Platelet Endothelial Cell Adhesion Molecule-1 Is Phosphorylatable by c-Src, Binds Src-Src homology 2 Domain, and Exhibits Immunoreceptor Tyrosine-based Activation Motif-like Properties*
Abstract
Platelet endothelial cell adhesion molecule-1 (PECAM-1) is 130-kDa member of the immunoglobulin gene superfamily that localizes to cell-cell borders of confluent endothelial cells and has been shown to play a role in the control of endothelial sheet migration and leukocyte transmigration through the endothelium. The cytoplasmic tail plays an important role in the modulation of PECAM-1 function. Mutation of tyrosine 663 or 686 in the cytoplasmic tail reduces phosphorylation and mutation of 686 is associated with a reduction in PECAM-1-mediated inhibition of cell migration (1). We have previously noted that these two tyrosine residues are surrounded by consensus sequences for Src homology 2 (SH2) domain binding (1, 2), and the experiments presented explore the potential for PECAM-1-Src and PECAM-1-SH2 domain interactions. PECAM-1 is more highly phosphorylated in endothelial cells overexpressing c-Src, and inin vitro kinase assays, c-Src can phosphorylate a glutathione S-transferase (GST)-PECAM cytoplasmic tail fusion protein. The phosphorylated fusion protein associates with the bead-bound c-Src. This association appears to be mediated by Src-SH2 domain, because PECAM-1 can be precipitated by a GST-Src-SH2 affinity matrix. The binding to the GST-Src-SH2 affinity matrix correlates directly with the level of PECAM-1 phosphorylation, because more PECAM-1 is precipitated from c-Src overexpressors and from wild-type rather than Tyr663 → Phe and Tyr686 → Phe mutant PECAM-1 expressors. Yet unidentified phosphoproteins can also be coimmunoprecipitated with wild-type but not mutant PECAM-1. Finally, we note the similarity of the PECAM-1 cytoplasmic domain sequence to the immunoreceptor tyrosine-based activation motif. Our data begin to delineate how tyrosines 663 and 686 may play a role in mediating PECAM-1 signal transduction.
Footnotes
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↵* This work was supported in part by U. S. Public Health Service Grants RO1-HL-28373, PO1-KD-38979, and RO1-HL-51018 and a gift from Genzyme, Inc. (to J. A. 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.
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↵§ Partially supported by a Medical Scientist Training Program training grant awarded to Yale University School of Medicine. Present address: Dept. of Pediatrics, Children’s Hospital of Philadephia, Philadelphia, PA 19104.
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↵¶ Partially supported by an Experimental Pathology Training Grant TE-DK-07556 awarded to Yale University School of Medicine.
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↵** To whom correspondence should be addressed: Dept. of Pathology, Yale University School of Medicine, 310 Cedar St., New Haven, CT 06510. Tel.: 203-785-2763; Fax: 203-785-7303.
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↵1 The abbreviations used are: PECAM-1, platelet endothelial cell adhesion molecule-1; BAEC, bovine aortic endothelial cell; GST, glutathione S-transferase; HUVEC, human umbilical vein endothelial cell; ITAM, immunoreceptor tyrosine-based activation motif; SH2, Src homology 2; PECAM-1cyto, recombinant cytoplasmic domain of PECAM-1; PAGE, polyacrylamide gel electrophoresis; DFP, diisopropylphosphofluoridate.
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↵2 M. Barreuther, T. Lu, S. Davis, and J. A. Madri, unpublished observations.
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- Received February 25, 1997.
- Revision received April 9, 1997.
