Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53

Mdm2 has been shown to regulate p53 stability by targeting the p53 protein for proteasomal degradation. We now report that Mdm2 is a ubiquitin protein ligase (E3) for p53 and that its activity is dependent on its RING finger. Furthermore, we show that Mdma mediates its own ubiquitination in a RING f inger-dependent manner, which requires no eukaryotic proteins other than ub iquitin-activating enzyme (El) and an ubiquitin-conjugating enzyme (E2). It is apparent, therefore, that Mdma manifests an intrinsic capacity to media te ubiquitination, Mutation of putative zinc coordination residues abrogate d this activity, as did chelation of divalent cations. After cation chelati on, the full activity could be restored by addition of zinc. We further dem onstrate that the degradation of p53 and Mdma in cells requires additional potential zinc coordinating residues beyond those required for the intrinsi c activity of Mdm2 in vitro. Replacement of the Mdm2 RING with that of anot her protein (Praja1) reconstituted ubiquitination and proteasomal degradati on of Mdma. However, this RING was ineffective in ubiquitination and protea somal targeting of p53, suggesting that there may be specificity at the lev el of the RING in the recognition of heterologous substrates.