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• W2017481487 abstract "Guanosine 5′-O-(3-thiotriphosphate) (GTPγS) treatment of permeabilized adipocytes results in GLUT4 translocation similar to that elicited by insulin treatment. However, although the selective phosphatidylinositol 3-kinase inhibitor, wortmannin, completely prevented insulin-stimulated GLUT4 translocation, it was without effect on GTPγS-stimulated GLUT4 translocation. In addition, insulin was an effective stimulant, whereas GTPγS was a very weak activator of the downstream Akt serine/threonine kinase. Consistent with an Akt-independent mechanism, guanosine 5′-O-2-(thio)diphosphate inhibited insulin-stimulated GLUT4 translocation without any effect on the Akt kinase. Surprisingly, two functionally distinct tyrosine kinase inhibitors, genistein and herbimycin A, as well as microinjection of a monoclonal phosphotyrosine specific antibody, inhibited both GTPγS- and insulin-stimulated GLUT4 translocation. Phosphotyrosine immunoblotting and specific immunoprecipitation demonstrated that GTPγS did not elicit tyrosine phosphorylation of insulin receptor or insulin receptor substrate-1. In contrast to insulin, proteins in the 120–130-kDa and 55–75-kDa range were tyrosine-phosphorylated following GTPγS stimulation. Several of these proteins were identified and include protein-tyrosine kinase 2 (also known as CAKβ, RAFTK, and CADTK), pp125 focal adhesion tyrosine kinase, pp130 Crk-associated substrate, paxillin, and Cbl. These data demonstrate that the GTPγS-stimulated GLUT4 translocation utilizes a novel tyrosine kinase pathway that is independent of both the phosphatidylinositol 3-kinase and the Akt kinase. Guanosine 5′-O-(3-thiotriphosphate) (GTPγS) treatment of permeabilized adipocytes results in GLUT4 translocation similar to that elicited by insulin treatment. However, although the selective phosphatidylinositol 3-kinase inhibitor, wortmannin, completely prevented insulin-stimulated GLUT4 translocation, it was without effect on GTPγS-stimulated GLUT4 translocation. In addition, insulin was an effective stimulant, whereas GTPγS was a very weak activator of the downstream Akt serine/threonine kinase. Consistent with an Akt-independent mechanism, guanosine 5′-O-2-(thio)diphosphate inhibited insulin-stimulated GLUT4 translocation without any effect on the Akt kinase. Surprisingly, two functionally distinct tyrosine kinase inhibitors, genistein and herbimycin A, as well as microinjection of a monoclonal phosphotyrosine specific antibody, inhibited both GTPγS- and insulin-stimulated GLUT4 translocation. Phosphotyrosine immunoblotting and specific immunoprecipitation demonstrated that GTPγS did not elicit tyrosine phosphorylation of insulin receptor or insulin receptor substrate-1. In contrast to insulin, proteins in the 120–130-kDa and 55–75-kDa range were tyrosine-phosphorylated following GTPγS stimulation. Several of these proteins were identified and include protein-tyrosine kinase 2 (also known as CAKβ, RAFTK, and CADTK), pp125 focal adhesion tyrosine kinase, pp130 Crk-associated substrate, paxillin, and Cbl. These data demonstrate that the GTPγS-stimulated GLUT4 translocation utilizes a novel tyrosine kinase pathway that is independent of both the phosphatidylinositol 3-kinase and the Akt kinase. It has been well established that the insulin stimulation of glucose uptake primarily results from the translocation of the GLUT4 1The abbreviations used are: GLUT4, the insulin-responsive glucose transporter isoform; PI 3-kinase, phosphatidylinositol 3-kinase; IRS1, insulin receptor substrate-1; SH2, Src homology 2; PYK2, protein-tyrosine kinase 2 (also known as CAKβ, RAFTK, and CADTK); pp125FAK, pp125 focal