Cantley L.C. J. Biol. Chem. 1999; 274: 8347-8350Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar, 4Czech M.P. Corvera S. J. Biol. Chem. 1999; 274: 1865-1868Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). We and others (5Ishihara 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, 6Pesesse X. Deleu S. De Smedt F. Drayer L. Erneux C. Biochem. Biophys. Res. Commun. 1997; 239: 697-700Crossref PubMed Scopus (200) Google Scholar) have recently cloned SH2-containing inositol phosphatase 2 (SHIP2), which has 5′-phosphatase activity toward the PI3-kinase product, PI(3,4,5)P3, in the target tissues of insulin. Overexpression of SHIP2 inhibited insulin-induced metabolic signaling leading to glucose uptake and glycogen synthesis via 5′-phosphatase activity hydrolyzing the PI3-kinase product PI(3,4,5)P3 to phosphatidylinositol 3,4-diphosphate in 3T3-L1 adipocytes and L6 myotubes (7Wada T. Sasaoka T. Funaki M. Hori H. Murakami S. Ishiki M. Haruta T. Asano T. Ogawa W. Ishihara H. Kobayashi M. Mol. Cell Biol. 2001; 21: 1633-1646Crossref PubMed Scopus (153) Google Scholar, 8Sasaoka T. Hori H. Wada T. Ishiki M. Haruta T. Ishihara H. Kobayashi M. Diabetologia. 2001; 44: 1258-1267Crossref PubMed Scopus (80) Google Scholar). Importantly, targeted disruption of the SHIP2 gene in mice increased insulin sensitivity without affecting other biological systems (9Clement S. Krause U. Desmedt 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. 2001; 409: 92-97Crossref PubMed Scopus (319) Google Scholar). These reports indicate that SHIP2 is a physiologically important negative regulator relatively specific to the insulin signaling. This prompted us to clarify the molecular mechanism by which SHIP2 specifically regulates the metabolic actions of insulin. Among the effector molecules downstream of PI3-kinase, Akt is strongly implicated in the metabolic action of insulin including glucose uptake and glycogen synthesis (10Calera M.R. Martinez C. Liu H. El Jack A.K. Birnbaum M.J. Pilch P.F. J. Biol. Chem. 1998; 273: 7201-7204Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 11Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1096) Google Scholar, 12Cong L.-N. Chen H. Li Y. Zhou L. McGibbon M.A. Taylor S.I. Quon M.J. Mol. Endocrinol. 1997; 11: 1881-1890Crossref PubMed Google Scholar). Upon insulin treatment, Akt is known to translocate from the cytosol to the plasma membrane where it is primarily activated by phosphorylation at Thr308/309 and Ser473/474 (13Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J.C. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 14Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1308) Google Scholar, 15Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R.J. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (915) Google Scholar, 16Stokoe D. Stephens L.R. Copeland T. Gaffney P.R.J. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1052) Google Scholar). Because Akt1 and Akt2 are the predominant isoforms expressed in 3T3-L1 adipocytes (17Katome T. Obata T. Matsushima R. Masuyama N. Cantley L.C. Gotoh Y. Kishi K. Shiota H. Ebina Y. J. Biol. Chem. 2003; 278: 28312-28323Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), the role of SHIP2 in the insulin-induced phosphorylation of Akt1 and Akt2 at various subcellular locations was examined by expressing the wild-type SHIP2 (WT-SHIP2) and a 5′-phosphatase-defective SHIP2 (ΔIP-SHIP2) into 3T3-L1 adipocytes using adenovirus-mediated gene transfer (7Wada T. Sasaoka T. Funaki M. Hori H. Murakami S. Ishiki M. Haruta