PROTEOMIC STUDY OF BRASSINOSTEROID SIGNAL TRANSDUCTION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 甾体类化合物是一类在植物中具有重要作用的甾体激素。BR信号由细胞表面受体激酶BRI 1感知，其启动蛋白磷酸化级联反应，导致核基因表达和细胞反应。下游BR信号传导涉及细胞质GSK 3/SHAGGY样激酶BIN 2。BIN 2磷酸化转录因子BZR 1和BZR 2/BES 1。转录因子的磷酸化抑制它们的DNA结合和核定位，并促进它们被蛋白酶体降解。本研究的目标是在生化和蛋白质组水平上了解BR信号传导。首先，我们将使用质谱（MS）鉴定BZR 1的BIN 2磷酸化位点。一些磷酸化位点的重要性将通过定点诱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BIN 2活性，我们将纯化BIN 2相互作用蛋白，并使用MS分析鉴定它们。第三，我们正在使用二维差异凝胶电泳（2-D DIGE）和iTRAQ质谱鉴定BR调节蛋白。我们将通过对总蛋白、质膜和磷蛋白组分进行蛋白质组学分析来鉴定BR调控蛋白。第四，我们计划使用固定化金属亲和层析和iTRAQ方法来研究BR调节的蛋白磷酸化。将使用遗传和转基因实验来研究所鉴定的蛋白质的功能。该研究项目将促进我们对植物中类固醇反应的分子机制的理解，这将对我们对类固醇作用的理解产生广泛的影响。

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.