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• W2041199058 abstract "Protein phosphatase-1 (PP-1) in heart and skeletal muscle binds to a glycogen-targeting subunit (GM) in the sarcoplasmic reticulum. Phosphorylation of GM has been postulated to govern activity of PP1 in response to adrenaline and insulin. In this study, we used biochemical assays and GM expression in living cells to examine the effects of insulin on the phosphorylation of GM, and the binding of PP-1 to GM. We also assayed glycogen synthase activation in cells expressing wild type GM and GM mutated at the phosphorylation sites. In biochemical assays kinase(s) prepared from insulin-stimulated Chinese hamster ovary (CHO-IR) cells and C2C12 myotubes phosphorylated a glutathioneS-transferase (GST) fusion protein, GST-GM(1–240), at both site 1 (Ser48) and site 2 (Ser67). Phosphorylation of both sites was dependent on activation of the mitogen-activated protein kinase pathway, involving in particular ribosomal protein S6 kinase. Full-length GMwas expressed in CHO-IR cells and metabolic 32P labeling at sites 1 and 2 was increased by insulin treatment. The GMexpressed in CHO-IR cells or in C2C12 myotubes co-immunoprecipitated endogenous PP-1, and association was transiently lost following treatment of the cells with insulin. In contrast PP-1 binding to GM(S67T), a version of GM not phosphorylated at site 2, was unaffected by insulin treatment. Expression of GM increased basal activity of endogenous glycogen synthase in CHO-IR cells. Insulin stimulated glycogen synthase activity the same extent in cells expressing wild type GM or GMmutated to eliminate phosphorylation site 1 and/or site 2. Phosphorylation of GM is stimulated by insulin, but this phosphorylation is not involved in insulin control of glycogen metabolism. We speculate that other functions of GM at the sarcoplasmic reticulum membrane might be affected by insulin. Protein phosphatase-1 (PP-1) in heart and skeletal muscle binds to a glycogen-targeting subunit (GM) in the sarcoplasmic reticulum. Phosphorylation of GM has been postulated to govern activity of PP1 in response to adrenaline and insulin. In this study, we used biochemical assays and GM expression in living cells to examine the effects of insulin on the phosphorylation of GM, and the binding of PP-1 to GM. We also assayed glycogen synthase activation in cells expressing wild type GM and GM mutated at the phosphorylation sites. In biochemical assays kinase(s) prepared from insulin-stimulated Chinese hamster ovary (CHO-IR) cells and C2C12 myotubes phosphorylated a glutathioneS-transferase (GST) fusion protein, GST-GM(1–240), at both site 1 (Ser48) and site 2 (Ser67). Phosphorylation of both sites was dependent on activation of the mitogen-activated protein kinase pathway, involving in particular ribosomal protein S6 kinase. Full-length GMwas expressed in CHO-IR cells and metabolic 32P labeling at sites 1 and 2 was increased by insulin treatment. The GMexpressed in CHO-IR cells or in C2C12 myotubes co-immunoprecipitated endogenous PP-1, and association was transiently lost following treatment of the cells with insulin. In contrast PP-1 binding to GM(S67T), a version of GM not phosphorylated at site 2, was unaffected by insulin treatment. Expression of GM increased basal activity of endogenous glycogen synthase in CHO-IR cells. Insulin stimulated glycogen synthase activity the same extent in cells expressing wild type GM or GMmutated to eliminate phosphorylation site 1 and/or site 2. Phosphorylation of GM is stimulated by insulin, but this phosphorylation is not involved in insulin control of glycogen metabolism. We speculate that other functions of GM at the sarcoplasmic reticulum membrane might be affected by insulin. cAMP-dependent