Critical roles for the serine 20, but not the serine 15, phosphorylation site and for the polyproline domain in regulating p53 turnover

The p53 tumour suppressor protein is a short-lived transcription factor tha t becomes stabilized in response to a wide range of cellular stresses. Ubiq uitination and the targeting of p53 for degradation by the proteasome are m ediated by Mdm2 (mouse double minute clone 2), a negative regulatory partne r of p53. Previous studies have suggested that DNA-damage-induced phosphory lation of p53 at key N-terminal sites has a pivotal role in regulating the interaction with Mdm2 but the precise role of phosphorylation of serines 15 and 20 is still unclear. Here we show that replacement of serine 15 and a range of other key N-terminal phosphorylation sites with alanine, which can not be phosphorylated, has little effect on the ubiquitination and degradat ion of full-length human p53. In contrast, replacement of serine 20 makes p 53 highly sensitive to Mdm2-mediated turnover. These results define distinc t roles for serines 15 and 20, two sites previously demonstrated to be depe ndent on phosphorylation through mechanisms mediated by DNA damage and ATM (ataxia telangiectasia mutated). We also show that the polyproline region o f p53, a domain that has a key role in p53-induced apoptosis, exerts a crit ical influence over the Mdm2-mediated turnover of p53.