Redox Regulation of β-Actin during Integrin-mediated Cell Adhesion

Abstract

Redox sensitivity of actin toward an exogenous oxidative stress has recently been reported. We report here the first evidence of in vivo actin redox regulation by a physiological source of reactive oxygen species, specifically those species generated by integrin receptors during cell adhesion. Actin oxidation takes place via the formation of a mixed disulfide between cysteine 374 and glutathione; this modification is essential for spreading and for cytoskeleton organization. Impairment of actin glutathionylation, either through GSH depletion or expression of the C374A redox-insensitive mutant, greatly affects cell spreading and the formation of stress fibers, leading to inhibition of the disassembly of the actinomyosin complex. These data suggest that actin glutathionylation is essential for cell spreading and cytoskeleton organization and that it plays a key role in disassembly of actinomyosin complex during cell adhesion.Actin is the main component of the cytoskeleton and exists as monomeric G-actin, able upon extracellular stimuli to polymerize into filamentous F-actin. Different structures of F-actin are produced by elongation of preformed filaments by accessory proteins, such as profilin, actin-related protein 2/3, actindepolymerizing factor/cofilin complexes, and many others. These actin-interacting proteins may act through different mechanisms, as forming transversal branching filaments or blocking the extensive elongation at the cell margins (1, 2).Actin cytoskeleton is modified by integrin signal in response to extracellular matrix (ECM) 3 transforming actin architecture from a cortical ring to a plain structure of stress fibers in complete adherent cells. Integrin-mediated cell adhesion proceeds in a step-like program: cells contact the substratum, integrins ligate to the ECM proteins and clusterize, focal contacts form, the cell spreads and finally organizes the actin filaments in stress fibers for mechanical resistance. The control of cytoskeleton organization is mainly due by the action of the members of the family of the small GTPases Rho like Cdc42, Rho, and Rac. In response to growth or chemotactic factors and cell-cell or cell-matrix interaction, Rho is activated thus inducing the formation of actin stress fibers. In contrast, activated Rac provokes actin-dependent membrane ruffling and Cdc42 causes protrusion of actin-rich microspikes from the cell surface (1).Much evidence supports the idea that reactive oxygen species (ROS) act as second messengers thus modulating the activity of signaling proteins upon growth factors and cytokines stimulation. The downstream effect of ROS production is the reversible oxidation of redox-sensible proteins through a direct modification of the thiol group of reactive cysteines (3). Redoxregulated proteins include tyrosine phosphatases, several transcription factors, p53, and the p21Ras family of proto-oncogenes (4). Recently, Chiarugi et al. (5) proposed a redox regulation of integrin signaling during cell adhesion. In this new model, integrin activation is join...

The Requirement of Reversible Cysteine Sulfenic Acid Formation for T Cell Activation and Function

“…We also observed that actin oxidation occurred with differential kinetics compared with the PTPs. It has been previously shown in mouse fibroblasts that cysteine 374 in actin is sensitive to oxidation and that this modification plays a role in leading to glutathionylation of the protein (36). Glutathionylation of actin is required for spreading and cytoskeleton organization.…”

“…2F). We also measured the oxidation of actin, because glutathionylation is important for cell spreading and cytoskeleton reorganization (36). In contrast to the PTPs, sulfenic acid levels in actin did not peak until 120 min after activation (Fig.…”

Cocaine-Induced Adaptations in Cellular Redox Balance Contributes to Enduring Behavioral Plasticity

“…For example, a number of proteins known to be affected by cocaine, including actin, JNK, nuclear kinase kappaB, and cyclic AMP-dependent protein kinase (Hyman et al, 2006;Kalivas and O'Brien, 2008), are also regulated by S-glutathionylation (Fiaschi et al, 2006;Humphries et al, 2005;Klatt and Lamas, 2002;Reynaert et al, 2006). Indeed, kinases such as JNK and ASK1 are susceptible to direct inhibition by GSTpi or thioredoxin, respectively (Adler et al, 1999).…”

“…Importantly, S-glutathionylation of proteins is a significant signaling event in the cellular response to oxidative stress. For example, S-glutathionylation of c-Jun and c-Jun aminoterminal kinase (JNK) is a necessary antecedent for protein phosphorylation and subsequent gene regulation by these proteins (Klatt and Lamas, 2002) and S-glutathionylation of actin promotes the formation of G-actin, thereby affecting cell morphology (Fiaschi et al, 2006). In addition, S-glutathionylation regulates many proteins involved in endoplasmic reticulum (ER) stress and the unfolded protein response that accompanies oxidative stress (Townsend, 2007).…”

Common and Diverging Integrin Signals Downstream of Adhesion and Mechanical Stimuli and Their Interplay with Reactive Oxygen Species

“…Other targets include transcription factors (41,46), kinases (e.g. Src, (47)), small GTPases (48-52), matrix metalloproteinases (41,53), actin (54,55) and actin-associated molecules (56)(57)(58). ROS acting on these targets will give rise to numerous feedback and feedforward reactions and is likely responsible for providing signal amplification and mediating cross-talk between different signal cascades (59).…”