Integrated Genomewide Analysis of the lpL1 Kinase Signaling Network

描述(由申请人提供)：这项提案概述了一套完整的基于酵母的技术，用于研究激酶信号网络。我们详细介绍了能够研究依赖于磷酸化的蛋白质-蛋白质相互作用图谱控制的方法。虽然范围一般，但本提案中应用的方法将重点放在酵母极光激酶Ipl1上。Aurora激酶的过度表达在许多癌症中得到证实，小分子抑制会导致表型，如细胞凋亡和有丝分裂灾难，从而导致肿瘤的退化。我们的综合方法解决了这样一个事实，即磷酸化影响蛋白质-蛋白质相互作用，因此我们将在构建动态相互作用组图中贡献一个重要因素。细胞的动态行为在有丝分裂期间最为明显，并依赖于蛋白质-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因子。我们假设，磷酸化位点将特异性地调节蛋白质之间的相互作用，这些将与Ipl1的各种有丝分裂作用相关联。我们将通过以下方式对Ipl1信号网络进行分析：i)使用基于合成剂量致死性原则的优化方法确定与Ipl1相关的底物和细胞通路；ii)通过磷突变扫描酵母双杂交方法确定Ipl1启动的磷酸化对蛋白质间相互作用的影响；iii)将Ipl1磷酸化位点和相应的蛋白质相互作用与Ipl1的有丝分裂途径和作用相关联；以及iv)根据我们的合成剂量致死率和磷突变实验，验证我们对胰腺癌细胞模型中抗癌靶点的预测。磷酸调节的相互作用与Ipl1在协调染色体分离中的作用的关联将与动粒和纺锤体的生物学和结构研究相结合。这项建议解决了蛋白质组学研究中的一个瓶颈--确定与目前正在确定的数千个磷酸化事件相关的生物学后果和途径。在治疗层面上，这项研究调查了极光激酶错误调节如何引发细胞周期缺陷，如染色体不稳定。 公共卫生相关性：我们正在研究极光蛋白在细胞增殖和分裂中的作用，以及这对癌症的影响。我们的目标是在面包师酵母中开发技术，使我们能够了解这种蛋白质和其他相关蛋白质是如何与细胞分裂机制进行沟通的。更好地了解这些复杂的蛋白质网络将使我们能够找到治疗方法，并设计出更好的药物，优先针对癌细胞。

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.