Tsujimura K, Ogawara M, Takeuchi Y, Imajoh-Ohmi S, Ha M H, Inagaki M
Department of Neurophysiology, Tokyo Metropolitan Institute of Gerontology, Japan.
J Biol Chem. 1994 Dec 9;269(49):31097-106.
To investigate the role of intermediate filament (IF) protein phosphorylation by cdc2 kinase during mitosis, we developed a monoclonal antibody 4A4 recognizing Ser55-phosphorylated vimentin. Western blotting indicated that this antibody reacted with vimentin phosphorylated by cdc2 kinase but not with non-phosphorylated vimentin or with vimentin phosphorylated by other kinases such as cAMP-dependent protein kinase, protein kinase C, or Ca(2+)-calmodulin-dependent protein kinase II. Immunofluorescence and immunoelectron microscopy showed that vimentin Ser55 residues distributed in the entire cytoplasmic vimentin filament system are phosphorylated when the cells enter mitosis and dephosphorylated in cytokinesis. All cell lines examined showed a similar appearance of immunoreactivity with antibody 4A4. Fractionation of mitotic cell extracts on Mono-Q Sepharose revealed a single peak of vimentin Ser55 kinase activity, and the anti-p34cdc2 antibody reacted with the 34 kDa band in the kinase containing fractions. Vimentin Ser55 kinase activities were nil in the interphase cell extract. Immunofluorescent evidence using antibody 4A4 and biochemical analysis using vimentin Ser55 peptide showed that the degree of disassembly of vimentin filament of various cell types at early mitotic phase correlated well with the amount of mitotically activated cdc2 kinase.
为了研究中间丝（IF）蛋白在有丝分裂过程中被细胞分裂周期蛋白2（cdc2）激酶磷酸化的作用，我们制备了一种识别丝氨酸55磷酸化波形蛋白的单克隆抗体4A4。蛋白质免疫印迹法表明，该抗体能与被cdc2激酶磷酸化的波形蛋白发生反应，但不与未磷酸化的波形蛋白或被其他激酶（如环磷酸腺苷依赖性蛋白激酶、蛋白激酶C或钙/钙调蛋白依赖性蛋白激酶II）磷酸化的波形蛋白发生反应。免疫荧光和免疫电子显微镜显示，当细胞进入有丝分裂时，分布在整个细胞质波形蛋白丝系统中的波形蛋白丝氨酸55残基被磷酸化，而在胞质分裂时则去磷酸化。所有检测的细胞系与抗体4A4的免疫反应外观相似。用单Q琼脂糖凝胶对有丝分裂细胞提取物进行分级分离，发现波形蛋白丝氨酸55激酶活性呈现单一峰值，抗p34cdc2抗体与含激酶组分中的34 kDa条带发生反应。间期细胞提取物中波形蛋白丝氨酸55激酶活性为零。使用抗体4A4的免疫荧光证据以及使用波形蛋白丝氨酸55肽的生化分析表明，各种细胞类型在有丝分裂早期波形蛋白丝的解聚程度与有丝分裂激活的cdc2激酶量密切相关。
