Ota S, Kizaka-Kondoh S, Hashimoto Y, Nishihara H, Nagashima K, Kurata T, Okayama H, Matsuda M
Department of Pathology, National Institute of Infectious Diseases, Tokyo, Japan.
Cell Signal. 1998 Apr;10(4):283-90. doi: 10.1016/s0898-6568(97)00130-7.
Crk belongs to the adapter proteins that participate in many signalling pathways from cell surface receptors. We have characterised the CrkII-23 mutant that inhibits the transformation of NRK cells induced by epidermal growth factor (EGF) and transforming growth factor (TGF)-beta. To study the biochemical difference, cDNAs of the wild-type CrkII and the CrkII-23 mutant were introduced stably into NIH 3T3 cells expressing EGF receptor (EGFR). Both CrkII and CrkII-23 were phosphorylated on tyrosine upon EGF simulation with similar time course and dose dependency. Whereas the wild-type CrkII bound to EGFR only after EGF stimulation, CrkII-23 bound to EGFR from before stimulation. Mutation in the Src homology (SH) 2 or amino-terminal SH3 domain did not abolish the binding of CrkII-23 to EGFR in the quiescent cells, suggesting that the binding is mediated by a novel mechanism. These CrkII-23-derived mutants, however, did not suppress transformation of NRK cells by EGF and TGF-beta. Hence, both the SH2 and amino-terminal SH3 domains are required to inhibit transformation of NRK cells. These results suggest that persistent signalling from CrkII-23 bound to EGFR suppresses transformation by EGF and TGF-beta in NRK23 cells.
Crk属于参与许多细胞表面受体信号通路的衔接蛋白。我们已对CrkII - 23突变体进行了表征，该突变体可抑制表皮生长因子（EGF）和转化生长因子（TGF）-β诱导的NRK细胞转化。为研究其生化差异，将野生型CrkII和CrkII - 23突变体的cDNA稳定导入表达EGF受体（EGFR）的NIH 3T3细胞中。在用EGF刺激后，CrkII和CrkII - 23的酪氨酸均发生磷酸化，且具有相似的时间进程和剂量依赖性。野生型CrkII仅在EGF刺激后才与EGFR结合，而CrkII - 23在刺激前就与EGFR结合。Src同源（SH）2或氨基末端SH3结构域的突变并未消除静止细胞中CrkII - 23与EGFR的结合，这表明这种结合是由一种新机制介导的。然而，这些源自CrkII - 23的突变体并不能抑制EGF和TGF -β对NRK细胞的转化。因此，SH2和氨基末端SH3结构域对于抑制NRK细胞的转化都是必需的。这些结果表明，与EGFR结合的CrkII - 23持续发出的信号抑制了NRK23细胞中EGF和TGF -β介导的转化。
