Phosphorylation of CrkII adaptor protein at tyrosine 221 by epidermal growth factor receptor
Phosphorylation of CrkII adaptor protein at tyrosine 221 by epidermal growth factor receptor
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DOI：
10.1074/jbc.273.27.17186
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发表时间：
1998-07-03
影响因子：
4.8
通讯作者：
Matsuda, M
中科院分区：
文献类型：
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作者：
Hashimoto, Y;Katayama, H;Matsuda, M
在不同类型的刺激下，CrkII接头蛋白发生酪氨酸磷酸化。我们研究了已被c-Abl磷酸化的酪氨酸221是否也被其他酪氨酸激酶，如表皮生长因子(EGF)受体所磷酸化。为此，我们开发了一种特异性识别Tyr(221)磷酸化的CrkII的抗体，并证明了在EGF刺激下，CrkII在Tyr(221)上被磷酸化。当NRK细胞受到EGF刺激时，在细胞外围检测到酪氨酸磷酸化的CrkII，那里的褶皱突出，提示CrkII的信号可能参与了EGF依赖的细胞骨架重组。在c-Abl缺失的细胞系中也检测到依赖于EGF的CrkII的磷酸化。此外，重组的CrkII蛋白在体外被EGF受体磷酸化。这些结果有力地表明，EGF受体直接磷酸化CrkII。突变分析表明，src同源2结构域是EGF受体磷酸化CrkII所必需的，但不是c-Abl所必需的，这表明这些蛋白通过不同的磷酸化机制使CrkII磷酸化。最后，我们发现，在EGF刺激下被磷酸化的CrkII蛋白不能与含有磷酸酪氨酸的多肽结合，并且CrkII在3min内开始与EGF受体解离，即使EGF受体持续的酪氨酸磷酸化也是如此。这一结果表明，Tyr(221)的磷酸化参与了src同源α介导的CrkII与EGF受体结合的负调控。
CrkII adaptor protein becomes tyrosine-phosphorylated upon various types of stimulation. We examined whether tyrosine 221, which has been shown to be phosphorylated by c-Abl, was phosphorylated also by other tyrosine kinases, such as epidermal growth factor (EGF) receptor. For this purpose, we developed an antibody that specifically recognizes Tyr(221)-phosphorylated CrkII, and we demonstrated that CrkII was phosphorylated on Tyr(221) upon EGF stimulation. When NRK cells were stimulated with EGF, the tyrosine-phosphorylated CrkII was detected at the periphery of the cells, where ruffling is prominent, suggesting that signaling to CrkII may be involved in EGF-dependent cytoskeletal reorganization. The EGF-dependent phosphorylation of CrkII was also detected in a c-Abl-deficient cell line. Moreover, recombinant CrkII protein was phosphorylated in vitro by EGF receptor. These results strongly suggest that EGF receptor directly phosphorylates CrkII. Mutational analysis revealed that the src homology 2 domain was essential for the phosphorylation of CrkII by EGF receptor but not by c-Abl, arguing that these kinases phosphorylate CrkII by different phosphorylation mechanisms. Finally, we found that the CrkII protein phosphorylated upon EGF stimulation did not bind to the phosphotyrosine-containing peptide and that CrkII initiated dissociation from EGF receptor within 3 min even with the sustained tyrosine phosphorylation of EGF receptor. This result implicated phosphorylation of Tyr(221) in the negative regulation of the src homology a-mediated binding of CrkII to EGF receptor.