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

丝裂原活化蛋白激酶（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反应，同时放大传输信号。到目前为止，作为MPK底物的蛋白质的身份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蛋白和磷酸化位点的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。这些尝试通常偏向于鉴定高度丰富的磷酸肽。在这里，我们采用了一个强大的和高选择性的方法来识别和定量的位点特异性磷酸化的低丰度蛋白质底物MPKs在拟南芥。该技术采用磷蛋白和肽的连续富集。该策略结合了蛋白质提取变性条件下，磷蛋白富集Al（OH）3为基础的金属氧化物亲和层析（MOAC），胰蛋白酶消化的富集磷蛋白馏分，和随后的TiO 2为基础的MOAC的磷酸肽。在我们的建议中，我们展示了试点实验证明这种磷酸化蛋白质组学策略的能力，通过鉴定和定量一些已知的和新的推测体内MPK 3/6底物在拟南芥中的独特磷酸化位点。我们建议确定，并在功能上表征底物的拟南芥MPKs。由于MPK参与调节所有真核生物中的多种细胞反应，因此拟议工作的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.