protein kinase protein phosphatase 1 skeletal muscle glycogen-targeting subunit glycogen associated form of protein phosphatase 1 protein kinase inhibitor glutathione S-transferase epitope tag of hemagglutinin A with sequence YPYDVPDYA triple HA tagged glycogen synthase kinase-3 ribosomal protein S6 kinase mitogen-activated protein MAP kinase MAP kinase kinase epidermal growth factor phosphatidylinositol 3-kinase protein kinase B Dulbecco's modified Eagle's medium glucose 6-phosphate polyacrylamide gel electrophoresis uridine 5′-diphosphoglucose wild type Chinese hamster ovary-insulin receptor phosphate-buffered saline Insulin stimulates glycogen synthesis in mammalian striated muscle by promoting the multisite dephosphorylation and activation of glycogen synthase, the rate-limiting enzyme in this process (1.Villar-Palasi C. Larner J. Biochim. Biophys. Acta. 1960; 39: 171-173Crossref PubMed Scopus (96) Google Scholar, 2.Lawrence J.C. Annu. Rev. Physiol. 1992; 54: 177-193Crossref PubMed Scopus (55) Google Scholar, 3.Cohen P. Boyer P. Krebs E.G. The Enzymes. Academic Press, Orlando, FL1986: 461-497Google Scholar). Based on biochemical studies, glycogen synthase is inactivated primarily by cAMP-dependent protein kinase (PKA)1 and glycogen synthase kinase-3 (GSK-3) that together phosphorylate 7 separate sites in glycogen synthase (4.Roach P.J. Takeda Y. Larner J. J. Biol. Chem. 1976; 251: 1913-1919Abstract Full Text PDF PubMed Google Scholar, 5.Roach P.J. Larner J. Trends Biochem. Sci. 1976; 1: 110-112Abstract Full Text PDF Scopus (6) Google Scholar, 6.Roach P.J. FASEB J. 1990; 4: 2961-2968Crossref PubMed Scopus (194) Google Scholar, 7.Parker P.J. Caudwell F.B. Cohen P. Eur. J. Biochem. 1983; 130: 227-234Crossref PubMed Scopus (208) Google Scholar, 8.Rylatt D.B. Aitken A. Bilham T. Condon G.D. Cohen P. Eur. J. Biochem. 1980; 107: 529-537Crossref PubMed Scopus (182) Google Scholar), and activated by PP-1G, a glycogen-bound form of protein phosphatase-1 that shows specificity for dephosphorylation of these sites in glycogen synthase, relative to other substrates (9.Strålfors P. Hiraga A. Cohen P. Eur. J. Biochem. 1985; 149: 295-303Crossref PubMed Scopus (200) Google Scholar, 10.Hubbard M.J. Cohen P. Eur. J. Biochem. 1989; 186: 711-716Crossref PubMed Scopus (87) Google Scholar, 11.Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (792) Google Scholar). The subcellular location, substrate specificity, and regulation of PP-1 activity are determined by its interaction with targeting subunits. In skeletal and cardiac muscle the major glycogen-associated form of PP-1 (PP-1G) is a heterodimer composed of the phosphatase catalytic subunit (PP-1) and a subunit called GM or RGl. GM directs PP-1 to glycogen-protein particles as well as membranes of the sarcoplasmic reticulum (11.Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (792) Google Scholar, 12.Tang P.M. Bondor J.A. Swinderek K.M. DePaoli-Roach A.A. J. Biol. Chem. 1991; 266: 15782-15789Abstract Full Text PDF PubMed Google Scholar), by directly binding glycogen and by virtue of a putative transmembrane segment near the C terminus. This localization presumably facilitates the dephosphorylation of glycogen-metabolizing enzymes and sarcoplasmic reticulum proteins, such as phospholamban (11.Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (792) Google Scholar,13.Hubbard M.J. Dent P. Smythe C. Cohen P. Eur. J. Biochem. 1990; 189: 243-249Crossref PubMed Scopus (62) Google Scholar, 14.Berman H.K. O'Doherty R.M. Anderson P. Newgard C.B. J. Biol. Chem. 1998; 273: 26421-26425Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 15.MacDougall L.K. Jones L.R. Cohen P. Eur. J. Biochem. 