The tumor suppressor p53 is degraded by the ubiquitin-proteasome system. p53 was polyubiquitinated in the presence of E1, UbcH5 as E2 and MDM2 oncoprotein. A ubiquitin molecule bound MDM2 through sulfhydroxy bond which is characteristic of ubiquitin ligase (E3)-ubiquitin binding. The cysteine residue in the carboxyl terminus of MDM2 was essential for the activity. These data suggest that the MDM2 protein, which is induced by p53, functions as a ubiquitin ligase, E3, in human papillomavirus-uninfected cells which do not have E6 protein.z 1997 Federation of European Biochemical Societies.

We have demonstrated previously that the oncoprotein Mdm2 has a ubiquitin ligase activity for the tumor suppressor p53 protein. In the present study, we characterize this ubiquitin ligase activity of Mdm2. We first demonstrate the ubiquitination of several p53 point mutants and deletion mutants by Mdm2. The point mutants, which cannot bind to Mdm2, are not ubiquitinated by Mdm2. The ubiquitination of the C-terminal deletion mutants, which contain so-called Mdm2-binding sites, is markedly decreased, compared with that of wild-type p53. The binding of Mdm2 to p53 is essential for ubiquitination, but p53's tertiary structure and/or C-terminal region may also be important for this reaction. DNA-dependent protein kinase is known to phosphorylate p53 on Mdm2-binding sites, where DNA damage induces phosphorylation, and p53 phosphorylated by this kinase is not a good substrate for Mdm2. This suggests that DNA damage-induced phosphorylation stabilizes p53 by inhibiting its ubiquitination by Mdm2. We further investigated whether the tumor suppressor p19(ARF) affects the ubiquitin ligase activity of Mdm2 for p53. The activity of p19(ARF)-bound Mdm2 was found to be lower than that of free Mdm2, suggesting that p19(ARF) promotes the stabilization of p53 by inactivating Mdm2.

Sumoylation of p53 by the ubiquitin-like protein, SUMO-1/sentrin/PIC1, has been shown to stimulate its transcriptional activation activity. The SUMO E3 ligase, a key enzyme in the recognition of substrates to be sumoylated, has not yet been identified. We isolated PIAS1 (protein inhibitor of activated STAT1) as a SUMO-1 binding protein by yeast two-hybrid screening. In addition, PIAS1 bound p53 and Ubc9, the E2 for SUMO. PIAS1 that was mutated in the RING finger-like domain bound p53 and SUMO-1, but not Ubc9. PIAS1 catalyzed the sumoylation of p53 both in U2OS cells and in vitro in a domain-dependent manner. These data suggest that PIAS1 functions as a SUMO ligase, or possibly as a tightly bound regulator of it, toward p53.

It has been postulated that reactive oxygen species (ROS) may act as second messengers leading to nuclear factor (NF)-kB activation. This hypothesis is mainly based on the ®ndings that N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), compounds recognized as potential antioxidants, can inhibit NF-kB activation in a wide variety of cell types. Here we reveal that both NAC and PDTC inhibit NF-kB activation independently of antioxidative function. NAC selectively blocks tumor necrosis factor (TNF)-induced signaling by lowering the af®nity of receptor to TNF. PDTC inhibits the IkB± ubiquitin ligase activity in the cell-free system where extracellular stimuli-regulated ROS production does not occur. Furthermore, we present evidence that endogenous ROS produced through Rac/NADPH oxidase do not mediate NF-kB signaling, but instead lower the magnitude of its activation.

We previously showed that oncoprotein MDM2 has ubiquitin ligase activity toward tumor suppressor p53. In that paper, we showed very weak homology in the carboxyl terminal portion between MDM2 and E6AP (HECT domain). We mutated the cysteine residue (C464) corresponding to the residue essential for the ubiquitin ligase activity of E6AP and this mutation diminished the ligase activity of MDM2. The cysteine residue described above is also one of the cysteine residues that form the RING ®nger domain of MDM2. We tried to ®nd out whether the diminishing of the activity by the mutation is attributable to the disruption of the RING ®nger domain or not. When the ring ®nger domain of MDM2 was deleted, the truncation mutant did not have the ubiquitin ligase activity. When we mutated the seven cysteine residues of RING ®nger domain of MDM2 in the carboxyl terminus, the disruption of each residue in the RING ®nger completely diminished the ubiquitin ligase activity of MDM2 toward MDM2 itself and toward tumor suppressor p53. These data indicate that the RING ®nger domain in MDM2 is essential for its ubiquitin ligase activity toward p53 and itself. Oncogene (2000) 19, 1473 ± 1476.Keywords: MDM2; p53; ubiquitin ligase; E3; RING ®nger domain The p53 tumor suppressor gene product induces the expression of various genes (Gadd45, Waf1/p21/Cip1, cyclinG, Bax and MDM2) regulating cell-cycle arrest and apoptosis (Ko et al., 1996). The degradation of p53 is regulated by the ubiquitin-proteasome system (Maki et al., 1996). In the ubiquitin conjugation system, there are three enzymatic steps, i.e., E1, E2, and E3 (Ciechanover, 1998). The E3 enzyme, ubiquitin ligase, recognizes the speci®c substrate and ubiquitinates the target protein in the presence of E1 and E2. In cells infected with human papilloma virus, HPV16 or 18, papilloma virus E6 and cellular E6AP (E6 associated protein) form complex and function as ubiquitin ligase, E3 toward p53 (Schener et al., 1993). In non infected cells, MDM2 works as ubiquitin ligase, E3, in the presence of E1 and UbcH5 as E2 (Honda et al., 1997). The transfection of the cells with the MDM2 gene accelerates the p53 degradation (Haupt et al., 1997;Kubbutat et al., 1997). When the cells are exposed to genotoxic stress, the p53 is phosphorylated at ser15 through ATM kinase activation (Banin et al., 1999; Christine et al., 1999). The phosphorylated p53 seems not to be good substrate for MDM2 (Honda et al., 1999) because of its defective binding to MDM2 (Shieh et al., 1997). As a result, the amount of p53 in the cell increases.There are several types of E3 or ubiquitin ligase in mammals. One of them, E6AP, works as ubiquitin ligase toward p53 in the presence of E6 protein of papilloma virus 16 or 18. The catalytic site of the ubiquitin ligase is located in the carboxyl terminal portion of E6AP and this portion is named as HECT domain. (Ciechanover, 1998). The MDM2 may also be the HECT-type E3, because in its carboxyl terminus, there is a weak homologous sequence with HECT domain sequence (Figure 1a). When we...