title: Dephosphorylation of cAMP-dependent protein kinase regulatory subunit in stimulated parietal cells.
abstract: The phosphorylation of endogenous proteins was investigated in subcellular fractions prepared from isolated rabbit parietal cells incubated with either cimetidine (unstimulated) or a combination of histamine and forskolin (maximally stimulated). Phosphorylation of endogenous proteins in subfractions was then assessed in a post hoc assay using [gamma-32P]ATP as a phosphate donor in vitro. The Mg(2+)-dependent incorporation of [32P]phosphate into a 52-kDa protein (pp52M) was observed in the 4,000 g membrane fraction from stimulated but not unstimulated cells. The pp52M protein was identified as the type II regulatory subunit of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (RII) by isoelectric focusing, comigration with cAMP-binding proteins, and immunoprecipitation. Incorporation of [32P]phosphate into RII in the in vitro assay in the presence of Zn2+ was apparent in the 4,000 g membrane from stimulated but not unstimulated cells. The results thus suggested that, on stimulation, RII in membrane was dephosphorylated. Incorporation of [32P]phosphate into membrane-associated RII was completely abolished in the presence of 10 microM cAMP. The decrease in RII phosphorylation in membrane from stimulated cells assayed in the presence of cAMP was due to a phosphoprotein phosphatase activity that was completely inhibited by okadaic acid (1 microM). The results indicate that stimulation of parietal cells with histamine and forskolin results in the dephosphorylation of membrane bound RII by a protein phosphatase that is also membrane associated. Furthermore, okadaic acid inhibited histamine-stimulated accumulation of [14C]aminopyrine into isolated parietal cells without altering stimulated increases in cAMP. Thus protein phosphatase may be a significant regulator of parietal cell function.
authors.affiliation: Department of Surgery, Yale University School of Medicine, New Haven, Connecticut.
authors.forename: J R
authors.initials: JR
authors.lastname: Goldenring
authors.forename: V A
authors.initials: VA
authors.lastname: Asher
authors.forename: M F
authors.initials: MF
authors.lastname: Barreuther
authors.forename: J J
authors.initials: JJ
authors.lastname: Lewis
authors.forename: S M
authors.initials: SM
authors.lastname: Lohmann
authors.forename: U
authors.initials: U
authors.lastname: Walter
authors.forename: I M
authors.initials: IM
authors.lastname: Modlin
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D000242:Cyclic AMP / Q000494:pharmacology; D004988:Ethers, Cyclic / Q000494:pharmacology; D017506:H(+)-K(+)-Exchanging ATPase; D019319:Okadaic Acid; D010295:Parietal Cells, Gastric / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D010766:Phosphorylation; D011233:Precipitin Tests; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution
chemical_list: D004988:Ethers, Cyclic; D010710:Phosphates; D011506:Proteins; D019319:Okadaic Acid; D000242:Cyclic AMP; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D000251:Adenosine Triphosphatases; D017506:H(+)-K(+)-Exchanging ATPase