title: Role of Rho, Rac, and Rho-kinase in phosphorylation of myosin light chain, development of polarity, and spontaneous migration of Walker 256 carcinosarcoma cells.
abstract: As previously shown, constitutive activation of the small GTPase Rho and its downstream target Rho-kinase is crucial for spontaneous migration of Walker carcinosarcoma cells. We now show that after treatment of cells with either the Rho inhibitor C3 exoenzyme or the Rho-kinase inhibitor Y-27632, constitutive myosin light chain (MLC) phosphorylation is significantly decreased, correlating with inhibition of cell polarization and migration. Transfection with a dominant-negative Rho-kinase mutant similarly inhibits cell polarization and MLC phosphorylation. Transfection with a dominant-active Rho-kinase mutant leads to significantly increased MLC phosphorylation, membrane blebbing, and inhibition of cell polarization. This Rho-kinase-induced membrane blebbing can be inhibited by Y-27632, ML-7, and blebbistatin. Unexpectedly, overactivation of RhoA has similar effects as its inhibition. Introduction of a bacterially expressed constitutively activated mutant protein (but not of wild-type RhoA) into the cells or transfection of cells with a constitutively active RhoA mutant both inhibit polarization and decrease MLC phosphorylation. Transfection of cells with constitutively active or dominant-negative Rac both abrogate polarity, and the latter inhibits MLC phosphorylation. Our findings suggest an important role of Rac, Rho/Rho-kinase, and MLCK in controlling myosin activity in Walker carcinosarcoma cells and show that an appropriate level of RhoA, Rac, and Rho-kinase activity is required to regulate cell polarity and migration.
authors.affiliation: Department of Pathology, University of Bern, Murtenstr. 31, CH-3010 Bern, Switzerland.
authors.forename: Marc C
authors.initials: MC
authors.lastname: Gutjahr
authors.forename: Jérémie
authors.initials: J
authors.lastname: Rossy
authors.forename: Verena
authors.initials: V
authors.lastname: Niggli
mesh_terms: D000818:Animals; D002279:Carcinoma 256, Walker / Q000378:metabolism*; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology*; D016764:Cell Polarity / Q000502:physiology*; D022081:Cell Surface Extensions / Q000201:enzymology; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D009154:Mutation / Q000502:physiology; D018994:Myosin Light Chains / Q000378:metabolism*; D024462:Myosin Type II / Q000378:metabolism; D009361:Neoplasm Invasiveness / Q000503:physiopathology; D009362:Neoplasm Metastasis / Q000503:physiopathology; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D020744:rac GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D054460:rho-Associated Kinases
chemical_list: D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D018994:Myosin Light Chains; C491772:RND1 protein, human; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D024462:Myosin Type II; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins