Rapid and Long Term Effects of Protein Kinase C on Receptor Tyrosine Kinase Phosphorylation and Degradation

doi:10.1074/jbc.270.32.18953

Rapid and Long Term Effects of Protein Kinase C on Receptor Tyrosine Kinase Phosphorylation and Degradation (*)

From theDepartment of Molecular Biology, Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, 82152 Martinsried, Germany

* To whom all correspondence should be addressed. Tel.: 49-89-8578-2513; Fax: 49-89-857-7866.

↵^§ Present address: Hagedorn Research Institute, Niels Steensens Vej 6, 2820 Gentofte, Denmark.
↵^¶ Present address: Merck Sharpe & Dohme Research Laboratories, Neuroscience Research Centre, Terlings Park, Harlow, Essex CM20 2QR United Kingdom.

Abstract

Rapid and long term effects of protein kinase Cα activation on receptor tyrosine kinase signaling parameters were investigated in human 293 embryonic fibroblasts and mouse NIH 3T3 cells. Within minutes of phorbol 12-myristate 13-acetate treatment, epidermal growth factor receptor and HER2 tyrosine phosphorylation was decreased, while platelet-derived growth factor receptor and insulin receptor autophosphorylation was up-regulated. These effects are not mediated by protein kinase C-dependent receptor tyrosine kinase phosphorylation but apparently by activation or inactivation of receptor tyrosine kinase-specific phosphatases, as indicated by neutralization of these phenomena upon treatment of cells with sodium orthovanadate. In contrast to these short term effects, sustained activation of protein kinase Cα by phorbol 12-myristate 13-acetate results in translocation of protein kinase C from the cytosol to the membrane fraction where it forms stable complexes with all receptor tyrosine kinases investigated. Ligand-induced receptor tyrosine kinase/protein kinase C association in NIH 3T3 fibroblasts is accompanied by a mobility shift of the receptor, indicating phosphorylation by activated protein kinase C. This phenomenon correlates with the disappearance of receptor tyrosine kinases from the cell surface, implying that this interaction plays a role in the process of receptor internalization and degradation. Interestingly, ligand-stimulated receptor down-regulation is also enhanced by overexpression of phospholipase C, which strongly indicates a role for this common receptor tyrosine kinase substrate in negative regulation of growth factor signals.

Footnotes

↵* This work was supported by a grant from Sugen, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

RTK

receptor tyrosine kinase

PLC

phospholipase C

GAP

GTPase-activating protein

PI 3′-kinase

phosphatidylinositol 3′-kinase

PKC

protein kinase C

PMA

phorbol 12-myristate 13-acetate

DAG

diacylglycerol

PAGE

polyacrylamide gel electrophoresis

EGF

epidermal growth factor

EGF-R

epidermal growth factor receptor

PDGF

platelet-derived growth factor

PDGF-R

platelet-derived growth factor receptor

mAb

monoclonal antibodies

DMEM

Dulbecco's modified Eagle's medium

CMV

cytomegalovirus

PIP

phosphatidylinositol monophosphate

BES

2-[bis(2-hydroxyethyl)amino]ethanesulfonic acid.
↵²K. Seedorf, M. Shearman, and A. Ullrich, unpublished results.
↵³K. Seedorf, M. Shearman, and A. Ullrich, unpublished observation.
- Received November 21, 1994.
- Revision received May 10, 1995.