Regulation of MAPK phosphatase 1 (AtMKP1) by calmodulin in Arabidopsis.
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 Ca(2+)-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 Ca(2+)/CaM-dependent enzyme, and a split-ubiquitin assay. The peptides for two CaMBDs, CaMBDI and CaMBDII, bound CaM in a Ca(2+)-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, Trp(453) and Leu(456) in CaMBDI and Trp(678) and Ile(684) in CaMBDII, play a pivotal role in CaM binding. Moreover, the phosphatase activity of AtMKP1 was increased by CaM in a Ca(2+)-dependent manner. Our results suggest that two important signaling pathways, Ca(2+) 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.
Lee
Kyunghee
K
Division of Applied Life Science (BK21 Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Korea.
Song
Eun Hyeon
EH
Kim
Ho Soo
HS
Yoo
Jae Hyuk
JH
Han
Hay Ju
HJ
Jung
Mi Soon
MS
Lee
Sang Min
SM
Kim
Kyung Eun
KE
Kim
Min Chul
MC
Cho
Moo Je
MJ
Chung
Woo Sik
WS
D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D054638:Dual Specificity Phosphatase 1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015723:Gene Library; D020935:MAP Kinase Signaling System / Q000502:physiology*; D009154:Mutation; D012275:Oryza / Q000201:enzymology / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D014026:Nicotiana / Q000201:enzymology / Q000235:genetics
D002147:Calmodulin; D010455:Peptides; D048049:Extracellular Signal-Regulated MAP Kinases; D054638:Dual Specificity Phosphatase 1; D002118:Calcium