Internalized Epidermal Growth Factor Receptors Participate in the Activation of p21 ras in Fibroblasts

doi:10.1074/jbc.274.48.34350

Internalized Epidermal Growth Factor Receptors Participate in the Activation of p21^ras in Fibroblasts*

From the ^‡Department of Chemical Engineering and the ^‡Division of Bioengineering & Environmental Health, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, the ^‖Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294, and the ^¶Department of Pathology, University of Utah, Salt Lake City, Utah 84132

Abstract

Regulated activation of the highly conserved Ras GTPase is a central event in the stimulation of cell proliferation, motility, and differentiation elicited by receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR). In fibroblasts, this involves formation and membrane localization of Shc·Grb2·Sos complexes, which increases the rate of Ras guanine nucleotide exchange. In order to control Ras-mediated cell responses, this activity is regulated by receptor down-regulation and a feedback loop involving the dual specificity kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK). We investigated the role of EGFR endocytosis in the regulation of Ras activation. Of fundamental interest is whether activated receptors in endosomes can participate in the stimulation of Ras guanine nucleotide exchange, because the constitutive membrane localization of Ras may affect its compartmentalization. By exploiting the differences in postendocytic signaling of two EGFR ligands, epidermal growth factor and transforming growth factor-α, we found that activated EGFR located at the cell surface and in internal compartments contribute equally to the membrane recruitment and tyrosine phosphorylation of Shc in NR6 fibroblasts expressing wild-type EGFR. Importantly, both the rate of Ras-specific guanine nucleotide exchange and the level of Ras-GTP were depressed to near basal values on the time scale of receptor trafficking. Using the selective MEK inhibitor PD098059, we were able to block the feedback desensitization pathway and maintain activation of Ras. Under these conditions, the generation of Ras-GTP was not significantly affected by the subcellular location of activated EGFR. In conjunction with our previous analysis of the phospholipase C pathway in the same cell line, this suggests a selective continuation of specific signaling activities and cessation of others upon receptor endocytosis.

Footnotes

↵* Financial support was provided by the National Science Foundation Biotechnology Program in the Division of Biological & Environmental Systems and NCI, National Institutes of Health.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.
↵§ Supported by graduate fellowships from the National Science Foundation and the Merck/Massachusetts Institute of Technology Collaboration.
↵** Current address: Dept. of Pathology, University of Pittsburgh, Pittsburgh, PA 15261.
↵§§ To whom correspondence should be addressed. Tel.: 617-252-1629; Fax: 617-258-0204; E-mail: lauffen@mit.edu.
Abbreviations:

EGFR

epidermal growth factor receptor

EGF

epidermal growth factor

TGFα

transforming growth factor-α

Grb2

growth factor receptor-binding protein 2

Sos

son of sevenless

GEF

guanine nucleotide exchange factor

MEK

mitogen-activated protein kinase and extracellular signal-regulated kinase kinase

PLC

phospholipase C

PIP₂

phosphatidylinositol (4,5)-bisphosphate

SH2

Src homology 2

Erk

extracellular signal-regulated kinase

MEM

minimum essential medium

PIPES

1,4-piperazinediethanesulfonic acid

WT

wild-type
- Received June 14, 1999.
- Revision received August 20, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.