PROTEOMIC: BRASSINOSTEROID SIGNAL TRANSDUCTION

蛋白质组学：油菜素类固醇信号转导

• 批准号: 7369050
• 负责人: ZHIYONG WANG
• 金额: $ 1.89万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369050
• 关键词: PROTEOMIC; BRASSINOSTEROID; SIGNAL; TRANSDUCTION

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.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。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。所列机构为中心，不一定是研究者所在机构。乙二甾醇（BR）是植物生长调节的重要激素。BR信号由细胞表面受体激酶BRI 1感知，其启动导致核基因表达和细胞应答的信号级联。BR信号传导涉及下游蛋白质的丰度和活性的翻译后调节。例如，BR信号负调节细胞质GSK 3/SHAGGY样激酶BIN 2。BIN 2使核蛋白BZR 1磷酸化，并靶向BZR 1以供蛋白酶体降解。首先，我们尝试使用质谱法确定BZR 1的磷酸化位点。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，我们正在分离BR调节蛋白质，使用二维差异凝胶电泳（2-D DIGE）和质谱。这些包括其丰度或翻译后修饰受BR调节或在BR信号转导突变体中受影响的蛋白质。我们进行了系统的二维DIGE分析，并确定了约300 BR调控蛋白点，其中约60已被MS鉴定。结合BR调控RNA的微阵列分析，我们确定了3个蛋白质pupillary调控BR在转录后水平。我们还进行了亚细胞分级分离2-D DIGE之前，并确定了大量的质膜蛋白，快速响应BR治疗。使用磷蛋白富集试剂盒，我们已经确定了几个BR调节磷蛋白。这些蛋白质正在使用质谱法进行鉴定。我们计划使用基于β-消除/迈克尔加成（BEMAD）的方法研究BR调节的蛋白磷酸化和O-GlcNAc修饰。该研究项目将推进我们对植物中类固醇反应的分子机制的理解，这将对我们理解类固醇的作用产生广泛的影响。