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Volume 272, Number 38, Issue of September 19, 1997 pp. 23653-23658
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

Role of Serine/Threonine Protein Phosphatases in Insulin Regulation of Na⁺/K⁺-ATPase Activity in Cultured Rat Skeletal Muscle Cells

(Received for publication, April 28, 1997, and in revised form, June 30, 1997)

Louis Ragolia ^§ , Basil Cherpalis ^§ , Malathi Srinivasan ^§ and Najma Begum ^§

From  The Diabetes Research Laboratory, Winthrop University Hospitol, Mineola, New York 11501 and the ^§ School of Medicine, State University of New York, Stony Brook, New York 11704

In this study, we examined the potential role of serine/threonine protein phosphatase-1 (PP-1) and PP-2A in the mechanism of Na⁺/K⁺-ATPase activation by insulin in the rat skeletal muscle cell line L6. Incubation of L6 cells with insulin caused a time- and dose-dependent stimulation of ouabain-sensitive plasma membrane Na⁺/K⁺-ATPase activity. Pretreatment with okadaic acid (OA; 0.1-1 µM) or calyculin A (1 µM) blocked insulin's effect on Na⁺/K⁺-ATPase activation. Low concentrations of OA that specifically inhibit PP-2A were ineffective. Immunoprecipitation of the enzyme from ³²P-labeled cells with an antibody directed against the -1 subunit of the enzyme revealed a 60% decrease in 110-kDa protein phosphorylation in insulin-treated cells. The presence of calyculin A blocked insulin-mediated dephosphorylation of Na⁺/K⁺-ATPase, whereas low concentrations of OA were ineffective. To further confirm the role of PP-1, we used L6 cell lines that overexpress the glycogen/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 catalytic activity. This was accompanied by a 30% increase in basal Na⁺/K⁺-ATPase activity and a >2-fold increase in insulin's effect on pump activity. Inhibition of phosphatidylinositol-3 kinase with wortmannin blocked insulin-stimulated PP-1 activation as well as the dephosphorylation and activation of Na⁺/K⁺-ATPase. We conclude that insulin regulates the activity of Na⁺/K⁺-ATPase by promoting dephosphorylation of the  subunit via an insulin-stimulated PP-1 and that phosphatidylinositol-3 kinase-generated signals may mediate insulin activation of PP-1 and Na⁺/K⁺-ATPase.

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