Myosin Light Chain Kinase / Actin Interaction in Phorbol Dibutyrate–Stimulated Smooth Muscle Cells

• Main Authors: Sean E. Thatcher, Mike E. Fultz, Hideyuki Tanaka, Haruo Hagiwara, Hou-Li Zhang, Ying Zhang, Kohichi Hayakawa, Shinji Yoshiyama, Akio Nakamura, Hong Hui Wang, Takeshi Katayama, Masaru Watanabe, Yuan Lin, Gary L. Wright, Kazuhiro Kohama
• Format: Article
• Language: English
• Published: Elsevier 2011-01-01
• Series: Journal of Pharmacological Sciences
• Online Access: http://www.sciencedirect.com/science/article/pii/S1347861319307285

Abstract.: 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. Keywords:: actin-remodeling, podosome, phorbol 12,13-dibutyrate (PDBu), FRET analysis, myosin light chain kinase (MLCK)-deficient cell