MUTATIONAL ANALYSIS OF THE NF-xB p65 (RelA) SUBUNIT AND ISOLATION OF A DOMINANT-POSITIVE MUTANT of p65

Abstract

The Rel/NF-κB family of transcription factors, whose members share the conserved Rel homology domain (RHD), are involved in specific DNA binding interactions in the promoter/enhancer regions of a variety of different genes especially those involved in immune responses (M. Lenardo and D. Baltimore, 1989; Bours, et al. 1990; Meyer, et al. 1991; Ruben, et al. 1991; Ryseck, et al. 1992 ). The predominant inducible form of the NF-KB transcription factor is a heterodimeric complex containing two Rel-related DNA-binding subunits, termed p50 and p65. The p50 subunit is the major DNA-binding component of the heterodimeric complex (reviewed by Grilli, et al. 1993; Siebenlist, et al. 1994; A. Baldwin, 1996) while the p65 subunit provides the transactivation activity in this complex and binds the cytoplasmic inhibitor of NF-κB, IκB-α (Ganchi, et al. 1992; Beg, et al. 1992; Ganchi, et al. 1993). The first objective of this study was to investigate regions in the p65 RHD that are important for DNA binding, dimerization and nuclear localization using site-directed mutagenesis. Results from expression experiments using p65 DNA-binding mutants identified three regions of the p65 RHD that are important for DNA binding and showed that these regions correlate with analogous regions in the homologous p50 RHD (Bressler, et al. 1993). One of these p65 DNA-binding mutations at amino acids 245-246 in the p65 RHD also blocked dimerization with p50 and with wild-type p65. This suggested that these amino acids were important not only for DNA binding but for dimerization and confirmed reports that p65 dimerization is an obligatory event for DNA binding (Ganchi, et al 1992; Ruben, et al. 1992; Ganchi, et al. 1993). The functional data from DNA-binding and dimerization mutants showed that they were unable to transactivate in vivo. This data correlated with the function of mutants made in the p50 RHD. Together, these findings suggest that there are analogous amino acids within the p50 and p65 Rel homology domain that are critical for DNA binding and dimerization. The second objective of this study was to isolate a dominant-positive mutant by altering the nuclear localization signal (NLS) of p65. It had been previously shown that the NLS of p65 is the binding site for IκB-α which allows IκB to inhibit NF-κB complexes at the point of translocation and DNA binding (P. Baeuerle and D. Baltimore, 1989; Beg, et al. 1992; Ganchi, et al. 1992; Ganchi, et al. 1993; N. Rice and M. Ernst, 1995). It was postulated in this study that mutating the NLS of p65 would block I-κB-α-mediated inhibition of translocation and DNA binding and potentially alter the transactivation function of the mutated p65. In order to act in a dominant-positive manner (i.e., to over-ride the wild-type phenotype in heterozygotes), a p65 NLS mutant would be resistant to IκB-α-mediated inhibition of DNA binding and alter the duration of transcriptional activity compared to wildtype p65. Two p65 NLS mutants were analyzed in vitro and in vivo and found to be affected in their ability to bind IκB-α. One of these mutants containing a four base pair substitution of the NLS sequence when co-transfected with wild-type p50, was able to transactivate in vivo at a higher level than wild-type p50/p65. This suggests that this mutant could act as a potential dominant-positive mutant by producing an altered and enhanced transcriptional signal. This mutant would be useful in determining the affects of prolonged transcriptional activation of specific genes on cell function especially during immune cell activation.