Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration
Crk adaptor protein-induced phosphorylation of Gab1 on tyrosine 307 via Src is important for organization of focal adhesions and enhanced cell migration
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DOI：
10.1038/cr.2009.40
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发表时间：
2009-05-01
期刊：
影响因子：
44.1
通讯作者：
Tanaka, Shinya
中科院分区：
文献类型：
--
作者：
Watanabe, Takuya;Tsuda, Masumi;Tanaka, Shinya
在生长因子刺激下，支架蛋白Gab 1被酪氨酸磷酸化，随后接头蛋白Crk从Gab 1传递信号。我们以前已经表明，Crk过表达，这是在各种人类癌症中检测到的，诱导酪氨酸磷酸化的Gab 1没有细胞外刺激。在本研究中，进一步研究了其潜在的机制。CrkII的突变分析表明，SH 2结构域，而不是SH 3（N）或调控的Y221残基的CrkII，是关键的诱导Gab 1-Y307磷酸化。CrkII的SH 2突变也降低了与Gab 1的相互作用。在GST pull-down分析中，Crk-SH 2与野生型Gab 1结合，而Crk-SH 3（N）与缺少聚集的酪氨酸区域（残基242-410）的Gab 1突变体相互作用。酪氨酸磷酸化的Gab 1诱导的所有Crk家族蛋白质，但不是其他含SH 2的信号转导衔接子。Src-家族激酶抑制剂PP 2可抑制Gab 1的酪氨酸磷酸化。Y307磷酸化在缺乏Src、Yes和Fyn的成纤维细胞中检测不到，即使在Crk过表达时也是如此，而仅缺乏Yes和Fyn的细胞仍然含有具有磷酸化Y307的Gab 1。Crk诱导Src-Y 416磷酸化，从而增强了Crk与Csk的相互作用。Gab 1-Y307 F突变体未能定位在质膜附近，即使在HGF刺激和细胞迁移减少。此外，Gab 1-Y307 F干扰了局部粘连的典型成分Crk、FAK和桩蛋白的定位。综上所述，这些结果表明，Crk通过Src促进Gab 1-Y307的酪氨酸磷酸化，有助于组织粘着斑和增强细胞迁移，从而可能促进人类癌症的发展。
Upon growth factor stimulation, the scaffold protein, Gab1, is tyrosine phosphorylated and subsequently the adaptor protein, Crk, transmits signals from Gab1. We have previously shown that Crk overexpression, which is detectable in various human cancers, induces tyrosine phosphorylation of Gab1 without extracellular stimuli. In the present study, the underlying mechanisms were further investigated. Mutational analyses of CrkII demonstrated that the SH2 domain, but not the SH3(N) or the regulatory Y221 residue of CrkII, is critical for the induction of Gab1-Y307 phosphorylation. SH2 mutation of CrkII also decreased the interaction with Gab1. In GST pull-down assay, Crk-SH2 bound to wild-type Gab1, whereas Crk-SH3(N) interacted with the Gab1 mutant, which lacks the clustered tyrosine region (residues 242-410). Tyrosine phosphorylation of Gab1 was induced by all Crk family proteins, but not other SH2-containing signalling adaptors. Src-family kinase inhibitor, PP2, abrogates Crk-induced tyrosine phosphorylations of Gab1. Y307 phosphorylation was undetectable in fibroblasts lacking Src, Yes, and Fyn, even upon overexpression of Crk, whereas cells lacking only Yes and Fyn still contained Gab1 with phosphorylated Y307. Furthermore, Crk induced the phosphorylation of Src-Y416; accordingly the interaction between Crk and Csk was increased. The Gab1-Y307F mutant failed to localize near the plasma membrane even upon HGF stimulation and decreased cell migration. Moreover, Gab1-Y307F disturbed the localization of Crk, FAK, and paxillin, which are the typical components of focal adhesions. Taken together, these results indicate that Crk facilitates tyrosine phosphorylation of Gab1-Y307 through Src, contributing to the organization of focal adhesions and enhanced cell migration, thereby possibly promoting human cancer development.