The Urokinase-type Plasminogen Activator Receptor Mediates Tyrosine Phosphorylation of Focal Adhesion Proteins and Activation of Mitogen-activated Protein Kinase in Cultured Endothelial Cells

doi:10.1074/jbc.273.29.18268

The Urokinase-type Plasminogen Activator Receptor Mediates Tyrosine Phosphorylation of Focal Adhesion Proteins and Activation of Mitogen-activated Protein Kinase in Cultured Endothelial Cells*

From the Departments of ^¶Medicine,^‡Pharmacology, and ^‖Biochemistry, Vanderbilt University School of Medicine and Nashville Veterans Affairs Medical Center, Nashville, Tennessee 37232

Abstract

Urokinase-type plasminogen activator (uPA) binds to cells via a specific glycosylphosphatidylinositol-anchored receptor. Although occupancy of the uPA receptor (uPAR) has been shown to alter cellular function and to induce gene expression, the signaling mechanism has not been characterized. Urokinase induced an increase in the tyrosine phosphorylation of multiple proteins in bovine aortic endothelial cells. In contrast, low molecular weight uPA did not induce this response. Analysis by immunoblotting demonstrated tyrosine phosphorylation of focal adhesion kinase (FAK), the focal adhesion-associated proteins paxillin and p130^cas ,and mitogen-activated protein kinase (MAPK) following the occupancy of the uPAR by uPA. Treatment of cells with phosphatidylinositol-specific phospholipase C, which cleaves glycosylphosphatidylinositol-linked proteins from the cell surface, blocked the uPA-induced tyrosine phosphorylation of FAK, indicating the requirement of an intact uPAR on the cell surface. The uPA-induced activation of MAPK was completely inhibited by genistein, but not by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, a specific inhibitor of Src family kinases. Thus, this study demonstrates a novel role for the uPAR in endothelial cell signal transduction that involves the activation of FAK and MAPK, which are mediated by the receptor-binding domain of uPA. This may have important implications for the mechanism through which uPA influences cell migration and differentiation.

Footnotes

↵* This work was supported in part by a New Investigator Award from the Tennessee Affiliate of the American Heart Association (to D. M. K.), by National Institutes of Health Grants HL14192 and HL35323 (to T. I.) and Grants HL51387 and HL50878 (to D. E. V.), and by a Merit Award from the Department of Veterans Affairs Research Service (to D. E. V.).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.
↵§ These authors contributed equally to this study.
↵** Recipient of a Clinical Investigator Award from the Department of Veterans Affairs Research Service. To whom correspondence should be addressed: Div. of Cardiology, Vanderbilt University Medical Center, Rm. 315 MRBII, Nashville, TN 37232. Tel.: 615-936-1720; Fax: 615-936-1872; E-mail: doug.vaughan{at}mcmail.vanderbilt.edu.
↵1 The abbreviations used are: uPA, urokinase-type plasminogen activator; uPAR, uPA receptor; BAE, bovine aortic endothelial; GPI, glycosylphosphatidylinositol; ATF, amino-terminal fragment of uPA; FAK, focal adhesion kinase; MAPK, mitogen-activated protein kinase; mAb, monoclonal antibody; PP₁, 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine; PBS, phosphate-buffered saline.
- Received April 16, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.