FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2053232597> ?p ?o ?g. }

• W2053232597 endingPage "1464" @default.
• W2053232597 startingPage "1458" @default.
• W2053232597 abstract "Glut4-containing vesicles immunoadsorbed from primary rat adipocytes possess endogenous protein kinase activity and phosphorylation substrates. Phosphorylation of several vesicle proteins including Glut4 itself is rapidly activated by insulin. Wortmannin blocks the effect of insulin when added to cells in vivoprior to insulin administration. By means of MonoQ chromatography and Western blot analysis, vesicle-associated protein kinase is identified as Akt-2, a lipid-binding protein kinase involved in insulin signaling. Akt-2 is found to be recruited to Glut4-containing vesicles in response to insulin. Glut4-containing vesicles immunoadsorbed from primary rat adipocytes possess endogenous protein kinase activity and phosphorylation substrates. Phosphorylation of several vesicle proteins including Glut4 itself is rapidly activated by insulin. Wortmannin blocks the effect of insulin when added to cells in vivoprior to insulin administration. By means of MonoQ chromatography and Western blot analysis, vesicle-associated protein kinase is identified as Akt-2, a lipid-binding protein kinase involved in insulin signaling. Akt-2 is found to be recruited to Glut4-containing vesicles in response to insulin. The regulation of postprandial blood glucose levels by insulin is achieved mainly by increased glucose transport into skeletal and cardiac muscle and fat (1DeFronzo R.A. Jacot E. Jequier E. Maeder E. Wahren J. Felber J.P. Diabetes. 1981; 30: 1000-1007Crossref PubMed Scopus (1397) Google Scholar, 2James D.E. Jenkins A.B. Kraegen E.W. Am. J. Physiol. 1985; 248: E567-E574PubMed Google Scholar). These are the only tissues that express a specific isoform of the glucose transporter, Glut4, which mediates the hormonal effect (for recent reviews see Refs. 3Birnbaum M.J. Int. Rev. Cytol. 1992; 137A: 239-297Crossref Scopus (118) Google Scholar, 4Bell G.I. Burant C.F. Takeda J. Gould G.W. J. Biol. Chem. 1993; 268: 19161-19164Abstract Full Text PDF PubMed Google Scholar, 5James D.E. Piper R.C. J. Cell Biol. 1994; 126: 1123-1126Crossref PubMed Scopus (104) Google Scholar, 6Mueckler M. Eur. J. Biochem. 1994; 219: 713-725Crossref PubMed Scopus (955) Google Scholar, 7Stephens J.M. Pilch P.F. Endocr. Rev. 1994; 16: 529-546Google Scholar, 8Rea S. James D.E. Diabetes. 1997; 46: 1667-1677Crossref PubMed Google Scholar). It has been shown that in adipocytes under normal conditions, Glut4 is localized in an intracellular microsomal compartment, “Glut4-containing vesicles,” which are translocated to the plasma membrane in response to insulin. Because total glucose uptake into insulin-sensitive tissues is, in general, proportional to the amount of Glut4 molecules at the cell surface, this translocation process is usually considered as the major mechanism of insulin action on glucose transport. The protein composition of Glut4-containing vesicles is now rather well characterized. Besides Glut4, they include a novelinsulin-regulatedaminopeptidase (IRAP), the IGFII/Man 6-phosphate 1The abbreviations used are: IGF II, insulin-like growth factor; Glut4-vesicle, Glut4-containing vesicle; PI, phosphatidylinositol; PBS, phosphate-buffered saline; LM, light microsome; MBP, myelin basic protein. receptor, the transferrin receptor, and a recently cloned protein, sortilin (reviewed in Refs. 9Kandror K.V. Pilch P.F. Am. J. Physiol. 1996; 271: E1-E14Crossref PubMed Google Scholar and 10Kandror K.V. Pilch P.F. Semin. Cell Dev. Biol. 1996; 7: 269-278Crossref Scopus (8) Google Scholar; see also Refs. 11Lin B.-Z. Pilch P.F. Kandror K.V. J. Biol. Chem. 1997; 272: 24145-24147Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar and 12Morris N.J. Ross S.A. Lane W.S. Moestrup S.K. Petersen C.M. Keller S.R. Lienhard G.E. J. Biol. Chem. 1998; 273: 3582-3587Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). These proteins, which have extracellular functional domains, represent major constituents of Glut4-containing vesicles as shown by silver and Coomassie staining (13Kandror K.V. Pilch P.F. J. Biol. Chem. 1994; 269: 138-142Abstract Full Text PDF PubMed Google Scholar, 14Kandror K.V. Pilch P.F. Biochem. J. 1998; 331: 829-835Crossref PubMed Scopus (62) Google Scholar). In addition to these major “cargo” proteins, Glut4-vesicles are enriched with peripheral and integral membrane proteins that are thought to be involved in membrane trafficking and fusion, such as vesicle-associatedmembrane protein-2 (VAMP2), cellubrevin, secretorycarrier-associated membraneproteins (SCAMPs), low molecular mass GTP-binding proteins, phosphatidylinositol kinases, and several others (reviewed in Refs. 9Kandror K.V. Pilch P.F. Am. J. Physiol. 