Regulation of annexin I-dependent aggregation of phospholipid vesicles by protein kinase C

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...

Section: Discussionsupporting

confidence: 56%

Regulation of Annexin A2 by Reversible Glutathionylation

Caplan

Filipenko

Fitzpatrick

et al. 2004

“…Ax II has also been localized to adhesions between confluent Madin-Darby canine kidney cells (25). Ax II has been found to bind directly to anionic liposomes (11,12), and cholesterol has been reported to enhance this binding (26), suggesting that an Ax II/SHP-2 complex could bind directly to membrane lipid domains enriched in cholesterol. Membrane anchoring could also occur through an intrinsic membrane protein such as CD44, which was recently found to anchor Ax II to detergentresistant, cholesterol-rich membranes (rafts) in mammary epithelial EpH4 cells (27).…”

Section: Localization Of Shp-2 and Ax II To Adhesion Bands And Stressmentioning

confidence: 99%

“…Here, we report that extracts prepared with digitonin from confluent, but not subconfluent endothelial cells, contained the tyrosine phosphatase SHP-2. SHP-2 was found in a complex with Ax II, a phospholipidbinding protein (11,12) implicated in cholesterol trafficking (13), and both SHP-2 and Ax II were visualized at intercellular junctions in confluent endothelial monolayers. Our observations indicate that, as endothelial cells reach confluence, a complex containing SHP-2 and Ax II is recruited to intercellular junctions by a novel, cholesterol-dependent, mechanism.…”

mentioning

confidence: 99%

Regulation of the SHP-2 Tyrosine Phosphatase by a Novel Cholesterol- and Cell Confluence-dependent Mechanism

Burkart

Samii

Corvera

et al. 2003

Endothelial cells approaching confluence exhibit marked decreases in tyrosine phosphorylation of receptor tyrosine kinases and adherens junctions proteins, required for cell cycle arrest and adherens junctions stability. Recently, we demonstrated a close correlation in endothelial cells between membrane cholesterol and tyrosine phosphorylation of adherens junctions proteins. Here, we probe the mechanistic basis for this correlation. We find that as endothelial cells reach confluence, the tyrosine phosphatase SHP-2 is recruited to a low-density membrane fraction in a cholesterol-dependent manner. Binding of SHP-2 to this fraction was not abolished by phenyl phosphate, strongly suggesting that this binding was mediated by other regions of SHP-2 beside its SH2 domains. Annexin II, previously implicated in cholesterol trafficking, was associated in a complex with SHP-2, and both proteins localized to adhesion bands in confluent endothelial monolayers. These studies reveal a novel, cholesterol-dependent mechanism for the recruitment of signaling proteins to specific plasma membrane domains via their interactions with annexin II.Endothelial cells approaching confluence in culture undergo profound morphological and functional alterations, including the formation of intercellular junctions and the cessation of cell growth. These changes are associated with reductions in tyrosine phosphorylation levels of both receptor tyrosine kinases (1, 2) and cadherins and catenins (3), the major components of adherens junctions. The dephosphorylation of adherens junctions proteins appears to be necessary for the formation of stable, confluent endothelial monolayers, since increased tyrosine phosphorylation of adherens junctions proteins leads to the disruption of adherens junctions (4 -6). The mechanisms by which cell density decreases tyrosine phosphorylation of endothelial membrane proteins are unknown.One possible mechanism is that increased cell confluence modulates the local environment of signaling proteins in the plasma membrane, thereby altering their response to extracellular ligands. Recently (7), we identified membrane cholesterol as a potential determinant of tyrosine phosphorylation levels in growing endothelial monolayers. Membrane cholesterol increased markedly with cell density in cultures of growing endothelial cells, exhibiting levels 3-4-fold higher upon reaching confluence. Depletion of cholesterol from confluent monolayers of CPAE 1 cells induced the tyrosine phosphorylation of multiple membrane proteins, including the adherens junctions proteins ␥-catenin and pp120, and disrupted adherens junctions.Cholesterol might regulate tyrosine phosphorylation levels through the localization of tyrosine kinases or phosphatases to specific plasma membrane loci. To begin to identify such kinases or phosphatases, we have used very low (0.01%) levels of the sterol-containing detergent digitonin to identify proteins bound to membranes in a cholesterol-dependent manner (8). At these concentrations, digitonin complexes specifica...

