Stimulus-induced phosphorylation of vacuolar H(+)-ATPase by protein kinase A.
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.
Voss
Martin
M
Institut für Biochemie und Biologie, Zoophysiologie, Universität Potsdam, Karl-Liebknecht-Strasse 24/25, D-14476 Potsdam, Germany.
Vitavska
Olga
O
Fachbereich Biologie und Chemie, Tierphysiologie, Universität Osnabrück, Barbarastrasse 11, D-49076 Osnabrück, Germany.
Walz
Bernd
B
Institut für Biochemie und Biologie, Zoophysiologie, Universität Potsdam, Karl-Liebknecht-Strasse 24/25, D-14476 Potsdam, Germany.
Wieczorek
Helmut
H
Fachbereich Biologie und Chemie, Tierphysiologie, Universität Osnabrück, Barbarastrasse 11, D-49076 Osnabrück, Germany.
Baumann
Otto
O
Institut für Biochemie und Biologie, Zoophysiologie, Universität Potsdam, Karl-Liebknecht-Strasse 24/25, D-14476 Potsdam, Germany. Electronic address: obaumann@uni-potsdam.de.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D002384:Catalysis; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D007546:Isoquinolines / Q000494:pharmacology; D018402:Manduca; D010766:Phosphorylation; D020033:Protein Isoforms; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry; D012469:Salivary Glands / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D014617:Vacuoles / Q000378:metabolism
D007546:Isoquinolines; D020033:Protein Isoforms; D011994:Recombinant Proteins; D013449:Sulfonamides; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D000251:Adenosine Triphosphatases; D006180:Proton-Translocating ATPases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide