Phosphoproteomic analysis of ethylene-regulated protein phosphorylation in etiolated seedlings of Arabidopsis mutant ein2 using two-dimensional separations coupled with a hybrid quadrupole time-of-flight mass spectrometer
Phosphoproteomic analysis of ethylene-regulated protein phosphorylation in etiolated seedlings of Arabidopsis mutant ein2 using two-dimensional separations coupled with a hybrid quadrupole time-of-flight mass spectrometer
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DOI：
10.1002/pmic.200800420
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发表时间：
2009-03-01
期刊：
影响因子：
3.4
通讯作者：
Li, Ning
中科院分区：
文献类型：
--
作者：
Li, Hao;Wong, Wai Shing;Li, Ning
乙烯调节植物的多种胁迫反应和发育适应。本研究采用磷酸化蛋白质组学方法研究拟南芥乙烯不敏感2 （ein2）突变体中乙烯对差异蛋白的磷酸化作用。共鉴定出224个磷酸肽，其中检测三次及以上的有64个。乙烯诱导ein2中磷酸化蛋白的普遍减少。共鉴定出3个乙烯增强型和3个乙烯抑制型独特磷酸肽。这些磷酸化蛋白的细胞功能分类表明，55.5%的磷酸化蛋白与信号传导和基因表达有关。肽序列比对显示了两个高度保守的磷酸化基序，barx下的PRVD/G (S)和bar PDYxx下的PRVD/G (S)。这些磷酸化肽与拟南芥蛋白的比对揭示了五个磷酸化基序。在这些基序中存在乙烯增强型和抑制型磷酸肽。预计il -1， ERF110转录因子和Hua增强子4 （HEN4）包含一个磷酸化基序。含基序肽的磷酸化已通过体外激酶测定和质谱分析得到验证。Western dot blot分析证实了乙烯对磷酸化的差异调控。总的来说，这些。结果表明，乙烯信号可能通过受体的磷酸化转导，通过ein2依赖性和非依赖性途径转导到转录事件。
Ethylene regulates a variety of stress responses and developmental adaptation in plants. In the present study, the phosphoproteomics is adopted to investigate the differential protein phosphorylation by ethylene in Arabidopsis ethylene-insensitive 2 (ein2) mutant. A total of 224 phosphopeptides were identified, of which 64 phosphopeptides were detected three or more times. Ethylene induces a general reduction in phosphorylated proteins in ein2. Totally, three ethylene-enhanced and three ethylene-repressible unique phosphopeptides were identified, respectively. Classification of the cellular functions of these phosphoproteins revealed that 55.5% of them are related to signaling and gene expression. Peptide sequence alignment reveals two highly conserved phosphorylation motifs, PRVD/G (S) under barx and (S) under bar PDYxx. Alignment of these phosphopeptides with Arabidopsis proteins reveals five phosphorylation motifs. Both ethylene-enhanced and -repressible phosphopeptides present in these motifs. EIL-1, ERF110 transcription factors and Hua enhancer 4 (HEN4) are predicted to contain one of the phosphorylation motifs. The phosphorylation of the motif-containing peptides has been validated by the in vitro kinase assays coupled with MS analysis. The differential regulation of phosphorylation by ethylene is substantiated by Western dot blot analysis. Taken together, these. results suggest that ethylene signals may be transduced by a phosphor-relay from receptors to transcriptional events via both ein2-dependent and -independent pathways.