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. 2007 Aug;13(2):177-89.
doi: 10.1016/j.devcel.2007.06.009.

An essential role for 14-3-3 proteins in brassinosteroid signal transduction in Arabidopsis

Srinivas S Gampala  1 , Tae-Wuk Kim, Jun-Xian He, Wenqiang Tang, Zhiping Deng, Mingyi-Yi Bai, Shenheng Guan, Sylvie Lalonde, Ying Sun, Joshua M Gendron, Huanjing Chen, Nakako Shibagaki, Robert J Ferl, David Ehrhardt, Kang Chong, Alma L Burlingame, Zhi-Yong Wang
Affiliations

An essential role for 14-3-3 proteins in brassinosteroid signal transduction in Arabidopsis

Srinivas S Gampala et al. Dev Cell. 2007 Aug.

Abstract

Brassinosteroids (BRs) are essential hormones for plant growth and development. BRs regulate gene expression by inducing dephosphorylation of two key transcription factors, BZR1 and BZR2/BES1, through a signal transduction pathway that involves cell-surface receptors (BRI1 and BAK1) and a GSK3 kinase (BIN2). How BR-regulated phosphorylation controls the activities of BZR1/BZR2 is not fully understood. Here, we show that BIN2-catalyzed phosphorylation of BZR1/BZR2 not only inhibits DNA binding, but also promotes binding to the 14-3-3 proteins. Mutations of a BIN2-phosphorylation site in BZR1 abolish 14-3-3 binding and lead to increased nuclear localization of BZR1 protein and enhanced BR responses in transgenic plants. Further, BR deficiency increases cytoplasmic localization, and BR treatment induces rapid nuclear localization of BZR1/BZR2. Thus, 14-3-3 binding is required for efficient inhibition of phosphorylated BR transcription factors, largely through cytoplasmic retention. This study demonstrates that multiple mechanisms are required for BR regulation of gene expression and plant growth.

