Tsai Fu-Ming, Shyu Rong-Yaun, Jiang Shun-Yuan
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan.
Cell Signal. 2006 Mar;18(3):349-58. doi: 10.1016/j.cellsig.2005.05.005. Epub 2005 Jul 6.
The retinoid-inducible gene 1 (RIG1) protein is a retinoid-inducible growth regulator. Previous studies have shown that the RIG1 protein inhibits the signaling pathways of Ras/mitogen-activated protein kinases. However, neither the mode of action nor the site of inhibition of RIG1 is known. This study investigated the effects of RIG1, and the mechanisms responsible for these effects, on the activation of Ras proteins in HtTA cervical cancer cells. RIG1 reduced the levels of activated Ras (Ras-GTP) and total Ras protein in cells transfected with mutated H-, N-, or K-Ras(G12V), or in cells transfected with the wild type H- or N-Ras followed by stimulation with epidermal growth factor. The half-life of Ras protein decreased from more than 36 h in control cells to 18 h in RIG1-transfected cells. RIG1 immunoprecipitated with the Ras protein in co-transfected cellular lysates. In contrast to the predominant plasma membrane localization in control cells, the H-Ras fusion protein EGFP-H-Ras was localized within a discrete cytoplasmic compartment where it co-localized with RIG1. RIG1 inhibited more than 93% of the Elk- and CHOP-mediated transactivation induced by H- or K-Ras(G12V). However, RIG1 did not inhibit the transactivation induced by MEK1 or MEK3, and failed to suppress the phosphorylation of extracellular signal-regulated kinases 1 and 2 induced by the constitutively activated B-Raf(V599E). The RIG1 with carboxyl terminal truncation (RIG1DeltaC) did not immunoprecipitate with Ras and had no effect on Ras activation or transactivation of the downstream signal pathways. These data indicate that RIG1 exerts its inhibitory effect at the level of Ras activation, which is independent of Ras subtype but dependent on the membrane localization of the RIG1 protein. This inhibition of Ras activation may be mediated through downregulation of Ras levels and alteration of Ras subcellular distribution.
视黄酸诱导基因1（RIG1）蛋白是一种视黄酸诱导的生长调节因子。先前的研究表明，RIG1蛋白可抑制Ras/丝裂原活化蛋白激酶的信号通路。然而，RIG1的作用方式和抑制位点均不清楚。本研究调查了RIG1对HtTA宫颈癌细胞中Ras蛋白激活的影响及其作用机制。RIG1降低了用突变的H-、N-或K-Ras（G12V）转染的细胞，或用野生型H-或N-Ras转染后再用表皮生长因子刺激的细胞中活化Ras（Ras-GTP）和总Ras蛋白的水平。Ras蛋白的半衰期从对照细胞中的超过36小时降至RIG1转染细胞中的18小时。在共转染的细胞裂解物中，RIG1与Ras蛋白进行免疫沉淀。与对照细胞中主要定位于质膜不同，H-Ras融合蛋白EGFP-H-Ras定位于一个离散的细胞质区室，在那里它与RIG1共定位。RIG1抑制了由H-或K-Ras（G12V）诱导的超过93%的Elk和CHOP介导的反式激活。然而，RIG1不抑制由MEK1或MEK3诱导的反式激活，也未能抑制由组成型激活的B-Raf（V599E）诱导的细胞外信号调节激酶1和2的磷酸化。羧基末端截短的RIG1（RIG1DeltaC）不与Ras进行免疫沉淀，对Ras激活或下游信号通路的反式激活没有影响。这些数据表明，RIG1在Ras激活水平发挥其抑制作用，这与Ras亚型无关，但依赖于RIG1蛋白的膜定位。这种对Ras激活的抑制可能是通过下调Ras水平和改变Ras亚细胞分布来介导的。
