MdmX is a RING finger ubiquitin ligase capable of synergistically enhancing Mdm2 ubiquitination.
It has been well documented that Mdm2 and its homologue MdmX not only are critical negative regulators of the tumor suppressor p53 but that both Mdm2 and MdmX interact to affect the function of the other. The mechanisms through which these effects are manifested, however, remain unclear. Although Mdm2 has been established as a RING finger ubiquitin ligase, MdmX has not been shown to possess this activity despite the extensive sequence homology between their respective RING finger domains. Here we demonstrate that MdmX acts as a ubiquitin ligase in vitro, being capable of autoubiquitination, as well as mediating the ubiquitination of p53. The addition of Mdm2 to in vitro ubiquitination assays containing MdmX results in a synergistic increase of ubiquitin conjugation. Analysis of the resulting ubiquitin conjugates reveals that this observed synergy reflects an increase in Mdm2 ubiquitination. This study also suggests that ubiquitination of Mdm2 and MdmX may not serve as a signal for degradation, as we show that each are capable of synthesizing non-lysine 48 polyubiquitin chains and, in fact, utilize multiple lysine linkages. Taken together, these findings suggest a more active role for MdmX in the Mdm2-MdmX-p53 regulatory network than has been proposed previously.
Badciong
James C
JC
Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226, USA.
Haas
Arthur L
AL
D000818:Animals; D002384:Catalysis; D002417:Cattle; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008025:Ligases / Q000378:metabolism* / Q000502:physiology*; D008239:Lysine / Q000737:chemistry; D009687:Nuclear Proteins; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000378:metabolism* / Q000502:physiology*; D051736:Proto-Oncogene Proteins c-mdm2; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*
D018076:DNA, Complementary; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D025801:Ubiquitin; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D005982:Glutathione Transferase; D008025:Ligases; D008239:Lysine