Abstract
In Arabidopsis, mitogen-activated protein kinases MPK3, MPK4 and MPK6 constitute essential relays for a variety of functions including cell division, development and innate immunity. While some substrates of MPK3, MPK4 and MPK6 have been identified, the picture is still far from complete. To identify substrates of these MAPKs likely involved in cell division, growth and development we compared the phosphoproteomes of wild-type and mpk3, mpk4 and mpk6. To study the function of these MAPKs in innate immunity, we analyzed their phosphoproteomes following microbe-associated molecular pattern (MAMP) treatment. Partially overlapping substrates were retrieved for all three MAPKs, showing target specificity to one, two or all three MAPKs in different biological processes. More precisely, our results illustrate the fact that the entity to be defined as a specific or a shared substrate for MAPKs is not a phosphoprotein but a particular (S/T)P phosphorylation site in a given protein. As a whole, 152 peptides were identified to be differentially phosphorylated in response to MAMP treatment and/or when compared between genotypes and 70 of them could be classified as putative MAPK targets. Biochemical analysis of a number of putative MAPK substrates by phosphorylation and interaction assays confirmed the global phosphoproteome approach. Our study also expands the set of MAPK substrates to involve other protein kinases, including calcium-dependent (CDPK) and sugar non-fermenting (SnRK) protein kinases.
This work was supported by the Agence Nationale de la Recherche (ANR-2010-JCJC-1608 to D. P.) and by the King Abdullah University of Science and Technology. The IPS2 benefits from the support of the LabEx Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS). We would like to thank Veronique Legros and Huoming Zhang for technical assistance in MS, and Christophe Bruley, Véronique Dupierris and Emmanuelle Lastrucci (EDyP) for (83)informatics support. We would like to thank Carlos Xavier Pita and Heno Hwang from KAUST for scientific Illustrations.