Extracellular leucine-rich repeats as a platform for receptor/coreceptor complex formation

Abstract

Receptor kinases with leucine-rich repeat (LRR) extracellular domains form the largest family of receptors in plants. In the few cases for which there is mechanistic information, ligand binding in the extracellular domain often triggers the recruitment of a LRRcoreceptor kinase. The current model proposes that this recruitment is mediated by their respective kinase domains. Here, we show that the extracellular LRR domain of BRI1-ASSOCIATED KINASE1 (BAK1), a coreceptor involved in the disparate processes of cell surface steroid signaling and immunity in plants, is critical for its association with specific ligand-binding LRR-containing receptors. The LRRs of BAK1 thus serve as a platform for the molecular assembly of signalcompetent receptors. We propose that this mechanism represents a paradigm for LRR receptor activation in plants. -5). Ligand perception at the cell surface by either BRI1 or FLS2 induces the subsequent recruitment of BAK1 to a ligand-bound receptor complex (6-10). This process triggers transphosphorylation at multiple serines and threonines of the respective kinase domains inside the cell (11-13). Perhaps because BRI1 is a long-lived protein that apparently cycles between the plasma membrane and endosomes (14), there are multiple mechanisms to maintain the kinase domain in a basal state. BRI1 kinase is auto-inhibited by its C-terminal tail (15), by auto-phosphorylation on threonine 872 (11), and by a protein, BRI1 KINASE INHIBITOR 1 (BKI1), which associates with BRI1's kinase domain (10, 16). BKI1 inhibits BR signaling by binding to the BRI1's kinase domain, thereby inhibiting the interaction between BRI1-and BAK1-kinase (10, 16). Upon ligand binding, BRI1 phosphorylates BKI1 on a tyrosine within its membrane-targeting region, which dissociates BKI1 from the cell membrane and targets it to the cytoplasm, where it is inactive (10). Dissociation of BKI1 from BRI1 allows formation of a stable BRI1-BAK1 complex that is competent to induce downstream signaling (17).The interplay between BRI1 and BAK1 kinase domains is further regulated by BAK1 autophosphorylation on tyrosine 610 (tyr-610), which is required to stimulate BRI1 kinase activity in vitro and for proper BR signaling in vivo (18). Of note, BAK1 tyr-610 phosphorylation is not required for flagellin response and it is possible that tyr-610 phosphorylation might be involved in the proper interaction with its cognate receptors. However, tyr-610 mutations affect only BRI1 kinase activation but not its interaction with BRI1 intracellular domain (18). Therefore, a critical unanswered question is how ligand-bound LRR-RKs selectively recruit BAK1. Here, we report that the LRR domain of BAK1 is required for its recruitment to a ligand-bound LRR-RK and allows the kinase domains to be in physical contact for subsequent reciprocal transphosphorylation. Furthermore, our data indicate that the extracellular domain (ECD) of BAK1 is critical for the high affinity formation of the correct receptor/coreceptor pair.

Cautionary Notes on the Use of C-Terminal BAK1 Fusion Proteins for Functional Studies

“…Strikingly, all BAK1 fusion proteins were fully functional for BR-mediated responses, as previously reported for BAK1-GFP, BAK1-YFP, BAK1-FLAG, and BAK1-CIT-RINE (Albrecht et al, 2008;Wang et al, 2008;Jaillais et al, 2011). In only one case did we find that BAK1-HA 3 was not fully complementing the hyporesponsiveness of the null bak1-4 mutant to BR in the root inhibition assay.…”

“…The recent elucidation of the crystal structure of the extracellular domain of BRI1 in complex with its ligand suggests that it may act as a platform for binding other proteins, such as the extracellular domain of BAK1 (Hothorn et al, 2011;Jaillais et al, 2011;She et al, 2011). BR binds to the extracellular domain of BRI1, which activates the BRI1 intracellular kinase domain leading to autophosphorylation and transphosphorylation of BAK1 on residues in its kinase domain.…”

Visualization of BRI1 and BAK1(SERK3) Membrane Receptor Heterooligomers during Brassinosteroid Signaling

“…Subsequently, BRI1 can transphosphorylate BKI1, leading to the release of the kinase inhibitor into the cytosol (Jaillais et al, 2011b). The dissociation of BKI1 in turn may enable the recruitment of BAK1(SERK3) into the BRI1 receptor complex Kim and Wang, 2010;Jaillais et al, 2011aJaillais et al, , 2011b. Via sequential transphosphorylation events within the BRI1-BAK1 (SERK3) heterooligomers, BRI1 eventually gains full kinase activity and downstream BR signaling is initiated Kim and Wang, 2010;Clouse, 2011;Jaillais et al, 2011a).…”

“…Current models of BR signaling assume that in the absence of ligands, BRI1 resides in a homodimeric configuration in the PM Kim and Wang, 2010;Jaillais et al, 2011a) and that a doublelock mechanism prevents aberrant signaling activity (Jaillais et al, 2011a;Wang et al, 2012). This inhibitory mechanism involves the BRI1 KINASE INHIBITOR1 (BKI1), which binds to the kinase domain of unliganded BRI1 receptors and thereby keeps the LRR-RLK inactive (Jaillais et al, 2011b).…”

“…In vitro, BKI1 precludes BRI1 from associating with BRI1-ASSOCIATED KINASE1 (BAK1; also known as SOMATIC EMBRYO-GENESIS RECEPTOR-LIKE KINASE3 [SERK3]; Wang and Chory, 2006;Jaillais et al, 2011b), another LRR-RLK required for BR signal transduction Nam and Li, 2002;Gou et al, 2012). Binding of BRs to the extracellular LRR domain of BRI1 homodimers is thought to result in conformational changes, which trigger basal BRI1 kinase activity and autophosphorylation Kim and Wang, 2010;Jaillais et al, 2011a). Subsequently, BRI1 can transphosphorylate BKI1, leading to the release of the kinase inhibitor into the cytosol (Jaillais et al, 2011b).…”