Omics approaches to explore cellular signaling networks

受体酪氨酸激酶(RTK)是基本的分子传感器，使细胞能够解释他们的环境，并决定是否生长、移动、分化或死亡。RTK驻留在质膜上，但也可以进入细胞，积聚在特定的细胞质区域，称为内小体，并返回到细胞表面。这一过程被称为受体交易。从质膜向质膜的运输改变了RTK传递环境信号的方式，这些信号允许细胞解释他们的环境。具体地说，受体聚集到内吞体内(内化)，然后再循环到细胞表面，调节细胞内的信号级联，导致特定的长期细胞反应的激活。重要的是，受体贩运的这种不平衡与人类疾病(即乳腺癌)有关，使这一过程成为生物科学研究的关键焦点。该项目采用高度多学科的方法，旨在揭示RTK贩运的关键调控因素及其对信号特异性和细胞决策的影响。聚焦于成纤维细胞生长因子受体2b(FGFR2b)，它在发育和癌症进展中起着重要作用，我们已经确定激酶RSK2是一种新的调节FGFR2b运输和某些配体刺激后输出的调节因子。“功能蛋白质组学”集成了基于定量质谱学(MS)的蛋白质组学、生物信息学、功能分析和成像技术，以及RNA-SEQ将被用于产生依赖RSK2的签名，以在FGFR2b运输和2D和3D细胞培养的细胞分析中得到验证。该项目的具体目标是：1)在RSK2表达水平将受到操纵的上皮细胞中，使用免疫荧光和生化检测来剖析RSK2在FGFR2b运输中的作用(即CRISPR/Cas9技术)。2)在早期时间点刺激时执行基于MS的磷酸蛋白质组学，在晚期时间点刺激时执行定量蛋白质组学和RNA-SEQ，然后进行生物信息学分析以整合所有这些数据集，以在上皮细胞中产生全面的RSK2依赖的信号。选定的候选人将使用贩运和功能分析进行验证。3)探讨RSK2在缺失RSK2的细胞中如何影响细胞输出，即细胞迁移。通过结合尖端组学技术、生物信息学和功能分析，学生将对我们理解依赖回收的输出如何被操纵来重定向细胞决策做出重大贡献。这一高度多学科的项目将尖端的基于质谱学(MS)的蛋白质组学和转录组学与生物信息学和功能分析相结合，解决了BBSRC在生物科学中的“系统生物科学方法”和“数据驱动生物学”的任务。对涉及受体酪氨酸激酶运输的蛋白质的组学研究将产生复杂的数据集，将使用先进的生物信息学工具进行分析，然后在各种基于细胞的分析中进行验证。通过这种“新的工作方式”，该项目将探索预测系统生物学方法如何在与已定义的生物学终点相结合时促进科学发现的进步。此外，还将提供定量蛋白质组学、转录组学、生物信息学和核心研究学科(分子和细胞生物学)方面的培训，从而形成下一代蛋白质科学家。

Receptor Tyrosine Kinases (RTKs) are fundamental molecular sensors that enable cells to interpret their environment and to decide whether to grow, move, differentiate, or die. RTKs reside at the plasma membrane, but can also enter into the cell, accumulate in specific cytoplasmic areas named endosomes and go back to the cell surface. This process is called receptor trafficking. Trafficking from and to the plasma membrane alters the way in which RTKs transmit the environmental signals that allow cells to interpret their surroundings. In particular, receptor accumulation into endosomes (internalization) followed by recycling to the cell surface regulates intracellular signaling cascades leading to the activation of specific long-term cellular responses. Importantly, such imbalances in receptor trafficking have been associated with human diseases (i.e. breast cancer), making this process a key focal point for study in biosciences. Using a highly multidisciplinary approach this project aims to uncover the key regulators of RTK trafficking and their influence on signaling specificity and cellular decisions. Focusing on Fibroblast Growth Factor Receptor 2b (FGFR2b), which plays major roles during development and cancer progression, we have identified the kinase RSK2 as a novel regulator of FGFR2b trafficking and outputs upon stimulation with certain ligands. 'Functional proteomics', which integrates quantitative Mass Spectrometry (MS)-based proteomics, bioinformatics, functional assays, and imaging techniques, and RNA-seq will be used to generate RSK2-dependent signatures to be validated in FGFR2b trafficking and cellular assays in 2D and 3D cell culture. The specific aims of this project are:1) To dissect the role of RSK2 in FGFR2b trafficking using immunofluorescence- and biochemical-based assays in epithelial cells where the expression level of RSK2 will be manipulated (i.e. CRISPR/Cas9 technology). 2) To generate a comprehensive RSK2-dependent signature in epithelial cells performing MS-based phosphoproteomics upon early time points stimulation, and quantitative proteomics and RNA-seq upon late time points stimulation, followed by bioinformatics analysis to integrate all these data sets. Selected candidates will be validated using trafficking and functional assays. 3) To explore how RSK2 affects cellular outputs, i.e. cell migration, in cells depleted of RSK2. By combining cutting-edge -omics technology, bioinformatics, and functional assays, the student will make a significant contribution to our understanding of how recycling-dependent outputs could be manipulated to re-direct cellular decisions. This highly multidisciplinary project integrates cutting-edge mass spectrometry (MS)-based (phospho) proteomics and transcriptomics with bioinformatics, and functional assays, addressing BBSRC remits for `systems approaches to the biosciences' and 'data driven biology' in bioscience. Omics investigation of proteins involved in trafficking of receptor tyrosine kinases will generate complex datasets to be analyzed with advanced bioinformatics tools followed by validation in a variety of cell-based assays. Through this 'new way of working', this project will exploit how a predictive systems biology approach enables advancements in scientific discoveries when integrated with defined biological endpoints. Furthermore, training will be provided in quantitative proteomics, transcriptomics, bioinformatics, and core research disciplines (molecular and cell biology), thus forming the next generation of protein scientists.