Novel Type of Receptor-like Protein Kinase from a Higher Plant (Catharanthus roseus)

Abstract

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 Mn 2؉ than with Mg 2؉ and achieved half-maximal rates at 2-2.5 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 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 ␤-casein. This Thr is conserved in other related kinases, suggesting a subfamily sharing common autophosphorylation mechanisms. Animal receptor protein kinases (RPK)1 located in the membranes play an important role in the perception and transmittance of external signals. All RPK contain an extracellular domain connected by a membrane-spanning amino acid (AA) stretch to the intracellular kinase domain. The proteins can be subdivided into two groups that autophosphorylate on Ser/Thr or on Tyr residues, and oligomerization appears to play an important role in the regulation of enzyme activity (1).Several plant cDNAs and genes for RPK homologs sharing the same basic domain structure have been described. They were called receptor-like kinases (RLK), because the ligands are unknown and their function as receptors has not yet been demonstrated. Several groups are presently distinguished, and the classification is usually based on the sequences in the extracellular domain (reviewed in Ref.2): (a) the S-domain type, with similarities to the S-locus glycoproteins in Brassica; these proteins also contain ten or more cysteines in conserved positions; (b) the leucine-rich repeat type, with 9 -26 Leu-rich repeats; and (c) the epidermal growth factor (EGF)-like type that contains several EGF-like repeats. Very recent data suggest two additional types, with the extracellular region related either to plant defense proteins (3) or to lectins (4), but the investigations have not been extended much beyond the sequences. The physiological functions of plant RLKs are unknown, except for the S-domain type RLKs from Brassica that appear to be involved in the self-incompatibility phenotype (5-7).

Biochemical Characterization of the Kinase Domain of the Rice Disease Resistance Receptor-like Kinase XA21

“…For instance, the MBP-XA21K K m for ATP (0.3 M) is comparable with the values obtained for the epidermal growth factor receptor (0.2-3 M), the Catharanthus roseus CrRLK1 (2-2.5 M), and the Arabidopsis RLK5 kinases (15.2-17.8 M) (19,21). Similar to CrRLK1 and the Arabidopsis BRI1 kinases, the activity of XA21K requires Mn 2ϩ and Mg 2ϩ but not Ca 2ϩ (21,22). These results suggest that autophosphorylation of XA21K may be an important step in the XA21-mediated signaling.…”

The integral membrane S-locus receptor kinase of Brassica has serine/threonine kinase activity in a membranous environment and spontaneously forms oligomers in planta

“…The kinase domains of SRK (8,30) and several other RLKs (38)(39)(40)(41) have been shown to possess serine͞threonine kinase activity when expressed as soluble polypeptides in E. coli. Currently, the only exception among the plant RLK family is PRK1, a pollen-specific RLK from Petunia inflata, which has been shown to autophosphorylate on serine and tyrosine residues (42).…”

Recombinant Brassinosteroid Insensitive 1 Receptor-Like Kinase Autophosphorylates on Serine and Threonine Residues and Phosphorylates a Conserved Peptide Motif in Vitro

“…Two methods have been employed to study the mechanism of autophosphorylation in plant receptor kinases: incubation of active and mutant KDs with different molecular mass vector tags to allow differentiation by SDS-PAGE and examination of reaction order with respect to enzyme concentration. By using one or the other of these methods, it was shown that RLK4 (Coello et al, 1999), RLK5 , and OsTMK (van der Knaap et al, 1999) followed intermolecular autophosphorylation, whereas CrRLK1 exhibited intramolecular autophosphorylation (Schulze-Muth et al, 1996). We employed both methods to show that BRI1-KD, like CrRLK1, followed an intramolecular autophosphorylation mechanism.…”

“…Pairwise alignment of BRI1-KD with other receptor-like kinases did not reveal any obvious motif that would predict whether the kinases autophosphorylated primarily on Ser or Thr. For example, based on BLASTP alignment scores and sequence identity, CrRLK1 (Schulze-Muth et al, 1996) and TMK1 (Chang et al, 1992), which autophosphorylate predominantly on Thr, were as closely related to BRI1 as RLK5 and CLV1 (Stone et al, 1998), both of which have a preference for Ser. Nor was the type of extracellular domain any predictor of Ser versus Thr autophosphorylation, with both Leu-rich repeat and S locus-type glycoprotein domains occurring in receptor-like kinases that preferred either Ser or Thr.…”

“…To our knowledge, only one previous study has identified a specific site of autophosphorylation in a plant receptor-like kinase. Using formic acid digests and in vitro mutagenesis, it was shown that Thr-720 was required for autophosphorylation of CRLK1, a novel receptor-like kinase from Catharanthus roseus (Schulze-Muth et al, 1996). We used MALDI-MS analysis to show that FLAG-BRI1-KD autophosphorylates on at least 12 residues within the cytoplasmic domain.…”