Identification of in vivo Substrates of MEK-MPK Modules in Arabidopsis

有丝分裂原激活的蛋白激酶(MPK)存在于所有真核生物中。它们将细胞外信号传递给细胞内的反应，同时放大传递的信号。到目前为止，作为MPK底物的蛋白质的特性在很大程度上仍然难以捉摸；据推测，其中许多是低丰度蛋白质。从复杂的生物样品中鉴定磷酸蛋白和磷酸化位点的一种常见策略是从消化的细胞裂解物中富集磷酸肽，然后进行质谱分析。这些尝试往往偏向于鉴定高度丰富的磷酸肽。在这里，我们应用一种稳健和高选择性的方法来鉴定和定量拟南芥中MPKs的低丰度蛋白底物的位点特异性磷酸化。这项技术使用了对磷酸蛋白和多肽的连续浓缩。该策略结合了变性条件下的蛋白质提取，使用Al(OH)3为基础的金属氧化物亲和层析(MOAC)浓缩磷蛋白，浓缩的磷蛋白部分的胰酶消化，以及随后的基于二氧化钛的MOAC的磷酸肽。在我们的提案中，我们展示了通过鉴定和量化一些已知的和新的体内假定的MPK3/6底物的独特的磷酸化位点来证明这一磷酸蛋白质组策略的力量的初步实验。我们建议鉴定拟南芥MPK的底物，并对其进行功能表征。由于MPK参与了所有真核生物不同细胞反应的调节，因此拟议的工作结果将对植物生物学以及生命科学的其他领域产生重大影响。

Mitogen-activated protein kinases (MPKs) are found in all eukaryotes. They transmit extracellular signals to intracellular responses while at the same time amplifying the transmitting signal. By now, the identity of proteins that serve as MPK substrates remained largely elusive; presumably many of them are low abundant proteins. A common strategy to identify phosphoproteins and phosphorylation sites from complex biological samples is the enrichment of phosphopeptides from digested cellular lysates followed by mass spectrometry. These attempts are often biased towards identification of highly abundant phosphopeptides. Here we apply a robust and highly selective approach for the identification and quantification of site-specific phosphorylation of low abundant protein substrates for MPKs in Arabidopsis thaliana. The technique employs successive enrichment of phosphoproteins and –peptides. The strategy combines protein extraction under denaturing conditions, phosphoprotein enrichment using Al(OH)3-based metaloxide affinity chromatography (MOAC), tryptic digestion of the enriched phosphoprotein fraction, and subsequent TiO2-based MOAC of phosphopeptides. In our proposal we demonstrate pilot experiments proving the power of this phosphoproteomic strategy by the identification and quantification of unique phosphorylation sites of a number of known and novel presumed in vivo MPK3/6 substrates in Arabidopsis. We propose to identify, and functionally characterize substrates of Arabidopsis MPKs. Since MPKs are involved in the regulation of diverse cellular responses in all eukaryotes, results of the proposed work will have a significant impact on plant biology as well as other areas in the life sciences.