Isotani Eiji, Zhi Gang, Lau Kim S, Huang Jian, Mizuno Yusuke, Persechini Anthony, Geguchadze Ramaz, Kamm Kristine E, Stull James T
Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9040, USA.
Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):6279-84. doi: 10.1073/pnas.0308742101. Epub 2004 Apr 7.
Ca(2+)/calmodulin (CaM)-dependent phosphorylation of myosin regulatory light chain (RLC) by myosin light chain kinase (MLCK) initiates smooth muscle contraction and regulates actomyosin-based cytoskeletal functions in nonmuscle cells. The net extent of RLC phosphorylation is controlled by MLCK activity relative to myosin light chain phosphatase activity. We have constructed a CaM-sensor MLCK where Ca(2+)-dependent CaM binding increases the catalytic activity of the kinase domain, whereas coincident binding to the biosensor domain decreases fluorescence resonance energy transfer between two fluorescent proteins. We have created transgenic mice expressing this construct specifically in smooth muscle cells to perform real-time evaluations of the relationship between smooth muscle contractility and MLCK activation in intact tissues and organs. Measurements in intact bladder smooth muscle demonstrate that MLCK activation increases rapidly during KCl-induced contractions but is not maximal, consistent with a limiting amount of cellular CaM. Carbachol treatment produces the same amount of force development and RLC phosphorylation, with much smaller increases in Ca(2+) and MLCK activation. A Rho kinase inhibitor suppresses RLC phosphorylation and force but not MLCK activation in carbachol-treated tissues. These observations are consistent with a model in which the magnitude of an agonist-mediated smooth muscle contraction depends on a rapid but limited Ca(2+)/CaM-dependent activation of MLCK and Rho kinase-mediated inhibition of myosin light chain phosphatase activity. These studies demonstrate the feasibility of producing transgenic biosensor mice for investigations of signaling processes in intact systems.
肌球蛋白轻链激酶（MLCK）介导的肌球蛋白调节轻链（RLC）的Ca²⁺/钙调蛋白（CaM）依赖性磷酸化启动平滑肌收缩，并调节非肌肉细胞中基于肌动球蛋白的细胞骨架功能。RLC磷酸化的净程度由相对于肌球蛋白轻链磷酸酶活性的MLCK活性控制。我们构建了一种CaM传感器MLCK，其中Ca²⁺依赖性CaM结合增加了激酶结构域的催化活性，而与生物传感器结构域的同时结合降低了两种荧光蛋白之间的荧光共振能量转移。我们创建了在平滑肌细胞中特异性表达该构建体的转基因小鼠，以实时评估完整组织和器官中平滑肌收缩性与MLCK激活之间的关系。在完整膀胱平滑肌中的测量表明，在KCl诱导的收缩过程中，MLCK激活迅速增加，但未达到最大值，这与细胞内CaM的有限量一致。卡巴胆碱处理产生相同程度的力发展和RLC磷酸化，细胞内Ca²⁺浓度（[Ca²⁺]i）和MLCK激活的增加要小得多。在卡巴胆碱处理的组织中，Rho激酶抑制剂可抑制RLC磷酸化和力，但不抑制MLCK激活。这些观察结果与一种模型一致，即激动剂介导的平滑肌收缩幅度取决于MLCK的快速但有限的Ca²⁺/CaM依赖性激活以及Rho激酶介导的肌球蛋白轻链磷酸酶活性抑制。这些研究证明了生产转基因生物传感器小鼠用于完整系统中信号传导过程研究的可行性。
