"In Vitro" Phosphorylation of Annexin 2 Heterotetramer by Protein Kinase C

Annexin II is a member of a multigene family of Ca 2؉ -regulated, membrane-binding proteins implicated through biochemical and perforated cell experiments in Ca 2؉ -triggered secretion. Within most cells annexin II resides in a tight heterotetrameric complex with a cellular protein ligand, p11, and complex formation is mediated via the N-terminal 14 residues of annexin II including the N-terminal acetyl group. To analyze at the single cell level whether the annexin II-p11 complex is involved in regulated secretion, we used membrane capacitance measurements to follow exocytotic fusion events in bovine aortic endothelial cells manipulated with respect to their annexin II-p11 complex formation. Upon guanosine 5-O-(thiotriphosphate) (GTP␥S) stimulation, the endothelial cells show a significant increase in membrane capacitance which is generally preceded by a transient rise in intracellular Ca 2؉ and thus indicative of the occurrence of Ca 2؉ -regulated secretion. The GTP␥S-induced capacitance increase is markedly reduced in cells loaded with a synthetic peptide, Ac1-14, which corresponds in sequence to the N-terminal 14 residues of annexin II in their correctly acetylated form and which is capable of disrupting preformed annexin II-p11 complexes. The effect of the peptide is highly specific as the nonacetylated variant, N1-14, which is incapable of disrupting annexin II-p11, does not interfere with the GTP␥S-induced increase in membrane capacitance. These data show that intact annexin II-p11 complexes are indispensable for regulated exocytosis to occur in an efficient manner in endothelial cells.Changes in the intracellular Ca 2ϩ levels are known to regulate a variety of cellular processes ranging from the control of cell architecture to intracellular communication, cellular differentiation, and cell death. These cellular responses are mediated through a large number of Ca 2ϩ

Section: Discussionmentioning

confidence: 99%

The Annexin II-p11 Complex Is Involved in Regulated Exocytosis in Bovine Pulmonary Artery Endothelial Cells

König¹,

Prenen²,

Nilius³

et al. 1998

“…Moreover, regulation via the N-terminal domain occurs through several post-translational modifications within the Nterminal sequences of some annexins and includes phosphorylation (27), transglutamination (28), proteolytic cleavage (29), and attachment to other proteins (30,31). The above modifications influence the annexin-induced aggregation of phospholipid vesicles and chromaffin granules, together with the calcium requirement for these reactions (32)(33)(34)(35)(36). In addition, they control the interaction with some S100 calcium-binding proteins (34 -36).…”

Section: Discussionmentioning

confidence: 99%

Histidine Phosphorylation of Annexin I in Airway Epithelia

Muimo

Hornickova

Riemen

et al. 2000

Although [Cl؊ ] i regulates many cellular functions including cell secretion, the mechanisms governing these actions are not known. We have previously shown that the apical membrane of airway epithelium contains a 37-kDa phosphoprotein (p37) whose phosphorylation is regulated by chloride concentration. Using metal affinity (chelating Fe 3؉ -Sepharose) and anion exchange (PO-ROS HQ 20) chromatography, we have purified p37 from ovine tracheal epithelia to electrophoretic homogeneity. Sequence analysis and immunoprecipitation using monoclonal and specific polyclonal antibodies identified p37 as annexin I, a member of a family of Ca We have previously shown that [Cl Ϫ ]/[anions]/ATP/GTP differentially determine the profile of phosphorylation of a number of apical membrane proteins in airway epithelia (3, 4). We identified two of the phosphoproteins, a 19/21-kDa doublet that showed chloride concentration-dependent phosphorylation, as two isoforms of nucleoside-diphosphate kinase (3, 5). We also observed that phosphorylation of a 37-kDa protein (p37) from both human nasal and sheep tracheal epithelia was differentially modulated by nucleotides (GTP, ATP, and GDP), [Cl Ϫ ], and other anion species (3, 4). We found that GTP was the principal phosphate donor for p37 from both species; and in the presence of ATP/GDP, phosphorylation of p37 was enhanced 20-fold. However, this phosphorylation could not be inhibited by known serine/threonine and tyrosine protein kinase inhibitors, suggesting that phosphorylation was not due to the activity of these enzymes.In this study, our objective was to establish the identity of the ion-sensitive phosphoprotein p37 and to characterize its phosphorylated amino acid residue. By selectively enhancing phosphorylation of p37 (3) and by applying metal affinity chromatography, we show that p37 is identical to annexin I and provide evidence for novel annexin phosphorylation on histidine residues. Our data suggest that annexin is a component of an intracellular signaling system involving histidine phosphorylation, which is regulated by chloride concentration. EXPERIMENTAL PROCEDURESSample Preparation-A membrane fraction was prepared from ovine airway epithelia as described previously (3). Briefly, tracheal epithelia were scraped and dislodged into homogenization buffer (250 mM sucrose, 10 mM triethanolamine; one protease inhibitor tablet from Roche Molecular Biochemicals per 50 ml buffer). Pooled scrapings were homogenized and spun at 600 ϫ g for 15 min. The post-nuclear supernatant was re-spun at 100,000 ϫ g for 2 h. The pellet was resuspended in homogenization buffer and spun for 30 min at 16,000 ϫ g (this procedure was repeated three times). All procedures were conducted at 4°C. Use of lactate dehydrogenase as a cytosol marker showed that the pellet contained no cytosol (3). Aliquots of the cytosol and membrane pellet were stored in liquid nitrogen.Phosphorylation and Electronic Autoradiography-Phosphorylation and detection of phosphoproteins were performed as described previously (3). Briefl...

