Brassinosteroid signal transduction from cell surface receptor kinases to nuclear transcription factors

Steroid hormones are critical for development of all multicellular organisms and in plants brassinosteroids (BRs) play essential roles in a wide range of developmental and physiological processes³. Unlike animal steroid hormones, which act through nuclear receptors, BRs bind to a receptor kinase (BRI1) at the cell surface to activate the BR response transcription factors named BZR1 and BZR2 (also known as BES1) through a signal transduction pathway^{1, 4}. Although many components have been identified and studied in detail, our understanding of the BR signaling pathway remains incomplete, with major gaps between the receptor kinases at the cell surface and downstream components in the cytoplasm and nucleus (Supplementary Information, Fig. S1a)^{1, 2}.

The upstream BR-signaling components at the plasma membrane include BRI1^{5, 6} and BAK1^{7, 8} receptor kinases, a novel protein (BKI1) that inhibits BRI1⁹, and the plasma membrane associated BR-signaling kinases (BSKs)¹⁰. BR binding to the extracellular domain of BRI1 causes disassociation of BKI1 from BRI1^{9, 11} and induces association and trans-phosphorylation between BRI1 and its co-receptor BAK1¹², leading to activation of BRI1 kinase and phosphorylation of its substrates BSKs¹⁰. Proteomic and genetic studies demonstrated an essential role for BSKs in transducing the signal to the downstream components, but their direct target remains unknown¹⁰.

Downstream BR signaling involves the GSK3-like kinase BIN2¹³, the Kelch-repeats-containing phosphatase BSU1¹⁴, the 14-3-3 family of phosphopeptide-binding proteins¹⁵, and BZR1 and BZR2, which directly bind to DNA and regulate BR-responsive gene expression^16–19. As a negative regulator of BR signaling, BIN2 phosphorylates BZR1 and BZR2 at numerous sites to inhibit their activities through multiple mechanisms². These include accelerating proteasome-mediated degradation²⁰, promoting nuclear export and cytoplasmic retention by the 14-3-3 proteins^{15, 21}, and inhibiting DNA binding and transcriptional activity^{2, 15, 22}. By contrast, the BSU1 phosphatase is a positive regulator of BR signaling¹⁴. Overexpression of BSU1 increases the dephosphorylated BZR2/BES1 and activates BR responses^{4, 14}. However, BSU1 does not interact with or effectively dephosphorylate BZR2/BES1 in vitro and the biochemical function of BSU1 remains unknown^{4, 14}. It is believed that BR induces rapid dephosphorylation of BZR1 and BZR2 by inhibiting BIN2 and/or activating BSU1. However, the mechanisms by which upstream BR signaling regulates BIN2 and BSU1 remain unclear (Supplementary Information, Fig. S1a)^{2, 23}.

To understand how BR signaling regulates BIN2, we analyzed BR-induced changes of BIN2 using immunoblotting of 2-dimensional gel electrophoresis and found that treatment with brassinolide (BL, the most active BR) caused disappearance of the acidic forms and an increase of the basic forms of an epitope-tagged BIN2 protein (Fig. 1a), suggesting that BR induced dephosphorylation of BIN2. This result led us to test the possible role of BSU1 phosphatase in BR regulation of BIN2. Both BSU1 and its closest homolog BSL1 promote BR signaling in vivo (Supplementary Information, Fig. S2a)¹⁴ and show manganese-dependent phosphatase activities (Supplementary Information, Fig. S2b, c). BSU1 only partially reduced the phosphorylation of BZR1 when co-incubated with BIN2 and BZR1 (Supplementary Information, Fig. S3a, b)¹⁴, and failed to dephosphorylate BZR1 and BZR2 when added after BIN2 and ATP were removed from the kinase reaction (Fig. 1b; Supplementary Information, Fig. S3c, d). On the other hand, BSU1 most effectively reduced the BZR1 phosphorylation when pre-incubated with BIN2 before adding BZR1 (Fig. 1c). The BZR1 protein partially phosphorylated by BIN2 using radioactive ³²P-γATP can be further phosphorylated by BIN2 using non-radioactive ATP, causing a mobility shift of the pre-labeled BZR1. Addition of BSU1 did not reduce the radioactivity of ³²P-labeled BZR1 but abolished the mobility shift of the BZR1 band caused by BIN2 (Fig. 1d). These results indicate that BSU1 inhibits BIN2 kinase activity but does not dephosphorylate pre-phosphorylated BZR1 in vitro. The phosphatase domain of BSU1 reduced BIN2 phosphorylation of BZR1 whereas the Kelch repeat domain showed no effect (Supplementary Information, Fig. S3e)

