Lee W C, Yu J S, Yang S D, Lai Y K
Institute of Life Science, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
J Cell Biochem. 1992 Aug;49(4):378-93. doi: 10.1002/jcb.240490408.
Okadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26-fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphorylated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno-staining with anti-vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two-dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent-solubility of the protein. In untreated cells, the detergent-soluble and -insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure-function regulation of cytoskeleton in the cell.
冈田酸（OA）是一种蛋白磷酸酶抑制剂，已发现它能诱导9L大鼠脑肿瘤细胞中波形蛋白中间丝的过度磷酸化和重排。该过程呈剂量依赖性。波形蛋白的磷酸化最初在30分钟内被400 nM OA增强，当细胞用400 nM OA处理90分钟时达到最大水平（约26倍）。去除OA后，观察到过度磷酸化的波形蛋白发生去磷酸化，并且在细胞在正常生长条件下恢复11小时后，磷酸化水平恢复到对照水平。OA诱导的波形蛋白的磷酸化和去磷酸化同时导致波形蛋白丝的可逆重排和细胞形态的改变。在OA存在下，细胞在进入有丝分裂时变圆，并在恢复11小时后恢复正常外观。用抗波形蛋白抗体进行免疫染色显示，当细胞用OA处理时，波形蛋白丝被拆解并聚集在细胞核周围，但在去除OA后随后又恢复到丝状状态。二维电泳分析进一步表明，波形蛋白的过度磷酸化产生了至少七种具有不同等电点的异构体。此外，波形蛋白磷酸化的增强伴随着该蛋白去污剂溶解性的变化。在未处理的细胞中，去污剂可溶和不可溶的波形蛋白数量相等，但当波形蛋白在OA存在下过度磷酸化时，其溶解性会增加。综上所述，结果表明OA可能参与波形蛋白中间丝的可逆过度磷酸化和重排，这可能在细胞骨架的结构 - 功能调节中起重要作用。