adhesion tyrosine kinase; pp130Cas, pp130 Crk-associated substrate; DMEM, Dulbecco's modified Eagle's medium; SL-O, streptolysin-O; PBS, phosphate-buffered saline; GTPγS, guanosine 5′-O-(3-thiotriphosphate); GDPβS, guanosine 5′-O-2-(thio)diphosphate; IC, intracellular; MOPS, 4-morpholinepropanesulfonic acid; MBP, maltose-binding protein; GST, glutathione S-transferase.1The abbreviations used are: GLUT4, the insulin-responsive glucose transporter isoform; PI 3-kinase, phosphatidylinositol 3-kinase; IRS1, insulin receptor substrate-1; SH2, Src homology 2; PYK2, protein-tyrosine kinase 2 (also known as CAKβ, RAFTK, and CADTK); pp125FAK, pp125 focal adhesion tyrosine kinase; pp130Cas, pp130 Crk-associated substrate; DMEM, Dulbecco's modified Eagle's medium; SL-O, streptolysin-O; PBS, phosphate-buffered saline; GTPγS, guanosine 5′-O-(3-thiotriphosphate); GDPβS, guanosine 5′-O-2-(thio)diphosphate; IC, intracellular; MOPS, 4-morpholinepropanesulfonic acid; MBP, maltose-binding protein; GST, glutathione S-transferase. glucose transporter isoform from intracellular storage sites to the cell surface membrane in muscle and adipose tissues. Although the molecular mechanism and signaling cascade(s) regulating the intracellular trafficking of GLUT4-containing vesicles have not been completely elucidated, several important effector molecules have recently been identified. Insulin binding to the insulin receptor results in tyrosine autophosphorylation of the β-subunit and activation of its intrinsic tyrosine kinase (1Cheatham B. Kahn C.R. Endocr. Rev. 1995; 16: 117-142Crossref PubMed Google Scholar). Subsequently, the insulin receptor tyrosine kinase phosphorylates several intracellular proteins on tyrosine residues, most notably insulin receptor substrate 1 (IRS1) (1Cheatham B. Kahn C.R. Endocr. Rev. 1995; 16: 117-142Crossref PubMed Google Scholar). The phosphorylation of these substrates creates recognition sites for additional effector proteins containing Src homology 2 (SH2) domains, thereby generating multisubunit signaling complexes (1Cheatham B. Kahn C.R. Endocr. Rev. 1995; 16: 117-142Crossref PubMed Google Scholar, 2White M.F. Kahn C.R. J. Biol. Chem. 1994; 269: 1-4Abstract Full Text PDF PubMed Google Scholar). In particular, the tyrosine phosphorylation of IRS1 induces the association and activation of phosphatidylinositol (PI) 3-kinase (3Myers M.G.J. Backer J.M. Sun X.J. Shoelson S. Hu P. Schlessinger J. Yoakim M. Schaffhausen B. White M.F. Proc. Natl. Acad. Sci. U. S. A. 1992; 21: 10350-10354Crossref Scopus (383) Google Scholar). The targeting and/or activation of PI 3-kinase is well documented to be necessary for GLUT4 translocation (4Czech M.P. Annu. Rev. Nutr. 1995; 15: 441-471Crossref PubMed Scopus (95) Google Scholar, 5Shepherd P.R. Siddle K. Nave B.T. Biochem. Soc. Trans. 1997; 25: 978-981Crossref PubMed Scopus (16) Google Scholar). Recently, it has been shown that insulin stimulates the Akt kinase, also known as protein kinase B or RAC-PK (6Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (318) Google Scholar). Insulin-stimulated Akt kinase activity is dependent on PI 3-kinase activation, and expression of a membrane-targeted and constitutively active Akt kinase results in persistent GLUT4 translocation (7Tanti J.F. Grillo S. Gremeaux T. Coffer P.J. Obberghen E.V. Marchand-Brustel Y.L. Endocrinology. 