PROTEOMIC STUDY OF BRASSINOSTEROID SIGNAL TRANSDUCTION

该子项目是利用 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来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 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使转录因子BZR 1和BZR 2/BES 1磷酸化，这种磷酸化抑制它们的DNA结合和核定位，并促进它们被蛋白酶体降解。本研究的目的是在生化和蛋白质组学水平上了解BR信号转导。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，我们将确定BZR 1相互作用的蛋白质，使用串联亲和纯化和质谱分析。第三，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。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, and this phosphorylation 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, we will identify the BZR1-interacting proteins using tandem affinity purification and mass spectrometry analysis. Third, in order to understand how BR signaling regulates BIN2 activity, we will purify BIN2-interacting proteins and identify them using MS analysis. 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.