Determination of primary sequence specificity of Arabidopsis MAPKs MPK3 and MPK6 leads to identification of new substrates

MAPKs (mitogen-activated protein kinases) are signalling components highly conserved among eukaryotes. Their diverse biological functions include cellular differentiation and responses to different extracellular stress stimuli. Although some substrates of MAPKs have been identified in plants, no information is availableaboutwhetheraminoacidsintheprimarysequenceotherthan proline-directed phosphorylation (pS-P) contribute to kinase specificity towards substrates. In the present study, we used a random positionalpeptidelibrarytosearchforconsensusphosphorylation sequences for Arabidopsis MAPKs MPK3 and MPK6. These experiments indicated a preference towards the sequence L/PP/X-S-P-R/K for both kinases. After bioinformatic processing, a number of novel candidate MAPK substrates were predicted and subsequentlyconfirmedbyinvitrokinaseassaysusingbacterially expressed native Arabidopsis proteins as substrates. MPK3 and MPK6phosphorylatedallproteinstestedmoreefficientlythandid another MAPK, MPK4. These results indicate that the amino acid residuesintheprimarysequencesurroundingthephosphorylation site of Arabidopsis MAPK substrates can contribute to MAPK specificity. Furthercharacterization ofoneofthesenewsubstrates confirmed that At1g80180.1 was phosphorylated in planta in a MAPK-dependent manner. Phenotypic analyses of Arabidopsis expressing phosphorylation site mutant forms of At1g80180.1 showedclusteredstomataandhigherstomatalindexincotyledons expressing the phosphomimetic form of At1g80180.1, providing a link between this new MAPK substrate and the defined role for MPK3 and MPK6 in stomatal patterning.