Secretagogue regulation of pancreatic acinar cell protein phosphorylation shown by large-scale 2D-PAGE.
High-resolution large-scale two-dimensional polyacrylamide gel electrophoresis (2D-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. Isolated acini were labeled with 32Pi, exposed to hormones and other test agents, and subjected to large-scale 2D-PAGE and autoradiography. This procedure resolved 500 phosphoproteins in pancreatic acinar whole cell lysates, approximately 90% of which were localized in the soluble fraction of centrifuged samples. Soluble proteins were further characterized 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 phosphorylated in response to phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA), calcium ionophore A-23187, and adenosine 3',5'-cyclic monophosphate (cAMP) analogue 8-bromo-cAMP. One of these proteins was identified as the myristoylated, alanine-rich, C-kinase substrate (MARCKS) protein by immunoprecipitation. The time course and dose response of phosphorylation changes due to CCK showed considerable variation between proteins, although a temporal hierarchy of phosphorylation events was clearly exhibited. Particularly striking was the rapid dephosphorylation 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.
Wishart
M J
MJ
Department of Physiology, University of Michigan Medical School, Ann Arbor 48109-0622.
Groblewski
G
G
Göke
B J
BJ
Wagner
A C
AC
Williams
J A
JA
D000818:Animals; D002766:Cholecystokinin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010179:Pancreas / Q000166:cytology / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015290:Second Messenger Systems
D047908:Intracellular Signaling Peptides and Proteins; C000619712:Marcks protein, rat; D008565:Membrane Proteins; D010750:Phosphoproteins; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D002766:Cholecystokinin; D011493:Protein Kinase C