1991; 196: 725-734Crossref PubMed Scopus (186) Google Scholar). Since the discovery of GM a family of glycogen-targeting subunits have been found, and these are closely related in sequence to the N-terminal domain of GM. All members of this family bind glycogen and PP-1. Overexpression of one of these subunits, called PTG, promotes the activation of glycogen synthase (14.Berman H.K. O'Doherty R.M. Anderson P. Newgard C.B. J. Biol. Chem. 1998; 273: 26421-26425Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Curiously, although the structure and function seems to be conserved among this family of proteins, only GM is phosphorylated. Purified preparations of rabbit skeletal muscle GM were phosphorylated at Ser48 (site 1) and Ser67(site 2) by PKA, which prevented binding of PP-1, as a possible way of reducing PP-1 activity toward glycogen-metabolizing enzymes (11.Hubbard M.J. Cohen P. Trends Biochem. Sci. 1993; 18: 172-177Abstract Full Text PDF PubMed Scopus (792) Google Scholar, 16.MacKintosh C. Campbell D.G. Hiraga A. Cohen P. FEBS Lett. 1988; 234: 189-194Crossref PubMed Scopus (32) Google Scholar). As expected, phosphorylation of GM at both sites 1 and 2 occurs in intact muscle in response to adrenaline (17.Walker K.S. Watt P.W. Cohen P. FEBS Lett. 2000; 466: 121-124Crossref PubMed Scopus (47) Google Scholar). However, the phosphorylation of GM in response to insulin, and the possible role of GM phosphorylation in activation of glycogen synthase, has been a subject of controversy for 10 years. Insulin reportedly increased phosphorylation of Ser48 (site 1) in GM by activation of an insulin-stimulated protein kinase (18.Dent P. Lavoinne A. Nakielny S. Caudwell F.B. Watt P. Cohen P. Nature. 1990; 348: 302-308Crossref PubMed Scopus (404) Google Scholar). Site 1 phosphorylation was said to produce a 2-fold increase in the PP-1G-catalyzed dephosphorylation of glycogen synthase (18.Dent P. Lavoinne A. Nakielny S. Caudwell F.B. Watt P. Cohen P. Nature. 1990; 348: 302-308Crossref PubMed Scopus (404) Google Scholar). This offered a pathway for insulin activation of glycogen synthase, through site 1 phosphorylation of GM. With the discovery that insulin-stimulated protein kinase was RSK-2 (19.Lavoinne A. Erikson E. Maller J.L. Price D.J. Avruch J. Cohen P. Eur. J. Biochem. 1991; 199: 723-728Crossref PubMed Scopus (74) Google Scholar), a downstream substrate for MAP kinase (20.Sturgill T.W. Ray L.B. Erikson E. Maller J.L. Nature. 1988; 334: 715-718Crossref PubMed Scopus (754) Google Scholar), site 1 phosphorylation of GM was linked to insulin activation of the MAP kinase pathway. However, multiple groups provided convincing evidence that MAP kinase was not involved in glycogen synthase activation. Experiments used the selective MEK inhibitor, PD98059, or the potent MAP kinase activator, EGF, and showed that activation of MAP kinase and RSK was neither necessary nor sufficient for the activation of glycogen synthase (21.Lin T.-A. Lawrence Jr., J.C. J. Biol. Chem. 1994; 269: 21255-21261Abstract Full Text PDF PubMed Google Scholar, 22.Azpiazu I. Saltiel A.R. DePaoli-Roach A.A. Lawrence Jr., J.C. J. Biol. Chem. 1996; 271: 5033-5039Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 23.Dudley D.T. Dang L. Decker S.J. Bridges A.J. Saltiel A.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7686-7689Crossref PubMed Scopus (2593) Google Scholar, 24.Lazar D.F. Wiese R.J. Brady M.J. Mastick C.C. Waters S.B. Yamauchi K. Pessin J.E. Cuatrecasas P. Saltiel A.R. J. Biol. Chem. 1995; 270: 20801-20807Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar). This was shown in both skeletal muscle cells and adipocytes. The role for MAP kinase in activation of glycogen synthase through activation of PP1G was discounted, but there remained the possibility that other insulin-stimulated kinases phosphorylated site 1 in