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
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
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DOI：
10.1074/jbc.272.40.24868
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发表时间：
1997-10-03
影响因子：
4.8
通讯作者：
Newman, PJ
中科院分区：
文献类型：
--
作者：
Jackson, DE;Kupcho, KR;Newman, PJ
最近的研究表明，Src同源性-2（SH 2）结构域含有蛋白酪氨酸磷酸酶，SHP-2，与PECAM-1的胞质结构域相关，因为它在血小板聚集过程中变得酪氨酸磷酸化：这一过程可以部分地通过与PECAM-1的胞质结构域对应的小合成磷酸肽模拟，包括酪氨酸残基Tyr-663或Tyr-686。为了进一步研究PECAM-1/SHP-2相互作用的分子要求，我们产生了稳定表达携带酪氨酸至苯丙氨酸的PECAM-1突变形式的人胚肾（HEK）-293细胞系胞质结构域中的（Tyr -> Phe）突变，Y 663 F和Y 686 F形式的PECAM-1的酪氨酸磷酸化程度略低于野生型PECAM-1，而双取代的Y 663，686 F形式的PECAM-1不能变成酪氨酸磷酸化的，这表明PECAM-1胞质结构域酪氨酸残基596，636和701不作为细胞激酶的底物。有趣的是，当Tyr-663或Tyr-686变为苯丙氨酸时，SHP-2结合丧失，表明这两个残基是SHP-2/PECAM-1结合所必需的。尽管PECAM-1磷酸肽NSDVQpY（663）TEVQV和DTETVpY（686）SEVRK以相似的程度刺激磷酸酶的催化活性，表面等离子体共振研究显示，含Tyr-663的肽对SHP-2的亲和力比Tyr-663的肽高约10倍。686肽。最后，肽沉淀分析显示SHP-2的NH 2-末端SH 2结构域优先与Tyr-663 PECAM-1磷酸肽反应，而Tyr-686磷酸肽仅与磷酸酶的COOH-末端SH 2结构域结合。这些数据提供了PECAM-1/SHP-2相互作用的分子模型，可以阐明PECAM-1-SHP-2的下游事件。介导的血管细胞的相互作用。
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 nob 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 NSDVQpY(663)TEVQV and DTETVpY(686)SEVRK 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.