In this study, we examined the distribution of protein serine/threonine phosphatase-1 (PP-1) and analyzed the effect of insulin on PP-1 and its mechanism of activation in freshly isolated rat adipocytes. The adipocyte particulate fraction (PF) constituted 80% of cellular PP-1 activity, while PP-2A was entirely cytosolic. Insulin rapidly stimulated PF PP-1 in a time- and dose-dependent manner (maximum stimulation at 5 min with 4 nM insulin). Immunoprecipitation of PF with an antibody against the site-1 sequence of rabbit skeletal muscle glycogen-associated PP-1 (PP-1G) subunit indicated that 40% of adipocyte PP-1 activity was due to PP-1G form of the enzyme. Insulin stimulated PP-1G (120% over basal levels) without affecting the other forms of PP-1 in the PF. Insulin activation of PP-1 was accompanied by >2-fold increase in the phosphorylation state of the 160-kDa regulatory subunit of PP-1. Stimulation of p21/mitogen-activated protein kinase pathway (MAP) with GTP analogues also resulted in stimulation of PP-1 similar to insulin. The insulin effect on MAP kinase and PP-1 activation was blocked by a GTP antagonist, guanyl-5`-yl thiophosphate. The inhibitors of MAP kinase activation (viz. cAMP agonists, SpcAMP and ML-9) also blocked PP-1 stimulation by insulin. The time course of MAP kinase activation preceded the phosphorylation of PP-1 regulatory subunit and PP-1 activation. We conclude that insulin rapidly activates a membrane-associated PP-1 in adipocytes, which may be similar to rabbit skeletal muscle PP-1G, and the activation is mediated by p21/MAP kinase pathway.

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