A receptor-like kinase from Arabidopsis thaliana is a calmodulin-binding protein.
Screening a cDNA expression library with a radiolabelled calmodulin (CaM) probe led to the isolation of AtCaMRLK, a receptor-like kinase (RLK) of Arabidopsis thaliana. AtCaMRLK polypeptide sequence shows a modular organization consisting of the four distinctive domains characteristic of receptor kinases: an amino terminal signal sequence, a domain containing seven leucine-rich repeats, a single putative membrane-spanning segment and a protein kinase domain. Using truncated versions of the protein and a synthetic peptide, we demonstrated that a region of 23 amino acids, located near the kinase domain of AtCaMRLK, binds CaM in a calcium-dependent manner. Real-time binding experiments showed that AtCaMRLK interacted in vitro with AtCaM1, a canonical CaM, but not with AtCaM8, a divergent isoform of the Ca2+ sensor. The bacterially expressed kinase domain of the protein was able to autophosphorylate and to phosphorylate the myelin basic protein, using Mn2+ preferentially to Mg2+ as an ion activator. Site-directed mutagenesis of the conserved lysine residue (Lys423) to alanine, in the kinase subdomain II, resulted in a complete loss of kinase activity. CaM had no influence on the autophosphorylation activity of AtCaMRLK. AtCaMRLK was expressed in reproductive and vegetative tissues of A. thaliana, except in leaves. Disruption in the AtCaMRLK coding sequence by insertion of a DsG transposable element in an Arabidopsis mutant did not generate a discernible phenotype. The CaM-binding motif of AtCaMRLK was found to be conserved in several other members of the plant RLK family, suggesting a role for Ca2+/CaM in the regulation of RLK-mediated pathways.
Charpenteau
Martine
M
UMR 5546 CNRS/Université Paul Sabatier, Pôle de Biotechnologie Végétale, BP 17 Auzeville, 31326 Castanet-Tolosan cedex, France.
Jaworski
Krzysztof
K
Ramirez
Bertha C
BC
Tretyn
Andrzej
A
Ranjeva
Raoul
R
Ranty
Benoît
B
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy; D024202:Electrophoretic Mobility Shift Assay; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008555:Melitten / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding / Q000187:drug effects; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020794:Receptor Protein-Tyrosine Kinases
D029681:Arabidopsis Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D018076:DNA, Complementary; D012333:RNA, Messenger; D008555:Melitten; D008345:Manganese; D011494:Protein Kinases; C489144:CaMRLK protein, Arabidopsis; D020794:Receptor Protein-Tyrosine Kinases; D008274:Magnesium; D002118:Calcium