The non-ankyrin C terminus of Ikappa Balpha physically interacts with p53 in vivo and dissociates in response to apoptotic stress, hypoxia, DNA damage, and transforming growth factor-beta 1-mediated growth suppression.
Transforming growth factor beta (TGF-beta1) suppresses the growth of mink lung Mv1Lu epithelial cells, whereas testicular hyaluronidase abolishes the growth inhibition. Exposure of Mv1Lu cells to TGF-beta1 rapidly resulted in down-regulation of cytosolic IkappaBalpha and hyaluronidase prevented this effect, suggesting a possible role of IkappaBalpha in the growth regulation. Ectopic expression of wild-type and dominant negative IkappaBalpha prevented TGF-beta1-mediated growth suppression. Nonetheless, the blocking effect of IkappaBalpha is not related to regulation of NF-kappaB 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 IkappaBalpha function, and the C terminus alone blocked the TGF-beta1 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 IkappaBalpha physically interacted with p53. In contrast, Mdm2, an inhibitor of p53, was barely detectable in the immunoprecipitates. The cytosolic p53 x IkappaBalpha complex rapidly dissociated in response to apoptotic stress, etoposide- and UV-mediated DNA damage, hypoxia, and TGF-beta1-mediated growth suppression. Also, a rapid increase in the formation of the nuclear p53 x IkappaBalpha complex was observed during exposure to etoposide and UV. In contrast, TGF-beta1-mediated promotion of fibroblast growth failed to mediate p53 x IkappaBalpha dissociation. Mapping by yeast two-hybrid showed that the non-ankyrin C terminus of IkappaBalpha 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 x IkappaBalpha interaction. Alteration to threonine retained the binding interaction, suggesting that serine 46 phosphorylation is involved in the p53 x IkappaBalpha complex formation. Functionally, enhancement of p53 apoptosis was observed when p53 and IkappaBalpha were transiently co-expressed in cells. Together, the IkappaBalpha x p53 complex plays an important role in responses involving growth regulation, apoptosis, and hypoxic stress.
Chang
Nan-Shan
NS
Guthrie Research Institute, Laboratory of Molecular Immunology, Guthrie Medical Center, Sayre, Pennsylvania, 18840, USA. nschang@inet.guthrie.org
D000818:Animals; D017487:Ankyrins / Q000378:metabolism*; D017209:Apoptosis; D019556:COS Cells; D002455:Cell Division; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation; D005047:Etoposide / Q000494:pharmacology; D005799:Genes, Dominant; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000378:metabolism; D000860:Hypoxia; D020672:I-kappa B Proteins; D008099:Liver / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D000072000:NF-KappaB Inhibitor alpha; D010766:Phosphorylation; D011233:Precipitin Tests; D011392:Proline / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013154:Spleen / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013912:Threonine / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*; D053773:Transforming Growth Factor beta1; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D014466:Ultraviolet Rays
D017487:Ankyrins; D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; C000606206:Nfkbia protein, rat; D011994:Recombinant Proteins; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; C508962:Tgfb1 protein, rat; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D016159:Tumor Suppressor Protein p53; D000072000:NF-KappaB Inhibitor alpha; D013912:Threonine; D012694:Serine; D005047:Etoposide; D011392:Proline; D006821:Hyaluronoglucosaminidase; D019311:Staurosporine