Identification of the role of p33ING1 protein in the cellular activities of p53 tumor suppressor protein
Emre, N. C. Tolga
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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 are transactivated by p53 code for proteins that are implicated in the negative regulation of cell proliferation (via apoptosis or cell cycle arrest) and DNA damage repair. 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 (for "Inhibitor of Growth 1"), was identified as a candidate tumor suppressor gene due to its functions in apoptosis and cell cycle arrest. p33ING1, the protein product of ING1, was shown to enhance the growth suppressive functions of p53. Furthermore, a physical association between p53 and p33ING1 proteins has been detected by immunoprecipitation. In this study, we investigated the physical interaction between p53 and p33ING1 using in vitro methods in order to determine the region of p53 that enabled this interaction. As a preliminary step for the study, the ING1 cDNA was amplified from total cDNA of a cell line by PCR and cloned into expression vectors. The recombinant p33ING1 protein was overexpressed in E.coli and purified as a fusion protein with GST. The wild-type p53 cDNA and its several deletion mutant constructs were subcloned into a suitable expression vector to enable subsequent in vitro transcription-translation reactions. In vitro transcription-translation products of these constructs were used in GST pulldown assays with purified GST p53 protein to map the interacting region on the human p53 protein. The results of the study suggest that the primary determinant on p53 protein in its interaction with p33ING1, is the C-terminal domain while there may be other regions that are involved in the interaction.