Tumusiime Sylvester, Rana Manish K, Kher Swapnil S, Kurella Vinodh B, Williams Kelly A, Guidry Jessie J, Worthylake David K, Worthylake Rebecca A
Department of Pharmacology and Experimental Therapeutics, LSU Health Sciences Center, 1901 Perdido St., 5th Floor MEB, New Orleans, LA 70112, USA.
Biochem Biophys Res Commun. 2009 Apr 10;381(3):393-6. doi: 10.1016/j.bbrc.2009.02.056. Epub 2009 Feb 15.
ROCKII kinase activity is known to be regulated by Rho GTPase binding; however, the context-specific regulation of ROCKII is not clearly understood. We pursued the C-terminal PH domain as a candidate domain for regulating ROCKII function. A proteomics-based screen identified potential ROCKII signaling partners, a large number of which were associated with membrane dynamics. We used subcellular fractionation to demonstrate that ROCKII is localized to both the plasma membrane and internal endosomal membrane fractions, and then used microscopy to show that the C-terminal PH domain can localize to internal or peripheral membrane compartments, depending on the cellular context. Co-immunoprecipitation demonstrated that Dynamin1 is a novel ROCKII binding partner. Furthermore, blocking Dynamin function with a dominant negative mutant mimicked the effect of inhibiting ROCK activity on the actin cytoskeleton. Our data suggest that ROCKII is regulated by localization to specific membrane compartments and its novel binding partner, Dynamin1.
已知ROCKII激酶活性受Rho GTPase结合调控；然而，ROCKII在特定环境下的调控机制尚不清楚。我们将C末端PH结构域作为调节ROCKII功能的候选结构域进行研究。基于蛋白质组学的筛选确定了潜在的ROCKII信号转导伙伴，其中大量伙伴与膜动力学相关。我们通过亚细胞分级分离证明ROCKII定位于质膜和内部内体膜部分，然后利用显微镜观察表明，C末端PH结构域可根据细胞环境定位于内部或外周膜区室。免疫共沉淀表明发动蛋白1是一种新型的ROCKII结合伙伴。此外，用显性负性突变体阻断发动蛋白功能可模拟抑制ROCK活性对肌动蛋白细胞骨架的影响。我们的数据表明，ROCKII受定位于特定膜区室及其新型结合伙伴发动蛋白1的调控。
