Proximal Protein Interaction Landscape of RAS Paralogs

SIMPLE SUMMARY: RAS paralogs (HRAS, NRAS and KRAS) are of major interest in biology because they are involved in developmental disorders (e.g., Costello and Noonan syndromes) and in a broad variety of human neoplasia. Many research groups have devoted tremendous efforts to deepen our understanding of the RAS proteins functions and regulations, notably through identifying their functional protein partners. However, while most of these studies were focused on pathogenic RAS mutants, much less research has been dedicated to deciphering the normal activities of RAS paralogs. However, such characterization appears as a prerequisite to clearly identify pathogenic features. We delineated and compared the wild type RAS paralogs proximal interactomes. We detected more than 800 RAS high confident proximal interactors, either shared between paralogs or unique, and validated a subset of data through proximity ligation assays-based validation. Our results describe differential interactors enrichment between RAS paralogs and uncover novel ties between RAS signaling and cellular metabolism. We believe that our findings will support further studies aiming at better understanding how RAS paralogs could be differentially involved in discrete cellular processes and could serve as a basis to template oncogenic mechanism investigations. ABSTRACT: RAS proteins (KRAS, NRAS and HRAS) are frequently activated in different cancer types (e.g., non-small cell lung cancer, colorectal cancer, melanoma and bladder cancer). For many years, their activities were considered redundant due to their high degree of sequence homology (80% identity) and their shared upstream and downstream protein partners. However, the high conservation of the Hyper-Variable-Region across mammalian species, the preferential activation of different RAS proteins in specific tumor types and the specific post-translational modifications and plasma membrane-localization of each paralog suggest they could ensure discrete functions. To gain insights into RAS proteins specificities, we explored their proximal protein–protein interaction landscapes using the proximity-dependent biotin identification technology (BioID) in Flp-In T-REx 293 cell lines stably transfected and inducibly expressing wild type KRAS4B, NRAS or HRAS. We identified more than 800 high-confidence proximal interactors, allowing us to propose an unprecedented comparative analysis of wild type RAS paralogs protein networks. These data bring novel information on poorly characterized RAS functions, e.g., its putative involvement in metabolic pathways, and on shared as well as paralog-specific protein networks that could partially explain the complexity of RAS functions. These networks of protein interactions open numerous avenues to better understand RAS paralogs biological activities.