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.