School of Healthcare Science, Manchester Metropolitan University, Manchester, United Kingdom.
PLoS One. 2013 Sep 30;8(9):e75538. doi: 10.1371/journal.pone.0075538. eCollection 2013.
Cyclin-dependent kinase-5 (Cdk5) is over-expressed in both neurons and microvessels in hypoxic regions of stroke tissue and has a significant pathological role following hyper-phosphorylation leading to calpain-induced cell death. Here, we have identified a critical role of Cdk5 in cytoskeleton/focal dynamics, wherein its activator, p35, redistributes along actin microfilaments of spreading cells co-localising with p(Tyr15)Cdk5, talin/integrin beta-1 at the lamellipodia in polarising cells. Cdk5 inhibition (roscovitine) resulted in actin-cytoskeleton disorganisation, prevention of protein co-localization and inhibition of movement. Cells expressing Cdk5 (D144N) kinase mutant, were unable to spread, migrate and form tube-like structures or sprouts, while Cdk5 wild-type over-expression showed enhanced motility and angiogenesis in vitro, which was maintained during hypoxia. Gene microarray studies demonstrated myocyte enhancer factor (MEF2C) as a substrate for Cdk5-mediated angiogenesis in vitro. MEF2C showed nuclear co-immunoprecipitation with Cdk5 and almost complete inhibition of differentiation and sprout formation following siRNA knock-down. In hypoxia, insertion of Cdk5/p25-inhibitory peptide (CIP) vector preserved and enhanced in vitro angiogenesis. These results demonstrate the existence of critical and complementary signalling pathways through Cdk5 and p35, and through which coordination is a required factor for successful angiogenesis in sustained hypoxic condition.
周期蛋白依赖性激酶-5（Cdk5）在缺氧性中风组织的神经元和微血管中过度表达，在过度磷酸化导致钙蛋白酶诱导的细胞死亡后，具有重要的病理作用。在这里，我们已经确定了 Cdk5 在细胞骨架/焦点动力学中的关键作用，其激活剂 p35 沿着伸展细胞中的肌动蛋白微丝重新分布，与极化细胞中的 p（Tyr15）Cdk5、桩蛋白/整合素β-1 共定位。Cdk5 抑制（罗昔洛韦）导致肌动蛋白细胞骨架紊乱、蛋白质共定位的预防和运动的抑制。表达 Cdk5（D144N）激酶突变体的细胞无法伸展、迁移和形成管状结构或芽，而 Cdk5 野生型过表达在体外显示出增强的运动性和血管生成能力，在缺氧时仍然保持。基因微阵列研究表明肌细胞增强因子（MEF2C）是 Cdk5 介导的体外血管生成的底物。MEF2C 与 Cdk5 发生核共免疫沉淀，siRNA 敲低后几乎完全抑制分化和芽形成。在缺氧条件下，插入 Cdk5/p25 抑制肽（CIP）载体可保存并增强体外血管生成。这些结果表明，通过 Cdk5 和 p35 存在关键且互补的信号通路，并且协调是在持续缺氧条件下成功血管生成的必需因素。