“…Phosphorylation of serine 11 has been suggested to interfere with p11 binding (20,30,31). To test this hypothesis, we isolated a membrane fraction of endothelial cells following treatment with or without plasmin (Fig.…”

Section: Plasmin Induces Activation Of Classical Pkc In Endothelialmentioning

confidence: 99%

Feedback Regulation of Endothelial Cell Surface Plasmin Generation by PKC-dependent Phosphorylation of Annexin A2

He¹,

Sui

Xiong

et al. 2011

In response to blood vessel injury, hemostasis is initiated by platelet activation, advanced by thrombin generation, and tempered by fibrinolysis. The primary fibrinolytic protease, plasmin, can be activated either on a fibrin-containing thrombus or on cells. Annexin A2 (A2) heterotetramer (A2⅐p11) 2 is a key profibrinolytic complex that assembles plasminogen and tissue plasminogen activator and promotes plasmin generation. We now report that, in endothelial cells, plasmin specifically induces activation of conventional PKC, which phosphorylates serine 11 and serine 25 of A2, triggering dissociation of the (A2⅐p11) 2 tetramer. The resulting free p11 undergoes ubiquitinmediated proteasomal degradation, thus preventing further translocation of A2 to the cell surface. In vivo, pretreatment of A2 ؉/؉ but not A2 ؊/؊ mice with a conventional PKC inhibitor significantly reduced thrombosis in a carotid artery injury model. These results indicate that augmentation of fibrinolytic vascular surveillance by blockade of serine phosphorylation is A2-dependent. We also demonstrate that plasmin-induced phosphorylation of A2 requires both cleavage of A2 and activation of Toll-like receptor 4 on the cell surface. We propose that plasmin can limit its own generation by triggering a finely tuned "feedback" mechanism whereby A2 becomes serine-phosphorylated, dissociates from p11, and fails to translocate to the cell surface.The human hemostatic system represents a coordinated balance between procoagulant and anticoagulant/profibrinolytic activities. Together, these systems prevent blood loss while maintaining blood flow. Plasmin, the major protease of the fibrinolytic system, acts to digest insoluble fibrin into soluble fibrin degradation products. Plasmin generation is modulated by plasminogen activators and plasminogen activator inhibitors and is further regulated by binding of plasminogen and its activators to cell surface receptors (1). Membrane-associated annexin A2 (A2) 3 is part of a heterotetrameric complex in which the N termini of two copies of A2 bind to two copies of the S100 family protein, S100A10 (p11) (2). A2, a member of the annexin superfamily, consists of a highly conserved C-terminal phospholipid-binding core domain and an N-terminal ligandinteracting domain (3). Complex formation with p11 increases the affinity of A2 for calcium and phospholipids, thereby directing it to cellular membranes (4). Components of the (A2⅐p11) 2 tetramer, moreover, specifically bind tissue plasminogen activator (t-PA) and plasminogen and strongly enhance plasmin generation (5-8, 52).Several lines of evidence identify the (A2⅐p11) 2 complex as a significant regulator of fibrin balance in vivo. First, A2 Ϫ/Ϫ microvascular endothelial cells lack t-PA cofactor activity, and the fibrin content of highly perfused A2 Ϫ/Ϫ tissues is ϳ2-fold greater than that observed in wild type controls (9). In addition, arterial injury in A2 Ϫ/Ϫ mice is followed by a 2-fold increase in thrombotic vascular occlusion with an equivalent reduction in blood flo...