A Proteomics Screen Identifies MDM2 Binding Partners Specific to Ribosomal Protein Haploinsufficiency

Ribosomopathies such as 5q- syndrome and Diamond-Blackfan anemia are broadly characterized by haploinsufficiency of one or more ribosome-associated proteins leading to an aberrant activation of the p53 pathway. The prevailing hypothesis for the mechanism of p53 stabilization and activation in the context of ribosomal protein haploinsufficiency suggests that increased binding of a subset of free ribosomal proteins (including RPL5, RPL11, RPL23a, and RPS7) to MDM2 abrogates the negative regulatory control of MDM2 on p53. In order to understand more fully how the binding partner profile of MDM2 changes as a consequence of ribosome dysfunction, we performed a SILAC-based IP/MS proteomics screen on MDM2 immunoprecipitates to identify proteins that were differentially bound to MDM2 with and without shRNA-mediated knockdown of RPS14, a commonly haploinsufficient gene in 5q- syndrome. A V5-tagged MDM2 was expressed in A549 cells, and lentivirally-delivered shRNAs were expressed that targeted either luciferase (for control) or RPS14. Immunoprecipitations were performed on whole cell lysates using a V5 antibody and analyzed by mass spectrometry to identify peptides associated with MDM2 in each condition. We found that RPL5, RPL11, and RPL23a were strongly associated with MDM2 in the context of RPS14 knockdown, but were also abundant in MDM2 immunoprecipitates when a control hairpin was used. Another ribosomal protein, RPL38, was also strongly associated with MDM2 in both conditions. In contrast, RPS14 knockdown appeared to abrogate binding of RPS7 to MDM2, which was strongly associated in the control knockdown condition only. These data corroborate previous findings that a subset of 60S subunit ribosomal proteins can physically associate with MDM2, and they further suggest that this binding may commonly occur even in the absence of ribosomal haploinsufficiency. While differential binding of free ribosomal proteins to MDM2 may indeed contribute to p53 pathway activation in ribosomopathies, these data suggest that the difference in binding is subtle and may be a part of a larger set of p53-activating signals. Additional non-ribosomal proteins were identified that were specifically associated with MDM2 in the context of ribosomal protein deficiency, and follow-up experiments are ongoing to interrogate the biological consequences of these context-specific interactions.