Functional characterisation of T329 phosphorylation in the tetramerisation domain of p53

Abstract

As a tumor suppressor the transcription factor p53 plays an essential role in maintaining genome stability due to its ability to repair the genome and induce apoptosis upon DNA damage. Phosphorylations play an important role in the activation and regulation of the p53 response. Many PTMs have already been identified as important regulators, such as the phosphorylation at S329 by CK2 and the phosphorylation at S46 by HIPK2 or PKCδ. This study identifies and characterises the phosphorylation of T329 in the tetramerisation domain of p53 as a priming modification, which mediates further PTMs and thus contribute to the p53 activation. Initially, pT329 was identified by mass spectrometry as a result of DNA damage. With a pT329-specific antibody, the phosphorylation under various genotoxic stimulants could be confirmed. To investigate the pT329 functions, HCT116 p53 knockout cells were reconstituted with p53 WT, p53 T329A and p53 T329E in the safe harbour gene locus AAVS1. The T329A mutant represents the non-phosphorylated T329 and the T329E mutant the state of the phosphorylated T329. The T329A mutation leads to a significant reduction in p53-dependent gene expression, which results in impaired G1 cell cycle arrest and reduced induction of apoptosis. Chromatin immunoprecipitation showed that the T329A mutant does not bind inductively to the DNA after stimulation of the p53 response. An examination of the p53 conformation showed that p53 T329A has a lower thermodynamic stability than p53 WT or p53 T329E. The phosphorylation at T329 thus supports the folding and thereby the stability of the p53 protein. As a result, pT329 plays an important role in protein-protein interactions, and thus also in the modification of p53 with PTMs. The interactome confirmed that both mutations of T329 have an evident influence on the association of p53 with other proteins. Thus, the T329A mutant stronger binds chaperones, while ribosomal proteins interact primarily with the p53 WT protein. In addition, the visualisation of other activating PTMs showed that activating modifications such as pS392, pS46 and acK373/K382 are missing or are significantly reduced in p53 T329A. Therefore, pT329 influences the p53 activation probably by stabilising the conformation and the mediation of the activating PTM network. Through kinase inhibitors and knockdown experiments, Chk1, CK2 and PKCδ could be identified as possible kinases for T329 phosphorylation. In summary, phosphorylation of T329 is a new detail in p53 activation, thus improving the understanding of p53 activation in relation to cancer prevention and therapy.