Localization of Epidermal Growth Factor-stimulated Ras/Raf-1 Interaction to Caveolae Membrane

doi:10.1074/jbc.271.20.11930

Localization of Epidermal Growth Factor-stimulated Ras/Raf-1 Interaction to Caveolae Membrane (*)

From the (1)Departments of Cell Biology and
(2)Neuroscience and Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235

^¶ To whom correspondence should be addressed.

Abstract

An essential step in the epidermal growth factor (EGF)-dependent activation of MAP kinase is the recruitment of Raf-1 to the plasma membrane. Here we present evidence that caveolae are the membrane site where Raf-1 is recruited. Caveolae fractions prepared from normal Rat-1 cells grown in the absence of serum were highly enriched in both EGF receptors and Ras. Thirty seconds after EGF was added to these cells Raf-1 began to appear in caveolae but not in non-caveolae membrane fractions. The maximum concentration was reached at 3 min followed by a decline over the next 60 min. During this time EGF receptors disappeared from the caveolae fraction while the concentration of Ras remained constant. The Raf-1 in this fraction was able to phosphorylate MAP kinase kinase, whereas cytoplasmic Raf-1 in the same cell was inactive. Elevation of cellular cAMP blocked the recruitment of Raf-1 to caveolae. Overexpression of Ha-Ras caused the recruitment of Raf-1 to caveolae independently of EGF stimulation, and this was blocked by the farnesyltransferase inhibitor BZA-5B. Finally, prenylation appeared to be required for localization of Ras to caveolae.

Footnotes

↵^§ Supported by a postdoctoral fellowship from the Helen Hay Whitney Foundation.
↵* This work was supported by National Institutes of Health Grants HL 20948, GM 43169, and GM 15631 and the Perot Family Foundation. 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:

EGF

epidermal growth factor

DMEM

Dulbecco's modified Eagle's medium

mAb

monoclonal antibody

DMSO

dimethyl sulfoxide

DTT

dithiothreitol

Tricine

N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine

MAP

mitogen-activated protein.
- Received January 19, 1996.
- Revision received March 13, 1996.