Protein Disulfide Isomerase Directly Interacts with β-Actin Cys374 and Regulates Cytoskeleton Reorganization

Background: PDI regulates cytoskeleton reorganization by the thiol-disulfide exchange in -actin. Results: PDI directly binds to Cys(374) of -actin during cell adhesion and spreading. Conclusion: Interaction of PDI with -actin is induced by integrin-mediated cell adhesion and promotes cytoskeleton reorganization. Significance: PDI is a new regulator of the intramolecular disulfide-thiol rearrangement of -actin in response to IIb3 integrin engagement. 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 -actin molecule of adhering cells. First, we showed that PDI forms a disulfide-bonded complex with -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 -actin could be abolished by the membrane-permeable sulfhydryl blocker, N-ethylmaleimide, by the RGD peptide, and by anti-IIb3 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 -actin Cys(374) thiol. Formation of the -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 -actin when MEG-01 cells adhere via the IIb3 integrin to fibronectin. Thus, PDI appears to regulate cytoskeletal reorganization by the thiol-disulfide exchange in -actin via a redox-dependent mechanism.