Xiao J, Monteiro M J
Molecular and Cell Biology Graduate Program, University of Maryland School of Medicine, Baltimore 21201.
J Neurosci. 1994 Mar;14(3 Pt 2):1820-33. doi: 10.1523/JNEUROSCI.14-03-01820.1994.
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).
神经丝（NF）是神经元细胞中主要的细胞骨架成分，是大脑中表达的磷酸化程度最高的蛋白质之一。除了NF在维持神经元结构中所起的结构作用外，人们对其功能知之甚少。我们在此描述的证据表明，NF可能在神经元轴浆中支持许多其他蛋白质，包括蛋白激酶。为了分离与NF结合的蛋白质，我们首先在细菌中表达小鼠高分子量NF亚基（NF-H）的羧基末端尾域作为融合蛋白，然后将NF-H的这一部分用作亲和色谱中的配体。从小鼠脑裂解物中分离出许多不同的蛋白质，它们特异性地结合到NF-H柱上，而不结合以牛血清白蛋白（BSA）作为配体结合的对照柱。从NF-H柱上洗脱的蛋白质含有能够在体外有效磷酸化NF蛋白的激酶。我们通过在变性聚丙烯酰胺凝胶上分离蛋白质并原位重建激酶活性，进一步对这些激酶进行了表征。使用该测定法，我们鉴定了许多单个激酶，包括一种115 kDa的多肽，该多肽对NF蛋白作为底物表现出明显的偏好。发现天然NF是115 kDa激酶的最佳底物，其次是细菌表达的NF-H非融合蛋白和NF-H融合蛋白。然而，低分子量NF亚基（NF-L）是一种较差的底物。使用两种不同的NF单克隆抗体SMI31和SMI32（Sternberger单克隆公司）进一步证明，115 kDa激酶在体内与NF相关。该激酶与两种NF单克隆抗体一起与NF共免疫沉淀，但似乎优先与磷酸化形式的NF相关。我们在此讨论我们称为NAK115（分子量为115 kDa的NF相关激酶）的115 kDa NF相关激酶的一些新特性。
