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. 2007 Sep;19(9):2749-62.
doi: 10.1105/tpc.107.053728. Epub 2007 Sep 14.

Nucleocytoplasmic shuttling of BZR1 mediated by phosphorylation is essential in Arabidopsis brassinosteroid signaling

Hojin Ryu  1 , Kangmin Kim, Hyunwoo Cho, Joonghyuk Park, Sunghwa Choe, Ildoo Hwang
Affiliations

Nucleocytoplasmic shuttling of BZR1 mediated by phosphorylation is essential in Arabidopsis brassinosteroid signaling

Hojin Ryu et al. Plant Cell. 2007 Sep.

Abstract

Phytohormone brassinosteroids (BRs) play critical roles in plant growth and development. BR acts by modulating the phosphorylation status of two key transcriptional factors, BRI1 EMS SUPPRESSOR1 and BRASSINAZOLE RESISTANT1 (BZR1), through the action of BRASSINOSTEROID INSENSITIVE1/BRI1 ASSOCIATED RECEPTOR KINASE1 receptors and a GSK3 kinase, BRASSINOSTEROID INSENSITIVE2 (BIN2). It is still unknown how the perception of BR at the plasma membrane connects to the expression of BR target genes in the nucleus. We show here that BZR1 functions as a nucleocytoplasmic shuttling protein and GSK3-like kinases induce the nuclear export of BZR1 by modulating BZR1 interaction with the 14-3-3 proteins. BR-activated phosphatase mediates rapid nuclear localization of BZR1. Besides the phosphorylation domain for 14-3-3 binding, another phosphorylation domain in BZR1 is required for the BIN2-induced nuclear export of BZR1. Mutations of putative phosphorylation sites in two distinct domains enhance the nuclear retention of BZR1 and BR responses in transgenic plants. We propose that the spatial redistribution of BZR1 is critical for proper BR signaling in plant growth and development.

