Signaling by the EGF Receptor from Endosomes

细胞内转运调节信号转导过程的机制仍有待阐明 对于理解不同的刺激如何通过细胞表面传播信号非常重要 保守的胞浆机制和通向细胞核的信号导致刺激和上下文特定的信号 结果。表皮生长因子受体(EGFR)是一种典型的受体酪氨酸激酶，已成为 研究信令网络时空调控的主要实验模型。EGFR发挥着至关重要的作用 在成年哺乳动物发育和组织动态平衡中的作用，并参与人类的发病机制 尤其是癌症。然而，尽管EGFR信号网络的主要组成部分是已知的，但它们是如何 在EGFR内吞和随后的受体靶向降解到 内体确保信号过程的适当强度和持续时间在很大程度上是未知的。 解决这一根本问题现在已经成为可能，因为有了新的切割机-- EDGE方法。用时间分辨定量质谱法分析细胞磷蛋白质组 发现在大多数活性的EGFR被内化到内吞体内后，大量的 已知参与调节生长、存活、细胞运动和黏附信号的蛋白质是 由EGFR维护。因此，我们假设涉及这些假定的信号效应器的通路 EGFR通过内体EGFR的持续活性在内体中发挥作用。我们进一步假设 通过维持某些信号通路的活性，同时下调其他通路，EGFR 内吞作用决定了EGFR信号的整体功能结果。 为了在生理实验系统中检验这些假设(依赖于生长的细胞 我们将：1)检查内吞的EGFR的假定底物和信号效应分子是否已确定 通过磷蛋白组学分析，通过标记内源性效应分子，定位于含有EGFR的内体 通过基因编辑和解剖其随时间变化的定位动态 三维荧光显微镜成像；以及2)检查细胞吞噬作用和定位在 内小体控制可能的磷酸化底物和效应物的下游信号活性 内体EGFR，并定义这种调节的机制。高通量方法的组合 用各种定量活细胞光学成像方法进行基因编辑标记内源蛋白质 分辨率的水平将允许我们应用系统生物学方法来解开整个内体信号。 程序。

Elucidation of the mechanisms by which endocytic trafficking regulates signal transduction processes remains to be profoundly important for understanding how diverse stimuli propagate signals from the cell surface through the conserved cytosolic machinery and to the nucleus leading to a stimulus- and context-specific signaling outcome. Epidermal growth factor (EGF) receptor (EGFR), a prototypic receptor tyrosine kinase, has been the major experimental model to study spatiotemporal regulation of signaling networks. EGFR plays an essential role in mammalian development and tissue homeostasis in the adult, and is involved in human pathogenesis, in particular, cancer. However, while the main constituents of the EGFR signaling network are known, how they coordinately function during EGFR endocytosis and subsequent targeting of receptors for degradation to endosomes to ensure proper intensity and duration of signaling processes is for the large part unknown. Addressing this fundamental question has now become possible owing to the availability of new cutting- edge methodologies. Using time-resolved quantitative mass-spectrometry of cellular phosphoproteomes we found that after the majority of active EGFRs are internalized into endosomes, phosphorylation of a multitude of proteins, known to be involved in the regulation of signaling to growth, survival, cell motility and adhesion, is maintained by EGFR. Therefore, we hypothesize that the pathways involving these putative signaling effectors of EGFR operate in endosomes through the sustained activity of endosomal EGFR. We further hypothesize that by maintaining the activity along some signaling pathways while down-regulating other pathways, EGFR endocytosis shapes the overall functional outcome of EGFR signaling. To test these hypothesis in the physiological experimental system (cells that are growth-dependent on EGFR) we will: 1) examine whether putative substrates and signaling effectors of endocytosed EGFR identified by the phosphoproteomic analysis are located in EGFR-containing endosomes by labeling endogenous effectors with fluorescent proteins by gene-editing and dissecting their time-dependent localization dynamics using multi- dimensional fluorescence microscopy imaging; and 2) examine whether endocytosis and localization in endosomes control downstream signaling activity of putative phosphorylation substrates and effectors of endosomal EGFR, and define the mechanisms of this regulation. Combination of the high-throughput method of labeling endogenous proteins by gene-editing with various methods of quantitative live-cell optical imaging at all levels of resolution will allow us to apply a systems biology approach to untangling the entire endosomal signaling program.