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A more recent version of this article appeared on April 1, 2003
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Submitted on April 26, 2002
Revised on November 27, 2002
Accepted on December 9, 2002
1 Le Centre de recherche en cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, Québec G1R-2J6, Canada
2 MRC Phosphorylation Unit, School of Life Sciences, University of Dundee, MS1/WTB Complex, Dow Street, Dundee DD1 5EH, United Kingdom
3 Department of Cell and Developmental Biology, Upstate Medical University, Syracuse, New York 13210, USA
4 Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Tokyo 113-0033, Japan
5 Faculté de Pharmacie, Université de Montréal, 2900 Edouard-Montpetit, Montréal H3C- 3J7, Canada
* Corresponding author. E-mail address: Jacques.Huot{at}phc.ulaval.ca.
Oxidative stress induces in endothelial cells a quick and transient co-activation of both SAPK2/p38 and ERK MAP kinases. We found that inhibiting the ERK pathway resulted, within five minutes of oxidative stress, in a misassembly of focal adhesions characterized by mislocalization of key proteins such as paxillin. The focal adhesion misassembly that followed ERK inhibition with the MEK inhibitor PD098059 or with a kinase negative mutant of ERK in the presence of H2O2 resulted in a quick and intense membrane blebbing that was associated with important damage to the endothelium. We isolated by 2D-gel electrophoresis a PD098059-sensitive phosphoprotein of 38 kDa that we identified, by mass spectrometry, as tropomyosin-1. In fact, H2O2 induced a time-dependent phosphorylation of tropomyosin that was sensitive to inhibition by PDO98059 and UO126. Tropomyosin phosphorylation was also induced by expression of a constitutive activated form of MEK1 (MEKCA), which confirms that its phosphorylation resulted from the activation of ERK. In unstimulated cells, tropomyosin-1 was found diffuse in the cells whereas it quickly co-localized with actin and stress fibers upon stimulation of ERK by H2O2 or by expression of MEKCA. We propose that phosphorylation of tropomyosin-1 downstream of ERK by contributing to formation of actin filaments increases cellular contractility and promotes the formation of focal adhesions. Incidentally, ML-7, an inhibitor of cell contractility, inhibited phosphorylation of tropomyosin and blocked the formation of stress fibers and focal adhesions, which also led to membrane blebbing in the presence of oxidative stress. Our finding that tropomyosin-1 is phosphorylated downstream of ERK, an event that modulates its interaction with actin, may lead to further understand the role of this protein in regulating cellular functions associated with cytoskeletal remodeling.
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