Saito T, Shima H, Osawa Y, Nagao M, Hemmings B A, Kishimoto T, Hisanaga S
Laboratory of Cell and Developmental Biology, Faculty of Biosciences, Tokyo Institute of Technology, Yokohama, Japan.
Biochemistry. 1995 Jun 6;34(22):7376-84. doi: 10.1021/bi00022a010.
Neurofilament phosphatase (NF-phosphatase) activity, which dephosphorylates NF proteins phosphorylated by cyclic AMP-dependent protein kinase (A-kinase), was detected in NF fractions prepared from bovine spinal cords. This phosphatase was suggested to be associated with NFs by gel filtration and sedimentation analysis and was further demonstrated by dephosphorylation-dependent binding assay of NFs to microtubules. The NF-associated NF-phosphatase was identified as a type of protein phosphatase 2A (PP2A) by (i) its complete inhibition with 100 nM okadaic acid, at which concentration the purified type 1 protein phosphatase (PP1) was inhibited only 25%; (ii) the absence of effect of inhibitor-2, a specific inhibitor of PP1, on the NF-phosphatase activity; and (iii) the detection of 38-kDa catalytic and 65-kDa regulatory subunits of PP2A by immunoblotting. The NF-associated PP2A was partially solubilized from NFs by a high concentration of MgSO4, and the solubilized PP2A was suggested by gel filtration to be a dimeric holoenzyme consisting of a 38-kDa catalytic and a 65-kDa regulatory subunit. Phosphorylated NF-L, which is assembly incompetent, was induced to assemble into filaments by dephosphorylation with PP2A. These results suggest a role of NF-associated PP2A in preserving filamentous forms of NF in neurons.
在从牛脊髓制备的神经丝（NF）组分中检测到了神经丝磷酸酶（NF-磷酸酶）活性，该酶可使由环磷酸腺苷依赖性蛋白激酶（A激酶）磷酸化的NF蛋白去磷酸化。通过凝胶过滤和沉降分析表明这种磷酸酶与神经丝相关，并且通过神经丝与微管的去磷酸化依赖性结合试验进一步得到证实。通过以下几点将与神经丝相关的NF-磷酸酶鉴定为蛋白磷酸酶2A（PP2A）的一种类型：（i）用100 nM冈田酸可完全抑制其活性，在此浓度下纯化的1型蛋白磷酸酶（PP1）仅被抑制25%；（ii）PP1的特异性抑制剂抑制剂-2对NF-磷酸酶活性无影响；（iii）通过免疫印迹检测到PP2A的38 kDa催化亚基和65 kDa调节亚基。通过高浓度的MgSO4可使与神经丝相关的PP2A从神经丝中部分溶解，凝胶过滤表明溶解的PP2A是由一个38 kDa催化亚基和一个65 kDa调节亚基组成的二聚体全酶。无组装能力的磷酸化NF-L通过用PP2A去磷酸化被诱导组装成丝。这些结果表明与神经丝相关的PP2A在维持神经元中神经丝的丝状形式方面发挥作用。
