Journal of Plant Research

, Volume 126, Issue 6, pp 833–840 | Cite as

Phosphorylation of Arabidopsis thaliana MEKK1 via Ca2+ signaling as a part of the cold stress response

  • Tomoyuki Furuya
  • Daisuke Matsuoka
  • Takashi Nanmori
Regular Paper


The Arabidopsis mitogen activated protein kinase kinase kinase (MEKK1) plays an important role in stress signaling. However, little is known about the upstream pathways of MEKK1. This report describes the regulation of MEKK1 activity during cold signaling. Immunoprecipitated MEKK1 from cold-treated Arabidopsis seedlings showed elevated kinase activity towards mitogen activated protein kinase kinase2 (MKK2), one of the candidate MEKK1 substrates. To clarify how MEKK1 becomes active in response to cold stress signaling, MEKK1 phosphorylation was monitored by an enzyme extracted from the seedlings grown under cold stress with or without EGTA. MEKK1 was phosphorylated after cold stress, but EGTA inhibited the phosphorylation. MKK2 was also phosphorylated by the same extract, but only when EGTA was absent. These results suggested that Ca2+ signaling occurred upstream of the MEKK1–MKK2 pathway. Full-length MEKK1 showed almost no activity but MEKK1 without the N-terminal region (MEKK1 KD) that retained the kinase domain had a strong ability to phosphorylate MKK2, demonstrating the inhibitory role of the N-terminal region of MEKK1. In addition, MEKK1 was phosphorylated by calcium/calmodulin-regulated receptor-like kinase (CRLK1), which suggested that CRLK1 is one of candidates located upstream of MEKK1.


