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J. Biol. Chem., Vol. 283, Issue 35, 23581-23588, August 29, 2008

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Regulation of MAPK Phosphatase 1 (AtMKP1) by Calmodulin in Arabidopsis^*

Kyunghee Lee¹², Eun Hyeon Song¹, Ho Soo Kim², Jae Hyuk Yoo, Hay Ju Han, Mi Soon Jung, Sang Min Lee², Kyung Eun Kim², Min Chul Kim, Moo Je Cho, and Woo Sik Chung³

From the Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center and the Environmental Biotechnology National Core Research Center, Gyeongsang National University, Jinju 660-701, Korea

The mitogen-activated protein kinases (MAPKs) are key signal transduction molecules, which respond to various external stimuli. The MAPK phosphatases (MKPs) are known to be negative regulators of MAPKs in eukaryotes. We screened an Arabidopsis cDNA library using horseradish peroxidase-conjugated calmodulin (CaM), and isolated AtMKP1 as a CaM-binding protein. Recently, tobacco NtMKP1 and rice OsMKP1, two orthologs of Arabidopsis AtMKP1, were reported to bind CaM via a single putative CaM binding domain (CaMBD). However, little is known about the regulation of phosphatase activity of plant MKP1s by CaM binding. In this study, we identified two Ca²⁺-dependent CaMBDs within AtMKP1. Specific binding of CaM to two different CaMBDs was verified using a gel mobility shift assay, a competition assay with a Ca²⁺/CaM-dependent enzyme, and a split-ubiquitin assay. The peptides for two CaMBDs, CaMBDI and CaMBDII, bound CaM in a Ca²⁺-dependent manner, and the binding affinity of CaMBDII was found to be higher than that of CaMBDI. CaM overlay assays using mutated CaMBDs showed that four amino acids, Trp⁴⁵³ and Leu⁴⁵⁶ in CaMBDI and Trp⁶⁷⁸ and Ile⁶⁸⁴ in CaMBDII, play a pivotal role in CaM binding. Moreover, the phosphatase activity of AtMKP1 was increased by CaM in a Ca²⁺-dependent manner. Our results suggest that two important signaling pathways, Ca²⁺ signaling and the MAPK signaling cascade, are connected in plants via the regulation of AtMKP1 activity. To our knowledge, this is the first report to show that the biochemical activity of MKP1 in plants is regulated by CaM.

Received for publication, February 26, 2008 , and in revised form, June 25, 2008.

^* This work was supported in part by the Plant Diversity Research Center of the 21st Century Frontier Research Program (Grant #PF06303-01) and the Environmental Biotechnology National Core Research Center (Grant #R15-2003-012-02003-0) funded by the Ministry of Education and Science Technology in Korea. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Supported by scholarships from the BK21 program of the Ministry of Education and Science Technology.

³ To whom correspondence should be addressed: Division of Applied Life Science (BK21 program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, 660-701, Korea. Tel.: 82-55-751-6254; Fax: 82-55-759-9363; E-mail: chungws{at}gnu.ac.kr.

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