Biomedical subjects

Mithua Ghosh

Publications and source records attributed to Mithua Ghosh.

3 recordsLinked to original sources

PubMed2005

MdmX inhibits ARF mediated Mdm2 sumoylation.

Mdm2, by virtue of an intrinsic E3 ubiquitin ligase activity, is capable of autoubiquitination and the ubiquitination of the p53 tumor suppressor protein. Additionally, Hdm2 has been reported to undergo a p14ARF-dependent sumoylation with concurrent Hdm2 stabilization. In this present work, we report that MdmX can undergo ARF-mediated sumoylation similar to that reported for Mdm2. When coexpressed, MdmX overexpression results in a dose-dependent inhibition of Mdm2 sumoylation and a concurrent increase in Mdm2 ubiquitination. This switch from Mdm2 sumoylation to Mdm2 ubiquitination may explain the destablization of Mdm2 previously observed in cells overexpressing both ARF and MdmX. Given that MdmX can heterodimerize with Mdm2 and separately associate with ARF we employed a series of MdmX mutants to examine how MdmX blocks Mdm2 sumoylation. A MdmX miniprotein capable of binding to ARF, but not p53 or Mdm2 was able to competitively inhibit Mdm2 sumoylation and reverse ARF mediated activation of p53 transactivation. Taken together, these results demonstrate that MdmX can affect post-translational modification and stability of Mdm2 and p53 activity through interaction with ARF.

Blotting, Western↗

PubMed2004

MdmX represses E2F1 transactivation.

Based on knockout mouse studies, Mdm2 and MdmX have been identified as critical regulators of the p53 tumor suppressor protein, at least during early development. While many of the functions attributed to Mdm2 and MdmX involve p53 and overexpression of each gene appears to have oncogenic activities, a number of studies have suggested that each protein also possesses p53-independent functions. While examining the effect of Mdm2 overexpression on E2F1 transactivation we uncovered a novel MdmX function, the ability to inhibit E2F1 transactivation in a p53 and Mdm2 independent manner. Using a series of MdmX deletion mutants the central region of MdmX, amino acids 128-444 appears to possess the repressive domain. While an in vivo association of MdmX with either E2F1 or DP1 was not observed, a slight reduction in DP1 and an increased cytoplasmic localization of E2F1 were seen in cells overexpressing MdmX. These results suggest that elevated MdmX expression may repress E2F1-regulated genes like p14ARF and thus represent another regulatory mechanism in the Rb-p53 signaling pathway.

Blotting, Western↗

PubMed2003

Overexpression of Mdm2 and MdmX fusion proteins alters p53 mediated transactivation, ubiquitination, and degradation.

Mdm2 and MdmX function as cellular regulators of the p53 tumor suppressor protein. Mdm2, a p53 inducible protein, negatively regulates p53 by inhibiting p53 transcriptional activity and promoting ubiquitin mediated proteasome degradation. The Mdm2 ring finger domain has been shown to possess E3 ligase activity and to be a necessary domain for targeting p53 degradation. MdmX, a p53 binding protein sharing a high degree of structural homology with Mdm2, has emerged as another negative regulator of the p53 tumor suppressor. MdmX has also been shown to block p53 transactivation but unlike Mdm2 cannot induce p53 degradation. Since MdmX also possesses a ring finger domain that allows MdmX to associate with Mdm2, this study focused on elucidating how the ring and zinc fingers of these two proteins affected p53 function. We have generated a series of fusion proteins between Mdm2 and MdmX by swapping the ring finger domains with or without the zinc finger domains and examined how these fusions regulated p53 induced transactivation, ubiquitination, and degradation. All fusions inhibited the transcriptional activity of p53. In the absence of Mdm2, none of the fusion proteins could trigger p53 ubiquitination or degradation. However, in a cell line with endogenous Hdm2, Mdm2:X fusions containing the ring finger domain with or without the zinc finger domain demonstrated p53 ubiquitination presumably through stabilization of Hdm2. Additionally, an Mdm2:XZFRF fusion also degraded p53 when endogenous Hdm2 was present. Results from immunofluorescence studies suggest that p53 is colocalized to the cytoplasm when coexpressed with a Mdm2:X fusion (Mdm2:XZFRF) and that this fusion is capable of stabilizing endogenous Hdm2. Since none of the fusions triggered p53 ubiquitination in cells lacking Mdm2, these results indicate that the E3 ligase domain within the ring finger of Mdm2 when part of MdmX and the MdmX ring finger fused to Mdm2 were not sufficient to trigger p53 ubiquitination, in vivo.

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