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

蛋白质翻译起始因子的磷酸化作为油菜素类固醇信号转导的新成分

• 批准号: 0742411
• 负责人: Steven Clouse
• 金额: $ 54万
• 依托单位: North Carolina State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0742411&HistoricalAwards=false
• 关键词: Phosphorylation; Protein; Translation; Initiation; Factors

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.

类甾醇（BR）是调节植物生长和发育的多个方面的必需植物激素，并且需要两种受体激酶，类甾醇不敏感1（BRI 1）和BRI 1相关受体激酶1（BAK 1），用于激素感知和信号转导。主要研究人员确定了拟南芥BRI 1和BAK 1在植物中的特定BR依赖性磷酸化位点，并分离出了与哺乳动物TGF-β受体相互作用蛋白（TRIP-1）同源的BRI 1的推定细胞质底物。TRIP-1（也称为eIF 3 i）是一种双重功能蛋白，其调节哺乳动物中的TGF-β信号传导，并且还在动物、酵母和植物中的eIF 3蛋白翻译起始复合物中起关键作用。拟南芥BRI 1在植物体内与TRIP-1相互作用，并在体外磷酸化TRIP-1的三个特定残基。起始是真核生物蛋白质翻译的限速步骤，通常受特异性起始因子亚基磷酸化的调控。新的BRI 1和BAK 1相互作用物的蛋白质组学筛选鉴定了另外四个eIF亚基：eIF 2B、eIF 3g、eIF 4A和eIF 5，作为BRI 1和/或BAK 1的推定激酶结构域底物。综上所述，初步证据表明，BR依赖的TRIP-1（和其他eIF亚基）的磷酸化BRI 1可能会影响起始因子的活性和/或组装，从而影响蛋白质翻译的全球细胞现象，为BR调节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研究BR信号转导和蛋白质翻译起始的交叉点。目的1.通过多种质谱方法表征拟南芥TRIP-1体内磷酸化位点，并分析其在BR信号传导和eIF 3活性方面的功能意义。目标2. eIF 3g和eIF 5与BRI 1和BAK 1的推定相互作用的详细体内和体外表征。目的3.建立多个eIF亚基的体内磷酸化位点数据库，然后使用无标记质谱方法定量研究这些蛋白中BR依赖的磷酸化。关于BR信号传导的基因组效应和BR调节的基因表达，我们知道很多，但是关于BR可能直接调节细胞生理学的非基因组途径，例如通过磷酸化细胞质蛋白如翻译起始因子亚基。鉴定eIF亚基中特异性的体内磷酸化位点及其功能特性将增强我们对植物蛋白质翻译调控分子机制的理解。更广泛的影响：拟议的研究将为各个层次的生物化学、分子生物学和质谱学提供优秀的培训，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开提供，对研究界有用的载体构建体和转基因植物将通过拟南芥生物资源中心分发。BR现已被牢固地确立为植物生长和发育的重要调节剂，影响广泛的发育过程。番茄、水稻、大麦和豌豆中BR生物合成和不敏感突变体的鉴定清楚地将这些化合物的重要性从实验植物拟南芥扩展到作物植物，并且最近的田间实验已经显示水稻BRI 1表达的轻微改变可以改变水稻产量高达30%。因此，了解BR信号转导的分子细节可以在调节农业植物生长方面具有实际应用。