Vascular Endothelial Cell Growth Factor Promotes Tyrosine Phosphorylation of Mediators of Signal Transduction That Contain SH2 Domains

doi:10.1074/jbc.270.12.6729

Vascular Endothelial Cell Growth Factor Promotes Tyrosine Phosphorylation of Mediators of Signal Transduction That Contain SH2 Domains

ASSOCIATION WITH ENDOTHELIAL CELL PROLIFERATION (*)

From the (1)Department of Physiology and Biophysics and the Walther Oncology Center, Indiana University School of Medicine, Indianapolis, Indiana 46202 and the
(2) Department of Surgery, University of California, San Francisco, California 94143

** To whom correspondence and reprint requests should be addressed:
Dept. of Physiology and Biophysics and The Walther Oncology Center, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202.
Tel.: 317-278-2155; Fax: 317-274-3318.

Abstract

Vascular endothelial cell growth factor (VEGF), an endothelial cell-specific mitogen that plays an important role in angiogenesis, promotes the tyrosine phosphorylation of at least 11 proteins in bovine aortic endothelial cells (BAEC). Proteins immunoprecipitated from lysates of control- and VEGF-stimulated BAEC with antisera to phospholipase C- (PLC-) were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P. Evaluation of the Western blots with antisera to phosphotyrosine demonstrated that PLC- and two proteins (100 and 85 kDa) that associate with PLC- were phosphorylated in response to VEGF. By using antisera specific to other mediators of signal transduction that contain SH2 domains for immunoprecipitation, it was demonstrated that VEGF promotes phosphorylation of phosphatidylinositol 3-kinase, Ras GTPase activating protein (GAP), and the oncogenic adaptor protein NcK. Proteins of M_r consistent with the VEGF receptors Flt-1 and Flk-1/KDR were also tyrosine phosphorylated in stimulated cells. Tyrosine-phosphorylated Nck, PLC-, and two GAP-associated proteins, p190 and p62, were in GAP immunoprecipitates of VEGF-stimulated BAEC, and tyrosine-phosphorylated NcK was in phosphatidylinositol 3-kinase immunoprecipitates. These observations suggest that VEGF promotes formation of multimeric aggregates of VEGF receptors with proteins that contain SH2 domains and activate various signaling pathways. VEGF-promoted proliferation of endothelial cells and tyrosine phosphorylation of SH2 domain containing signaling molecules were inhibited by the tyrosine kinase inhibitor genistein.

Footnotes

↵^§ Supported by a predoctoral fellowship from the Indiana affiliate of the American Heart Association.
↵^¶ Supported by a postdoctoral fellowship from the Indiana affiliate of the American Heart Association.
↵* This work was supported by a grant from the Indiana affiliate of the American Diabetes Association (to D. B. D.). 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:

VEGF

vascular endothelial cell growth factor

BAEC

bovine aortic endothelial cells

PLC-

phospholipase C-

PI-3 kinase

phosphatidylinositol 3-kinase

GAP

GTPase activating protein

PTyr Ab

antisera to phosphotyrosine

PAGE

polyacrylamide gel electrophoresis

PDGF

platelet-derived growth factor

EGF

epidermal growth factor

PAEC

porcine aortic endothelial cells.
↵²D. Guo and D. B. Donner, unpublished observations.
- Received September 27, 1994.
- Revision received December 20, 1994.