Multiple roles for acetylation in the interaction of p300 HAT with ATF-2
Multiple roles for acetylation in the interaction of p300 HAT with ATF-2
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DOI：
10.1021/bi7000054
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发表时间：
2007-07-17
期刊：
影响因子：
2.9
通讯作者：
Cole, Philip A.
中科院分区：
文献类型：
--
作者：
Karanam, Balasubramanyam;Wang, Ling;Cole, Philip A.
转录共激活因子同源物p300和CBP含有乙酰转移酶结构域（HAT），并催化组蛋白和其他蛋白质的赖氨酸乙酰化，这是它们功能的一个重要方面。先前的研究表明，转录因子ATF - 2（也称为CRE - BP1）的碱性亮氨酸拉链结构域（b - ZIP）可与CBP的HAT结构域相互作用。在本研究中，我们检测了ATF - 2的b - ZIP结构域与p300的HAT结构域的相互作用，并表明p300的HAT结构域的自身乙酰化可增强结合亲和力。下拉实验表明，高乙酰化的p300的HAT结构域比低乙酰化的p300的HAT结构域更能有效地被固定的ATF - 2的b - ZIP结构域所保留。缺失自身乙酰化环的p300的HAT结构域的保留情况与高乙酰化的p300的HAT结构域大致相同，这表明该环和ATF - 2竞争与p300的HAT结构域的结合。虽然ATF - 2的b - ZIP结构域是低乙酰化的p300的HAT结构域对组蛋白H4肽乙酰化的弱抑制剂，但高乙酰化的p300的HAT结构域受到ATF - 2的b - ZIP结构域的抑制作用更强。此外，我们表明ATF - 2的b - ZIP结构域可作为p300的HAT结构域的乙酰转移酶底物。利用质谱法，在ATF - 2的b - ZIP结构域中定位了两个p300的HAT结构域的赖氨酸乙酰化位点。用抗乙酰化赖氨酸抗体进行的免疫沉淀 - 蛋白质印迹分析表明，ATF - 2在体内可发生可逆的乙酰化。对ATF - 2的b - ZIP结构域的两个乙酰化位点的突变分析揭示了它们对ATF - 2介导的转录激活的潜在贡献。综上所述，这些研究表明蛋白质乙酰化在p300/CBP和ATF - 2对转录的调控中具有多种作用。
The transcriptional coactivator paralogues p300 and CBP contain acetyltransferase domains (HAT) and catalyze the lysine acetylation of histones and other proteins as an important aspect of their functions. Prior studies revealed that the basic leucine zipper domain (b-ZIP) of transcription factor ATF-2 (also called CRE-BP1) can interact with the CBP HAT domain. In this study, we have examined the ATF-2 b-ZIP interaction with the p300 HAT domain and shown that p300 HAT autoacetylation can enhance the binding affinity. Pull-down assays revealed that hyperacetylated p300 HAT is more efficiently retained by immobilized ATF-2 b-ZIP than hypoacetylated p300 HAT. Loop deleted p300 HAT lacking autoacetylation was retained about as well as hyperacetylated p300 HAT, suggesting that the loop and ATF-2 compete for p300 HAT binding. While ATF-2 b-ZIP is a weak inhibitor of hypoacetylated p300 HAT acetylation of a histone H4 peptide, hyperacetylated p300 HAT is much more potently inhibited by ATF-2 b-ZIP. Moreover, we showed that ATF-2 b-ZIP could serve as an acetyltransferase substrate for p300 HAT. Using mass spectrometry, two p300 HAT lysine acetylation sites were mapped in ATF-2 b-ZIP. Immunoprecipitation-Western blot analysis with anti-acetyl-lysine antibody revealed that ATF-2 can undergo reversible acetylation in vivo. Mutational analysis of the two ATF-2 b-ZIP acetylation sites revealed their potential contributions to ATF-2-mediated transcriptional activation. Taken together, these studies suggest multiple roles for protein acetylation in the regulation of transcription by p300/CBP and ATF-2.