title: Membrane enzyme systems responsible for the Ca(2+)-dependent phosphorylation of Ser(27), the independent phosphorylation of Tyr(10) and Tyr(7), and the dephosphorylation of these phosphorylated residues in the alpha-chain of H/K-ATPase.
abstract: H/K-ATPase preparations (the G1 membrane) from pig stomach contain both kinases and phosphatases and show reversible phosphorylation of Tyr(7), Tyr(10), and Ser(27) residues of the alpha-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 Ca(2)(+) (K(0.5) = 0.9 microM), sensitive to a PKC inhibitor, and recognized by antibodies against PKCalpha and PKCbetaII. 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 alpha-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 G1 membrane and others indicate the apparent molecular weight of the Src-kinase to be approximately 60 kDa, the PKCalpha and/or PKCbII to be approximately 80 kDa, the Tyr-phosphatase to be 200 kDa, and PP-1 to be approximately 35 kDa. These data show that these membrane-bound enzyme systems are in sufficiently close proximity to be responsible for reversible phosphorylation of Tyr(7), Tyr(10), and Ser(27) of the catalytic subunit of membrane H/K-ATPase in parietal cells, the physiological role of which is unknown.
authors.affiliation: Biological Chemistry, Graduate School of Science, and Institute for Immunological Science Hokkaido University, Sapporo 060-0810, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Kanagawa
authors.forename: S
authors.initials: S
authors.lastname: Watanabe
authors.forename: S
authors.initials: S
authors.lastname: Kaya
authors.forename: K
authors.initials: K
authors.lastname: Togawa
authors.forename: T
authors.initials: T
authors.lastname: Imagawa
authors.forename: A
authors.initials: A
authors.lastname: Shimada
authors.forename: K
authors.initials: K
authors.lastname: Kikuchi
authors.forename: K
authors.initials: K
authors.lastname: Taniguchi
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D005744:Gastric Acid / Q000378:metabolism; D017506:H(+)-K(+)-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D054645:Protein Phosphatase 1; D011499:Protein Processing, Post-Translational; D017346:Protein Serine-Threonine Kinases / Q000302:isolation & purification / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013270:Stomach / Q000201:enzymology; D013379:Substrate Specificity; D013552:Swine
chemical_list: D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases; D017506:H(+)-K(+)-Exchanging ATPase; D002118:Calcium