Lazer Galit, Pe'er Liron, Schapira Vered, Richard Stéphane, Katzav Shulamit
The Hubert H. Humphrey Center for Experimental Medicine and Cancer Research, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel.
Cell Signal. 2007 Dec;19(12):2479-86. doi: 10.1016/j.cellsig.2007.07.022. Epub 2007 Aug 8.
Vav1 functions in the hematopoietic system as a specific GDP/GTP nucleotide exchange factor regulated by tyrosine phosphorylation. An intact C-terminal SH3 domain of Vav1 (Vav1SH3C) was shown to be necessary for Vav1-induced transformation, yet the associating protein(s) necessary for this activity have not yet been identified. Using a proteomics approach, we identified Sam68 as a Vav1SH3C-associating protein. Sam68 (Src-associated in mitosis of 68 kD) belongs to the heteronuclear ribonucleoprotein particle K (hnRNP-K) homology (KH) domain family of RNA-binding proteins. The Vav1/Sam68 interaction was observed in vitro and in vivo. Mutants of Vav1SH3C previously shown to lose their transforming potential did not associate with Sam68. Co-expression of Vav1 and Sam68 in Jurkat T cells led to increased localization of Vav1 in the nucleus and changes in cell morphology. We then tested the contribution of Sam68 to known functions of Vav1, such as focus-forming in NIH3T3 fibroblasts and NFAT stimulation in T cells. Co-expression of oncogenic Vav1 with Sam68 in NIH3T3 fibroblasts resulted in a dose-dependent increase in foci, yet no further enhancement of NFAT activity was observed in Jurkat T cells, as compared to cells overexpressing only Vav1 or Sam68. Our results strongly suggest that Sam68 contributes to transformation by oncogenic Vav1.
Vav1在造血系统中作为一种受酪氨酸磷酸化调节的特异性GDP/GTP核苷酸交换因子发挥作用。Vav1完整的C端SH3结构域（Vav1SH3C）被证明是Vav1诱导转化所必需的，然而尚未鉴定出该活性所需的相关蛋白。我们采用蛋白质组学方法，鉴定出Sam68是一种与Vav1SH3C相关的蛋白。Sam68（有丝分裂中与Src相关的68kD蛋白）属于RNA结合蛋白的异核核糖核蛋白颗粒K（hnRNP-K）同源（KH）结构域家族。在体外和体内均观察到Vav1与Sam68的相互作用。先前显示失去转化潜能的Vav1SH3C突变体不与Sam68结合。Vav1和Sam68在Jurkat T细胞中共表达导致Vav1在细胞核中的定位增加以及细胞形态改变。然后我们测试了Sam68对Vav1已知功能的贡献，例如在NIH3T3成纤维细胞中形成集落以及在T细胞中刺激NFAT。与仅过表达Vav1或Sam68的细胞相比，致癌性Vav1与Sam68在NIH3T3成纤维细胞中共表达导致集落呈剂量依赖性增加，但在Jurkat T细胞中未观察到NFAT活性的进一步增强。我们的结果强烈表明Sam68有助于致癌性Vav1的转化。