GM. Experiments reported after original submission of this paper contradicted earlier results from the same group, because phospho-specific antibodies against sites in GM did not detect significant increase in phosphorylation of GM from freeze-clamped rat hindlimb, in response to insulin injection (17.Walker K.S. Watt P.W. Cohen P. FEBS Lett. 2000; 466: 121-124Crossref PubMed Scopus (47) Google Scholar). Therefore, two key questions remain: does insulin stimulate phosphorylation of GM, and is phosphorylation of GM required for insulin activation of glycogen synthase? In the present study, we examined the phosphorylation of GMin response to insulin using: 1) extracts of insulin-stimulated cells plus recombinant GST-GM and 2) expression of full-length tagged GM in CHO-IR and C2C12 muscle cells. Our data shows that insulin-stimulated phosphorylation of GM at both site 1 and site 2, and this is dependent on the activation of MAP kinase pathway. Insulin-stimulated phosphorylation of site 2 in GMdecreased the association of PP-1 in intact cells. Furthermore, we found the stimulation of glycogen synthase activity by insulin remained unchanged in cells expressing GM. Expression of wild type GM or GM mutated in sites 1 and 2 produced the same activation of glycogen synthase in response to insulin, suggesting that phosphorylation of GM is not required for glycogen synthase activation, parallel to the function of PTG (14.Berman H.K. O'Doherty R.M. Anderson P. Newgard C.B. J. Biol. Chem. 1998; 273: 26421-26425Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 25.Printen J.A. Brady M.J. Saltiel A.R. Science. 1997; 275: 1475-1478Crossref PubMed Scopus (243) Google Scholar). CHO cells overexpressing insulin receptors (CHO-IR cells) were from Dr. J. E. Pessin (University of Iowa), and C2C12 myoblasts were from Dr. L. Mei (University of Virginia). Tissue culture reagents and the kit for reverse transcription (Preamplification System for First Strand cDNA Synthesis) were purchased from Life Technologies (Grand Island, NY). pGEX vectors and glutathione-Sepharose were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). [U-14C]Uridine diphosphoglucose ([U-14C]UDPG) was purchased from NEN Life Science Products Inc. (Boston, MA). Bovine insulin, PD98059, and Microcystin-LR were purchased from Calbiochem-Novabiochem Corp. (La Jolla, CA). EGF and PKI were purchased from Sigma. Mouse anti-PP-1 monoclonal antibodies were purchased from Transduction Laboratories (Lexington, KY), and mouse anti-phospho-MAPK antibodies were from Upstate Biotechnology, Inc. (Lake Placid, NY). Goat anti-RSK-2 and mouse anti-HA antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). GM antisera were generated by immunizing rabbits with recombinant GST-GM(1–240) fusion protein. Restriction enzymes were from Promega Life Science (Madison, WI). All the oligonucleotides were synthesized by ITD (Coralville, IA). Rabbit cDNA encoding GM(1–240) was cloned and inserted into pGEX4T2 vector to produce recombinant GST-GM(1–240) fusion protein as described before (26.Wu J. Kleiner U. Brautigan D.L. Biochemistry. 1996; 35: 13858-13864Crossref PubMed Scopus (30) Google Scholar). GM(1–240) fragment was then subcloned into pKH3 mammalian expression vector byBamHI/EcoRI double digestion to produce (HA)3-tagged GM(1–240) protein. Mutated forms of GM(1–240) used in the following experiments were made by the polymerase chain reaction method described elsewhere (26.Wu J. Kleiner U. Brautigan D.L. Biochemistry. 