V-ATPase deactivation in blowfly salivary glands is mediated by protein phosphatase 2C.
The activity of vacuolar H(+)-ATPase (V-ATPase) in the apical membrane of blowfly (Calliphora vicina) salivary glands is regulated by the neurohormone serotonin (5-HT). 5-HT induces, via protein kinase A, the phosphorylation of V-ATPase subunit C and the assembly of V-ATPase holoenzymes. The protein phosphatase responsible for the dephosphorylation of subunit C and V-ATPase inactivation is not as yet known. We show here that inhibitors of protein phosphatases PP1 and PP2A (tautomycin, ocadaic acid) and PP2B (cyclosporin A, FK-506) do not prevent V-ATPase deactivation and dephosphorylation of subunit C. A decrease in the intracellular Mg(2+) level caused by loading secretory cells with EDTA-AM leads to the activation of proton pumping in the absence of 5-HT, prolongs the 5-HT-induced response in proton pumping, and inhibits the dephosphorylation of subunit C. Thus, the deactivation of V-ATPase is most probably mediated by a protein phosphatase that is insensitive to okadaic acid and that requires Mg(2+), namely, a member of the PP2C protein family. By molecular biological techniques, we demonstrate the expression of at least two PP2C protein family members in blowfly salivary glands.
Voss
Martin
M
Institute of Biochemistry and Biology, University of Potsdam, 14476 Potsdam, Germany.
Blenau
Wolfgang
W
Walz
Bernd
B
Baumann
Otto
O
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004175:Diptera; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D000071636:Protein Phosphatase 2C; D012469:Salivary Glands / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D014617:Vacuoles / Q000201:enzymology*
D017931:DNA Primers; D004791:Enzyme Inhibitors; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C; D000251:Adenosine Triphosphatases