T. Asano T. Ogawa W. Ishihara H. Kobayashi M. Mol. Cell Biol. 2001; 21: 1633-1646Crossref PubMed Scopus (153) Google Scholar). Although PI3-kinase is activated by a number of growth factors, only insulin elicits the physiologically important metabolic action via the PI3-kinase pathway (18Anai M. Ono H. Funaki M. Fukushima Y. Inukai K. Ogihara T. Sakoda H. Onishi Y. Yazaki Y. Kikuchi M. Oka Y. Asano T. J. Biol. Chem. 1998; 273: 29686-29692Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 19Clark S.F. Martin S. Carozzi A.J. Hill M.M. James D.E. J. Cell Biol. 1998; 140: 1211-1225Crossref PubMed Scopus (159) Google Scholar, 20Hill M.M. Clark S.F. Tucker D.F. Birnbaum M.J. James D.E. Macaulay S.L. Mol. Cell Biol. 1999; 19: 7771-7781Crossref PubMed Google Scholar). In this regard, we investigated the impact of SHIP2 expression on the translocation and phosphorylation of Akt induced by the constitutively active form of PI3-kinase, myr-p110 (7Wada T. Sasaoka T. Funaki M. Hori H. Murakami S. Ishiki M. Haruta T. Asano T. Ogawa W. Ishihara H. Kobayashi M. Mol. Cell Biol. 2001; 21: 1633-1646Crossref PubMed Scopus (153) Google Scholar, 21Kitamura T. Kitamura Y. Kuroda S. Hino Y. Ando M. Kotani K. Konishi H. Matsuzaki H. Kikkawa U. Ogawa W. Kasuga M. Mol. Cell Biol. 1999; 19: 6286-6296Crossref PubMed Scopus (311) Google Scholar). Furthermore, to clarify whether SHIP2 specifically or non-specifically regulates the metabolic signaling of insulin mediated via IRS-1 and IRS-2, the effect of SHIP2 expression on the insulin-induced phosphorylation of Akt was studied in embryonic fibroblasts lacking either IRS-1 or IRS-2 (22Miki H. Yamauchi T. Suzuki R. Komeda K. Tsuchida A. Kubota N. Terauchi Y. Kamon J. Kaburagi Y. Matsui J. Akanuma Y. Nagai R. Kimura S. Tobe K. Kadowaki T. Mol. Cell Biol. 2001; 21: 2521-2532Crossref PubMed Scopus (176) Google Scholar). Here, we show that SHIP2 negatively regulates the insulin-induced translocation and phosphorylation of Akt2 at the PM mediated via both the IRS-1 and IRS-2 pathway. Materials—Human crystal insulin was provided by Novo Nordisk Pharmaceutical Co., (Copenhagen, Denmark). The two polyclonal anti-SHIP2 antibodies were described previously (5Ishihara 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). The anti-SHIP2 antibodies raised against the C terminus and N terminus were used for the immunoprecipitation and immunoblotting, respectively. A monoclonal anti-phosphotyrosine antibody (PY20) was purchased from Transduction Laboratories (Lexington, KY). A polyclonal anti-Thr308 phospho-specific Akt antibody and a polyclonal anti-Ser473 phospho-specific Akt antibody were obtained from New England Biolabs, Inc. (Beverly, MA). A polyclonal anti-Akt antibody and a polyclonal anti-Akt1-specific antibody were from Santa Cruz Biotechnology (Santa Cruz, CA). A polyclonal anti-Akt2-specific antibody was from Calbiochem. Enhanced chemiluminescence reagents were from Amersham Biosciences. Dulbecco's modified Eagle's medium (DMEM), minimum essential medium vitamin mixtures, and minimum essential medium amino acid solutions were from Invitrogen. All other reagents were of analytical grade and purchased from Sigma or Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Construction of Adenoviral Vectors—cDNAs encoding rat WT-SHIP2 and ΔIP-SHIP2 were subcloned into the vector pAxCAwt and transferred to recombinant adenovirus by homologous recombination utilizing an adenovirus expression vector kit (Takara Biomedicals, Tokyo, Japan) as described previously (7Wada T. Sasaoka