title: Extracellular signal-regulated kinase mediates phosphorylation of tropomyosin-1 to promote cytoskeleton remodeling in response to oxidative stress: impact on membrane blebbing.
abstract: Oxidative stress induces in endothelial cells a quick and transient coactivation of both stress-activated protein kinase-2/p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases. We found that inhibiting the ERK pathway resulted, within 5 min 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 mitogen-activated protein kinase kinase (MEK) inhibitor PD098059 (2'-amino-3'-methoxyflavone) or with a kinase negative mutant of ERK in the presence of H(2)O(2) resulted in a quick and intense membrane blebbing that was associated with important damage to the endothelium. We isolated by two-dimensional gel electrophoresis a PD098059-sensitive phosphoprotein of 38 kDa that we identified, by mass spectrometry, as tropomyosin-1. In fact, H(2)O(2) induced a time-dependent phosphorylation of tropomyosin that was sensitive to inhibition by PD098059 and UO126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butanediane). Tropomyosin phosphorylation was also induced by expression of a constitutively activated form of MEK1 (MEK(CA)), 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 colocalized with actin and stress fibers upon stimulation of ERK by H(2)O(2) or by expression of MEK(CA). 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 (1-[5iodonaphthalene-1-sulfonyl]homopiperazine, HCl), 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 understanding of the role of this protein in regulating cellular functions associated with cytoskeletal remodeling.
authors.affiliation: Le Centre de Recherche en Cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, Québec G1R 2J6, Canada.
authors.forename: François
authors.initials: F
authors.lastname: Houle
authors.forename: Simon
authors.initials: S
authors.lastname: Rousseau
authors.forename: Nick
authors.initials: N
authors.lastname: Morrice
authors.forename: Mario
authors.initials: M
authors.lastname: Luc
authors.forename: Sébastien
authors.initials: S
authors.lastname: Mongrain
authors.forename: Christopher E
authors.initials: CE
authors.lastname: Turner
authors.forename: Sakae
authors.initials: S
authors.lastname: Tanaka
authors.forename: Pierre
authors.initials: P
authors.lastname: Moreau
authors.forename: Jacques
authors.initials: J
authors.lastname: Huot
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000378:metabolism*; D029721:Drosophila Proteins; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D022001:Focal Adhesions; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D020935:MAP Kinase Signaling System; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D018384:Oxidative Stress; D010766:Phosphorylation; D014335:Tropomyosin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D029721:Drosophila Proteins; C490775:TPM1 protein, human; C097163:Tm2 protein, Drosophila; D014335:Tropomyosin; D006861:Hydrogen Peroxide; D020928:Mitogen-Activated Protein Kinases