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

Abstract

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 ...

Oncogenes, growth factors and phorbol esters regulate Raf-1 through common mechanisms

“…Surprisingly, replacement of Raf-1 S499 with alanine resulted in a protein which was super inducible by PMA. This is consistent with a recently published report suggesting that PKCa phosphorylation of S499 may decrease activity of Raf-1 (Schonwasser et al, 1998). These results suggest that induction of Raf-1 activity by either Ras or Src does not require phosphorylation of the activation loop.…”

“…PKCa, PKCZ and PKCe have been reported as being Raf activating kinases (Cai et al, 1997;Schonwasser et al, 1998). PKCa has been reported to activate Raf-1 via phosphorylation of serine 499 in the activation loop, although this mechanism has been disputed (Dent et al, 1995b;MacDonald et al, 1993;Schonwasser et al, 1998).…”

“…PKCa has been reported to activate Raf-1 via phosphorylation of serine 499 in the activation loop, although this mechanism has been disputed (Dent et al, 1995b;MacDonald et al, 1993;Schonwasser et al, 1998). PKC-dependent phosphorylation of serine 497 has also been detected in hematopoetic cells in response to IL-3 (Carroll and May, 1994).…”

“…Phorbol ester-dependent activation of Raf-1 has been reported as being both Ras-dependent and Ras-independent (Arai and Escobedo, 1996;Burgering and Bos, 1995;Cai et al, 1993Cai et al, , 1997Ueda et al, 1996;Zou et al, 1996). One hypothesis is that certain PKC isoforms function as Raf kinase kinases, which together with Ras-GTP, promotes activation of Raf-1 (Burgering and Bos, 1995;Schonwasser et al, 1998). Additional support for an activation mechanism requiring discrete phosphorylation of the Raf-1 activation loop is provided by the observation that oligomerization of Raf-1 results in catalytic activation (Farrar et al, 1996;Luo et al, 1996).…”

The protein kinase C-η isoform induces proliferation in glioblastoma cell lines through an ERK/Elk-1 pathway

“…However, it is now known that members of all three classes of PKC isoforms can activate extracellular signal-regulated kinase (ERK) and MAPK/ERK kinase-1 (MEK-1) (Schonwasser et al, 1998b). This latter pathway is of particular interest in this study, in that the ERK signaltransduction pathway is activated by EGFR.…”