Thrombin Inactivates Myosin Light Chain Phosphatase via Rho and Its Target Rho Kinase in Human Endothelial Cells*
- Markus Essler‡§,
- Mutsuki Amano¶,
- Hans-Joachim Kruse‡‖,
- Kozo Kaibuchi¶,
- Peter C. Weber‡ and
- Martin Aepfelbacher‡§**
- From ‡Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Universität München, Pettenkoferstrasse 9, 80336 München, Germany, **Max von Pettenkofer Institut für Medizinische Mikrobiologie, Pettenkoferstrasse 9a, 80336 München, Germany, and the¶Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma, 630-01, Japan
Abstract
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.
Footnotes
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↵* This study was supported by Deutsche Forschungsgemeinschaft Grants Ae11/5–1 and SFB413, by August Lenz Stiftung, and by Wilhelm Sander Stiftung.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence and reprint requests may be addressed. Markus Essler: Tel.: 49-89-5160-4371; E-mail:messler{at}klp.med.uni-muenchen.de. Martin Aepfelbacher: Tel.: 49-89-5160-5264; E-mail:aepfelbacher{at}m3401.mpk.med.uni-muenchen.de.
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↵‖ Present address: Universitätsklinikum Carl Gustav Carus der TU Dresden, Med. Klinik III/Angiologie, Fetscherstr. 74, 01307 Dresden, Germany.
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↵2 M. Essler, M. Amano, H.-J. Kruse, K. Kaibuchi, P. C. Weber, and M. Aepfelbacher, unpublished observation.
- Abbreviations:
- MLCK
-
myosin light chain kinase
- HUVEC
-
human umbilical vein endothelial cells
- MLC
-
myosin light chain
- MBS
-
myosin binding subunit of myosin light chain phosphatase
- PP1
-
protein phosphatase 1
- PP2
-
protein phosphatase 2
- PP1C
-
catalytic subunit of PP1
- PP1M
-
myosin-bound PP1
- RBD
-
Rho-binding domain of Rho kinase
- PAGE
-
polyacrylamide gel electrophoresis
- PBS
-
phosphate-buffered saline
- GTPγS
-
guanosine 5′-3-O-(thio)triphosphate.
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- Received August 27, 1997.
- Revision received May 11, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
