Abstract
We studied the biochemical properties of a plant receptor-like kinase to gain insights into the regulatory mechanism of this largest class of plant kinases. SYMRK (symbiosis receptor kinase) is required for early signal transduction leading to plant root symbioses with nitrogen-fixing rhizobia and phosphate-acquiring arbuscular mycorrhizal fungi. Amino acid substitutions in positions critical for activity of other related kinases cause a nonsymbiotic plant phenotype, suggesting that SYMRK kinase activity is required for symbiosis. SYMRK is capable of intermolecular autophosphorylation. Nonphosphorylated SYMRK is less active than the phosphorylated version, suggesting the phosphorylation status of SYMRK determines its activity. Three Ser/Thr residues were identified as residues required for full kinase activation through targeted mutagenesis. Using quadrupole time-of-flight mass spectrometry analysis, two of these were confirmed to be phosphorylated in vitro. These crucial phosphorylation sites are conserved among various plant receptor-like kinases as well as animal Pelle/interleukin-1 receptor associated kinase. Despite the distinct domain architecture of receptor-like kinases versus Pelle/interleukin-1 receptor associated kinase, our results suggest the existence of conserved activation mechanisms.
Highlights
Cesses, such as clavata 1, brassinosteroid-insensitive 1 (BRI1), and erecta, and genes involved in plant defense, such as FLS2 and Xa21 [2, 3]
It is known that autophosphorylation of animal receptor tyrosine kinases, induced by dimerization or oligomerization upon ligand binding, leads to activation of their catalytic activity followed by signal transduction
A critical role for the kinase activity is suggested by several missense and nonsense point mutation alleles in the symbiosis receptor kinase (SYMRK) kinase domain from L. japonicus and P. sativum (Refs. 5 and 19 and Table I), which result in plants that cannot establish root nodule symbio
Summary
Plasmid Construction—cDNA encoding the entire SYMRK intracellular domain was amplified by PCR using the forward primer TGCCGCTACAGACAA and the reverse primer ATGCATTTACTATCTCGG with additional appropriate restriction endonuclease sites. The following combinations of restriction enzymes and vectors were used: SphI and SalI with pQE30 (Qiagen, Crawley, UK) for His-SYMRK; EcoRI and SalI with pET32a (Novagen, Nottingham, UK) for TRX-SYMRK; EcoRI and SalI with pGEX4T-1 (Amersham Biosciences) for GST-SYMRK. GST-tagged proteins were purified using glutathione-Sepharose 4B (Amersham Biosciences) according to the manufacturer’s instructions. The CIP-treated GST-SYMRK protein was incubated with glutathione-Sepharose (Amersham Biosciences) at 4 °C for 2 h. Candidate peptides with probability-based Mowse scores exceeding the threshold (p Ͻ 0.05), and indicating a significant or extensive homology, were referred to as “hits.” Protein scores were derived from peptide ion scores as a non-probabilistic basis for ranking proteins
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
