Multiple myosin II heavy chain kinases:: Roles in filament assembly control and proper cytokinesis in Dictyostelium
Multiple myosin II heavy chain kinases:: Roles in filament assembly control and proper cytokinesis in Dictyostelium
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DOI：
10.1091/mbc.e05-03-0219
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发表时间：
2005-09-01
影响因子：
3.3
通讯作者：
Egelhoff, TT
中科院分区：
文献类型：
--
作者：
Yumura, S;Yoshida, M;Egelhoff, TT
盘基网柄菌中的肌球蛋白 II 丝组装是通过位于肌球蛋白 II 重链 (MHC) 尾部羧基末端部分的残基的磷酸化来调节的。该系统中的一系列新型蛋白激酶能够在体外磷酸化这些残基，从而驱动细丝分解。先前的研究表明，这些激酶中至少有三种（MHCK A、MHCK B 和 MHCK C）在活细胞中表现出不同的定位模式。我们为该激酶家族创建了一系列单基因、双基因和三基因敲除细胞系。对这些细胞系的分析表明，三种 MHC 激酶似乎代表了能够驱动肌球蛋白 II 丝分解的大部分细胞活性，并揭示了胞质分裂缺陷随着被破坏的激酶数量的增加而增加。使用细胞骨架的生化分级分离和通过光漂白后荧光恢复 (FRAP) 进行的体内测量，我们发现突变体中肌球蛋白 II 过度组装逐渐增加，MHCK A(-)/B-/C- 三重突变体显示出严重的肌球蛋白 II 过度组装。这些研究表明，现已鉴定出对该生物体的生长期和胞质分裂肌球蛋白 II 分解有显着贡献的全部 MHC 激酶。
Myosin II filament assembly in Dictyostelium discoideum is regulated via phosphorylation of residues located in the carboxyl-terminal portion of the myosin II heavy chain (MHC) tail. A series of novel protein kinases in this system are capable of phosphorylating these residues in vitro, driving filament disassembly. Previous studies have demonstrated that at least three of these kinases (MHCK A, MHCK B, and MHCK C) display differential localization patterns in living cells. We have created a collection of single, double, and triple gene knockout cell lines for this family of kinases. Analysis of these lines reveals that three MHC kinases appear to represent the majority of cellular activity capable of driving myosin II filament disassembly, and reveals that cytokinesis defects increase with the number of kinases disrupted. Using biochemical fractionation of cytoskeletons and in vivo measurements via fluorescence recovery after photobleaching (FRAP), we find that myosin II overassembly increases incrementally in the mutants, with the MHCK A(-)/B-/C- triple mutant showing severe myosin II overassembly. These studies suggest that the full complement of MHC kinases that significantly contribute to growth phase and cytokinesis myosin II disassembly in this organism has now been identified.