T cell activation stimulates the association of enzymatically active tyrosine-phosphorylated ZAP-70 with the Crk adapter proteins
T cell activation stimulates the association of enzymatically active tyrosine-phosphorylated ZAP-70 with the Crk adapter proteins
复制标题
DOI：
10.1074/jbc.274.31.21519
复制
发表时间：
1999-07-30
影响因子：
4.8
通讯作者：
Isakov, N
中科院分区：
文献类型：
--
作者：
Gelkop, S;Isakov, N
T 细胞抗原受体的结合启动信号转导，涉及多个效应分子的酪氨酸磷酸化以及受体位点多分子复合物的形成。具有 SH2 和 SH3 蛋白-蛋白相互作用结构域的接头蛋白参与细胞激活诱导的信号复合物的组装。我们发现 Crk 接头蛋白与人 T 细胞系 Jurkat 中的 zeta 链相关蛋白 (ZAP-70) 酪氨酸激酶发生激活诱导的相互作用。将各种谷胱甘肽 S-转移酶融合蛋白与活化的 Jurkat 细胞裂解物一起孵育，导致 ZAP-70 与 Crk 选择性结合，但不与 Grb2 或 Nck、接头结合。 蛋白质。此外，酪氨酸磷酸化的ZAP-70与来自活化的Jurkat细胞裂解物的Crk进行免疫共沉淀，并且在活化的人外周血T细胞的裂解物中观察到ZAP-70与GST-Crk结合。两个分子之间的关联是通过直接物理相互作用介导的，并涉及 Crk-SH2 结构域和 ZAP-70 上含有磷酸酪氨酰的序列。这种关联需要完整的 Lck，被认为是 ZAP-70 的上游调节因子，因为它不能在激活的 JCaM1 细胞中发生，该细胞表达正常水平的 ZAP-70 但缺乏 Lck。最后，发现谷胱甘肽 S-转移酶-Crk 融合蛋白主要与膜上酪氨酸磷酸化 ZAP-70 相互作用，ZAP-70 表现出自身磷酸化活性以及外源底物 CFB3 的磷酸化。这些发现表明，Crk 接头蛋白在 T 淋巴细胞的早期激活事件中发挥作用，显然是通过与膜上的 ZAP-70 蛋白酪氨酸激酶直接相互作用并对其进行调节。
Engagement of the T cell antigen receptor initiates signal transduction involving tyrosine phosphorylation of multiple effector molecules and the formation of multimolecular complexes at the receptor site. Adapter proteins that possess SH2 and SH3 protein-protein interaction domains are implicated in the assembly of cell activation-induced signaling complexes. We found that Crk adapter proteins undergo activation-induced interaction with the zeta-chain associated protein (ZAP-70) tyrosine kinase in the human T cell line, Jurkat, Incubation of various glutathione S-transferase fusion proteins with a lysate of activated Jurkat cells resulted in selective association of ZAP-70 with Crk, but not Grb2 or Nck, adapter proteins. In addition, tyrosine-phosphorylated ZAP-70 co-immunoprecipitated with Crk from a lysate of activated Jurkat cells, and ZAP-70 association with GST-Crk was observed in a lysate of activated human peripheral blood T cells. Association between the two molecules was mediated by direct physical interaction and involved the Crk-SH2 domain and phosphotyrosyl-containing sequences on ZAP-70. The association required intact Lck, considered to be an upstream regulator of ZAP-70, because it could not take place in activated JCaM1 cells, which express normal levels of ZAP-70 but are devoid of Lck. Finally, glutathione S-transferase-Crk fusion proteins were found to interact predominantly with membrane-residing tyrosine-phosphorylated ZAP-70 that exhibited autophosphorylation activity as well as phosphorylation of an exogenous substrate, CFB3. These findings suggest that Crk adapter proteins play a role in the early activation events of T lymphocytes, apparently, by direct interaction with, and regulation of, the membrane-residing ZAP-70 protein tyrosine kinase.