The non-ankyrin C terminus of IκBα physically interacts with p53 in vivo and dissociates in response to apoptotic stress, hypoxia, DNA damage, and transforming growth factor-β1-mediated growth suppression
The non-ankyrin C terminus of IκBα physically interacts with p53 in vivo and dissociates in response to apoptotic stress, hypoxia, DNA damage, and transforming growth factor-β1-mediated growth suppression
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DOI：
10.1074/jbc.m106607200
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发表时间：
2002-03-22
影响因子：
4.8
通讯作者：
Chang, NS
中科院分区：
文献类型：
--
作者：
Chang, NS
转化生长因子β (tgf - β 1)抑制水貂肺Mv1Lu上皮细胞的生长，而睾丸透明质酸酶则消除这种生长抑制作用。Mv1Lu细胞暴露于tgf - β后，细胞内ikappabα迅速下调，透明质酸酶阻止了这种作用，提示ikappabα可能在生长调节中起作用。野生型和显性阴性IkappaBalpha的异位表达阻止了tgf - β 1介导的生长抑制。尽管如此，IkappaBalpha的阻断作用与其n端锚蛋白重复区域（氨基酸1-243）对NF-kappaB功能的调节无关。去除含脯氨酸-谷氨酸-丝氨酸-苏氨酸结构域C端（氨基酸244-314）可使IkappaBalpha功能消失，C端单独阻断tgf - β 1的生长抑制作用。利用Mv1Lu和其他类型的细胞以及大鼠肝脏和脾脏进行抗p53抗体共免疫沉淀，发现部分细胞质IkappaBalpha与p53发生物理相互作用。相反，p53抑制剂Mdm2在免疫沉淀中几乎检测不到。胞质p53-IkappaBalpha复合物在凋亡应激、依托泊苷和紫外线介导的DNA损伤、缺氧和tgf - β - 1介导的生长抑制下迅速解离。此外，暴露于依托泊苷和紫外线下，细胞核p53-IkappaBalpha复合物的形成迅速增加。相比之下，tgf - β 1介导的成纤维细胞生长促进未能介导p，53-IkappaBalpha解离。酵母双杂交图谱显示，IkappaBalpha的非锚蛋白C端与p53富含脯氨酸的区域和磷酸化位点丝氨酸46发生物理相互作用。删除46号丝氨酸或将46号丝氨酸改变为甘氨酸可消除p53-IkappaBalpha相互作用。苏氨酸的改变保留了结合相互作用，这表明丝氨酸46磷酸化参与了p53-IkappaBalpha复合物的形成。在功能上，当p53和IkappaBalpha在细胞中短暂共表达时，可以观察到p53凋亡的增强。总之，ikappabα -p53复合物在包括生长调节、细胞凋亡和缺氧应激的反应中起重要作用。
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-betal 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 wildtype 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-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-IkappaBalpha complex was observed during exposure to etoposide and UV. In contrast, TGF-beta1-mediated promotion of fibroblast growth failed to mediate p,53-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-IkappaBalpha interaction. Alteration to threonine retained the binding interaction, suggesting that serine 46 phosphorylation is involved in the p53-IkappaBalpha complex formation. Functionally, enhancement of p53 apoptosis was observed when p53 and IkappaBalpha were transiently co-expressed in cells. Together, the IkappaBalpha-p53 complex plays an important role in responses involving growth regulation, apoptosis, and hypoxic stress.