1997; 138: 2005-2010Crossref PubMed Google Scholar, 8Kohn 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 (1087) Google Scholar). Consistent with a role for GLUT4 trafficking, expression of a dominant interfering Akt mutant inhibited insulin-stimulated GLUT4 translocation (9Cong 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). In addition to insulin, various other stimuli display insulinomimetic properties and can induce the translocation of GLUT4-containing vesicles to the plasma membrane. For example, introduction of guanosine 5′-O-(3-thiotriphosphate) (GTPγS), a nonhydrolyzable GTP analogue, into adipocytes rapidly stimulates GLUT4 translocation to a similar extent as insulin (10Baldini G. Hohman R. Charron M.J. Lodish H.F. J. Biol. Chem. 1991; 266: 4037-4040Abstract Full Text PDF PubMed Google Scholar, 11Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (257) Google Scholar). In addition, GTPγS can stimulate GLUT4 translocation in the absence of ATP, suggesting that ATP is required at an early step(s) in the insulin-signaling pathway and that a GTP-binding protein(s) functions at a more distal step(s) (11Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (257) Google Scholar). The stimulatory effect of GTPγS can also be mimicked by treatment with AlF4−, which is characteristic of the involvement of a heterotrimeric GTP binding protein (12Obermaier-Kusser B. Muhlbacher C. Mushack J. Rattenhuber E. Fehlmann M. Haring H.U. Biochem. J. 1988; 256: 515-520Crossref PubMed Scopus (26) Google Scholar, 13Kanai F. Nishioka Y. Hayashi H. Kamohara S. Todaka M. Ebina Y. J. Biol. Chem. 1993; 268: 14523-14526Abstract Full Text PDF PubMed Google Scholar). Consistent with this interpretation, adrenergic stimulation can induce GLUT4 translocation and glucose uptake in both cardiac myocytes (14Slot J.W. Geuze H.J. Gigengack S. James D.E. Lienhard G.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7815-7819Crossref PubMed Scopus (351) Google Scholar,15Fischer Y. Kamp J. Thomas J. Popping S. Rose H. Carpene C. Kammermeier H. Am. J. Physiol. 1996; 270: C1211-C1220Crossref PubMed Google Scholar) and brown adipocytes (16Shimizu Y. Shimizu T. Biochem. Biophys. Res. Commun. 1994; 202: 660-665Crossref PubMed Scopus (43) Google Scholar, 17Omatsu-Kanbe M. Kitasato H. FEBS Lett. 1992; 314: 246-250Crossref PubMed Scopus (34) Google Scholar). Furthermore, in transfected Chinese hamster ovary cells and 3T3L1 adipocytes, activation of receptors coupled to Gq also stimulate GLUT4 translocation (18Kishi K. Hayashi H. Wang L. Kamohara S. Tamaoka K. Shimizu T. Ushikubi F. Narumiya S. Ebina Y. J. Biol. Chem. 1996; 271: 26561-26568Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). In this regard, skeletal muscle appears to have two distinct pathways mediating GLUT4 translocation. Similar to adipocytes, insulin-stimulated glucose transport in muscle is PI 3-kinase dependent (19Elmendorf J.S. Damrau-Abney A. Smith T.R. David T.S. Turinsky J. Biochem. Biophys. Res. Commun. 1995; 208: 1147-1153Crossref PubMed Scopus (29) Google Scholar, 20Berger J. Hayes N. Szalkowski D.M. Zhang B. Biochem. Biophys. Res. Commun. 1994; 205: 570-576Crossref PubMed Scopus (36) Google Scholar, 21Tsakiridis T. McDowell H.E. Walker T. Downes C.P. Hundal H.S. Vranic M. Klip A. Endocrinology. 1995; 136: 4315-4322Crossref PubMed Google Scholar). In contrast, muscle contraction/exercise or hypoxia stimulates glucose transport through a PI 3-kinase-independent pathway, which may utilize a distinct and separate pool of GLUT4 intracellular vesicles (22Yeh J.-I. Gulve E.A. Rameh L. Birnbaum M.J. J. Biol. Chem. 1995; 270: 2107-2111Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar). It has been suggested that this alternative pathway leading to GLUT4 translocation may be mediated by an increase in cytoplasmic calcium levels (23Cartee G.D. Douen A.G. Ramlal T. Klip A. Holloszy J.O. J. Appl. Physiol. 1991; 70: 1593-1600Crossref PubMed Scopus (258) Google Scholar, 24Clausen T. Elbrink J. Dahl-Hansen A.B. Biochim. Biophys. Acta. 1975; 375: 292-308Crossref PubMed Scopus (59) Google Scholar, 25Holloszy J.O. Narahara H.T. Science. 