T. Funaki M. Hori H. Murakami S. Ishiki M. Haruta T. Asano T. Ogawa W. Ishihara H. Kobayashi M. Mol. Cell Biol. 2001; 21: 1633-1646Crossref PubMed Scopus (153) Google Scholar). The adenoviral vector encoding the constitutively active form of bovine p110 with a Src myristration signal sequence at the N terminus (myr-p110) was reported previously (21Kitamura T. Kitamura Y. Kuroda S. Hino Y. Ando M. Kotani K. Konishi H. Matsuzaki H. Kikkawa U. Ogawa W. Kasuga M. Mol. Cell Biol. 1999; 19: 6286-6296Crossref PubMed Scopus (311) Google Scholar). Cell Culture and Infection with Adenovirus—3T3-L1 fibroblasts were grown and passaged in DMEM supplemented with 10% newborn calf serum. Cells at 2 to 3 days post-confluence were used for differentiation. The differentiation medium contained 10% fetal calf serum (FCS), 250 nm dexamethasone, 0.5 mm isobutyl methylxanthine, and 500 nm insulin. After 3 days, the differentiation medium was replaced with postdifferentiation medium containing 10% FCS and 500 nm insulin. After 3 more days, the post-differentiation medium was replaced with DMEM supplemented with 10% FCS (7Wada T. Sasaoka T. Funaki M. Hori H. Murakami S. Ishiki M. Haruta T. Asano T. Ogawa W. Ishihara H. Kobayashi M. Mol. Cell Biol. 2001; 21: 1633-1646Crossref PubMed Scopus (153) Google Scholar). Preparation of IRS-1(–/–) and IRS-2(–/–) embryonic fibroblasts from IRS-1- and IRS-2-deficent mice was described previously (22Miki H. Yamauchi T. Suzuki R. Komeda K. Tsuchida A. Kubota N. Terauchi Y. Kamon J. Kaburagi Y. Matsui J. Akanuma Y. Nagai R. Kimura S. Tobe K. Kadowaki T. Mol. Cell Biol. 2001; 21: 2521-2532Crossref PubMed Scopus (176) Google Scholar). Embryonic fibroblasts were cultured with α-minimum essential medium supplemented with 10% FCS. WT-SHIP2, ΔIP-SHIP2, and myr-p110 were transiently expressed in differentiated 3T3-L1 adipocytes and embryonic fibroblasts by means of adenovirus-mediated gene transfer. A multiplicity of infection (m.o.i.) of 10–40 pfu/cell was used to infect 3T3-L1 adipocytes and embryonic fibroblasts in DMEM containing 2% FCS, with the virus being left on the cells for 16 h prior to removal. Subsequent experiments were conducted 24 to 48 h after initial addition of the virus. The efficiency of the adenovirus-mediated gene transfer of WT-SHIP2, ΔIP-SHIP2, and myr-p110 was ∼95%. Subcellular Fractionation—3T3-L1 adipocytes were washed twice with phosphate-buffered saline and once with HES buffer (255 mm sucrose, 20 mm HEPES, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, 2 μg/ml aprotinin, and 50 ng/ml okadaic acid, pH 7.4) and immediately homogenized by 20 strokes with a motor-driven homogenizer in HES buffer at 4 °C. The homogenates (two 10-cm-diameter dishes per condition) were subjected to subcellular fractionation as described previously to isolate PM, high density microsomes, low density microsomes (LDM), and cytosol (23Heller-Harrison R.A. Morin M. Czech M.P. J. Biol. Chem. 1995; 270: 24442-24450Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 24Inoue G. Cheatham B. Emkey R. Kahn C.R. J. Biol. Chem. 1998; 273: 11548-11555Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In brief, the homogenates were centrifuged at 19,000 × g for 20 min. The resulting supernatant was centrifuged at 41,000 × g for 20 min, yielding a pellet of high density microsomes. The supernatant from this spin was centrifuged at 250,000 × g for 90 min, yielding a pellet of LDM. Remaining supernatant was concentrated by Centricon-30 (Amicon Inc., Beverly, Mass.) and used as cytosol. The