Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT 06030-6403, USA.
Oncogene. 2010 Dec 2;29(48):6378-89. doi: 10.1038/onc.2010.369. Epub 2010 Aug 23.
The v-Crk oncogene product consists of two protein interaction modules, a Src homology 2 (SH2) domain and a Src homology 3 (SH3) domain. Overexpression of CrkI, the cellular homolog of v-Crk, transforms mouse fibroblasts, and elevated CrkI expression is observed in several human cancers. The SH2 and SH3 domains of Crk are required for transformation, but the identity of the critical cellular binding partners is not known. A number of candidate Crk SH3-binding proteins have been identified, including the nonreceptor tyrosine kinases c-Abl and Arg, and the guanine nucleotide exchange proteins C3G, SOS1 and DOCK180. The aim of this study is to determine which of these are required for transformation by CrkI. We found that short hairpin RNA-mediated knockdown of C3G or SOS1 suppressed anchorage-independent growth of NIH-3T3 cells overexpressing CrkI, whereas knockdown of SOS1 alone was sufficient to suppress tumor formation by these cells in nude mice. Knockdown of C3G was sufficient to revert morphological changes induced by CrkI expression. By contrast, knockdown of Abl family kinases or their inhibition with imatinib enhanced anchorage-independent growth and tumorigenesis induced by Crk. These results show that SOS1 is essential for CrkI-induced fibroblast transformation, and also reveal a surprising negative role for Abl kinases in Crk transformation.
v-Crk 癌基因产物由两个蛋白相互作用模块组成，一个Src 同源 2（SH2）结构域和一个 Src 同源 3（SH3）结构域。CrkI 的过表达，即 v-Crk 的细胞同源物，可转化小鼠成纤维细胞，并且在几种人类癌症中观察到 CrkI 表达升高。Crk 的 SH2 和 SH3 结构域是转化所必需的，但关键的细胞结合伴侣的身份尚不清楚。已经鉴定出许多候选 Crk SH3 结合蛋白，包括非受体酪氨酸激酶 c-Abl 和 Arg，以及鸟嘌呤核苷酸交换蛋白 C3G、SOS1 和 DOCK180。本研究的目的是确定这些蛋白中哪些是 CrkI 转化所必需的。我们发现，短发夹 RNA 介导的 C3G 或 SOS1 敲低抑制了过表达 CrkI 的 NIH-3T3 细胞的锚定非依赖性生长，而单独敲低 SOS1 足以抑制这些细胞在裸鼠中的肿瘤形成。C3G 的敲低足以逆转 CrkI 表达诱导的形态变化。相比之下，Abl 家族激酶的敲低或用伊马替尼抑制它们增强了 Crk 诱导的锚定非依赖性生长和肿瘤发生。这些结果表明 SOS1 是 CrkI 诱导的成纤维细胞转化所必需的，并且还揭示了 Abl 激酶在 Crk 转化中的惊人负作用。
