Cdk5/p25(nck5a) interaction with synaptic proteins in bovine brain.
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.
Rosales
J L
JL
Department of Cell Biology and Anatomy, University of Calgary, Alberta, Canada. rosales@ucalgary.ca
Nodwell
M J
MJ
Johnston
R N
RN
Lee
K Y
KY
D000818:Animals; D001921:Brain / Q000378:metabolism*; D002417:Cattle; D002850:Chromatography, Gel; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases / Q000302:isolation & purification / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D010766:Phosphorylation; D013569:Synapses / Q000378:metabolism*
D009419:Nerve Tissue Proteins; C089495:neuronal Cdk5 activator (p25-p35); C075637:amphiphysin; D051360:Cyclin-Dependent Kinase 5; D018844:Cyclin-Dependent Kinases