THE ROLE OF C-REL IN TRANSACTIVATION OF THE IκBα GENE

Abstract

The feedback inhibition loop involving p65 and its inhibitor IκBα, has been well established (Scott, et al. 1993; Sun, et al. 1993). Overexpressed p65 is localized in the cytoplasm due to transactivation of its inhibitor gene, IκBα. The resultant increased levels of IκBα proteins bind the overexpressed p65 and retain the transactivator in the cytoplasm. The thesis data confirms that c-Re1 also increases levels of IκBα protein, though not as well as p65 and that this is consistent with transactivation. This activity was initially shown by overexpressing the c-Re1 protein in either canine D17 cells or human 293 cells and analyzing for increased IκBα levels by immunofluorescence using an anti-c-Re1 serum. In both cell lines, c-Re1 localization was strictly cytoplasmic in about 70% of transfected cells. This result implies that c-Re1 caused the levels of IκBα to be increased sufficiently to retain the overexpressed c-Re1 in the cytoplasm. Further evidence of increased IκBα in c-re1 transfected cells was shown by subjecting the transfected cell lysates to western analysis and detecting IκBα using an anti-IκBα serum (1309). c-Re1 clearly caused the levels of IκBα to be increased over mocktransfected cells. By making C-terminal deletions of c-Re1, transfecting them into D17 and 293 cells, and then subjecting the cell lysates to western analysis using anti-IκBα serum, it was shown that none of the deletion mutants increase the levels of IκBα to a detectable level. Even the smallest deletion of 37 amino acids (Re1-551) lost most of the ability to increase IκBα levels. These results were substantiated by immunofluorescence data from cells transfected with the deletion mutants. The 37 amino acid deleted protein localized only in the cytoplasm in about 50% of transfected cells while full-length c-Re1 was located only in the cytoplasm in about 70% of the cells. By deleting 166 amino acids from c-Re1 (Re1-422), the ability to increase IκBα protein levels was lost. The levels of IκBα were insufficient to retain the overexpressed Re1-422 entirely in the cytoplasm. These results were not due to the loss of an ability to bind IκBα because all of the deletion mutants were bound and retained in the cytoplasm of cells cotransfected with a re/-deletion and an IκBα expression vector. The subcellular localization was detected by immunofluorescence. The ability to bind its inhibitor was also tested by bandshift to determine the ability of IκBα to inhibit the Re1 deletion mutants from binding DNA. All deletion mutants were inhibited by IκBα from binding DNA. By these two assays each of the deletion mutants was able to bind IκBα as well as full-length c-Re1. Thus it has been shown that overexpression of c-Re1 leads to increased levels of bd3a, and this results from c-Re1 transactivating the gene for its inhibitor, IκBα. This increased level of Mk causes c-Re1 to be retained in the cytoplasm of cells. These results also show that a 37 amino acid deletion at the C terminus of c-Re1 is sufficient to lose the ability to increase IκBα levels. Additional residues within the C-terminus of c-Re1 (AA #422-551) may contribute to transactivation activity but additional data is necessary to establish this more precisely.