Characterization of Phosphotyrosine Binding Motifs in the Cytoplasmic Domain of Platelet/Endothelial Cell Adhesion Molecule-1 (PECAM-1) That Are Required for the Cellular Association and Activation of the Protein-tyrosine Phosphatase, SHP-2*
- From the ‡Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee, Wisconsin 53233 and the§Departments of Cellular Biology and Pharmacology, Medical College of Wisconsin, Milwaukee, Wisconsin 53266
Abstract
Recent studies have shown that the Src homology-2 (SH2) domain-containing protein-tyrosine phosphatase, SHP-2, associates with the cytoplasmic domain of PECAM-1 as it becomes tyrosine-phosphorylated during platelet aggregation: a process that can be mimicked in part by small synthetic phosphopeptides corresponding to the cytoplasmic domain of PECAM-1 encompassing tyrosine residues Tyr-663 or Tyr-686. To further examine the molecular requirements for PECAM-1/SHP-2 interactions, we generated human embryonic kidney (HEK)-293 cell lines that stably expressed mutant forms of PECAM-1 harboring tyrosine to phenylalanine (Tyr → Phe) mutations in the cytoplasmic domain. Y663F and Y686F forms of PECAM-1 were tyrosine-phosphorylated to a somewhat lesser extent than wild-type PECAM-1, and a doubly substituted Y663,686F form of PECAM-1 failed to become tyrosine-phosphorylated, suggesting that the PECAM-1 cytoplasmic domain tyrosine residues 596, 636 and 701 do not serve as substrates for cellular kinases. Interestingly, SHP-2 binding was lost when either Tyr-663 or Tyr-686 were changed to phenylalanine, indicating that both residues are required for SHP-2/PECAM-1 association. Although PECAM-1 phosphopeptides NSDVQpY663TEVQV and DTETVpY686SEVRK stimulated the catalytic activity of the phosphatase to a similar extent, surface plasmon resonance studies revealed that the Tyr-663-containing peptide had approximately 10-fold higher affinity for SHP-2 than did the Tyr-686 peptide. Finally, peptido-precipitation analysis showed that the NH2-terminal SH2 domain of SHP-2 reacted preferentially with the Tyr-663 PECAM-1 phosphopeptide, while the Tyr-686 phosphopeptide associated only with the COOH-terminal SH2 domain of the phosphatase. Together, these data provide a molecular model for PECAM-1/SHP-2 interactions that may shed light on the downstream events that follow PECAM-1-mediated interactions of vascular cells.
Footnotes
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↵* This work was supported by Program Project Grant HL-44612 (to P. J. N.) from the National Institutes of Health, American Heart Association (Wisconsin Affiliate) Postdoctoral Fellowship Award 96F-Post-34 (to D. E. J.), and Established Investigatorship 92001390 of the American Heart Association (P. J. N.).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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↵¶ To whom correspondence should be addressed: Blood Research Inst., The Blood Center of Southeastern Wisconsin, 638 N. 18th St., Milwaukee, WI 53233-2194. Tel.: 414-937-6237; Fax: (414) 937-6284 E-mail: pjn{at}smtpgate.bcsew.edu.
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↵1 The abbreviations used are: SH2, Src homology 2; PAGE, polyacrylamide gel electrophoresis; RCM, reduced carboxyamidomethylated and maleylated; PECAM-1, platelet endothelial cell adhesion molecule-1; IRS-1, insulin receptor substrate-1; BIT, brain immunoglobulin-like molecule; pY or Tyr(P), phosphotyrosine; TAM, tyrosine-based activation motif; N-SH2, NH2-terminal SH2 domain; C-SH2, COOH-terminal SH2 domain; GST, glutathioneS-transferase; PDGF, platelet-derived growth factor.
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- Received June 25, 1997.
