Investigation of phosphorylation of p53-TAD by DNA-PK enzyme

TP53 gene is one of the most frequently mutated genes in human cancers (30-50%). The cellular level of p53 is regulated depending on HDM2 (Mouse Double Minute 2 Homolog, human homolog of MDM2). Under normal physiological conditions, TAD interacts with the HDM2 protein and directs p53 to proteasomal degradation. Ser15 and Ser37 residues of TAD are phosphorylated by DNA-PK (DNA-dependent protein kinase). This phosphorylation event prevents the effective interaction of p53 with HDM2, and thus p53 is stabilized. Stabilized p53 initiates the activation of various genes as a transcription factor. Previous studies have reported that the Leu22Gln-Trp23Ser mutation in the TAD domain of p53 reduces the transactivation function of p53 to different degrees. It has also been reported that mutation of Phe19 to arginine negatively affects the transactivation function of p53. These reported mutations are located close to the Ser15 and Ser37 phosphorylation sites, which are phosphorylated by the DNA-PK enzyme. In this study, Leu22Gln-Trp23Ser and Phe19Ala mutations of p53 were generated by site-directed mutagenesis, and the effect of these mutations on the phosphorylation of Ser15 and Ser 37 by DNA-PK was determined by western blotting using antibodies specific for Ser15P and Ser37P. It was determined that the Phe19Ala mutant reduced Ser15 phosphorylation by 2.3 times and the Leu22Gln-Trp23Ser mutant by 1.6 times compared to the wild type. The Phe19Ala mutant reduced Ser37 phosphorylation by 1.6 times compared to the wild type, and the Leu22Gln-Trp23Ser mutant decreased by 2.0 times. When comparing wild-type Ser15 and Ser37 phosphorylation, it was determined that DNA-PK phosphorylates Ser37 1.9 times faster. Besides, it was predicted by I-TASSER that the mutants caused conformational changes in the TAD. In addition, it was determined by HADDOCK 2.4 that this conformational change changed the orientation of the TAD - (DNA-PKcs) interaction. De novo design data suggest that the decrease in phosphorylation in the mutants is due to the conformational change caused by the mutations, which changes the orientation of the enzyme-substrate interaction.