Role of SPRED1 and endocytosis in the regulation of the Ras/Erk pathwayand

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The Ras/Erk pathway is arguably the signaling pathway most often found aberrantly activated in human pathologies. This includes not just cancer but also a recently characterized class of developmental syndromes known as RASopathies. The molecular mechanisms causing many of the known RASopathies are reasonably well understood, since the detected mutations often affect known players of the Ras/Erk pathway. However, this does not apply to LEGIUS syndrome, a RASopathy caused by loss-of-function mutations in SPRED1, a protein of dubious function that only recently was reported to prevent activation of the Ras/Erk pathway. However, how and at which level SPRED1 inhibits the Ras/Erk pathway remains obscure. We have collected preliminary evidence disclosing hitherto unknown biochemical properties of SPRED1 that open up a new perspective for understanding the function of SPRED1. Firstly, we observed that SPRED1 blocks the Ras/Erk pathway directly at the level of Ras by preventing Ras-GTP loading by its activator Sos. Secondly, we found that SPRED1 upregulates early endocytosis, a process traditionally ascribed to the removal and silencing of signaling constituents from the cell surface. Importantly, we find that LEGIUS SPRED1-mutants lack these effects, arguing that these are properties relevant to the biological and pathological function of SPRED1. Given that Sos-dependent Ras activation proceeds at the plasma membrane (PM) and since Ras proteins leave the PM via endocytosis it is plausible to speculate that the regulation of Ras activity and early endocytosis by SPRED1 are not independent but functionally connected processes. Summing up, we hypothesize that SPRED1 regulates Ras activity by promoting its recruitment into early endosomes, concomitantly removing Ras from the vicinity of its activator Sos at the PM. This model could explain the mechanism of Ras/Erk pathway activity control by SPRED1 and the molecular mechanism underlying LEGIUS syndrome. We propose to put this hypothesis to scrutiny and test if the regulation of endocytosis by SPRED1 is linked to the inhibition of Ras. In addition to addressing this hypothesis directly with biochemical and microscopy approaches, we propose to run an unbiased screen for the identification of SPRED1 interactors, which shall provide additional information on the mode of action of SPRED1. In conclusion, we predict that this study will shed light on the role and mechanism of SPRED1 as an inhibitor of the Ras/Erk pathway and by extension, on the molecular causes of LEGIUS syndrome. More fundamentally, this project will provide insight into the reciprocal regulation and interdependence of Ras activation and endocytosis, a long-standing unsettled controversy in cell biology research.