Abstract
The receptor kinase BRI1 (BRASSINOSTEROID-INSENSITIVE 1) is a key component in BR (brassinosteroid) perception and signal transduction, and has a broad impact on plant growth and development. In the present study, we demonstrate that Arabidopsis CaM (calmodulin) binds to the recombinant cytoplasmic domain of BRI1 in a Ca2+-dependent manner in vitro. In silico analysis predicted binding to Helix E of the BRI1 kinase subdomain VIa and a synthetic peptide based on this sequence interacted with Ca2+/CaM. Co-expression of CaM with the cytoplasmic domain of BRI1 in Escherichia coli strongly reduced autophosphorylation of BRI1, in particular on tyrosine residues, and also reduced the BRI1-mediated transphosphorylation of E. coli proteins on tyrosine, threonine and presumably serine residues. Several isoforms of CaM and CMLs (CaM-like proteins) were more effective (AtCaM6, AtCaM7 and AtCML8, where At is Arabidopsis thaliana) than others (AtCaM2, AtCaM4 and AtCML11) when co-expressed with BRI1 in E. coli. These results establish a novel assay for recombinant BRI1 transphosphorylation activity and collectively uncover a possible new link between Ca2+ and BR signalling.
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