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Infection and Immunity, September 2003, p. 5188-5193, Vol. 71, No. 9
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.9.5188-5193.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Cytotoxic Necrotizing Factor 1 of Escherichia coli Stimulates Rho/Rho-Kinase-Dependent Myosin Light-Chain Phosphorylation without Inactivating Myosin Light-Chain Phosphatase in Endothelial Cells

Markus Essler,1 Stefan Linder,1 Barbara Schell,1 Katharina Hüfner,1 Agnès Wiedemann,2 Katharina Randhahn,1 James M. Staddon,3 and Martin Aepfelbacher2*

Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten,1 Max von Pettenkofer-Institut für Medizinische Mikrobiologie, LMU München, 80336 Munich, Germany,2 Eisai London Research Laboratories, University College London, London WC1E 6BT, United Kingdom3

Received 8 April 2003/ Returned for modification 13 May 2003/ Accepted 28 May 2003

Cytotoxic necrotizing factor 1 (CNF-1) is an exotoxin of Escherichia coli that constitutively activates the GTPases Rho, Rac, and CDC42. Stimulation of Rho was shown to enhance myosin light-chain (MLC) phosphorylation via Rho kinase-mediated inhibition of MLC phosphatase in endothelial cells. Here we report that 3 h after CNF stimulation of endothelial cells, RhoA was activated and MLC phosphorylation was increased in a Rho/Rho-kinase-dependent manner, but no decrease in MLC phosphatase activity could be detected. Despite continuous RhoA activation, MLC phosphatase activity was doubled after 24 h of CNF stimulation, and this coincided with decreased MLC phosphorylation and cell spreading. Rac was also activated at 3 to 24 h but did not contribute to MLC phosphorylation, and its amount gradually decreased in the CNF-stimulated cells. CDC42Hs was not activated above control values by CNF. These results suggest that CNF can induce specific decoupling (Rho kinase from MLC phosphatase) and deactivation events in Rho GTPase signaling, potentially reflecting cellular protection mechanisms against permanently active Rho GTPases.

* Corresponding author. Mailing address: Max von Pettenkofer-Institut für Medizinische Mikrobiologie, Pettenkoferstr. 9a, 80336 Munich, Germany. Phone: 49 89 5160-5264. Fax: 49 89 5160-5223. E-mail: aepfelbacher{at}m3401.mpk.med.uni-muenchen.de.

Editor: J. T. Barbieri

Infection and Immunity, September 2003, p. 5188-5193, Vol. 71, No. 9
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.9.5188-5193.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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