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J. Biol. Chem., Vol. 282, Issue 46, 33735-33742, November 16, 2007

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

Martin Voss¹, Olga Vitavska¹, Bernd Walz, Helmut Wieczorek, and Otto Baumann²

From the Institut für Biochemie und Biologie, Zoophysiologie, Universität Potsdam, Karl-Liebknecht-Strasse 24/25, D-14476 Potsdam, Germany and the Fachbereich Biologie und Chemie, Tierphysiologie, Universität Osnabrück, Barbarastrasse 11, D-49076 Osnabrück, Germany

Eukaryotic vacuolar-type H⁺-ATPases (V-ATPases) are regulated by the reversible disassembly of the active V₁V₀ holoenzyme into a cytosolic V₁ complex and a membrane-bound V₀ complex. The signaling 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 V₁V₀ 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 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 V₁V₀ holoenzyme.

Received for publication, April 23, 2007 , and in revised form, August 15, 2007.

^* This work was supported by Deutsche Forschungsgemeinschaft Grants SFB 431 (to H. W.) and WA463/9-5 (to B. W. and O. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² To whom correspondence should be addressed: Institut für Biochemie und Biologie, Zoophysiologie, Universität Potsdam, Karl-Liebknecht-Str. 24/25, 14476 Potsdam, Germany. Tel.: 49-331-977-5525; Fax: 49-331-977-5522; E-mail: obaumann{at}uni-potsdam.de.

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