An activated mutant of R-Ras inhibits cell death caused by cytokine deprivation in BaF3 cells in the presence of IGF-I
An activated mutant of R-Ras inhibits cell death caused by cytokine deprivation in BaF3 cells in the presence of IGF-I
复制标题
R-Ras 的激活突变体在 IGF-I 存在的情况下抑制 BaF3 细胞中因细胞因子剥夺引起的细胞死亡
作者：
Jotaro Suzuki;Y. Kaziro;H. Koide
R-Ras 属于低分子量 GTP 结合蛋白家族，与 H-Ras 具有 55% 的氨基酸同一性。已经证明，H-Ras 可抑制 BaF3 细胞中白细胞介素 3 (IL-3) 撤退引起的细胞死亡（Kinoshita 等人 (1995b)；Terada 等人 (1995)）。在本研究中，我们检查了 R-Ras 是否也能将 BaF3 细胞从因子缺失的细胞死亡中拯救出来。为此，建立了几种 BaF3 转染子，其中野生型和突变型 R-Ras 的表达受到诱导型启动子的调节。使用这些转染子，我们发现激活的 R-Ras 突变体 R-Ras (Q87L) 的表达抑制了 IL-3 缺失的 BaF3 细胞的死亡。另一方面，R-Ras野生型和显性失活突变体的表达对细胞死亡没有显示出抑制作用，表明R-RasGTP消除了因IL-3剥夺而导致的细胞死亡。此外，还发现血清中的IGF-I是R-Ras的抗凋亡活性所必需的。 R-Ras(Q87L) 对细胞死亡的抑制作用可被渥曼青霉素、LY294002（磷脂酰肌醇 3-激酶 (PI3K) 抑制剂）或 PD98059（MEK 抑制剂，丝裂原激活蛋白激酶 (MAPK) 的特异性激活剂）抑制。此外，我们还发现，在HEK293细胞中，R-Ras和IGF-I可以协同激活MAPK。此外，PI3K 活性与 R-Ras 的激活突变体进行共免疫沉淀。这些结果表明，R-Ras 与 IGF-I 合作抑制了由 IL-3 剥夺引起的 BaF3 凋亡细胞死亡，可能是通过调节 MAPK 和 PI3K 的活性来实现的。
R-Ras belongs to a family of low molecular weight GTP-binding proteins and exhibits 55% amino acid identity to H-Ras. It has been demonstrated that H-Ras inhibits cell death caused by interleukin-3 (IL-3) withdrawal in BaF3 cells (Kinoshita et al. (1995b); Terada et al. (1995)). In the present study, we examined whether R-Ras also rescues BaF3 cells from the factor-deprived cell death. To do this, several BaF3 transfectants were established, in which expression of wild-type as well as mutant R-Ras was regulated by an inducible promoter. Using these transfectants, we found that expression of an activated R-Ras mutant, R-Ras (Q87L), suppressed the death of IL-3-deprived BaF3 cells. On the other hand, expression of the wild-type and the dominant-negative mutant of R-Ras showed no inhibitory effect on cell death, indicating that R-RasGTP abrogated cell death caused by deprivation of IL-3. Furthermore, it was found that IGF-I in serum was required for the anti-apoptotic activity of R-Ras. Suppression of cell death by R-Ras(Q87L) was inhibited by wortmannin, LY294002 (phosphatidylinositol 3-kinase (PI3K) inhibitors), or PD98059 (inhibitor for MEK, a specific activator of mitogen-activated protein kinase (MAPK)). In addition, we have shown that, in HEK293 cells, R-Ras and IGF-I could activate MAPK synergistically. Also, PI3K activity was co-immunoprecipitated with an activated mutant of R-Ras. These results suggest that R-Ras in collaboration with IGF-I suppressed apoptotic cell death of BaF3 caused by IL-3 deprivation, presumably by modulating the activitites of MAPK and PI3K.