Tyrosine phosphorylation of the BRI1 receptor kinase emerges as a component of brassinosteroid signaling in Arabidopsis

Abstract

Brassinosteroids (BRs) are essential growth-promoting hormones that regulate many aspects of plant growth and development. Two leucine-rich repeat receptor-like kinases (LRR-RLKs) are involved in BR perception and signal transduction: brassinosteroid insensitive 1 (BRI1), which is the BR receptor, and its coreceptor BRI1-associated kinase 1 (BAK1). Both proteins are classified as serine/threonine protein kinases, but here we report that recombinant cytoplasmic domains of BRI1 and BAK1 also autophosphorylate on tyrosine residues and thus are dual-specificity kinases. With BRI1, Tyr-831 and Tyr-956 are identified as autophosphorylation sites in vitro and in vivo. Interestingly, Tyr-956 in kinase subdomain V is essential for activity, because the Y956F mutant is catalytically inactive and thus this site cannot be simply manipulated by mutagenesis. In contrast, Tyr-831 in the juxtamembrane domain is not essential for kinase activity but plays an important role in BR signaling in vivo, because expression of BRI1(Y831F)-Flag in transgenic bri1-5 plants results in plants with larger leaves (but altered leaf shape) and early flowering relative to plants expressing wild-type BRI1-Flag. Acidic substitutions of Tyr-831 restored normal leaf size (but not shape) and normal flowering time. This is an example where a specific tyrosine residue has been shown to play an important role in vivo in plant receptor kinase function. Interestingly, 6 additional LRR-RLKs (of the 23 tested) were also found to autophosphorylate on tyrosine in addition to serine and threonine, suggesting that tyrosine signaling should be considered with other plant receptor kinases as well.

Fig. 1.

BRI1 is phosphorylated on tyrosine residues in vitro and in vivo. (A) Detection of phosphotyrosine residues in recombinant BRI1 and BAK1 CDs by immunoblotting with antiphosphotyrosine (pY) antibodies. Preincubation of the antibodies with pY-BSA eliminated the immunoblot signal indicating specificity of the detection. Protein abundance was visualized by Coomassie Brilliant Blue (CBB) staining. (B) The kinase-inactive K911E mutant, mBRI1 (6), does not autophosphorylate as indicated by the lack of immunoblot signal with antiphosphotyrosine (pY) antibodies, antiphosphothreonine (pT) antibodies, or staining with Pro-Q Diamond phosphoprotein stain. (C) Treatment of the recombinant BRI1-CD with recombinant PTP1B dephosphosphorylates tyrosine residues without substantially reducing phosphorylation on serine/threonine residues, as indicated by staining with Pro-Q Diamond phosphoprotein stain. (D) Phosphorylation of BRI1-Flag on tyrosine residues in vivo. Nontransgenic wild-type plants served as a control to demonstrate specificity of the immunoprecipitation of BRI1-Flag from transgenic plants, and mBRI1-Flag served as a control to demonstrate specificity of the interaction with the antiphosphotyrosine antibodies.

Fig. 2.

Identification of tyrosine residues on BRI1 that affect autophosphorylation and transphosphorylation of a synthetic peptide substrate. (A) Effect of site-directed mutagenesis of tyrosine residues in the BRI1-CD on autophosphorylation of the recombinant protein. (B) Densitometry analysis of antiphosphotyrosine immunoblots similar to those shown in A. (C) Tyrosine mutagenesis inhibits transphosphorylation of the SP11 (5) synthetic peptide.

Fig. 3.

The JM domain is a positive regulator of BRI1 kinase activity and is required for tyrosine phosphorylation. (A) Schematic representation of the Flag-BRI1-CD and the deletion mutants used in these experiments. J, K, and C refer to the JM, kinase, and C-terminal domains, respectively, and the hatch marks identify the location of the 10 tyrosine residues in BRI1-CD. The shaded portion of the KD depicts the activation loop. (B) Autophosphorylation of the recombinant proteins shown in A as measured with Pro-Q Diamond stain and immunoblotting with antiphosphotyrosine (pY) or antiphosphothreonine (pT) antibodies. (C) Transphosphorylation activity of the recombinant full-length and truncated proteins using the SP11 synthetic peptide as substrate.

Fig. 4.

Identification of Tyr-831, Tyr-956, and Tyr-1072 as phosphorylation sites on BRI1 using modification-specific antibodies. (A) Immunoblot analysis of recombinant Flag-BRI1-CD expressed in E. coli. The kinase-inactive mBRI1, wild-type BRI1 (treated or not with PTP1B), and the site-directed mutants Y831F, Y956F, and Y1072F were affinity purified and analyzed by immunoblotting (IB) with the indicated antibodies. (B) Immunoblot analysis of full-length BRI1-Flag constructs expressed in transgenic Arabidopsis plants. Protein was immunoprecipitated (IP) from nontransgenic wild-type plants (Ws-2) and transgenic plants using immobilized anti-Flag antibodies, and the purified protein was analyzed by immunoblot analysis as in A. Seedlings were grown in liquid culture and treated with 10⁻⁷ M brassinolide for 2 h before harvest.

Fig. 5.

Effect of mutating Tyr-831 or Tyr-1052 in the BRI1-CD on rescue of the weak bri1–5 mutant. (A) Three independent transgenic lines (T2 generation) for each construct are shown after 30 days of growth under short days (8-h photoperiod). Immunoblotting with anti-Flag antibodies was used to demonstrate the level of transgenic protein in each line. (B) Tyr-831 plays an important role in BR signaling in vivo as revealed by rescue of some, but not all, growth abnormalities of the bri1–5 mutant. Plants were grown for 32 days under long day conditions (16-h photoperiod). (C) Impact of tyrosine mutations of BRI1-Flag on leaf area (cm²·rosette leaf⁻¹), leaf shape (L/D, length/width), and timing of transition from vegetative to reproductive growth of plants grown on a long day (16 h) photoperiod for 32 days. The bri1–5, BRI1-Flag, BRI1(Y831D/E)-Flag, and BRI1(Y1052F)-Flag plants involved 10 independent transgenic lines (10 plants total) except for BRI1(Y831F)-Flag, which involved 12 transgenic lines (24 plants total). Transgenic lines, all in the bri1–5 background, were used at the T2 stage and identical results were obtained with plants at the T1 stage (data not shown).