Phosphorylation of Protein Translation Initiation Factors as a Novel Component of Brassinosteroid Signal Transduction

油菜素类固醇(BRs)是一种重要的植物激素，调节植物生长发育的多个方面，需要两种受体激酶，即油菜素类固醇不敏感1(BRI1)和BRI1相关受体激酶1(BAK1)，进行激素感知和信号转导。主要研究人员在植物中鉴定了拟南芥BRI1和BAK1依赖于BR的特异性磷酸化位点，并分离到一个与哺乳动物转化生长因子-β受体相互作用蛋白(TRIP-1)同源的BRI1胞质底物。Trip-1(也称为eIF3i)是一种双功能蛋白，在哺乳动物中调节转化生长因子-β信号转导，也在动物、酵母和植物中的eIF3蛋白翻译起始复合体中发挥关键作用。拟南芥BRI1在植物中与TRIP-1相互作用，并在体外使TRIP-1在三个特定残基上磷酸化。起始是真核蛋白质翻译过程中的限速步骤，通常由特定起始因子亚基的磷酸化来调节，以响应各种信号。对新的BRI1和BAK1相互作用蛋白的蛋白质组筛选发现，另外四个eIF亚基：eIF2B、eIF3G、eIF4A和eIF5可能是BRI1和/或BAK1的激酶域底物。综上所述，初步证据表明，BRI1依赖于BR对TRIP-1(和其他eIF亚基)的磷酸化可能影响启动因子的活性和/或组装，从而影响蛋白质翻译的全球细胞现象，为BR调控植物生长提供了一种新的机制。这项研究将通过三个目标来考察BR信号转导和蛋白质翻译启动的交叉点。目的1.用多种质谱学方法鉴定拟南芥体内TRIP-1的磷酸化位点，并分析其在BR信号和eIF3活性中的功能意义。目的：详细研究eIF3g和eIF5与BRI1和BAK1的相互作用。目的3.建立体内多个eIF亚基的磷酸化位点数据库，并利用无标记质谱仪对这些蛋白质中BR依赖的磷酸化进行定量研究。智力优势：BR信号的基因组效应和BR调控的基因表达知道得很多，但对BRs可能直接调节细胞生理的非基因组途径知之甚少，例如通过磷酸化细胞质蛋白，如翻译起始因子亚基。识别eIF亚基中特定的体内磷酸化位点，结合它们的功能表征，将增强我们对植物蛋白质翻译调控分子机制的理解。广泛影响：拟议的研究将提供各级生物化学、分子生物学和质谱学方面的优秀培训，包括博士后科学家、研究生和本科生以及高中生暑期实习生。EIF磷酸化位点的数据库将公之于众，对研究社区有用的载体构建物和转基因植物将通过拟南芥生物资源中心分发。BRs现在被牢固地确立为植物生长和发育的基本调节因子，影响着广泛的发育过程。在番茄、水稻、大麦和豌豆中鉴定BR生物合成和不敏感突变体，显然将这些化合物的重要性从实验植物拟南芥扩展到农作物，最近的田间实验表明，水稻BRI1表达的轻微变化可以使水稻产量增加30%。因此，了解BR信号转导的分子细节对于调控农业植物的生长具有实际应用价值。

Brassinosteroids (BRs) are essential plant hormones that regulate multiple aspects of plant growth and development and require two receptor kinases, Brassinosteroid Insensitive 1 (BRI1) and BRI1-Associated Receptor Kinase 1 (BAK1), for hormone perception and signal transduction. The principal investigators identified specific BR-dependent phosphorylation sites of Arabidopsis BRI1 and BAK1 in planta and isolated a putative cytoplasmic substrate of BRI1 with homology to the mammalian TGF-beta receptor interacting protein (TRIP-1). TRIP-1 (also known as eIF3i) is a dual function protein that regulates TGF-beta signaling in mammals and also plays a critical role in the eIF3 protein translation initiation complex in animals, yeast and plants. Arabidopsis BRI1 interacts with TRIP-1 in planta and phosphorylates TRIP-1 on three specific residues in vitro. Initiation is the rate-limiting step in eukaryotic protein translation and is often regulated by phosphorylation of specific initiation factor subunits in response to various signals. A proteomic screen for novel BRI1 and BAK1 interactors identified an additional four eIF subunits; eIF2B, eIF3g, eIF4A and eIF5, as putative kinase domain substrates for BRI1 and/or BAK1. Taken together, the preliminary evidence suggests that BR-dependent phosphorylation of TRIP-1 (and other eIF subunits) by BRI1 may affect initiation factor activity and/or assembly and thus impact the global cellular phenomenon of protein translation, providing a novel mechanism for BR regulation of plant growth. The research will examine the intersection of BR signal transduction and protein translation initiation by focusing on three objectives. Objective 1. Characterization of Arabidopsis TRIP-1 in vivo phosphorylation sites by a variety of mass spectrometry approaches and analysis of their functional significance with respect to BR signaling and eIF3 activity. Objective 2. Detailed in vivo and in vitro characterization of the putative interaction of eIF3g and eIF5 with BRI1 and BAK1. Objective 3.Generation of an in vivo phosphorylation site database of multiple eIF subunits followed by quantitative studies of BR-dependent phosphorylation in these proteins using label-free mass spectrometry methodologies.Intellectual merit: A great deal is known about genomic effects of BR signaling and BR regulated gene expression, but little is known about non-genomic pathways through which BRs might regulate cellular physiology directly, e.g. by phosphorylating cytoplasmic proteins such as translation initiation factor subunits. Identification of specific in vivo phosphorylation sites in eIF subunits coupled with their functional characterization will enhance our understanding of the molecular mechanisms regulating protein translation in plants.Broader impacts: The proposed research will provide excellent training in biochemistry, molecular biology and mass spectrometry at all levels, including postdoctoral scientists, graduate and undergraduate students and high school student summer interns. A database of eIF phosphorylation sites will be made publicly available and vector constructs and transgenic plants useful to the research community will be distributed via the Arabidopsis Biological Resource Center. BRs are now firmly established as essential regulators of plant growth and development affecting a broad spectrum of developmental processes. The identification of BR biosynthetic and insensitive mutants in tomato, rice, barley and pea, clearly extends the importance of these compounds from the experimental plant Arabidopsis thaliana to crop plants and recent field experiments have shown slight alterations in rice BRI1 expression can alter rice yields by up to 30%. Understanding the molecular details of BR signal transduction can thus have practical application in regulating the growth of agricultural plants.