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

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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.