KINASE SUPPRESSOR OF RAS-EFFECTOR OF TNF IN INTESTINAL C

肠 C 中 TNF 的 RAS 激酶抑制剂

• 批准号: 6207613
• 负责人: FANG YAN
• 金额: $ 4.63万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6207613
• 关键词: binding proteins; biological signal transduction; cell growth regulation; ceramides; chimeric proteins; complementary DNA; enzyme activity; gastrointestinal epithelium; gene expression; genetic library; guanine nucleotide binding protein; immunoprecipitation; inflammatory bowel diseases; mitogen activated protein kinase; protein purification; protein sequence; protein structure function; site directed mutagenesis; tissue /cell culture; tumor necrosis factor alpha; western blottings; yeast two hybrid system

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.

肿瘤坏死因子（tumor necrosis factor，TNF）α通过细胞外信号调节激酶（extracellular signal regulated kinase，ERK 1/ERK 2）/丝裂原活化蛋白（mitogen activated protein，MAP）激酶等多种信号转导途径，在肠道生长发育和炎症性肠病的发病机制中发挥重要作用。 初步研究表明，神经酰胺调节的显性负性、激酶失活（ki）的Ras激酶抑制因子（KSR）的表达抑制TNF α诱导的ERK 1/ERK 2活化。此外，kiKSR表达将肠上皮细胞中的TNF α从抗增殖转化为凋亡，暗示KSR是TNF α信号传导的关键介质。该提议旨在验证KSR是TNF α刺激的ERK/MAP激酶活化中的重要调节分子的假设。提出了三个具体的研究目标。目标1. KSR上的神经酰胺调控域是什么？目标二。在肠上皮细胞中，TNF α激活ERK 1/ERK 2需要KSR的哪些调节结构域？ 目标3。哪些蛋白质与TNF α激活ERK 1/ERK 2中的KSR调节结构域相关？将采用多种方法来实现这些特定目的，包括定点突变和缺失突变、蛋白激酶测定、酵母双杂交测定和GST融合蛋白的表达。这些研究的发现将对肠道生物学产生广泛的影响，从正常的生长和发育到炎症性肠病的发病机制。