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 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。油菜甾醇（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 BRI1 感知，启动信号级联反应，导致核基因表达和细胞反应。 BR 信号传导涉及下游蛋白质丰度和活性的翻译后调节。例如，BR 信号传导负向调节细胞质 GSK3/SHAGGY 样激酶 BIN2。 BIN2 磷酸化核蛋白 BZR1 并靶向 BZR1 以供蛋白酶体降解。首先，我们尝试使用质谱法确定 BZR1 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，我们使用二维差异凝胶电泳 (2-D DIGE) 和质谱法分离 BR 调节蛋白。这些包括丰度或翻译后修饰受 BR 调节或受 BR 信号突变体影响的蛋白质。我们进行了系统的 2-D DIGE 分析，鉴定了约 300 个 BR 调节的蛋白点，其中约 60 个已通过 MS 鉴定。通过结合 BR 调节 RNA 的微阵列分析，我们鉴定了 3 种可能在转录后水平受 BR 调节的蛋白质。我们还在 2-D DIGE 之前进行了亚细胞分级分离，并鉴定了大量对 BR 处理快速响应的质膜蛋白。使用磷蛋白富集试剂盒，我们鉴定了几种 BR 调节的磷蛋白。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调节的蛋白质磷酸化和O-GlcNAc修饰。该研究项目将增进我们对植物类固醇反应分子机制的理解，这将对我们对类固醇作用的总体理解产生广泛的影响。