Turning the RING Domain Protein MdmX into an Active Ubiquitin-Protein Ligase*

Alejandro Rojas-Fernandez,Saravanakumar Iyappan,Andreas Marquardt,Rajesh K. Singh,Hans-Peter Wollscheid,Martin Scheffner,Hao-Cheng Tang

doi:10.1074/jbc.m110.115113

Alejandro Rojas-Fernandez, Saravanakumar Iyappan + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m110.115113

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Abstract

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.

Highlights

Via Adamello 16, 20139 Milan, Italy. 3 To whom correspondence should be addressed: Dept. of Biology, Box 642, has MdmX-independent functions in p53 regulation and vice versa
In silico comparisons with structures of RING domains of other E3s with their cognate E2 enzymes indicate that the amino acid residues of the Mdm2 RING domain that are presumably involved in E2 interaction are at least in part conserved in the MdmX RING domain [27, 33]
As we previously reported that Mdm2 detectably interacts with UbcH5b in the yeast two-hybrid system but not under the conditions of an in vitro coprecipitation experiment [14], the ability of the RING domain of MdmX to interact with UbcH5b was tested in yeast

Summary

Introduction

Via Adamello 16, 20139 Milan, Italy. 3 To whom correspondence should be addressed: Dept. of Biology, Box 642, has MdmX-independent functions in p53 regulation and vice versa. A chimeric protein consisting of the RING domain of Mdm2 fused to amino acid residues 1– 434 of MdmX was not able to target p53 for degradation in coexpression experiments (Fig. 2C, X/2), whereas the respective chimera containing the central region of Mdm2 (Fig. 2C, AD-X/2) was active (note that in the in vitro system, the central region of Mdm2 is not required for p53 ubiquitination; see under “Discussion”).

Results

Conclusion