Goi T, Rusanescu G, Urano T, Feig L A
Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Mol Cell Biol. 1999 Mar;19(3):1731-41. doi: 10.1128/MCB.19.3.1731.
Ras proteins can activate at least three classes of downstream target proteins: Raf kinases, phosphatidylinositol-3 phosphate (PI3) kinase, and Ral-specific guanine nucleotide exchange factors (Ral-GEFs). In NIH 3T3 cells, activated Ral-GEFs contribute to Ras-induced cell proliferation and oncogenic transformation by complementing the activities of Raf and PI3 kinases. In PC12 cells, activated Raf and PI3 kinases mediate Ras-induced cell cycle arrest and differentiation into a neuronal phenotype. Here, we show that in PC12 cells, Ral-GEF activity acts opposite to other Ras effectors. Elevation of Ral-GEF activity induced by transfection of a mutant Ras protein that preferentially activates Ral-GEFs, or by transfection of the catalytic domain of the Ral-GEF Rgr, suppressed cell cycle arrest and neurite outgrowth induced by nerve growth factor (NGF) treatment. In addition, Rgr reduced neurite outgrowth induced by a mutant Ras protein that preferentially activates Raf kinases. Furthermore, inhibition of Ral-GEF activity by expression of a dominant negative Ral mutant accelerated cell cycle arrest and enhanced neurite outgrowth in response to NGF treatment. Ral-GEF activity may function, at least in part, through inhibition of the Rho family GTPases, CDC42 and Rac. In contrast to Ras, which was activated for hours by NGF treatment, Ral was activated for only approximately 20 min. These findings suggest that one function of Ral-GEF signaling induced by NGF is to delay the onset of cell cycle arrest and neurite outgrowth induced by other Ras effectors. They also demonstrate that Ras has the potential to promote both antidifferentiation and prodifferentiation signaling pathways through activation of distinct effector proteins. Thus, in some cell types the ratio of activities among Ras effectors and their temporal regulation may be important determinants for cell fate decisions between proliferation and differentiation.
Ras蛋白可激活至少三类下游靶蛋白：Raf激酶、磷脂酰肌醇-3磷酸（PI3）激酶和Ral特异性鸟嘌呤核苷酸交换因子（Ral-GEFs）。在NIH 3T3细胞中，活化的Ral-GEFs通过补充Raf和PI3激酶的活性，促进Ras诱导的细胞增殖和致癌转化。在PC12细胞中，活化的Raf和PI3激酶介导Ras诱导的细胞周期停滞并分化为神经元表型。在此，我们表明，在PC12细胞中，Ral-GEF活性与其他Ras效应器的作用相反。通过转染优先激活Ral-GEFs的突变Ras蛋白，或转染Ral-GEF Rgr的催化结构域所诱导的Ral-GEF活性升高，抑制了神经生长因子（NGF）处理诱导的细胞周期停滞和神经突生长。此外，Rgr减少了优先激活Raf激酶的突变Ras蛋白诱导的神经突生长。此外，通过表达显性负性Ral突变体抑制Ral-GEF活性，加速了细胞周期停滞，并增强了对NGF处理的神经突生长反应。Ral-GEF活性可能至少部分地通过抑制Rho家族GTP酶CDC42和Rac发挥作用。与NGF处理激活数小时的Ras不同，Ral仅激活约20分钟。这些发现表明，NGF诱导的Ral-GEF信号传导的一个功能是延迟其他Ras效应器诱导的细胞周期停滞和神经突生长的开始。它们还证明，Ras有潜力通过激活不同的效应蛋白来促进抗分化和促分化信号通路。因此，在某些细胞类型中，Ras效应器之间的活性比例及其时间调节可能是细胞增殖和分化之间细胞命运决定的重要决定因素。