1996; 271: E1-E14Crossref PubMed Google Scholarand 10Kandror K.V. Pilch P.F. Semin. Cell Dev. Biol. 1996; 7: 269-278Crossref Scopus (8) Google Scholar). Although the biochemical mechanism of translocation of Glut4-containing vesicles to the cell surface is still unknown, there is evidence suggesting that these vesicles represent a subcompartment of the endosomal system in insulin-sensitive cells in which recycling is inhibited under basal conditions, i.e. in the absence of hormone (14Kandror K.V. Pilch P.F. Biochem. J. 1998; 331: 829-835Crossref PubMed Scopus (62) Google Scholar). Insulin administration to cells may release this trafficking block by, for example, removal of an endogenous inhibitor from Glut4-containing vesicles, or by disassociating these vesicles from an intracellular anchor, thus leading to their default fusion with the plasma membrane. Indirect support for this hypothesis derives from recent data showing that the introduction of the cytoplasmic portion of several vesicular proteins, such as Glut4 (15Lee W. Jung C.Y. J. Biol. Chem. 1997; 272: 21427-21431Crossref PubMed Scopus (25) Google Scholar) or IRAP (16Waters S.B. D'Auria M. Martin S.S. Nguyen C. Kozma L.M. Luskey K.L. J. Biol. Chem. 1997; 272: 23323-23327Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), causes Glut4 translocation to the plasma membrane, presumably as a result of competing with the endogenous proteins for the putative inhibitor or anchoring protein. After the trafficking block is released, Glut4-vesicles fuse with the plasma membrane, most likely, via a v-SNARE/t-SNARE-mediated process (reviewed in Ref.8Rea S. James D.E. Diabetes. 1997; 46: 1667-1677Crossref PubMed Google Scholar). A major question in the field that still remains to be resolved is the signal transduction pathway that couples activated insulin receptor with the Glut4-containing compartment (vesicles) and triggers its recruitment to the plasma membrane. At present, we know only about the upstream part of this pathway: insulin receptor, insulin receptor substrates, PI 3-kinase, PDK1, and Akt/PK B (17Cohen P. Alessi D.R. Cross D.A.E. FEBS Lett. 1997; 410: 3-10Crossref PubMed Scopus (235) Google Scholar). The downstream signaling components, proximal to Glut4-vesicles, remain unknown. We show here that Glut4-containing vesicles immunoisolated from rat adipocytes possess a tightly associated protein kinase activity and several phosphorylation substrates. The vesicle-associated protein kinase has been identified as Akt-2 by MonoQ chromatography and Western blot analysis. Phosphorylation of vesicle component proteins as well as artificial substrates by the vesicle-associated protein kinase is rapidly increased by insulin in a wortmannin-sensitive fashion. These data may provide a missing link in the insulin signal transduction pathway by directly coupling Glut4-containing vesicles to the previously established enzymatic cascade. In the present study, we used the monoclonal anti-GLUT4 antibody 1F8 (18James D.E. Brown R. Navarro J. Pilch P.F. Nature. 1988; 333: 183-185Crossref PubMed Scopus (466) Google Scholar), the anti-SCAMPs antibody 3F8 (19Thoidis G. Kotliar N. Pilch P.F. J. Biol. Chem. 1993; 268: 11691-11696Abstract Full Text PDF PubMed Google Scholar), and the anti-Akt-2 sheep antibody (Upstate Biotechnology, Lake Placid, NY). Adipocytes were isolated from the epididymal fat pads of male Sprague-Dawley rats (200–250 g) by collagenase digestion (20Rodbell M. J. Biol. Chem. 1964; 239: 375-380Abstract Full Text PDF PubMed Google Scholar) and transferred to KRP buffer (12.5 mm HEPES, 120 mm NaCl, 6 mm KCl, 1.2 mm MgSO4, 1 mm CaCl2, 0.6 mmNa2HPO4, 0.4 mmNaH2PO4, 2.5 mmd-glucose, 2% bovine serum albumin, pH 7.4). Insulin was administered to cells (where indicated) to a final