pellet obtained from the initial spin was resuspended in HES buffer, layered onto a 1.12 m sucrose cushion, and centrifuged at 100,000 × g in a swing rotor for 60 min. A white fluffy band at the interface was collected and resuspended in HES buffer and centrifuged at 40,000 × g for 20 min, yielding a pellet of PM. All fractions were adjusted to a final protein concentration of 1 to 3 mg/ml, which was measured by the Bradford method, and stored at –80 °C until use. Immunoprecipitation and Western Blotting—3T3-L1 adipocytes and embryonic fibroblasts grown in 6-well multiplates were serum-starved for 16 h in DMEM. The cells were treated with 17 nm insulin at 37 °C for various periods. They were then lysed in a buffer containing 20 mm Tris, 150 mm NaCl, 1 mm EDTA, 1 mm EGTA, 2.5 mm sodium deoxycholate, 1 mm β-glycerophosphate, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, 1 mm Na3VO4, 50 mm sodium fluoride, 10 μg/ml aprotinin, and 10 μm leupeptin, pH 7.4, for 15 min at 4 °C. Lysates obtained from the same number of cells were centrifuged to remove insoluble materials. The supernatants (100 μg of protein) were immunoprecipitated with antibodies for 2 h at 4 °C. The precipitates or whole cell lysates were then separated by 7.5% SDS-PAGE and transferred onto polyvinylidene difluoride membranes using a Bio-Rad Transblot apparatus. The membranes were blocked in a buffer containing 50 mm Tris, 150 mm NaCl, 0.1% Tween 20, and 2.5% bovine serum albumin or 5% non-fat milk, pH 7.5, for 2 h at 20 °C. The membranes were then probed with antibodies for 2 h at 20 °C or for 16 h at 4 °C. After the membranes were washed in a buffer containing 50 mm Tris, 150 mm NaCl, and 0.1% Tween 20, pH 7.5, blots were incubated with a horseradish peroxidase-linked secondary antibody and subjected to enhanced chemiluminescence detection using ECL reagent according to the manufacturer's instructions (Amersham Biosciences) (5Ishihara 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, 7Wada T. Sasaoka T. Funaki M. Hori H. Murakami S. Ishiki M. Haruta T. Asano T. Ogawa W. Ishihara H. Kobayashi M. Mol. Cell Biol. 2001; 21: 1633-1646Crossref PubMed Scopus (153) Google Scholar). Statistical Analysis—The data are represented as means ± S.E. p values were determined using a Student's t test, and p < 0.05 was considered statistically significant. Structures of SHIP2 Constructs and the Expression in 3T3-L1 Adipocytes—SHIP2 is a 140-kDa protein composed of an SH2 domain at the N terminus, a central 5′-phosphatase catalytic domain, and a proline-rich region including the phosphotyrosine binding domain binding consensus at the C terminus. Three amino acids, located within the catalytic domain of SHIP2, that are highly conserved among known 5′-phosphatases were mutated to generate ΔIP-SHIP2 (7Wada T. Sasaoka T. Funaki M. Hori H. Murakami S. Ishiki M. Haruta T. Asano T. Ogawa W. Ishihara H. Kobayashi M. Mol. Cell Biol. 2001; 21: 1633-1646Crossref PubMed Scopus (153) Google Scholar) (Fig. 1A). WT-SHIP2 and ΔIP-SHIP2 were transiently expressed in 3T3-L1 adipocytes by adenovirus-mediated gene transfer. Endogenous SHIP2 was seen in control 3T3-L1 adipocytes transfected with LacZ alone. On transfection with either WT-SHIP2 or ΔIP-SHIP2 at an m.o.i. of 40 pfu/cell, we observed similar levels of expression of WT-SHIP2 and ΔIP-SHIP2, which were 5-fold greater than the levels of endogenous SHIP2. Insulin treatment did not affect the expression of WT-SHIP2 and ΔIP-SHIP2 (Fig. 1B). Effect of SHIP2 Expression on Insulin-induced Phosphorylation of Akt in Whole Cell Lysates—Akt is a downstream target of PI3-kinase important for mediation of the metabolic actions of insulin (1Virkamäki A. Ueki K. Kahn C.R. J. Clin. Invest. 