Protein kinase C mediates cholinergically regulated protein phosphorylation in a Cl(-)-secreting epithelium.
T84 cell monolayers were used to study the cholinergic regulation of protein phosphorylation in epithelial cells. When T84 cell monolayers are labeled with 32Pi and stimulated with carbachol, six proteins exhibit altered phosphorylation. The most prominent response is a fivefold increase in labeling of p83, an acidic protein of Mr 83,000. Increasing labeling of p83 parallels stimulated secretion with respect to the onset of agonist action, agonist potency, and antagonism by atropine. However, the p83 and secretory responses differ in that the p83 response is more sustained. When T84 cell fractions are incubated with [gamma-32P]ATP, Ca2(+)-phospholipid stimulates p83 labeling. Phosphorylation of p83 also occurs when a T84 cell extract is incubated with purified protein kinase C and when intact cells are exposed to phorbol myristate acetate. p83 does not become phosphorylated in cell fractions incubated with adenosine 3',5'-cyclic monophosphate (cAMP) or in monolayers stimulated with agonists acting via cAMP. Thus carbachol stimulates the phosphorylation of an endogenous substrate for protein kinase C in T84 cells. The duration of this phosphorylation response suggests that protein kinase C may mediate a sustained response to carbachol, possibly acting to limit the duration of stimulated secretion.
Cohn
J A
JA
Department of Medicine, Duke University, Durham, North Carolina 27710.
D000818:Animals; D001692:Biological Transport; D002217:Carbachol / Q000494:pharmacology*; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D007700:Kinetics; D008970:Molecular Weight; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000096:biosynthesis / Q000302:isolation & purification; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D013379:Substrate Specificity
D002712:Chlorides; D010710:Phosphates; D010750:Phosphoproteins; D002217:Carbachol; D011493:Protein Kinase C