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。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。 Brassinosteriod（BR）是植物生长调节的重要激素。细胞表面受体激酶BRI1感知到BR信号，该细胞表面受体激酶BRI1启动了信号级联反应，导致核基因表达和细胞反应。 BR信号传导涉及下游蛋白丰度和活性的翻译后调节。例如，BR信号对细胞质GSK3/毛茸茸的激酶BIN2进行负调节。 BIN2磷酸化核蛋白BZR1，并靶向BZR1降解蛋白酶体。首先，我们尝试使用质谱法确定BZR1的磷酸化位点。阳期位点的重要性将通过位置定向的 - 刺激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调节的蛋白质。其中包括其丰度或翻译后修饰的蛋白质受到BR调节或在BR信号突变体中受影响的蛋白质。我们已经进行了系统的二维DIGE分析，并确定了约300个BR调节的蛋白质斑点，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，我们已经确定了3种受BR在转录后水平调节的蛋白质。我们还在2-D DIGE之前进行了亚细胞分级，并确定了大量对BR治疗迅速反应的质膜蛋白。使用磷蛋白富集试剂盒，我们已经确定了几种BR调节的磷蛋白。这些蛋白质正在使用质谱法鉴定。我们计划使用基于β-淘汰/迈克尔添加（BEMAD）方法研究BR调节的蛋白质磷酸化和O-GLCNAC修饰。该研究项目将提高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具有广泛的影响。