1999; 103: 931-943Crossref PubMed Scopus (726) Google Scholar, 2Cantley L.C. Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4678) Google Scholar, 3Rameh L.E. Cantley L.C. J. Biol. Chem. 1999; 274: 8347-8350Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar, 4Czech M.P. Corvera S. J. Biol. Chem. 1999; 274: 1865-1868Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). Because Akt is primarily activated as a result of its phosphorylation at the Thr308 (Akt2 at Thr309) and Ser473 (Akt2 at Ser474) residues (13Andjelkovic M. Alessi D.R. Meier R. Fernandez A. Lamb N.J.C. Frech M. Cron P. Cohen P. Lucocq J.M. Hemmings B.A. J. Biol. Chem. 1997; 272: 31515-31524Abstract Full Text Full Text PDF PubMed Scopus (903) Google Scholar, 16Stokoe D. Stephens L.R. Copeland T. Gaffney P.R.J. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1052) Google Scholar, 17Katome T. Obata T. Matsushima R. Masuyama N. Cantley L.C. Gotoh Y. Kishi K. Shiota H. Ebina Y. J. Biol. Chem. 2003; 278: 28312-28323Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 18Anai M. Ono H. Funaki M. Fukushima Y. Inukai K. Ogihara T. Sakoda H. Onishi Y. Yazaki Y. Kikuchi M. Oka Y. Asano T. J. Biol. Chem. 1998; 273: 29686-29692Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 19Clark S.F. Martin S. Carozzi A.J. Hill M.M. James D.E. J. Cell Biol. 1998; 140: 1211-1225Crossref PubMed Scopus (159) Google Scholar, 20Hill M.M. Clark S.F. Tucker D.F. Birnbaum M.J. James D.E. Macaulay S.L. Mol. Cell Biol. 1999; 19: 7771-7781Crossref PubMed Google Scholar, 25Scheid M.P. Marignani P.A. Woodgett J.R. Mol. Cell Biol. 2002; 22: 6247-6260Crossref PubMed Scopus (271) Google Scholar), we examined the effect of SHIP2 expression on the insulin-induced phosphorylation of Akt in 3T3-L1 adipocytes. Treatment with insulin induced phosphorylation of Akt at Thr308 and Ser473 in a time-dependent manner in LacZ-transfected control 3T3-L1 adipocytes. Transfection of WT-SHIP2 decreased insulin-induced phosphorylation of Akt at both Thr308 and Ser473. In contrast, insulin-induced phosphorylation of Akt at Thr308 and Ser473 was increased by transfection with ΔIP-SHIP2 (Fig. 2, A and B). These results are summarized in Fig. 2, E and F. Following 15 min of insulin treatment, the phosphorylation of Akt at Thr308 was significantly decreased 30.1 ± 4.9% by the expression of WT-SHIP2 and increased 34.6 ± 5.7% by the expression of ΔIP-SHIP2. Similarly, the phosphorylation of Akt at Ser473 was decreased 27.9 ± 3.4% by the expression of WT-SHIP2 and increased 31.8 ± 4.4% by the expression of ΔIP-SHIP2 following 5 min of insulin stimulation compared with that in control 3T3-L1 adipocytes transfected with LacZ. To assure equal amounts of protein were loaded among the samples, the cell lysates were immunoblotted with anti-Akt antibody (Fig. 2C). Similar expression levels of WT-SHIP2 and ΔIP-SHIP2 were detected on the immunoblotting of the cell lysates with anti-SHIP2 antibody (Fig. 2D). Effect of SHIP2 Expression on Insulin-induced Phosphorylation of Akt at Subcellular Locations—Because it is known that Akt is localized in the cytosol, PM, and LDM fractions (10Calera M.R. Martinez C. Liu H. El Jack A.K. Birnbaum M.J. Pilch P.F. J. Biol. Chem. 1998; 273: 7201-7204Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), we next examined the effect of SHIP2 expression on the insulin-induced