Wang H G, Millan J A, Cox A D, Der C J, Rapp U R, Beck T, Zha H, Reed J C
La Jolla Cancer Research Foundation, California 92037, USA.
J Cell Biol. 1995 May;129(4):1103-14. doi: 10.1083/jcb.129.4.1103.
The Bcl-2 protein is an important regulator of programmed cell death, but the biochemical mechanism by which this protein prevents apoptosis remains enigmatic. Recently, Bcl-2 has been reported to physically interact with a member of the Ras superfamily of small GTPases, p23-R-Ras. To examine the functional significance of R-Ras for regulation of cell death pathways, the IL-3-dependent cells 32D.3 and FL5.12 were stably transfected with expression plasmids encoding an activated form (38 Glycine-->Valine) of R-Ras protein. R-Ras(38V)-producing 32D.3 and FL5.12 cells experienced increased rates of apoptotic cell death relative to control transfected cells when deprived of IL-3. Analysis of several independent clones of transfected 32D.3 cells revealed a correlation between higher levels of R-Ras protein and faster rates of cell death upon withdrawal of IL-3 from cultures. 32D.3 cells cotransfected with R-Ras(38V) and Bcl-2 exhibited prolonged cell survival in the absence of IL-3, equivalent to 32D.3 cells transfected with Bcl-2 expression plasmids alone. R-Ras(38V) also increased rates of cell death in serum-deprived NIH-3T3 cells, and Bcl-2 again abrogated most of this effect. The ratio of GTP and GDP bound to R-Ras(38V) was not significantly different in control 32D.3 cells vs those that overexpressed Bcl-2, indicating that Bcl-2 does not abrogate R-Ras-mediated effects on cell death by altering R-Ras GDP/GTP regulation. Moreover, purified Bcl-2 protein had no effect on the GTPase activity of recombinant wild-type R-Ras in vitro. When expressed in Sf9 cells using recombinant baculoviruses, R-Ras(38V) bound to and induced activation of Raf-1 kinase irrespective of whether Bcl-2 was coproduced in these cells, suggesting that Bcl-2 does not nullify R-Ras effects by interfering with R-Ras-mediated activation of Raf-1 kinase. Taken together, these findings suggest that R-Ras enhances the activity of a cell death pathway in growth factor-deprived cells and imply that Bcl-2 acts downstream of R-Ras to promote cell survival.
Bcl-2蛋白是程序性细胞死亡的重要调节因子，但其阻止细胞凋亡的生化机制仍不清楚。最近，有报道称Bcl-2与小GTP酶Ras超家族的成员p23-R-Ras发生物理相互作用。为了研究R-Ras对细胞死亡途径调节的功能意义，用编码R-Ras蛋白激活形式（38位甘氨酸突变为缬氨酸）的表达质粒稳定转染了依赖白细胞介素-3（IL-3）的细胞32D.3和FL5.12。与对照转染细胞相比，产生R-Ras(38V)的32D.3和FL5.12细胞在缺乏IL-3时凋亡细胞死亡速率增加。对转染的32D.3细胞的几个独立克隆进行分析发现，R-Ras蛋白水平较高与从培养物中撤除IL-3后细胞死亡速率较快之间存在相关性。与R-Ras(38V)和Bcl-2共转染的32D.3细胞在缺乏IL-3的情况下表现出延长的细胞存活，相当于仅用Bcl-2表达质粒转染的32D.3细胞。R-Ras(38V)也增加了血清饥饿的NIH-3T3细胞的死亡速率，而Bcl-2再次消除了大部分这种效应。在对照32D.3细胞与过表达Bcl-2的细胞中，与R-Ras(38V)结合的GTP与GDP的比例没有显著差异，这表明Bcl-2不会通过改变R-Ras的GDP/GTP调节来消除R-Ras介导的对细胞死亡的影响。此外，纯化的Bcl-2蛋白在体外对重组野生型R-Ras的GTP酶活性没有影响。当使用重组杆状病毒在Sf9细胞中表达时，无论这些细胞中是否共产生Bcl-2，R-Ras(38V)都能结合并诱导Raf-1激酶的激活，这表明Bcl-2不会通过干扰R-Ras介导的Raf-1激酶激活来消除R-Ras的作用。综上所述，这些发现表明R-Ras增强了生长因子缺乏细胞中细胞死亡途径的活性，并暗示Bcl-2在R-Ras的下游发挥作用以促进细胞存活。
