Journal of Neuroscience, Vol 14, 1820-1833, Copyright © 1994 by Society for Neuroscience
Identification and characterization of a novel (115 kDa) neurofilament- associated kinase
J Xiao and MJ Monteiro
Molecular and Cell Biology Graduate Program, University of Maryland School of Medicine, Baltimore 21201.
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).
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