Identification of another calmodulin-binding domain at the C-terminal region of AtCBP63
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Calcium signals can be transduced by binding calmodulin (CaM), a Ca2+ sensor in eukaryotes, is known to be involved in the regulation of diverse cellular functions. We isolated a CaM-binding protein 63 kD (AtCBP63) from the pathogen-treated Arabidopsis cDNA expression library. Recently, AtCBP63 was identified as a CaM bining protein. The CaM binding domain of AtCBP63 was reported to be located in its N-terminal region. In this study, however, we showed that ACaM2 could specifically bind to second CaM-binding domain (CaMBD) of AtCBP63 at the C-terminal region. The specific binding of CaM to CaM binding domain was confirmed by a gel mobility shift assay, a split ubiquitin assay, site- directed mutagenesis, and a competition assay using a Ca2+/CaM-dependent enzyme. The gene expression of AtCBP63 was induced by pathogens and pathogens related second messengers. This result suggests that a CaM binding protein, AtCBP63, may play role in pathogen defense signaling pathway.
Abstract
Calcium signals can be transduced by binding calmodulin (CaM), a Ca2+ sensor in eukaryotes, is known to be involved in the regulation of diverse cellular functions. We isolated a CaM-binding protein 63 kD (AtCBP63) from the pathogen-treated Arabidopsis cDNA expression library. Recently, AtCBP63 was identified as a CaM bining protein. The CaM binding domain of AtCBP63 was reported to be located in its N-terminal region. In this study, however, we showed that ACaM2 could specifically bind to second CaM-binding domain (CaMBD) of AtCBP63 at the C-terminal region. The specific binding of CaM to CaM binding domain was confirmed by a gel mobility shift assay, a split ubiquitin assay, site- directed mutagenesis, and a competition assay using a Ca2+/CaM-dependent enzyme. The gene expression of AtCBP63 was induced by pathogens and pathogens related second messengers. This result suggests that a CaM binding protein, AtCBP63, may play role in pathogen defense signaling pathway.
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