DNA-dependent acetylation of p53 by the transcription coactivator p300
DNA-dependent acetylation of p53 by the transcription coactivator p300
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DOI：
10.1074/jbc.m211460200
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发表时间：
2003-04-11
影响因子：
4.8
通讯作者：
Hupp, TR
中科院分区：
文献类型：
--
作者：
Dornan, D;Shimizu, H;Hupp, TR
p300.p53转录复合物组装阶段的重建已经确定了一种新型的DNA依赖性调节p300催化的乙酰化。在p53的N-末端活化结构域中的CHK 2位点（Ser（20））处的磷酸化稳定了p300结合。p300的磷酸肽结合活性在体外被定位到两个结构域：C-末端IBiD结构域和N-末端IHD结构域（IBiD同源结构域）。如使用哺乳动物双杂交VP 16-GAL 4荧光素酶报告基因测定所确定的，IHD或IBiD小域可以在体内结合p53活化结构域。在体内，p300的IHD和IBiD minidomain也作为p53依赖性转录的显性负抑制剂发挥作用。在研究p300结合对底物乙酰化的影响后，我们发现p53共有位点DNA在体外促进p53乙酰化的显著增加。将p53基因和含有p53的共有DNA结合位点的质粒DNA共转染到细胞中激活了体内p53的DNA依赖性乙酰化。p300的磷酸肽结合活性对于DNA依赖性乙酰化是关键的，因为磷酸-Ser（20）肽抑制p53乙酰化。p53的一致位点DNA依赖性乙酰化稳定了p300-p53蛋白复合物，而p300在非特异性DNA存在下对p53的基础乙酰化导致p300解离。这些数据鉴定了p300-p53复合物组装中的至少三个不同阶段：1）p300通过IBiD和/或IHD结构域对接至p53的活化结构域; 2）p53的DNA依赖性乙酰化;和3）乙酰化后p300.p53（AC）复合物的稳定化。DNA作为变构配体激活底物乙酰化的能力确定了可以对p300-乙酰化反应施加的构象约束，其可能是扩增信号并影响启动子处的蛋白质-蛋白质接触。
Reconstitution of the stages in the assembly of the p300.p53 transcription complex has identified a novel type of DNA-dependent regulation of p300-catalyzed acetylation. Phosphorylation at the CHK2 site (Ser(20)) in the N-terminal activation domain of p53 stabilized p300 binding. The phosphopeptide binding activity of p300 was mapped in vitro to two domains: the C-terminal IBiD domain and the N-terminal IHD domain (IBiD homology domain). The IHD or IBiD minidomains can bind to the p53 activation domain in vivo as determined using the mammalian two-hybrid VP16-GAL4 luciferase reporter assay. The IHD and IBiD minidomains of p300 also functioned as dominant negative inhibitors of p53-dependent transcription in vivo. Upon examining the affects of p300 binding on substrate acetylation, we found that the p53 consensus site DNA promotes a striking increase in p53 acetylation in vitro. Co-transfection into cells of the p53 gene and plasmid DNA containing the consensus DNA binding site of p53 activated DNA-dependent acetylation of p53 in vivo. The phosphopeptide binding activity of p300 is critical for DNA-dependent acetylation, as p53 acetylation was inhibited by phospho-Ser(20) peptides. Consensus site DNA-dependent acetylation of p53 stabilized the p300-p53 protein complex, whereas basal acetylation of p53 by p300 in the presence of nonspecific DNA resulted in p300 dissociation. These data identify at least three distinct stages in the assembly of a p300-p53 complex: 1) p300 docking to the activation domain of p53 via the IBiD and/or IHD domains; 2) DNA-dependent acetylation of p53; and 3) stabilization of the p300.p53(AC) complex after acetylation. The ability of DNA to act as an allosteric ligand to activate substrate acetylation identifies a conformational constraint that can be placed on the p300-acetylation reaction that is likely to be an amplification signal and influence protein-protein contacts at a promoter.