TRANSFORMATION OF CELLS BY THE V-REL ONCOGENE

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This proposal describes experiments that will further our understanding of transcriptional control and oncogenesis caused by the Rel family of proteins and proteins that associate with Rel proteins. Although the primary focus is on the vRel retroviral oncoprotein, other Rel (cRel, NF- kappaB p50/p105, p49/p100, etc.) and IkappaB-like proteins (p40, MAD-3, etc.) will also be characterized. Most studies will be performed with avian proteins, since the avian system is still the only system in which frank transformation of cells by Rel proteins has been seen. Our long- range goal is an understanding of the mechanism by which Rel proteins control normal and abnormal cell growth. As such, there are several related Specific Aims. First, using approaches that have previously been successful for us, we will isolate additional avian rel-related cDNAs. For example, we will clone a chicken p49/p100 cDNA. Using this cDNA, we will determine whether p100 is the remaining unidentified vRel-associated protein. We will characterize vRel protein complexes in vitro and in vivo. Using Gal4 fusion proteins, in vitro mutagenesis, and genetic selections in yeast, we will characterize sequences important for transcriptional activation in Rel and IkappaB proteins. In addition, we will attempt to identify the proteins with which these activation domains interact in chicken cells and in yeast. We will determine the effect of phosphorylation on chicken cRel, specifically in terms of regulation of subcellular location, DNA binding and dimerization. Finally, experiments will be performed to address the mechanism of transformation by vRel. First, we will characterize the mechanism by which vRel represses the c-rel promoter and activates Sp1 site-containing promoters. Second, we will characterize sequences in vRel that are necessary for its transforming activity, and the changes that can occur to make cRel transforming. After creating conditional mutants of vRel, we will attempt to identify genes controlled directly by vRel. The experiments proposed here will have relevance to gene control, oncogenesis, development, perhaps the control of HIV replication, and are meant to extend and expand ongoing studies in the laboratory aimed at understanding the mechanism of action of Rel proteins.