PROTEOMIC: BRASSINOSTEROID SIGNAL TRANSDUCTION

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。油菜素类固醇(BR)是植物生长调节的重要激素。BR信号是由细胞表面受体激酶BRI1感知的，它启动了导致核基因表达和细胞反应的信号级联反应。BR信号涉及下游蛋白丰度和活性的翻译后调节。例如，BR信号负性调节细胞质GSK3/Saggy样激酶BIN2。BIN2使核蛋白BZR1磷酸化，并以BZR1为靶标，由蛋白酶体降解。首先，我们尝试用质谱仪确定BZR1的磷酸化位点。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，我们正在使用二维差示凝胶电泳法(2-D DGE)和质谱仪分离BR调节的蛋白质。这些蛋白质包括其丰度或翻译后修饰受BR调节或在BR信号突变中受影响的蛋白质。我们进行了系统的2-D DGE分析，鉴定了约300个BR调控的蛋白质点，其中约60个已被MS鉴定。通过结合BR调控RNA的微阵列分析，我们在转录后水平鉴定了3个可能受BR调控的蛋白质。我们还在2-D DGE之前进行了亚细胞分离，并鉴定了大量对BR处理快速反应的质膜蛋白。使用磷蛋白浓缩试剂盒，我们已经鉴定了几个受BR调控的磷蛋白。这些蛋白质正在使用质谱学进行鉴定。我们计划使用基于β-消除/迈克尔加成(BEMAD)的方法来研究BR调节的蛋白质磷酸化和O-GlcNAc修饰。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. Brassinosteriod (BR) is an important hormone for growth regulation in plants. BR signal is perceived by the cell-surface receptor kinase BRI1, which initiates a signaling cascade leading to nuclear gene expression and cellular responses. BR signaling involves posttranslational regulation of the abundance and activity of downstream proteins. For example, BR signaling negatively regulates the cytoplasmic GSK3/SHAGGY-like kinase BIN2. BIN2 phosphorylates the nuclear protein BZR1 and targets BZR1 for degradation by the proteasome. First, we have attempted to determine the phosphorylation sites of BZR1 using mass spectrometry. The importance of the phosporylation sites will be further tested by site-directed-mutagenesis and transgenic experiments. Second, we are isolating BR-regulated proteins using two-dimensional difference gel electrophoresis (2-D DIGE) and mass spectrometry. These include the proteins whose abundance or posttranslational modification is regulated by BR or affected in BR signaling mutants. We have performed systematic 2-D DIGE analysis and identified about 300 BR-regulated protein spots and about 60 of them have been identified by MS. By combining microarray analysis of BR regulated RNAs, we have identified 3 proteins putatively regulated by BR at the posttranscriptional level. We have also performed subcellular fractionation before 2-D DIGE and have identified large number of plasma membrane proteins that respond quickly to BR treatment. Using a phosphoprotein enrichment kit, we have identified several BR regulated phosphoproteins. These proteins are being identified using mass spectrometry. We plan to study BR regulated protein phosphorylation and O-GlcNAc modification using beta-elimination/Michael addition (BEMAD) based method. This research project will advance our understanding of the molecular mechanism of steroid responses in plants, which will have broad implications in our understanding of steroid actions in general.