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Figures

Figure 1.
Figure 1.
Nucleocytoplasmic Distribution of BZR1 Negatively Follows BIN2 Activity. (A) Nuclear localization of BZR1-CFP is decreased in tissues in which BIN2 is actively expressed. The expression of BIN2 was monitored by the BIN2 promoter–driven GUS activity at the maturation (top panel) and elongation (bottom panel) zones of 2-week-old roots. Subcellular localization of BZR1-CFP in the epidermis and vasculature of the corresponding tissues was examined by optical sectioning using confocal laser scanning microscopy. The CFP signal at the epidermis of the root tip is also presented. Note that autofluorescence in wild-type plants (ecotype Columbia [Col-0]) was nearly undetected. Top, the top of the confocal layers; Middle, an internal layer with the vasculature. The scanning depth from the tissue surface was designated Z. (B) BZR1 is a multiphosphorylated and nucleocytoplasmic protein. The cytoplasmic and nuclear fractions from ProBZR1:BZR1-HA plants were separated by centrifugation. W, whole leaf extract; P, protoplast extract; N, nuclear fraction; C, cytosolic fraction. Histone H2B and RHA1 (Sohn et al., 2003) were used as nuclear and cytoplasmic markers, respectively. (C) BZR1-HA is localized in the nucleus and cytoplasm in protoplasts. The cytoplasmic and nuclear fractions from protoplasts transfected with the indicated genes were separated by centrifugation. The proteins were detected with an anti-HA antibody. N, nuclear fraction; C, cytosolic fraction. ARR2-HA and At MPK3-HA were used as nuclear and cytosolic markers, respectively. (D) The hyperphosphorylated BZR1 by BIN2 accumulates mainly in the cytoplasm. Protoplasts were transfected with BRI1-Myc/BZR1-HA or BIN2-Myc/BZR1-HA. The nuclear and cytosolic fractions were probed with an anti-HA antibody. ARR2-HA was cotransfected as a nuclear marker (Hwang and Sheen, 2001).
Figure 2.
Figure 2.
BIN2 Expression Leads to BZR1 Transport from the Nucleus to the Cytosol. (A) BIN2 increases the cytoplasmic localization of BZR1. BZR1-GFP was cotransfected into protoplasts with BIN2-HA, bin2-1-HA, or BIN2K69R-HA and examined by confocal microscopy. RFP-ARR2 was used as a nuclear marker. The fluorescent signal intensities of BZR1-GFP and RFP-ARR2 were determined along a line drawn on the confocal images using LSM Image Browser Rel. 4.0 software. (B) BIN2-HA expression is specifically induced by DEX in a time- and concentration-dependent manner. Rubisco large subunit (RbcL) was used as a loading control. (C) BIN2 induces the nuclear export of BZR1. Protoplasts were cotransfected with 8OP:BIN2-HA, Pro35S:VP16-GR, and BZR1-GFP. After incubation for 4 h to express GFP-tagged BZR1, BIN2 expression was induced by DEX treatment. 0 hr, time of DEX induction. (D) Leptomycin B (LMB) blocks the BIN2-mediated nuclear export of BZR1. Protoplasts were transfected with the plasmids and incubated for 4 h as described for (C). The transformed protoplasts were treated with leptomycin B for 1 h and then further treated with DEX.
Figure 3.
Figure 3.
Nuclear Translocation of BZR1 Is Induced by BR-Activated PP2A Phosphatase. (A) BR induces the nuclear translocation of BZR1. BZR1-GFP was cotransfected with BRI1-HA and BIN2-HA into protoplasts and expressed for 5 h. The protoplasts were then treated with epi-BL for 2 h. 0 hr, time of BR treatment. Note that BRI1 expression facilitates the BR-induced nuclear localization of BZR1. (B) The expression levels of BRI1-HA and BIN2-HA were similar in transformed cells. (C) BRI1 facilitates the BR-mediated dephosphorylation of BZR1 in protoplasts. HA-tagged BES1 and BZR1 were cotransfected into protoplasts with the indicated amounts of BRI1-HA and 4 μg of BIN2-HA. After 4 h of transfection, the protoplasts were incubated with cycloheximide for 30 min before treatment with BR for 1 h. (D) Okadaic acid, a PP2A phosphatase inhibitor, blocks BR-induced nuclear translocation of BZR1. Protoplasts were transfected with BIN2-HA and BZR1-GFP and incubated for 5 h to express the effectors. The transfected protoplasts were treated with 1 μM epi-BL alone and 10 or 30 nM okadaic acid (OA) together for 3 h. (E) BSU1 accelerates the BR-induced dephosphorylation of BZR1. After incubation for 4 h, protoplasts transfected with BSU-HA or BSU1-YFP and BZR1-HA were treated with or without epi-BL for 1 h in the presence of cycloheximide. BSU-HA and BZR1-HA proteins were detected using an anti-HA antibody. (F) BSU1 enhances nuclear localization. Four hours after transfection, protoplasts expressing BSU1-HA, BIN2-HA, and BZR1-GFP were incubated without or with epi-BL for 1 h in the presence of cycloheximide. Subcellular localization of BZR1-GFP was followed by fluorescence microscopy.
