Incubation of Swiss mouse 3T3-D1 cells with physiological concentrations of insulin resulted in a rapid and transient activation of protein phosphatase activity as measured by using [32P]phosphorylase a as substrate. Activation reached a maximum level (140% of control value) within 5 min of addition and returned to control levels within 20 min. The effect of insulin was dose-dependent with half-maximal activation occurring at %5 nM insulin. This activity could be completely inhibited by addition of the heat-stable protein inhibitor 2, which suggests the presence of an activated type-i phosphatase. Similar effects on phQsphatase activity were seen when epidermal growth factor aid platelet-derived growth factor were tested. These results suggest that some of the intracellular effects caused by insulin and growth factors are mediated through the activation of a protein phosphatase.Insulin triggers a complex array of metabolic processes in a variety of mammalian cell types. These effects range from increased uptake of ions and nutrients, altered flux pattern of carbon in synthetic and degradative pathways of glucose, lipid, and protein metabolism to changes in specific gene expression (for reviews, see refs. 1 and 2). Although these biochemical events have been under intense investigation for many years, the detailed signaling mechanisms by which insulin functions remain to be elucidated. A major mode of action of insulin involves changes of the phosphorylation state of various proteins. The first event initiated upon binding of the hormone to its cell-surface receptor is the induction ofan intrinsic protein-tyrosine kinase activity in the cytoplasmic domain of the receptor. Based on site-specific mutagenesis studies of the human insulin receptor, it has been suggested that this kinase activity is essential for at least some of the metabolic actions of insulin, such as an increase in ribosomal protein S6 phosphorylation and in glycogen synthesis (3). These and many other intracellular enzyme systems are known to be regulated through phosphorylation cascades involving serine/threonine residues, but the steps connecting the target proteins to tyrosine phosphorylation by the receptor are still unknown.In glycogen metabolism, the rate-limiting enzymes of breakdown and synthesis are regulated by phosphorylation/ dephosphorylation mechanisms. (7,8). From studies carried out with purified enzymes, various mechanisms for regulating protein phosphatase activities in vitro have been identified; however, to date little is known about the regulation of these enzymes in vivo.Our finding that insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) activate glycogen synthase (11, 12) in cultured 3T3 cells has prompted an investigation of the effect of these polypeptide factors on phosphoprotein phosphatase activity. Since glycogen synthase is activated by dephosphorylation (6, 13, 14), it seemed probable that phosphatases would have a critical role in the regulation of this enzyme. In this initi...

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