The GSK3-like kinase BIN2 phosphorylates and destabilizes BZR1, a positive regulator of the brassinosteroid signaling pathway in Arabidopsis

Abstract

Brassinosteroids (BRs) are a class of steroid hormones essential for normal growth and development in plants. BR signaling involves the cell-surface receptor BRI1, the glycogen synthase kinase-3-like kinase BIN2 as a negative regulator, and nuclear proteins BZR1 and BZR2/BES1 as positive regulators. The interactions among these components remain unclear. Here we report that BRs induce dephosphorylation and accumulation of BZR1 protein. Experiments using a proteasome inhibitor, MG132, suggest that phosphorylation of BZR1 increases its degradation by the proteasome machinery. BIN2 directly interacts with BZR1 in yeast two-hybrid assays, phosphorylates BZR1 in vitro, and negatively regulates BZR1 protein accumulation in vivo. These results strongly suggest that BIN2 phosphorylates BZR1 and targets it for degradation and that BR signaling causes BZR1 dephosphorylation and accumulation by inhibiting BIN2 activity.

Figure 1

Immunoblot analysis of BR-induced dephosphorylation and accumulation of BZR1 protein. (A) 35S-BZR1-CFP and mBZR1-CFP transgenic plants grown in the dark for 5 days were treated with 1 μM BL for various times, and the BZR1-CFP proteins were analyzed by immunoblotting. (B) BZR1-CFP protein in BL-untreated sample was immunoprecipitated and treated with protein phosphatase. (C) Dark-grown BZR1-CFP (W) and mBZR1-CFP (m) plants were treated with BL or water for 1 h. (D) The 35S-BZR1-CFP plants were grown in light for 3 weeks, and the leaf tissues were treated with abscisic acid (ABA, 10 μM), gibberellic acid 3 (GA, 10 μM), cytokinin (BA, 10 μM), auxin (IAA, 10 μM), and BL (1 μM) for 1 h. (E and F) Leaf tissues of BZR1-CFP (E) or mBZR1-CFP (F) transgenic plants grown in light for 3 weeks were treated with 10 μM MG132, 1 μM BL, or 0.05% DMSO (solvent) for the time shown. (G) BZR1-CFP and mBZR1-CFP seedlings were grown in the dark for 5 days and pretreated with H₂O or 10 μM MG132 for 20 min before 1 μM BL was added. The samples were harvested at various time points of BL treatment. Control samples (0 min) were incubated in pretreatment solution for 30 min without BL. The protein samples were analyzed on 4–8% Nu-PAGE gels (A and B) or 7.5% Laemmli SDS/PAGE gels (C–G), blotted to nitrocellulose membranes, and probed with anti-GFP antibody.

Figure 2

BIN2 interacts with and phosphorylates BZR1. (A) BIN2 interacts with BZR1 in yeast. Yeast was transformed with a bait and a prey construct. The bait constructs contain GAL4-DNA binding domain fused with either wild-type BIN2, mutant bin2, or kinase domain of BRI1 and the prey constructs contain GAL4-activation domain fused with wild-type BZR1 or mutant bzr1 peptides (BZR1 and bzr1, amino acids 21–336; BZR1N, amino acids 21–104; BZR1C, amino acids 90–336). Interactions between each pair of test proteins were determined by selection for growth on histidine dropout (−His) medium and by 5-bromo-4-chloro-3-indolyl β-d-galactoside (X-Gal) assay. (B) In vitro kinase assays using GST-BIN2 (Upper) and GST-BRI1K (Lower) kinase proteins. (Left) Autoradiographs showing protein phosphorylation; (Right) the same gels stained with Coomassie brilliant blue for total protein. Asterisks mark the substrate protein bands. Arrows show phosphorylated MBP-BZR1 and autophosphorylated kinases. (C) In vitro kinase assay using 100 ng of GST-BIN2 and 20 ng of MBP-BZR1 in each reaction incubated for various times. Arrows show the partially (Lower) and completely (Upper) phosphorylated MBP-BZR1. (D) mBZR1-CFP protein was immunoprecipitated (IP) and then treated with a protein phosphatase (PP) or GST-BIN2 kinase for 1.5 h.

Figure 3

BIN2 negatively regulates BZR1 accumulation. (A) bzr1–1D is epistatic to bin2. Six-day-old dark-grown seedlings (Upper) and 6-week-old light-grown plants of wild-type, bzr1–1D, bin2, and bzr1–1D/bin2 double mutants. (B) Heterozygous bin2+/− mutant, heterozygous bin2+/− mutant containing the 35S-BZR1-CFP transgene (bin2/BZR1-ox), and wild-type (WT) plants were grown in light for 5 weeks. (C and D) Heterozygous bin2+/− plants were crossed with a homozygous 35S-BZR1-CFP line (C) or an mBZR1-CFP line (D). The wild-type BIN2+/+ and heterozygous bin2+/− F₁ plants were treated with 1 μM BL for the time shown and analyzed by immunoblotting using anti-GFP antibody.

Figure 4

A diagram of the BR signal transduction pathways. In the absence of BR, BRI1 is inactive, and BIN2 phosphorylates BZR1 and BZR2/BES1 and targets them for degradation by the proteasome. BR binding to the cell-surface receptor BRI1 activates BRI1 kinase and leads to inhibition of the BIN2 kinase, allowing dephosphorylation and accumulation of BZR1 and BZR2/BES1 proteins in the nucleus. BZR2/BES1 activates BR-induced genes and growth responses. BZR1 mediates BR-regulated gene expression to confer growth response and feedback regulation of BR biosynthesis.