Integrating Phosphoproteomics and Bioinformatics to Study Brassinosteroid-Regulated Phosphorylation Dynamics in Arabidopsis.
Integrating Phosphoproteomics and Bioinformatics to Study Brassinosteroid-Regulated Phosphorylation Dynamics in Arabidopsis.
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DOI：
10.1186/s12864-015-1753-4
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发表时间：
2015-07-18
期刊：
影响因子：
4.4
通讯作者：
Juan HF
中科院分区：
文献类型：
--
作者：
Lin LL;Hsu CL;Hu CW;Ko SY;Hsieh HL;Huang HC;Juan HF
植物激素调控的蛋白质磷酸化参与植物基本发育的协调。类甾醇（BR）是一类植物激素，其磷酸化调控机制尚不清楚。在这项研究中，我们进行了基于质谱（MS）的磷酸化蛋白质组学，在5 min至12 h的时间段内，在BR处理的5个时间点进行全局和动态磷酸化蛋白质组分析。质谱结合磷酸肽富集技术已成为蛋白质磷酸化分析的有力工具。然而，基于MS的方法往往具有数据一致性和覆盖范围问题。为了解决这些问题，使用生物信息学方法来补充未检测到的蛋白质并恢复磷酸化事件的动力学。从739个独特的磷蛋白中共鉴定出1104个独特的磷酸化肽。时间依赖的基因本体（GO）分析显示了生物过程从信号转导到形态发生和应激反应的转变。蛋白质相互作用分析发现，大多数已鉴定的磷蛋白与已知的BR信号组分有很强的联系。使用Motif-X进行分析，以鉴定15个富集的基序，其中11个对应于6个已知的激酶家族。为了揭示激酶的动态活性，将富集的基序与磷酸化谱相结合，发现酪蛋白激酶2和丝裂原活化蛋白激酶的底物在BR处理的初始时间分别被显著磷酸化和去磷酸化。构建了时间依赖的激酶-底物相互作用网络，发现许多底物是生长素和阿坝信号的下游。在比较BR反应性磷酸化蛋白质组和基因表达数据时，我们发现大多数磷酸化变化不是由基因表达变化引起的。我们的研究结果表明，BR信号的许多下游蛋白是通过各种激酶磷酸化诱导的，而不是通过转录调控。通过大规模的磷酸化蛋白质组的动态分析，结合生物信息学，揭示了一个与BR调控生长相关的复杂的以激酶为中心的网络。本研究所鉴定的磷蛋白和磷酸化位点为揭示BR调控的信号网络提供了有用的数据集，同时也拓展了我们对植物蛋白磷酸化修饰的认识，为进一步解决植物生长问题提供了依据。本文的在线版本（doi：10.1186/s12864-015-1753-4）包含补充材料，可供授权用户使用。
Protein phosphorylation regulated by plant hormone is involved in the coordination of fundamental plant development. Brassinosteroids (BRs), a group of phytohormones, regulated phosphorylation dynamics remains to be delineated in plants. In this study, we performed a mass spectrometry (MS)-based phosphoproteomics to conduct a global and dynamic phosphoproteome profiling across five time points of BR treatment in the period between 5 min and 12 h. MS coupling with phosphopeptide enrichment techniques has become the powerful tool for profiling protein phosphorylation. However, MS-based methods tend to have data consistency and coverage issues. To address these issues, bioinformatics approaches were used to complement the non-detected proteins and recover the dynamics of phosphorylation events. A total of 1104 unique phosphorylated peptides from 739 unique phosphoproteins were identified. The time-dependent gene ontology (GO) analysis shows the transition of biological processes from signaling transduction to morphogenesis and stress response. The protein-protein interaction analysis found that most of identified phosphoproteins have strongly connections with known BR signaling components. The analysis by using Motif-X was performed to identify 15 enriched motifs, 11 of which correspond to 6 known kinase families. To uncover the dynamic activities of kinases, the enriched motifs were combined with phosphorylation profiles and revealed that the substrates of casein kinase 2 and mitogen-activated protein kinase were significantly phosphorylated and dephosphorylated at initial time of BR treatment, respectively. The time-dependent kinase-substrate interaction networks were constructed and showed many substrates are the downstream of other signals, such as auxin and ABA signaling. While comparing BR responsive phosphoproteome and gene expression data, we found most of phosphorylation changes were not led by gene expression changes. Our results suggested many downstream proteins of BR signaling are induced by phosphorylation via various kinases, not through transcriptional regulation. Through a large-scale dynamic profile of phosphoproteome coupled with bioinformatics, a complicated kinase-centered network related to BR-regulated growth was deciphered. The phosphoproteins and phosphosites identified in our study provide a useful dataset for revealing signaling networks of BR regulation, and also expanded our knowledge of protein phosphorylation modification in plants as well as further deal to solve the plant growth problems. The online version of this article (doi:10.1186/s12864-015-1753-4) contains supplementary material, which is available to authorized users.