phosphorylation of Akt at subcellular locations (Fig. 3). Insulin induced the phosphorylation of Akt at Thr308 and Ser473 in the cytosol, PM, and LDM fractions. Although a large amount of Akt resides in the cytosol, the insulin-induced phosphorylation of Akt at Thr308 and Ser473 in the cytosol was not significantly affected by the expression of either WT-SHIP2 or ΔIP-SHIP2. In contrast, the phosphorylation of Akt in the PM and LDM was affected by the expression of SHIP2. Notably, the insulin-induced phosphorylation of Akt at both Thr308 and Ser473 in the PM fraction was markedly decreased by the expression of WT-SHIP2, whereas it was increased by the expression of ΔIP-SHIP2. Densitometric analysis revealed that insulin-induced phosphorylation of Akt at Thr308 and Ser473 was decreased by 47.3 ± 1.2% and 45.7 ± 3.1%, respectively, in WT-SHIP2-expressing cells, whereas it was enhanced by 44.3 ± 5.6% and 45.3 ± 6.6% in ΔIP-SHIP2-expressing cells. Effect of SHIP2 Expression on Insulin-induced Phosphorylation of Akt1 and Akt2 Isoforms—Because Akt1 and Akt2 are the main isoforms expressed in 3T3-L1 adipocytes (17Katome T. Obata T. Matsushima R. Masuyama N. Cantley L.C. Gotoh Y. Kishi K. Shiota H. Ebina Y. J. Biol. Chem. 2003; 278: 28312-28323Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar), we next examined the effect of SHIP2 expression on the insulin-induced phosphorylation of Akt1 and Akt2. The cell lysates were immunoprecipitated with anti-Akt1 antibody, and the precipitates were immunoblotted with anti-phosphospecific Akt antibody. As shown in Fig. 4C, Akt1 is efficiently immunoprecipitated by this procedure, and the Akt2 isoform is not present in the precipitates. Insulin induced phosphorylation of the Akt1 isoform at Thr308 and Ser473 in anti-Akt1 immunoprecipitates, and this phosphorylation was not affected by the expression of either WT-SHIP2 or ΔIP-SHIP2 (Fig. 4, A and B). Because an anti-Akt2 antibody was not available for the immunoprecipitation, we performed an immunodepletion experiment. After the cell lysates were effectively immunoprecipitated with anti-Akt1 antibody, the supernatants were used for the experiment with Akt2. As can be seen in Fig. 4F, only Akt2, not Akt1, is present in the sample obtained by this procedure. Importantly, insulin-induced phosphorylation of Akt2 at Thr309 and Ser474 was markedly decreased by the expression of WT-SHIP2, whereas it was increased by the expression of ΔIP-SHIP2 (Fig. 4, D and E). These results indicate that SHIP2 regulates the insulin-induced phosphorylation of Akt2, and not Akt1, in 3T3-L1 adipocytes. Effect of SHIP2 Expression on the Insulin-induced Subcellular Distribution of Akt Isoforms—It is known that growth factor induces a subcellular relocalization of Akt to the plasma membrane to be phosphorylated (10Calera M.R. Martinez C. Liu H. El Jack A.K. Birnbaum M.J. Pilch P.F. J. Biol. Chem. 1998; 273: 7201-7204Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 20Hill M.M. Clark S.F. Tucker D.F. Birnbaum M.J. James D.E. Macaulay S.L. Mol. Cell Biol. 1999; 19: 7771-7781Crossref PubMed Google Scholar, 25Scheid M.P. Marignani P.A. Woodgett J.R. Mol. Cell Biol. 2002; 22: 6247-6260Crossref PubMed Scopus (271) Google Scholar). Although SHIP2 negatively regulates insulin-induced Akt2 phosphorylation, it is unclear whether SHIP2 affects the phosphorylation of Akt directly or via its translocation to the PM. To address this issue, we next examined the effect of SHIP2 expression on the insulin-induced subcellular redistribution of Akt1 and