PROTEOMIC STUDY OF BRASSINOSTEROID SIGNAL TRANSDUCTION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 油菜素类固醇(Brassinosteriods，BRs)是一类在植物体内具有重要作用的类固醇激素。BR信号是由细胞表面受体激酶BRI1感知的，它启动了一系列蛋白质磷酸化，导致核基因表达和细胞反应。BR下游信号涉及细胞质GSK3/Saggy样激酶BIN2。BIN2使转录因子BZR1和BZR2/BES1磷酸化。转录因子的磷酸化抑制了它们的DNA结合和核定位，并促进了它们被蛋白酶体降解。本研究的目的是在生物化学和蛋白质组水平上了解BR信号转导。首先，我们将使用质谱学(MS)鉴定BZR1的BIN2-磷酸化位点。一些磷酸化位点的重要性将通过定点突变和转基因实验进一步测试。其次，为了了解BR信号如何调节BIN2活性，我们将纯化与BIN2相互作用的蛋白，并用MS分析鉴定它们。第三，我们正在使用二维差示凝胶电泳(2-D DGE)和iTRAQ质谱学来鉴定BR调节的蛋白质。我们将通过对总蛋白、质膜和磷蛋白部分进行蛋白质组学分析来鉴定BR调节的蛋白质。第四，我们计划利用固定化金属亲和层析和iTRAQ方法研究BR调节的蛋白质磷酸化。识别出的蛋白质的功能将通过遗传和转基因实验进行研究。这项研究将促进我们对植物中类固醇反应的分子机制的理解，这将对我们从总体上理解类固醇的作用具有广泛的意义。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Brassinosteriods (BRs) are steroid hormones with important roles in plants. BR signal is perceived by the cell-surface receptor kinase BRI1, which initiates a cascade of protein phosphorylation leading to nuclear gene expression and cellular responses. Downstream BR signaling involves the cytoplasmic GSK3/SHAGGY-like kinase BIN2. BIN2 phosphorylates the transcription factors BZR1 and BZR2/BES1. Phosphorylation of the transcription factors inhibits their DNA binding and nuclear localization and promotes their degradation by the proteasome. The goal of this study is to understand BR signal transduction at the biochemical and proteomic levels. First, we will identify the BIN2-phosphorylation sites of BZR1 using mass spectrometry (MS). The importance of some of the phosphorylation sites will be further tested by site-directed-mutagenesis and transgenic experiments. Second, in order to understand how BR signaling regulates BIN2 activity, we will purify BIN2-interacting proteins and identify them using MS analysis. Third, we are identifying BR-regulated proteins using two-dimensional difference gel electrophoresis (2-D DIGE) and iTRAQ mass spectrometry. We will identify BR-regulated proteins by performing proteomic analysis of total protein, plasma membrane, and phosphoprotein fractions. Forth, we plan to study BR regulated protein phosphorylation using immobilized metal affinity chromatography and the iTRAQ method. The functions of the identified proteins will be studied using genetic and transgenic experiments. This research project will advance our understanding of the molecular mechanism for steroid responses in plants, which will have broad implications in our understanding of steroid actions in general.