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 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 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 V1 complex but not as a part of the V 1V0 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 V1V0 holoenzyme. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.