Constitutive Activation of Phosphatidylinositol 3-Kinase by a Naturally Occurring Mutant Epidermal Growth Factor Receptor

doi:10.1074/jbc.273.1.200

Constitutive Activation of Phosphatidylinositol 3-Kinase by a Naturally Occurring Mutant Epidermal Growth Factor Receptor*

From the Departments of ^‡Microbiology and Immunology and ^§Biochemistry and Molecular Pharmacology, Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, Pennsylvania 19107 and the ^¶Division of Oncology, Seattle Veterans Administration Medical Center, Seattle, Washington 98108

Abstract

The most frequently found alteration of the epidermal growth factor receptor (EGFR) in human tumors is a deletion of exons 2–7. This receptor, termed EGFRvIII, can transform NIH 3T3 cells, and the frequent expression of this variant implies that it confers a selective advantage upon tumor cells in vivo. Although EGFRvIII is a constitutively activated tyrosine kinase, there is no increase in Ras·GTP levels and low levels of mitogen-activated protein kinase activity in NIH 3T3 cells expressing this variant. We investigated whether phosphatidylinositol (PI) 3-kinase was an effector in transformation by the EGFRvIII. High levels of PI 3-kinase activity were constitutively present in EGFRvIII-transformed cells and were dependent upon the kinase activity of the receptor. While mitogen-activated protein kinase activity was quickly down-regulated to basal levels after 12 h of continuous EGFR activation, there was a 3-fold increase in PI 3-kinase activity in cells expressing normal EGFR and an 8-fold increase in cells expressing EGFRvIII after 48 h. This increased activity may reflect enhanced binding to EGFRvIII and the presence of novel PI 3-kinase isoforms. Treatment with the PI 3-kinase inhibitors wortmannin and LY294002 blocked both anchorage-independent growth and growth in low serum media and also resulted in morphological reversion of EGFRvIII-transformed cells. These results support an essential role for PI 3-kinase in transformation by this EGFR variant.

Footnotes

↵* This work was supported by National Institutes of Health Grants CA 69495 and NS34514 (to A. J. W. and M. H. M), a grant from the American Cancer Society (to A. J. W.), and National Institutes of Health Training Grant 5-T32-CA09678–03 (to D. K. M.).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.: 215-503-4650; Fax: 215-923-4498; E-mail: a wong{at}lac.jci.tju.edu.
↵1 The abbreviations used are: EGFR, epidermal growth factor receptor; CS, calf serum; DMEM, Dulbecco’s modified Eagle’s medium; EGFRvIII, type III EGF receptor variant; GST, glutathione S-transferase; MAP, mitogen-activated protein; PI, phosphatidylinositol; PDGF, platelet-derived growth factor; SH2, Src homology region 2; pTyr, phosphotyrosine.
- Received April 3, 1997.
- Revision received September 18, 1997.
The American Society for Biochemistry and Molecular Biology, Inc.