DEPHOSPHORYLATION OF CAMP-DEPENDENT PROTEIN-KINASE REGULATORY SUBUNIT IN STIMULATED PARIETAL-CELLS

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-P-32]ATP as a phosphate donor in vitro. The Mg2+-dependent incorporation of [P-32]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 (R(II)) by isoelectric focusing, comigration with cAMP-binding proteins, and immunoprecipitation. Incorporation of [P-32]phosphate into R(II) 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, R(II) in membrane was dephosphorylated. Incorporation of [P-32]phosphate into membrane-associated R(II) was completely abolished in the presence of 10-mu-M cAMP. The decrease in R(II) 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-mu-M). The results indicate that stimulation of parietal cells with histamine and forskolin results in the dephosphorylation of membrane bound R(II) by a protein phosphatase that is also membrane associated. Furthermore, okadaic acid inhibited histamine-stimulated accumulation of [C-14]aminopyrine into isolated parietal cells without altering stimulated increases in cAMP. Thus protein phosphatase may be a significant regulator of parietal cell function.