Akt2. The Akt1 isoform mainly resides in the cytosol fraction, and insulin treatment did not appear to induce apparent subcellular redistribution. In addition, overexpression of neither WT-SHIP2 nor ΔIP-SHIP2 appeared to affect the subcellular localization of Akt1 (Fig. 5A). Thus, the amount of Akt1 in the cytosol did not significantly alter in response to insulin. The Akt2 isoform is also mainly localized in the cytosol fraction in the basal state. Compared with the results obtained with Akt1, insulin efficiently elicited a subcellular redistribution of the Akt2 isoform from the cytosol and LDM to the PM. Importantly, the redistribution was markedly decreased by the expression of WT-SHIP2, whereas it was enhanced by that of ΔIP-SHIP2 (Fig. 5B). These results indicate that SHIP2 appears to regulate the subcellular redistribution of Akt2, and not Akt1, in 3T3-L1 adipocytes. Insulin-induced Subcellular Redistribution of SHIP2—Our previous study (26Ishihara H. Sasaoka T. Ishiki M. Wada T. Hori H. Kagawa S. Kobayashi M. Mol. Endocrinol. 2002; 16: 2371-2381Crossref PubMed Scopus (25) Google Scholar) indicated that the membrane localization of SHIP2 is important for its functioning via the 5′-phosphatase activity. Expression of SHIP2 with the myristoylation signal efficiently inhibited insulin-induced phosphorylation of Akt in Rat1 fibroblasts (26Ishihara H. Sasaoka T. Ishiki M. Wada T. Hori H. Kagawa S. Kobayashi M. Mol. Endocrinol. 2002; 16: 2371-2381Crossref PubMed Scopus (25) Google Scholar). Given this, we reasoned that SHIP2 might elicit this function by changing the subcellular localization to efficiently regulate the phosphorylation of Akt in the PM fraction. We examined whether insulin induces the subcellular redistribution of SHIP2 (Fig. 6A). WT-SHIP2 resides largely in the cytosol and partly in the LDM and PM fractions in the basal state. Insulin treatment significantly induced a redistribution of some of the expressed WT-SHIP2 and ΔIP-SHIP2 to the PM fraction. We further assessed the role of PI3-kinase in the insulin-induced redistribution of SHIP2. Pretreatment of the cells with the PI3-kinase inhibitor LY294002 partly, but significantly, inhibited the insulin-induced redistribution of both WT-SHIP2 and ΔIP-SHIP2 to the PM. Densitometric analysis demonstrated that the redistribution of WT-SHIP2 and ΔIP-SHIP2 to the PM was inhibited 41.3 ± 7.2 and 51.7" @default.
• W2076523960 created "2016-06-24" @default.
• W2076523960 creator A5016735166 @default.
• W2076523960 creator A5024658973 @default.
• W2076523960 creator A5040548657 @default.
• W2076523960 creator A5048136133 @default.
• W2076523960 creator A5051757814 @default.
• W2076523960 creator A5056021133 @default.
• W2076523960 creator A5062136115 @default.
• W2076523960 creator A5070737875 @default.
• W2076523960 creator A5072998209 @default.
• W2076523960 date "2004-04-01" @default.
• W2076523960 modified "2023-10-04" @default.
• W2076523960 title "SH2-containing Inositol Phosphatase 2 Predominantly Regulates Akt2, and Not Akt1, Phosphorylation at the Plasma Membrane in Response to Insulin in 3T3-L1 Adipocytes" @default.
• W2076523960 cites W1674259800 @default.
• W2076523960 cites W1697523131 @default.
• W2076523960 cites W1967709747 @default.
• W2076523960 cites W1969396185 @default.
• W2076523960 cites W1973837499 @default.
• W2076523960 cites W1975987716 @default.
• W2076523960 cites W1978777057 @default.
• W2076523960 cites W1983566655 @default.
• W2076523960 cites W1985782864 @default.
• W2076523960 cites W1989294105 @default.
