Quantitative measurements of Ca2+/calmodulin binding and activation of myosin light chain kinase in cells
Quantitative measurements of Ca2+/calmodulin binding and activation of myosin light chain kinase in cells
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DOI：
10.1016/s0014-5793(03)01456-x
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发表时间：
2004-01-16
期刊：
影响因子：
3.5
通讯作者：
Stull, JT
中科院分区：
文献类型：
--
作者：
Geguchadze, R;Zhi, G;Stull, JT
肌球蛋白II调节轻链(RLC)由钙/钙调蛋白(CaM)依赖的肌球蛋白轻链激酶(MLCK)磷酸化，参与多种细胞肌动蛋白的细胞骨架功能。我们用荧光生物传感器MLCK定量检测了MLCK的活性，在体外，依赖于钙离子的激酶活性的增加与荧光共振能量转移(FRET)的减少是一致的。在稳定转染CaM传感器MLCK的细胞中，增加[Ca~(2+)](I)可同时增加MLCK的活性和RLC的磷酸化。在低[Ca~(2+)](I)时，未发现CaM结合但不激活MLCK的证据。在饱和[Ca~(2+)](I)时，MLCK未被完全激活，可能是由于细胞内Ca~(2+)/CaM的有效性有限。(C)2003年欧洲生化学会联合会。爱思唯尔出版，版权所有。
Myosin II regulatory light chain (RLC) phosphorylation by Ca2+/calmodulin (CaM)-dependent myosin light chain kinase (MLCK) is implicated in many cellular actin cytoskeletal functions. We examined MLCK activation quantitatively with a fluorescent biosensor MLCK where Ca2+-dependent increases in kinase activity were coincident with decreases in fluorescence resonance energy transfer (FRET) in vitro. In cells stably transfected with CaM sensor MLCK, increasing [Ca2+](i) increased MLCK activation and RLC phosphorylation coincidently. There was no evidence for CaM binding but not activating MLCK at low [Ca2+](i). At saturating [Ca2+](i), MLCK was not fully activated probably due to limited availability of cellular Ca2+/CaM. (C) 2003 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.