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Mol. Cells 2007; 24(2): 276-282

Published online October 31, 2007

© The Korean Society for Molecular and Cellular Biology

Isolation and Characterization of a Novel Calcium/Calmodulin-Dependent Protein Kinase, AtCK, from Arabidopsis

Jae Cheol Jeong, Dongjin Shin, Jiyoung Lee, Chang Ho Kang, Dongwon Baek, Moo Je Cho, Min Chul Kim and Dae-Jin Yun

Abstract

Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin (Ca2+/CaM)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about Ca2+/CaM-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative Ca2+-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a Ca2+-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM Mn2+. The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other Ca2+/CaM-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and Ca2+/CaM-dependent protein kinase), increasing the concentration of calmodulin to more than 3 ?M suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis Ca2+/CaM-dependent protein kinase which is presumably involved in CaM-mediated signaling.

Keywords Arabidopsis thaliana; Calcium; Calmodulin; Protein Kinase; Signaling

Article

Research Article

Mol. Cells 2007; 24(2): 276-282

Published online October 31, 2007

Copyright © The Korean Society for Molecular and Cellular Biology.

Isolation and Characterization of a Novel Calcium/Calmodulin-Dependent Protein Kinase, AtCK, from Arabidopsis

Jae Cheol Jeong, Dongjin Shin, Jiyoung Lee, Chang Ho Kang, Dongwon Baek, Moo Je Cho, Min Chul Kim and Dae-Jin Yun

Abstract

Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin (Ca2+/CaM)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about Ca2+/CaM-dependent protein kinases (CaMKs) in plants. From an Arabidopsis expression library screen using a horseradish peroxidase-conjugated soybean calmodulin isoform (SCaM-1) as a probe, we isolated a full-length cDNA clone that encodes AtCK (Arabidopsis thaliana calcium/calmodulin-dependent protein kinase). The predicted structure of AtCK contains a serine/threonine protein kinase catalytic domain followed by a putative calmodulin-binding domain and a putative Ca2+-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a Ca2+-dependent manner. The ability of CaM to bind to AtCK was confirmed by gel mobility shift and competition assays. AtCK exhibited its highest levels of autophosphorylation in the presence of 3 mM Mn2+. The phosphorylation of myelin basic protein (MBP) by AtCK was enhanced when AtCK was under the control of calcium-bound CaM, as previously observed for other Ca2+/CaM-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and Ca2+/CaM-dependent protein kinase), increasing the concentration of calmodulin to more than 3 ?M suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis Ca2+/CaM-dependent protein kinase which is presumably involved in CaM-mediated signaling.

Keywords: Arabidopsis thaliana, Calcium, Calmodulin, Protein Kinase, Signaling

Mol. Cells
Aug 31, 2022 Vol.45 No.8, pp. 513~602
COVER PICTURE
Cryo-EM structure of human porphyrin transporter ABCB6 (main figure) shows that binding of hemin (inset, magenta) in concert with two glutathione molecules (cyan) primes ABCB6 for high ATP turnover (Kim et al., pp. 575-587).

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