The E3 ubiquitin-protein ligase MDM2 is a novel interactor of the von Hippel-Lindau tumor suppressor

Abstract

Mutations of the von Hippel-Lindau (pVHL) tumor suppressor are causative of a familiar predisposition to develop different types of cancer. pVHL is mainly known for its role in regulating hypoxia-inducible factor 1-α (HIF-1α) degradation, thus modulating the hypoxia response. There are different pVHL isoforms, including pVHL30 and pVHL19. However, little is known about isoform-specific functions and protein-protein interactions. Integrating in silico predictions with in vitro and in vivo assays, we describe a novel interaction between pVHL and mouse double minute 2 homolog (MDM2). Importantly, we found that pVHL30, and not pVHL19, forms a complex with MDM2, and that the N-terminal acidic tail of pVHL30 is required for its association with MDM2.Further, we demonstrate that an intrinsically disordered region upstream of the tetramerization domain of MDM2 is responsible for its isoform-specific association with pVHL30. This region is highly conserved in higher mammals, including primates, similarly to what has been already proposed for the N-terminal tail of pVHL30. Finally, we show that overexpression of pVHL30 and MDM2 together reduces cell proliferation, suggesting a synergistic effect of these E3 ubiquitin ligases. Collectively, our data support the idea that pVHL30 plays a role in MDM2 regulation, suggesting a wider interplay among hypoxia sensing and cell cycle regulation. mainly localized in the nucleus and participating in the regulation of transcription in response to hypoxia and cellular stress. The second cluster was composed of seven proteins, including pVHL, and, as expected, it was linked to protein degradation. The last cluster was the most heterogeneous and included proteins associated with both hypoxia-and stress-response, e.g. nutrient deprivation and viral aggression. Of note, we found a number of experimentally validated interactions connecting all of these three clusters, suggesting that the hypoxia response is functionally interconnected with cell cycle regulation. Manually curated enrichment of protein-protein interactions across network nodes further extended the connection between clusters I and II.Considering the concept of "interactors of interactors", a well-established property of proteinprotein interaction networks 28,29 and the already validated physical interaction between pVHL, p53, and p14ARF, our in silico analysis predicts that MDM2 is a novel direct interactor of pVHL.