Thrombin inactivates myosin light chain phosphatase via Rho and its target Rho kinase in human endothelial cells.
The role of Rho GTPase and its downstream targets Rho kinase and myosin light chain phosphatase in thrombin-induced endothelial cell contraction was investigated. The specific Rho inactivator C3-transferase from Clostridium botulinum as well as microinjection of the isolated Rho-binding domain of Rho kinase or active myosin light chain phosphatase abolished thrombin-stimulated endothelial cell contraction. Conversely, microinjection of constitutively active V14Rho, constitutively active catalytic domain of Rho kinase, or treatment with the phosphatase inhibitor tautomycin caused contraction. These data are consistent with the notion that thrombin activates Rho/Rho kinase to inactivate myosin light chain phosphatase in endothelial cells. In fact, we demonstrate that thrombin transiently inactivated myosin light chain phosphatase, and this correlated with a peak in myosin light chain phosphorylation. C3-transferase abolished the decrease in myosin light chain phosphatase activity as well as the subsequent increase in myosin light chain phosphorylation and cell contraction. These data suggest that thrombin activates the Rho/Rho kinase pathway to inactivate myosin light chain phosphatase as part of a signaling network that controls myosin light chain phosphorylation/contraction in human endothelial cells.
Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Universität München, Pettenkoferstrasse 9, 80336 München, Germany. messler@klp.med.uni-muenchen.de
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M
Essler
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Amano
H J
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Kruse
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Kaibuchi
P C
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Weber
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Aepfelbacher
D036002:ADP Ribose Transferases / Q000378:metabolism; D000470:Alkaloids / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology; D001905:Botulinum Toxins; D002227:Carbazoles; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007211:Indoles; D047908:Intracellular Signaling Peptides and Proteins; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism; D043263:Myosin-Light-Chain Phosphatase; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011714:Pyrans; D013141:Spiro Compounds; D013917:Thrombin / Q000378:metabolism*; D054460:rho-Associated Kinases
D000470:Alkaloids; D000935:Antifungal Agents; D002227:Carbazoles; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D007211:Indoles; D047908:Intracellular Signaling Peptides and Proteins; D011714:Pyrans; D013141:Spiro Compounds; C053079:tautomycin; C063769:KT 5926; D019319:Okadaic Acid; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase; D010749:Phosphoprotein Phosphatases; D043263:Myosin-Light-Chain Phosphatase; D013917:Thrombin; D001905:Botulinum Toxins