Rapid Heteromerization and Phosphorylation of Ligand-activated Plant Transmembrane Receptors and Their Associated Kinase BAK1

Abstract

In plants leucine-rich repeat receptor kinases (LRR-RKs) located at the plasma membrane play a pivotal role in the perception of extracellular signals. For two of these LRR-RKs, the brassinosteroid receptor BRI1 and the flagellin receptor FLS2, interaction with the LRR receptor-like kinase BAK1 (BRI1-associated receptor kinase 1) was shown to be required for signal transduction. Here we report that FLS2⅐BAK1 heteromerization occurs almost instantaneously after perception of the ligand, the flagellin-derived peptide flg22. Flg22 can induce formation of a stable FLS2⅐BAK1 complex in microsomal membrane preparations in vitro, and the kinase inhibitor K-252a does not prevent complex formation. A kinase dead version of BAK1 associates with FLS2 in a flg22-dependent manner but does not restore responsiveness to flg22 in cells of bak1 plants, demonstrating that kinase activity of BAK1 is essential for FLS2 signaling. Furthermore, using in vivo phospholabeling, we are able to detect de novo phosphorylation of both FLS2 and BAK1 within 15 s of stimulation with flg22. Similarly, brassinolide induces BAK1 phosphorylation within seconds. Other triggers of plant defense, such as bacterial EF-Tu and the endogenous AtPep1 likewise induce rapid formation of heterocomplexes consisting of de novo phosphorylated BAK1 and proteins representing the ligand-specific binding receptors EF-Tu receptor and Pep1 receptor 1, respectively. Thus, we propose that several LRR-RKs form tight complexes with BAK1 almost instantaneously after ligand binding and that the subsequent phosphorylation events are key initial steps in signal transduction.One of the central themes in cell biology is the sensing of extracellular chemical signals through cell surface receptors: How does the event of receptor-ligand interaction on the outside of the cell activate a signal transduction chain in the inside of the cell? In higher plants, the most prominent class of membrane receptors is formed by proteins with intracellular serine/ threonine-type protein kinases. These receptors account for ϳ2.5-4% of all proteins encoded by the genome of a plant (1). Despite their importance, there is still little experimental evidence on the molecular activation mechanisms of plant transmembrane receptor kinases. Current models are based on the precedent of animal receptor tyrosine kinases where ligand binding causes receptor tyrosine kinases to form homo-or hetero-oligomers, followed by transphosphorylation (2). In the case of the epidermal growth factor receptor, these phosphorylation events occur within 60 s of receptor activation (3).The best studied plant transmembrane receptor kinase is BRI1, the receptor for the brassinosteroid growth hormones (4). BRI1 is one of the 224 members of LRR-RKs 2 in Arabidopsis (5). Upon ligand binding BRI1 interacts with a second LRR receptor-like kinase named BAK1 (6, 7). Two further well characterized plant LRR-RKs are the flagellin receptor FLS2 (flagellin sensing 2) (8) and the EF-Tu receptor (EFR) (9). FLS2 perceives a generally conser...

Structural basis for recognition of an endogenous peptide by the plant receptor kinase PEPR1

“…Previous studies showed that AtPep1 binding induced PEPR1-BAK1 heteromerization [23,24]. However, in vitro reconstitution of an AtPep1-induced complex using purified proteins has not yet been reported.…”

“…Ca 2+ has been shown to be important for the AtPep/ PEPR signaling [21,22]. Similar to FLS2 and EFR, PEPR1 stably associates with BAK1 in response to treatment with AtPeps [23,24]. A number of studies suggest that PEPR-mediated immune responses serve to amplify PTI signaling [25][26][27][28] via the JA/ET (jasmonic acid-ethylene) and SA (salicylic acid) pathways [25,28].…”

Arabidopsis thaliana Pattern Recognition Receptors for Bacterial Elongation Factor Tu and Flagellin Can Be Combined to Form Functional Chimeric Receptors

“…EFR and FLS2, although detecting different ligands, share all functional aspects of receptor activation and signal output. Activation of both receptors occurs according to the address-message concept, presumably involving ligand-induced, conformational changes that allow heteromerization with co-receptors such as BAK1 (18,21,39,40). Thus, for substitutions with parts of FLS2 that fully retain elf binding, one would rather expect functionality of receptor activation to be retained as well.…”

“…Upon activation with their respective ligands, FLS2, EFR, and AtPEPR1 seem to form heteromeric complexes with the co-receptor BAK1. Thus, intriguingly, BAK1 appears to function in the transmembrane signaling of plant RLKs with very different signal output programs (17)(18)(19)(20).The receptor kinase EFR from A. thaliana responds to bacterial elongation factor Tu (EF-Tu) with the induction of defense and increased resistance (14, 21). Mutant plants lacking this perception system show increased susceptibility to infection by Agrobacterium tumefaciens.…”

“…Upon activation with their respective ligands, FLS2, EFR, and AtPEPR1 seem to form heteromeric complexes with the co-receptor BAK1. Thus, intriguingly, BAK1 appears to function in the transmembrane signaling of plant RLKs with very different signal output programs (17)(18)(19)(20).…”