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

Ras/Erk途径可以说是在人类病理学中最常发现异常激活的信号传导途径。这不仅包括癌症，还包括最近被称为RASopathies的一类发育综合征。引起许多已知RASopathies的分子机制是合理的，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SPRED 1功能缺失突变引起的RAS病，SPRED 1是一种功能可疑的蛋白质，最近才报道可以阻止Ras/Erk通路的激活。然而，SPRED 1如何以及在哪个水平抑制Ras/Erk通路仍然不清楚。我们已经收集了初步证据，揭示了SPRED 1迄今未知的生化特性，为理解SPRED 1的功能开辟了新的视角。首先，我们观察到SPRED 1通过阻止其激活剂Sos加载Ras-GTP而直接在Ras水平阻断Ras/Erk通路。其次，我们发现SPRED 1上调早期内吞作用，这一过程传统上归因于从细胞表面去除和沉默信号成分。重要的是，我们发现LEGIUS SPRED 1突变体缺乏这些效应，认为这些特性与SPRED 1的生物学和病理学功能相关。考虑到依赖于Sos的Ras激活在质膜（PM）处进行，并且由于Ras蛋白通过内吞作用离开PM，因此推测Ras活性的调节和SPRED 1的早期内吞作用不是独立的而是功能上相关的过程是合理的。综上所述，我们假设SPRED 1通过促进Ras向早期内体的募集来调节Ras活性，同时在PM处将Ras从其激活剂Sos附近移除。该模型可以解释SPRED 1调控Ras/Erk通路活性的机制以及LEGIUS综合征的分子机制。我们建议将这一假设进行审查和测试，如果由SPRED 1的内吞调节与Ras的抑制有关。除了解决这个假设直接与生化和显微镜的方法，我们建议运行一个公正的屏幕识别SPRED 1的相互作用，这将提供更多的信息SPRED 1的作用模式。总之，我们预测这项研究将阐明SPRED 1作为Ras/Erk通路抑制剂的作用和机制，并进一步阐明LEGIUS综合征的分子原因。更重要的是，该项目将深入了解Ras激活和内吞作用的相互调节和相互依赖性，这是细胞生物学研究中一个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.