Rosales J L, Nodwell M J, Johnston R N, Lee K Y
Department of Cell Biology and Anatomy, University of Calgary, Alberta, Canada.
J Cell Biochem. 2000 Apr;78(1):151-9. doi: 10.1002/(sici)1097-4644(20000701)78:1<151::aid-jcb14>3.0.co;2-l.
Cyclin-dependent kinase 5 (Cdk5) exists in large multimeric complexes, but its function and binding partners in these complexes are unclear. We explored these issues by chromatographic and immunochemical analyses of Cdk5 and p25(nck5a) (a neuronal Cdk5 activator) and their associated proteins from bovine brain. Mono-S column enzyme eluates were divided into three fractions and analyzed by gel filtration. The majority of p25(nck5a) from Mono-S fractions I, II, and III eluted from the gel filtration column at approximately 60, 200, and 400 kDa, respectively, and Cdk5 was abundant in fractions >400 kDa. We characterized these macromolecular structures by immunoprecipitating p25(nck5a), followed by a second immunoprecipitation of remaining unbound proteins using a Cdk5 antibody. The p25(nck5a) immunoprecipitates showed association with Cdk5. Amphiphysin was detected in the 400-kDa complex and synapsin I in the >400 kDa structure. The Cdk5 immunoprecipitates, however, revealed abundant retained Cdk5 but no remaining p25(nck5a), indicating that Cdk5 in macromolecular structures is mostly unassociated with p25(nck5a). Thus, we demonstrate: an amphiphysin-associated 400-kDa Cdk5/p25(nck5a) complex, a synapsin I-associated >400-kDa Cdk5/p25(nck5a) complex, and nck5a-free Cdk5 complexes (200 to >400 kDa). Amphiphysin acts as a Cdk5/p25(nck5a) substrate in the 400-kDa complex and we speculate that Cdk5/p25(nck5a) participates in amphiphysin-mediated endocytosis.
细胞周期蛋白依赖性激酶5（Cdk5）以大型多聚体复合物的形式存在，但其在这些复合物中的功能及结合伴侣尚不清楚。我们通过对来自牛脑的Cdk5和p25（nck5a，一种神经元Cdk5激活剂）及其相关蛋白进行色谱分析和免疫化学分析，来探究这些问题。单磺酸柱酶洗脱液被分为三个组分，并通过凝胶过滤进行分析。单磺酸组分I、II和III中的大部分p25（nck5a）分别在凝胶过滤柱上以约60 kDa、200 kDa和400 kDa的分子量被洗脱下来，而Cdk5在分子量大于400 kDa的组分中含量丰富。我们通过免疫沉淀p25（nck5a），然后使用Cdk5抗体对剩余未结合蛋白进行第二次免疫沉淀，来对这些大分子结构进行表征。p25（nck5a）免疫沉淀物显示与Cdk5相关。在400 kDa的复合物中检测到了发动蛋白，在分子量大于400 kDa的结构中检测到了突触素I。然而，Cdk5免疫沉淀物显示保留了大量的Cdk5，但没有剩余的p25（nck5a），这表明大分子结构中的Cdk5大多与p25（nck5a）不相关。因此，我们证明了：一种与发动蛋白相关的400 kDa的Cdk5/p25（nck5a）复合物、一种与突触素I相关的分子量大于400 kDa的Cdk5/p25（nck5a）复合物以及不含nck5a的Cdk5复合物（200 kDa至大于400 kDa）。发动蛋白在400 kDa的复合物中作为Cdk5/p25（nck5a）的底物，我们推测Cdk5/p25（nck5a）参与发动蛋白介导的内吞作用。