1967; 155: 573-575Crossref PubMed Scopus (26) Google Scholar), consistent with the known functional role of G proteins in stimulating increases in intracellular calcium (26Dolphin A.C. Forda S.R. Scott R.H. J. Physiol. 1986; 373: 47-61Crossref PubMed Scopus (186) Google Scholar, 27Bean B.P. Nature. 1989; 340: 153-156Crossref PubMed Scopus (671) Google Scholar, 28Mintz I.M. Bean B.P. Neuron. 1993; 10: 889-898Abstract Full Text PDF PubMed Scopus (215) Google Scholar, 29Rhim H. Miller R.J. J. Neurosci. 1994; 14: 7608-7615Crossref PubMed Google Scholar, 30Bourinet E. Soong T.W. Stea A. Snutch T.P. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1486-1491Crossref PubMed Scopus (217) Google Scholar). In the present study, we have examined the mechanism by which GTPγS stimulates GLUT4 translocation by comparing its signaling properties with those of insulin. Our data demonstrate that GTPγS-stimulated GLUT4 translocation is independent of the PI 3-kinase, the Akt kinase, and changes in intracellular calcium ion concentration. However, GTPγS-stimulated GLUT4 translocation occurred through the activation of a novel tyrosine kinase pathway, which does not involve the insulin receptor or IRS1 but may require the tyrosine phosphorylation of protein-tyrosine kinase 2 (PYK2, also known as CAKβ, RAFTK, and CADTK), pp125 focal adhesion tyrosine kinase (pp125FAK), pp130 Crk-associated substrate (pp130Cas), paxillin, and Cbl. Murine 3T3L1 preadipocytes were obtained from the American Type Tissue Culture repository and were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 25 mm glucose and 10% calf serum at 37 °C. Confluent cultures were induced to differentiate into adipocytes by incubation of the cells with DMEM containing 25 mm glucose, 10% fetal bovine serum, 1 μg/ml insulin, 1 mm dexamethasone, and 0.5 mmisobutyl-1-methylxanthine. After 4 days, the medium was changed to DMEM containing 25 mm glucose, 10% fetal bovine serum, and 1 μg/ml insulin for an additional 4 days. The medium was then changed to DMEM containing 25 mm glucose and 10% fetal bovine serum. Under these conditions, more than 95% of the cell population morphologically differentiated into adipocytes. All studies were performed on adipocytes between 8 and 12 days after initiation of differentiation (day 0). Prior to all experimental treatments, the differentiated adipocytes were serum-starved in DMEM containing 25 mm glucose and 0.1% bovine serum albumin for 2 h at 37 °C. 3T3L1 adipocytes were permeabilized with streptolysin-O (SL-O) as described by Robinson et al. (11Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (257) Google Scholar) with minor modifications. Briefly, 3T3L1 adipocytes were washed three times with intracellular (IC) buffer (140 mm potassium glutamate, 20 mm Hepes, pH 7.15, 7.5 mm MgCl2, 5 mm EGTA, 5 mm NaCl, 2 mm CaCl2) and incubated in IC buffer containing 0.8 IU/ml of SL-O (Murex Diagnostics Inc., Atlanta, GA) for 5 min at 37 °C. Under these conditions, more than 95% of the cells were permeabilized based upon incorporation of propidium iodide. Following SL-O permeabilization, the cells were washed two times with ICR buffer (IC buffer containing 1 mg/ml bovine serum albumin, 1 mm dithiothreitol and enriched with either 10 mm MgATP or an ATP-regenerating system: 40 IU/ml creatine phosphokinase, 5 mm creatine phosphate, and 1 mm ATP). Unless otherwise indicated, all experimental treatments were performed by incubating the cells in ICR buffer containing various additions for 15 min at 37 °C. 3T3L1 adipocytes used for microinjection were grown on 60-mm tissue culture dishes. The cells were incubated in Krebs-Ringer bicarbonate Hepes buffer (pH 7.4), containing 2 mm pyruvate, 0.5% bovine serum albumin, and 2.5 mm glucose for 45 min prior to microinjection. Adipocytes were microinjected with antibodies over a 45-min period using an Eppendorf model 5171 micromanipulator and given injections of approximately 0.1 pl directly into the cell cytoplasm with an Eppendorf model 5246 transjector. Following microinjection of approximately 100–300 cells/dish, the buffer was changed to fresh DMEM containing 0.1% bovine serum albumin, and the cells were allowed to recover for 90 min at 37 °C, prior to permeabilization and treatment. Preparation of plasma membrane sheets from the adipocytes was performed essentially by the method of Robinson et al. (11Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (257) Google Scholar). Briefly, cells cultured on 35- or 60-mm dishes, following the appropriate treatment as described in each figure legend, were rinsed once in ice-cold phosphate-buffered saline (PBS) and incubated with 0.5 mg/ml of poly-l-lysine in PBS for 30 s. The cells were then swollen in a hypotonic buffer (23 mm KCl, 10 mm Hepes, pH 7.5, 2 mmMgCl2, 1 mm EGTA) by three successive rinses. The swollen cells were sonicated for 3 s at power setting 4.5 with a model 550 Fisher sonic dismembrator fitted with a 5-mm microtip set 1 cm above the surface of the cell monolayer in 10 ml of sonication buffer (70 mm KCl, 30 mm Hepes, pH 7.5, 5 mm MgCl2, 3 mm EGTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride). The bound plasma membrane sheets were washed two times with sonication buffer and used for either indirect immunofluorescence or immunoblot analysis as described below. Total cell extracts were prepared from 60- or 150-mm plates of 3T3L1 adipocytes following the appropriate treatment as described in each figure legend. Cells from each plate were washed two times with ice-cold PBS and scraped into 1 ml of lysis buffer (50 mm HEPES, pH 7.8, 1% Triton X-100, 100 mm NaF, 10 mm Na3P2O7, 2.5 mm EDTA) containing 1.0 mm phenylmethylsulfonyl fluoride, 2 mm Na3VO4, 1 mg/ml aprotinin, 10 mm leupeptin, and 1 mm pepstatin A by rotation for 15 min at 4 °C. Insoluble material was separated from the soluble extract by microcentrifugation for 15 min at 4 °C. Protein concentration was determined, and samples were either subjected directly to SDS-polyacrylamide electrophoresis (as described below) or immunoprecipitated for IRS1 or PYK2. Briefly, 3–5 mg of cellular protein were immunoprecipitated with 5 μg of IRS1 polyclonal antibody (αIRS1; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or PYK2 polyclonal antibody (31Chen D. Elmendorf J.S. Olson A.L. Li X. Earp H.S. Pessin J.E. J. Biol. Chem. 1997; 272: 27401-27410Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) for 2 h at 4 °C. Immune complexes were recovered by the addition of protein A-Sepharose (Amersham Pharmacia Biotech) and subjected to SDS-polyacrylamide electrophoresis (as described below). GST and GST-CrkII were prepared as described previously (32Okada S. Pessin J.E. J. Biol. Chem. 1997; 272: 28179-28182Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Soluble GST fusion proteins were covalently linked to agarose beads with Amino-Link (Pierce). 