Characterisation of new cellular signalling systems using novel, rapid, high resolution protein electrophoresis

使用新型、快速、高分辨率蛋白质电泳表征新的细胞信号系统

• 批准号: 1907426
• 金额: --
• 依托单位: University College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1907426
• 关键词: Characterisation; new; cellular; signalling; systems

A very large fraction of the human genome, and the genomes of other metazoans, encodes proteins that respond rapidly to changes in external and internal cellular environments. For example about 5% is given over to signaling systems that operate through GTP binding proteins and about 2.5% accounts for protein kinases. In addition, many hundreds more genes code proteins that support or respond to these two systems. In a series of experiments (unpublished) we can demonstrate an unusual and unexpected link between a major G-protein signaling system - the Arf family G-proteins that control vesicular trafficking and intracellular transport, cytoskeletal dynamics and phosphoinositide metabolism - and the Src family of non-receptor tyrosine kinases that contains major cellular oncogenes with roles in cell growth, differentiation, cell shape, migration and survival, and specialised cell signals. We can show that Arfs are tyrosine phosphorylated by Src kinases in a manner dependent on the Arf activation state - in the GDP bound "inactive" cytosolic form Arfs are poor substrates and in the GTP bound, membrane associated "active" form they are excellent substrates for the the kinases. Proofs in place (and relevant molecular biological tools and methods available) include: i) in vitro phosphorylation of pure recombinant GTP-Arf isoforms by pure recombinant Src and Lck enzymes but not GDP Arf formsii) demonstration of Arf protein tyrosine phosphorylation using transfection and immunoprecipitation (IP) of HA-tagged GTP-Arf1 and GTP-Arf6 but not GDP forms.iii) mass spectrometric identification of the phosphorylation site on Arf1 and Arf6 following IPiv) IP of native, wild-type Arfs and demonstration of tyrosine phosphorylation.v) differences in membrane association dynamics of wild-type Arf GFP fusion proteins and those with YF mutants (phosphorylation site eliminated) using FRAP confocal microscopy. We propose to use newly developed advanced, rapid protein separation and quantification techniques to fully investigate the importance of combined kinase-G protein signalling in the new coupled Src-Arf system. The project will have a manageable remit and a realistic prospect for success within the very tight 4-year timeframe. This project is technically, conceptually and theoretically very demanding. For example the dynamic properties of the GDP/GTP, GEF/GAP switch of G-proteins is (in reality) poorly understood (mathematical proofs and models in Stanley and Thomas 2016) compared to the kinase/phosphatase phosphorylation/dephosphorylaton cycle. As a result the dynamics of the combined system are not intuitive since the four-way interconversion of GDP-Arf, GTP-Arf, GDP-phospho Arf and GTP-phospho Arf forms is entirely novel in the cell signalling field. We therefore need methods to simultaneously isolate and quantify all four states of the Arf proteins at sufficient speed and very high species resolution (Appendix 1) to allow dynamics to be studied over highly sampled experimental time courses. Time series data will underpin mathematical models completely describing the dynamics of the coupled system that will supplement the cell biological and biochemical data described and allow a complete systems biological understanding of this unique combined cell signalling motif. The mathematical and computational approach will allow the complete description of the system for transparent and unambiguous communication to other researchers.

人类基因组的很大一部分，以及其他后生动物的基因组，编码对外部和内部细胞环境变化迅速反应的蛋白质。例如，约5%被给予通过GTP结合蛋白操作的信号系统，约2.5%占蛋白激酶。此外，还有数百个基因编码支持或响应这两个系统的蛋白质。在一系列的实验中（未发表）我们可以证明一个不寻常的和意想不到的联系之间的主要G蛋白信号系统-Arf家族G蛋白，控制囊泡运输和细胞内运输，细胞骨架动力学和磷酸肌醇代谢-和Src家族的非受体酪氨酸激酶，含有主要的细胞癌基因与细胞生长，分化，细胞形状，迁移和生存的作用，和专门的细胞信号我们可以证明，Arf是酪氨酸磷酸化的Src激酶的方式依赖于Arf的活化状态-在GDP结合的“无活性”的胞质形式Arf是穷人的底物和GTP结合，膜相关的“活性”形式，他们是极好的底物的激酶。证据到位（以及可用的相关分子生物学工具和方法）包括：i）通过纯重组Src和Lck酶而非GDP Arf形式体外磷酸化纯重组GTP-Arf同种型ii）使用HA标记的GTP-Arf 1和GTP-Arf 6而非GDP形式的转染和免疫沉淀（IP）证明Arf蛋白酪氨酸磷酸化。在IPiv）IP天然，野生型Arf和酪氨酸磷酸化的证明。v）野生型Arf GFP融合蛋白和具有YF突变体的融合蛋白的膜结合动力学差异使用FRAP共聚焦显微镜观察（磷酸化位点消除）。我们建议使用新开发的先进的，快速的蛋白质分离和定量技术，充分调查的重要性，结合激酶G蛋白信号在新的耦合Src-Arf系统。该项目将有一个可管理的职权范围和一个现实的前景成功的非常紧迫的4年时间表。这个项目在技术上、概念上和理论上都要求很高。例如，与激酶/磷酸酶磷酸化/去磷酸化循环相比，G蛋白的GDP/GTP、GEF/GAP开关的动态特性（实际上）知之甚少（Stanley和托马斯2016中的数学证明和模型）。因此，组合系统的动力学不是直观的，因为GDP-Arf、GTP-Arf、GDP-磷酸Arf和GTP-磷酸Arf形式的四向相互转换在细胞信号传导领域中是完全新颖的。 因此，我们需要的方法，同时分离和量化的所有四个国家的Arf蛋白质在足够的速度和非常高的物种分辨率（附录1），使动力学研究在高度采样的实验时间过程。时间序列数据将支撑完全描述耦合系统动态的数学模型，这些模型将补充所描述的细胞生物学和生化数据，并允许对这种独特的组合细胞信号基序进行完整的系统生物学理解。数学和计算方法将允许系统的完整描述，以便与其他研究人员进行透明和明确的通信。