Transforming growth factor beta (TGF-β1) suppresses the growth of mink lung Mv1Lu epithelial cells, whereas testicular hyaluronidase abolishes the growth inhibition. Exposure of Mv1Lu cells to TGF-β1 rapidly resulted in down-regulation of cytosolic IκBα and hyaluronidase prevented this effect, suggesting a possible role of IκBα in the growth regulation. Ectopic expression of wild-type and dominant negative IκBα prevented TGF-β1-mediated growth suppression. Nonetheless, the blocking effect of IκBα is not related to regulation of NF-κB function by its N-terminal ankyrin-repeat region (amino acids 1–243). Removal of the PEST (proline-glutamic acid-serine-threonine) domain-containing C terminus (amino acids 244–314) abolished the IκBα function, and the C terminus alone blocked the TGF-β1 growth-inhibitory effect. Co-immunoprecipitation by anti-p53 antibody using Mv1Lu and other types of cells, as well as rat liver and spleen, revealed that a portion of cytosolic IκBα physically interacted with p53. In contrast, Mdm2, an inhibitor of p53, was barely detectable in the immunoprecipitates. The cytosolic p53·IκBα complex rapidly dissociated in response to apoptotic stress, etoposide- and UV-mediated DNA damage, hypoxia, and TGF-β1-mediated growth suppression. Also, a rapid increase in the formation of the nuclear p53·IκBα complex was observed during exposure to etoposide and UV. In contrast, TGF-β1-mediated promotion of fibroblast growth failed to mediate p53·IκBα dissociation. Mapping by yeast two-hybrid showed that the non-ankyrin C terminus of IκBα physically interacted with the proline-rich region and a phosphorylation site, serine 46, in p53. Deletion of serine 46 or alteration of serine 46 to glycine abolished the p53·IκBα interaction. Alteration to threonine retained the binding interaction, suggesting that serine 46 phosphorylation is involved in the p53·IκBα complex formation. Functionally, enhancement of p53 apoptosis was observed when p53 and IκBα were transiently co-expressed in cells. Together, the IκBα·p53 complex plays an important role in responses involving growth regulation, apoptosis, and hypoxic stress.
