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

点击翻译按钮获取中文摘要

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.