PROTEOMIC: BRASSINOSTEROID SIGNAL TRANSDUCTION

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。油菜素内酯（Brassinosteriod， BR）是植物重要的生长调节激素。BR信号被细胞表面受体激酶BRI1感知，引发信号级联，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控。例如，BR信号负调控细胞质GSK3/SHAGGY-like激酶BIN2。BIN2磷酸化核蛋白BZR1，并靶向BZR1被蛋白酶体降解。首先，我们尝试用质谱法确定BZR1的磷酸化位点。磷酸化位点的重要性将通过位点定向诱变和转基因实验进一步验证。其次，我们使用二维凝胶电泳（2-D DIGE）和质谱法分离br调节蛋白。这些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。我们进行了系统的二维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个推测在转录后水平上受BR调节的蛋白。我们还在二维DIGE之前进行了亚细胞分离，并鉴定了大量对BR处理反应迅速的质膜蛋白。使用磷蛋白富集试剂盒，我们已经鉴定了几个BR调节的磷蛋白。这些蛋白质正在用质谱法进行鉴定。我们计划使用基于β消除/Michael加成（BEMAD）的方法研究BR调节的蛋白磷酸化和O-GlcNAc修饰。该研究项目将促进我们对植物类固醇反应的分子机制的理解，这将对我们理解类固醇的一般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.