The RING domain E3 ubiquitin ligase Mdm2 is the master regulator of the tumor 
suppressor p53. It targets p53 for proteasomal degradation, restraining the 
potent activity of p53 and enabling cell survival and proliferation. Like most 
E3 ligases, Mdm2 can also ubiquitinate itself. How Mdm2 auto-ubiquitination may 
influence its substrate ubiquitin ligase activity is undefined. Here we show 
that auto-ubiquitination of Mdm2 is an activating event. Mdm2 that has been 
conjugated to polyubiquitin chains, but not to single ubiquitins, exhibits 
substantially enhanced activity to polyubiquitinate p53. Mechanistically, 
auto-ubiquitination of Mdm2 facilitates the recruitment of the E2 
ubiquitin-conjugating enzyme. This occurs through noncovalent interactions 
between the ubiquitin chains on Mdm2 and the ubiquitin binding domain on E2s. 
Mutations that diminish the noncovalent interactions render auto-ubiquitination 
unable to stimulate Mdm2 substrate E3 activity. These results suggest a model in 
which polyubiquitin chains on an E3 increase the local concentration of E2 
enzymes and permit the processivity of substrate ubiquitination. They also 
support the notion that autocatalysis may be a prevalent mode for turning on the 
activity of latent enzymes.