“…Serine or tyrosine residues near the N terminus of annexin II can be phosphorylated by protein kinase C and tyrosine kinase pp60 c-Src , respectively. In vitro phosphorylation of these sites reduces the ability of this protein to aggregate vesicles without affecting its membrane binding capacity (21,22). We have shown recently (23,24) that the modification of cysteine or tyrosine residues of annexin II by nitric oxide or peroxynitrite leads to the loss of its liposome aggregation activity.…”

mentioning

confidence: 99%

“…Other groups also used liposomes to mimic granule membrane or plasma membrane (10,15,16,21,22,25). However, a direct biochemical analysis of the ability of AIIt to fuse plasma membrane with lamellar bodies or other granule membranes has not been reported.…”

mentioning

confidence: 99%

Fusion of Lamellar Body with Plasma Membrane Is Driven by the Dual Action of Annexin II Tetramer and Arachidonic Acid

Chattopadhyay

Sun

Wang

et al. 2003

Annexin II has been implicated in membrane fusion during the exocytosis of lamellar bodies from alveolar epithelial type II cells. Most previous studies were based on the fusion assays by using model membranes. In the present study, we investigated annexin II-mediated membrane fusion by using isolated lamellar bodies and plasma membrane as determined by the relief of octadecyl rhodamine B (R18) self-quenching. Immunodepletion of annexin II from type II cell cytosol reduced its fusion activity. Purified annexin II tetramer (AIIt) induced the fusion of lamellar bodies with the plasma membrane in a dose-dependent manner. This fusion is Ca 2؉ -dependent and is highly specific to AIIt because other annexins (I and II monomer, III, IV, V, and VI) were unable to induce the fusion. Modification of the different functional residues of AIIt by N-ethylmaleimide, nitric oxide, or peroxynitrite abolished AIIt-mediated fusion. Arachidonic acid enhanced AIIt-mediated fusion and reduced its Ca 2؉ requirement to an intracellularly achievable level. This effect is due to membranebound arachidonic acid, not free arachidonic acid. Other fatty acids including linolenic acid, palmitoleic acid, myristoleic acid, stearic acid, palmitic acid, and myristic acid had little effect. AIIt-mediated fusion was suppressed by the removal of arachidonic acid from lamellar body and plasma membrane using bovine serum albumin. The addition of arachidonic acid back to the arachidonic acid-depleted membranes restored its fusion activity. Our results suggest that the fusion between lamellar bodies with the plasma membrane is driven by the synergistic action of AIIt and arachidonic acid.Lung surfactant is a surface active material synthesized and secreted by cuboidal alveolar type II cells (1-3). It is composed of phospholipids, mainly dipalmitoylphosphatidylcholine, and surfactant proteins A-D. It minimizes the surface tension at the air-liquid interface by inserting phospholipids between water molecules. As a result, surfactant disrupts the cohesive hydrogen bonds and prevents them from exerting high molecular forces at the alveolar surface. Deficiency of dipalmitoylphosphatidylcholine in the alveolar surface has been associated with infant respiratory distress syndrome.The secretion of surfactant by type II cells involves the translocation, docking, and fusion of lamellar bodies to the plasma membrane. For the lamellar body content to reach its extracellular destination, a continuity of the vesicular lumen and extracellular fluid must be established. This continuity is maintained through the formation of a narrow pore similar to membrane channels (4 -6). The formation of this pore is a complex event involving physical and chemical factors and is regulated by intracellular Ca 2ϩ and proteins (7). Studies on mast cells and chromaffin cells revealed that the opening of fusion pore and extrusion of granule content is influenced by an osmotic force depending upon the osmolarity of the surrounding solution (8).Annexin II plays multidimensional roles in dif...