title: Protein disulfide isomerase directly interacts with β-actin Cys374 and regulates cytoskeleton reorganization.
abstract: Recent studies support the role of cysteine oxidation in actin cytoskeleton reorganization during cell adhesion. The aim of this study was to explain whether protein disulfide isomerase (PDI) is responsible for the thiol-disulfide rearrangement in the β-actin molecule of adhering cells. First, we showed that PDI forms a disulfide-bonded complex with β-actin with a molecular mass of 110 kDa. Specific interaction of both proteins was demonstrated by a solid phase binding assay, surface plasmon resonance analysis, and immunoprecipitation experiments. Second, using confocal microscopy, we found that both proteins colocalized when spreading MEG-01 cells on fibronectin. Colocalization of PDI and β-actin could be abolished by the membrane-permeable sulfhydryl blocker, N-ethylmaleimide, by the RGD peptide, and by anti-αIIbβ3 antibodies. Consequently, down-regulation of PDI expression by antisense oligonucleotides impaired the spreading of cells and initiated reorganization of the cytoskeleton. Third, because of transfection experiments followed by immunoprecipitation and confocal analysis, we provided evidence that PDI binds to the β-actin Cys(374) thiol. Formation of the β-actin-PDI complex was mediated by integrin-dependent signaling in response to the adhesion of cells to the extracellular matrix. Our data suggest that PDI is released from subcellular compartments to the cytosol and translocated toward the periphery of the cell, where it forms a disulfide bond with β-actin when MEG-01 cells adhere via the αIIbβ3 integrin to fibronectin. Thus, PDI appears to regulate cytoskeletal reorganization by the thiol-disulfide exchange in β-actin via a redox-dependent mechanism.
authors.affiliation: From the Department of Molecular and Medical Biophysics, Medical University of Lodz, 92-215 Lodz, Poland and.
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Sobierajska
authors.forename: Szymon
authors.initials: S
authors.lastname: Skurzynski
authors.forename: Marta
authors.initials: M
authors.lastname: Stasiak
authors.forename: Jakub
authors.initials: J
authors.lastname: Kryczka
authors.forename: Czeslaw S
authors.initials: CS
authors.lastname: Cierniewski
authors.forename: Maria
authors.initials: M
authors.lastname: Swiatkowska
mesh_terms: D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000970:Antineoplastic Agents / Q000494:pharmacology; D002448:Cell Adhesion / Q000187:drug effects / Q000235:genetics; D002460:Cell Line; D003599:Cytoskeleton / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005353:Fibronectins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009842:Oligopeptides / Q000494:pharmacology; D010084:Oxidation-Reduction / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000235:genetics / Q000378:metabolism; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
chemical_list: D000199:Actins; D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D005353:Fibronectins; D009842:Oligopeptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; C047981:arginyl-glycyl-aspartic acid; D019704:Protein Disulfide-Isomerases; D005033:Ethylmaleimide
keywords: Actin; Adhesion; Cytoskeleton; Disulfide; Thiol