Rho-dependent agonist-induced spatio-temporal change in myosin phosphorylation in smooth muscle cells
Authors
Miyazaki, KojiYano, Takeo
Schmidt, David J.
Tokui, Toshiya
Shibata, Masao
Lifshitz, Lawrence M.
Kimura, Satoshi
Tuft, Richard A.
Ikebe, Mitsuo
UMass Chan Affiliations
Department of Physiology and Biomedical Imaging GroupDocument Type
Journal ArticlePublication Date
2001-10-24Keywords
AnimalsBiological Transport
COS Cells
Carbachol
Cells, Cultured
Muscle, Smooth
Myosin-Light-Chain Kinase
Myosins
Phosphorylation
Swine
Trachea
rhoA GTP-Binding Protein
Life Sciences
Medicine and Health Sciences
Metadata
Show full item recordAbstract
Agonist-induced translocation of RhoA and the spatio-temporal change in myosin regulatory light chain (MLC20) phosphorylation in smooth muscle was clarified at the single cell level. We expressed green fluorescent protein-tagged RhoA in the differentiated tracheal smooth muscle cells and visualized the translocation of RhoA in a living cell with three-dimensional digital imaging analysis. The stimulation of the cells by carbachol initiated the translocation of green fluorescent protein-tagged wild type RhoA to the plasma membrane within a minute. The change in MLC20 phosphorylation level after carbachol stimulation was monitored by using phospho-Ser-19-specific antibody recognizing the phosphorylated MLC20 in single cells. Cells expressing the dominant negative form (T19N) of RhoA significantly suppressed sustained MLC20 phosphorylation during the prolonged phase (>300 s), whereas the maximum phosphorylation level (reached at 10 s after stimulation) of these cells was not significantly different from the control cells. The kinetics of RhoA translocation was consistent with that of sustained myosin phosphorylation, suggesting the involvement of a RhoA pathway. Carbachol stimulation increased myosin phosphorylation within a minute both at the cortical and the central region. On the other hand, during prolonged phase, myosin phosphorylation was sustained at the cortical region of the cells but not at the central fibers. A myosin light chain kinase-specific inhibitor, ML-9, diminished myosin phosphorylation at the central region of the cells after the stimulation but not at the cortical area. On the other hand, Y-27632, a Rho kinase-specific inhibitor, diminished myosin phosphorylation at the cortical region but not the central region. The results clearly show that the myosin light chain kinase pathway and the Rho pathway distinctly change myosin phosphorylation in smooth muscle cells in both a temporal and spatial manner.Source
J Biol Chem. 2002 Jan 4;277(1):725-34. Epub 2001 Oct 22. Link to article on publisher's siteDOI
10.1074/jbc.M108568200Permanent Link to this Item
http://hdl.handle.net/20.500.14038/42379PubMed ID
11673466Related Resources
Link to Article in PubMedae974a485f413a2113503eed53cd6c53
10.1074/jbc.M108568200