Integrated Genomewide Analysis of the lpL1 Kinase Signaling Network

描述（由申请人提供）：该提案概述了一套基于酵母的综合技术，以研究激酶信号网络。我们详细的方法，使磷酸化依赖性控制的蛋白质-蛋白质相互作用概况的调查。虽然范围一般，但本提案中应用的方法将侧重于酵母极光激酶，Ipl1。在许多癌症中都存在极光激酶的过表达，小分子抑制会导致细胞凋亡和有丝分裂突变等表型，从而导致肿瘤消退。我们的综合方法解决了磷酸化影响蛋白质-蛋白质相互作用的事实，因此我们将为构建动态相互作用组图贡献重要因素。细胞的动态行为在有丝分裂期间最为明显，并且依赖于蛋白质-蛋白质相互作用的网络，越来越多的证据表明Ipl1激酶是一个主要的调节器。我们假设磷酸化位点会特异性地调节蛋白-蛋白相互作用，这些作用将与Ipl1的各种有丝分裂作用相关。我们对Ipl1信令网络的分析将由以下人员进行：i)利用基于合成剂量致死原理的优化方法鉴定与Ipl1相关的底物和细胞途径，ii)通过磷化体扫描酵母双杂交方法确定Ipl1启动的磷酸化对蛋白-蛋白相互作用的影响，iii)将Ipl1磷酸化位点和相应的蛋白-蛋白相互作用与Ipl1的有丝分裂途径和作用联系起来。iv)基于我们的合成剂量致死性和磷化剂实验，在胰腺癌细胞模型中测试我们对抗癌靶点的预测。磷酸化相互作用与Ipl1在协调染色体分离中的作用的关联将与着丝点和纺锤体的生物学和结构研究相结合。该提案解决了蛋白质组学研究的瓶颈-确定与目前已确定的数千个磷酸化事件相关的生物学后果和途径。在治疗水平上，本研究探讨了极光激酶调控不当如何引发细胞周期缺陷，如染色体不稳定性。

DESCRIPTION (provided by applicant): This proposal outlines an integrated set of yeast-based technologies to investigate a kinase signaling network. We detail methodology that enables the investigation of phosphorylation-dependent control of protein-protein interaction profiles. While general in scope, the methods applied in this proposal will focus on the yeast Aurora kinase, Ipl1. Overexpression of the Aurora kinase is established in many cancers and small molecule inhibition results in phenotypes such as apoptosis and mitotic catastrophe resulting in tumor regression. Our integrated methods address the fact that phosphorylation affects protein-protein interactions and hence we will contribute an important factor in building a dynamic interactome map. The dynamic behavior of the cell is most evident during mitosis and dependent on a network of protein-protein interactions with evidence accumulating that the Ipl1 kinase is a master regulator. We hypothesize that phosphorylation sites will specifically modulate protein- protein interactions and these will associate with the various mitotic roles of Ipl1. Our analysis of the Ipl1 signaling network will be conducted by: i) identifying substrates and cellular pathways associated with Ipl1 using optimized methods based on the principle of synthetic dosage lethality, ii) determine the effects of Ipl1- initiated phosphorylation on protein-protein interactions by a phosphomutant scanning yeast two-hybrid method, iii) associate the Ipl1 phosphorylation sites and corresponding protein-protein interactions with mitotic pathways and roles of Ipl1, and iv) test our predictions for anti-cancer targets in a pancreatic cancer cell model based on our synthetic dosage lethality and phosphomutant experiments. The association of phosphomodulated interactions with the role of Ipl1 in orchestrating chromosome segregation will interface with biological and structural studies of the kinetochore and spindle. This proposal addresses a bottleneck in proteomics research - identifying the biological consequence and pathways associated with the thousands of phosphorylation events currently being identified. On a therapeutic level, this research investigates how Aurora kinase misregulation can initiate cell cycle defects such as chromosomal instability. PUBLIC HEALTH RELEVANCE: We are investigating the role of the Aurora kinase protein in cell proliferation and division and the implications this has on cancer. Our goal is to develop technology in the baker's yeast that allows us to understand how this protein, and other related proteins, communicates with the cell division machinery. A better understanding of these complicated protein networks will allow us to find therapies and design better drugs that would preferentially target cancerous cells.