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Figures

Figure 1
Figure 1. BR Induces Nuclear Localization of BZR1
(A) Effect of brassinazole (BRZ) and brassinolide (BL) on the subcellular localization of BZR1-YFP. Transgenic Arabidopsis expressing BZR1-YFP were grown on MS or MS + 2 μM BRZ medium in the dark for 4 days. Seedlings grown on BRZ medium were treated with mock or 100 nM BL for 1 hr and images of the YFP signal (top) were obtained using confocal microscopy. Numbers in each image show the average ratios between nuclear and cytoplasmic signal intensities and standard errors calculated from seven cells for each treatment. The white lines inside the images show the areas used for line scan measurements that yielded plot profiles shown in the lower panels. N, nuclear signal, C, cytoplasmic signal. Fluorescent intensity is 1000x and scale bar is 10 μm. (B) Kinetics of the BL-induced nuclear accumulation of BZR1-YFP. BZR1-YFP seedlings grown on 2 μM BRZ were treated with BL or mock solution (−BL), and images were acquired at each time point to measure the nuclear/cytoplasmic ratios. Error bars are ± standard errors of mean. (C) Unphosphorylated BZR1 is enriched in the nuclear fraction. Immunoblot of unphosphorylated and phosphorylated BZR1-CFP proteins in total and nuclear fractions from mock- or BL-treated seedlings. (D) Phosphorylated BZR1 is more enriched in membrane fractions than unphosphorylated BZR1. Transgenic plants expressing the BZR1-CFP protein were treated with (+BL) or without (−BL) 1 μM BL, and the soluble, microsomal (MF), and plasma membrane (PM) fractions were analyzed by immunoblot. (E) BIN2 phosphorylation inhibits DNA binding activity of BZR1. Gel blot of unphosphorylated and BIN2-phosphorylated MBP-BZR1 proteins was probed with radiolabeled CPD promoter DNA. CBB, Coomassie Brilliant Blue stained gel. Unphosphorylated BZR1 (BZR1) and phosphorylated BZR1 (pBZR1) proteins are marked by arrows (C-E).
Figure 2
Figure 2. BIN2-Phosphorylated BZR1 Interacts With 14-3-3 Proteins
(A) BZR1 interacts with 14-3-3λ in yeast two-hybrid assays. Each yeast clone contains the pGAD-14-3-3λ prey construct and one of the following genes in the pGBKT7 vector: 1, BZR1, 2, bzr1-1D, 3, BZR1C, 4, bzr1-1DC, 5, BIN2, 6, bin2-1, 7, BRI1-KD, 8, p53, 9, no gene insert. Growth of yeast cells indicates interaction between the test protein and 14-3-3λ. (B) 14-3-3λ specifically interacts with phosphorylated BZR1. Recombinant MBP-BZR1 protein was phosphorylated by GST-BIN2, gel blotted, and probed with GST-14-3-3λ and anti-GST antibody. Right panel shows the Coomassie Brilliant Blue (CBB) stained gel. Asterisks mark the unphosphorylated (BZR1) and phosphorylated (pBZR1) MBPBZR1 bands. Dagger sign (δ) marks the GST-BIN2 band. (C) BZR1 contains a putative 14-3-3 binding site. Two known 14-3-3 binding site sequences (Mode I and Mode II) are aligned against BZR1 sequence. ‘x’ represents any given amino acid. Amino acids are presented as single letters namely: R, Arginine; I, Isoleucine; pS, phosphoSerine; N, Asparagine; C, Cysteine; P, Proline. Conserved Serine residue at -3 position that's crucial for 14-3-3 binding was numbered and marked in bold. Various mutations created in the 14-3-3 binding sites are also shown. Hyphen (-) represents deletion of an amino acid residue. (D) Effects of mutations of the 14-3-3-binding site of BZR1 on the binding of 14-3-3λ. Wild type and mutant MBP-BZR1 proteins were phosphorylated by GST-BIN2 and then mixed with unphosphorylated proteins before pull-down assay (right panel) with glutathione agarose beads containing GST-14-3-3λ (lanes 1 to 4) or GST (lane 5). Lanes 1 to 5: MBP-BZR1, MBP-BZR1Δ, MBP-BZR1S173A, MBP-BZR1ΔI170, and MBPBZR1. (E) In vivo interaction between BZR1 and 14-3-3λ detected by BiFC. The N-terminal and C-terminal halves of YFP were fused to 14-3-3λ and BZR1, respectively, and the constructs were co-transformed into tobacco leaf cells and fluorescence images were obtained using confocal microscopy. Scale bar is 10 μm. (F) The BZR1-cYFP and the 14-3-3λ-nYFP constructs were co-transformed into tobacco cells with 1 μM BL. YFP fluorescence images were obtained using a confocal microscope. Scale bar is 10 μm. (G) Quantification of BiFC signals. Each pair of indicated constructs was co-transformed into tobacco cells with (+BL) or without (−BL) 1 μM BL. Fluorescence signal intensity (x1000) was average of measurements from at least 7 transformed cells after background subtraction. Error bars are ± standard error of mean. (H) Mutations in 14-3-3 binding site abolish in vivo interaction of BZR1 with 14-3-3s. Co-Immunoprecipitation (Co-IP) was carried using anti-GFP antibody coupled to Protein A Sepharose beads with tissue material from either BZR1-YFP or ΔBZR1-YFP transgenic plants. Western blot was probed with α-GFP and α-14-3-3 antibodies.
Figure 3
Figure 3. 14-3-3 Binding Inhibits BZR1 Activity
(A-D) Phenotypes of BZR1Δ and BZR1S173A transgenic plants compared with wild type Columbia (WT) and bzr1-1D. Three-week old plants (A) and leaves (B), five-week old plants (C) and bending of the stems at branch junction (pointed by arrows) (D). (E) BZR1Δ and BZR1S173A mutations suppress BR-deficiency phenotypes. Seedlings were grown in the dark on MS medium with or without 2 μM BRZ for 4 days. Two representative seedlings for each treatment are shown. (F) Mutations in BZR1 that reduce 14-3-3 binding suppress bri1-5 phenotype. Plants shown from left to right are bri1-5, bri1-5/BZR1-YFP, bri1-5/BZR1Δ-YFP, bri1-5/BZR1S173A-YFP and bri1-5/BZR1ΔI170-YFP. (G) BR biosynthesis genes are down regulated in BZR1Δ plants. Expression levels of CPD and DWF4 genes were measured by Quantitative Real Time PCR. The data was normalized first to UBC and then to Col. Error bars are ± standard errors of mean.
