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

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.

The interaction of life forms with their environment is governed by the perception of a myriad of pieces of information and their transmission to a central processing level. One of the most important signal transduction relays is the Mitogen Associated Protein Kinase (MAPK) cascade. Essentially it consist of three protein modules in series, MAPK kinase kinases (MEKK), MAPK kinases (MEK) and MAP kinases (MPK). Following perception of stimuli, these protein modules are sequentially switched on (and off) by reversible covalent attachment of one or two phosphoryl groups. The final link in this signal transduction chain, the MPK then attaches one or more phosphoryl groups to a target protein which leads to signal processing and an appropriate cellular response. Organisms devoid of the most important MAP kinases for instance through gene mutation are not viable, clearly underscoring the tremendous importance of these proteins.In light of the central role of the MAPK signaling cascade, it is probable, that it activates a large number of cellular proteins with diverse functions. At the time of project begin however only a handful of MPK target proteins, also known as MPK substrates were known. Most of these are involved in the regulation of development and in plant in the response to pathogens and pests, plant innate immunity. The goal of this project was to discover the potential plethora of unknown yet suspected MPK substrates and concurrently to shed light on the roles of MAPK signal transduction in regulating manifold cellular processes. This was to be carried out in the model plant Arabidopsis thaliana.Activated MPK substrates carry one or more phosphoryl groups that induce a shift in their molecular mass compared to their quiescent counterparts. Therefore mass spectrometry, a physical technique that measures the mass of charged particles is ideal for this task. Directed switching on of MPK activity via a transgene will lead to preferential attachment of phosphoryl groups to MPK substrates in one plant compared to another plant with relatively weak MPK activity in normal development. A new technology was developed in the project to enrich activated MPK substrates by way of their phosphoryl groups and measure them using high resolution/accurate mass (HR/AM) mass spectrometry at the University of Vienna. This was the first work to decidedly identify more than 100 previously unknown MPK substrates in the living plant (in vivo). The study confirmed much of the prevalent speculation and lead to new hypotheses of the function of these central signal transduction relays in plants and other organisms.