concentration of 10 nm. After that, KCN was added to cells to final concentration of 2 mm for 5 min, cells were washed three or four times with HES buffer cooled to 14–16 °C (20 mmHEPES, 250 mm sucrose, 1 mm EDTA, 5 mm benzamidine, 1 mm phenylmethanesulfonyl fluoride, 1 μm pepstatin, 1 μm aprotinin, 1 μm leupeptin, pH 7.4), homogenized with a Potter-Elvehjem Teflon pestle, and subcellular fractions were prepared as described previously (21Simpson I.A. Yver D.R. Hissin P.J. Wardzala L.J. Karnieli E. Salans L.B. Cushman S.W. Biochim. Biophys. Acta. 1983; 763: 393-407Crossref PubMed Scopus (330) Google Scholar). In some experiments, phosphatase inhibitors (a mixture of 25 mm Na4P2O7, 50 mm NaF, 5 mm Na3VO4) were added to homogenization buffer with no significant effect. Isolated membrane fractions were resuspended in PBS, which contained all of the protease inhibitors listed above. Protein A-purified 1F8 antibody, as well as nonspecific mouse IgG (Sigma), were each coupled to acrylic beads (Reacti-gel GF 2000, Pierce) at a concentration 0.4 and 0.6 mg of antibody/ml of resin, respectively, according to the manufacturer's instructions. Before usage, the beads were saturated with 2% bovine serum albumin in PBS for at least 1 h and washed with PBS. The light microsomes (LMs) from rat adipocytes were incubated separately with each of the specific and nonspecific antibody-coupled beads overnight at 4 °C. The beads were washed twice with PBS and twice with protein kinase buffer (10 mmTris, 10 mm KCl, 100 mm NaCl, 5 mmMgCl2, pH 7.8), and the adsorbed material was used for phosphorylation experiments as described in the following paragraph. [γ-32P]ATP (50–100 μm, 300–5000 cpm/pmol) and phosphorylation substrates (as specified separately for each experiment) were added to Glut4-containing vesicles immunoadsorbed on the beads (see the previous paragraph) in protein kinase buffer. To provide an efficient mixing, the volume of the liquid phase exceeded the volume of settled beads 2-fold. This suspension was intensively shaken for 30 min at room temperature, and immunobeads were separated from the liquid phase. Beads were then washed twice from the excess of radioactive ATP with protein kinase buffer and 10 mm Tris, pH 7.4, and eluted with either 1% Triton X-100 in protein kinase buffer or Laemmli sample buffer without 2-mercaptoethanol. In the experiments when exogenous protein substrates were added to protein kinase assay, 50-μl aliquots of the liquid phase were applied on 2 × 2 cm squares of Whatman P81 chromatography paper, washed three times with 75 mmphosphoric acid, and counted in a scintillation counter by Cherenkov. Alternatively, phosphorylated proteins were electrophoresed, and dried gels were exposed in a storage phosphor screen cassette and quantitated in a PhosphorImager (Molecular Dynamics). In cases when the exogenous substrates were represented by short synthetic peptides, bovine serum albumin (final concentration, 1%) and trichloroacetic acid (final concentration, 10%) was added to samples, which were then applied on Whatman P81 paper and processed as described above. To separate proteins by anion exchange chromatography, membrane samples were solubilized in 1% Triton X-100 for at least 2 h at 4 °C, centrifuged, and applied to a 1-ml Amersham Pharmacia Biotech MonoQ column equilibrated with 20 mm Tris, 50 mm NaCl, 0.1% Triton X-100, pH 8.0. Elution was carried out with a linear gradient of NaCl (final concentration, 0.5 m; total volume of the gradient, 30 ml) at a flow rate of 0.5 ml/min. Thirty 1-ml fractions were collected and analyzed for the total protein content and protein kinase activity. Concentration of NaCl in the gradient fractions was re-evaluated with the help of a digital conductivity meter (VWR). No Triton X-100 was present in the samples or in the buffers upon fractionation of cytosolic fractions. Proteins were separated in SDS-polyacrylamide gels according to Laemmli (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207231) Google Scholar), but without reducing agents, and were transferred to Immobilon-P membrane (Millipore) in 25 mm Tris, 192 mm glycine. Following transfer, the membrane was blocked with 10% nonfat dry milk in PBS for 2 h at 37 °C. Proteins were visualized with specific antibodies, horseradish