Cancer-Associated Mutations in the MDM2 Zinc Finger Domain Disrupt Ribosomal Protein Interaction and Attenuate MDM2-Induced p53 Degradation

Abstract

The p53-inhibitory function of the oncoprotein MDM2 is regulated by a number of MDM2-binding proteins, including ARF and ribosomal proteins L5, L11, and L23, which bind the central acidic domain of MDM2 and inhibit its E3 ubiquitin ligase activity. Various human cancer-associated MDM2 alterations targeting the central acidic domain have been reported, yet the functional significance of these mutations in tumor development has remained unclear. Here, we show that cancer-associated missense mutations targeting MDM2's central zinc finger disrupt the interaction of MDM2 with L5 and L11. We found that the zinc finger mutant MDM2 is impaired in undergoing nuclear export and proteasomal degradation as well as in promoting p53 degradation, yet retains the function of suppressing p53 transcriptional activity. Unlike the wild-type MDM2, whose p53-suppressive activity can be inhibited by L11, the MDM2 zinc finger mutant escapes L11 inhibition. Hence, the MDM2 central zinc finger plays a critical role in mediating MDM2's interaction with ribosomal proteins and its ability to degrade p53, and these roles are disrupted by human cancer-associated MDM2 mutations.The mammalian p53 transcription factor mediates a major tumor suppression pathway that is negatively controlled by the proto-oncoprotein MDM2 (HDM2 in humans; henceforth denoted MDM2) and is altered in most, if not all, human cancers. The gene for mouse Mdm2 (murine double minute 2) was originally identified in a spontaneously transformed mouse BALB/c cell line (13). The Mdm2 protein was found to be responsible for transformation of NIH 3T3 and Rat2 cells when overexpressed (13), and this transforming function is believed to stem from its ability to bind with and inhibit the transactivation activity of p53 (39). Subsequently, the HDM2 gene, the human homologue of Mdm2, was found to be amplified in over one-third of those human sarcomas that still retain wild-type p53 (41), suggesting that overexpression of MDM2 could be a common mechanism by which cells inactivate p53. Mice with targeted deletion of the Mdm2 gene die during early embryonic development, and this lethality can be rescued by concomitant deletion of p53, indicating that a major in vivo function of MDM2 is to keep p53 activity in check (27,33).It is believed that MDM2 controls p53 through two mechanisms: inhibition of the transcriptional activity of p53 (39) and promotion of p53 ubiquitination and degradation (18,29). Mdm2 binds to and masks the N-terminal transactivation domain of p53 by directly interfering with the interaction between p53 and the basal transcriptional machinery (42, 54). Mdm2 belongs to a large family of RING finger ubiquitin ligases (25). Studies have demonstrated that Mdm2 is a ubiquitin ligase (19) and that the ubiquitin ligase activity of Mdm2 is responsible for degradation of p53 both in vitro (14,20) and in transfected cells (14). MDM2-mediated p53 degradation also depends on its ability to promote p53 nuclear export (46). Mutation of a nuclear export signal (NES) in MDM2 abolishes ...

Association of v-ErbA with Smad4 Disrupts TGF-β Signaling

“…The zinc finger domain is known to mediate other protein-protein interactions. A recent publication confirms that mutations in the zinc finger domain of the MDM2 oncoprotein disrupts ribosomal protein interaction and attenuates MDM2-induced p53 degradation (Lindstrom et al, 2007). Both the delZF and C308A mutations were unable to associate with Smad4 and cells stably expressing either delZF or C308A regained TGF-␤-induced cell growth inhibition and TGF-␤-responsive promoter activity.…”

Ribosome biogenesis surveillance: probing the ribosomal protein-Mdm2-p53 pathway

“…Mutation in the zinc-finger of Mdm2 disrupt the binding of Rpl5 and Rpl11, but not Rpl23, to bypass RP-mediated inhibition of Mdm2 (Lindstrom et al, 2007). In general, most cancers ramp up the production of new ribosomes to support elevated levels of protein synthesis, so in this context, the RP-Mdm2-p53 pathway could have a more prominent role in tumor suppression.…”

Ribosomal protein–Mdm2–p53 pathway coordinates nutrient stress with lipid metabolism by regulating MCD and promoting fatty acid oxidation

“…Detailed analysis revealed that several RPs require the Mdm2 central zinc finger motif for efficient Mdm2 binding. Interestingly, cancer-associated MDM2 mutations have been reported to affect the central zinc finger motif (4,5) and can specifically disrupt RP binding (6). To study the physiological functions of the RP-Mdm2-p53 pathway, we generated mice carrying a single human cancer-associated phenylalanine substitution at cysteine residue 305, which is structurally critical for the central zinc finger of Mdm2 (Mdm2 C305F ).…”