We have been examining the function and regulation of the tumor suppressor protein p53. We focused on the possible role of phosphorylation on regulation of p53 function. We have taken a molecular biological approach utilizing site-directed mutagenesis to examine this problem. Site-directed mutagenesis was performed on potential phosphorylation sites (ex. ser315, ser392); this will produce mutant proteins with an alanine residue substituting for a serine and will thus eliminate a potential phosphorylation site. The wild type and mutated cDNA were then cloned into mammalian expression vector pRc/CMV which allows the expression of either wild type or mutant p53 in mammalian cells after transfection with the respective plasmids. It is known that colon cell line SW480 transfected with plasmid carrying the wild type p53 cDNA exhibits decreased colony formation. Utilizing this property of SW480 we assayed colony formation to assess the effects of alteration of phosphorylation sites. We observed, in preliminary experiments, that removal of these phosphorylation sites did not affect the inhibitory properties of p53. This suggests that phosphorylation may not be a positive control signal of p53 function. The possibility that it serves as a negative control signal is being examined. Currently we are in the process of developing assays using reporter genes to further substantiate our observation.
