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

• Published in: The Journal of biological chemistry Vol. 272; no. 40; pp. 24868 - 24875
• Main Authors: Jackson, Denise E., Kupcho, Kevin R., Newman, Peter. J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 03.10.1997; American Society for Biochemistry and Molecular Biology
• Subjects: Binding Sites; Cell Line; Cytoplasm - metabolism; DNA Primers; Enzyme Activation; Humans; Intracellular Signaling Peptides and Proteins; Kinetics; Mutagenesis, Site-Directed; Peptide Fragments - chemistry; Phenylalanine; Phosphopeptides - chemistry; Phosphotyrosine; Platelet Endothelial Cell Adhesion Molecule-1 - chemistry; Platelet Endothelial Cell Adhesion Molecule-1 - metabolism; Point Mutation; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases - chemistry; Protein Tyrosine Phosphatases - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; SH2 Domain-Containing Protein Tyrosine Phosphatases; src Homology Domains; Transfection
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.272.40.24868

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.