Regulation of p53 function and stability by phosphorylation

The p53 tumor suppressor protein can be phosphorylated at several sites wit hin the N- and C-terminal domains, and several protein kinases have been sh own to phosphorylate p53 in vitro. In this study, we examined the activity of p53 proteins with combined mutations at all of the reported N-terminal p hosphorylation sites (p53N-term), all of the C-terminal phosphorylation sit es (p53C-term), or all of the phosphorylation sites together (p53N/C-term). Each of these mutant proteins retained transcriptional transactivation fun ctions, indicating that phosphorylation is not essential for this activity of p53, although a subtle contribution of the C-terminal phosphorylation si tes to the activation of expression of the endogenous p21(Waf1/Cip1)-encodi ng gene aas detected. Mutation of the phosphorylation sites to alanine did not affect the sensitivity of p53 to binding to or degradation by Mdm2, alt hough alteration of residues 15 and 37 to aspartic acid, which could mimic phosphorylation, resulted in a slight resistance to Mdm2-mediated degradati on, consistent with recent reports that phosphorylation at these sites inhi bits the p53-Mdm2 interaction. However, expression of the phosphorylation s ite mutant proteins in both wild-type p53-expressing and p53-null lines sho wed that all of the mutant proteins retained the ability to be stabilized f ollowing DNA damage. This indicates that phosphorylation is not essential f or DNA damage-induced stabilization of p53, although phosphorylation could clearly contribute to p53 stabilization under some conditions.