DNA Binding and Phosphorylation Regulate the Core Structure of the NF-κB p50 Transcription Factor.
DNA Binding and Phosphorylation Regulate the Core Structure of the NF-κB p50 Transcription Factor.
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DOI：
10.1007/s13361-018-1984-0
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发表时间：
2019-01
影响因子：
3.2
通讯作者：
Eyers CE
中科院分区：
文献类型：
--
作者：
Vonderach M;Byrne DP;Barran PE;Eyers PA;Eyers CE
众所周知，核因子-κB转录因子被广泛的磷酸化，通过动态的位点特异性修饰调节它们与DNA二聚体和相互作用的能力。P50是p105(NF-κB1)的蛋白水解物，形成同源二聚体，与κ结合，但缺乏内在的反式激活功能，作为转录抑制因子来自DNA B启动子。在这里，我们研究了由蛋白激酶A(PKAc)或Chk1催化的特定磷酸化事件在调节p50同源二聚体功能中的作用。通过对体外磷酸化后的蛋白水解酶p50进行LC-MS/MS分析，我们可以将Ser328和Ser337定义为PKAc和Chk1介导的修饰，并精确定位了另外四个Chk1亚基：Ser65、Thr152、Ser242和Ser248。天然质谱仪(MS)显示Chk1和PKAc通过Ser337调节p50同源二聚体的形成。此外，我们将Chk1介导的亚磷酸盐Ser242描述为DNA结合的调节因子，而S242D p50模拟磷脂酶使 与DNA的结合亲和力降低了10倍。用离子迁移率MS(IM-MS)进一步研究了包括S242D在内的仿磷p50突变体的构象动力学。最后，与实验观察到的p50构象的比较理论建模，在没有和存在DNA的情况下，揭示了p50同源二聚体在电喷雾电离过程中经历构象收缩，这是通过与κB DNA形成络合物而稳定的。ᅟ本文的在线版本(10.1007/s13361-0181984-0)包含向授权用户提供的补充材料。
The NF-κB transcription factors are known to be extensively phosphorylated, with dynamic site-specific modification regulating their ability to dimerize and interact with DNA. p50, the proteolytic product of p105 (NF-κB1), forms homodimers that bind DNA but lack intrinsic transactivation function, functioning as repressors of transcription from κB promoters. Here, we examine the roles of specific phosphorylation events catalysed by either protein kinase A (PKAc) or Chk1, in regulating the functions of p50 homodimers. LC-MS/MS analysis of proteolysed p50 following in vitro phosphorylation allows us to define Ser328 and Ser337 as PKAc- and Chk1-mediated modifications, and pinpoint an additional four Chk1 phosphosites: Ser65, Thr152, Ser242 and Ser248. Native mass spectrometry (MS) reveals Chk1- and PKAc-regulated disruption of p50 homodimer formation through Ser337. Additionally, we characterise the Chk1-mediated phosphosite, Ser242, as a regulator of DNA binding, with a S242D p50 phosphomimetic exhibiting a > 10-fold reduction in DNA binding affinity. Conformational dynamics of phosphomimetic p50 variants, including S242D, are further explored using ion-mobility MS (IM-MS). Finally, comparative theoretical modelling with experimentally observed p50 conformers, in the absence and presence of DNA, reveals that the p50 homodimer undergoes conformational contraction during electrospray ionisation that is stabilised by complex formation with κB DNA. ᅟ The online version of this article (10.1007/s13361-018-1984-0) contains supplementary material, which is available to authorized users.
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影响因子：
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DOI：
10.1073/pnas.0908797106
发表时间：
2009-11-17
影响因子：
11.1
作者：
Bergqvist, Simon;Alverdi, Vera;Komives, Elizabeth A.
通讯作者：
Komives, Elizabeth A.