Guerrero C, Fernandez-Medarde A, Rojas J M, Font de Mora J, Esteban L M, Santos E
Laboratory of Cellular and Molecular Biology, NCI, DBS, NIH, Bethesda, Maryland 20892, USA.
Oncogene. 1998 Feb 5;16(5):613-24. doi: 10.1038/sj.onc.1201569.
The guanine nucleotide releasing protein C3G was initially identified as a Crk SH3-binding protein and recently shown to exhibit exchange activity on Rap1 proteins. Overexpression in NIH3T3 cells of a full-length C3G cDNA isolated from human placenta markedly reduced the focus forming activity of cotransfected, malignantly activated, ras oncogenes (5-7-fold). C3G also had a reverting effect on sis-mediated transformation, decreasing the number of c-sis-induced foci by a factor of 5-10-fold. The observed inhibitory effect of C3G on focus-forming activity of Ras and Sis was always higher than that observed with Rap1A, a known target of C3G. The inhibition of focus formation observed in the presence of C3G was not due to toxic effects on cell viability, since transfected C3G cells exhibited the same survival and growth rates as untransfected NIH3T3 cells or cells transfected with plasmid vector alone. Surprisingly, as opposed to Rap1A, which has no effect on Raf-1 oncogene-mediated transformation, C3G also reduced dramatically (6-8-fold) the number of v-raf-induced foci in transfected NIH3T3 cells. The inhibitory effect on Raf-induced transformation suggests that C3G has other functional targets in addition to Rap1. A C3G mutant (C3G deltaCat) lacking the catalytic domain (CDC25-H) but retaining the rest of the N-terminal sequences, including the Crk-binding domain, exhibited similar ability than full length C3G to inhibit focus formation. In contrast, a C3G mutant (C3G Cat), containing the catalytic domain only but lacking the rest of the N-terminal sequences, did not have any inhibitory effect on transformation mediated by the oncogenes tested. The C3G-derived gene products overexpressed in our transfected cell lines localized to the cytoplasm and did not change the basal MAPK or JNK activity of those cell lines nor their ability to activate the kinases in response to agonists. Our results suggest that the N-terminal region of C3G, and not its catalytic domain, may be responsible for the inhibitory effects observed.
鸟嘌呤核苷酸释放蛋白C3G最初被鉴定为一种Crk SH3结合蛋白，最近发现它对Rap1蛋白具有交换活性。从人胎盘中分离出的全长C3G cDNA在NIH3T3细胞中过表达，显著降低了共转染的恶性激活的ras癌基因的集落形成活性（5 - 7倍）。C3G对sis介导的转化也有逆转作用，使c-sis诱导的集落数量减少5 - 10倍。观察到C3G对Ras和Sis集落形成活性的抑制作用总是高于其已知靶点Rap1A所观察到的抑制作用。在C3G存在下观察到的集落形成抑制并非由于对细胞活力的毒性作用，因为转染C3G的细胞与未转染的NIH3T3细胞或仅用质粒载体转染的细胞具有相同的存活和生长速率。令人惊讶的是，与对Raf-1癌基因介导的转化无影响的Rap1A不同，C3G也显著减少了（6 - 8倍）转染的NIH3T3细胞中v-raf诱导的集落数量。对Raf诱导转化的抑制作用表明，除了Rap1之外，C3G还有其他功能靶点。一种缺乏催化结构域（CDC25-H）但保留包括Crk结合结构域在内的其余N端序列的C3G突变体（C3G deltaCat），与全长C3G相比，具有相似的抑制集落形成的能力。相反，一种仅包含催化结构域但缺乏其余N端序列的C3G突变体（C3G Cat），对所测试的癌基因介导的转化没有任何抑制作用。在我们的转染细胞系中过表达的C3G衍生基因产物定位于细胞质，并且没有改变这些细胞系的基础MAPK或JNK活性，也没有改变它们响应激动剂激活激酶的能力。我们的结果表明，C3G的N端区域而非其催化结构域可能是观察到的抑制作用的原因。
