Insulin provoked rapid increases in enzyme activity of immunoprecipitable protein kinase C-(PKC-) in rat adipocytes. Concomitantly, insulin provoked increases in 32 P labeling of PKC-both in intact adipocytes and during in vitro assay of immunoprecipitated PKC-; the latter probably reflected autophosphorylation, as it was inhibited by the PKC-pseudosubstrate. Insulin-induced activation of immunoprecipitable PKC-was inhibited by LY294002 and wortmannin; this suggested dependence upon phosphatidylinositol (PI) 3-kinase. Accordingly, activation of PI 3-kinase by a pYXXM-containing peptide in vitro resulted in a wortmannin-inhibitable increase in immunoprecipitable PKC-enzyme activity. Also, PI-3,4-(PO 4 ) 2 , PI-3,4,5-(PO 4 ) 3 , and PI-4,5-(PO 4 ) 2 directly stimulated enzyme activity and autophosphoralytion in control PKC-immunoprecipitates to levels observed in insulin-treated PKC-immunoprecipitates. In studies of glucose transport, inhibition of immunoprecipitated PKC-enzyme activity in vitro by both the PKC-pseudosubstrate and RO 31-8220 correlated well with inhibition of insulin-stimulated glucose transport in intact adipocytes. Also, in adipocytes transiently expressing hemagglutinin antigentagged GLUT4, co-transfection of wild-type or constitutive PKC-stimulated hemagglutinin antigen-GLUT4 translocation, whereas dominant-negative PKC-partially inhibited it. Our findings suggest that insulin activates PKC-through PI 3-kinase, and PKC-may act as a downstream effector of PI 3-kinase and contribute to the activation of GLUT4 translocation.The atypical protein kinase C (PKC), 1 PKC-, is ubiquitously expressed, but little is known about its activation or actions. This ignorance partly derives from the fact that PKC-is not activated by membrane-associated diacylglycerol (DAG) or phorbol esters, generally does not translocate appreciably from cytosol to membrane when activated, and is not depleted by prolonged phorbol ester treatment. Consequently, methods used to evaluate DAG-sensitive conventional (␣, ␤, and ␥) and novel (␦, ⑀, , and ) PKCs are not relevant to PKC-and other DAG-insensitive, atypical PKCs. Although not activated by DAG, PKC-is activated in vitro by phosphatidylserine and polyphosphoinositides, including D3-PO 4 derivatives of phosphatidylinositol (PI) (1, 2). Because of its activation by polyphosphoinositides, PKC-has been suspected to operate downstream of PI 3-kinase; however, direct experimental evidence for this suspicion is lacking, particularly in intact cells. Since insulin increases total polyphosphoinositide levels (3-6), probably largely through PI 3-kinase activation (7), we examined the possibility that insulin activates PKC-by a PI 3-kinasedependent mechanism. To this end, we assayed immunoprecipitable PKC-(a) following treatment of intact adipocytes with insulin in the presence and absence of PI 3-kinase inhibitors; (b) following PI 3-kinase activation in vitro by a pYXXMcontaining peptide; and (c) in response to polyphosphoinositides added directly to the assay of PKC-in vitro. Also...