• W2076523960 cites W1989324173 @default.
• W2076523960 cites W2002127663 @default.
• W2076523960 cites W2002398354 @default.
• W2076523960 cites W2012079492 @default.
• W2076523960 cites W2018662191 @default.
• W2076523960 cites W2033429522 @default.
• W2076523960 cites W2037308821 @default.
• W2076523960 cites W2037387433 @default.
• W2076523960 cites W2040143421 @default.
• W2076523960 cites W2047531477 @default.
• W2076523960 cites W2053073281 @default.
• W2076523960 cites W2064242629 @default.
• W2076523960 cites W2070732456 @default.
• W2076523960 cites W2074186032 @default.
• W2076523960 cites W2082770517 @default.
• W2076523960 cites W2091495230 @default.
• W2076523960 cites W2094068658 @default.
• W2076523960 cites W2095252238 @default.
• W2076523960 cites W2107573527 @default.
• W2076523960 cites W2112009693 @default.
• W2076523960 cites W2117290383 @default.
• W2076523960 cites W2120138780 @default.
• W2076523960 cites W2121042865 @default.
• W2076523960 cites W2124208226 @default.
• W2076523960 cites W2124615128 @default.
• W2076523960 cites W2124880593 @default.
• W2076523960 cites W2128842224 @default.
• W2076523960 cites W2140439371 @default.
• W2076523960 cites W2145545657 @default.
• W2076523960 cites W2148785089 @default.
• W2076523960 cites W2153977812 @default.
• W2076523960 cites W2164340474 @default.
• W2076523960 cites W2166039829 @default.
• W2076523960 cites W2166324877 @default.
• W2076523960 cites W2398276155 @default.
• W2076523960 cites W4245804658 @default.
• W2076523960 cites W4298301507 @default.
• W2076523960 doi "https://doi.org/10.1074/jbc.m311534200" @default.
• W2076523960 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/14744864" @default.
• W2076523960 hasPublicationYear "2004" @default.
• W2076523960 type Work @default.
• W2076523960 sameAs 2076523960 @default.
• W2076523960 citedByCount "59" @default.
• W2076523960 countsByYear W20765239602012 @default.
• W2076523960 countsByYear W20765239602013 @default.
• W2076523960 countsByYear W20765239602015 @default.
• W2076523960 countsByYear W20765239602016 @default.
• W2076523960 countsByYear W20765239602019 @default.
• W2076523960 countsByYear W20765239602020 @default.
• W2076523960 crossrefType "journal-article" @default.
• W2076523960 hasAuthorship W2076523960A5016735166 @default.
• W2076523960 hasAuthorship W2076523960A5024658973 @default.
• W2076523960 hasAuthorship W2076523960A5040548657 @default.
• W2076523960 hasAuthorship W2076523960A5048136133 @default.
• W2076523960 hasAuthorship W2076523960A5051757814 @default.
• W2076523960 hasAuthorship W2076523960A5056021133 @default.
• W2076523960 hasAuthorship W2076523960A5062136115 @default.
• W2076523960 hasAuthorship W2076523960A5070737875 @default.
• W2076523960 hasAuthorship W2076523960A5072998209 @default.
• W2076523960 hasBestOaLocation W20765239601 @default.
• W2076523960 hasConcept C11960822 @default.
• W2076523960 hasConcept C134018914 @default.
• W2076523960 hasConcept C154137905 @default.
• W2076523960 hasConcept C160160445 @default.
• W2076523960 hasConcept C170493617 @default.
• W2076523960 hasConcept C178666793 @default.
• W2076523960 hasConcept C181199279 @default.
• W2076523960 hasConcept C185592680 @default.
• W2076523960 hasConcept C2777427919 @default.
• W2076523960 hasConcept C2779306644 @default.
• W2076523960 hasConcept C55493867 @default.
• W2076523960 hasConcept C75217442 @default.
• W2076523960 hasConcept C86803240 @default.
• W2076523960 hasConcept C95444343 @default.

• next