Functional importance of BAK1 tyrosine phosphorylation in vivo

Abstract

The plant receptor kinase BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) is known as a partner of several ligand-binding leucine-rich repeat receptor kinases, including BRASSINOSTEROID INSENSITIVE 1 (BRI1) and the flagellin receptor FLS2. Autophosphorylation of receptor kinases is recognized to be an important process in receptor kinase signaling, and at least with the recombinant protein, BAK1 was shown to autophosphorylate on Tyr residues in addition to numerous Ser/Thr residues documented previously. We recently identified Tyr-610 in the carboxy-terminal domain of BAK1 as a major site of autophosphorylation and showed that phosphorylation of this residue is essential for at least some functions of BAK1 in vivo. In particular, the function of BAK1 as co-receptor with BRI1 in brassinosteroid (BR) signaling is impaired in transgenic plants expressing the BAK1(Y610F)-Flag directed mutant. Recombinant cytoplasmic domains of BRI1 and BAK1 interact and transphosphorylate each other in vitro in a manner that mimics their interaction in vivo; while BAK1(Y610F) binds normally to BRI1 its ability to transphosphorylate and activate the kinase domain of BRI1 is severely compromised. To further elaborate on this earlier model, we present additional results showing that the interaction between BAK1 and BRI1 in vitro is Mg(2+) dependent, suggesting that cytosolic [Mg(2+)] may play some role in receptor kinase signaling in vivo. We also compare the primary structures of BRI1 and BAK1 in terms of the occurrence of Tyr residues in the cytoplasmic domain, and identify differences in which residues are essential for kinase activity. Finally, transgenic plants expressing the BAK1(Y610F) directed mutant have alterations in the transcriptome that extend beyond the genes that are BR regulated in nontransgenic plants. In particular, the basal expression of many defense genes is significantly reduced in Y610F plants, which is consistent with the earlier report in reference 4, that BAK1 controls the expression of a number of genes associated with microbial infection. The present results establish a site-specific role for Tyr phosphorylation of BAK1 in BR signaling and regulation of plant defense mechanisms, which may have implications for enhancing agricultural productivity.

Figure 1

Clustal W alignment of the cytoplasmic domains of BRI1 and BAK1 identifying Tyr residues essential for kinase activity or identified as sites of autophosphorylation. The predicted transmembrane domains are underlined and the juxtamembrane and carboxy-terminal domains are in blue. Tyrosine residues essential for kinase activity are shown in red, and identified sites of Tyr autophosphorylation are in bold italics and include BRI1 Tyr-831 and Tyr-956 and BAK1 Tyr-610. An in vitro site of SER K1 Tyr phosphorylation that was identified is shown projected onto the BAK1 sequence (underlined Tyr residue).

Figure 2

Rescue of the seedling-lethal phenotype of bak1 bkk1 double mutants by expression of wild type BAK1-Flag or directed mutants of Tyr-610. Plants expressing the BAK1(Y610F)-Flag directed mutant in the bak1 bkk1 double mutant background have reduced hypocotyl elongation and the phosphomimetic Y610E substantially increases hypocotyl growth. Measurements were made 5 days after germination. Values are means ± SEM; n = 3.

Figure 3

(A) Sequential transphosphorylations between BRI1 and BAK1 occur normally and are required for enhanced BR signaling in vivo leading to growth and other responses. (B) In the absence of phosphorylation of Tyr-610 of BAK1 (or in the Y610F directed mutant) transphosphorylation of BRI1 is diminished and phosphorylation of target sites is attenuated (light grey symbols), which results in reduced BR signaling and the dwarf phenotype observed for plants expressing the BAK1(Y610F) directed mutant. Basic concepts adapted from Wang et al.

Figure 4

Label-free binding of Flag-BRI1 cytoplasmic domain to immobilized GST-BAK1 cytoplasmic domain requires millimolar concentrations of free Mg²⁺. Binding was measured using the ForteBio Octet, which uses fiberoptic sensors to detect protein:protein interactions via biolayer interferometry.

Figure 5

Phosphorylation of Tyr-610 is (A) required for enhanced BR signaling and the basal expression of many defense genes, but (B) is not required for flg22-induced inhibition of seedling growth or inhibition of cell death. It remains to be determined whether other flg22-induced responses, such as pattern-triggered immunity (PTI), are affected by the phosphorylation status of Tyr-610. (C) Working model showing a link between BL signaling and plant defense involving Tyr-610 phosphorylation, which we speculate will also be impacted by plant protein tyrosine phosphatases (PTPs) and possibly bacterial effector proteins such as Pseudomonas HopAO1.