In rat adipocytes, insulin provoked rapid increases in (a) endogenous immunoprecipitable combined protein kinase C (PKC)-/ activity in plasma membranes and microsomes and (b) immunoreactive PKC-and PKCin GLUT4 vesicles. Activity and autophosphorylation of immunoprecipitable epitope-tagged PKC-and PKCwere also increased by insulin in situ and phosphatidylinositol 3,4,5-(PO 4 ) 3 (PIP 3 ) in vitro. Because phosphoinositide-dependent kinase-1 (PDK-1) is required for phosphorylation of activation loops of PKC-and protein kinase B, we compared their activation. Both RO 31-8220 and myristoylated PKC-pseudosubstrate blocked insulin-induced activation and autophosphorylation of PKC-/ but did not inhibit PDK-1-dependent (a) protein kinase B phosphorylation/activation or (b) threonine 410 phosphorylation in the activation loop of PKC-. Also, insulin in situ and PIP 3 in vitro activated and stimulated autophosphorylation of a PKC-mutant, in which threonine 410 is replaced by glutamate (but not by an inactivating alanine) and cannot be activated by PDK-1. Surprisingly, insulin activated a truncated PKC-that lacks the regulatory (presumably PIP 3 -binding) domain; this may reflect PIP 3 effects on PDK-1 or transphosphorylation by endogenous full-length PKC-. Our findings suggest that insulin activates both PKCand PKC-in plasma membranes, microsomes, and GLUT4 vesicles by a mechanism requiring increases in PIP 3 , PDK-1-dependent phosphorylation of activation loop sites in PKC-and , and subsequent autophosphorylation and/or transphosphorylation.Insulin has been reported to activate atypical forms of protein kinase C (PKC), 1 i.e. PKC-and/or PKC-, in 3T3/L1 adipocytes (1, 2), rat adipocytes (3), L6 myotubes (4), and 32D cells (5). These increases in atypical PKC enzyme activity appear to be largely dependent upon activation of phosphatidylinositol (PI) 3-kinase (1-5) and subsequent increases in D3-PO 4 polyphosphoinositides, i.e. PI 3,4,5-(PO 4 ) 3 and PI 3,4-(PO 4 ) 2 (3). Moreover, transfection studies suggest that PKC-and/or PKC-is/are required for and may be sufficient for insulin stimulation of GLUT4 translocation and subsequent glucose transport (1-4). At present, there is only limited information on the mechanism whereby D3-PO 4 polyphosphoinositides activate atypical PKCs and little or no information on the subcellular compartments in which atypical PKCs are activated or, for that matter, whether one or both atypical PKCs are activated by insulin in specific cell types. With respect to the first point, recent findings (6, 7) suggest that PI 3,4,5-(PO 4 ) 3 and PI 3,4-(PO 4 ) 2 activate, or allow access for, 3-phosphoinositide-dependent kinase-1 (PDK-1), which phosphorylates threonine 410 in the activation loop of PKC-, thereby initiating the activation of this atypical PKC. Indeed, in other studies, we have found that PDK-1 action is required for insulin-induced activation of PKC-in rat adipocytes.2 However, it is uncertain whether this requirement reflects a permissive effect of PDK-1 or whether PDK-1 mediates acute a...

We presently studied (a) insulin effects on protein kinase C (PKC) and (b) effects of transfection-induced, stable expression of PKC isoforms on glucose transport in 3T3/L1 cells. In both fibroblasts and adipocytes, insulin provoked increases in membrane PKC enzyme activity and membrane levels of PKC-alpha and PKC-beta. However, insulin-induced increases in PKC enzyme activity were apparent in both non-down-regulated adipocytes and adipocytes that were down-regulated by overnight treatment with 5 microM phorbol ester, which largely depletes PKC-alpha, PKC-beta, and PKC-epsilon, but not PKC-zeta. Moreover, insulin provoked increases in the enzyme activity of immunoprecipitable PKC-zeta. In transfection studies, stable overexpression of wild-type or constitutively active forms of PKC-alpha, PKC-beta1, and PKC-beta2 failed to influence basal or insulin-stimulated glucose transport (2-deoxyglucose uptake) in fibroblasts and adipocytes, despite inhibiting insulin effects on glycogen synthesis. In contrast, stable overexpression of wild-type PKC-zeta increased, and a dominant-negative mutant form of PKC-zeta decreased, basal and insulin-stimulated glucose transport in fibroblasts and adipocytes. These findings suggested that: (a) insulin activates PKC-zeta, as well as PKC-alpha and beta; and (b) PKC-zeta is required for, and may contribute to, insulin effects on glucose transport in 3T3/L1 cells.