Unusual Regulation of the NF-kappaB/IKK Pathway by Ras

Ras 对 NF-kappaB/IKK 通路的异常调控

• 批准号: 6890991
• 负责人: ALBERT Sidney BALDWIN
• 金额: $ 26.28万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-08-15 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6890991
• 关键词: JUN kinase; apoptosis; biological signal transduction; cell growth regulation; chromatin immunoprecipitation; enzyme activity; gene expression; gene mutation; gene targeting; genetic transduction; genetically modified animals; growth inhibitors; guanine nucleotide binding protein; laboratory mouse; neoplasm /cancer chemotherapy; neoplasm /cancer genetics; neoplasm /cancer remission /regression; neoplasm /cancer therapy; neoplastic transformation; nuclear factor kappa beta; oncoproteins; phosphorylation; protein kinase; protooncogene; tumor necrosis factor alpha

DESCRIPTION (provided by applicant): Oncogenic transformation by oncoproteins such as H-Ras is increasingly understood at the level of signal transduction cascades but nuclear controls associated with transformation remain relatively obscure. Previously, we and others have provided evidence that the transcription factor NF-kappaBeta is required for oncogenic H-Ras to transform cells and required for tumor formation induced by certain oncoproteins. Our new preliminary studies now demonstrate that the p65 subunit of NF-kappaBeta along with the Ikappabeta kinase (IKK) subunit IKappabeta are essential for Ras to induce cellular transformation in vitro. Interestingly, Ras induces p65 accumulation in a manner which does not involve Ikappabetaalpha degradation, does not involve traditional IKK activity, and does not result in enhanced DNA binding activity as measured in our standard EMSA assay. We show that Ras induces cyclin D1 gene expression in a manner partially dependent on the p65 subunit of NF-kappaBeta and that p65 and IKK are recruited to the cyclin D1 promoter as measured in a ChIP assay. However, other NF-kappabeta-dependent genes (for example, iNOS) are not activated by Ras. Curiously, Ras-expressing cells actually suppress the ability of TNF to activate NF-kappabeta, apparently through the suppression of IKK activity. Preliminary data also indicate that a newly identified form of IKK (IKKepsilon) is critically involved with the ability of PI3K/Akt to activate NF-kappabeta. Thus our hypothesis is that oncogenic Ras activates a selective arm of the NF-kappabeta pathway while concomitantly suppressing the traditional NF-kappabeta system. We hypothesize that this unusual regulation (simultaneously activating one form of NF-kappabeta while suppressing other forms) is required for oncogenic transformation by Ras. Additionally, we have found that IKKalpha functions to modify chromatin through regulating histone phosphorylation and it is known that Ras-induces both histone H3 and H1 phosphorylation. In order to address our overall hypothesis and to study the potential involvement of IKK subunits in regulating Ras-induced chromatin modifications, we propose to: (1) identify Ras-induced genes controlled by the non-traditional NF-kappabeta/IKK pathway and determine if IKK subunits control chromatin modification induced by Ras, (2) characterize the signal transduction pathways induced by oncogenic Ras and its effectors which target NF-(( activation and determine how Ras-induced signaling suppresses the traditional, cytokine-induced NF-(( activation pathway while maintaining JNK activation, and (3) utilize animal models to address the requirement of NF-((/IKK specific pathways as well as TNF-specific signals in oncogenic transformation.

描述（由申请人提供）：越来越多地在信号转导级联的水平上了解了肿瘤蛋白（例如H-RAS）的致癌转化，但是与转化相关的核控制仍然相对晦涩。以前，我们和其他人提供了证据，表明转录因子NF-kappabeta是致癌性H-RAS转化细胞所必需的，并且需要某些癌蛋白诱导的肿瘤形成所必需的。我们的新初步研究现在表明，NF-kappabeta的p65亚基以及Ikappabeta激酶（IKK）亚基Ikappabeta对于RAS对于Ras在体外诱导细胞转化至关重要。有趣的是，RAS以不涉及Ikappabetaalpha降解的方式诱导P65的积累，不涉及传统的IKK活性，并且不会导致在我们的标准EMSA分析中测得的DNA结合活性增强。我们表明，RAS以一种依赖于NF-Kappabeta的p65亚基的方式诱导细胞周期蛋白D1基因表达，并且p65和IKK被募集到Cyclin D1启动子中，如芯片测定中所测量。但是，其他NF-kappabeta依赖性基因（例如，iNOS）并未被RA激活。奇怪的是，表达RAS的细胞实际上抑制了TNF激活NF-Kappabeta的能力，显然是通过抑制IKK活性。初步数据还表明，新鉴定的IKK（Ikkepsilon）形式与PI3K/AKT激活NF-Kappabeta的能力非常重要。因此，我们的假设是致癌性RA会激活NF-Kappabeta途径的选择性臂，同时同时抑制传统的NF-Kappabeta系统。我们假设这种不寻常的调节（同时激活一种NF-kappabeta同时抑制其他形式）是RAS的致癌转化所必需的。此外，我们发现Ikkalpha通过调节组蛋白磷酸化来修饰染色质，并且众所周知，RAS诱导组蛋白H3和H1磷酸化。为了解决我们的总体假设并研究IKK亚基在调节RAS诱导的染色质修饰中的潜在参与，我们建议：（1）确定由非传统NF-Kappabeta/IKK途径控制RAS诱导的基因，并确定IKK亚基通过chatryation ryas的传播（2）（2）（2） oncogenic Ras and its effectors which target NF-(( activation and determine how Ras-induced signaling suppresses the traditional, cytokine-induced NF-(( activation pathway while maintaining JNK activation, and (3) utilize animal models to address the requirement of NF-((/IKK specific pathways as well as TNF-specific signals in oncogenic transformation.