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. 2002 Nov;130(3):1221-9.
doi: 10.1104/pp.102.010918.

Two putative BIN2 substrates are nuclear components of brassinosteroid signaling

Jun Zhao  1 , Peng Peng, Robert J Schmitz, Adria D Decker, Frans E Tax, Jianming Li
Affiliations

Two putative BIN2 substrates are nuclear components of brassinosteroid signaling

Jun Zhao et al. Plant Physiol. 2002 Nov.

Abstract

GSK3 is a highly conserved kinase that negatively regulates many cellular processes by phosphorylating a variety of protein substrates. BIN2 is a GSK3-like kinase in Arabidopsis that functions as a negative regulator of brassinosteroid (BR) signaling. It was proposed that BR signals, perceived by a membrane BR receptor complex that contains the leucine (Leu)-rich repeat receptor-like kinase BRI1, inactivate BIN2 to relieve its inhibitory effect on unknown downstream BR-signaling components. Using a yeast (Saccharomyces cerevisiae) two-hybrid approach, we discovered a potential BIN2 substrate that is identical to a recently identified BR-signaling protein, BES1. BES1 and its closest homolog, BZR1, which was also uncovered as a potential BR-signaling protein, display specific interactions with BIN2 in yeast. Both BES1 and BZR1 contain many copies of a conserved GSK3 phosphorylation site and can be phosphorylated by BIN2 in vitro via a novel GSK3 phosphorylation mechanism that is independent of a priming phosphorylation or a scaffold protein. Five independent bes1 alleles containing the same proline-233-Leu mutation were identified as semidominant suppressors of two different bri1 mutations. Over-expression of the wild-type BZR1 gene partially complemented bin2/+ mutants and resulted in a BRI1 overexpression phenotype in a BIN2(+) background, whereas overexpression of a mutated BZR1 gene containing the corresponding proline-234-Leu mutation rescued a weak bri1 mutation and led to a bes1-like phenotype. Confocal microscopic analysis indicated that both BES1 and BZR1 proteins were mainly localized in the nucleus. We propose that BES1/BZR1 are two nuclear components of BR signaling that are negatively regulated by BIN2 through a phosphorylation-initiated process.

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Figures

Figure 1
Figure 1
Identification of BES1 and BZR1 as potential BIN2 substrates. A, BES1 displays a specific interaction with BIN2 measured by growth on medium lacking His (second strip) and blue color on 5-bromo-4-chloro-3-indolyl-β-d-galactoside-containing medium (third strip). B, The hypothetical product of the BES1 gene. The Ala stretch, the bipartite nuclear localization sequence, and putative GSK3 phosphorylation sites are denoted by blue, pink, and red underlines, respectively. The arrows indicate the N-terminal positions where partial BES1 proteins of the original yeast two-hybrid clones are fused with the GAL4 DNA activation domain. C, Alignment of BES1 protein and its homologs. Aligned with BES1 are BZR1 (AAL57684), four other Arabidopsis hypothetical proteins (NP_190644, NP_193624, AAK91411, and NP_565187), a tomato mature anther-specific protein (AAK71662), and an unknown rice protein (BAB33003). The multiple sequence alignment was conducted using the Lasergene sequence analysis software package (DNAStar, Inc., Madison, WI). Absolutely conserved amino acids are indicated by the pink box, whereas homologous amino acids are shaded with blue color. D, BIN2 can also interact with BZR1 in the yeast two-hybrid assay.
Figure 2
Figure 2
Phosphorylation of BES1/BZR1 by BIN2 in vitro. A, BIN2 can phosphorylate both BES1 and BZR1 in vitro. GST-BES1 (top) or GST-BZR1 (bottom) fusion proteins were mixed with the wild-type BIN2 (lane 1), the Lys-69-Arg-mutated GST-BIN2 (lane 2), or the bin2-1-mutated GST-BIN2 (lane 3) to measure protein phosphorylation activity as described in “Materials and Methods.” B, GST-BRI1CK was unable to phosphorylate BES1 or BZR1 protein. C, The phosphorylation of BES1 or BZR1 by BIN2 can be inhibited by lithium. For A through C, the amount of proteins used in the kinase assays is indicated by Coomassie Blue staining in the top strip above the white dividing line, whereas the level of proteins phosphorylation is shown by autoradiography in the bottom strip.
Figure 3
Figure 3
BIN2 phosphorylation of BES1 and BZR1 via a novel mechanism. A, Phosphorylation of BES1 and BZR1 by BIN2 does not require priming phosphorylation. CIP-treated GST-BES1 or GST-BZR1 was incubated with GST-BIN2 or with a mutated GST-BIN2 fusion protein containing Arg-80-Ala mutation, and assayed for protein phosphorylation as described in the “Materials and Methods.” B, Both GST-BES1 and GST-BZR1 proteins can be phosphorylated by an MBP-BIN2 fusion kinase. C, FRATide has no inhibitory effect on the BIN2 phosphorylation of either BES1 or BZR1. For all panels, the amount of protein used for the kinase assays are indicated in the top strip, whereas the levels of protein phosphorylation are shown in the bottom strip.
Figure 4
Figure 4
Identification of semidominant bes1 mutations as suppressors for two different bri1 mutations. A and B, Suppression of bri1-9 mutant phenotypes by a semidominant suppressor mutation m9-1. Shown in A and B (from left to right) are a wild-type plant, a bri1-9 mutant, and a bri1-9 m9-1 double mutant. C, Molecular nature of the five semidominant bri1 suppressor mutations that suppress bri1-5 or bri1-9 mutation. A C-T mutation (indicated by an arrow) in the BES1 gene was detected in the five independently isolated bri1 suppressor mutants, resulting in a missense mutation of Pro-233 to Leu.
Figure 5
Figure 5
Overexpression of the BZR1 gene suppressed bin2/+ and bri1 mutant phenotypes. A, Overexpression of the wild-type BZR1 rescued the short-petiole phenotype of the bin2/+ mutant. B, The cabbage-like rosette phenotype of the bin2/+ mutant was rescued by BZR1 overexpression. C, BZR1 overexpression can rescue the overall growth defect and the silique phenotype of the bin2/+ mutant. Shown in A through C (from left to right) are a wild-type plant, the bin2-1/+ mutant, and transgenic bin2-1/+ plants overexpressing the BZR1 gene. D, The overexpression of the wild-type BZR1 gene in a BIN2+ background leads to a phenotype that resembles that of BRI1 overexpression transgenic plants. E, Overexpression of a mutated BZR1 gene containing the Pro-234-Leu mutation rescued the bin2/+ phenotype. Shown here (from left to right) are a wild-type seedling, a bin2-1/+ mutant, and a transgenic bin2-1/+ mutant expressing the mutated BZR1 gene. F, Overexpression of the mBZR1 gene rescued the weak bri1-301 mutation and gives rise to a bes1-like phenotype. From left to right are a bes1-103 bri1-9 double mutant, a transgenic bri1-301 plant expressing the mBZR1 gene, a bri1-301 mutant, and a wild-type control plant.
Figure 6
Figure 6
Nuclear localization of BES1 and BZR1. A, Root tip from a wild-type control plant. B, Root tip from a BRI1-BES1:GFP transgenic seedling. C, Root tip from a BRI1-BZR1:GFP transgenic seedling. The localization patterns of BES1:GFP and BZR1:GFP were analyzed by examining root tips after 1 min of treatment with 10 μg mL−1 propidium iodide (red signal to visualize cell walls) using a confocal microscope (LSM510, Zeiss, Welwyn Garden City, UK) filtered with FITC10 set (excitation 488 nm with emission 505–530 and 530–560 nm).
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