Hisanaga S, Yasugawa S, Yamakawa T, Miyamoto E, Ikebe M, Uchiyama M, Kishimoto T
Department of Anatomy and Cell Biology, Faculty of Medicine, University of Tokyo.
J Biochem. 1993 Jun;113(6):705-9. doi: 10.1093/oxfordjournals.jbchem.a124107.
The dephosphorylation-induced interaction of neurofilaments (NFs) with microtubules (MTs) was investigated by using several phosphatases. Escherichia coli alkaline and wheat germ acid phosphatases increased the electrophoretic mobility of NF-H and NF-M by dephosphorylation, and induced the binding of NF-H to MTs. The binding of NFs to MTs was observed only after the electrophoretic mobility of NF-H approached the exhaustively dephosphorylated level when alkaline phosphatase was used. The number of phosphate remaining when NF-H began to bind to MTs was estimated by measuring phosphate bound to NF-H. NF-H did not bind to MTs even when about 40 phosphates from the total of 51 had been removed by alkaline phosphatase. The removal of 6 further phosphates finally resulted in the association of NF-H with MTs. A similar finding, that the restricted phosphorylation sites in the NF-H tail domain, but not the total amount of phosphates, were important for binding to MTs, was also obtained with acid phosphatases. In contrast to alkaline and acid phosphatases, four classes of protein phosphatases (protein phosphatases 1, 2A, 2B, and 2C) were ineffective for shifting the electrophoretic mobility of NF proteins and for inducing the association of NFs to MTs.
通过使用几种磷酸酶研究了去磷酸化诱导的神经丝（NFs）与微管（MTs）之间的相互作用。大肠杆菌碱性磷酸酶和小麦胚芽酸性磷酸酶通过去磷酸化增加了NF-H和NF-M的电泳迁移率，并诱导了NF-H与MTs的结合。当使用碱性磷酸酶时，只有在NF-H的电泳迁移率接近完全去磷酸化水平后，才观察到NFs与MTs的结合。通过测量与NF-H结合的磷酸盐来估计NF-H开始与MTs结合时剩余的磷酸盐数量。即使碱性磷酸酶已经从总共51个磷酸盐中去除了约40个磷酸盐，NF-H也没有与MTs结合。再去除6个磷酸盐最终导致NF-H与MTs结合。使用酸性磷酸酶也得到了类似的结果，即NF-H尾部结构域中受限的磷酸化位点而非磷酸盐的总量对与MTs的结合很重要。与碱性和酸性磷酸酶不同，四类蛋白磷酸酶（蛋白磷酸酶1、2A、2B和2C）在改变NF蛋白的电泳迁移率以及诱导NFs与MTs结合方面无效。
