Stimulus-induced phosphorylation of V-ATPase by protein kinase A

doi:10.1074/jbc.M703368200

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Stimulus-induced phosphorylation of V-ATPase by protein kinase A

Martin Voss, Olga Vitavska, Bernd Walz, Helmut Wieczorek, and Otto Baumann

Institut fuer Biochemie und Biologie, Universität Potsdam, Golm D-14476

Corresponding Author: obaumann{at}uni-potsdam.de

Eukaryotic vacuolar-type H⁺-ATPases (V-ATPases) are regulated by the reversible disassembly of the active V[sub1}V_O holoenzyme into a cytosolic V₁ complex and a membrane bound V_O complex. The signalling cascades that trigger these events in response to changing cellular conditions are largely unknown. We report that the V₁ 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 V₁ complex, but not as a part of the V₁V_O holoenzyme. Both the phosphorylated and the unphosphorylated form of subunit C are able to reassociate with the V₁ 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-analogue 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 V₁V_O holoenzyme.

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