Cdk5/p25nck5a interaction with synaptic proteins in bovine brain
✍ Scribed by Jesusa L. Rosales; Michael J. Nodwell; Randal N. Johnston; Ki-Young Lee
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 178 KB
- Volume
- 78
- Category
- Article
- ISSN
- 0730-2312
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✦ Synopsis
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 ϳ60, 200, and 400 kDa, respectively, and Cdk5 was abundant in fractions Ͼ400 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 Ͼ400 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 Ͼ400-kDa Cdk5/ p25 nck5a complex, and nck5a-free Cdk5 complexes (200 to Ͼ400 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.