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Mol Cell Biol, February 1998, p. 790-798, Vol. 18, No. 2
Imperial Cancer Research Fund,
Received 24 December 1996/Returned for modification 17 March
1997/Accepted 28 October 1997
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/mitogen-activated protein
kinase (ERK/MAPK) cascade. It is shown that dominant-negative PKC-
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Activation of the Mitogen-Activated Protein Kinase/Extracellular
Signal-Regulated Kinase Pathway by Conventional, Novel, and
Atypical Protein Kinase C Isotypes
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-O-tetradecanoylphorbol-13-acetate induced mitogenesis
and provide evidence for multiple PKC controls acting on this MAPK
cascade.
*
Corresponding author. Mailing address: Imperial Cancer
Research Fund, P.O. Box 123, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom. Phone: 171-269 3388. Fax: 171-269 3092. E-mail: parkerp{at}icrf.icnet.uk.
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