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

描述(申请人提供)：H-RAS等癌蛋白的致癌转化在信号转导级联的水平上得到越来越多的理解，但与转化相关的核控制仍然相对模糊。此前，我们和其他人已经提供了证据，证明转录因子NF-kappaBeta是致癌H-RAS转化细胞所必需的，也是某些癌蛋白诱导肿瘤形成所必需的。我们新的初步研究表明，NF-kappaBeta的p65亚基和Ikappabeta激酶(IKK)亚基IKappabeta是RAS在体外诱导细胞转化所必需的。有趣的是，RAS诱导p65积聚的方式不涉及IkappabetaAlpha降解，不涉及传统的IKK活性，也不会导致我们标准EMSA检测的DNA结合活性增强。我们发现RAS通过部分依赖于NF-kappaBeta的p65亚基的方式诱导细胞周期蛋白d1基因的表达，芯片检测表明p65和ikk被招募到细胞周期蛋白d1启动子。然而，其他依赖于NF-kappabeta的基因(例如，iNOS)不被RAS激活。奇怪的是，表达RAS的细胞实际上抑制了肿瘤坏死因子激活核因子-kappabeta的能力，显然是通过抑制IKK活性来实现的。初步数据还表明，一种新发现的IKK形式(IKKepsilon)与PI3K/Akt激活NF-kappabeta的能力密切相关。因此，我们的假设是，致癌的RAS激活了NF-kappabeta途径的一个选择性臂，同时抑制了传统的NF-kappabeta系统。我们推测，这种不寻常的调控(同时激活一种形式的核因子-kappabeta，同时抑制其他形式)是RAS致癌转化所必需的。此外，我们还发现，IKKalpha通过调控组蛋白的磷酸化来修饰染色质，并且已知RAS诱导组蛋白H3和H1的磷酸化。为了解决我们的总体假设和研究IKK亚基在调节RAS诱导的染色质修饰中的潜在作用，我们建议：(1)鉴定由非传统的NF-kappabeta/ikk途径控制的RAS诱导的基因，并确定IKK亚基是否控制RAS诱导的染色质修饰；(2)表征致癌RAS诱导的信号转导通路及其以NF-(活化)为靶点的效应器，并确定RAS诱导的信号如何抑制传统的细胞因子诱导的NF-(活化)途径，同时保持JNK的激活，(3)利用动物模型研究肿瘤转化过程中对核因子-(/IKK)特异性通路以及肿瘤坏死因子-特异性信号的需求。

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.