KINASE SUPPRESSOR OF RAS-EFFECTOR OF TNF IN INTESTINAL C

肿瘤坏死因子(TNF) α通过包括细胞外信号调节激酶(ERK1/ERK2)/丝裂原活化蛋白（MAP）激酶在内的几种关键信号转导途径，在肠道生长发育和炎症性肠病的发病机制中发挥关键作用。初步研究表明，神经酰胺调控的显性阴性，激酶失活（ki） Ras激酶抑制因子（KSR）的表达抑制tnfalpha诱导的ERK1/ERK2活化。此外，kiKSR的表达将肠上皮细胞中的TNFalpha从抗增殖转化为凋亡，这表明KSR是TNFalpha信号传导的关键介质。本研究旨在验证KSR是TNFalpha刺激的ERK/MAP激酶激活的重要调控分子的假设。提出了三个具体的研究目标。目的1。KSR的神经酰胺调控域是什么？目标2。肠上皮细胞中TNFalpha激活ERK1/ERK2需要KSR的哪些调控结构域？目标3。在被TNFalpha激活的ERK1/ERK2中，哪些蛋白与KSR调控域相关？将采用多种方法来实现这些特定目标，包括位点定向和缺失诱变、蛋白激酶试验、酵母双杂交试验和gst融合蛋白的表达。这些研究结果将对肠道生物学，从正常生长发育到炎症性肠病的发病机制具有广泛的意义。

Tumor necrosis factor (TNF)alpha plays a key role in regulation of intestinal growth, development and the pathogenesis of inflammatory bowel diseases by several critical signal transduction pathways, including the extracellular signal regulated kinase (ERK1/ERK2)/mitogen activated protein (MAP) kinase. Preliminary studies have shown expression of the ceramide-regulated dominant-negative, kinase inactive (ki) kinase suppressor of Ras (KSR) inhibits TNFalpha-induced ERK1/ERK2 activation. Furthermore, kiKSR expression converts TNFalpha from anti-proliferative to apoptotic in intestinal epithelial cells, implicating KSR as a key mediator of TNFalpha signaling. This proposal is designed to test the hypothesis that KSR is an essential regulatory molecule in TNFalpha stimulated ERK/MAP kinase activation. Three specific aims are proposed for study. Aim 1. What is the ceramide regulatory domain on KSR? Aim 2. What regulatory domains of KSR are required for ERK1/ERK2 activation by TNFalpha in intestinal epithelial cells? Aim 3. What proteins associate with the KSR regulatory domains in ERK1/ERK2 activation by TNFalpha? A variety of methods will be employed to achieve these Specific Aims, including site-directed and deletion mutagenesis, protein kinase assay, yeast two-hybrid assay and expression of GST-fusion proteins. The findings from these studies will have broad implications on intestinal biology, from normal growth and development to the pathogenesis of inflammatory bowel disease.