Regulation of Mouse PECAM-1 Tyrosine Phosphorylation by the Src and Csk Families of Protein-tyrosine Kinases

doi:10.1074/jbc.273.25.15765

Regulation of Mouse PECAM-1 Tyrosine Phosphorylation by the Src and Csk Families of Protein-tyrosine Kinases*

From the ^tOaMcGill Cancer Centre and the Departments of ^tOdBiochemistry, ^tOeMedicine and ^tOfOncology, McGill University, Montréal, Québec H3G 1Y6, Canada, the Departments of ⁱMedicine and Oncology, Montreal General Hospital, Montréal, Québec H3G 1A4, Canada, and the ^tOhDepartment of Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19014-4283

Abstract

PECAM-1 is an adhesion molecule expressed on hemopoietic and endothelial cells. Recently, it was observed that PECAM-1 becomes tyrosine-phosphorylated in response to a variety of physiological stimuli. Furthermore, tyrosine-phosphorylated PECAM-1 was shown to associate with SHP-2, a Src homology 2 (SH2) domain-containing protein-tyrosine phosphatase expressed ubiquitously. In light of the significance of tyrosine protein phosphorylation as a regulatory mechanism, we wished to understand better the nature and impact of the protein-tyrosine kinases (PTKs) mediating PECAM-1 tyrosine phosphorylation. Through reconstitution experiments in COS-1 cells, we determined that mouse PECAM-1 could be tyrosine-phosphorylated by Src-related PTKs and Csk-related PTKs, but not by other kinases such as Syk, Itk, and Pyk2. Using site-directed mutagenesis and peptide phosphorylation studies, we found that these PTKs were efficient at phosphorylating Tyr-686, but not Tyr-663, of PECAM-1. Src-related enzymes also phosphorylated mouse PECAM-1 at one or more yet to be identified sites. In other studies, we demonstrated that phosphorylation of PECAM-1 by Src or Csk family kinases was sufficient to trigger its association with SHP-2. Moreover, it was able to promote binding of PECAM-1 to SHP-1, a SHP-2-related protein-tyrosine phosphatase expressed in hemopoietic cells. Taken together, these findings indicated that the Src and Csk families of kinases are strong candidates for mediating tyrosine phosphorylation of PECAM-1 and triggering its association with SH2 domain-containing phosphatases under physiological circumstances.

Footnotes

↵* This work was supported in part by grants from the National Cancer Institute of Canada (to A. V.), the Medical Research Council of Canada (to N. B. and A. V.), and National Institutes of Health Grant HL-46311 (to S. M. A.).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.
↵FNb Holds a Joseph Kaufmann Fellowship.
↵FNc Supported by a Fellowship from the Medical Research Council of Canada.
↵FNg Senior Scientist of the “Fonds de la Recherche en Santé du Québec.”
↵FNj Scientist of the Medical Research Council of Canada. To whom correspondence should be addressed: Rm. 715, McIntyre Medical Sciences Bld., McGill University, 3655 Drummond St., Montréal, Québec H3G 1Y6, Canada. Tel.: 514-398-8936; Fax: 514-398-4438; E-mail: VEILLETTE{at}MEDCOR.MCGILL.CA.
↵1 The abbreviations used are: PECAM-1, platelet endothelial cell adhesion molecule-1; PTP, protein-tyrosine phosphatase; PTK, protein-tyrosine kinase; SH2, Src homology domain 2; GST, glutathione S-transferase; mAb, monoclonal antibody; Tricine,N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
↵2 F. Gervais and A. Veillette, unpublished data.
↵3 A. Veillette and N. Beauchemin, unpublished data.
↵4 M. Huber, A. Veillette, and N. Beauchemin, manuscript in preparation.
↵5 A. Veillette, unpublished results.
↵6 M. Fournel and A. Veillette, unpublished results.
- Received December 30, 1997.
- Revision received April 8, 1998.
The American Society for Biochemistry and Molecular Biology, Inc.