peroxidase-conjugated secondary antibodies (Sigma), and an enhanced chemiluminescent substrate kit (NEN Life Science Products). Autoradiograms were normally exposed overnight in a storage phosphor screen cassette and quantitated in a PhosphorImager (Molecular Dynamics). Protein content was determined with a BCA kit (Pierce) according to manufacturer's instructions. Glut4-containing vesicles were immunoadsorbed from intracellular membranes of rat adipose cells treated and not treated with insulin, and [γ-32P]ATP was added directly to the material adsorbed on the beads as described under “Materials and Methods.” Under these conditions, radioactive phosphate is incorporated into several proteins in Glut4-vesicles. Although the pattern of minor phosphorylated proteins varies to some extent in different experiments, we consistently detect phosphorylation of polypeptides with molecular masses 110, 50, 39, and 25 kDa (Fig.1 A). Insulin stimulation of adipocytes for 15 min results in the significant (p < 0.01) increase in the incorporation of radioactive phosphate into these proteins 1.8 ± 0.3, 2.1 ± 0.5, 2.8 ± 0.4, and 3.2 ± 1.3-fold, correspondingly. The electrophoretic mobility of three of the phosphorylated substrates completely matches that of the major constituents of Glut4-vesicles, gp110, or sortilin (11Lin B.-Z. Pilch P.F. Kandror K.V. J. Biol. Chem. 1997; 272: 24145-24147Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), Glut4, and the high molecular mass isoform of the SCAMP triplet, p39 (19Thoidis G. Kotliar N. Pilch P.F. J. Biol. Chem. 1993; 268: 11691-11696Abstract Full Text PDF PubMed Google Scholar) (Fig. 1 A). To further identify these proteins, 1F8-bound material from insulin-stimulated cells was subsequently eluted with PBS containing 1% Triton X-100 and, after that, with Laemmli sample buffer. As we have shown earlier (13Kandror K.V. Pilch P.F. J. Biol. Chem. 1994; 269: 138-142Abstract Full Text PDF PubMed Google Scholar), under these conditions, all vesicular proteins with the exception for Glut4 can be recovered in the Triton eluate, whereas Glut4 is resistant to Triton elution and can be removed from immunobeads only with SDS-containing Laemmli sample buffer. Triton eluate from 1F8 beads was used for immunoprecipitation of SCAMPs according to the previously published protocol (19Thoidis G. Kotliar N. Pilch P.F. J. Biol. Chem. 1993; 268: 11691-11696Abstract Full Text PDF PubMed Google Scholar). As expected, phosphorylated Glut4 was detected in the SDS eluate from 1F8 beads, whereas phosphorylated SCAMPs were solubilized in 1% Triton X-100 and were immunoprecipitated with the specific antibodies (Fig.1 B). The nature of the low molecular mass phosphorylated protein (p25) remains unknown (see “Discussion”). Several other phosphorylated bands can be noticed on the autoradiogram shown in Fig.1 A, including those with the molecular masses over 200 kDa and below 20 kDa. Electrophoretic mobilities of these proteins correspond to that of the known components of Glut4-vesicles: the IGFII/Man 6-phosphate receptor (p230) (23Kandror K.V. Pilch P.F. J. Biol. Chem. 1996; 271: 21703-21708Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) and VAMP (p18) (24Cain C.C. Trimble W.S. Lienhard G.E. J. Biol. Chem. 1992; 267: 11681-11684Abstract Full Text PDF PubMed Google Scholar). We are currently trying to identify these proteins. Along with protein kinase(s), Glut4-containing vesicles may also contain an endogenous phosphatase activity, which may alter the results of the in vitro phosphorylation. To check this possibility, Glut4-vesicles were immunoadsorbed from insulin-treated and untreated cells, phosphorylated in vitro as described above, thoroughly washed of radioactive ATP, and incubated under the same conditions for another hour. This additional incubation in the absence of ATP does not change the total pattern of phosphorylation or specific incorporation of radioactive phosphate into individual proteins (not shown). In the next experiments, we explored the substrate specificity of the vesicle-associated protein kinase. As is shown in Fig.2, myelin basic protein (MBP) was phosphorylated to a greater extent than other substrates analyzed. A small amount incorporation of radioactive phosphate into total fraction of histones (Sigma) was also detected. On the other hand, neither casein nor a synthetic peptide corresponding to the phosphorylation site on the regulatory p85 subunit of PI 3-kinase (25Dhand R. Hiles I. Panayotou G. Roche S. Fry M.J. Gout I. Totty N.F. Truong O. Vicendo P. Yonezawa K. Kasuga M. Courtneidge S.A. Waterfield M.D. EMBO J. 1994; 13: 522-533Crossref PubMed Scopus (415) Google Scholar) was phosphorylated by a vesicle-associated protein kinase (Fig. 2), although it could be phosphorylated by immunoprecipitated PI 3-kinase. 