Phorbol ester treatment of quiescent Swiss 3T3 cells leads to cell proliferation, a response thought to be mediated by protein kinase C (PKC), the major cellular receptor for this class of agents. We demonstrate here that this proliferation is dependent on the activation of the extracellular signal-regulated kinase/mitogenactivated protein kinase (ERK/MAPK) cascade. It is shown that dominant-negative PKC-␣ inhibits stimulation of the ERK/MAPK pathway by phorbol esters in Cos-7 cells, demonstrating a role for PKC in this activation. To assess the potential specificity of PKC isotypes mediating this process, constitutively active mutants of six PKC isotypes (␣, ␤ 1 , ␦, , , and ) were employed. Transient transfection of these PKC mutants into Cos-7 cells showed that members of all three groups of PKC (conventional, novel, and atypical) are able to activate p42 MAPK as well as its immediate upstream activator, the MAPK/ERK kinase MEK-1. At the level of Raf, the kinase that phosphorylates MEK-1, the activation cascade diverges; while conventional and novel PKCs (isotypes ␣ and ) are potent activators of c-Raf1, atypical PKC-cannot increase c-Raf1 activity, stimulating MEK by an independent mechanism. Stimulation of c-Raf1 by PKC-␣ and PKC-was abrogated for RafCAAX, which is a membrane-localized, partially active form of c-Raf1. We further established that activation of Raf is independent of phosphorylation at serine residues 259 and 499. In addition to activation, we describe a novel Raf desensitization induced by PKC-␣, which acts to prevent further Raf stimulation by growth factors. The results thus demonstrate a necessary role for PKC and p42 MAPK activation in 12-Otetradecanoylphorbol-13-acetate induced mitogenesis and provide evidence for multiple PKC controls acting on this MAPK cascade.To date, 11 members of the protein kinase C (PKC) superfamily have been identified (for reviews, see references 13, 28, 45, and 52). On the basis of their biochemical properties and sequence homologies, they have been divided into three groups: the conventional PKCs (cPKC-␣, -␤ 1 , -␤ 2 , and -␥), which are activated in a diacylglycerol (DAG)-and calciumdependent manner; the calcium-independent but DAG-dependent novel PKCs (nPKC-␦, -ε, -, -, and -, also termed PKD); and a third group consisting of atypical PKCs (aPKCand -/). The members of this last group of isotypes are unresponsive to DAG and calcium and, in contrast to c-and nPKCs, do not respond to phorbol esters. The existence of this large family of PKC isotypes suggests that individual PKC isotypes likely have specific roles in signal transduction. We have been interested in determining if such specificity exists in the case of the extracellular signal-regulated kinase/mitogenactivated protein kinase (ERK/MAPK) cascade, by which PKC may mediate some of its effects on cell growth and differentiation.The MAPK cascade, which involves the kinases Raf, MAPK/ ERK kinase (MEK), and ERK/MAPK, is ubiquitously expressed in mammalian cells and serves to couple various cell surface ...

The 80kDa Myristolated Alanine-Rich C-Kinase Substrate (MARCKS) is a major in vivo substrate of protein kinase C (PKC). Here we report that MARCKS is a major substrate for the lipid-activated PKC-related kinase (PRKI) in cell extracts. Furthermore, PRK1 is shown to phosphorylate MARCKS on the same sites as PKC in vitro. Thus, control of MARCKS phosphorylation on these previously identified 'PKC' sites may be regulated under certain circumstances by PRK as well as PKC mediated signalling pathways. The implications for MARCKS as a marker of PKC activation and as a point of signal convergence are discussed.Key words." Protein kinase C; PKC; PRK; MARCKS; Phosphorylation both the calmodulin and actin binding properties of MARCKS as well as loss of membrane binding. In addition to phosphorylation by PKC isotypes, MARCKS has been reported recently to be phosphorylated in vivo by proline-directed protein kinases [12], the effect of these phosphorylations on MARCKS function has not yet been elucidated.In order to determine the potential role of PRKs in signal transduction pathways we have investigated cellular PRK substrates. In this paper we show that purified PRK1 is able to phosphorylate MARCKS. Moreover the resulting phosphopeptides are identical to those produced when MARCKS is subjected to phosphorylation by PKC. The results 1 2 3 4