Figure 4.
Figure 4.
Identification of Putative Phosphorylation Residues Required for Nuclear Export of BZR1 by BIN2. (A) Ten putative phosphorylation residues in two distinct domains, marked by asterisks, are involved in BIN2-medated cytoplasmic translocation of BZR1. The Ser-130/Ser-134 and Ser-173/Thr-177 residues in red were selected in this study as representatives for the first and second domains, respectively. (B) Mutations of putative phosphorylation sites blocked the nuclear export of BZR1. BIN2 was cotransfected into protoplasts with BZR1-GFP or mutated BZR1-GFP as indicated. (C) Mutations of putative phosphorylation sites in BZR1 partly abolished BIN2-mediated phosphorylation. GFP-tagged BZR1 or its mutants were expressed in protoplasts with or without BIN2 proteins. Protein expression patterns of BZR1 and its mutants were analyzed with an anti-GFP antibody. BIN2 expression was probed with an anti-HA antibody.
Figure 5.
Figure 5.
14-3-3 Proteins Interact with BZR1 and Prevent BZR1 from Being Constitutively Active by Mediating Nuclear Export. (A) Putative 14-3-3 binding site of BZR1. Two 14-3-3 binding site sequences (mode I and mode II) are aligned against the BZR1 sequence. X, any given amino acid; Y, Tyr; R, Arg; I, Ile; pS, phospho-Ser; N, Asn; C, Cys; P, Pro; –, absence of an amino acid residue. (B) BZR1 interacts with the 14-3-3 proteins in yeast two-hybrid assays. BZR1 and 14-3-3κ/14-3-3ɛ were cloned into pGADT7 and pGBKT7 vectors, respectively. BIN2 was used for a positive interaction control. Transformed yeast cells were examined for interaction on synthetic medium lacking Leu, Trp, and His with 3 mM 3-aminotriazole. (C) Mutations at Ser-173 and Thr-177 of the 14-3-3 binding site of BZR1 abolish in vivo interaction with 14-3-3κ and 14-3-3ɛ proteins. HA-tagged BZR1, BZR1S173A, or BZR1T177A was cotransfected into protoplasts with myc-tagged 14-3-3κ or 14-3-3ɛ. 14-3-3 proteins were immunoprecipitated with anti-myc monoclonal antibody, and BZR1 proteins were detected with an anti-HA antibody. The asterisk and arrowheads indicate nonspecific bands and 14-3-3-myc, respectively.
Figure 6.
Figure 6.
Mutations of Putative Phosphorylation Residues Enhance BR Responses in Transgenic Plants. (A) BZR1S130/134A, BZR1S173A, and BZR1T177A mutations suppress the BR-deficiency phenotype. Transgenic seedlings harboring HA-tagged bzr1-1D, BZR1S130/134A, BZR1S173A, or BZR1T177A driven by the 35S promoter display a BRZ-resistant phenotype similar to that of the bzr1-1D mutant. Seedlings were grown without or with BRZ (top panel) for 5 d in the dark, and the hypocotyl length of each seedling was measured. Error bars indicate sd (n = 10). (B) The BZR1 mutations result in increased BR sensitivity in hypocotyl elongation. Transgenic lines that overexpress BZR1S130/134A-HA, BZR1S173A-HA, or BZR1T177A-HA show BR hypersensitivity similar to the Pro35S:bzr1-1D and ProBRI1:BRI1-GFP plants. The bri1-5 mutant is insensitive to BR. Seedlings were grown on Murashige and Skoog medium containing various concentration of epi-BL for 6 d, and the hypocotyl length of each seedling was measured. Error bars indicate sd (n = 10). (C) Phenotypes of mutated BZR1 transgenic leaves (top panel) and plants (bottom panel) grown for 3 weeks under long-day conditions in soil. The mutants show BR-response phenotypes similar to that of the bzr1-1D mutant. (D) The BZR1T177A mutation abolishes the cytoplasmic localization of hyperphosphorylated BZR1. Subcellular fractionation was performed with protoplasts isolated from Pro35S:BZR1-HA and Pro35S:BZR1T177A-HA transgenic lines. Note that the ectopic expression of BZR1 does not affect its subcellular distribution in the transgenic line. W, whole leaf extract; P, protoplast extract; N, nuclear fraction; C, cytosolic fraction. Histone H2B and RHA1 were used as the nuclear and cytoplasmic markers, respectively. (E) The BZR1T177A mutation in the 14-3-3 binding site does not affect the DNA binding capacity of BZR1. ChIP assays of the CPD promoter were performed with BZR1-HA or BZR1T177A-HA transgenic plants using anti-HA monoclonal antibody. The specific primer set (He et al., 2005) was used to analyze the CPD promoter bound to the proteins. (F) Expression of BR biosynthesis genes is suppressed in BZR1S130/134A-HA, BZR1S173A-HA, and BZR1T177A-HA transgenic lines. The transcript levels of CPD and DWF4 were quantified using quantitative real-time PCR. UBQ10 was used for normalization of the data. Error bars indicate sd (n = 3).
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