2S. Heydrick and K. V. Kandror, unpublished observations. This, together with other evidence (see Fig. 5), suggests that PI 3-kinase which, in a recent study, was found to be associated with Glut4-containing vesicles in an insulin-dependent manner (26Heller-Harrison R.A. Morin M. Guilherme A. Czech M.P. J. Biol. Chem. 1996; 271: 10200-10204Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), does not phosphorylate their component proteins in vitro. It was also found that the vesicle-associated protein kinase is fairly specific for ATP and cannot use GTP, taken at an equal concentration (data not shown).Figure 5Wortmannin inhibits insulin-dependent activation of the vesicle-associated protein kinase in vivo, but not in vitro. Left panel, LMs (0.15 mg of protein) from adipocytes untreated and treated with insulin for 2 min were immunoadsorbed with 50 μl of 1F8 beads and phosphorylated in vitro in the absence or in the presence of 5 μm wortmannin.Right panel, adipocytes were pre-incubated with 100 nm wortmannin (where indicated) for 20 min before insulin administration for 2 min, and Glut4-vesicles were immunoadsorbed and phosphorylated in vitro as described in the legend to theleft panel. The positions of the molecular mass standards are shown on the right. A representative result of four independent experiments is shown. Quantitation of the results shown in the right panel of the figure is presented in Table I.View Large Image Figure ViewerDownload (PPT) As an additional control for phosphatase activity, in vitrophosphorylated MBP was incubated with Glut4-containing vesicles immunoadsorbed from insulin-treated and not treated cells. No dephosphorylation of 32P-labeled MBP was detected under these conditions (not shown). Because cytosolic proteins can, theoretically, be nonspecifically associated with the immunoadsorbed material and dissociate with an increase in the ionic strength of the washing buffer, it seemed essential to determine how tightly the endogenous protein kinase is associated with Glut4-containing vesicles. Fig.3 shows that extensive wash of immunoadsorbed Glut4-containing vesicles with a high ionic strength buffer cannot remove endogenous protein kinase activity from this compartment and does not change the pattern of phosphorylated proteins. As is shown in Figs. Figure 1, Figure 2, Figure 3, some increase in the activity of the vesicle-associated protein kinase always takes place after adipocytes are stimulated by insulin for 15 min. This time point was chosen because the effect of insulin on Glut4 recruitment to the plasma membrane reaches the maximum at about this time (for recent study see Ref. 27Kublaoui B. Lee J. Pilch P.F. J. Biol. Chem. 1995; 270: 59-65Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). However, activation of the vesicle-associated protein kinase may precede vesicle translocation. Thus, we immunoadsorbed Glut4-containing vesicles from adipocytes treated with insulin for 2, 4, 8, and 16 min and determined the level of protein kinase activity in this material. Fig. 4 A shows that phosphorylation of the vesicle proteins by the endogenous protein kinase is rapidly increased by insulin and then gradually declines after 2 min of insulin stimulation. In this and other (Figs. 1, 3, and 5) experiments, however, the effect of insulin on phosphorylation of Glut4-containing vesicles in vitro may and should depend on the phosphorylation status of the substrate proteins in living cells, which is hard if not impossible to measure. In other words, if insulin causes dephosphorylation of Glut4-containing vesicles in vivo, we may see an increase in their phosphorylation in vitro even if the level of the vesicle-associated protein kinase activity stays the same. To distinguish between these two possibilities, we measured