Association of neurofilament proteins with neuronal Cdk5 activator

Cdk5 exists in brain extracts in multiple forms, one of which is a macromolecular protein complex comprising Cdk5, neuron-specific Cdk5 activator p35(nck5a) and other protein components (Lee, K.-Y., Resales, J. L., Tang, D., and Wang, J. H. (1996) J. Biol. Chem. 271, 1538-1543). The yeast two-hybrid system was employed to identify p35(nck5a)-interacting proteins from a human brain cDNA library. One of the isolated clones encodes a fragment of glial fibrillary acidic protein, which is a glial-specific protein. Sequence alignment revealed significant homology between the p35(nck5a)-binding fragment of glial fibrillary acidic protein and corresponding regions in neurofilaments. The association between p35(nck5a) and neurofilament medium molecular weight subunit (NF-M) was confirmed by both the yeast two-hybrid assay and direct binding of the bacteria-expressed proteins. The p35(nck5a) binding site on NF-M was mapped to a carboxyl-terminal region of the rod domain, in close proximity to the putative Cdk5 phosphorylation sites in NF-M, A region immediately amino-terminal to the kinase-activating domain in p35(nck5a) is required for its binding with NF-M. In in vitro binding assays, NF-M binds both monomeric p35(nck5a) and the Cdk5/p35(nck5a) complex. The binding of NF-M has no effect on the kinase activity of Cdk5/p35(nck5a).