Stable Transfectants of Smooth Muscle Cell Line Lacking the Expression of Myosin Light Chain Kinase and Their Characterization with Respect to the Actomyosin System

Kishi, Hiroko; Matsukawa, Takashi; Seto, Minoru; Sasaki, Yasuharu; Kanayasu-Toyoda, Toshie; Yamaguchi, Teruhide; Imamura, Michihiro; M, Ito; Karaki, Hideaki; Bao, Jianjun; Nakamura, Akio; Ishikawa, Ryoki; Kohama, Kazuhiro

doi:10.1074/jbc.275.2.1414

Cited by 37 publications

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“…The failure to detect changes in MLC phosphorylation is in apparent contradiction to the role of MLC kinase. However, we experienced a similar contradiction when we allowed VSMCs to migrate towards platelet-derived growth factor (PDGF); the extent of MLC phosphorylation was not elevated even if PDGF existed at a concentration sufficient to cause migration (11). MLC kinase regulates myosin of VSMCs not only by its kinase activity but also by its non-kinase activity as reviewed by Gao et al (12).…”

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“…We cultured GbaSM-4 cells with and without the presence of 6´10 -6 M nicotine; the culture was stopped at various times and processed to detect the phosphorylation using the method described by Kishi et al (11). The extent of phosphorylation expressed as mono-and di-phosphorylated MLC per total MLC was 33.7% in the absence of nicotine and 26.5% -31.6% throughout the treatment of nicotine up to 10 h. Furthermore, neither Fig.…”

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“…The culture was kept at 37°C in an atmosphere of 5% CO2 in air. A cell migratory assay was performed using the Boyden chamber method as described by Kishi et al (11). We used a 96-well microplate chamber with a 3.2-mm-diameter well containing polycarbonate filters with 8-m m pores (Neuro Probe, Inc., Gaithersburg, MD, USA).…”

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Nicotinic Acetylcholine Receptor α7 Subunit Mediates Migration of Vascular Smooth Muscle Cells Toward Nicotine

Li¹,

Zhao²,

Hong³

et al. 2004

J Pharmacol Sci

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Abstract. GbaSM-4 cells, vascular smooth muscle cells (VSMCs) derived from brain basilar arteries, were shown to migrate toward d-nicotine by augmenting the actin cytoskeleton in their cell bodies and lamellipodia, and expression of nicotinic acetylcholine receptor (a 7-nAChR) was detected in GbaSM-4 cells. Their chemotaxis was antagonized by an a 7-nAChR antagonist of methyllycaconitine. It was also antagonized by inhibiting myosin light chain (MLC) kinase and by down-regulating MLC kinase. However, the changes in MLC phosphorylation were not associated with the nicotine treatment, suggesting the involvement of non-kinase activity of MLC kinase as reviewed by Gao et al. (IUBMB Life. 2001;51:337). This plot may work to induce arteriosclerosis during cigarette smoking.

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Nicotinic Acetylcholine Receptor α7 Subunit Mediates Migration of Vascular Smooth Muscle Cells Toward Nicotine

Li¹,

Zhao²,

Hong³

et al. 2004

J Pharmacol Sci

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“…The larger 210-kDa isoform is thought to serve primarily in cytokinesis and cell division and interacts not only with actin but with microtubules as well (5). Down-regulation of the smaller isoform has suggested that this kinase is directly involved in smooth muscle contraction (6,7). In the presence of the Ca 2+ /calmodulin (CaM) complex, MLCK phosphorylates the serine-19 residue of the 20-kDa myosin light chain (MLC), activating myosin ATPase activity with subsequent force development (8).…”

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

et al. 2011

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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.

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“…Moreover, in our literature survey, we found no reports regarding the roles of MLCK other than its kinase activity in the cytokinesis. Therefore, in order to elucidate the functions of MLCK, we knocked-out the MLCK expression in vascular smooth muscle (VSM) cells of vertebrate origin (5,6). The cultured VSM cells were able to proliferate rapidly in spite of the lack of MLCK.…”

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Down-Regulation of Myosin Light Chain Kinase Expression in Vascular Smooth Muscle Cells Accelerates Cell Proliferation: Requirement of Its Actin-binding Domain for Reversion to Normal Rates

et al. 2012

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J Pharmacol Sci 119, 91 -96 (2012) Myosin light-chain kinase (MLCK) is a key regulatory protein of smooth muscle contraction, non-muscle migration, and cytokinesis. MLCK is a calmodulin-dependent kinase that phosphorylates smooth muscle myosin regulatory light-chain (reviewed in ref. 1). MLCK is a multi-domain protein composed of an actin-binding domain at its N-terminal and a myosin-binding domain at its C-terminal, in addition to a kinase domain in its central core. We have been investigating the function of MLCK under conditions where the kinase activity has been deleted (reviewed in ref. 2).Many investigations have shown the importance of the kinase activity of MLCK in cytokinesis (reviewed in ref.3). The contractile ring is formed during cytokinesis by the assembly of myosin with actin filaments. When the myosin light chain is phosphorylated by MLCK, cell division is induced by actin-myosin interaction to originate daughter cells. On the other hand, contrary views that do not support a role for MLCK in cytokinesis, like in the case of C. elegans (4), were also reported. Moreover, in our literature survey, we found no reports regarding the roles of MLCK other than its kinase activity in the cytokinesis. Therefore, in order to elucidate the functions of MLCK, we knocked-out the MLCK expression in vascular smooth muscle (VSM) cells of vertebrate origin (5, 6). The cultured VSM cells were able to proliferate rapidly in spite of the lack of MLCK. In the above reports, we described the functions of MLCK other than its kinase activity in smooth muscle contraction, but on that occasion we did not address its role in pro liferation.Recently, the role of the long isoform of MLCK was identified, and its regulatory role in the cytokinesis was elucidated (7). Because our previous studies have examined only the short isoform of MLCK, we decided herein to down-regulate the expressions of both MLCK isoforms, to readdress the roles of both MLCKs in cytokinesis, by using cell proliferation as a marker.We examined two types of vascular smooth muscle cell lines, GbaSM-4 cells of guinea pig basilar artery origin (8) and SM3 cells of rabbit aorta origin (9). We Nishitokyo, Tokyo 202-8585, Japan Received November 10, 2011; Accepted March 2, 2012 Abstract. Myosin light-chain kinase (MLCK) is a multi-domain protein with kinase and actinbinding domains, among others. Deficiency of MLCK expression in GBaSM-4 vascular smooth muscle cells enhanced cell proliferation rate and shortened cell doubling time. Transient transfection of the MLCK-deficient cells with cDNA constructs of either wild-type MLCK or its mutant lacking the kinase activity reverted the cell proliferation rate to that of wild-type cells, whereas that of MLCK lacking the actin-binding domain maintained cell proliferation at an elevated rate similar to the MLCK-deficient cells. Thus, the actin-binding domain of MLCK seems to play a role in regulating cell proliferation.

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Stable Transfectants of Smooth Muscle Cell Line Lacking the Expression of Myosin Light Chain Kinase and Their Characterization with Respect to the Actomyosin System

Cited by 37 publications

References 44 publications

Nicotinic Acetylcholine Receptor α7 Subunit Mediates Migration of Vascular Smooth Muscle Cells Toward Nicotine

Nicotinic Acetylcholine Receptor α7 Subunit Mediates Migration of Vascular Smooth Muscle Cells Toward Nicotine

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

Down-Regulation of Myosin Light Chain Kinase Expression in Vascular Smooth Muscle Cells Accelerates Cell Proliferation: Requirement of Its Actin-binding Domain for Reversion to Normal Rates

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