SECRETAGOGUE REGULATION OF PANCREATIC ACINAR CELL PROTEIN-PHOSPHORYLATION SHOWN BY LARGE-SCALE 2D-PAGE

High-resolution large-scale two-dimensional polyacrylamide gel electro phoresis (ED-PAGE) combined with computer-assisted image analysis was used to construct a database of secretagogue/second messenger-induced phosphoprotein modifications in intact rat pancreatic acinar cells. Is olated acini were labeled with P-32(i), exposed to hormones and other test agents, and subjected to large-scale 2D-PAGE and autoradiography. This procedure resolved 500 phosphoproteins in pancreatic acinar whol e cell lysates, similar to 90% of which were localized in the soluble fraction of centrifuged samples. Soluble proteins were further charact erized as to heat and acid stability. Cholecystokinin (CCK), carbachol , and bombesin altered the phosphorylation state of about 27 proteins with both increases and decreases observed. Subsets of proteins were p hosphorylated in response to phorbol ester 12-O-tetradecanoylphorbol 1 3-acetate (TPA), calcium ionophore A-23187, and adenosine 3',5'-cyclic monophosphate (cAMP) analogue 8-bromo-cCAMP. One of these proteins wa s identified as the myristoylated, alanine-rich, C-kinase substrate (M ARCKS) protein by immunoprecipitation. The time course and dose respon se of phosphorylation changes due to CCK showed considerable variation between proteins, although a temporal hierarchy of phosphorylation ev ents was clearly exhibited. Particularly striking was the rapid dephos phorylation within 30 s of a 19-kDa soluble protein to a minimum of 20 +/- 1% of control. Increased phosphorylation of the MARCKS and other TPA-regulated proteins suggests that CCK, carbachol, bombesin, and the CCK partial agonist, JMV-180, all activate protein kinase C in intact acini.