Eukaryotic vacuolar-type H؉ -ATPases (V-ATPases) are regulated by the reversible disassembly of the active V 1 V 0 holoenzyme into a cytosolic V 1 complex and a membrane-bound V 0 complex. The signaling cascades that trigger these events in response to changing cellular conditions are largely unknown. We report that the V 1 subunit C of the tobacco hornworm Manduca sexta interacts with protein kinase A and is the only V-ATPase subunit that is phosphorylated by protein kinase A. Subunit C can be phosphorylated as single polypeptide as well as a part of the V 1 complex but not as a part of the V 1 V 0 holoenzyme. Both the phosphorylated and the unphosphorylated form of subunit C are able to reassociate with the V 1 complex from which subunit C had been removed before. Using salivary glands of the blowfly Calliphora vicina in which V-ATPase reassembly and activity is regulated by the neurohormone serotonin via protein kinase A, we show that the membrane-permeable cAMP analog 8-(4-chlorophenylthio)adenosine-3,5-cyclic monophosphate (8-CPT-cAMP) causes phosphorylation of subunit C in a tissue homogenate and that phosphorylation is reduced by incubation with antibodies against subunit C. Similarly, incubation of intact salivary glands with 8-CPT-cAMP or serotonin leads to the phosphorylation of subunit C, but this is abolished by H-89, an inhibitor of protein kinase A. These data suggest that subunit C binds to and serves as a substrate for protein kinase A and that this phosphorylation may be a regulatory switch for the formation of the active V 1 V 0 holoenzyme. Vacuolar type Hϩ -ATPases (V-ATPases) 3 are the most versatile proton pumps, being common to all eukaryotic organisms, and are found in endomembrane systems and in the plasma membrane (1-3). V-ATPases are multi-subunit transporters composed of a catalytic ATP-hydrolyzing V 1 complex (Ϸ550 kDa), which resides on the cytoplasmic side of the membrane, and a membrane-bound proton-translocating V 0 complex (Ϸ250 kDa). V-ATPase-dependent proton pumping is essential for cellular pH homeostasis and creates an electrochemical proton gradient that energizes secondary transport mechanisms in a wide variety of organelles and membrane systems. Acidification of organelles by V-ATPase activity is crucial to various cellular processes such as neurotransmitter uptake into synaptic vesicles, intracellular protein trafficking, and the secretion and activation of lysosomal enzymes for protein processing and degradation (4 -7). Located in the plasma membrane of specialized cells, V-ATPases are involved in processes such as cation secretion, bone resorption, renal acidification, and osmoregulation (8 -16). With respect to this diversity of function, mutations in genes encoding V-ATPase subunits obviously lead to several diseases, e.g. osteopetrosis (17) or renal tubular acidosis (18).Several mechanisms have been proposed for the regulation of V-ATPase activity (3). The most prominent and physiologically relevant mechanism is the reversible disassembly of the V-ATPase holoenzyme i...

Reversible assembly of the V0V1 holoenzyme from V0 and V1 subcomplexes is a widely used mechanism for regulation of vacuolar-type H ؉ -ATPases (V-ATPases) in animal cells. In the blowfly (Calliphora vicina) salivary gland, V-ATPase is located in the apical membrane of the secretory cells and energizes the secretion of a KCl-rich saliva in response to the hormone serotonin. We have examined whether the cAMP pathway, known to be activated by serotonin, controls V-ATPase assembly and activity. Fluorescence measurements of pH changes at the luminal surface of isolated glands demonstrate that cAMP, Sp-adenosine-3 ,5 -cyclic monophosphorothioate, or forskolin, similar to serotonin, cause VATPase-dependent luminal acidification. In addition, V-ATPasedependent ATP hydrolysis increases upon treatment with these agents. Immunofluorescence microscopy and pelleting assays have demonstrated further that V1 components become translocated from the cytoplasm to the apical membrane and V-ATPase holoenzymes are assembled at the apical membrane during conditions that increase intracellular cAMP. Because these actions occur without a change in cytosolic Ca 2؉ , our findings suggest that the cAMP pathway mediates the reversible assembly and activation of V-ATPase molecules at the apical membrane upon hormonal stimulus.regulation ͉ translocation ͉ secretion T he vacuolar-type H ϩ -ATPases (V-ATPases) are multisubunit heteromeric complexes that are organized into two domains, designated V 0 and V 1 (1-4). V 0 forms a membranespanning proton-translocating complex; in yeast, it is composed of at least five different subunits termed a, c, cЈ, cЉ, and d, of which subunit c binds bafilomycin A 1 , a specific inhibitor of V-ATPases (5-8). The V 1 sector is attached to the cytoplasmic side of the V 0 sector, consists of at least eight different subunits termed A-H, and contains catalytic and noncatalytic ATPbinding sites. V-ATPase is vital for almost every eukaryotic cell and fulfils a variety of functions. On intracellular acidic membrane systems, such as endosomes, lysosomes, and synaptic vesicles, these proton pumps are involved in protein sorting during biosynthetic and endocytotic pathways, zymogen activation, and transmitter uptake, respectively (3, 4). V-ATPase molecules in the plasma membrane of animal cells, especially on the apical plasma membrane of epithelial cells, contribute to intracellular pH homeostasis, extracellular acidification, or alkalinization, or they energize the plasma membrane for secondary transport processes (3, 4).In some cells, V-ATPase requires a considerable amount of energy. For reasons of economy, it is thus favorable if V-ATPase activity is adapted to the physiological needs of the cell. Several regulatory mechanisms have been identified (1,2,4,9). One of these is the reversible dissociation of the V 1 sector from the V 0 sector, as revealed by experiments performed in yeast, midgut epithelial cells of the tobacco hornworm Manduca sexta, mammalian dendritic cells, and renal epithelial cells (10-16). In th...

