Functional dissection of novel protein kinases and their roles in diurnally regulated signal transduction in Arabidopsis thaliana

植物利用光能从空气中收集二氧化碳来制造复杂的分子。检测(光信号)和/或预测(生物钟)光可用性的能力是植物生长和存活的主要决定因素。这些能力共同形成了一个昼夜调节电路，使植物能够动态适应日常环境。在分子水平上，昼夜调节是通过转录、翻译和翻译后机制的组合实现的。尤其是翻译后蛋白质修饰(PTM)，这是真核细胞正常功能的核心。PTM代表了一种快速、微调的蛋白质机制，可以产生不同的结果，如酶活性或细胞定位的变化。在PTM中，蛋白质的磷酸化最为丰富，调控着超过70%的真核蛋白质。然而，由于我们目前对植物细胞昼夜调控的理解主要来自于转录水平的变化，蛋白质水平和PTM变化对调控的程度仍然知之甚少。*我研究的长期目标是利用定量蛋白质组学在模式植物拟南芥中发现新的昼夜信号蛋白，并了解这些蛋白如何发挥调节植物细胞过程的功能。*目前，我们对蛋白质磷酸化如何昼夜调节细胞过程的了解仅限于少数几项研究。此外，昼夜磷酸化事件和负责催化这些事件的蛋白激酶之间的联系是有限的。利用磷蛋白质组学，我们最近发现，在多种组织类型中，拟南芥的日终和夜终磷蛋白质组存在着重大的数量差异。在这里，我们鉴定了一些蛋白激酶，它们的磷酸化状态每天都在变化；特别是一组独特的植物特有的包含Phox/BemP1结构域的蛋白激酶(PXK)。鉴于磷酸化事件在控制蛋白质功能方面可能有不同的结果，我们假设通过我们的多组织磷蛋白质组筛查观察到的每日磷酸化变化表明每个PXK都更广泛地参与了日常植物细胞过程的调节。为了评估这一点，我们将(1)进行PXK亚家族的生化和生物学特性研究，(2)鉴定和量化PXK蛋白的昼夜相互作用网络，(3)进行PXK蛋白复合体的功能特性研究。总之，这三个目标将揭示每个PXK在植物细胞昼夜调控中所起的作用，同时也有助于进一步弥合植物磷酸蛋白质组学和催化这些反应的特定蛋白激酶之间存在的差距。**

Plants utilize light energy to harvest carbon dioxide from the air to build complex molecules. The ability to detect (light signaling) and/or anticipate (circadian clock) the availability of light represents a major determinant of plant growth and survival. Together, these abilities form a diurnal regulatory circuit which allows plants to dynamically adapt to their daily environment. At the molecular level, diurnal regulation is achieved through a combination of transcriptional, translational and post-translational mechanisms. In particular, post-translational protein modifications (PTMs), which are central to the proper functionality of eukaryotic cells. PTMs represent a fast-acting, fine-tuning mechanism for proteins that can have diverse outcomes such as a change in enzyme activity or cellular localization. Of the PTMs, protein phosphorylation is the most abundant, regulating over 70% of all eukaryotic proteins. However, with our current understanding of diurnal plant cell regulation largely derived from changes in transcript levels, the extent of regulation by protein level and PTM changes remains poorly understood. ******The long term goal of my research is to utilize quantitative proteomics to uncover new diurnal signaling proteins in the model plant Arabidopsis and to understand how these proteins function to regulate plant cell processes.******Currently, our understanding of how protein phosphorylation diurnally regulates cellular processes is limited to a handful of studies. Furthermore, connections between diurnal phosphorylation events and the protein kinases responsible for catalyzing these events is limited. Using phosphoproteomics, we have recently shown major quantitative differences exist in the end-of-day and end-of-night phosphoproteome of Arabidopsis thaliana across multiple tissue types. Here, we identified a number of protein kinases undergoing diurnal changes in their phosphorylation status; in particular, an unique set of plant specific Phox/BemP1 domain containing protein kinases (PXKs). Given the diverse outcomes phosphorylation events can have in controlling protein function, we hypothesize that the diurnal phosphorylation changes we observed through our multi-tissue phosphoproteomic screen are indicative of each PXKs broader involvement in the regulation of diurnal plant cell processes. To evaluate this, we will (1) undertake biochemical and biological characterization of the PXK sub-family, (2) identify and quantify diurnal PXK protein interaction networks and (3) undertake functional characterization of PXK protein complexes. Together, these 3 objectives will uncover the role each PXK plays in diurnal plant cell regulation, while also helping to further bridge the gap that exists between plant phosphoproteomics and the specific protein kinases that catalyze these reactions. **