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Oncogenesis. 2015 Jun 1;4:e150. doi: 10.1038/oncsis.2015.13.

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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1
Graduate School of Medical Life Science, Yokohama City University, Yokohama, Japan.

Abstract

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.

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