Figure 4
Figure 4. Mutations in 14-3-3 Binding Site Do Not Affect Stability or DNA Binding Activity of BZR1
(A) Immunoblot of BZR1-YFP, BZR1Δ-YFP, and BZR1S173A-YFP (two independent lines) proteins in the transgenic plants. BZR1-YFP was detected using a α-GFP antibody. (B) Mutations in 14-3-3 binding site do not alter the DNA binding activity of BZR1. Gel blot of unphosphorylated and BIN2-phosphorylated MBP-BZR1 proteins was probed with radio-labeled CPD promoter DNA. CBB, Coomassie Brilliant Blue stained gel.
Figure 5
Figure 5. 14-3-3 Binding Reduces Nuclear Localization of BZR1
(A-B) Subcellular localization of BZR1-, BZR1Δ-, BZR1S173A-, and BZR1ΔI170-YFP proteins in the leaves of transgenic Arabidopsis plants. Representative images of YFP fluorescence (A) and quantification of nuclear/cytoplasmic signal ratios (B) of three independent lines from BZR1-YFP and BZR1Δ-YFP are shown. Error bars are ± standard errors of mean. (C) BZR1Δ-CFP protein predominantly localizes in the nucleus. Pairs of indicated YFP and CFP fusion constructs were co-transformed into tobacco leaves with or without 1 μM BL. Images in CFP and YFP channels were false-colored as green and red, respectively, in overlay. Scale bar is 10 μm. (D) AICAR treatment decreases cytoplasmic localization of BZR1. Transgenic plants expressing BZR1-YFP were treated with either mock solution or 20 mM AICAR for 1 hr, and images were obtained using confocal microscope. Scale bar is 10 μM. Numbers in each image show the average ratios between nuclear and cytoplasmic signal intensities and standard errors calculated from seven cells for each treatment. (E) AICAR treatment down regulates BZR1-target gene expression. Transgenic plants expressing BZR1-YFP were treated with either mock solution or 20 mM AICAR for 3 hr and expression of CPD and DWF4 was measured using qRT-PCR.
Figure 6
Figure 6. BR Treatment and Mutation of 14-3-3 Binding Site Increase Nuclear Localization of BZR2/BES1
(A) 14-3-3λ interacts with BZR2 in yeast two-hybrid assays. pACT2 is the empty prey vector as a negative control. (B) BL treatment increases the nuclear localization of BZR2. BZR2-GFP transgenic seedlings were grown on media containing 2 μM BRZ or 10 nM BL in the dark for 4 days, and BZR2-GFP subcellular localization was visualized by confocal microscopy. Numbers in each image show the average ratios between nuclear and cytoplasmic signal intensities and standard errors calculated from seven cells for each treatment. Error bars are ± standard errors of mean. (C) YFP fusion constructs of BZR1, BZR1S173A, BZR2 or BZR2S171A were transiently expressed in tobacco leaves and subcellular localization of YFP was observed. Scale bar is 10 μm.
Figure 7
Figure 7. A Model for the BR Signaling Pathway in Arabidopsis
BR signaling involves a cell-surface receptor complex (BRI1/BAK1), a GSK3 kinase (BIN2), a phosphatase (BSU1) and two homologous transcription factors (BZR1 and BZR2/BES1). Green and red colors represent positive and negative functional roles in BR signaling, respectively. Arrows and bars represent actions of promotion and inhibition, respectively. In the absence of BR, BKI1 suppresses BRI1, and BIN2 phosphorylates and inhibits BZR1 and BZR2/BES1 (not shown). BR binds to the extracellular domain of BRI1 to activate its kinase, which leads to disassociation of BKI1 from BRI1 and dimerization with and activation of BAK1. Then, through an unknown mechanism, the activated receptor kinases inhibit BIN2 or activate BSU1, yielding dephosphorylated BZR1 and BZR2/BES1, which directly regulate BR-responsive gene expression. BIN2 phosphorylation negatively regulates BZR1 by increasing proteasomal degradation and BZR2/BES1 by inhibiting DNA binding. The current study shows that BR promotes nuclear localization of BZR1 and BZR2/BES1. BIN2 phosphorylation of BZR1 not only inhibits its DNA binding but also promotes its binding with 14-3-3 proteins, which is required for cytoplasmic retention and efficient inhibition of phosphorylated BZR1 in vivo. 14-3-3 proteins also regulate subcellular localization of BZR2/BES1. We propose that the large numbers of BIN2-phosphorylation sites in BZR1 and BZR2/BES1 allow BIN2 to inhibit BZR1/BZR2 through multiple mechanisms, conferring an efficient control of gene expression and plant growth by BR signaling.
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References

    1. Aitken A. 14-3-3 proteins: a historic overview. Semin Cancer Biol. 2006;16:162–172. - PubMed
    1. Bae MS, Cho EJ, Choi EY, Park OK. Analysis of the Arabidopsis nuclear proteome and its response to cold stress. Plant J. 2003;36:652–663. - PubMed
    1. Beck T, Hall MN. The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature. 1999;402:689–692. - PubMed
    1. Biggin MD. To bind or not to bind. Nat Genet. 2001;28:303–304. - PubMed
    1. Bridges D, Moorhead GB. 14-3-3 proteins: a number of functions for a numbered protein. Sci STKE. 2005;2005:re10. - PubMed

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