100 μg of fusion proteins bound to the beads were incubated for 2 h at 4 °C with 4 mg of cell extracts isolated from control, insulin-stimulated, or GTPγS-stimulated cells. The beads were subsequently pelleted, washed three times with washing buffer (10 mm Tris, pH 7.4, 2 mm EDTA, 150 mmNaCl, 0.2% Triton X-100, 0.1% Nonidet P-40, 0.2 mmphenylmethylsulfonyl fluoride, and 0.2 mm sodium vanadate), and boiled in Laemmli sample buffer. The precipitated proteins were analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting (as described below). Plasma membrane sheets were scraped into a buffer containing 250 mm sucrose, 20 mm Tris, pH 7.40, 1 mm EDTA and pelleted by centrifugation at 200,000 × g for 1 h at 4 °C. The pelleted fraction was solubilized in 100 μl of Laemmli sample buffer, and 5 μg of the total solubilized fraction was separated by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide). The resolved proteins were then transferred to nitrocellulose membranes and immunoblotted with polyclonal rabbit GLUT4 antibody (IRGT; Charles River). Whole cell lysates, GST precipitates, and immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis (7.5% polyacrylamide). The resolved proteins were then transferred to Immobilon P membrane (Millipore Corp.) and immunoblotted with a monoclonal phosphotyrosine antibody (PY20:HRPO; Transduction Laboratories), a monoclonal paxillin antibody (Transduction Laboratories), a monoclonal p130Cas antibody (Transduction Laboratories), and a polyclonal Cbl antibody (Santa Cruz Biotechnology). All immunoblots were subjected to enhanced chemiluminescence detection (Amersham Pharmacia Biotech). Sonicated plasma membrane sheets were fixed for 20 min at 4 °C in a solution containing 2% paraformaldehyde, 70 mm KCl, 30 mm HEPES, pH 7.5, 5 mm MgCl2, and 3 mm EGTA. The fixed plasma membrane sheets were quenched for 15 min at 25 °C in 100 mm glycine-PBS (pH 7.5). After three rinses in PBS, the sheets were blocked overnight at 4 °C in PBS containing 5% donkey serum (Sigma). The blocked sheets were incubated at 4 °C overnight with a 1:100 dilution of polyclonal rabbit GLUT4 antibody (IRGT; Charles River). For the microinjection studies, this incubation was done in combination with a 1:5000 dilution of polyclonal sheep anti-maltose binding protein antiserum (generously provided by Dr. Morris Birnbaum, University of Pennsylvania). The plasma membrane sheets were then washed for 30 min with PBS (six changes of PBS) and incubated overnight at 4 °C with a 1:50 dilution of lissamine-rhodamine-conjugated donkey anti-rabbit IgG (Jackson Immunoresearch, Inc.). For the microinjection studies, this incubation was done in combination with a 1:100 dilution of fluorescein isothiocyanate-conjugate donkey anti-sheep IgG (Jackson Immunoresearch Inc.). Following incubation with the secondary antibodies, the membrane sheets were washed for 30 min with PBS (six changes of PBS) and mounted for microscopic analysis with Vectashield mounting medium (Vector Laboratories Inc.). Confocal images were obtained on a Bio-Rad MRC 600 laser confocal microscope (University of Iowa Central Microscopy Research Facility). Akt protein kinase activity was determined as described by Moule et al. (33Moule S.K. Welsh G.I. Edgell N.J. Foulstone E.J. Proud C.G. Denton R.M. J. Biol. Chem. 1997; 272: 7713-7719Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar). 3T3L1 adipocyte detergent cell extracts were prepared as described above, and 600 μg of cellular protein were immunoprecipitated with 5 μg of Akt antiserum (kindly provided by Dr. Kelly Moule, University of Bristol) for 2 h at 4 °C. Immune complexes were recovered by the addition of protein A-Sepharose (Amersham Pharmacia Biotech). The protein A-Sepharose beads were washed and resuspended in 40 μl of assay buffer (20 mm MOPS, pH 7.0, 1 mm EDTA, 1 mm EGTA, 0.01% Brij 35, 5% glycerol) containing 0.1% 2-mercaptoethanol, 2.5 μm cAMP-dependent protein kinase inhibitor peptide. The activity of Akt in the immunoprecipitate was measured using histone H2B (0.5 mg/ml) as anin vitro substrate. The reaction was initiated by the addition of 100 μm [γ-32P]ATP (10 μCi) for 20 min at 30 °C. The reaction was stopped by the addition of 2× Laemmli sample buffer (25 