Stabilization of the MDM2 Oncoprotein by Interaction with the Structurally Related MDMX Protein

Abstract

The MDM2 oncoprotein has transforming potential that can be activated by overexpression, and it represents a critical regulator of the p53 tumor suppressor protein. To identify other factors with a potential role in influencing the expression and/or function of MDM2, we utilized a yeast two-hybrid screening protocol. Here we report that MDM2 physically interacts with a structurally related protein termed MDMX. The results obtained in these studies provide evidence that C-terminal RING finger domains, contained within both of these proteins, play an important role in mediating the association between MDM2 and MDMX. The interaction of these proteins interferes with MDM2 degradation, leading to an increase in the steady-state levels of MDM2. MDMX also inhibits MDM2-mediated p53 degradation, with subsequent accumulation of p53. Taken together, these data indicate that MDMX has the potential to regulate the expression and function of the MDM2 oncoprotein.An accumulating number of observations have implicated aberrant expression of the MDM2 oncogene in the pathogenesis of human neoplasias. This mammalian gene has transforming potential that can be activated by overexpression (1, 2). Originally identified as a gene amplified and overexpressed in a spontaneously transformed mouse 3T3 cell line (3), MDM2 is now known to be amplified in a variety of human tumors, particularly soft tissue sarcomas (4 -7). Additionally, there are several cases reported of tumor cells having an elevated expression of MDM2 that results from mechanisms other than gene amplification, including enhanced translation of MDM2 transcripts (8 -10).The MDM2 gene encodes a key negative regulator of the p53 tumor suppressor protein, and the role of MDM2 overexpression in cell transformation has been attributed, at least in part, to its disruption of the biological activities of p53 (11,12). MDM2 tightly associates with the N-terminal region of the p53 protein, inhibiting the trans-activation and G 1 growth arrest functions of p53 (13-16). Moreover, binding of MDM2 targets p53 for rapid degradation via the ubiquitin-proteasome pathway (17,18). In recent reports, evidence has been obtained suggesting that MDM2 can function as an E3 1 ubiquitin ligase and is responsible for targeting both itself, and p53, for degradation (19,20). Interestingly, the MDM2 gene itself is a transcriptional target of p53. When activated as a transcription factor, p53 binds to a promoter region within the first intron of the MDM2 gene and up-regulates its expression (21-24). Thus, there is evidence for an autoregulatory feedback loop involving the expression and function of MDM2 and p53 (23). Although the best characterized activities of MDM2 concern its functional interactions with p53, MDM2 also associates with other proteins. Some of these include E2F1 (25), pRb (26), and p300 (27). Such interactions could contribute to the transforming potential of MDM2 or may be concerned with modulating MDM2 function. Recently, a link between MDM2 and yet another tumor suppressor protein, ...

The contribution of the RING finger domain of MDM2 to cell cycle progression

“…The novel function does not appear to be binding to RNA (Elenbaas et al, 1996), since mutants with di erent e ects on cell cycle bind RNA with similar a nity (poly-G agarose beads, data not shown). The MDM2 RING ®nger has been reported to bind to TAF II 250 (Leveillard and Wasylyk, 1997) and MDMX (Sharp et al, 1999;Tanimura et al, 1999). It may also interact with speci®c E2s, in analogy with a number of RING ®nger proteins that have recently been shown to have E3 ubiquitin ligase activity and to bind to E2 ubiquitin conjugating enzymes (Hu and Fearon, 1999;Joazeiro et al, 1999;Lorick et al, 1999).…”

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

“…One possible scenario, based on the observation that Mdm2 and Mdm4 heterodimerize, is that the two proteins work together to inhibit p53. 39,40 However, the molecular mechanisms underlining the abilities of both Mdm proteins to regulate p53 activity have yet to be clarified in vivo. 10 Conditional alleles have now been developed that yield further insight into how and in what cell types Mdm2 and Mdm4 regulate p53 (Figure 1).…”

Activation and activities of the p53 tumour suppressor protein

“…Binding of MDMX inhibits p53 transactivation function (Shvarts et al, 1996), although MDMX does not appear to target p53 for degradation. It is possible that MDMX does not show ubiquitin ligase activity, and hetero-oligomerization of MDMX with MDM2 through their RING finger domains results in the stabilization of MDM2 (Sharp et al, 1999;Tanimura et al, 1999). Furthermore, when overexpressed, MDMX protected p53 from MDM2-mediated degradation while still maintaining suppression of p53 transactivation (Jackson and Berberich, 2000).…”