Protein Kinase C Regulates Integrin-induced Activation of the Extracellular Regulated Kinase Pathway Upstream of Shc

doi:10.1074/jbc.274.15.10571

Protein Kinase C Regulates Integrin-induced Activation of the Extracellular Regulated Kinase Pathway Upstream of Shc*

From the ^‡Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115 and the ^§Department of Molecular Biology, Yokohama City University School of Medicine, 3-9, Fuku-ura, Kanazawa-ku, Yokohama 236, Japan

Abstract

Adhesion of fibroblasts to extracellular matrices via integrin receptors is accompanied by extensive cytoskeletal rearrangements and intracellular signaling events. The protein kinase C (PKC) family of serine/threonine kinases has been implicated in several integrin-mediated events including focal adhesion formation, cell spreading, cell migration, and cytoskeletal rearrangements. However, the mechanism by which PKC regulates integrin function is not known. To characterize the role of PKC family kinases in mediating integrin-induced signaling, we monitored the effects of PKC inhibition on fibronectin-induced signaling events in Cos7 cells using pharmacological and genetic approaches. We found that inhibition of classical and novel isoforms of PKC by down-regulation with 12-0-tetradeconoyl-phorbol-13-acetate or overexpression of dominant-negative mutants of PKC significantly reduced extracellular regulated kinase 2 (Erk2) activation by fibronectin receptors in Cos7 cells. Furthermore, overexpression of constitutively active PKCα, PKCδ, or PKCε was sufficient to rescue 12-0-tetradeconoyl-phorbol-13-acetate-mediated down-regulation of Erk2 activation, and all three of these PKC isoforms were activated following adhesion. PKC was required for maximal activation of mitogen-activated kinase kinase 1, Raf-1, and Ras, tyrosine phosphorylation of Shc, and Shc association with Grb2. PKC inhibition does not appear to have a generalized effect on integrin signaling, because it does not block integrin-induced focal adhesion kinase or paxillin tyrosine phosphorylation. These results indicate that PKC activity enhances Erk2 activation in response to fibronectin by stimulating the Erk/mitogen-activated protein kinase pathway at an early step upstream of Shc.

Footnotes

↵* This work was funded by grants CA 78773, CA27951 (J.S.B.) and CA72203 (C.K.M.) from the National Cancer Institute.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ To whom correspondence should be addressed. Tel.: 617-432-3974; Fax: 617-432-3969; E-mail:Joan_Brugge{at}hms.harvard.edu.
↵2 W. King, unpublished results.
↵3 C. Miranti, unpublished results.
Abbreviations:

MAPK

mitogen-activated protein kinase

PKC

protein kinase C

TPA

12-0-tetradecanoyl-phorbol-13-acetate

Erk2

extracellular regulated kinase 2

MEK

mitogen-activated kinase kinase

FAK

focal adhesion kinase

PI 3-K

phosphatidylinositol 3-kinase

PLC

phospholipase C

EGF

epidermal growth factor

PTP

phosphotyrosine phosphatase

PBS

phosphate-buffered saline

DMEM

Dulbecco’s modified Eagle’s medium

RBD

Ras-binding domain

HA

hemagglutinin

IP

immunoprecipitation(s)

GAP

GTPase-activating protein
- Received November 10, 1998.
- Revision received January 20, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.