Novel type of receptor-like protein kinase from a higher plant (Catharanthus roseus). cDNA, gene, intramolecular autophosphorylation, and identification of a threonine important for auto- and substrate phosphorylation.
We characterize CrRLK1, a novel type of receptor-like kinase (RLK), from the plant Catharanthus roseus (Madagascar periwinkle). The protein (90.2 kDa) deduced from the complete genomic and cDNA sequences is a RLK by predicting a N-terminal signal peptide, a large extracytoplasmic domain, a membrane-spanning hydrophobic region followed by a transfer-stop signal, and a C-terminal cytoplasmic protein kinase with all 11 conserved subdomains. It is a novel RLK type because the predicted extracytoplasmic region shares no similarity with other RLKs. The autophosphorylation was investigated with affinity-purified proteins expressed in Escherichia coli. The activity was higher with Mn2+ than with Mg2+ and achieved half-maximal rates at 2-2.5 microM ATP. The phosphorylation was predominantly on Thr, less on Ser, and not on Tyr. In contrast to other plant RLK, the kinase used an intra- rather than an intermolecular phosphorylation mechanism. After protein cleavage with formic acid, most of the radioactivity was in a 14.1-kDa peptide located at the end of the kinase domain. Mutagenesis of the four Thr residues in this peptide identified Thr-720 in the subdomain XI as important for autophosphorylation and for phosphorylation of beta-casein. This Thr is conserved in other related kinases, suggesting a subfamily sharing common autophosphorylation mechanisms.
Institut für Biologie II, Universität Freiburg, Schänzlestrasse 1, D-79104 Freiburg, Federal Republic of Germany.
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P
Schulze-Muth
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S
Irmler
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G
Schröder
J
J
Schröder
D000595:Amino Acid Sequence; D002478:Cells, Cultured; D018076:DNA, Complementary; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D010944:Plants / Q000201:enzymology*; D011494:Protein Kinases / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; D010940:Plant Proteins; D011494:Protein Kinases