1996; 35: 13858-13864Crossref PubMed Scopus (30) Google Scholar). Mutations were all confirmed by DNA sequencing. The entire coding sequence of rabbit GM was cloned into pGEX-4T2 vector by reverse transcriptase-polymerase chain reaction and subcloning methods as described previously (27.Liu J. Wu J. Oliver C. Shenolikar S. Brautigan D.L. Biochem. J. 2000; 346: 77-82Crossref PubMed Scopus (28) Google Scholar). (HA)3-tagged GM was constructed by insertion of GM cDNA into pKH3 mammalian expression vector. Point mutations in full-length GM were made by using Quick-Change Mutagenesis kit according to the manufacturer's protocol. Mutations were all confirmed by DNA sequencing. CHO-IR cells were maintained in minimal Eagle's medium containing nucleotides and 10% fetal bovine serum. C2C12 myoblasts were maintained in Dulbecco's modified Eagle's medium containing 20% fetal bovine serum until confluent, at which point differentiation was initiated by conversion to Dulbecco's modified Eagle's medium with 4% horse serum. Cell fusion was apparent after 48 h. CHO-IR cells were routinely deprived of serum for 3 h prior to analysis using Ham's F-12 medium. C2C12 myotubes were serum starved for 4 h in Krebs-Ringer buffer with 30 mmHepes (pH 7.4) containing 0.5% bovine serum albumin and 2.5 mm glucose. CHO-IR cells and C2C12 myoblasts were grown to 50–60% confluency and transfected by FuGene reagent according to the manufacturer's instructions (Roche Molecular Biochemicals). Plasmid DNA (6 μg/60-mm dish) was usually transiently transfected for 36 h in the complete medium. While CHO-IR cells were then used for experiments, the confluent C2C12 myoblasts were induced to form myotubes by switching to Dulbecco's modified Eagle's medium with 4% horse serum. The cells were serum starved as described above before the treatment with insulin ± PD98059 or EGF. To obtain a higher degree of transfection efficiency for glycogen synthase activity assay, CHO-IR cells were electroporated with 40 μg of plasmid DNA as described previously (28.Yamauchi K. Pessin J.E. Mol. Cell Biol. 1994; 14: 4427-4434Crossref PubMed Scopus (50) Google Scholar). Under these conditions, approximately 25% of the cells remained viable, and over 80% of the surviving cells were transfected as determined by parallel transfection with a green fluorescent protein construct. CHO-IR cells and C2C12 myotubes were serum-starved and incubated with or without PD98059, wortmannin, and rapamycin for 30 min prior to the addition of 100 nm insulin. After 10 min of insulin treatment, cells (150-mm dishes) were washed twice with ice-cold PBS, then lysed in 0.5 ml of lysis/kinase buffer (50 mm Tris, pH 7.4, 50 mm sodium fluoride, 100 mm NaCl, 1% Nonidet P-40, 5 mm MgCl2, 2 mm EGTA, 20 mm β-glycerolphosphate, 1 mm sodium vanadate, 1 μm Microcystin-LR, 1 mmphenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin). Lysates were clarified at 10,000 × g for 10 min. GST-GM fusion proteins (7.5 μg) were mixed with 22 μl of cell lysate in a final volume of 45 μl containing 50 mm Tris (pH 7.4), 50 mm sodium fluoride, 100 mm NaCl, 0.5% Nonidet P-40, 5 mmMgCl2, 2 mm EGTA, 20 mmβ-glycerolphosphate, 1 mm sodium vanadate, 1 μm Microcystin-LR, 10 μg/ml PKI, and 0.5 mm[γ-32P]ATP (2000 dpm/pmol). The mixture was incubated for 30 min at 30 °C before the reaction was stopped by addition of 500 μl of ice-cold PBS containing 1 mm dithiothreitol and 1 μm Microcystin-LR. 50 μl of glutathione-Sepharose slurry (1:1) was added and the mixture was shaken constantly for 30 min at room temperature. The beads were then pelleted by centrifugation at 20,000 × g for 20–30 s and washed 3 times with ice-cold PBS. Samples were analyzed by SDS-PAGE and autoradiography. Phosphopeptide mapping experiments were performed as described previously (29.West M.H.P. Wu R.S. Bonner W.M. Electrophoresis. 1984; 5: 133-138Crossref Scopus (73) Google Scholar, 30.Dadd C.A. Cook R.G. Allis C.D. BioTechniques. 