title: Molecular characterization of the effects of Y-27632.
abstract: Many key cellular functions, such as cell motility and cellular differentiation are mediated by Rho-associated protein kinases (ROCKs). Numerous studies have been conducted to examine the ROCK signal transduction pathways involved in these motile and contractile events with the aid of pharmacological inhibitors such as Y-27632. However the molecular mechanism of action of Y-27632 has not been fully defined. To assess the relative contribution of these Rho effectors to the effects of Y-27632, we compared the cytoskeletal phenotype, wound healing and neurite outgrowth in cells treated with Y-27632 or subjected to knockdown with ROCK-I, ROCK-II or PRK-2- specific siRNAs. Reduction of ROCK-I enhances the formation of thin actin-rich membrane extensions, a phenotype that closely resembles the effect of Y-27632. Knockdown of ROCK II or PRK-2, leads to the formation of disc-like extensions and thick actin bundles, respectively. The effect of ROCK-I knockdown also mimicked the effect of Y-27632 on wound closer rates. ROCK-I knockdown and Y-27632 enhanced wound closure rates, while ROCK-II and PRK-2 were not appreciably different from control cells. In neurite outgrowth assays, knockdown of ROCK-I, ROCK-II or PRK-2 enhances neurite lengths, however no individual knockdown stimulated neurite outgrowth as robustly as Y-27632. We conclude that several kinases contribute to the global effect of Y-27632 on cellular responses.
authors.affiliation: Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.
authors.forename: Hassina
authors.initials: H
authors.lastname: Darenfed
authors.forename: Bama
authors.initials: B
authors.lastname: Dayanandan
authors.forename: Tong
authors.initials: T
authors.lastname: Zhang
authors.forename: Sidney H-K
authors.initials: SH
authors.lastname: Hsieh
authors.forename: Alyson E
authors.initials: AE
authors.lastname: Fournier
authors.forename: Craig A
authors.initials: CA
authors.lastname: Mandato
mesh_terms: D000577:Amides / Q000494:pharmacology*; D000818:Animals; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D003599:Cytoskeleton / Q000187:drug effects*; D005347:Fibroblasts / Q000166:cytology; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D016501:Neurites / Q000187:drug effects / Q000502:physiology; D009474:Neurons / Q000166:cytology; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D014162:Transfection; D014945:Wound Healing / Q000187:drug effects; D054460:rho-Associated Kinases
chemical_list: D000577:Amides; D047908:Intracellular Signaling Peptides and Proteins; D011725:Pyridines; D034741:RNA, Small Interfering; C108830:Y 27632; C061570:protein kinase N; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D011493:Protein Kinase C