Kolman M F, Futey L M, Egelhoff T T
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970, USA.
J Cell Biol. 1996 Jan;132(1-2):101-9. doi: 10.1083/jcb.132.1.101.
Phosphorylation of the Dictyostelium myosin II heavy chain (MHC) has a key role in regulating myosin localization in vivo and drives filament disassembly in vitro. Previous molecular analysis of the Dictyostelium myosin II heavy chain kinase (MHCK A) gene has demonstrated that the catalytic domain of this enzyme is extremely novel, showing no significant similarity to the known classes of protein kinases (Futey, L. M., Q. G. Medley, G. P. Côté, and T. T. Egelhoff. 1995. J. Biol. Chem. 270:523-529). To address the physiological roles of this enzyme, we have analyzed the cellular consequences of MHCK A gene disruption (mhck A- cells) and MHCK A overexpression (MHCK A++ cells). The mhck A- cells are viable and competent for tested myosin-based contractile events, but display partial defects in myosin localization. Both growth phase and developed mhck A- cells show substantially reduced MHC kinase activity in crude lysates, as well as significant overassembly of myosin into the Triton-resistant cytoskeletal fractions. MHCK A++ cells display elevated levels of MHC kinase activity in crude extracts, and show reduced assembly of myosin into Triton-resistant cytoskeletal fractions. MHCK A++ cells show reduced growth rates in suspension, becoming large and multinucleated, and arrest at the mound stage during development. These results demonstrate that MHCK A functions in vivo as a protein kinase with physiological roles in regulating myosin II localization and assembly in Dictyostelium cells during both growth and developmental stages.
盘基网柄菌肌球蛋白II重链（MHC）的磷酸化在体内调节肌球蛋白定位中起关键作用，并在体外驱动丝状体解体。先前对盘基网柄菌肌球蛋白II重链激酶（MHCK A）基因的分子分析表明，该酶的催化结构域极其新颖，与已知的蛋白激酶类别没有显著相似性（Futey, L. M., Q. G. Medley, G. P. Côté, and T. T. Egelhoff. 1995. J. Biol. Chem. 270:523 - 529）。为了探究该酶的生理作用，我们分析了MHCK A基因敲除细胞（mhck A-细胞）和MHCK A过表达细胞（MHCK A++细胞）的细胞后果。mhck A-细胞能够存活，并且对于所测试的基于肌球蛋白的收缩事件具有能力，但在肌球蛋白定位方面表现出部分缺陷。生长阶段和发育后的mhck A-细胞在粗裂解物中均显示出MHC激酶活性大幅降低，以及肌球蛋白在抗Triton细胞骨架组分中的显著过度组装。MHCK A++细胞在粗提取物中显示出MHC激酶活性升高，并且肌球蛋白在抗Triton细胞骨架组分中的组装减少。MHCK A++细胞在悬浮培养中显示出生长速率降低，变得大且多核，并在发育过程中的丘状体阶段停滞。这些结果表明，MHCK A在体内作为一种蛋白激酶发挥作用，在盘基网柄菌细胞生长和发育阶段调节肌球蛋白II的定位和组装方面具有生理作用。
