Differential association and localization of myosin phosphatase subunits during agonist-induced signal transduction in smooth muscle.
It has been known for some time that agonist-induced contractions of vascular smooth muscle are often associated with a sensitization of the contractile apparatus to intracellular Ca2+. One mechanism that has been suggested to explain Ca2+ sensitization is inhibition of myosin phosphatase activity. In the present study, we tested the hypothesis that differential localization of the phosphatase might be associated with its inhibition. Quantitative confocal microscopy of freshly dissociated, fully contractile smooth muscle cells was used in parallel with measurements of myosin light chain and myosin phosphatase phosphorylation. The results indicate that, in the smooth muscle cells, the catalytic and targeting subunits of the phosphatase are dissociated from each other in an agonist-specific manner and that the dissociation is accompanied by a slower rate of myosin phosphorylation. Targeting of myosin phosphatase to the cell membrane precedes the dissociation of subunits and is associated with phosphorylation of the targeting subunit at a Rho-associated kinase (ROK) phosphorylation site. The phosphorylation and membrane translocation of the targeting subunit are inhibited by a ROK inhibitor. This dissociation of subunits may provide a mechanism for the decreased phosphatase activity of phosphorylated myosin phosphatase.
Shin
Heung-Mook
HM
Boston Biomedical Research Institute, Watertown, Mass 02472, USA.
Je
Hyun-Dong
HD
Gallant
Cynthia
C
Tao
Terence C
TC
Hartshorne
David J
DJ
Ito
Masaaki
M
Morgan
Kathleen G
KG
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D020134:Catalytic Domain / Q000502:physiology; D002469:Cell Separation; D015237:Dinoprost / Q000494:pharmacology; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005289:Ferrets; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D009119:Muscle Contraction / Q000187:drug effects; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000201:enzymology*; D018994:Myosin Light Chains / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011169:Portal Vein; D011485:Protein Binding / Q000187:drug effects / Q000502:physiology; D054645:Protein Phosphatase 1; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D021122:Protein Subunits; D021381:Protein Transport / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D054460:rho-Associated Kinases
D000316:Adrenergic alpha-Agonists; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; D021122:Protein Subunits; D015237:Dinoprost; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D043263:Myosin-Light-Chain Phosphatase