μl), after which the samples were boiled for 5 min at 100 °C and electrophoresed on a 16% SDS-polyacrylamide gel. The gel was stained with Coomassie Blue, dried, and subjected to autoradiography. As previously observed (10Baldini G. Hohman R. Charron M.J. Lodish H.F. J. Biol. Chem. 1991; 266: 4037-4040Abstract Full Text PDF PubMed Google Scholar, 11Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (257) Google Scholar), treatment of SL-O permeabilized 3T3L1 adipocytes with insulin resulted in the translocation of GLUT4 to the plasma membrane as detected by GLUT4 immunoblotting of isolated plasma membrane sheets (Fig. 1 A, lanes 1and 2). Similarly, the addition of GTPγS to the permeabilized cells also induced the translocation of GLUT4 (Fig.1 A, lanes 3 and 4). Since it is well established that activation and/or appropriate intracellular targeting of the PI 3-kinase is necessary for insulin-stimulated GLUT4 translocation, we next examined the role of PI 3-kinase in GTPγS-stimulated GLUT4 translocation. In the control, unstimulated cells, there was a low level of GLUT4 immunofluorescence detected from the isolated plasma membrane sheets (Fig. 1 B, panel 1). Pretreatment with the selective PI 3-kinase inhibitor wortmannin (100 nm) slightly reduced the amount of GLUT4 present in the isolated plasma membrane sheets from unstimulated cells (Fig. 1 B, panel 4). As expected, insulin stimulated a large increase in the translocation of GLUT4 to the plasma membrane, which was completely inhibited by pretreatment with wortmannin (Fig. 1 B, panels 2 and 5). In contrast, although GTPγS stimulated a similar extent of GLUT4 translocation compared with insulin, pretreatment with wortmannin was without effect (Fig. 1 B, panels 3 and6). In addition, pretreatment of the 3T3L1 adipocytes with higher concentrations of wortmannin (1 μm) also inhibited insulin-stimulated GLUT4 translocation but did not reduce the translocation induced by GTPγS (data not shown). Recently, it has been reported that insulin stimulation increases Akt protein kinase activity in a PI 3-kinase-dependent manner, and stable overexpression of a constitutively active membrane-bound form of Akt kinase resulted in the persistent translocation of GLUT4 (7Tanti J.F. Grillo S. Gremeaux T. Coffer P.J. Obberghen E.V. Marchand-Brustel Y.L. Endocrinology. 1997; 138: 2005-2010Crossref PubMed Google Scholar, 8Kohn 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 (1087) Google Scholar). We therefore examined the ability of GTPγS to stimulate Akt kinase as a potential common point of convergence between the insulin and GTPγS signaling pathways leading to GLUT4 translocation. Activation of Akt protein kinase activity was assessed by immunoprecipitation and analysis of in vitro protein kinase activity (Fig.2). Isolation of Akt from unstimulated cells demonstrated a basal level of Akt protein kinase activity that was reduced by pretreatment with wortmannin (Fig. 2, lanes 1and 2). Insulin stimulation resulted in a marked activation of Akt protein kinase activity, which was also attenuated by wortmannin (Fig. 2, lanes 5 and 6). Treatment with GTPγS resulted in an intermediate stimulation of Akt protein kinase activity (Fig. 2, lane 3). Nevertheless, pretreatment with wortmannin completely inhibited the GTPγS-stimulated activation of Akt protein kinase activity (Fig. 2, lane 4). Since Akt kinase activation is accompanied by dual phosphorylation on serine and threonine residues (34Alessi D.R. Andjelkovic M. Caudwell B. Cron P. Morrice N. Cohen P. Hemmings B.A. EMBO J. 1996; 15: 6541-6551Crossref PubMed Scopus (2498) Google Scholar), we also examined the effect of insulin and GTPγS on the SDS-polyacrylamide gel electrophoretic mobility