Stimulation of glycogen synthesis by heat shock in L6 skeletal-muscle cells: regulatory role of site-specific phosphorylation of glycogen-associated protein phosphatase 1.
Recent evidence suggests that glycogen-associated protein phosphatase 1 (PP-1(G)) is essential for basal and exercise-induced glycogen synthesis, which is mediated in part by dephosphorylation and activation of glycogen synthase (GS). In the present study, we examined the potential role of site-specific phosphorylation of PP-1(G) in heat-shock-induced glycogen synthesis. L6 rat skeletal-muscle cells were stably transfected with wild-type PP-1(G) or with PP-1(G) mutants in which site-1 (S1) Ser(48) and site-2 (S2) Ser(67) residues were substituted with Ala. Cells expressing wild-type and PP-1(G) mutants, S1, S2 and S1/S2, were examined for potential alterations in glycogen synthesis after a 60 min heat shock at 45 degrees C, followed by analysis of [(14)C]glucose incorporation into glycogen at 37 degrees C. PP-1(G) S1 mutation caused a 90% increase in glycogen synthesis on heat-shock treatment, whereas the PP-1(G) S2 mutant was not sensitive to heat stress. The S1/S2 double mutant was comparable with wild-type, which showed a 30% increase over basal. Heat-shock-induced glycogen synthesis was accompanied by increased PP-1 and GS activities. The highest activation was observed in S1 mutant. Heat shock also resulted in a rapid and sustained Akt/ glycogen synthase kinase 3 beta (GSK-3 beta) phosphorylation. Wortmannin blocked heat-shock-induced Akt/GSK-3 beta phosphorylation, prevented 2-deoxyglucose uptake and abolished the heat-shock-induced glycogen synthesis. Muscle glycogen levels regulate GS activity and glycogen synthesis and were found to be markedly depleted in S1 mutant on heat-shock treatment, suggesting that PP-1(G) S1 Ser phosphorylation may inhibit glycogen degradation during thermal stimulation, as S1 mutation resulted in excessive glycogen synthesis on heat-shock treatment. In contrast, PP-1(G) S2 Ser phosphorylation may promote glycogen breakdown under stressful conditions. Heat-shock-induced glycogenesis appears to be mediated via phosphoinositide 3-kinase/Akt-dependent GSK-3 beta inactivation as well as phosphoinositide 3-kinase-independent PP-1 activation.
Moon
Byoung
B
The Diabetes Research Laboratory, Winthrop University Hospital, Mineola, NY 11501, USA.
Duddy
Noreen
N
Ragolia
Louis
L
Begum
Najma
N
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006003:Glycogen / Q000096:biosynthesis* / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000378:metabolism; D000076249:Heat Shock Transcription Factors; D018869:Heat-Shock Response; D018482:Muscle, Skeletal / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D014157:Transcription Factors; D000077191:Wortmannin
D000730:Androstadienes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D018840:HSP70 Heat-Shock Proteins; D000076249:Heat Shock Transcription Factors; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors; D006003:Glycogen; C494920:Akt1 protein, rat; D000071679:Glycogen Synthase Kinase 3 beta; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D038362:Glycogen Synthase Kinase 3; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D000077191:Wortmannin