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

Ras/Erk通路可以说是人类病理中最常见的异常激活的信号通路。这不仅包括癌症，还包括最近被称为Rasopathies的一类发育综合征。许多已知的Rasopathies的分子机制已经被很好地理解了，因为检测到的突变通常会影响RAS/Erk途径的已知参与者。然而，这并不适用于LEGIUS综合征，这是一种由SPRED1功能缺失突变引起的Ras病，SPRED1是一种功能可疑的蛋白质，最近才被报道阻止RAS/Erk途径的激活。然而，SPRED1如何以及在何种水平抑制RAS/Erk通路仍不清楚。我们收集的初步证据揭示了迄今未知的SPRED1的生化特性，为理解SPRED1的功能开辟了一个新的视角。首先，我们观察到SPRED1通过其激活剂SOS阻止RAS-GTP的加载，在RAS水平上直接阻断RAS/Erk通路。其次，我们发现SPRED1上调早期的内吞作用，这一过程传统上被归因于从细胞表面移除和沉默信号成分。重要的是，我们发现LEGIUS SPRED1-突变体缺乏这些作用，认为这些属性与SPRED1的生物学和病理功能有关。鉴于SOS依赖的RAS活性在质膜(PM)进行，由于RAS蛋白通过内吞作用离开质膜，因此可以推测SPRED1对RAS活性的调节和早期的内吞作用不是独立的，而是在功能上相互联系的过程。综上所述，我们假设SPRED1通过促进其在早期内体中的募集，同时在PM时从其激活剂SOS附近移除RAS来调节RAS的活性该模型可以解释SPRED1调控Ras/Erk通路活性的机制和LEGIUS综合征的分子机制。我们建议对这一假设进行审查，并测试SPRED1对内吞作用的调节是否与RAS的抑制有关。除了直接用生化和显微镜方法解决这一假说外，我们还建议进行无偏筛选以鉴定SPRED1相互作用子，这将提供有关SPRED1作用模式的更多信息。总之，我们预测，这项研究将有助于阐明SPRED1作为RAS/Erk途径抑制物的作用和机制，进而阐明LEGIUS综合征的分子原因。更根本的是，这个项目将提供对RAS激活和内吞作用的相互调节和相互依赖的洞察，这是细胞生物学研究中长期悬而未决的争议。

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.