We next examined whether BR and the bin2-1 mutation affect BSU1 inhibition of BIN2. A BSU1-YFP (yellow fluorescence protein) fusion protein was immunoprecipitated from transgenic Arabidopsis. Similar to recombinant GST-BSU1, BSU1-YFP from plants did not dephosphorylate the pre-phosphorylated BZR1 (Supplementary Information, Fig. S4a), but reduced BZR1 phosphorylation when co-incubated with BIN2 and BZR1 (Supplementary Information, Fig. S4b) or pre-incubated with BIN2 before adding to BZR1 (Fig. 1e). Moreover, BSU1-YFP from plants treated with BL more effectively inhibited BIN2 phosphorylation of BZR1 than that from untreated plants (Supplementary Information, Fig. S4b; Fig. 1e), suggesting that BR activates BSU1. The gain-of-function bin2-1 mutation that causes BR-insensitive phenotypes²³ blocked the regulation by BSU1-YFP in vitro (Fig. 1e).

Several experiments demonstrated that BSU1 directly interacts with BIN2. First, GST-BIN2 was detected on a gel blot by MBP-BSU1 and anti-MBP antibody (Fig. 1f). Second, in vivo interaction was demonstrated by Bi-molecular Fluorescence Complementation (BiFC) assays²⁴, in which tobacco cells co-transformed with BIN2 fused to the N-terminal half (nYFP) and BSU1 fused to C-terminal half (cYFP) of YFP showed a strong fluorescence signal (Fig. 1g). Furthermore, epitope-tagged BIN2 and BSU1 were co-immunoprecipitated and the amount of co-immunoprecipitation was increased by BR treatment (Fig. 1h, I; Supplementary Information, Fig. S5a), indicating that upstream BR signaling induces BSU1 binding to BIN2. Similarly, BSL1 also interacts with BIN2 in BiFC and co-immunoprecipitation assays (Fig. 1g, h). The bin2-1 mutant protein also interacted with BSU1 and BSL1 in these assays (Fig. 1f; Supplementary Information, Fig. S5a, b), suggesting that the bin2-1 mutation blocks BSU1 regulation of BIN2 without abolishing their physical interaction.

A BSU1-GFP protein was previously observed only in the nucleus¹⁴. In this study, the BSU1-YFP protein was detected predominantly in the nucleus but weakly in the cytoplasm. Interestingly, BSL1-YFP was excluded from the nucleus and localized exclusively in the cytoplasm and plasma membrane (Fig. 1g; Supplementary Information, Fig. S5c). In fact, BSL1 and its two other homologs have all been identified as plasma membrane proteins by recent proteomics studies²⁵, suggesting that members of the BSU family can mediate upstream BR signaling at the plasma membrane as well as act in the cytoplasm and nucleus.

We further examined whether BSU1 inhibits BIN2 activity in vivo. We previously reported that BIN2 phosphorylation of BZR1 promotes BZR1 cytoplasmic retention by the 14-3-3 proteins while unphosphorylated BZR1 accumulates in the nucleus¹⁵. Co-expression of BIN2 with BZR1-YFP increased phosphorylation and cytoplasmic retention of BZR1-YFP. Such an effect of BIN2, but not of bin2-1, was canceled by the co-expressed BSU1 but not by a mutant BSU1 (BSU1-D510N), which has reduced phosphatase activity but normal localization (Fig. 2a, b; Supplementary Information, Fig. S6a, b).

It was reported recently that BR treatment induces proteasome-mediated degradation of BIN2²³. We showed that BR treatment, or overexpression of BSU1-YFP but not the mutant BSU1-D510N, decreased the protein level of BIN2-myc but not of bin2-1 (Fig. 2c, d; Supplementary Information, Fig. S6c–f). Consistent with a BSU1 function upstream of BIN2 and downstream of BRI1, overexpression of BSU1 partly suppressed the dwarf phenotype of the bri1-116 null mutant but not that of the homozygous bin2-1 mutant (Fig. 2e, f; Supplementary Information, Fig. S7). Furthermore, expression of the BES1-target gene, SAUR-AC1¹⁹, is increased in BSU1-YFP/bri1-116 plants (Fig. 2g). These results demonstrate that BSU1 acts between BRI1 and BIN2 in the BR signal transduction pathway.

