Department of Life Science, Ewha Womans University, 11-1 Daehyun-Dong, Seodaemoon-Gu, Seoul 120-750, Korea.
Cardiovasc Res. 2013 Feb 1;97(2):302-10. doi: 10.1093/cvr/cvs311. Epub 2012 Oct 5.
Ahnak protein acts as a scaffold protein networking phospholipase C-γ and protein kinase C-α, which subsequently stimulate an extracellular signal-regulated kinase (Erk) pathway. In mouse aortic smooth muscle cells (ASMCs), the activation of the signalling cascade ultimately promotes the cell migration through an unknown mechanism. We aimed to dissect the Ahnak-mediated cell signalling network involved in the migration of ASMCs.
Migration of ASMCs from wild-type mice was significantly increased by platelet-derived growth factor (PDGF) stimulation in transwell chamber and wound-healing assays, whereas migration of ASMCs from Ahnak knockout mice was reduced. Consistently, stimulation of wild-type ASMCs with PDGF resulted in Rac activation-mediated lamellipodial protrusion in migrating cells. In contrast, Ahnak knockout ASMCs displayed lower activation of Rac in response to PDGF and slow lamellipodial protrusion rate and cell migration. Ahnak signalling complex was analysed by immunoprecipitation with antibody to p21-activated protein kinase (PAK). Ahnak protein was shown to function as the signalling scaffold interacting with the multiple protein complex of Erk, PAK, and p21-activated kinase-interacting exchange factor β. The proposed role of Ahnak in cell migration was examined using a restenosis model in which the carotid arteries of mice were subjected to post-ligation injury. We show neointimal formation and SMC migration after ligation injury in Ahnak knockout mice were significantly retarded compared with wild-type mice.
Ahnak protein plays an important scaffolding function connecting Erk and Rac activation in PDGF-dependent migration of ASMC.
Ahnak 蛋白作为一种支架蛋白，将磷酯酶 C-γ 和蛋白激酶 C-α 连接在一起，从而刺激细胞外信号调节激酶（Erk）途径。在小鼠主动脉平滑肌细胞（ASMCs）中，信号级联的激活最终通过未知机制促进细胞迁移。我们旨在剖析 Ahnak 介导的参与 ASMC 迁移的细胞信号网络。
在 Transwell 室和划痕愈合实验中，血小板衍生生长因子（PDGF）刺激下，来自野生型小鼠的 ASMC 迁移明显增加，而来自 Ahnak 敲除小鼠的 ASMC 迁移减少。一致地，PDGF 刺激野生型 ASMC 导致 Rac 激活介导的迁移细胞中的片状伪足突起。相比之下，Ahnak 敲除 ASMC 对 PDGF 的反应中 Rac 的激活较低，并且片状伪足突起的速率和细胞迁移较慢。通过针对 p21 激活蛋白激酶（PAK）的抗体进行免疫沉淀分析 Ahnak 信号复合物。结果表明，Ahnak 蛋白作为信号支架，与 Erk、PAK 和 p21 激活激酶相互作用的交换因子β的多种蛋白复合物相互作用。使用小鼠颈动脉结扎损伤后的再狭窄模型来研究 Ahnak 在细胞迁移中的作用。与野生型小鼠相比，Ahnak 敲除小鼠结扎损伤后的新生内膜形成和 SMC 迁移明显延迟。
Ahnak 蛋白在 PDGF 依赖性 ASMC 迁移中发挥重要的支架功能，连接 Erk 和 Rac 激活。
