Lin P j, Luby-Phelps K, Stull J T
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
J Biol Chem. 1997 Mar 14;272(11):7412-20. doi: 10.1074/jbc.272.11.7412.
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 nonmuscle cells.
肌球蛋白轻链激酶可与平滑肌和非肌肉细胞中含肌动球蛋白的细丝结合。然而，尽管有人提出该激酶的N端和C端在体外分别与肌动蛋白和肌球蛋白结合，但在体内实现这种高亲和力结合所需的激酶区域尚不清楚。在杆状病毒系统中表达并纯化了截短的肌球蛋白轻链激酶，这些激酶含有催化核心和钙调蛋白结合结构域，但缺少N端（氨基酸1 - 655）和/或C端（氨基酸1004 - 1147）。所有酶都具有催化活性且依赖Ca2+/钙调蛋白。C端截短的肌球蛋白轻链激酶与经去污剂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。全长激酶对含肌动球蛋白细丝及相关蛋白的表观亲和力，比对纯化的平滑肌F - 肌动蛋白或骨骼肌的肌动球蛋白细丝更高。相比之下，仅N端截短或N端和C端都截短则不会导致明显的结合。通过另外两种检测方法也观察到了类似的效果：荧光标记的肌球蛋白轻链激酶与经去污剂处理的成纤维细胞中含肌动蛋白的应力纤维的结合，以及将荧光标记的激酶显微注射到培养的原代平滑肌细胞后的定位。全长和C端截短的肌球蛋白轻链激酶可与细胞中的细丝结合，而N端截短或N端和C端都截短的肌球蛋白轻链激酶则不能。因此，肌球蛋白轻链激酶的N端而非C端，是在平滑肌和非肌肉细胞中与含肌动球蛋白细丝进行高亲和力结合所必需的。
