Stimulus-induced Phosphorylation of Vacuolar H+-ATPase by Protein Kinase A

Abstract

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...

Subunit Interactions at the V1-Vo Interface in Yeast Vacuolar ATPase

“…Indeed, a number of environmental signals have been linked to enzyme dissociation, mainly related to metabolism and specifically glycolysis (10). Changes in enzyme association have been linked to phosphorylation (47), extracellular conditions (48), pH changes (34,48), and subcellular localization (33,49). Further complicating the understanding of this mechanism are the number of cellular binding partners for subunits likely to be involved in enzyme regulation.…”

“…Further complicating the understanding of this mechanism are the number of cellular binding partners for subunits likely to be involved in enzyme regulation. Subunit C, for example, has been shown to interact with kinases (47,50), actin (51), ATP (52), and the Regulator of the H ϩ -ATPase of vacuoles and endosomal membranes complex (53). Interestingly, the C subunit x-ray crystal structure was solved in two different conformations, but it remains unclear whether these changes may be linked to release of subunit C from the enzyme during regulated disassembly (18).…”

Development of apical membrane organization and V-ATPase regulation in blowfly salivary glands

“…First, we have not noticed a dramatic difference in the expression level of various V-ATPase subunits in the salivary glands between eclosion and 4h p.e., the stage at which 5-HT-induced secretion reaches maximal rate. Notably, this also applies to subunit C, which seems to play a key role in regulating V-ATPase assembly and activity in the salivary glands (Voss et al, 2007;Baumann and Walz, 2012). Although we do not have antibodies against all V-ATPase subunits, we consider that any subunits not detected in our assay have a similar temporal expression pattern.…”

“…Cross-reactivity of these antibodies with the homologous proteins in C. vicina has been demonstrated previously (Zimmermann et al, 2003;Dames et al, 2006;Voss et al, 2007;Voss et al, 2009;Baumann and Walz, 2012). Monoclonal anti-actin (clone C4) was obtained from Merck (Darmstadt, Germany), rabbit antiserum (cat.…”

An update in the structure, function, and regulation of V-ATPases: the role of the C subunit

“…According to Voss et al (2007), the C subunit is responsible for V-ATPase dissociation into cytosolic V1 and membranous V0 complexes via interaction with protein kinase A. The C subunit appears to be phosphorylated as part of the V1 complex but not as part of the V1V0 holoenzyme.…”