the activity of this protein kinase toward an exogenous substrate, MBP (Fig.4 B). Because the pattern of MBP phosphorylation mirrors the incorporation of radioactive phosphate into component proteins of Glut4-vesicles (Fig. 4 A) and given the lack of the detectable phosphatase in this preparation, we conclude that protein kinase activity associated with Glut4-vesicles is indeed stimulated by insulin with the maximum at 2 min after insulin administration. Thus, in all following experiments, the effect of insulin on phosphorylation of Glut4-vesicles was measured after 2 min of insulin treatment. To determine whether vesicle-associated protein kinase may participate in the transduction of the hormonal signal from the cell surface to its final target: Glut4-containing vesicles, we carried out experiments with wortmannin, a specific inhibitor of PI 3-kinase and its downstream signaling. The left panel of Fig.5 demonstrates that the addition of wortmannin (5 μm) to the in vitro protein kinase assay has no effect on phosphorylation of vesicle proteins by the endogenous protein kinase. This result provides additional evidence that Ser/Thr kinase activity of PI 3-kinase is not responsible for phosphorylation of Glut4-vesicles. In analogous experiments, we have shown that neither PK I (an inhibitor of protein kinase A) nor calphostin C (an inhibitor of protein kinase C) has any effect on the pattern of phosphorylated proteins in Glut4-vesicles (not shown). In contrast, wortmannin in a much lower concentration (100 nm) was able to prevent insulin-stimulated increase in phosphorylation of different proteins in Glut4-vesicles when added to adipose cellsin vivo, 20 min prior to insulin administration (Fig. 5,right panel, and Table I).Table IThe effect of wortmannin on insulin-stimulated phosphorylation of Glut4-containing vesicles by an endogenous protein kinaseProtein− Wortmannin+ Wortmanninp1103.5 ± 2.10.9 ± 0.3p607.6 ± 0.91.1 ± 0.2p50 (Glut4)6.2 ± 1.81.1 ± 0.4p39 (SCAMP)4.8 ± 0.91.2 ± 0.3p255.3 ± 1.51.3 ± 0.4The table represents quantitation of the data shown in Fig. 5(right panel). It demonstrates the increase in phosphorylation of the individual proteins of Glut4-containing vesicles (fold stimulation) by an endogenous protein kinase after insulin administration to adipocytes for 2 min in the absence and in the presence of 100 nm wortmannin. The numbers are the mean values ± S.E. of four independent experiments. Open table in a new tab The table represents quantitation of the data shown in Fig. 5(right panel). It demonstrates the increase in phosphorylation of the individual proteins of Glut4-containing vesicles (fold stimulation) by an endogenous protein kinase after insulin administration to adipocytes for 2 min in the absence and in the presence of 100 nm wortmannin. The numbers are the mean values ± S.E. of four independent experiments. Data shown in Figs. 4 and 5 suggest that the vesicle-associated protein kinase may participate in the insulin signaling downstream of PI 3-kinase. Moreover, in these experiments we have detected a protein with the molecular mass of 60 kDa, the phosphorylation of which is transiently activated by insulin in a wortmannin-sensitive fashion. After 2 min of insulin stimulation, p60 represents one of the major phosphorylated proteins in Glut4-vesicles, whereas after 15 min of insulin treatment its phosphorylation is hardly detectable (compare Figs. 4 and 5 with Figs. 1 and 3). The molecular mass of this protein corresponds well to that of the newly described lipid-binding Ser/Thr protein kinase, Akt. As has recently been shown, Akt is activated by insulin in adipose and muscle cells (28Kohn A.D. Kovacina K.S. Roth R.A. EMBO J. 1995; 14: 4288-4295Crossref PubMed Scopus (319) Google Scholar, 29Cross D.A.E. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4376) Google Scholar, 30Moule S.K. Welsh G.I. Edgell N.J. Foulstone E.J. Proud C.G. Denton R.M. J. Biol. Chem. 1997; 272: 7713-7719Crossref PubMed Scopus (226) Google Scholar, 31Wijkander J. Holst L.S. Rahn T. Resjo S. Castan I. Manganiello V. Belfrage P. Degerman E. J. Biol. Chem. 1997; 272: 21520-21526Crossref PubMed Scopus (74) Google Scholar), is located downstream of PI 3-kinase (reviewed in Ref. 17Cohen P. Alessi D.R. Cross D.A.E. FEBS Lett. 1997; 410: 3-10Crossref PubMed Scopus (235) Google Scholar), and may mediate the effect of insulin on Glut4 translocation (32Kohn 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 (1093) Google Scholar, 33Tanti J.