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

Abstract

Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used coimmunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis thaliana followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1. MS analysis also strongly suggested that an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo. We also identified four specific BAK1 autophosphorylation sites in vitro using LC/MS/MS. Sitedirected mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that the highly conserved activation loop residue T-1049 and either S-1044 or T-1045 were essential for kinase function in vitro and normal BRI1 signaling in planta. Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro. These findings are consistent with many aspects of the animal receptor kinase model in which ligand-dependent autophosphorylation of the activation loop generates a functional kinase, whereas phosphorylation of noncatalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates.

RGF1 INSENSITIVE 1 to 5, a group of LRR receptor-like kinases, are essential for the perception of root meristem growth factor 1 in Arabidopsis thaliana

“…The best characterized RLK receptor and co-receptor are probably BRI1 and BAK1, the receptor and co-receptor of BRs, respectively [35,36,57]. Phosphorylation of BRI1 and BAK1 can be induced rapidly [37,58]. In fact, the activation of BRI1 and BAK1 is via a sequential phosphorylation mechanism [37].…”

The brassinosteroid signal transduction pathway

“…However, overexpression of a dominant negative mutant form of BAK1 caused a severe dwarf phenotype similar to strong bri1 mutants, suggesting that BAK1 plays a major role in transducing the BR signal and the weak phenotype of bak1 mutant is likely due to redundant functions of its homologs [24]. In vivo interaction between BAK1 and BRI1 has been demonstrated by co-immunoprecipitation assays and fluorescence life time imaging microscopy [23][24][25], and it has been shown recently that BR treatment increases the BRI1-BAK1 interaction [21].…”

“…BR binding activates the BRI1 kinase and causes autophosphorylation of BRI1, as demonstrated by both mobility shift of BRI1 in SDS-PAGE and by increased signal detected by anti-phospho-serine/threonine antibodies in BR treated samples [15,21]. Many phosphorylation sites of BRI1 have recently been identified by mass spectrometry analysis of recombinant BRI1 expressed in E. coli or BRI1 immunoprecipitated from plant extracts [21,22].…”

“…Many phosphorylation sites of BRI1 have recently been identified by mass spectrometry analysis of recombinant BRI1 expressed in E. coli or BRI1 immunoprecipitated from plant extracts [21,22]. Mutation studies of the phosphorylation residues demonstrated that some of the phosphorylation events increase and some reduce the BRI1 activity, as measured by the ability of the mutated BRI1 protein to phosphorylate a synthetic substrate and to rescue a bri1 mutant [21]. For example, mutations of Ser or Thr residues in the activation loop and the juxtamembrane and C-terminal domains reduced the kinase activity, whereas mutation of T872A increased the kinase activity [21].…”

“…In this study, deletion of the C-terminal 40 amino acids (CT) of BRI1 was shown to increase receptor activity, as indicated by increased phosphorylation of BRI1 and growth response in transgenic plants expressing such truncated BRI1 receptor [29]. The inhibitory function of the CT region appears to be reduced by phosphorylation, because the kinase activity was increased by mutation of several Ser/Thr residues in this region to Asp, which likely mimics phosphorylation [29], and reduced by mutations of Ser/Thr to Ala, which prevent phosphorylation [21]. BRI1 was also shown to interact with itself in vivo and the interaction appears to be increased by BR, suggesting that BR binding stabilizes the BRI1 homodimer.…”