1993; 14: 266-273PubMed Google Scholar). Briefly, following in vitro phosphorylation and 10% SDS-PAGE, Coomassie Blue-stained GST-GM proteins were excised from the gel and exhaustive trypsin digestion was performed. After repeated lyophilization, phosphopeptides were resolved on a 40% alkaline polyacrylamide gel (pH 9.0) and then analyzed by autoradiography. This assay used 7.5 μg of GST-GM(1–240) protein coupled to glutathione-Sepharose beads to pull-down PP-1 from NIH3T3 cell lysates as described previously (26.Wu J. Kleiner U. Brautigan D.L. Biochemistry. 1996; 35: 13858-13864Crossref PubMed Scopus (30) Google Scholar). The binding of PP-1 was analyzed by anti-PP-1 immunoblotting and PP-1 activity assay. To assay activity following in vitro PP-1 pull-down assay, the GST-GM(1–240) on glutathione beads were pelleted by centrifugation at 20,000 × g for 20–30 s. After washing twice with ice-cold PBS and once with PP-1 assay buffer, the beads were resuspended in 20 μl of PP-1 assay buffer. PP-1 activity was determined against 60 μg of 32P-labeled phosphorylasea in 20 μl of PP-1 assay buffer for 10 min at 30 °C as described previously (31.Brautigan D.L. Shriner C.L. Methods Enzymol. 1988; 159: 339-346Crossref PubMed Scopus (72) Google Scholar). Cell lysates were prepared by detergent solubilization in a lysis buffer (50 mm Tris-HCl, pH 8.0, 100 mm NaCl, 50 mm sodium fluoride, 1% Nonidet P-40, 1 mmdithiothreitol, 1 mm Na3VO4, 1 μm Microcystin-LR, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin) for 30 min at 4 °C. The resultant cell lysates were clarified at 10,000 ×g for 10 min. Immunoprecipitations were performed by incubating cell lysate with anti-HA antibodies for 1 h at 4 °C. The samples were then incubated with protein G-Sepharose for 1 h at 4 °C. The resulting immunoprecipitates were washed three times with the same lysis buffer and then subjected to 10% SDS-PAGE. For immunoblotting, proteins on the gel were electrophoretically transferred to nitrocellulose membranes, which were then blocked in Tris-buffered saline containing 0.1% Tween 20 and 5% non-fat milk. Membranes were incubated with the appropriate antibodies and washed as described previously (26.Wu J. Kleiner U. Brautigan D.L. Biochemistry. 1996; 35: 13858-13864Crossref PubMed Scopus (30) Google Scholar). Antibody binding was detected by enhanced chemiluminescence with horseradish peroxidase-conjugated secondary antibodies. 36 h post-transfection, confluent CHO-IR cells in 100-mm dishes were serum-starved for 3 h in phosphate-free Dulbecco's modified Eagle's medium supplemented with pyruvate. Cells were then incubated for 1 h in the same medium containing 1 mCi/ml [32P]orthophosphate. After stimulation with insulin, cells were washed three times with ice-cold PBS. Lysate preparation and anti-HA immunoprecipitation were performed as described above. Precipitates were resuspended in SDS sample buffer and resovled by SDS-PAGE. After Coomassie Blue stain, tryptic phosphopeptide mapping was performed essentially as described above. Glycogen synthase activity was measured as described previously (24.Lazar D.F. Wiese R.J. Brady M.J. Mastick C.C. Waters S.B. Yamauchi K. Pessin J.E. Cuatrecasas P. Saltiel A.R. J. Biol. Chem. 1995; 270: 20801-20807Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar, 32.Robinson L.J. Razzack Z.F. Lawrence Jr., J.C. James D.E. J. Biol. Chem. 1993; 268: 26422-26427Abstract Full Text PDF PubMed Google Scholar) with some modifications. After two washes with ice-cold PBS, confluent CHO-IR cells (100-mm dishes) were scraped into 500 μl of lysis/glycogen synthase assay buffer (50 mm Tris-HCl, pH 7.8, 10 mm EDTA, 100 mm NaCl, 50 mm sodium fluoride, 1 μm Microcystin-LR, 1% Nonidet P-40, and protease inhibitors as above). Lysates were clarified at 10,000 ×g for 10 min. Then 50 μl of the cell lysate was added to an equal volume of