NOVEL KINASE SIGNALING CASCADES IN PANCREATIC ACINI

几种主要的胰腺疾病，包括胰腺炎、癌症和 囊性纤维化涉及细胞调节的改变。分泌物 胰腺腺泡细胞的消化酶主要受 细胞内钙离子和二酰甘油的增加，这是由 磷脂酶C的激活，但这一机制并不能解释 促分泌剂和激素对细胞生长、蛋白质的所有影响 合成和代谢。最近发现了一些新的蛋白激酶 已经阐明了级联反应在生长中扮演着重要的角色， 多种细胞的分化和基因表达。我们有 已发表的和初步的研究表明，CCK激活了三个 丝裂原活化蛋白激酶(MAPK)在大鼠腺泡引导中的级联 激活ERKs(P42和P44MAPK)、Jun Kinase和 P38/重新激活Kinase。此外，CCK还激活了一条独特的途径 在腺泡中导致p70 S6激酶，该酶对雷帕霉素和 沃特曼宁。这项建议的总体目标是了解 新的激酶级联在腺泡细胞中被激活，这种特定的刺激 激活它们，以及它们的一些生物学功能。四个具体的 目的包括：1)确定CCK和EGF的作用机制 激活RAS-RAF-MEK-ERK级联；适配器的重要性 将对蛋白质Sch和Grb2以及RAS进行评估。2)确定 酪氨酸激酶被CCK激活，使Shc磷酸化。这 将涉及对Src和Src家族成员的激活进行分析 和粘着斑激酶(FAK)。3)确定激活 P38MAPK及其激活机制及其调控作用 小分子热休克蛋白的磷酸化；4)p70的作用机制 S6K激活及其在胰腺腺泡蛋白合成中的作用这个 研究将涉及激酶的免疫沉淀，Western blotting 和使用特定底物的激酶分析。选择性途径 使用特定抑制物的激活和抑制及其表达 腺病毒载体的显性负性突变蛋白将用于 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。虽然这项工作的目标是 了解胰腺腺泡细胞，它也将有意义 用于调节其他类型的胃肠道细胞。

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.