Graduate Student University of Kentucky Shelbyville, Kentucky, United States
Samuel White (University of Kentucky)| Haiying Liu (University of Kentucky)| Alex Rosa Campos (Sanford Burnham Prebys Medical Discovery Institute)| Douglas Andres (University of Kentucky)
RIT2 is a small Ras-like GTPase expressed exclusively in neurons. RIT2 expression is first detected late in embryonic development (~e16), increasing rapidly after birth and plateauing in the adult mouse brain, suggesting a more important role for RIT2 in adult neurological function than in developmental processes. Ras-related proteins serve as GDP/GTP-regulated binary switches which function to relay extracellular signals to signaling pathways that regulate diverse cellular processes, including essential roles in neuronal regulation. Genome-wide association studies have linked RIT2 to Parkinson’s disease, autism spectrum disorder, and schizophrenia; but its physiological function(s) remain obscure and known interaction partners are extremely limited. To begin to address these deficiencies, we employed proximity-dependent biotin identification technology (BioID) to define the proximal protein-protein interaction landscape of RIT2. This system allows weak or transient interactions in living cells to be captured. RIT2 was fused to a re-engineered version of the bacterial BirA biotin ligase, TurboID, which conjugates biotin to proteins within the immediate vicinity (~10nm) of its active site. Stable SK-N-DZ cells (a human neuroblastoma line known to express RIT2) expressing either active Turbo-RIT2 or unconjugated Turbo (control) were treated in triplicate with biotin. Biotinylated proteins were captured with streptavidin-resin and subjected to quantitative bioanalysis by LC-mass spectrometry. RIT2 interactors include Brn3a (POU4F1), a previously described RIT2 effector, as well as other transcription factors and chromatin modification factors. This study provides new information about the potential function(s) of the poorly characterized RIT2. These newly discovered protein interaction networks open many paths to greater understanding of RIT2 and cell signaling.