The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
Membrane Enzyme Systems Responsible for the Ca2+-Dependent Phosphorylation of Ser27, the Independent Phosphorylation of Tyr10 and Tyr7, and the Dephosphorylation of These Phosphorylated Residues in the α-Chain of H/K-ATPase
M. KanagawaS. WatanabeS. KayaK. TogawaT. ImagawaA. ShimadaK. KikuchiK. Taniguchi
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JOURNAL FREE ACCESS

2000 Volume 127 Issue 5 Pages 821-828

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Abstract

H/K-ATPase preparations (the G 1 membrane) from pig stomach contain both kinases and phosphatases and show reversible phosphorylation of Tyr7, Tyr10, and Ser27 residues of the α-chain of H/K-ATPase. The Tyr-kinase is sensitive to genistein and quercetin and recognized by anti-c-Src antibody. The Ser-kinase is dependent on Ca2+ (K0.5=0.9 μM), sensitive to a PKC inhibitor, and recognized by antibodies against PKCα and PKCβII. The addition of 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonic acid (CHAPS) caused a dramatic increase in the phosphorylation of added synthetic copolymer substrates and permitted the phosphorylation of maltose-binding proteins fused with the N-terminal domain of α-chains. The phosphotyrosine phosphatase was inhibited by vanadate. The phosphoserine phosphatase was inhibited by okadaic acid and by inhibitor-2. The presence of protein phosphatase-1 was immunologically detected. Column chromatographic separation of CHAPS-solubilized G 1 membrane and others indicate the apparent molecular weight of the Src-kinase to be _??_60 kDa, the PKCα and/or PKCβII to be _??_80 kDa, the Tyr-phosphatase to be 200 kDa, and PP-1 to be _??_35 kDa. These data show that these membrane-bound enzyme systems are in sufficiently close proximity to be responsible for reversible phosphorylation of Tyr7, Tyr10, and Ser27 of the catalytic subunit of membrane H/K-ATPase in parietal cells, the physiological role of which is unknown.

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