of Akt. Consistent with Akt kinase activity, insulin stimulation resulted in a marked reduction of Akt electrophoretic mobility, whereas GTPγS stimulation had little effect (data not shown). Furthermore, pretreatment of the cells with wortmannin completely prevented the reduction in Akt electrophoretic mobility. Thus, together these data demonstrate that GTPγS stimulation of GLUT4 translocation occurs in a pathway that is independent of both PI 3-kinase and Akt kinase activation. The data presented above suggest that the GTPγS stimulation of GLUT4 translocation occurs in a pathway(s) downstream to and/or in parallel with the PI 3-kinase and Akt kinase. To further characterize the relationship between insulin and GTPγS stimulation, we next incubated the SL-O-permeabilized cells with 100 μm GDPβS prior to insulin treatment (Fig.3). In the absence of insulin, GDPβS had no significant effect on GLUT4 translocation as detected by GLUT4 immunofluorescence of isolated plasma membrane sheets (Fig.3 A, panels 1 and 5). Insulin stimulation resulted in a dose-dependent increase in the amount of plasma membrane-associated GLUT4 protein (Fig. 3 A,panels 1–4). Pretreatment with GDPβS resulted in the inhibition of GLUT4 translocation at low (1 and 10 nm) insulin concentrations but not at high (100 nm) insulin concentrations (Fig. 3 A, panels 5–8). Insulin stimulation also resulted in a dose-dependent decrease in Akt SDS-polyacrylamide gel electrophoretic mobility (Fig.3 B, lanes 1, 2, 5, and8). However, pretreatment with GDPβS had no effect on the insulin stimulation of the Akt gel shift and, hence, phosphorylation and presumably protein kinase activation (Fig. 3 B,lanes 3, 6, and 9). These data further support a model in which GTPγS stimulation of GLUT4 translocation is mediated by a mechanism downstream of the Akt kinase. Recently, several studies have suggested that G protein-coupled receptors can modulate tyrosine kinase signaling pathways. Having ruled out any potential role for PI 3-kinase and Akt protein kinase, we next assessed whether GTPγS-stimulated GLUT4 translocation was mediated by a tyrosine kinase-dependent mechanism. This possibility was initially examined using the relatively specific tyrosine kinase inhibitors genistein and herbimycin A (Fig. 4). In unstimulated cells, there was little GLUT4 detectable in the isolated plasma membrane sheets, and pretreatment with either genistein or herbimycin A had no effect (Fig. 4, panels 1, 4, and 7). As expected, both genistein and herbimycin A were effective inhibitors of insulin-stimulated GLUT4 translocation (Fig. 4,panels 2, 5, and 8), as both of these agents inhibit the insulin receptor tyrosine kinase and IRS1 tyrosine phosphorylation (data not shown). However, to our surprise, pretreatment with these tyrosine kinase inhibitors also prevented the GTPγS-stimulated translocation of GLUT4 (Fig. 4, panels 3,6, and 9). Although genistein and herbimycin A are relatively specific tyrosine kinase inhibitors, it is possible that the prevention of GLUT4 translocation was due to some other nonspecific effect. To further confirm the requirement for tyrosine kinase activity, we next utilized single cell microinjection of a monoclonal phosphotyrosine antibody (Fig. 5). To id" @default.
• W2017481487 created "2016-06-24" @default.
• W2017481487 creator A5044973559 @default.
• W2017481487 creator A5075110027 @default.
• W2017481487 creator A5077728712 @default.
• W2017481487 date "1998-05-01" @default.
• W2017481487 modified "2023-09-30" @default.
• W2017481487 title "Guanosine 5′-O-(3-Thiotriphosphate) (GTPγS) Stimulation of GLUT4 Translocation is Tyrosine Kinase-dependent" @default.
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