Plant MAPK phosphatase interacts with calmodulins.
A mitogen-activated protein kinase (MAPK) phosphatase gene, designated NtMKP1, was isolated as a candidate gene for a calmodulin (CaM)-binding protein from tobacco. NtMKP1 protein has four characteristic domains conserved among plant MAPK phosphatases reported so far, namely a dual specificity protein phosphatase catalytic domain, gelsolin-like domain, putative CaM-binding domain (CaMBD), and serine-rich region, indicating that NtMKP1 is the ortholog of Arabidopsis MKP1. The bacterially expressed NtMKP1 protein physically interacted with three plant-specific types of CaM in an overlay assay with labeled CaMs, showing high affinity to NtCaM1 and NtCaM3 but lower affinity to NtCaM13. The peptide for the putative CaMBD bound both NtCaM1 and NtCaM3 significantly but bound NtCaM13 only slightly. Moreover, CaM overlay assays with mutated CaMBDs revealed that Trp440 and Leu443 in the middle of the basic amphiphilic alpha-helix motif (amino acids 436-453) are critical for binding CaM. In comparison with the transient accumulation of a wound-induced MAPK, WIPK transcript, a prolonged activation of NtMKP1 expression was found in response to wounding and tobacco mosaic virus-induced hypersensitive reaction. In transgenic tobacco plants overexpressing NtMKP1, wound-induced activation of SIPK, salicylic acid-induced MAPK, and WIPK was inhibited. These results suggest that plant CaMs are involved in these stress-activated MAPK cascades via NtMKP1.
Plant Physiology Department, National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
Hiromoto
H
Yamakawa
Shinpei
S
Katou
Shigemi
S
Seo
Ichiro
I
Mitsuhara
Hiroshi
H
Kamada
Yuko
Y
Ohashi
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002118:Calcium / Q000737:chemistry; D002147:Calmodulin / Q000378:metabolism*; D020134:Catalytic Domain; D016923:Cell Death; D018076:DNA, Complementary / Q000378:metabolism; D054638:Dual Specificity Phosphatase 1; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism; D018260:Gelsolin / Q000737:chemistry; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D020935:MAP Kinase Signaling System; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry; D010944:Plants / Q000378:metabolism; D030821:Plants, Genetically Modified; D011485:Protein Binding; D054645:Protein Phosphatase 1; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014026:Nicotiana / Q000378:metabolism; D019076:Transgenes
D000596:Amino Acids; D002147:Calmodulin; D018076:DNA, Complementary; D018260:Gelsolin; D010455:Peptides; D010940:Plant Proteins; D011994:Recombinant Proteins; D012694:Serine; D005982:Glutathione Transferase; D054645:Protein Phosphatase 1; D054638:Dual Specificity Phosphatase 1; D017027:Protein Tyrosine Phosphatases; D002118:Calcium