Identification and characterization of a novel (115 kDa) neurofilament-associated kinase

Neurofilaments (NF), the major cytoskeletal component in neuronal cells, are one of the most highly phosphorylated proteins expressed in brain. Apart from the structural role NFs play in maintaining neuronal architecture, little else is known of their function. We describe here evidence suggesting that NF may support many other proteins in the neuronal axoplasm including protein kinases. In order to isolate proteins that bind NF, we first expressed the carboxyl-terminal tail domain of the mouse high-molecular-weight NF subunit (NF-H) as a fusion protein in bacteria and then used this portion of NF-H as a ligand in affinity chromatography. A number of different proteins were isolated from mouse brain lysate that specifically bound to the NF-H column and that did not bind to a control column to which BSA was bound as a ligand. The proteins eluted from the NF-H column contained kinases able to phosphorylate NF proteins efficiently in vitro. We characterized these kinases further by separating proteins on denaturing polyacrylamide gels and reconstituting kinase activity in situ. Using this assay we identified a number of individual kinases including a 115 kDa polypeptide that showed a significant preference for NF proteins as substrate. Native NF was found to be the best substrate for the 115 kDa kinase, followed by a bacterially expressed NF-H nonfusion protein, and NF-H fusion protein. However, low-molecular-weight NF subunit (NF-L) was a poor substrate. Two different NF monoclonal antibodies, SMI31 and SMI32 (Sternberger Monoclonal Inc.), were used to demonstrate further that the 115 kDa kinase is associated with NF in vivo. The kinase was coimmunoprecipitated along with NF by the two NF monoclonal antibodies but appeared to be preferentially associated with phosphorylated forms of NF. We discuss here some of the novel properties of the 115 kDa NF-associated kinase we have termed NAK115 (for NF-associated kinase with a molecular weight of 115 kDa).