Department of Pharmacology, Physiology, and Toxicology, The Joan C. Edwards School of Medicine, Marshall University, Huntington, WV 25704, USA.
J Pharmacol Sci. 2011;116(1):116-27. doi: 10.1254/jphs.10296fp. Epub 2011 May 3.
Previous work has suggested that in addition to its kinase activity, myosin light chain kinase (MLCK) exhibits non-kinase properties within its N-terminus that could influence cytoskeletal organization of smooth muscle cells (A. Nakamura et al. Biochem Biophys Res Commun. 2008;369:135-143). Myosin ATPase activity measurements indicate that the 26-41 peptide of MLCK significantly decreases ATPase activity as the concentration of this peptide increases. Sliding velocity of actin-filaments on myosin and stress responses in skinned smooth muscle tissue are also inhibited. Peptide-mediated uptake and the microinjection technique in cells indicate that the peptide was necessary for actin-filament stabilization. Fluorescence resonance energy transfer analysis indicated that in the presence of MLCK, α-actin but not β-actin remodeled during phorbol 12,13-dibutyrate (PDBu)-induced contractions. PDBu also induced podosomes in the cell. When MLCK expression was down-regulated by introduction of RNAi for MLCK by lentivirus vector into the cells, we failed to observe the podosome induction upon PDBu stimulation. Rescue experiments indicate that the non-kinase activity of MLCK plays an important role in maintaining actin stress fibers and in the PDBu-induced reorganization of actin-filaments in smooth muscle cells.
先前的研究表明，除了激酶活性外，肌球蛋白轻链激酶（MLCK）在其 N 端还表现出非激酶特性，这些特性可能影响平滑肌细胞的细胞骨架组织（A. Nakamura 等人，《生物化学与生物物理研究通讯》。2008 年；369:135-143）。肌球蛋白 ATPase 活性测量表明，MLCK 的 26-41 肽显著降低 ATPase 活性，随着该肽浓度的增加而降低。肌球蛋白上的肌动蛋白丝的滑动速度和去皮平滑肌组织的应激反应也受到抑制。肽介导的摄取和细胞内的微注射技术表明，该肽对于肌动蛋白丝的稳定是必需的。荧光共振能量转移分析表明，在 MLCK 存在的情况下，α-肌动蛋白而不是β-肌动蛋白在佛波醇 12,13-二丁酸酯（PDBu）诱导的收缩过程中发生重塑。PDBu 还会在细胞中诱导足突。当通过慢病毒载体将 MLCK 的 RNAi 引入细胞中下调 MLCK 的表达时，我们未能观察到 PDBu 刺激后足突的诱导。挽救实验表明，MLCK 的非激酶活性在维持肌动蛋白应力纤维和 PDBu 诱导的平滑肌细胞中肌动蛋白丝重排方面起着重要作用。