glycogen synthase assay buffer (50 mmTris-HCl, pH 7.8, 10 mm EDTA, 50 mm sodium fluoride, and 1 μm Microcystin-LR) containing 10 mm UDP-[14C]glucose (0.15 μCi/μmol) and 15 mg/ml glycogen, in the presence or absence of 10 mmglucose 6-phosphate. After 15 min of incubation at 37 °C, assay tubes were chilled on ice for 15 min. The entire contents of the tubes were spotted on a Whatman filter paper (GF/A; 2.4 cm) which was immediately immersed in 25 ml of ice-cold 70% ethanol, mixed 1 h, then washed one more time with 25 ml of 70% ethanol for 30 min. Filters were air-dried, and radioactivity was counted with 5 ml of scintillation mixture. The recombinant fusion protein GST-GM(1–240) was used as a substrate for kinases from CHO-IR cells that we treated with insulin. Lysates were prepared and incubated with the fusion protein plus [γ-32P]ATP and PKI, a protein that specifically inhibits PKA activity. As seen in Fig. 1, GST-GM(1–240) was phosphorylated in this assay and insulin stimulation of the CHO-IR cells produced more than a 5-fold increase in the kinase activity in the lysate. Pretreatment of cells with the MEK inhibitor PD98059 nearly eliminated insulin stimulation of GST-GM(1–240) phosphorylation. However, pretreatment of cells with either wortmannin to block PI 3-kinase or rapamycin to inhibit the mammalian target of rapamycin did not change insulin stimulation of kinase activity (Fig.1, upper panel). Coomassie staining showed that a comparable amount of the substrate GST-GM(1–240) was present in each reaction (Fig. 1, lower panel) and the same amount of lysate protein was added. These results showed that insulin stimulated the specific activity of a kinase(s) on the MAP kinase pathway that phosphorylated GST-GM(1–240). Phosphopeptide mapping was performed following 32P labeling of GST-GM(1–240) to determine the site(s) of phosphorylation. Wild type and mutated GST fusion proteins were incubated with cell lysates in the presence of γ-[32P]ATP and an excess of PKI to block PKA activity. The GST fusion proteins were resolved by SDS-PAGE, digested exhaustively by trypsin, and phosphopeptides were resolved by alkaline PAGE and detected by autoradiography. The identity of site 1 and site 2 phosphopeptides were deduced by comparing migration of phosphopeptides using the following collection of proteins: GST-GM(1–240), GST-GM(1–240)/S67A, GST-GM(1–240)/S67T, GST-GM(1–240)/S48A, and GST-GM(51–240) (Fig.2 A). Next, GST-GM(1–240) was phosphorylated by lysates from (i) control cells, (ii) cells treated with insulin, and (iii) cells treated with PD98059 plus insulin. Insulin treatment increased phosphorylation of site 1 and 2 in parallel, and pretreatment of cells with PD98059 significantly inhibited insulin-stimulated phosphorylation of both sites (Fig. 2 B). Site 2 is known as a substrate for PKA, but the kinases here were active in the presence of PKI to block PKA. Similar results were obtained using a skeletal muscle model, C2C12 myotubes differentiated in culture (Fig. 2 C). Lysates of control, insulin-treated, or EGF-treated C2C12 myotubes were used to phosphorylate GST-GM(1–240). Insulin treatment of C2C12 myotubes activated a kinase(s) that equally phosphorylated site 1 and site 2. EGF is a more effective activator of the MAP kinase pathway in muscle cells compared with insulin, and indeed EGF enhanced phosphorylation of site 1 and site 2 in GST-GM(1–240) even more than insulin (Fig. 2 C). These data showed that insulin or EGF-stimulated kinases that phosphorylated site 1 as well as site 2, and activation of MAP kinase was necessary for the stimulation. We found that phosphorylation of GM was catalyzed by RSK-2, a kinase activated by MAP kinase (Fig. 3). A specific RSK-2 antibody was used to immunodeplete endogenous