Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses.
Quantitative phosphoproteomic analysis of plasma membrane proteins reveals regulatory mechanisms of plant innate immune responses.
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DOI：
10.1111/j.1365-313x.2007.03192.x
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发表时间：
2007-09
期刊：
影响因子：
7.2
通讯作者：
Peck, Scott C
中科院分区：
文献类型：
--
作者：
Nuhse, Thomas S;Bottrill, Andrew R;Jones, Alexandra M E;Peck, Scott C
蛋白质组学技术的进步使得大规模鉴定复杂蛋白质样品中的磷酸化位点成为可能，但新的生物学见解需要了解它们在体内的动力学。在这里，我们展示了使用稳定的同位素为基础的定量方法的途径发现和结构功能研究的拟南芥细胞处理细菌激发子鞭毛蛋白。定量比较确定了质膜(PM)蛋白上经历快速磷酸化或去磷酸化的个别位置。这些数据既揭示了同一蛋白质中不同位点的差异动态，也揭示了相关蛋白质中同源位点的协调调节，如PM H+-ATPase AHA1、2和3。强激发子反应的磷酸化位点可能反映了蛋白质活性的直接调节。我们证实了RbohD的这一预测，RbohD是一种NADPH氧化酶，它介导了激发子和病原体反应中活性氧物种(ROS)的快速产生。植物NADPH氧化酶在结构上不同于它们的哺乳动物同源物，对植物酶的调控知之甚少。在RbohD上，我们发现了不变的和强烈诱导的磷酸化位点。通过用非磷酸化形式的RbohD补充RbohD突变植物，我们表明只有那些经历差异调节的位置才是激活蛋白质所必需的。这些实验证明了使用定量磷酸蛋白质组学在分子水平上确定调控机制的潜力，并为天然免疫反应提供了新的见解。
Advances in proteomic techniques have allowed the large-scale identification of phosphorylation sites in complex protein samples, but new biological insight requires an understanding of their in vivo dynamics. Here, we demonstrate the use of a stable isotope-based quantitative approach for pathway discovery and structure–function studies in Arabidopsis cells treated with the bacterial elicitor flagellin. The quantitative comparison identifies individual sites on plasma membrane (PM) proteins that undergo rapid phosphorylation or dephosphorylation. The data reveal both divergent dynamics of different sites within one protein and coordinated regulation of homologous sites in related proteins, as found for the PM H+-ATPases AHA1, 2 and 3. Strongly elicitor-responsive phosphorylation sites may reflect direct regulation of protein activity. We confirm this prediction for RbohD, an NADPH oxidase that mediates the rapid production of reactive oxygen species (ROS) in response to elicitors and pathogens. Plant NADPH oxidases are structurally distinct from their mammalian homologues, and regulation of the plant enzymes is poorly understood. On RbohD, we found both unchanging and strongly induced phosphorylation sites. By complementing an RbohD mutant plant with non-phosphorylatable forms of RbohD, we show that only those sites that undergo differential regulation are required for activation of the protein. These experiments demonstrate the potential for use of quantitative phosphoproteomics to determine regulatory mechanisms at the molecular level and provide new insights into innate immune responses.