NOVEL KINASE SIGNALING CASCADES IN PANCREATIC ACINI

几种主要的胰腺疾病，包括胰腺炎、癌症和 囊性纤维化涉及改变细胞调节。分泌 胰腺腺泡细胞的消化酶主要由 细胞内Ca 2+和甘油二酯的增加， 磷脂酶C的活化。然而，这一机制不能解释 促分泌素和激素对细胞生长、蛋白质 合成和代谢。 近年来，一些新的蛋白激酶 已经阐明了级联在生长中起重要作用， 分化和多种细胞的基因表达。 我们有 已发表的和初步的研究表明，CCK激活三个 大鼠腺泡中丝裂原活化蛋白激酶（MAPK）级联反应导致 ERK（p42和p44 MAPK）、Jun激酶和 p38/再活化激酶。 此外，CCK激活一种独特的途径， 在腺泡中导致对雷帕霉素敏感的p70 S6激酶， 渥曼青霉素 本提案的总体目标是了解 新的激酶级联在腺泡中被激活， 激活它们，以及它们的一些生物功能。 四个具体 目的包括：1）确定CCK和EGF 激活Ras-Raf-MEK-ERK级联;适配器的重要性 将评价蛋白质Sch和Grb 2以及Ras。2)以确定 由CCK激活的酪氨酸激酶磷酸化Shc。 这 将涉及Src和Src家族成员的激活分析 和粘着斑激酶（FAK）。3)为了确定 p38 MAPK的激活机制及其在调节细胞凋亡中的作用 小分子热休克蛋白的磷酸化; 4）p70的作用机制 S6 K激活及其在胰腺腺泡蛋白合成中的作用 的 研究将涉及激酶的免疫沉淀、蛋白质印迹 和使用特定底物的激酶测定。 选择途径 使用特异性抑制剂的激活和抑制以及 腺病毒载体的显性失活突变蛋白将用于 评估导致生物效应的途径，包括淀粉酶 分泌、生长和蛋白质合成。 虽然这项工作的目的是 了解胰腺腺泡细胞， 用于调节其他胃肠道细胞类型。

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.