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

SPRED1 和内吞作用在 Ras/Erk 通路调节中的作用

• 批准号: 399510179
• 负责人: Privatdozent Dr. Ignacio Rubio
• 金额: --
• 依托单位: Klinik für Anästhesiologie und Intensivmedizin
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/399510179?language=en
• 关键词: Role; SPRED1; endocytosis; regulation; Ras

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.

Ras/Erk通路可以说是人类病理中最常发现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。由于检测到的突变通常会影响Ras/Erk通路的已知参与者，因此导致许多已知ras病变的分子机制已经相当清楚。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RASopathy， SPRED1是一种功能可疑的蛋白质，直到最近才被报道可以阻止Ras/Erk通路的激活。然而，SPRED1如何以及在哪个水平上抑制Ras/Erk通路仍不清楚。我们收集到的初步证据揭示了SPRED1迄今未知的生化特性，为理解SPRED1的功能开辟了新的视角。首先，我们观察到SPRED1通过阻止其激活剂Sos加载Ras- gtp，直接在Ras水平上阻断Ras/Erk通路。其次，我们发现SPRED1上调早期内吞作用，这一过程通常被认为是细胞表面信号成分的去除和沉默。重要的是，我们发现LEGIUS SPRED1突变体缺乏这些作用，认为这些特性与SPRED1的生物学和病理功能有关。鉴于sos依赖的Ras激活在质膜（PM）进行，并且由于Ras蛋白通过内吞作用离开质膜，因此有可能推测SPRED1对Ras活性和早期内吞作用的调节不是独立的过程，而是功能上相互联系的过程。综上所述，我们假设SPRED1通过促进Ras招募到早期核内体来调节Ras活性，同时在PM处将Ras从其激活物Sos附近移除。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。我们建议仔细研究这一假设，并测试SPRED1对内吞作用的调节是否与Ras的抑制有关。除了用生化和显微镜方法直接解决这一假设外，我们建议对SPRED1相互作用物进行无偏筛选，这将为SPRED1的作用方式提供更多信息。总之，我们预测本研究将揭示SPRED1作为Ras/Erk通路抑制剂的作用和机制，进而揭示LEGIUS综合征的分子原因。更重要的是，该项目将深入了解Ras活化和内吞作用的相互调节和相互依赖，这是细胞生物学研究中一个长期悬而未决的争议。