Madigan James P, Bodemann Brian O, Brady Donita C, Dewar Brian J, Keller Patricia J, Leitges Michael, Philips Mark R, Ridley Anne J, Der Channing J, Cox Adrienne D
Department of Radiation Oncology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Biochem J. 2009 Oct 23;424(1):153-61. doi: 10.1042/BJ20082377.
The Rnd proteins (Rnd1, Rnd2 and Rnd3/RhoE) form a distinct branch of the Rho family of small GTPases. Altered Rnd3 expression causes changes in cytoskeletal organization and cell cycle progression. Rnd3 functions to decrease RhoA activity, but how Rnd3 itself is regulated to cause these changes is still under investigation. Unlike other Rho family proteins, Rnd3 is regulated not by GTP/GDP cycling, but at the level of expression and by post-translational modifications such as prenylation and phosphorylation. We show in the present study that, upon PKC (protein kinase C) agonist stimulation, Rnd3 undergoes an electrophoretic mobility shift and its subcellular localization becomes enriched at internal membranes. These changes are blocked by inhibition of conventional PKC isoforms and do not occur in PKCalpha-null cells or to a non-phosphorylatable mutant of Rnd3. We further show that PKCalpha directly phosphorylates Rnd3 in an in vitro kinase assay. Additionally, we provide evidence that the phosphorylation status of Rnd3 has a direct effect on its ability to block signalling from the Rho-ROCK (Rho-kinase) pathway. These results identify an additional mechanism of regulation and provide clarification of how Rnd3 modulates Rho signalling to alter cytoskeletal organization.
Rnd蛋白（Rnd1、Rnd2和Rnd3/RhoE）构成了小GTP酶Rho家族的一个独特分支。Rnd3表达的改变会导致细胞骨架组织和细胞周期进程的变化。Rnd3的功能是降低RhoA活性，但Rnd3自身如何被调控以引起这些变化仍在研究中。与其他Rho家族蛋白不同，Rnd3不是通过GTP/GDP循环来调控，而是在表达水平以及通过翻译后修饰如异戊二烯化和磷酸化来调控。我们在本研究中表明，在蛋白激酶C（PKC）激动剂刺激后，Rnd3发生电泳迁移率改变，其亚细胞定位在内膜处富集。这些变化被传统PKC同工型的抑制所阻断，并且在PKCα基因敲除细胞中或Rnd3的非磷酸化突变体中不会发生。我们进一步表明，在体外激酶试验中PKCα直接使Rnd3磷酸化。此外，我们提供证据表明Rnd3的磷酸化状态对其阻断Rho-ROCK（Rho激酶）途径信号传导的能力有直接影响。这些结果确定了一种额外的调控机制，并阐明了Rnd3如何调节Rho信号传导以改变细胞骨架组织。
