Properties of filament-bound myosin light chain kinase
Properties of filament-bound myosin light chain kinase
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DOI：
10.1074/jbc.274.9.5987
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发表时间：
1999-02-26
影响因子：
4.8
通讯作者：
Stull, JT
中科院分区：
文献类型：
--
作者：
Lin, PJ;Luby-Phelps, K;Stull, JT
肌球蛋白轻链激酶与细胞中含有肌动蛋白的纤维结合的亲和力大于与f -肌动蛋白的亲和力，然而，细胞中的这种结合是否像与f -肌动蛋白一样受到Ca2+/钙调蛋白的调节尚不清楚。因此，在平滑肌来源的A7r5细胞中，研究了该激酶与应激纤维的结合特性，全长肌球蛋白轻链激酶或缺乏残基2-142的截断突变体以C端含有绿色荧光蛋白的嵌合体表达。在完整的细胞中，全长激酶与应激纤维结合，而截断的激酶在细胞质中显示弥漫性荧光。经皂素渗透后，截尾激酶的荧光消失，而全长激酶的荧光在应力纤维上保留。对全长肌球蛋白轻链激酶在皂素可渗透细胞中光漂白后的荧光强度和荧光恢复的测量表明，Ca2+/钙调蛋白不会将该激酶从这些细丝上解离。然而，丝结合激酶足以实现肌球蛋白调节轻链的Ca2+依赖性磷酸化和应激纤维的收缩。因此，肌凝蛋白轻链激酶与含有肌动蛋白的细丝的解离对于粗丝中的肌凝蛋白轻链的磷酸化是不必要的。我们注意到，激酶的N端和催化核心之间的距离足以跨越细丝和粗丝之间的距离。
Myosin light chain kinase binds to actin-containing filaments from cells with a greater affinity than to F-actin, However, it is not known if this binding in cells is regulated by Ca2+/calmodulin as it is with F-actin. Therefore, the binding properties of the kinase to stress fibers were examined in smooth muscle-derived A7r5 cells, Full-length myosin light chain kinase or a truncation mutant lacking residues 2-142 was expressed as chimeras containing green fluorescent protein at the C terminus. In intact cells, the full-length kinase bound to stress fibers, whereas the truncated kinase showed diffuse fluorescence in the cytoplasm. After permeabilization with saponin, the fluorescence from the truncated kinase disappeared, whereas the fluorescence of the full-length kinase was retained on stress fibers. Measurements of fluorescence intensities and fluorescence recovery after photobleaching of the full-length myosin light chain kinase in saponin-permeable cells showed that Ca2+/calmodulin did not dissociate the kinase from these filaments. However, the filament-bound kinase was sufficient for Ca2+ dependent phosphorylation of myosin regulatory light chain and contraction of stress fibers. Thus, dissociation of myosin light chain kinase from actin-containing thin filaments is not necessary for phosphorylation of myosin light chain in thick filaments. We note that the distance between the N terminus and the catalytic core of the kinase is sufficient to span the distance between thin and thick filaments.