Activation Ca2+ signaling Cold EGTA Phosphorylation MAPKKK 


  1. Agell N, Bachs O, Rocamora N, Villalonga P (2002) Modulation of the Ras/Raf/MEK/ERK pathway by Ca2+, and calmodulin. Cell Signal 14:649–654. doi: 10.1016/S0898-6568(02)00007-4 PubMedCrossRefGoogle Scholar
  2. Asai T, Tena G, Plotnikova J, Willmann MR, Chiu WL, Gomez-Gomez L, Boller T, Ausubel FM, Sheen J (2002) MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415:977–983. doi: 10.1038/415977a PubMedCrossRefGoogle Scholar
  3. Gao M, Liu J, Bi D, Zhang Z, Cheng F, Chen S, Zhang Y (2008) MEKK1, MKK1/MKK2 and MPK4 function together in a mitogen-activated protein kinase cascade to regulate innate immunity in plants. Cell Res 18:1190–1198. doi: 10.1038/cr.2008.300 PubMedCrossRefGoogle Scholar
  4. Hadiarto T, Nanmori T, Matsuoka D, Iwasaki T, Sato KI, Fukami Y, Azuma T, Yasuda T (2006) Activation of Arabidopsis MAPK kinase kinase (AtMEKK1) and induction of AtMEKK1–AtMEK1 pathway by wounding. Planta 223:708–713. doi: 10.1007/s00425-005-0126-7 PubMedCrossRefGoogle Scholar
  5. Hattori K, Naguro I, Runchel C, Ichijo H (2009) The roles of ASK family proteins in stress responses and diseases. Cell Commun Signal 7:9. doi: 10.1186/1478-811X-7-9 PubMedCrossRefGoogle Scholar
  6. Ichimura K, Mizoguchi T, Irie K, Morris P, Giraudat J, Matsumoto K, Shinozaki K (1998) Isolation of ATMEKK1 (a MAP kinase kinase kinase)-interacting proteins and analysis of a MAP kinase cascade in Arabidopsis. Biochem Biophys Res Commun 253:532–543. doi: 10.1006/bbrc.1998.9796 PubMedCrossRefGoogle Scholar
  7. Ichimura K, Mizoguchi T, Yoshida R, Yuasa T, Shinozaki K (2000) Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6. Plant J 24:655–665. doi: 10.1046/j.1365-313x.2000.00913.x PubMedCrossRefGoogle Scholar
  8. Ichimura K, Casais C, Peck SC, Shinozaki K, Shirasu K (2006) MEKK1 is required for MPK4 activation and regulates tissue-specific and temperature-dependent cell death in Arabidopsis. J Biol Chem 281:36969–36976. doi: 10.1074/jbc.M605319200 PubMedCrossRefGoogle Scholar
  9. Knight H, Trewavas AJ, Knight MR (1996) Cold calcium signaling in Arabidopsis involves two cellular pools and a change in calcium signature after acclimation. Plant Cell 8:489–503. doi: 10.1105/tpc.8.3.489 PubMedGoogle Scholar
  10. MAPK Group (2002) Mitogen-activated protein kinase cascades in plants: a new nomenclature. Trends Plant Sci 7:301–308. doi: 10.1016/S1360-1385(02)02302-6 CrossRefGoogle Scholar
  11. Matsuoka D, Nanmori T, Sato KI, Fukami Y, Kikkawa U, Yasuda T (2002) Activation of AtMEK1, an Arabidopsis mitogen-activated protein kinase kinase, in vitro and in vivo: analysis of active mutants expressed in E. coli and generation of the active form in stress response in seedlings. Plant J 29:637–647. doi: 10.1046/j.0960-7412.2001.01246.x PubMedCrossRefGoogle Scholar
  12. Mizoguchi T, Irie K, Hirayama T, Hayashida N, Yamaguchi-Shinozaki K, Matsumoto K, Shinozaki K (1996) A gene encoding a mitogen-activated protein kinase kinase kinase is induced simultaneously with genes for a mitogen-activated protein kinase and S6 ribosomal protein kinase by touch, cold, and water stress in Arabidopsis thaliana. Proc Natl Acad Sci USA 93:765–769. doi: 10.1073/pnas.93.2.765 PubMedCrossRefGoogle Scholar
  13. Mizoguchi T, Ichimura K, Irie K, Morris P, Giraudat J, Matsumoto K (1998) Shinozaki K (1998) Identification of a possible MAP kinase cascade in Arabidopsis thaliana based on pairwise yeast two-hybrid analysis and functional complementation tests of yeast mutants. FEBS Lett 437:56–60. doi: 10.1016/S0014-5793(98)01197-1 PubMedCrossRefGoogle Scholar
  14. Nakagami H, Pitzschke A, Hirt H (2005) Emerging MAP kinase pathways in plant stress signaling. Trends Plant Sci 10:339–346. doi: 10.1016/j.tplants.2005.05.009 PubMedCrossRefGoogle Scholar
  15. Nakagami H, Soukupová H, Schikora A, Zársky V, Hirt H (2006) A mitogen-activated protein kinase kinase kinase mediates reactive oxygen species homeostasis in Arabidopsis. J Biol Chem 281:38697–38704. doi: 10.1074/jbc.M605293200 PubMedCrossRefGoogle Scholar
  16. Qiu JL, Zhou L, Yun BW, Nielsen HB, Fiil BK, Petersen K, MacKinlay J, Loake GJ, Mundy J, Morris PC (2008) Arabidopsis mitogen-activated protein kinase kinases MKK1 and MKK2 have overlapping functions in defense signaling mediated by MEKK1, MPK4, and MKS1. Plant Physiol 148:212–222. doi: 10.1104/pp.108.120006 PubMedCrossRefGoogle Scholar
  17. Su S, Suarez MC, Krysan P (2007) Genetic interaction and phenotypic analysis of the Arabidopsis MAP kinase pathway mutations mekk1 and mpk4 suggests signaling pathway complexity. FEBS Lett 581:3171–3177. doi: 10.1016/j.febslet.2007.05.083 PubMedCrossRefGoogle Scholar
  18. Suarez-Rodriguez MC, Adams-Phillips L, Liu Y, Wang H, Su SH, Jester PJ, Zhang S, Bent AF, Krysan PJ (2007) MEKK1 is required for flg22-induced MPK4 activation in Arabidopsis plants. Plant Physiol 143:661–669. doi: 10.1104/pp.106.091389 PubMedCrossRefGoogle Scholar
  19. Takahashi Y, Soyano T, Sasabe M, Machida Y (2004) A MAP kinase cascade that controls plant cytokinesis. J Biochem 136:127–132. doi: 10.1093/jb/mvh118 PubMedCrossRefGoogle Scholar
  20. Teige M, Scheikl E, Eulgem T, Dóczi R, Ichimura K, Shinozaki K, Dangl JL, Hirt H (2004) The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis. Mol Cell 15:141–152. doi: 10.1016/j.molcel.2004.06.023 PubMedCrossRefGoogle Scholar
  21. Yang T, Chaudhuri S, Yang L, Du L, Poovaiah BW (2009) A calcium/calmodulin-regulated member of the receptor-like kinase family confers cold tolerance in plants. J Biol Chem 285:7119–7126. doi: 10.1074/jbc.M109.035659 PubMedCrossRefGoogle Scholar
  22. Yang T, Shad Ali G, Yang L, Du L, Reddy AS, Poovaiah BW (2010) Calcium/calmodulin-regulated receptor-like kinase CRLK1 interacts with MEKK1 in plants. Plant Signal Behav 5:991–994. doi: 10.4161/psb.5.8.12225 PubMedCrossRefGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer Japan 2013

Authors and Affiliations

  • Tomoyuki Furuya
    • 1
  • Daisuke Matsuoka
    • 1
  • Takashi Nanmori
    • 1
    • 2
  1. 1.Graduate School of Agricultural ScienceKobe UniversityKobeJapan
  2. 2.Research Center for Environmental Genomics, Organization of Advanced Science and TechnologyKobe UniversityKobeJapan

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