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The liver responds to an increase in blood glucose levels in the postprandial state by uptake of glucose and conversion to glycogen. Liver glycogen synthase (GYS2), a key enzyme in glycogen synthesis, is controlled by a complex interplay between the allosteric activator glucose-6-phosphate (G6P) and reversible phosphorylation through glycogen synthase kinase-3 and the glycogen-associated form of protein phosphatase 1. Here, we initially performed mutagenesis analysis and identified a key residue (Arg582) required for activation of GYS2 by G6P. We then used GYS2 Arg582Ala knockin (+/R582A) mice in which G6P-mediated GYS2 activation had been profoundly impaired (60–70%), while sparing regulation through reversible phosphorylation. R582A mutant–expressing hepatocytes showed significantly reduced glycogen synthesis with glucose and insulin or glucokinase activator, which resulted in channeling glucose/G6P toward glycolysis and lipid synthesis. GYS2+/R582A mice were modestly glucose intolerant and displayed significantly reduced glycogen accumulation with feeding or glucose load in vivo. These data show that G6P-mediated activation of GYS2 plays a key role in controlling glycogen synthesis and hepatic glucose-G6P flux control and thus whole-body glucose homeostasis.

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Cardiomyocyte death as a result of viral infection is an excellent model for dissecting the inflammatory stress response that occurs in heart tissue. We reported earlier that a specific proteasome isoform, the immunoproteasome, prevents exacerbation of coxsackievirus B3 (CVB3)-induced myocardial destruction and preserves cell vitality in heart tissue inflammation. Following the aim to decipher molecular targets of immunoproteasome-dependent proteolysis, we investigated the function and regulation of the soluble PRR Pentraxin3 (PTX3). We show that the ablation of PTX3 in mice aggravated CVB3-triggered inflammatory injury of heart tissue, without having any significant effect on viral titers. Thus, there might be a role of PTX3 in preventing damageassociated molecular pattern-induced cell death. We found that the catalytic activity of the immunoproteasome subunit LMP7 regulates the timely availability of factors controlling PTX3 production. We report on immunoproteasome-dependent alteration of ERK1/2 and p38MAPKs, which were both found to be involved in PTX3 expression control. Our finding of a cardioprotective function of immunoproteasome-dependent PTX3 expression revealed a crucial mechanism of the stress-induced damage response in myocardial inflammation. In addition to antigen presentation and cytokine production, proteolysis by the immunoproteasome can also regulate the innate immune response during viral infection.Keywords: Infection r Inflammation r Innate immunity r Myocarditis r Pentraxin3 r Proteasome r Stress response r Virus Additional supporting information may be found in the online version of this article at the publisher's web-site IntroductionThe myocardium can be injured by various pathophysiological processes. Myocarditis is an excellent model of heart muscle Correspondence: Dr. Antje Voigt e-mail: antje.voigt@charite.de injury for dissecting processes of the heart stress response to inflammation. In developed countries, myocarditis most commonly results from a virus infection, often by coxsackievirus B3 (CVB3) and adenoviruses. Fulminant injury of the heart muscle in * These authors contributed equally to this work.C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu 620Anna Paeschke et al. Eur. J. Immunol. 2016. 46: 619-633 viral myocarditis is attributed to both, direct virus-mediated cytotoxic effects and destruction of the heart muscle by the immune system [1,2]. The balance of intrinsic and innate immune mechanisms determines whether there is repair of the heart muscle or progression to chronic inflammation. This process involves the engagement of pattern recognition receptors (PRRs) either with pathogen-associated molecular patterns (PAMPs) such as viral RNA, or, upon the release of endogenous material, with damageassociated molecular patterns (DAMPs) [3]. Our knowledge on disease course in patients comes mainly from the mouse model of CVB3 myocarditis. Cardiotropic CVB3 infects mice and replicates to high titers, causing acute myocarditis (ACM) that faithfully mirrors...

The vacuolar H(+)-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary gland cells energizes the secretion of a KCl-rich saliva in response to the neurohormone serotonin (5-HT). We have shown previously that exposure to 5-HT induces a cAMP-mediated reversible assembly of V(0) and V(1) subcomplexes to V-ATPase holoenzymes and increases V-ATPase-driven proton transport. Here, we analyze whether the effect of cAMP on V-ATPase is mediated by protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac), the cAMP target proteins that are present within the salivary glands. Immunofluorescence microscopy shows that PKA activators, but not Epac activators, induce the translocation of V(1) components from the cytoplasm to the apical membrane, indicative of an assembly of V-ATPase holoenzymes. Measurements of transepithelial voltage changes and microfluorometric pH measurements at the luminal surface of cells in isolated glands demonstrate further that PKA-activating cAMP analogs increase cation transport to the gland lumen and induce a V-ATPase-dependent luminal acidification, whereas activators of Epac do not. Inhibitors of PKA block the 5-HT-induced V(1) translocation to the apical membrane and the increase in proton transport. We conclude that cAMP exerts its effects on V-ATPase via PKA.