Activation of the Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Pathway by Conventional, Novel, and Atypical Protein Kinase C Isotypes (original) (raw)

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.