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

Abstract

The rice disease resistance gene, Xa21, encodes a receptor kinase-like protein consisting of leucine-rich repeats in the putative extracellular domain and a serine/ threonine kinase in the putative intracellular domain. The putative XA21 kinase domain was expressed as maltose-binding and glutathione S-transferase fusion proteins in Escherichia coli. The fusion proteins are capable of autophosphorylation. Phosphoamino acid analysis of the glutathione S-transferase fusion protein indicates that only serine and threonine residues are phosphorylated. The relative phosphorylation rate of the XA21 kinase against increasing enzyme concentrations follows a first-order rather than second-order kinetics, indicating an intramolecular phosphorylation mechanism. Moreover, the active XA21 kinase cannot phosphorylate a kinase-deficient mutant of XA21 kinase. The enzymatic activity of the XA21 kinase in a buffer containing Mn 2؉ is at least 15 times higher than that with Mg 2؉ . The K m and V max of XA21 kinase for ATP are 0.3 M and 8.4 nmol/mg/min, respectively. Tryptic phosphopeptide mapping reveals that multiple sites on the XA21 kinase are phosphorylated. Finally, our data suggest that the region of XA21 kinase corresponding to the RD kinase activation domain is not phosphorylated, revealing a distinct mode of action compared with the tomato Pto serine/threonine kinase conferring disease resistance.

Consequences of Lysine 72 Mutation on the Phosphorylation and Activation State of cAMP-dependent Kinase

“…Furthermore, since exogenous wild type C-subunit does not phosphorylate Ser 338 in the K72H mutant and since the rate of phosphorylation of the C-subunit is concentration-independent, the autophosphorylation must be intramolecular or cis (39,40). Without a structure for the C-subunit in its unphosphorylated state, we can only speculate that Ser 338 either must be positioned close to the active site before phosphorylation at Thr 197 occurs or is freely mobile and capable of docking to the active site cleft.…”

Identification and Functional Analysis of in Vivo Phosphorylation Sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 Receptor Kinase

“…Although providing a very valuable initial database, the biological function of these phosphorylation events was not examined in this broad proteomic approach. Tryptic phosphopeptide mapping and site-directed mutagenesis of Ser and Thr residues has been used to study the phosphorylation status of several recombinant RLK kinase domains in vitro, but no analysis of specific in vivo phosphorylation sites was reported in these RLK studies (Schulze-Muth et al, 1996;Shah et al, 2001;Liu et al, 2002;Yoshida and Parniske, 2005).…”

Molecular Characterization of the Cytoplasmic Interacting Protein of the Receptor Kinase IRK Expressed in the Inflorescence and Root Apices ofArabidopsis

“…1) In the past decade, analysis of RLKs and related signaling pathways has proceeded steadily, revealing a few of the molecular components of the pathway. These studies have clarified the functions of various RLKs, including the roles of S-receptor kinase (SRK) in selfincompatibility, 5) CLV1, 1) and ER 6,7) in meristem development, XA21 in disease resistance, 8) Brassinosteroid insensitive 1 (BRI1) 9) and Phytosulfokine receptor (PSKR) 10) in hormone perception, and Nod-factor receptor kinase (NFR5) in nodule development. 11) More than 400 RLKs are thought to function in Arabidopsis, and a leucine-rich repeat (LRR)-type receptor group is known to be the largest family, comprising more than 200 members.…”