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.
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.
Mesh Terms:
Binding Sites, Cell Line, Cytoplasm, DNA Primers, Enzyme Activation, Humans, Intracellular Signaling Peptides and Proteins, Kinetics, Mutagenesis, Site-Directed, Peptide Fragments, Phenylalanine, Phosphopeptides, Phosphotyrosine, Platelet Endothelial Cell Adhesion Molecule-1, Point Mutation, Protein Tyrosine Phosphatase, Non-Receptor Type 11, Protein Tyrosine Phosphatase, Non-Receptor Type 6, Protein Tyrosine Phosphatases, Recombinant Proteins, SH2 Domain-Containing Protein Tyrosine Phosphatases, Transfection, src Homology Domains
Binding Sites, Cell Line, Cytoplasm, DNA Primers, Enzyme Activation, Humans, Intracellular Signaling Peptides and Proteins, Kinetics, Mutagenesis, Site-Directed, Peptide Fragments, Phenylalanine, Phosphopeptides, Phosphotyrosine, Platelet Endothelial Cell Adhesion Molecule-1, Point Mutation, Protein Tyrosine Phosphatase, Non-Receptor Type 11, Protein Tyrosine Phosphatase, Non-Receptor Type 6, Protein Tyrosine Phosphatases, Recombinant Proteins, SH2 Domain-Containing Protein Tyrosine Phosphatases, Transfection, src Homology Domains
J. Biol. Chem.
Date: Oct. 03, 1997
PubMed ID: 9312087
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