Different effects of p14ARF on the levels of ubiquitinated p53 and Mdm2 invivo

Mdm2 has been shown to promote its own ubiquitination and the ubiquitinatio n of the p53 tumour suppressor by virtue of its E3 ubiquitin ligase activit y. This modification targets Mdm2 and p53 for degradation by the proteasome . The p14ARF tumour suppressor has been shown to inhibit degradation of p53 mediated by Mdm2. Several models have been proposed to explain this effect of p14ARF. Here we have compared the effects of p14ARF overexpression on t he in vivo ubiquitination of p53 and Mdm2. We report that the inhibition of the Mdm2-mediated degradation of p53 by p14ARF is associated with a decrea se in the proportion of ubiquitinated p53. The levels of polyubiquitinated p53 decreased preferentially compared to monoubiquitinated species. p14ARF overexpression increased the levels of Mdm2 but it did not reduce the overa ll levels of ubiquitinated Mdm2 in vivo. This is unexpected because p14ARF has been reported to inhibit the ubiquitination of Mdm2 in vitro. In additi on we show that like p14ARF, the proteasome inhibitor MG132 can promote the accumulation of Mdm2 in the nucleolus and that this can occur in the absen ce of p14ARF expression. We also show that the mutation of the nucleolar lo calization signal of Mdm2 does not impair the overall ubiquitination of Mdm 2 but is necessary for the effective polyubiquitination of p53. These studi es reveal important differences in the regulation of the stability of p53 a nd of Mdm2.