Cellular characterisation of p53 mutants with a single missense mutation in the β-strand 326-333 and correlation of their cellular activities with in vitro properties

Recent evidence has shown that missense mutations in the p53 tetramerisation domain can inactivate the protein. However, most of these studies have been done only in vitro or concern proteins whose physico-chemical properties have not been fully investigated. Alanine mutants of the P-strand 326-333 from the tetramerisation domain have been characterized in vitro and studied for their thermodynamic stability. They therefore offer a unique opportunity to establish a correlation between in vitro and cellular activities of proteins with a mutated tetramerisation domain. The eight mutant proteins resulting from the mutation of the eight residues of the P-strand 326-333 to alanine were analysed for their ability to stimulate transcription, to inhibit the growth of Saos-2 cells and to repress the promoter of the multidrug resistance gene 1. The experimental results show a perfect correlation between in vitro and cellular data. The Leu330Ala and Ile332Ala proteins are inactive, the Phe328Ala protein has a moderate activity. The Glu326Ala, Tyr327Ala, Thr329Ala, Gln331Ala and Arg333Ala proteins show activity similar to that of wildtype protein. This work is based on an exhaustive analysis of p53 mutants both in vitro and in cells and shows that mutations in the tetramerisation domain might be of importance in cancer development since they inactivate the p53 protein. (C) 1999 Academic Press.