Real-time and simultaneous monitoring of the phosphorylation and enhanced interaction of p53 and XPC acidic domains with the TFIIH p62 subunit.
Real-time and simultaneous monitoring of the phosphorylation and enhanced interaction of p53 and XPC acidic domains with the TFIIH p62 subunit.
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DOI：
10.1038/oncsis.2015.13
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发表时间：
2015-06-01
期刊：
影响因子：
6.2
通讯作者：
Nishimura Y
中科院分区：
文献类型：
--
作者：
Okuda M;Nishimura Y
翻译后修饰通过相互作用在多种生物学过程中起着关键作用。肿瘤抑制蛋白p53和核苷酸切除修复因子XPC各自含有酸性区域，分别称为酸性反式激活结构域（acidic transactivation domain，ACTA）和酸性片段（acidic fragment，AF），其结合转录因子TFIIH的p62亚基的普列克底物蛋白同源（pleckstrin homology，PH）结构域。人p53-p53含有7个丝氨酸和2个苏氨酸残基，所有这些残基都可以被磷酸化。类似地，XPC-AF含有6个丝氨酸和2个苏氨酸残基，其中Thr 117、Ser 122和Ser 129已被报道为体内磷酸化位点，尽管它们的磷酸化作用尚不清楚。p53-AF的Ser 46和Thr 55的磷酸化增加了其结合能力;然而，XPC-AF磷酸化的作用仍然难以捉摸。在这里，我们描述了一个系统的实时和同时监测的磷酸化和p62-PH的亲和力的p53-β和XPC-AF使用核磁共振（NMR）光谱。出乎意料的是，在7种可能磷酸化p53-kD的Ser 46和/或Thr 55的激酶中，仅鉴定出两种特异性和高效的酶：Ser 46的JNK 2 α2和Thr 55的GRK 5。在与p62-PH的相互作用期间，鉴定了四种不同的亲和复合物，所述复合物由激酶对p53-p53的各种磷酸化状态产生。使用NMR系统实时监测p53-p53的位点特异性磷酸化反应的动力学及其对p62-PH的亲和力。等温量热法显示XPC-AF的Ser 129磷酸化增加了与p62-PH的结合。尽管从其序列背景预测CK 2磷酸化Ser 122、Ser 129和Ser 140，但它仅特异性地且有效地磷酸化Ser 129。同时监测p62-PH结合的磷酸化和增强，鉴定了p62-PH接触磷酸化Ser 129的关键残基。总之，我们已经建立了一个NMR系统，用于实时和同时监测位点特异性磷酸化和增强磷酸化结构域与其靶标之间的亲和力。该系统也适用于其他翻译后修饰。
Posttranslational modifications have critical roles in diverse biological processes through interactions. Tumor-suppressor protein p53 and nucleotide excision repair factor XPC each contain an acidic region, termed the acidic transactivation domain (TAD) and acidic fragment (AF), respectively, that binds to the pleckstrin homology (PH) domain of the p62 subunit of the transcription factor TFIIH. Human p53-TAD contains seven serine and two threonine residues, all of which can be phosphorylated. Similarly, XPC-AF contains six serine and two threonine residues, of which Thr117, Ser122 and Ser129 have been reported as phosphorylation sites in vivo, although their phosphorylation roles are unknown. Phosphorylation of Ser46 and Thr55 of p53-TAD increases its binding ability; however, the role of XPC-AF phosphorylation remains elusive. Here we describe a system for real-time and simultaneous monitoring of the phosphorylation and p62-PH affinity of p53-TAD and XPC-AF using nuclear magnetic resonance (NMR) spectroscopy. Unexpectedly, among seven reported kinases that presumably phosphorylate Ser46 and/or Thr55 of p53-TAD, only two specific and high-efficiency enzymes were identified: JNK2α2 for Ser46 and GRK5 for Thr55. During interaction with p62-PH, four different affinity complexes resulting from various phosphorylation states of p53-TAD by the kinases were identified. The kinetics of the site-specific phosphorylation reaction of p53-TAD and its affinity for p62-PH were monitored in real-time using the NMR system. Isothermic calorimetry showed that phosphorylation of Ser129 of XPC-AF increases binding to p62-PH. Although CK2 was predicted to phosphorylate Ser122, Ser129 and Ser140 from its sequence context, it specifically and efficiently phosphorylated only Ser129. Simultaneous monitoring of the phosphorylation and augmentation in p62-PH binding identified a key residue of p62-PH for contacting phosphorylated Ser129. In summary, we have established an NMR system for real-time and simultaneous monitoring of site-specific phosphorylation and enhancement of affinity between phosphorylation domains and their target. The system is also applicable to other posttranslational modifications.