Role of Spred1 and endocytosis in the regulation of the Ras/Erk pathway

SPRED1 is a protein known as a negative regulator of the central signal transduction and prominent protooncoprotein RAS involved in cell survival, differentiation and other mechanisms important for cellular homeostasis. Its loss of function leads to the hereditary neurological disorder Legius syndrome, a RASopathy, defined by mild hyperactivation of the RAS/ERK pathway. SPRED1s mechanism of action is so far a topic of heavy discussion in current literature but an interaction with the RAS-GAP NEUROFIBROMIN 1 (NF1), also a negative regulator of the RAS/ERK pathway, has been widely accepted as essential for its activity. Preliminary data from our working group has additionally shown an NF1 independent effect on the activation of RAS through SPRED1 and found first evidence suggesting that this effect might relate to endosomal trafficking via the SNX3 retromer. I aimed to further investigate the role of SPRED1 in the potential SNX3-retromer related trafficking of RAS through proximity biotinylation screens and analysis of subcellular localization and trafficking via confocal microscopy imaging in different KO and siRNA cell lines. The findings of this Thesis support the hypothesis of a two-fold SPRED1 mechanism of action for the regulation of RAS, (1) via the recruitment of the RAS-GAP NF1 and (2) via the trafficking of RAS together with the SNX3-retromer. It is a completely novel thought however that while the first mechanism leads to a negative regulation of the RAS/ERK pathway the second actually does the opposite. This has great implications not just for our understanding of the function of SPRED1 and the pathology of the disease its absence causes (Legius syndrome) but also for our knowledge about the regulation of the central cellular regulator RAS at the level of endocytosis and endosomal trafficking control.