PKD2 IS ACTIVATED BY SECRETAGOGUE STIMULATION IN THE PANCREAS

Abstract

Protein kinase D (PKD), an important serine/threonine kinase mediating G-protein coupled receptor signaling, consists of three different but related isoforms: PKD1, PKD2, and PKD3. Recently, we have demonstrated rapid translocation, phosphorylation, and activation of PKD1 by the secretagogues CCK and carbachol in isolated pancreatic acini. Whether PKD2 is also involved in pancreatic secretagogue signaling is unknown. The purpose of our study was twofold: 1) to delineate the expression and activation of PKD2 in vitro by pancreatic secretagogues, and 2) to determine whether PKD2 maybe activated in vivo during secretagogue-induced pancreatitis. Methods: 1) Isolated mouse pancreatic acini or the acinar cell line, AR42J, transfected with GST-PKD2-wildtype or pcDNA3 (control) plasmids were stimulated with the secretagogues CCK or carbachol. Activation of PKD2 was assessed by Ser876 phosphorylation. 2) Female Swiss Webster mice were treated with either caerulein (50μg/kg) or vehicle (control) i.p. every hour for a total of 6 injections. Pancreata were harvested 30 min after each hourly injection, and protein was extracted to determine PKD2 activation. Serum was collected for measurement of amylase levels as an index of caerulein-induced pancreatic injury. Results: 1) PKD2 is rapidly phosphorylated in isolated pancreatic acini and in AR42J cells following stimulation with CCK or carbachol in a concentration-dependent manner. Furthermore, CCK stimulates the phosphorylation of both endogenous PKD2 and exogenous GST-PKD2-wildtype proteins in transfected AR42J cells. 2) PKD2 is phosphorylated 30 min following the initial caerulein injection and remained activated over the time course but began to decrease after the 4th hourly injection. Serum amylase levels were elevated 30 min following the first caerulein injection and reached maximum levels after the final injection. Conclusions: PKD2, a novel isoform of the PKD family, is activated by secretagogue stimulation of the pancreas both in vitro and in vivo. Moreover, the results suggest that PKD2 activation maybe an early intracellular event during caerulein-induced pancreatitis.