Calcium/calmodulin inhibition of the Arabidopsis BRASSINOSTEROID-INSENSITIVE 1 receptor kinase provides a possible link between calcium and brassinosteroid signalling

摘要

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.Abbreviations: At, Arabidopsis thaliana; AtCaMRLK, Arabidopsis thaliana calmodulin-binding receptor-like kinase; BoSRK, Brassica oleracea S locus receptor kinase; BR, brassinosteroid; BRI1, BRASSINOSTEROID-INSENSITIVE 1; BAK1, BRI1-ASSOCIATED KINASE 1; CaM, calmodulin; CaMKII, Ca2+/CaM-dependent protein kinase II; CLV1, CLAVATA1; CML, CaM-like; CNGC2, cyclic nucleotide-gated channel 2; IMAC, immobilized metal-affinity chromatography; LC, liquid chromatography; LRR, leucine-rich repeat; MS/MS, tandem MS; Ni-NTA, Ni2+-nitrilotriacetate; RLK, receptor-like kinase; RU, response unit(s); SPR, surface plasmon resonance