ROLE OF SERINE THREONINE PROTEIN PHOSPHATASES IN INSULIN REGULATION OF NA+/K+-ATPASE ACTIVITY IN CULTURED RAT SKELETAL-MUSCLE CELLS/

In this study, we examined the potential role of serine/threonine prot ein phosphatase-1 (PP-1) and PP-2A in the mechanism of Na+/K+-ATPase a ctivation by insulin in the rat skeletal muscle cell line L6. Incubati on of L6 cells with insulin caused a time-and dose-dependent stimulati on of ouabain-sensitive plasma membrane Na+/K+-ATPase activity, Pretre atment with okadaic acid (OA; 0.1-1 mu M) or calyculin A (1 mu M) bloc ked insulin's effect on Na+/K+-ATPase activation, Low concentrations o f OA that specifically inhibit PP-2A were ineffective, Immunoprecipita tion of the enzyme from P-32-labeled cells with an antibody directed a gainst the alpha-1 subunit of the enzyme revealed a 60% decrease in 11 0 kDa protein phosphorylation in insulin-treated cells. The presence o f calyculin A blocked insulin-mediated dephosphorylation of Na+/K+-ATP ase, whereas low concentrations of OA were ineffective, To further con firm the role of PP-1, we used L6 cell lines that overexpress the glyc ogen/SR-associated regulatory subunit of PP-1, PP-1(G). Overexpression of PP-1(G) resulted in a 3-fold increase in insulin-stimulated PP-1 c atalytic activity. This was accompanied by a 30% increase in basal Na/K+-ATPase activity and a > 2-fold increase in insulin's effect on pum p activity, Inhibition of phosphatidylinositol-3 kinase with wortmanni n blocked insulin-stimulated PP-1 activation as well as the dephosphor ylation and activation of Na+/K+-ATPase. We conclude that insulin regu lates the activity of Na+/K+ ATPase by promoting dephosphorylation of the alpha subunit via an insulin-stimulated PP-1 and that phosphatidyl inositol 3 kinase-generated signals may mediate insulin activation of PP-1 and Na+/K+-ATPase.