Interaction of the Flt-1 Tyrosine Kinase Receptor with the p85 Subunit of Phosphatidylinositol 3-Kinase

doi:10.1074/jbc.270.35.20254

Interaction of the Flt-1 Tyrosine Kinase Receptor with the p85 Subunit of Phosphatidylinositol 3-Kinase

MAPPING OF A NOVEL SITE INVOLVED IN BINDING (*)

From the (1) Department of Pharmacology, Texas Biotechnology Corporation, Houston, Texas 77030 and the Departments of
(2) Physiology and Cell Biology and
(3) Neurobiology and Anatomy, University of Texas Health Science Center, Houston, Texas 77225

^§ To whom correspondence should be addressed:
Texas Biotechnology, Suite 1920, 7000 Fannin, Houston, TX 77030.

Abstract

We have examined the interactions of the p85 regulatory subunit of phosphatidylinositol 3-kinase with the endothelium-specific Flt-1 receptor tyrosine kinase using the yeast two-hybrid system. We find that both the amino- and carboxyl-terminal SH2 domains of p85 bind to Flt-1. We have performed site-directed mutagenesis on the carboxyl-terminal tail of the Flt-1 receptor in order to identify the site(s) that is responsible for the p85 interactions. A single tyrosine to phenylalanine change at position 1213 inhibits the binding of both p85 SH2 domains. Phosphopeptide mapping of the wild type and mutant protein expressed in insect cells verifies that this amino acid is a target for autophosphorylation. The amino acids following this tyrosine are VNA and thus define a novel binding site for p85.

Footnotes

↵* 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 growth factor

PDGF

platelet-derived growth factor

HGFR

hepatocyte growth factor receptor

wt

wild type

SH2

Src homology 2

PLC

phospholipase C

PI 3-kinase

phosphatidylinositol 3-kinase

PtdIns

phosphatidylinositol

PCR

polymerase chain reaction

HPLC

high performance liquid chromatography.
↵² S. A. Cunningham, P. Arrate, T. A. Brock, and M. Neal Waxham, manuscript in preparation.
- Received May 10, 1995.
- Revision received June 12, 1995.