Regulation of protein phosphorylation in pancreatic acini. Distinct effects of Ca2+ ionophore A23187 and 12-O-tetradecanoylphorbol 13-acetate

Abstract

Regulation of protein phosphorylation in isolated pancreatic acini by the intracellular messengers Ca2+ and diacylglycerol was studied by using the Ca2+ ionophore A23187 and the tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate. As assessed by two-dimensional polyacrylamide-gel electrophoresis, the phorbol ester (1 microM) and Ca2+ ionophore (2 microM) altered the phosphorylation of distinct sets of proteins between Mr 83,000 and 23,000 in mouse and guinea-pig acini. The phorbol ester increased the phosphorylation of four proteins, whereas the ionophore increased the phosphorylation of two proteins and, in mouse acini, decreased the phosphorylation of one other protein. In addition, the phorbol ester and ionophore each caused the dephosphorylation of two proteins, of Mr 20,000 and 20,500. Administered together, these agents reproduced the changes in phosphorylation induced by the cholinergic agonist carbamoylcholine. The effects of the phorbol ester and ionophore on acinar amylase release were also studied. In mouse pancreatic acini, a maximally effective concentration of phorbol ester (1 microM) produced a secretory response that was only 28% of that produced by a maximally effective concentration of carbamoylcholine, whereas the ionophore (0.3 microM) stimulated amylase release to two-thirds of the maximal response to carbamoylcholine. In contrast, in guinea-pig acini, the phorbol ester and carbamoylcholine evoked similar maximal secretory responses, whereas the maximal secretory response to the ionophore was only 35% of that to carbamoylcholine. Combination of phorbol ester and ionophore resulted in a modest synergistic effect on amylase release in both species. It is concluded that cholinergic agonists act via both diacylglycerol and Ca2+ to regulate pancreatic protein phosphorylation, but that synergism between these intracellular messengers is of limited importance in stimulating enzyme secretion.

Immunohistochemical and immunoblotting identification of protein phosphatase 1γ1 in rat salivary glands

“…In addition to increased phosphorylation of specific proteins, increased dephosphorylation of other proteins can be observed [17,18]. These findings suggest that the degree of phosphorylation of a specific protein in the parotid gland might be affected by changes in the activities of protein phosphatases.…”

Purification and Identification of a 28-kDa Calcium-regulated Heat-stable Protein

“…This protein was initially described by 32 P-metabolic labeling studies in mouse and guinea pig (8) and more recently in rat acini (10) as a predominantly cytosolic, heatstable protein that is markedly phosphorylated by calciummobilizing secretagogues. CRHSP-28 phosphorylation has been shown to be stimulated in response to picomolar concentrations of CCK and also following treatment of cells with the high affinity CCK receptor agonist JMV-180 (10).…”

“…CRHSP-28 phosphorylation has been shown to be stimulated in response to picomolar concentrations of CCK and also following treatment of cells with the high affinity CCK receptor agonist JMV-180 (10). Phosphorylation of the protein appears to occur exclusively through a calcium-dependent mechanism, as maximal increases in phosphate content can be demonstrated by treatment of cells with the calcium ionophore A23187 (8,10). Furthermore, it does not appear to be regulated via protein kinase A (10) or protein kinase C (8) activation.…”

“…One approach at elucidating potential endogenous regulatory proteins in pancreas has been to examine the effects of various secretagogues and pharmacological agents on protein phosphorylation using autoradiographic analysis of electrophoretically separated proteins from 32 P-labeled acinar cells (2,8,9). Using this strategy, along with large format two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), we have developed a data base of approximately 30 phosphoproteins that are differentially regulated by CCK (10).…”

“…The present study describes the purification and identification of one of the most prominent CCK regulated phosphoproteins in pancreas, which we designate CRHSP-28: calcium-regulated heat-stable protein with a molecular mass of 28 kDa on SDS-PAGE. It was initially identified as a cytosolic protein labeled phosphoprotein 5 in mouse and guinea pig acinar cells (8), in which it was found to be highly phosphorylated following treatment of cells with carbamylcholine or the calcium ionophore A23187 but not by treatment with phorbol esters. Later studies using rat acini identified the molecule as iI-28/5.4 on our twodimensional maps, due to the fact that it has an apparent pI value of 5.4 and undergoes an immediate increase in phosphorylation in response to physiological concentrations of calcium-mobilizing secretagogues, as well as JMV-180, a high affinity CCK receptor agonist (10).…”