title: LIM-kinase 2 and cofilin phosphorylation mediate actin cytoskeleton reorganization induced by transforming growth factor-beta.
abstract: Reorganization of the actin cytoskeleton in response to growth factor signaling, such as transforming growth factor beta (TGF-beta), controls cell adhesion, motility, and growth of diverse cell types. In Swiss3T3 fibroblasts, a widely used model for studies of actin reorganization, TGF-beta1 induced rapid actin polymerization into stress fibers and concomitantly activated RhoA and RhoB small GTPases. Consequently, dominant-negative RhoA and RhoB mutants blocked TGF-beta1-induced actin reorganization. Because Rho GTPases are known to regulate the activity of LIM-kinases (LIMK), we found that TGF-beta1 induced LIMK2 phosphorylation with similar kinetics to Rho activation. Cofilin and LIMK2 co-precipitated and cofilin became phosphorylated in response to TGF-beta1, whereas RNA interference against LIMK2 blocked formation of new stress fibers by TGF-beta1. Because the kinase ROCK1 links Rho GTPases to LIMK2, we found that inhibiting ROCK1 activity blocked completely TGF-beta1-induced LIMK2/cofilin phosphorylation and downstream stress fiber formation. We then tested whether the canonical TGF-beta receptor/Smad pathway mediates regulation of the above effectors and actin reorganization. Adenoviruses expressing constitutively activated TGF-beta type I receptor led to robust actin reorganization and Rho activation, whereas the constitutively activated TGF-beta type I receptor with mutated Smad docking sites (L45 loop) did not affect either actin organization or Rho activity. In line with this, ectopic expression of the inhibitory Smad7 inhibited TGF-beta1-induced Rho activation and cytoskeletal reorganization. Our data define a novel pathway emanating from the TGF-beta type I receptor and leading to regulation of actin assembly, via the kinase LIMK2.
authors.affiliation: Department of Biochemistry, School of Medicine, University of Crete, GR-71110, Heraklion, Greece.
authors.forename: Lina
authors.initials: L
authors.lastname: Vardouli
authors.forename: Aristidis
authors.initials: A
authors.lastname: Moustakas
authors.forename: Christos
authors.initials: C
authors.lastname: Stournaras
mesh_terms: D051339:Actin Depolymerizing Factors; D000199:Actins / Q000502:physiology*; D000818:Animals; D003599:Cytoskeleton / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000502:physiology*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D054461:Lim Kinases; D051379:Mice; D008840:Microfilament Proteins / Q000502:physiology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology; D051906:Smad7 Protein; D015534:Trans-Activators / Q000502:physiology; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D020743:rhoB GTP-Binding Protein / Q000378:metabolism
chemical_list: D051339:Actin Depolymerizing Factors; D000199:Actins; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; C497806:SMAD7 protein, human; D051906:Smad7 Protein; C497807:Smad7 protein, mouse; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D011494:Protein Kinases; C102895:LIMK2 protein, human; D054461:Lim Kinases; C095325:Limk2 protein, mouse; D017346:Protein Serine-Threonine Kinases; C515851:ROCK1 protein, human; C515854:Rock1 protein, mouse; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein; D020743:rhoB GTP-Binding Protein