Hisanaga S, Gonda Y, Inagaki M, Ikai A, Hirokawa N
Department of Anatomy and Cell Biology, Faculty of Medicine, University of Tokyo, Japan.
Cell Regul. 1990 Jan;1(2):237-48. doi: 10.1091/mbc.1.2.237.
Effects of phosphorylation of the neurofilament L protein (NF-L) on the reassembly system were studied by both sedimentation experiments and low-angle rotary shadowing. Bovine spinal cord NF-L was phosphorylated with 3-4 mol/mol protein by either the catalytic subunit of cAMP-dependent protein kinase or protein kinase C. Phosphorylated NF-L could not assemble into filaments. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C inhibited the same step of the reassembly process. Phosphorylated NF-L remained as an 8-chain complex even in favorable conditions for reassembly. The extent of the effect of phosphorylation on the filamentous structure of NF-L was also investigated by using the catalytic subunit of cAMP-dependent protein kinase. The amount of unassembled NF-L increased linearly with increased phosphorylation in the sedimentation experiments. Structural observations indicated that 1 or 2 mol of phosphorylation is enough to inhibit reassembly and to induce disassembly, and the disassembly process was also observed. The filaments were shown to unravel with disassembly. Star-like clusters, which we reported as being the initial stage of reassembly, were also identified.
通过沉降实验和低角度旋转阴影技术研究了神经丝L蛋白（NF-L）磷酸化对重组系统的影响。用环磷酸腺苷依赖性蛋白激酶的催化亚基或蛋白激酶C将牛脊髓NF-L磷酸化，使其磷酸化程度达到3 - 4摩尔/摩尔蛋白。磷酸化的NF-L不能组装成细丝。环磷酸腺苷依赖性蛋白激酶或蛋白激酶C的磷酸化作用均抑制了重组过程的同一步骤。即使在有利于重组的条件下，磷酸化的NF-L仍保持为8链复合物。还使用环磷酸腺苷依赖性蛋白激酶的催化亚基研究了磷酸化对NF-L丝状结构的影响程度。在沉降实验中，未组装的NF-L量随磷酸化程度的增加而线性增加。结构观察表明，1或2摩尔的磷酸化足以抑制重组并诱导解聚，并且还观察到了解聚过程。细丝显示随着解聚而解开。还鉴定出了我们报道的作为重组初始阶段的星状簇。
