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首页> 外文期刊>Molecular and Cellular Biology >The Insert Region of RhoA Is Essential for Rho Kinase Activation and Cellular Transformation
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The Insert Region of RhoA Is Essential for Rho Kinase Activation and Cellular Transformation

机译：RhoA的插入区域对于Rho激酶激活和细胞转化至关重要

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RhoA is involved in multiple cellular processes, including cytoskeletal organization, gene expression, and transformation. These processes are mediated by a variety of downstream effector proteins. However, which effectors are involved in cellular transformation and how these proteins are activated following interaction with Rho remains to be established. A unique feature that distinguishes the Rho family from other Ras-related GTPases is the insert region, which may confer Rho-specific signaling events. Here we report that deletion of the insert region does not result in impaired effector binding. Instead, this insert deletion mutant (RhoΔRas, in which the insert helix has been replaced with loop 8 of Ras) acted in a dominant inhibitory fashion to block RhoA-induced transformation. Since RhoΔRas failed to promote stress fiber formation, we examined the ability of this mutant to bind to and subsequently activate Rho kinase. Surprisingly, RhoΔRas-GTP coprecipitated with Rho kinase but failed to activate it in vivo. These data suggested that the insert domain is not required for Rho kinase binding but plays a role in its activation. The constitutively active catalytic domain of Rho kinase did not promote focus formation alone or in the presence of Raf(340D) but cooperated with RhoΔRas to induce cellular transformation. This suggests that Rho kinase needs to cooperate with additional Rho effectors to promote transformation. Further, the Rho kinase catalytic domain reversed the inhibitory effect of RhoΔRas on Rho-induced transformation, suggesting that one of the downstream targets of Rho-induced transformation abrogated by RhoΔRas is indeed Rho kinase. In conclusion, we have demonstrated that the insert region of RhoA is required for Rho kinase activation but not for binding and that this kinase activity is required to induce morphologic transformation of NIH 3T3 cells.
机译：RhoA参与多个细胞过程，包括细胞骨架组织，基因表达和转化。这些过程由多种下游效应蛋白介导。然而，哪些效应子参与细胞转化以及这些蛋白质与Rho相互作用后如何活化尚待确定。插入区域可以将Rho家族与其他与Ras相关的GTPases区别开来，这是一个独特的功能，可以赋予Rho特定的信号事件。在这里我们报告插入区域的删除不会导致效应子结合受损。相反，该插入缺失突变体（RhoΔRas，其中插入螺旋已被Ras的环8取代）以显性抑制方式起作用，以阻断RhoA诱导的转化。由于RhoΔRas无法促进应激纤维的形成，因此我们检查了该突变体结合并随后激活Rho激酶的能力。出人意料的是，RhoΔRas-GTP与Rho激酶共沉淀，但未能在体内激活它。这些数据表明插入结构域不是Rho激酶结合所必需的，但在其激活中起作用。 Rho激酶的组成型活性催化域不能单独或在Raf（340D）存在下促进聚焦形成，而是与RhoΔRas协同诱导细胞转化。这表明Rho激酶需要与其他Rho效应子配合以促进转化。此外，Rho激酶催化结构域逆转了RhoΔRas对Rho诱导的转化的抑制作用，表明由RhoΔRas废除的Rho诱导的转化的下游靶标的确实是Rho激酶。总之，我们已经证明RhoA的插入区域是Rho激酶激活所必需的，而不是结合所必需的，并且该激酶活性是诱导NIH 3T3细胞形态转化所必需的。

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