Binding of myosin light chain kinase to cellular actin-myosin filaments
Binding of myosin light chain kinase to cellular actin-myosin filaments
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DOI：
10.1074/jbc.272.11.7412
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发表时间：
1997-03-14
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;LubyPhelps, K;Stull, JT
肌凝蛋白轻链激酶与平滑肌细胞和非肌细胞中含有肌动球蛋白的细丝结合，然而，这种高亲和力结合所需的激酶区域尚不清楚，尽管已经提出N和C端分别与肌动蛋白和肌凝蛋白在体外结合。杆状病毒系统中表达了含有催化核心和钙调素结合域但缺乏N（氨基酸1-655）和/或C（氨基酸1004-1147）末端的截断肌球蛋白轻链激酶，并纯化了这些酶。所有酶都具有催化活性，并且依赖于Ca2+/钙调素。c端切短的肌球蛋白轻链激酶与洗洁精洗涤的平滑肌收缩蛋白结合，类似于重组全长肌球蛋白轻链激酶或从平滑肌中纯化的酶，全长激酶对含有肌动球蛋白的纤维与相关蛋白的表观亲和力大于对纯化的平滑肌f -肌动蛋白或从骨骼肌中纯化的肌动蛋白纤维的表观亲和力。仅在N端或在N端和C端均被切断不会导致明显的结合。在另外两项试验中观察到类似的效果：在洗涤剂处理过的成纤维细胞中，荧光标记的肌球蛋白轻链激酶与含有肌动蛋白的应激纤维结合，以及在培养的原代平滑肌细胞中微量注射荧光标记的激酶定位。全长和C端截断的肌凝蛋白轻链激酶，而不是N端截断的肌凝蛋白轻链激酶，或N端和C端都截断的肌凝蛋白轻链激酶，与细胞中的细丝相结合，因此，肌凝蛋白轻链激酶的N端而不是C端是与平滑肌细胞和非肌肉细胞中含有肌动球蛋白的细丝高亲和力结合所必需的。
Myosin light chain kinase binds to the actomyosin-containing filaments in smooth and nonmuscle cells, However, the region of the kinase necessary for this high affinity binding in vivo is not known, although it has been proposed that the N and C termini bind to actin and myosin in vitro, respectively, Truncated myosin light chain kinases containing the catalytic core and calmodulin-binding domain but lacking N (amino acids 1-655) and/or C (amino acids 1004-1147) termini were expressed in the baculovirus system and purified, All enzymes were catalytically active and Ca2+/calmodulin-dependent, The C-terminal truncated myosin light chain kinase bound to detergent-washed smooth muscle contractile proteins similar to recombinant full-length myosin light chain kinase or enzyme purified from smooth muscle, The apparent affinity of the full-length kinase was greater for the actomyosin-containing filaments with associated proteins than for purified smooth muscle F-actin or actomyosin filaments from skeletal muscle, In contrast, truncations at the N terminus alone or at both N and C termini resulted in no significant binding, Similar effects were observed by two other assays: binding of fluorescently labeled myosin light chain kinases to actin-containing stress fibers in detergent-treated fibroblasts and localization of fluorescently labeled kinases after microinjection into primary smooth muscle cells in culture. The full-length and the C-terminal truncated myosin light chain kinases, but not myosin light chain kinases truncated at the N terminus or both N and C termini, associated with filaments in cells, Thus, the N terminus and not the C terminus of myosin light chain kinase is necessary for high affinity binding to actomyosin-containing filaments in smooth and non-muscle cells.