title: Regulation of protein phosphatase 1 and 2A activities by insulin during myogenesis in rat skeletal muscle cells in culture.
abstract: In this study, we examined protein phosphatase 1 (PP-1) and protein phosphatase 2A (PP-2A) activities during various stages of myogenesis and their regulation by insulin in rat skeletal muscle cells. Protein phosphatase activities were measured using 32P-labeled phosphorylase a, glycogen synthase, and phosphorylase kinase as substrates. Spontaneous PP-1 activity increased progressively in cultures from 2 to 5 days, PP-2A activities remained constant in days 2-4 cultures and increased sharply on day 5. Most of the times in culture, a significant proportion (approximately 65%) of PP-1 was in a form that could be activated by trypsin. Insulin stimulated PP-1 activity (40-80% increase over basal) in a time (t1/2 approximately 5 min)- and dose (EC50 approximately 0.1 nM)-dependent manner. Insulin activation of PP-1 was accompanied by a corresponding inhibition in PP-2A activity. The effects of insulin on PP-1 and PP-2A were differentiation dependent and were observed only in cells at fusion (day 5) and post-fusion. The insulin's effect on PP-1 correlated with the gradual appearance of PP-1 G subunit in cells at fusion. Immunoprecipitation of PP-1 from 32P-labeled cells with an antibody directed against the site 1 sequence of rabbit skeletal muscle PP-1G detected a 160-kDa protein, phosphorylation of which was significantly increased by insulin. This correlated well with the increase observed in immunoprecipitated PP-1G activity. Treatment of cells with a cAMP agonist (SpcAMP) completely blocked activation of PP-1 by insulin and diminished insulin-stimulated phosphorylation of the 160-kDa protein. The likely identity of the 160-kDa band as the regulatory subunit of PP-1 was confirmed by assay of PP-1 activity in the immunoprecipitates and by competition studies with the site 1 peptide against which the antibody was made. From these studies, we conclude that insulin activates PP-1 in L6 cells by increasing the phosphorylation of its regulatory subunit.
authors.affiliation: Diabetes Research Laboratory, Winthrop University Hospital, Mineola, New York 11501.
authors.forename: M
authors.initials: M
authors.lastname: Srinivasan
authors.forename: N
authors.initials: N
authors.lastname: Begum
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D004789:Enzyme Activation; D007328:Insulin / Q000502:physiology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009132:Muscles / Q000166:cytology / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D051381:Rats; D013379:Substrate Specificity
chemical_list: D007328:Insulin; D007527:Isoenzymes; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2