Regulation of Annexin A2 by Reversible Glutathionylation

Abstract

The annexin A2-S100A10 heterotetramer (AIIt) is a multifunctional Ca 2؉ -dependent, phospholipid-binding, and F-actin-binding phosphoprotein composed of two annexin A2 subunits and two S100A10 subunits. It was reported previously that oxidative stress from exogenous hydrogen peroxide or generated in response to tumor necrosis factor-␣ results in the glutathionylation of Cys 8 of annexin A2. In this study, we demonstrate that AIIt is an oxidatively labile protein whose level of activity is regulated by the redox status of its sulfhydryl groups. Oxidation of AIIt by diamide resulted in a timeand concentration-dependent loss of the ability of AIIt to interact with phospholipid liposomes and F-actin. The inhibitory effect of diamide on the activity of AIIt was partially reversed by dithiothreitol. In addition, incubation of AIIt with diamide and GSH resulted in the glutathionylation of AIIt in vitro. Mass spectrometry established the incorporation of 2 mol of GSH/mol of annexin A2 subunit at Cys 8 and Cys 132 . Glutathionylation potentiated the inhibitory effects of diamide on the activity of AIIt. Furthermore, AIIt could be deglutathionylated by glutaredoxin (thiol transferase). Thus, we show for the first time that AIIt can undergo functional reactivation by glutaredoxin, therefore establishing that AIIt is regulated by reversible glutathionylation.The molecular mechanisms by which the cell alleviates oxidative stress and achieves redox homeostasis are still a matter of considerable debate. However, the modulation of the thiol disulfide status of critical cysteine residues on proteins is being recognized as a critical mechanism of oxidative signal transduction as well as a cellular response to protect key regulatory molecules from oxidative insult (1-3). Recent evidence suggests that the reversible covalent modification of cysteine residues by the tripeptide glutathione (␥-Glu-Cys-Gly) plays a significant role in the antioxidant network of the cells and is involved in regulating individual aspects of cellular function (3, 4). Although proteins can bind cysteine, GSH, and homocysteine to generate mixed disulfides, GSH is the dominant ligand, as it occurs in the cell at concentrations between 1 and 10 mM (5, 6). S-Glutathionylation has been shown to alter the function of a number of discrete proteins under oxidant stress (7,8). Furthermore, the formation of a mixed disulfide with glutathione precludes the irreversible oxidation of the cysteine thiol to a sulfinic or sulfonic acid and enables reactivation of the protein by cellular thioreductases.Annexins compose a large multigene family of water-soluble proteins that can bind to negatively charged phospholipids and cellular membranes in a Ca 2ϩ -dependent fashion (9 -11). The annexin family is structurally characterized by two domains: a highly conserved ␣-helical protein core consisting of four 70-amino acid repeats (eight repeats in the case of annexin VI) and a variable N-terminal segment (12). Annexin A2 is unique among the annexins, for its N-terminal tail poss...

Glutathionylation of chloroplast thioredoxin f is a redox signaling mechanism in plants

“…However, our results indicate that the most efficient way to glutathionylate TRX f in vitro is the use of an oxidant in the presence of GSH, as already observed for other proteins (17). Indeed, exposure of cells to ROS appears to be the more effective mechanism in vivo (15,16). Our data suggest that glutathionylation could be a way of decreasing TRXf activity under conditions of enhanced ROS production.…”

“…This modification notably occurs in response to enhanced production of reactive oxygen species (ROS) and͞or increases in oxidized glutathione (GSSG). Glutathionylation can protect proteins from irreversible oxidation and͞or modulate their activity (12)(13)(14)(15)(16)(17). Despite its theoretical appeal as a mechanism transmitting oxidative signals under stress, very little is known about glutathionylation in plants.…”

Roles of Oxidative Stress and Redox Regulation in Atherosclerosis

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Serum Levels of S-Glutathionylated Proteins as a Risk-Marker for Arteriosclerosis Obliterans

“…In response, the sulfhydryl groups are oxidized to form disulfides in a reaction with the reduced form of glutathione disulfide (GSSG) or converted irreversibly to sulfenic, sulfinic, and sulfonic acid derivatives. 11 S-Glutathionylated proteins reported to date include glyceraldehyde-3-phosphate dehydrogenase, 12 annexin A2, 13 protein kinase C, 14 and carbonic anhydrase III. 15 The S-glutathionylation of proteins is initiated in the presence of GSSG.…”