Functional analysis ofp53 phosphorylation and ap53 hot spot mutation

Mingming Hao, The University of Texas Graduate School of Biomedical Sciences at Houston


The tumor suppressor p53 is a phosphoprotein which functions as a transcriptional activator. By monitoring the transcriptional activity, we studied how p53 functions is regulated in relation to cell growth and contact inhibition. When cells were arrested at G1 phase of the cell cycle by contact inhibition, we found that p53 transactivation function was suppressed. When contact inhibition was overridden by cyclin E overexpression which stimulates cell cycle progression, p53 function was restored. This observation led to the development of a cell density assay to study the regulation of p53 function during cell cycle for the functional significance of p53 phosphorylation. The murine p53 is phosphorylated at serines 7, 9, 12, 18, 37, 312 and 389. To understand the role of p53 phosphorylation, we generated p53 constructs encoding serine-to-alanine or serine-to-glutamate mutations at these codons. The transcriptional activity were measured in cells capable of contact inhibition. In low-density cycling cells, no difference in transcriptional activity was found between wild type p53 and any of the mutants. In contact-inhibited cells, however, only mutations of p53 at serine 389 resulted in altered responses to cell cycle arrest and to cyclin E overexpression. The mutant with serine-to-glutamate substitution at codon 389 retained its function in contact inhibited cells. Cyclin E overexpression in these cells induced p53 phosphorylation at serine 389. Furthermore, we showed that phosphorylation at serine 389 regulates p53 DNA binding activity. Our findings implicate that phosphorylation is an important mechanism for p53 activation. p53 is the most frequently mutated gene in human tumors. To study the mechanism of p53 inactivation by mutations, we carried out detailed analysis of a murine p53 mutation with an arginine-to-tryptophane substitution at codon 245. The corresponding human p53 mutation at amino acid 248 is the most frequently mutated codon in tumors. We showed that this mutant is inactive in suppressing focus formation, binding to DNA and transactivation. Structural analysis revealed that this mutant assumes the wild type protein conformation. These findings define a novel class of p53 mutations and help to understand structure-function relationship of p53.

Subject Area

Molecular biology|Genetics|Cellular biology

Recommended Citation

Hao, Mingming, "Functional analysis ofp53 phosphorylation and ap53 hot spot mutation" (1995). Texas Medical Center Dissertations (via ProQuest). AAI9532531.