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首页> 美国卫生研究院文献>The Journal of Biophysical and Biochemical Cytology >Expression of a myosin regulatory light chain phosphorylation site mutant complements the cytokinesis and developmental defects of Dictyostelium RMLC null cells
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Expression of a myosin regulatory light chain phosphorylation site mutant complements the cytokinesis and developmental defects of Dictyostelium RMLC null cells

机译：肌球蛋白调节性轻链磷酸化位点突变体的表达可补充Dictyostelium RMLC空细胞的胞质分裂和发育缺陷

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摘要

In a number of systems phosphorylation of the regulatory light chain (RMLC) of myosin regulates the activity of myosin. In smooth muscle and vertebrate nonmuscle systems RMLC phosphorylation is required for contractile activity. In Dictyostelium discoideum phosphorylation of the RMLC regulates both ATPase activity and motor function. We have determined the site of phosphorylation on the Dictyostelium RMLC and used site-directed mutagenesis to replace the phosphorylated serine with an alanine. The mutant light chain was then expressed in RMLC null Dictyostelium cells (mLCR-) from an actin promoter on an integrating vector. The mutant RMLC was expressed at high levels and associated with the myosin heavy chain. RMLC bearing a ser13ala substitution was not phosphorylated in vitro by purified myosin light chain kinase, nor could phosphate be detected on the mutant RMLC in vivo. The mutant myosin had reduced actin-activated ATPase activity, comparable to fully dephosphorylated myosin. Unexpectedly, expression of the mutant RMLC rescued the primary phenotypic defects of the mlcR- cells to the same extent as did expression of wild-type RMLC. These results suggest that while phosphorylation of the Dictyostelium RMLC appears to be tightly regulated in vivo, it is not essential for myosin-dependent cellular functions.
机译：在许多系统中，肌球蛋白的调节轻链（RMLC）的磷酸化调节肌球蛋白的活性。在平滑肌和脊椎动物非肌肉系统中，收缩活性需要RMLC磷酸化。在盘基网柄菌中，RMLC的磷酸化调节ATP酶活性和运动功能。我们已经确定了双歧杆菌RMLC上的磷酸化位点，并使用定点诱变用丙氨酸代替了磷酸化的丝氨酸。然后，突变轻链在整合载体上的肌动蛋白启动子上表达于RMLC空Dictyostelium细胞（mLCR-）中。突变体RMLC高水平表达并与肌球蛋白重链相关。纯化的肌球蛋白轻链激酶在体外未将带有ser13ala取代的RMLC磷酸化，在突变体RMLC体内也未检测到磷酸盐。与完全去磷酸化的肌球蛋白相比，突变的肌球蛋白具有降低的肌动蛋白激活的ATPase活性。出乎意料的是，突变体RMLC的表达挽救了mlcR-细胞的主要表型缺陷，其程度与野生型RMLC的表达相同。这些结果表明，虽然在体内对Dictyostelium RMLC的磷酸化作用进行了严格的调节，但对于依赖肌球蛋白的细胞功能并不是必需的。

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