Extensive Post-Translational Modification of Endogenous Human p53

Abstract

The p53 tumor suppressor protein is a central component of numerous cellular signaling pathways and frequently mutated in human cancers. p53 undergoes extensive post-translational modification (PTM) in response to specific stressors and other cellular events, resulting in a combinatorial PTM code that may direct its stability, localization, and activity within the cell. p53 accumulates dramatically when normal or transformed cells are subjected to infection by a human adenovirus type 5 (Ad5) E1B 55 kDa-null mutant, which lacks the virus-specific ubiquitin ligase that targets p53 for proteasomal degradation. This population of p53 has been shown to localize normally, but is unable to activate transcription of p53 target genes. The mechanism by which this repression of p53 transcriptional activity occurs is unknown.

One hypothesis was that the transcriptionally inert status of p53 might be due, in part, to PTM. The p53 protein that accumulates within adenoviral E1B 55 kDa null mutant-infected normal cells presented a unique opportunity to isolate an endogenous population of wild-type, transcriptionally inert p53 for analysis of PTM by mass spectrometry. In Chapter 2, this system was utilized to generate an expanded modification profile for transcriptionally inert human p53 and to compare this profile to one generated from an equivalent population of transcriptionally active human p53, in order to identify patterns of modifications potentially responsible for modulating p53 transcriptional function. While there were no significant differences in the PTM of transcriptionally inert and active p53, the wealth of PTMs identified suggested the possibility of a far greater degree of combinatorial regulation of p53 by PTM than previously anticipated.

Additionally, p53 was recently reported to exhibit transcriptional activity in normal human cells infected by an E1B 55 kDa/E4orf3 double null mutant. In Chapter 3, an E1B 55 kDa/E4orf3 double null mutant was constructed for the purpose of clarifying the role of E4orf3 in the inhibition of p53-dependent target gene transcription, in addition to ascertaining whether the differences in p53 transcriptional activity or selectivity observed during infection with an E1B 55 kDa- or E1B 55 kDa/E4orf3-null mutant were in any way due to differential patterns of p53 PTM.