Identification of the interacting domain of p53 family members with P33ING1 protein

Abstract

p53 is a tumor suppressor gene, which is mutated, in about 50% of human cancers. The product of p53 gene encodes a sequence specific transcription factor. The genes transactivated by p53 code for proteins that are implicated in the negative regulation of cell proliferation and DNA damage repair. Two proteins, p63 and p73 members of p53 family, show striking homology to p53. p53 protein interacts with several viral and cellular proteins and these interactions are important in the regulation and dysregulation of the functions of p53. Another gene named, ING1 was identified as a candidate tumor suppressor gene due to its functions in apoptosis and cell cycle arrest. p24ING1, one of the protein product of ING1, was shown to enhance the growth suppressor functions of p53. Furthermore a physical association between p53 and p33ING1, another ING1 transcript, proteins has been detected by immunoprecipitation. In this study, we investigated the physical interaction between p53 family proteins and p33ING1 using in vitro techniques in order to determine the region(s) of p53 family proteins and p33ING1 that enabled this interaction. As a preliminary step for the study, the wild-type p53 cDNA and its several deletion mutant constructs were used in GST pulldown assays and Far Western assays with purified GST-p33 protein to map the interacting region on p53 protein. New deletion mutant constructs of p53 protein were created and cloned into expression vectors for the detailed analysis of the interacting domain of p53 protein. Also the other members of p53 protein family, p63 and p73 were examined in vitro for interaction with p33ING1. Deletion mutants of these proteins were created and cloned into expression vectors for protein-protein interaction assays. The results of this study shows that p53 protein interacts with p33ING1 and suggests that oligomerization domain of p53 protein is needed for this interaction. In addition, for the first time, it was showed that p63 and p73 proteins interact with p33ING1 and in p63 the C-terminus region is the primary determinant region, involved in these interactions with p33ING1.