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 mu M 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 Tvas 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.