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Novel MAP kinase substrates identified by solid-phase phosphorylation screening in Arabidopsis thaliana

Park, Hyeong Cheol, Nguyen, Xuan Canh, Bahk, Sunghwa, Park, Byung Ouk, Kim, Ho Soo, Kim, Min Chul, Bohnert, Hans J., Chung, Woo Sik
Plant biotechnology reports 2016 v.10 no.6 pp. 415-423
Arabidopsis thaliana, abiotic stress, bacteriophages, genes, messenger RNA, mitogen-activated protein kinase, phosphorylation, recombinant fusion proteins, screening, seedlings
Phosphorylation of substrate proteins by mitogen-activated protein kinases (MPKs) determines the specific cellular responses elicited by a particular extracellular stimulus. However, downstream targets of plant MPKs remain poorly characterized. In this study, 29 putative substrates of AtMPK3, AtMPK4 and AtMPK6 were identified by solid-phase phosphorylation screening of a λ phage expression library constructed from combined mRNAs from salt-treated, pathogen-treated and mechanically wounded Arabidopsis seedlings. To test the efficiency of this screening, we performed in vitro kinase assay with 10 recombinant fusion proteins. All proteins were phosphorylated by AtMPK3, AtMPK4 and AtMPK6, indicating the efficiency of this screening procedure. To confirm phosphorylation of isolated substrates by plant MPKs, we performed in-gel kinase assays. All test substrates were strongly phosphorylated by wounding or H₂O₂-activated AtMPK3 and AtMPK6. Three substrates, encoded by genes At2g41430, At2g41900, and At3g16770, were strongly phosphorylated, suggesting a function as AtMPK substrates. The type of screening provides a powerful way for identifying potential substrates of MAP kinases responsive to biotic and abiotic stresses.