Activity of MDM2, a ubiquitin ligase, toward p53 or itself is dependent on the RING finger domain of the ligase

Abstract

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...

Mdm2, but not Mdm4, protects terminally differentiated smooth muscle cells from p53-mediated caspase-3-independent cell death

“…It was indeed shown that, besides the ability of Mdm2 to bind p53 in its transactivation domain and to interfere with p53 transcriptional activity, Mdm2 acts as an E3 ubiquitin ligase responsible for the ubiquitination of p53 and itself. [15][16][17] It was later proposed that Mdm2 mediates monomeric p53 ubiquitination on multiple lysine residues, rather than polyubiquitination, as previously thought. 18 Because chains of multiple ubiquitin molecules are necessary for efficient protein degradation, the data suggested that the enzymatic activity of Mdm2 might not be sufficient for optimal degradation of p53, and that other proteins must aid in polyubiquitination and degradation of p53 in vivo.…”

Keeping p53 in check: essential and synergistic functions of Mdm2 and Mdm4

“…13 The Mdm2 protein is the first p53 E3-ubiquitin ligase described, and induces p53 polyubiquitination and degradation when overexpressed. [14][15][16][17] More recently, the idea that Mdm2 mediates monomeric p53 ubiquitination on multiple lysine residues rather than poly-ubiquitination has been proposed. 18 As chains of multiple ubiquitin molecules are necessary for efficient protein degradation, the data suggested that Mdm2 might not be sufficient for optimal degradation of p53, and that other proteins must aid in polyubiquitination and degradation of p53 in vivo.…”

MdmX is a substrate for the deubiquitinating enzyme USP2a

“…Mdm2 can inhibit p53 activity by binding to its transactivation domain, and through promoting the proteasomal degradation of p53 by acting as an E3 ligase (Fang et al, 2000;Honda and Yasuda, 2000;Michael and Oren, 2003). In mice, knockout of Mdm2 increases p53 protein levels and overall transactivation activity in both proliferating and post-mitotic cells.…”