Turning the RING domain protein MdmX into an active ubiquitin-protein ligase.
The related RING domain proteins MdmX and Mdm2 are best known for their role as negative regulators of the tumor suppressor p53. However, although Mdm2 functions as a ubiquitin ligase for p53, MdmX does not have appreciable ubiquitin ligase activity. In this study, we performed a mutational analysis of the RING domain of MdmX, and we identified two distinct regions that, when replaced by the respective regions of Mdm2, turn MdmX into an active ubiquitin ligase for p53. Mdm2 and MdmX form homodimers as well as heterodimers with each other. One of the regions identified localizes to the dimer interface indicating that subtle conformational changes in this region either affect dimer stability and/or the interaction with the ubiquitin-conjugating enzyme UbcH5b. The second region contains the cryptic nucleolar localization signal of Mdm2 but is also assumed to be involved in the interaction with UbcH5b. Here, we show that this region has a significant impact on the ability of respective MdmX mutants to functionally interact with UbcH5b in vitro supporting the notion that this region serves two distinct functional purposes, nucleolar localization and ubiquitin ligase activity. Finally, evidence is provided to suggest that the RING domain of Mdm2 not only binds to UbcH5b but also acts as an allosteric activator of UbcH5b.
Iyappan
Saravanakumar
S
From the Department of Biology and Konstanz Research School Chemical Biology, 78457 Konstanz, Germany.
Wollscheid
Hans-Peter
HP
From the Department of Biology and Konstanz Research School Chemical Biology, 78457 Konstanz, Germany.
Rojas-Fernandez
Alejandro
A
From the Department of Biology and Konstanz Research School Chemical Biology, 78457 Konstanz, Germany.
Marquardt
Andreas
A
Proteomics Facility, University of Konstanz, 78457 Konstanz, Germany.
Tang
Hao-Cheng
HC
From the Department of Biology and Konstanz Research School Chemical Biology, 78457 Konstanz, Germany.
Singh
Rajesh K
RK
Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, Maryland 20742-3360.
Scheffner
Martin
M
From the Department of Biology and Konstanz Research School Chemical Biology, 78457 Konstanz, Germany. Electronic address: martin.scheffner@uni-konstanz.de.
D000494:Allosteric Regulation / Q000502:physiology; D000818:Animals; D002460:Cell Line; D004795:Enzyme Stability / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism*; D055503:Protein Multimerization / Q000502:physiology*; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2 / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
C461497:Mdm4 protein, mouse; D019913:Nuclear Localization Signals; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2; D044767:Ubiquitin-Protein Ligases