Wible B A, Smith K E, Angelides K J
Department of Physiology and Molecular Biophysics, Baylor College of Medicine, Houston, TX 77030.
Proc Natl Acad Sci U S A. 1989 Jan;86(2):720-4. doi: 10.1073/pnas.86.2.720.
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.
在体内和体外，神经丝（NFs）都是已知的磷酸化程度最高的蛋白质之一。大多数NF磷酸化位点位于蛋白质的羧基末端尾巴上。我们从牛脊髓中分离并鉴定了一种不依赖效应物的神经丝特异性蛋白激酶，它与NF复合物相关，并且对NF三联体中最大的亚基NF-H表现出明显的底物特异性。这种激酶活性从与NaDodSO4/聚丙烯酰胺凝胶上的67 kDa双峰同时出现的NF偶联亲和柱中出现，纯度大于90%。纯化后的酶仅使NF-H尾巴磷酸化，并且依赖于该分子的预先磷酸化。该酶也不会自身磷酸化。虽然NF激酶的分子特性和底物特异性使其与环磷酸腺苷依赖性蛋白激酶、蛋白激酶C、Ca2+/钙调蛋白激酶以及酪蛋白激酶I和II不同，但它表现出某些与生长相关的组蛋白H1激酶相似但又不同的特性。NF激酶的分子特性和特定序列要求表明，这种酶可能在正常和病理状态（如阿尔茨海默病，其中NFs过度磷酸化）下NFs的磷酸化过程中起关键作用。
