Two distinct motifs within the p53 transactivation domain bind to the Taz2 domain of p300 and are differentially affected by phosphorylation.
Two distinct motifs within the p53 transactivation domain bind to the Taz2 domain of p300 and are differentially affected by phosphorylation.
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DOI：
10.1021/bi801716h
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发表时间：
2009-02-17
期刊：
影响因子：
2.9
通讯作者：
Mazur, Sharlyn J.
中科院分区：
文献类型：
--
作者：
Jenkins, Lisa M. Miller;Yamaguchi, Hiroshi;Hayashi, Ryo;Cherry, Scott;Tropea, Joseph E.;Miller, Maria;Wlodawer, Alexander;Appella, Ettore;Mazur, Sharlyn J.
肿瘤抑制因子 p53 作为许多基因的转录激活因子，包括参与细胞周期停滞和细胞凋亡的几个关键基因。 DNA 损伤引起的应激后，p53 经历广泛的翻译后修饰，从而提高稳定性和活性。 p53 介导的反式激活的两个关键辅助因子是组蛋白乙酰转移酶旁系同源物 CREB 结合蛋白 (CBP) 和 p300。 p53 的 N 端反式激活结构域与 CBP/p300 的多个结构域相互作用，包括 Taz2 结构域。在这里，我们报告了特定 p53 磷酸化对其与 p300 Taz2 结构域相互作用的影响。使用竞争性荧光各向异性测定，我们确定 p53 Ser15 或 Thr18 处的单磷酸化增加了 p53(1-39) 对 Taz2 的亲和力，而 Ser15 和 Ser37 或 Thr18 和 Ser20 处的二磷酸化进一步增加了亲和力。此外，我们还在 p53 残基 35-59 内鉴定了 Taz2 的第二个结合位点。第二个位点以与第一个位点相似的亲和力结合 Taz2，但这种结合不受磷酸化的影响。因此，p53 翻译后修饰仅调节 p300 Taz2 的两个结合位点之一。通过等温滴定量热法对 Taz2 与 p53(1-39) 或 p53(35-59) 结合的进一步研究表明，在复合物形成时，两个位点的恒压热容变化 ΔCp 均为负值，这表明疏水相互作用的重要性。然而，与第二个位点 (-234 cal/mol K) 相比，Taz2 与第一个位点 (-330 cal/mol K) 结合的 ΔCp 负值更大，表明两个位点之间非极性和极性相互作用的重要性不同。
The tumor suppressor p53 functions as a transcriptional activator for many genes, including several key genes involved in cell cycle arrest and apoptosis. Following DNA damage-induced stress, p53 undergoes extensive post-translational modification, resulting in increased stability and activity. Two critical cofactors for p53-mediated transactivation are the histone acetyltransferase paralogs CREB-binding protein (CBP) and p300. The N-terminal transactivation domain of p53 interacts with several domains of CBP/p300, including the Taz2 domain. Here, we report the effects of specific p53 phosphorylations on its interaction with the Taz2 domain of p300. Using a competitive fluorescence anisotropy assay, we determined that monophosphorylation of p53 at Ser15 or Thr18 increased the affinity of p53(1-39) for Taz2, and diphosphorylations at Ser15 and Ser37 or Thr18 and Ser20 further increased the affinity. In addition, we identified a second binding site for Taz2 within p53 residues 35-59. This second site bound Taz2 with a similar affinity as the first site, but the binding was unaffected by phosphorylation. Thus, p53 post-translational modification modulates only one of the two binding sites for p300 Taz2. Further investigation of Taz2 binding to p53(1-39) or p53(35-59) by isothermal titration calorimetry indicated that upon complex formation the change in heat capacity at constant pressure, ΔCp, was negative for both sites, suggesting the importance of hydrophobic interactions. However, the more negative value of ΔCp for Taz2 binding to the first (−330 cal/mol K) compared to the second site (−234 cal/mol K) suggests that the importance of nonpolar and polar interactions differs between the two sites.
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