RSK-2 from lysates prepared from insulin-stimulated CHO-IR cells (Fig. 3 A). Immunoblotting of the control and RSK-2-depleted lysates with the same antibody showed almost complete removal of RSK-2 from the lysate. RSK-2 depletion caused about 50% decrease in the phosphorylation of GST-GM(1–240). Moreover, we immunoprecipitated RSK-2 from lysates of control and insulin-treated CHO-IR cells. The specific activity toward GST-GM(1–240) in this assay was 8-fold higher in immunoprecipitates from insulin-treated cells (Fig.3 B). Phosphopeptide mapping showed that the RSK-2 recovered from either control cells or insulin-treated cells phosphorylated both sites in GM, but had preferential activity toward site 1, compared with site 2 (Fig. 3 C). Therefore, using GST-GM(1–240) as substrate, RSK-2 was a prominent insulin-stimulated kinase in cell lysates. But, for insulin and EGF to stimulate phosphorylation of site 1 and site 2 to the same extent, there must have been another kinase(s) activated in concert with RSK-2. We phosphorylated GST-GM(1–240) with lysates from control or insulin-treated CHO-IR cells and recovered the GST fusion protein with glutathione-Sepharose to carry out a pull-down assay for binding of native PP-1(Fig. 4 A). Samples were split, and one-half was subjected to Western blotting for PP-1 and the other half used for assay of phosphatase activity. Immunoblotting showed that phosphorylation of GST-GM(1–240) by kinases from insulin-stimulated cells completely prevented the binding of PP-1 (Fig. 4 A, upper panel). Binding of PP-1 to GST-GM(1–240) was evident after incubation with MgATP plus lysates from unstimulated cells (Fig. 4 A, upper panel). The same amount of GST fusion protein was present in all the samples, based on Coomassie Blue staining (Fig. 4 A, lower panel) and the same amount of lysate protein was added. Consistent with this loss of PP-1 binding, as seen by immunoblotting, there was nearly a 10-fold decrease in PP-1 activity associated with GST-GM(1–240), measured with 32P-labeled phosphorylase a as substrate (Fig. 4 B). We concluded that phosphorylation of GST-GM(1–240) by a kinase(s) from insulin-stimulated cells caused loss of PP-1 binding, probably due to phosphorylation of site 2 (Ser67). Our biochemical experiments suggested that insulin-activated kinases that phosphorylate sites 1 and 2 in the N-terminal domain of GM. To test this we expressed the full-length GM protein in CHO-IR cells. The cells were metabolically labeled with 32Pi and stimulated with insulin, then the GM protein was recovered by immunoprecipitation. The same amount of GM protein was recovered (Fig. 5) and insulin increased its 32P labeling in two independent experiments by 67 and 89% (average 78%) (Fig. 5). Phosphopeptide mapping of the GM from unstimulated, control and from insulin-stimulated cells showed that sites 1 and 2 were phosphorylated in control cells and insulin increased the phosphorylation of both sites (Fig. 5). Phosphorylation of site 2 in GM in living cells should cause a loss of PP-1 association. To test this prediction, epitope-tagged full-length GM was transiently expressed in CHO-IR cells. The cells were treated with or without insulin and immunoprecipitates prepared with anti-HA antibodies (Fig. 6 A, upper panel). Immunoblotting showed insulin tr" @default.
• W2041199058 created "2016-06-24" @default.
• W2041199058 creator A5012242706 @default.
• W2041199058 creator A5074981380 @default.
• W2041199058 date "2000-05-01" @default.
• W2041199058 modified "2023-09-26" @default.
• W2041199058 title "Insulin-stimulated Phosphorylation of the Protein Phosphatase-1 Striated Muscle Glycogen-targeting Subunit and Activation of Glycogen Synthase" @default.
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