Isolation and characterization of a novel calcium/calmodulin-dependent protein kinase, AtCK, from arabidopsis.
Protein phosphorylation is one of the major mechanisms by which eukaryotic cells transduce extracellular signals into intracellular responses. Calcium/calmodulin (Ca(2+)/CaM)-dependent protein phosphorylation has been implicated in various cellular processes, yet little is known about Ca(2+)/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 Ca(2+)-binding domain. Recombinant AtCK was expressed in E. coli and bound to calmodulin in a Ca(2+)-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 Mn(2+). 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 Ca(2+)/CaM-dependent protein kinases. In contrast to maize and tobacco CCaMKs (calcium and Ca(2+)/CaM-dependent protein kinase), increasing the concentration of calmodulin to more than 3 microgram suppressed the phosphorylation activity of AtCK. Taken together our results indicate that AtCK is a novel Arabidopsis Ca(2+)/CaM-dependent protein kinase which is presumably involved in CaM-mediated signaling.
Jeong
Jae Cheol
JC
Division of Applied Life Sciences (BK21 program), Plant Molecular Biology & Biotechnology Research Center, Graduate School of Gyeongsang National University, Jinju 660-701, Korea.
Shin
Dongjin
D
Lee
Jiyoung
J
Kang
Chang Ho
CH
Baek
Dongwon
D
Cho
Moo Je
MJ
Kim
Min Chul
MC
Yun
Dae-Jin
DJ
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology*; D002118:Calcium / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D018076:DNA, Complementary / Q000302:isolation & purification; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation / Q000187:drug effects; D010802:Phylogeny; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity / Q000187:drug effects
D002147:Calmodulin; D018076:DNA, Complementary; D010455:Peptides; D008345:Manganese; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium