PEST-dependent cytoplasmic retention of v-Rel by I(kappa)B-alpha: evidence that I(kappa)B-alpha regulates cellular localization of c-Rel and v-Rel by distinct mechanisms.

Association of c-Rel with the inhibitor of kappaB-alpha (IkappaB-alpha) protein regulates both cellular localization and DNA binding. The ability of v-Rel, the oncogenic viral counterpart of avian c-Rel, to evade regulation by p40, the avian IkappaB-alpha protein, contributes to v-Rel-mediated oncogenesis. The yeast two-hybrid system was utilized to dissect Rel:IkappaB-alpha interactions in vivo. We find that distinct domains in c-Rel and v-Rel are required for association with p40. Furthermore, while the ankyrin repeat domain of p40 is sufficient for association with c-Rel, both the ankyrin repeat domain and the PEST domain are required for association with v-Rel. Two amino acid differences between c-Rel and v-Rel that are principally responsible for PEST-dependent association of v-Rel with p40 were identified. These same amino acids were principally responsible for PEST-dependent cytoplasmic retention of v-Rel by p40. The presence of mutations in c-Rel that were sufficient to confer PEST-dependent association of the mutant c-Rel protein with p40 did not increase the weak oncogenicity of c-Rel. However, the introduction of these two c-Rel-derived amino acids into v-Rel markedly reduced the oncogenicity of v-Rel. Deletion of the NLS of either c-Rel or v-Rel did not abolish association with p40, but did confer PEST-dependent association of c-Rel with p40. Surprisingly, deletion of the nuclear localization signal in v-Rel did not affect oncogenicity by v-Rel. Analysis of several mutant c-Rel and v-Rel proteins demonstrated that association of Rel proteins with p40 is necessary but not sufficient for cytoplasmic retention. These results are not consistent with the hypothesis that p40 regulates cellular localization of v-Rel and c-Rel by the same mechanism. Rather, these results support the hypothesis that p40 regulates cellular localization of v-Rel and c-Rel by distinct mechanisms.

I kappa B-alpha-mediated inhibition of nuclear transport and DNA-binding by Rel proteins are separable functions: phosphorylation of C-terminal serine residues of I kappa B-alpha is specifically required for inhibition of DNA-binding.

I kappa B-alpha inhibits both DNA-binding and nuclear translocation of dimeric Rel complexes that contain either the RelA or c-Rel proteins. These inhibitory functions of I kappa B-alpha proteins are regulated by both constitutive and inducible phosphorylation. We have mapped the constitutive phosphorylation sites of p40, the avian I kappa B-alpha protein, to a C-terminal acidic serine-rich region that contains four serine residues. Deletions or point mutations that significantly alter the overall negatively charged character of this region abolish association of p40 with Rel proteins in vitro. Serine-to-alanine amino acid substitutions in this region modulate the association of p40 with Rel proteins in vitro and abolish p40-mediated inhibition of DNA-binding by c-Rel. Substitution of aspartic acid residues for the phosphorylated serine residues has no effect on p40-mediated inhibition of DNA-binding. In contrast, the C-terminal acidic serine-rich region is not required for p40-mediated inhibition of nuclear translocation of Rel proteins. Our results demonstrate that p40-mediated inhibition of nuclear translocation and inhibition of DNA-binding by Rel proteins are separable functions. Our results suggest that the phosphorylation status of C-terminal serine residues of I kappa B-alpha proteins will be an important aspect of the autoregulatory feedback loop that enforces temporal control of Rel-regulated gene expression.