Department of Cellular and Integrative Physiology, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA.
J Biol Chem. 2012 Oct 5;287(41):33996-4008. doi: 10.1074/jbc.M112.369603. Epub 2012 Aug 13.
The activation of the small GTPase RhoA is necessary for ACh-induced actin polymerization and airway smooth muscle (ASM) contraction, but the mechanism by which it regulates these events is unknown. Actin polymerization in ASM is catalyzed by the actin filament nucleation activator, N-WASp and the polymerization catalyst, Arp2/3 complex. Activation of the small GTPase cdc42, a specific N-WASp activator, is also required for actin polymerization and tension generation. We assessed the mechanism by which RhoA regulates actin dynamics and smooth muscle contraction by expressing the dominant negative mutants RhoA T19N and cdc42 T17N, and non-phosphorylatable paxillin Y118/31F and paxillin ΔLD4 deletion mutants in SM tissues. Their effects were evaluated in muscle tissue extracts and freshly dissociated SM cells. Protein interactions and cellular localization were analyzed using proximity ligation assays (PLA), immunofluorescence, and GTPase and kinase assays. RhoA inhibition prevented ACh-induced cdc42 activation, N-WASp activation and the interaction of N-WASp with the Arp2/3 complex at the cell membrane. ACh induced paxillin phosphorylation and its association with the cdc42 GEFS, DOCK180 and α/βPIX. Paxillin tyrosine phosphorylation and its association with βPIX were RhoA-dependent, and were required for cdc42 activation. The ACh-induced recruitment of paxillin and FAK to the cell membrane was dependent on RhoA. We conclude that RhoA regulates the contraction of ASM by catalyzing the assembly and activation of cytoskeletal signaling modules at membrane adhesomes that initiate signaling cascades that regulate actin polymerization and tension development in response to contractile agonist stimulation. Our results suggest that the RhoA-mediated assembly of adhesome complexes is a fundamental step in the signal transduction process in response to agonist -induced smooth muscle contraction.
RhoA 的激活对于 ACh 诱导的肌动蛋白聚合和气道平滑肌 (ASM) 收缩是必要的，但它调节这些事件的机制尚不清楚。ASM 中的肌动蛋白聚合由肌动蛋白丝成核激活剂 N-WASp 和聚合催化剂 Arp2/3 复合物催化。小 GTPase cdc42 的激活，一种特定的 N-WASp 激活剂，也需要肌动蛋白聚合和张力产生。我们通过表达显性负突变体 RhoA T19N 和 cdc42 T17N，以及非磷酸化的 paxillin Y118/31F 和 paxillin ΔLD4 缺失突变体，评估了 RhoA 调节肌动蛋白动力学和平滑肌收缩的机制。在肌肉组织提取物和新鲜分离的 SM 细胞中评估了它们的作用。使用邻近连接测定 (PLA)、免疫荧光和 GTPase 和激酶测定分析蛋白质相互作用和细胞定位。RhoA 抑制阻止了 ACh 诱导的 cdc42 激活、N-WASp 激活以及 N-WASp 与 Arp2/3 复合物在细胞膜上的相互作用。ACh 诱导了 paxillin 的磷酸化及其与 cdc42 GEFS、DOCK180 和 α/βPIX 的结合。paxillin 酪氨酸磷酸化及其与 βPIX 的结合依赖于 RhoA，并且是 cdc42 激活所必需的。ACh 诱导的 paxillin 和 FAK 向细胞膜的募集依赖于 RhoA。我们得出结论，RhoA 通过在膜粘着斑处催化细胞骨架信号模块的组装和激活来调节 ASM 的收缩，这些信号模块启动信号级联反应，从而调节肌动蛋白聚合和张力的产生，以响应收缩激动剂的刺激。我们的结果表明，RhoA 介导的粘着斑复合物的组装是对激动剂诱导的平滑肌收缩的信号转导过程中的一个基本步骤。
