NOVEL KINASE SIGNALING CASCADES IN PANCREATIC ACINI

点击翻译按钮获取中文摘要

Several major pancreatic disease including pancreatitis, cancer and cystic fibrosis involve altered cellular regulation. Secretion of digestive enzymes by pancreatic acinar cells is largely controlled by increases in intracellular Ca2+ and diacylglycerol which result from activation of phospholipase C. However, this mechanism can not explain all the effects of secretagogues and hormones on cell growth, protein synthesis and metabolism. Recently a number of novel protein kinase cascades have been elucidated that play important roles in growth, differentiation and gene expression of a variety of cells. We have shown in published and preliminary studies that CCK activates three mitogen activated protein kinase (MAPK) cascades in rat acini leading to activation of ERKs (p42 and p44 MAPK), Jun Kinase and p38/Reactivating Kinase. In addition, CCK activates a distinct pathway in acini leading to p70 S6 Kinase which is sensitive to rapamycin and wortmannin. The overall aim of this proposal is to understand how the novel kinase cascades are activated in acini, the specific stimuli which activate them, and some of their biological functions. Four specific aims include: 1) to determine the mechanism by which CCK and EGF activate the Ras-Raf-MEK-ERK cascade; the importance of the adapter proteins Sch and Grb2 as well as Ras will be evaluated. 2) To determine the tyrosine kinase activated by CCK which phosphorylates Shc. This will involve analysis of the activation of Src and Src family members and focal adhesion kinase (FAK). 3) To determine the activation of p38MAPK, its mechanism of activation, and its role in regulating phosphorylation of small heat shock protein, and 4) the mechanism of p70 S6K activation and its role in pancreatic acinar protein synthesis. The studies will involve immunoprecipitation of kinases, Western blotting and kinase assays using specific substrates. Selective pathway activation and inhibition using specific inhibitors and expression of dominant negative mutant proteins by adenoviral vectors will be used to evaluate pathways leading to biological effects including amylase secretion, growth and protein synthesis. While the work is aimed at understanding the pancreatic acinar cell, it will also have implications for the regulation of other gastrointestinal cell types.