The small GTP binding protein Ras is a central mediator of signaling pathways that regulates cell proliferation and differentiation. The importance of Ras in regulating cell growth is indicated by the fact that tumors frequently contain mutations in the ras gene that result in an activated Ras protein. Activation of Ras leads to activation of signal transduction pathways that regulate the activity of transcription factors resulting in enhanced expression of genes required for regulating cell growth. However, how activated Ras induces cell transformation is not fully understood. Understanding how constitutive activation of Ras contributes to tumorigenesis requires an understanding of both the signaling pathways that Ras activates and how these pathways in turn regulate gene expression. The mammalian transcription factor NF-kappa B is important for the expression of several genes that important for cell proliferation, for example NF-kappa B is required for expression of the MGSA/groalpha gene which is deregulated in melanoma. NF-kappaB activity is regulated, at least in part, by subcellular localization so that the protein is inactive when localized in the cytoplasm through an interaction with the inhibitor IkappaB. The classic mode for activation of NF-kappa B involves enhanced NF-kappaB nuclear localization in response to activating signals. Preliminary data presented in this research proposal demonstrate that activated Ras leads to activation of expression from kB sites by a mechanism that does not involve the classic increase in nuclear NF-kappaB. The primary goal of this research proposal is to determine how activated Ras enhances NF-kappaB activity and to determine what role NF-kappaB plays in mediating cell transformation. This will be accomplished by addressing three specific aims: 1) Characterizing the mechanism by which Ras activation enhances expression from kappaB sites, 2)Analyzing the requirement for NF-kappaB activity in Ras transformation, and 3) Characterizing the kappaB dependent increase in activity in melanoma cells.
Aim 1 will employ transfection of dominant negative members of the Ras signaling pathway into Ras transformed cells to determine which members of the signaling pathway are involved in enhancing kappaB dependent gene expression. The possible phosphorylation of the p65 NF-kappaB subunit in response to activated Ras will also be examined.
Aim 2 will transfection of 3T3 cells with a super-repressive IkappaB construct or a dominant negative NF-kappaB p50 subunit to determine if blocking NF-kappaB can block Ras mediated transformation. The requirement for NF-kappaB enhancing c-myc expression in response to Ras activation will also be examined.
Aim 3 will examine NF-kappaB activity in melanoma cells and investigate the role of both Ras and the cyclinD cdk4 inhibitor, p16 ink4, in regulating NF-kappaB in these cells to further characterize the role of NF-kappaB in mediating cell growth control. In summary, these studies will determine how Ras regulates gene expression through NF-kappaB and how this activation of NF- kappaB may function in cell transformation.
| Norris, J L; Baldwin Jr, A S (1999) Oncogenic Ras enhances NF-kappaB transcriptional activity through Raf-dependent and Raf-independent mitogen-activated protein kinase signaling pathways. J Biol Chem 274:13841-6 |