-F. Grillo S. Gremeaux T. Coffer P.J. VanObberghen E. LeMarchand-Brustel Y. Endocrinology. 1997; 138: 2005-2010Crossref PubMed Google Scholar, 34Ueki K. Yamamoto-Honda R. Kaburagi Y. Yamauchi T. Tobe K. Burgering B.M.T. Coffer P.J. Komuro I. Akanuma Y. Yazaki Y. Kadowaki T. J. Biol. Chem. 1998; 273: 5315-5322Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, 35Cong 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). Therefore, we decided to check whether Akt is directly present in Glut4-containing vesicles. As is seen in Fig.6, Glut4-vesicles include visible amounts of Akt-2, an isoform of the enzyme that is predominant in adipocytes (36Walker K.S. Deak M. Paterson A. Hudson K. Cohen P. Alessi D.R. Biochem. J. 1998; 331: 299-308Crossref PubMed Scopus (241) Google Scholar). These data correspond well to recent results of Calera et al. (37Calera M.R. Martinez C. Liu H. Jack A.K. El Birnbaum M.J. Pilch P.F. J. Biol. Chem. 1998; 273: 7201-7204Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), who demonstrated by immunoadsorption and sucrose gradient centrifugation that Akt-2 may interact with Glut4-containing vesicles in an insulin- and wortmannin-sensitive fashion. Note, that according to Western blot analysis, only a small portion (5–7%) of the total cellular Akt-2 was recovered in the fraction of light microsomes with the major pool of the enzyme being localized in the cytosol. Of this membrane-associated enzyme, 3–4% was found in Glut4-containing vesicles (Fig. 6). However, it is still an open question as to what portion of the LM fraction represents membranes and what portion is accounted for by ribosomes, cytoskeleton, heavy protein complexes, etc., that are also pelleted under conditions used to purify so-called “light microsomes” (38Clark 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). Thus, Fig. 6 may considerably underrepresent the fraction of the total membrane-associated Akt-2 that is present in Glut4-containing vesicles. Glut4-containing vesicles from insulin-treated cells contain at least three times more Akt-2 than vesicles from basal cells (Fig. 6). This is consistent with our data about insulin-stimulated increase in phosphorylation of vesicle proteins (Figs. Figure 1, Figure 2, Figure 3, Figure 4, Figure 5) and suggests that this phenomenon is likely to be explained by recruiting more active Akt-2 onto Glut4-vesicles. To estimate to what extent Akt-2 associated with Glut4-containing vesicles can account for the total protein kinase activity present in this compartment, we immunoadsorbed Glut4-containing vesicles on 1F8 beads, solubilized their component proteins with 1% Triton, fractionated this material on a MonoQ column, and determined protein kinase activity in the chromatorgaphic fractions using MBP as a substrate (Fig. 7, top panel). In parallel, we fractionated Triton-solubilized total LMs from adipose cells on the same column under the same experimental conditions and determined the position of Akt-2 by Western blotting (Fig. 7,bottom panel). As is seen in Fig. 7, the major peak of the protein kinase activity associated with Glut4-containing vesicles is eluted in fractions 5 and 6, which exactly corresponds to the position of the membrane-associated Akt-2. Thus, we conclude that Akt-2 is responsible for the major part of the total MBP kinase activity present in Glut4-vesicles. To prove that Akt-2 can phosphorylate component proteins of Glut4-vesicles, we have purified this enzyme from the cytos" @default.
• W2053232597 created "2016-06-24" @default.
• W2053232597 creator A5015934826 @default.
• W2053232597 creator A5058516278 @default.
• W2053232597 date "1999-01-01" @default.
• W2053232597 modified "2023-10-15" @default.
• W2053232597 title "Akt-2 Binds to Glut4-containing Vesicles and Phosphorylates Their Component Proteins in Response to Insulin" @default.
• W2053232597 cites W1257903918 @default.
• W2053232597 cites W1491506353 @default.
• W2053232597 cites W1520078901 @default.
• W2053232597 cites W1520970475 @default.
• W2053232597 cites W1529141138 @default.
• W2053232597 cites W1537820722 @default.
