ORIGINAL PAPER Arabidopsis MAP kinase phosphatase 1 is phosphorylated and activated by its substrate AtMPK6 Hyeong Cheol Park • Eun Hyeon Song • Xuan Canh Nguyen • Kyunghee Lee • Kyung Eun Kim • Ho Soo Kim • Sang Min Lee • Sun Ho Kim • Dong Won Bae • Dae-Jin Yun • Woo Sik Chung Received: 4 January 2011 / Revised: 16 March 2011 / Accepted: 17 March 2011 / Published online: 1 April 2011 Ó Springer-Verlag 2011 Abstract Arabidopsis MAP kinase phosphatase 1 (At- MKP1) is a member of the mitogen-activated protein kinase (MPK) phosphatase family, which negatively reg- ulates AtMPKs. We have previously shown that AtMKP1 is regulated by calmodulin (CaM). Here, we examined the phosphorylation of AtMKP1 by its substrate AtMPK6. Intriguingly, AtMKP1 was phosphorylated by AtMPK6, one of AtMKP1 substrates. Four phosphorylation sites were identified by phosphoamino acid analysis, TiO 2 chromatography and mass spectrometric analysis. Site- directed mutation of these residues in AtMKP1 abolished the phosphorylation by AtMPK6. In addition, AtMKP1 interacted with AtMPK6 as demonstrated by the yeast two-hybrid system. Finally, the phosphatase activity of AtMKP1 increased approximately twofold following phosphorylation by AtMPK6. By in-gel kinase assays, we showed that AtMKP1 could be rapidly phosphorylated by AtMPK6 in plants. Our results suggest that the catalytic activity of AtMKP1 in plants can be regulated not only by Ca 2? /CaM, but also by its physiological substrate, AtMPK6. Keywords Arabidopsis Á Calmodulin Á Mitogen-activated protein kinase (MPK) Á Mitogen-activated protein kinase phosphatase (MKP) Á Phosphorylation Introduction In all organisms, a variety of signal transduction pathways are used to carry out developmental processes, obtain essential nutrients, control metabolism, and cope with environmental biotic and abiotic stimuli. Numerous signals are decoded through changes in the concentration of sec- ond messengers such as, for example, calcium, cGMP, cAMP, and cADP-R (Trewavas and Malho ´ 1997). The second messengers are transduced to various signal trans- duction pathways which regulate all processes in the cells. Of high importance among the categories of signaling pathways are the protein kinases which add phosphate groups on critical enzymes and regulatory proteins con- trolling cell activities. Among them, Mitogen-activated Protein Kinase (MPK) cascades are possibly the best- known and characterized signal transducers in yeast and animal cells (Widmann et al. 1999; Davis 2000). MPK cascades are composed of three kinase modules: a MAP kinase kinase kinase (MPKKK), a MAP kinase kinase (MKK), and a MAP kinase (MPK). MPKs are activated by the MKK via phosphorylation of the con- served T-X–Y consensus motif such that a dual- Communicated by J. R. Liu. H. C. Park, E. H. Song, and X. C. Nguyen contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s00299-011-1064-4) contains supplementary material, which is available to authorized users. H. C. Park Á E. H. Song Á X. C. Nguyen Á K. Lee Á K. E. Kim Á H. S. Kim Á S. M. Lee Á S. H. Kim Á D.-J. Yun Á W. S. Chung (&) Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660-701, Republic of Korea e-mail: chungws@gnu.ac.kr D. W. Bae Central Instrument Facility, Gyeongsang National University, Jinju 660-701, Republic of Korea 123 Plant Cell Rep (2011) 30:1523–1531 DOI 10.1007/s00299-011-1064-4