Recombinant Brassinosteroid Insensitive 1 Receptor-Like Kinase Autophosphorylates on Serine and Threonine Residues and Phosphorylates a Conserved Peptide Motif in Vitro

Abstract

BRASSINOSTEROID-INSENSITIVE 1 (BRI1) encodes a putative Leucine-rich repeat receptor kinase in Arabidopsis that has been shown by genetic and molecular analysis to be a critical component of brassinosteroid signal transduction. In this study we examined some of the biochemical properties of the BRI1 kinase domain (BRI1-KD) in vitro, which might be important predictors of in vivo function. Recombinant BRI1-KD autophosphorylated on serine (Ser) and threonine (Thr) residues with p-Ser predominating. Matrix-assisted laser desorption/ionization mass spectrometry identified a minimum of 12 sites of autophosphorylation in the cytoplasmic domain of BRI1, including five in the juxtamembrane region (N-terminal to the catalytic KD), five in the KD (one each in sub-domains I and VIa and three in sub-domain VIII), and two in the carboxy terminal region. Five of the sites were uniquely identified (Ser-838, Thr-842, Thr-846, Ser-858, and Thr-872), whereas seven were localized on short peptides but remain ambiguous due to multiple Ser and/or Thr residues within these peptides. The inability of an active BRI1-KD to transphosphorylate an inactive mutant KD suggests that the mechanism of autophosphorylation is intramolecular. It is interesting that recombinant BRI1-KD was also found to phosphorylate certain synthetic peptides in vitro. To identify possible structural elements required for substrate recognition by BRI1-KD, a series of synthetic peptides were evaluated, indicating that optimum phosphorylation of the peptide required R or K residues at P Ϫ 3, P Ϫ 4, and P ϩ 5 (relative to the phosphorylated Ser at P ϭ 0).

RETRACTED: Autophosphorylation of Tyr-610 in the receptor kinase BAK1 plays a role in brassinosteroid signaling and basal defense gene expression

“…As previously reported (15), BAK1 can bind to and transphosphorylate BRI1 in vivo, and this is thought to be essential to enhance signaling output in planta. Importantly, both components of this functional interaction can be examined in vitro by monitoring the ability of BAK1 to (i) bind to BRI1 and (ii) increase the transphosphorylation activity of BRI1 using the BR13 synthetic peptide as substrate (15,25). As shown in Fig.…”

“…1A). The important conclusion is that BAK1 Tyr-610 phosphorylation is required to fully activate BRI1, which is recognized to be one of the important factors that influences signaling leading to plant growth (1,15,25,26). To further explore the molecular basis for the dwarf phenotype of the Y610F transgenic plants, we used ATH1 GeneChips (Affymetrix) to monitor relative transcript levels in plants expressing native sequence BAK1-Flag or BAK1(Y610F)-Flag in the bak1-4 bkk1-1 double mutant background.…”

PhosphoThr Peptide Binding Globally Rigidifies Much of the FHA Domain from Arabidopsis Receptor Kinase-Associated Protein Phosphatase,

“…The peptide, dubbed CLV1 pT868, comprises residues 863-875 of the Arabidopsis CLAVATA1 receptor-like kinase (RLK): PEYAY(pT)LKVDEKS. Multiple sequence alignment of kinase domains of RLKs indicates that the threonine that we phosphorylated in this CLV1 pT868 peptide corresponds to Ser1060 of the BRI1 RLK, one of two neighboring residues phosphorylated at the C-terminal end of the activation loop of kinases (66). This suggests pT868 of CLV1 might also be phosphorylated in vivo, making it the most likely of our KI-FHA-binding phosphopeptide candidates to be physiologically relevant.…”

The brassinosteroid signal transduction pathway

“…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].…”