Abstract
Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology modeling revealed that the plant RLK contains structural features particular to this group of kinases, including the tyrosine gatekeeper and the N-terminal extension α-helix B. Functional analysis revealed the importance of these conserved features for kinase activity and suggests that kinase activity is essential for the biological role of LYK3 in the establishment of the root nodule nitrogen-fixing symbiosis with rhizobia bacteria. The kinase domain of LYK3 has dual serine/threonine and tyrosine specificity, and mass spectrometry analysis identified seven serine, eight threonine, and one tyrosine residue as autophosphorylation sites in vitro. Three activation loop serine/threonine residues are required for biological activity, and molecular dynamics simulations suggest that Thr-475 is the prototypical phosphorylated residue that interacts with the conserved arginine in the catalytic loop, whereas Ser-471 and Thr-472 may be secondary sites. A threonine in the juxtamembrane region and two threonines in the C-terminal lobe of the kinase domain are important for biological but not kinase activity. We present evidence that the structure-function similarities that we have identified between LYK3 and IRAK-4 may be more widely applicable to plant RLKs in general.
Highlights
The structure of the human interleukin-1 receptor-associated kinase-4 (IRAK-4) kinase has been solved and has revealed several novel features, including a Tyr gatekeeper in the catalytic pocket, an N-terminal helix B, and regulation by activation loop phosphorylation that resembles Tyr kinases [2, 3]
To determine whether kinase activity is required for the function of lysin motif domain-containing receptorlike kinase-3 (LYK3) in the development of root nodules in response to rhizobia bacteria, site-directed mutagenesis was used to create specific mutants that were tested for both kinase activity and nodulation activity
To check for correct subcellular localization, yellow fluorescent protein (YFP) fusions of the wild-type and mutant proteins were expressed in the N. benthamiana leaf expression system; all the proteins localized uniformly to a thin layer at the cell boundary, identical to the localization of a plasma membrane marker labeled with mCherry (PM mCherry)
Summary
The structure of the human IRAK-4 kinase has been solved and has revealed several novel features, including a Tyr gatekeeper in the catalytic pocket, an N-terminal helix B, and regulation by activation loop phosphorylation that resembles Tyr kinases [2, 3]. By functional analysis and by reference to animal receptor Tyr kinases [11], it has been suggested that the mechanism of activation of plant non-RD RLKs, such as Xa21, may be different from RD kinases, such as BRI1 and BAK1, where activation loop phosphorylation is important [8]. We are studying the perception of bacterial lipochitooligosaccharidic signals (Nod factors), which are important for establishing the nitrogen-fixing symbiosis between rhizobia bacteria and legume plants. We aimed to determine whether LYK3 kinase activity is essential for nodulation in M. truncatula and to identify regions of the kinase domain that are important for signal transduction. We further argue that this structural and functional homology extends to other members of the plant RLK/RLCK family
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