The direct interaction and the requirement of phosphatase activity of BSU1 suggest that BSU1 inhibits BIN2 by dephosphorylating BIN2. We analyzed the autophosphorylation sites of BIN2 in vitro using mass spectrometry, and identified Phospho-tyrosine 200 (pTyr200) of BIN2 as a major phosphorylation site (Supplementary Information, Fig. S8a). The same residue was recently detected as an in vivo phosphorylated site of BIN2 by a phosphoproteome analysis of Arabidopsis²⁶. Mutation of Tyr200 to Phe (Y200F) in BIN2 greatly reduced its substrate phosphorylation (Fig. 3a), indicating an essential role of its phosphorylation for full BIN2 activity. The amino acid sequence flanking Tyr200 of BIN2 is highly conserved in mammalian GSK3s (Supplementary Information, Fig. S8b), and a monoclonal antibody for phospho-Tyr216 of human GSK3β²⁷ specifically detected wild type GST-BIN2 but not the GST-BIN2 containing Y200A mutation or the kinase-inactivating M115A mutation (Fig. 4b; Supplementary Information, Fig. S8c), indicating specificity for the phospho-Tyr200 residue of BIN2. Immunoblot experiments using this antibody showed that BSU1 (Fig. 3b, c) greatly reduced Tyr200 phosphorylation of the wild type BIN2, but not that of the mutant bin2-1. BL treatment also reduced the in vivo phosphorylation of Tyr200 of BIN2 but not that of bin2-1 (Fig. 3d, e). These results demonstrate that BR signaling inhibits BIN2 through BSU1-mediated dephosphorylation of pTyr200, and the bin2-1 mutation causes BR insensitivity by blocking this dephosphorylation.

To further confirm the role of Tyr200 phosphorylation for BIN2 regulation, a Y200F mutation was created. While overexpression of wild type BIN2 or mutant bin2-1 causes BR-insensitive dwarf phenotypes in transgenic Arabidopsis plants, overexpression of BIN2 or bin2-1 containing the Y200F mutation did not (Fig. 3f, g), indicating that Tyr200 phosphorylation is essential for BIN2 to inhibit BR-dependent plant growth and that dephosphorylation of pTyr200 is sufficient to inactivate BIN2. Consistent with an essential role of BSU1 and its homologs in inhibiting BIN2, suppressing BSL2 and BSL3 expression in the bsu1;bsl1 double mutant causes a severe dwarf phenotype and reduced expression of the BES1-target gene SAUR-AC1 (Fig. 3h–j). Taken together, these results demonstrate that dephosphorylation by the BSU1-related phosphatases is the primary mechanism of BIN2 inactivation and an essential step of BR signal transduction.

The Arabidopsis genome encodes 10 GSK3/Shaggy-like kinases (AtSKs), which are classified into four subgroups (Fig. 4a). A triple knockout mutant for group II AtSKs including BIN2 shows increased cell elongation but still accumulates phosphorylated BES1 and responds to BL, indicating that other GSK3-like kinases also act in BR signaling^{2, 22}. We performed interaction study between BZR1 and nine AtSKs representing four subgroups, and found that all six AtSKs of subgroup I and II interact with BZR1 in yeast two-hybrid assays (Fig. 4b). We further examined the function of AtSK12 as a representative of subgroup I AtSKs in BR signaling. BiFC assays showed that AtSK12 interacts with BZR1 as does BIN2 in Arabidopsis, and deletion of the C-terminal 29 amino acids of AtSK12 abolished the interaction with BZR1 (Fig. 4c; Supplementary Information, Fig. S9a). Transgenic plants overexpressing AtSK12, or AtSK12-E297K corresponding to the bin2-1 gain-of-function mutation, displayed similar dwarf phenotypes as those overexpressing BIN2 or bin2-1 (Fig. 4d)¹³. Similar to BIN2, AtSK12 strongly phosphorylates BZR1 in vitro (Fig. 4e), is localized in both cytoplasm and nucleus independent of BR (Supplementary Information, Fig. S9b), is stabilized by the BR biosynthetic inhibitor brassinazole (BRZ) (Supplementary Information, Fig. S9c) and destabilized by BL or by overexpression of BSU1-YFP (Fig. 4f, g). Mass spectrometry analysis indicated that Tyr233 of AtSK12 (corresponding to Tyr200 of BIN2) was also phosphorylated (Supplementary Information, Fig. S9d). BR treatment greatly reduced phosphorylation of AtSK12 Tyr233, indicating similar mechanism of BR regulation as for BIN2 (Fig. 4h). These results indicate that BSU1-mediated tyrosine dephosphorylation is a common mechanism shared by at least two of six GSK3-like kinases that are likely involved in BR signaling.

