Protein Kinase C, but Not Tyrosine Kinases or Ras, Plays a Critical Role in Angiotensin II-induced Activation of Raf-1 Kinase and Extracellular Signal-regulated Protein Kinases in Cardiac Myocytes

doi:10.1074/jbc.271.52.33592

Protein Kinase C, but Not Tyrosine Kinases or Ras, Plays a Critical Role in Angiotensin II-induced Activation of Raf-1 Kinase and Extracellular Signal-regulated Protein Kinases in Cardiac Myocytes*

From the ^‡ Department of Medicine III, University of Tokyo School of Medicine and the
^¶ Health Service Center, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan

^§ To whom correspondence should be addressed. Tel.: 81-3-3815-5411 (ext. 3127); Fax: 81-3-3815-2087; E-mail: komuro-tky{at}umin.u-tokyo.ac.jp

Abstract

Angiotensin II (AngII) induces cardiac hypertrophy through activating a variety of protein kinases. In this study, to understand how cardiac hypertrophy develops, we examined AngII-evoked signal transduction pathways leading to the activation of extracellular signal-regulated protein kinases (ERKs), which are reportedly critical for the development of cardiac hypertrophy, in cultured cardiac myocytes isolated from neonatal rats. Inhibition of protein kinase C (PKC) with calphostin C or down-regulation of PKC by pretreatment with a phorbol ester for 24 h abolished AngII-induced activation of Raf-1 and ERKs, and addition of a phorbol ester conversely induced a marked increase in the activities of Raf-1 and ERKs. Pretreatment with two chemically and mechanistically dissimilar tyrosine kinase inhibitors, genistein and tyrphostin, did not attenuate AngII-induced activation of ERKs. In contrast, genistein strongly blocked insulin-induced ERK activation in cardiac myocytes. Although pretreatment with manumycin, a Ras farnesyltransferase inhibitor, or overexpression of a dominant-negative mutant of Ras inhibited insulin-induced ERK activation, neither affected AngII-induced activation of ERKs. Overexpression of a dominant-negative mutant of Raf-1 completely suppressed ERK2 activation by AngII, endothelin-1, and insulin. These results suggest that PKC and Raf-1, but not tyrosine kinases or Ras, are critical for AngII-induced activation of ERKs in cardiac myocytes.

Footnotes

↵* This work was supported by a grant-in-aid for scientific research, developmental scientific research, and scientific research on priority areas from the Ministry of Education, Science, Sports, and Culture of Japan and by grants from the Japan Cardiovascular Foundation and the Sankyo Life Science and Mochida Memorial Foundation for Medical and Pharmaceutical Research, Japan (to I. K.). 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.
↵¹ The abbreviations used are:

AngII

angiotensin II

ET-1

endothelin-1

PHE

phenylephrine

G protein

guanine nucleotide-binding protein

PKC

protein kinase C

ERK

extracellular signal-regulated protein kinase

Raf-1

Raf-1 kinase

MEK

mitogen-activated protein kinase/ERK kinase

HA

hemagglutinin

TPA

12-O-tetradecanoylphorbol-13-acetate

MBP

myelin basic protein

D.N.Ras

dominant negative mutant of Ras

D.N.Raf-1

dominant-negative mutant of Raf-1 kinase
↵² Y. Zou, I. Komuro, T. Yamazaki, R. Aikawa, S. Kudoh, I. Shiojima, Y. Hiroi, T. Mizuno, and Y. Yazaki, manuscript in preparation.
↵³ Y. Zou, I. Komuro, T. Yamazaki, R. Aikawa, S. Kudoh, I. Shiojima, Y. Hiroi, T. Mizuno, and Y. Yazaki, unpublished observation.
- Received August 2, 1996.
- Revision received September 19, 1996.