title: Resolution and purification of a neurofilament-specific kinase.
abstract: Both in vivo and in vitro, neurofilaments (NFs) are among the most highly phosphorylated proteins known. The majority of the NF phosphorylation sites reside on the carboxyl-terminal tails of the proteins. We have isolated and characterized an effector-independent neurofilament-specific protein kinase from bovine spinal cord that is associated with the NF complex and exhibits a marked substrate specificity for NF-H, the largest subunit of the NF triplet. This kinase activity emerges from a NF-conjugated affinity column coincident with a 67-kDa doublet on NaDodSO4/polyacrylamide gels and has a purity of greater than 90%. The purified enzyme exclusively phosphorylates NF-H tails and is dependent on prior phosphorylation of this molecule. The enzyme is also not autophosphorylated. While the molecular properties and substrate specificities of the NF kinase distinguish it from cAMP-dependent protein kinase, protein kinase C, Ca2+/calmodulin kinase, and casein kinases I and II, it exhibits certain properties similar to, but different from, the growth-associated histone H1 kinase. The molecular properties and specific sequence requirements of the NF kinase suggest that this enzyme could play a pivotal role in the phosphorylation of NFs in normal and pathological states such as Alzheimer disease, where NFs are hyperphosphorylated.
authors.affiliation: Department of Physiology and Molecular Biophysics, Baylor College of Medicine, Houston, TX 77030.
authors.forename: B A
authors.initials: BA
authors.lastname: Wible
authors.forename: K E
authors.initials: KE
authors.lastname: Smith
authors.forename: K J
authors.initials: KJ
authors.lastname: Angelides
mesh_terms: D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003599:Cytoskeleton / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D007382:Intermediate Filaments / Q000201:enzymology*; D010766:Phosphorylation; D010770:Phosphotransferases / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D013116:Spinal Cord / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D010770:Phosphotransferases