• W2053232597 cites W1569980220 @default.
• W2053232597 cites W1588687595 @default.
• W2053232597 cites W1643583901 @default.
• W2053232597 cites W1787672602 @default.
• W2053232597 cites W1876033686 @default.
• W2053232597 cites W1965890828 @default.
• W2053232597 cites W1970529208 @default.
• W2053232597 cites W1972307668 @default.
• W2053232597 cites W1972977742 @default.
• W2053232597 cites W1973837499 @default.
• W2053232597 cites W1974052445 @default.
• W2053232597 cites W1976177239 @default.
• W2053232597 cites W1983566655 @default.
• W2053232597 cites W1986381441 @default.
• W2053232597 cites W1989294105 @default.
• W2053232597 cites W19918496 @default.
• W2053232597 cites W2006358698 @default.
• W2053232597 cites W2011984083 @default.
• W2053232597 cites W2016135608 @default.
• W2053232597 cites W2024421873 @default.
• W2053232597 cites W2034251015 @default.
• W2053232597 cites W2043498565 @default.
• W2053232597 cites W2058138221 @default.
• W2053232597 cites W2063647604 @default.
• W2053232597 cites W2064242629 @default.
• W2053232597 cites W2066052044 @default.
• W2053232597 cites W2068063638 @default.
• W2053232597 cites W2087321541 @default.
• W2053232597 cites W2090458861 @default.
• W2053232597 cites W2092294261 @default.
• W2053232597 cites W2094068658 @default.
• W2053232597 cites W2100040836 @default.
• W2053232597 cites W2100837269 @default.
• W2053232597 cites W2110700383 @default.
• W2053232597 cites W2112009693 @default.
• W2053232597 cites W2117483382 @default.
• W2053232597 cites W2125547155 @default.
• W2053232597 cites W2133374963 @default.
• W2053232597 cites W2138391708 @default.
• W2053232597 cites W2143873575 @default.
• W2053232597 cites W2145545657 @default.
• W2053232597 cites W2165604272 @default.
• W2053232597 cites W2165863663 @default.
• W2053232597 cites W2167497319 @default.
• W2053232597 cites W2169401893 @default.
• W2053232597 cites W2407358803 @default.
• W2053232597 cites W2411687918 @default.
• W2053232597 cites W4234043763 @default.
• W2053232597 cites W67399661 @default.
• W2053232597 cites W89084389 @default.
• W2053232597 doi "https://doi.org/10.1074/jbc.274.3.1458" @default.
• W2053232597 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9880520" @default.
• W2053232597 hasPublicationYear "1999" @default.
• W2053232597 type Work @default.
• W2053232597 sameAs 2053232597 @default.
• W2053232597 citedByCount "115" @default.
• W2053232597 countsByYear W20532325972012 @default.
• W2053232597 countsByYear W20532325972013 @default.
• W2053232597 countsByYear W20532325972014 @default.
• W2053232597 countsByYear W20532325972015 @default.
• W2053232597 countsByYear W20532325972016 @default.
• W2053232597 countsByYear W20532325972017 @default.
• W2053232597 countsByYear W20532325972018 @default.
• W2053232597 countsByYear W20532325972019 @default.
• W2053232597 countsByYear W20532325972021 @default.
• W2053232597 countsByYear W20532325972022 @default.
• W2053232597 countsByYear W20532325972023 @default.
• W2053232597 crossrefType "journal-article" @default.
• W2053232597 hasAuthorship W2053232597A5015934826 @default.
• W2053232597 hasAuthorship W2053232597A5058516278 @default.
• W2053232597 hasBestOaLocation W20532325971 @default.
• W2053232597 hasConcept C11960822 @default.
• W2053232597 hasConcept C121332964 @default.
• W2053232597 hasConcept C130316041 @default.
• W2053232597 hasConcept C134018914 @default.
• W2053232597 hasConcept C161573976 @default.
• W2053232597 hasConcept C168167062 @default.
• W2053232597 hasConcept C185592680 @default.
• W2053232597 hasConcept C2776188179 @default.
• W2053232597 hasConcept C2779306644 @default.
• W2053232597 hasConcept C41625074 @default.
• W2053232597 hasConcept C55493867 @default.
• W2053232597 hasConcept C75217442 @default.
• W2053232597 hasConcept C86803240 @default.
• W2053232597 hasConcept C95444343 @default.

• next