The function of BSU1 upstream of BIN2 suggests that it might be directly regulated by upstream components on the plasma membrane. We tested direct interaction of BSU1 with BRI1, BAK1 and BSK1 in vitro and found that the MBP-BSU1 protein interacted with BSK1 but not with BRI1 or BAK1 (Fig. 5a), which is consistent with BSK1 being downstream of BRI1 in the signaling pathway. BiFC assays showed that BSK1 interacts with both BSU1 and BSL1 in vivo (Fig. 5b), and the interaction was further confirmed by co-immunoprecipitation assays (Fig. 5c). We have previously shown that BRI1 phosphorylates BSK1 at Ser230. We found that phosphorylation of BSK1 by BRI1 increased while mutation of S230A abolished the binding of BSK1 to BSU1 (Fig. 5d), indicating that BRI1 phosphorylation of BSK1 at Ser230 increases its interaction with BSU1. These results demonstrate that BRI1 phosphorylation of BSK1 Ser230 promotes BSK1 binding to BSU1. Such interaction with BSK1 is likely to mediate BR activation of BSU1 in vivo, although we did not detect an effect of BSK1 on BSU1 activity in vitro (data not shown).

Together our results bridge the last major gaps and elucidate a complete BR signaling pathway that involves multiple steps of sequential phosphorylation/dephosphorylation, transducing the signal from BRI1/BAK1 receptor kinase complex to BSK1, BSU1, BIN2, and BZR1 and BZR2 (Fig. 5e). In the absence of BR, BZR1 and BZR2 are inhibited by BIN2-catalyzed phosphorylation and consequent exclusion from the nucleus due to binding by the 14-3-3 proteins², loss of DNA binding activity^{15, 22}, and degradation by the proteasome²⁰. BR binding to the extracellular domain of BRI1 activates BRI1 kinase through ligand-induced association and trans-phosphorylation with its co-receptor kinase BAK1¹². BRI1 then phosphorylates the BSK1 kinase at Ser230¹⁰, and this phosphorylation promotes BSK1 interaction with and activation of BSU1. Upon activation, BSU1 dephosphorylates BIN2 at the pTyr200 residue to inhibit its kinase activity, allowing accumulation of unphosphorylated BZR1 and BZR2 in the nucleus, where they regulate BR responsive gene expression and plant growth (Fig. 5e; Supplementary Information, Fig. S1b).

Signal transduction through cell surface receptor kinases is a fundamental mechanism for cellular regulation in living organisms. BRI1 is a member of the large family of leucine-rich-repeat receptor-like kinases (LRR-RLK), with over 220 members in Arabidopsis and 400 in rice²⁸. Only a handful of these RLKs have been studied²⁹. The BR signaling pathway described in this study represents the first complete RLK-mediated signaling pathway in plants and provides a paradigm for understanding other signal transduction pathways in plants.

Each BR signaling component in Arabidopsis is encoded by a small gene family with three to six members that have similar biochemical functions. BRI1 is the only essential gene for BR signaling that was identified by recessive mutations, yet two BRI1 homologs, BRL1 and BRL3, appear to mediate BR signaling in a tissue specific manner^{11, 30, 31}. All the other components were identified either by gain-of-function mutations or by proteomic/biochemical approaches¹⁰. Single knockout of BIN2, BZR1, BES1, BSU1, and BSK1 caused no obvious phenotype or very subtle growth phenotypes, suggesting genetic redundancy among the members of each gene family. Our transgenic and biochemical studies provide strong evidence that six Arabidopsis GSK3s (Group I and II) are involved in BR signaling (Fig. 4), and loss-of-function data show that all four members of the BSU1 family contribute to BR signaling (Fig. 3h–j). As such, each step of BR signal transduction seems to be carried out by several members of the gene family, although only the founding member of each family is presented in the conceptual model of BR signal transduction (Fig. 5e). On the other hand, microarray data indicates very similar ubiquitous expression patterns for BRI1, BSK1, BSU1, BSL1, and BZR1 (Supplementary Information, Fig. S10)^{14, 31}, supporting their major roles in BR responses.

Our study reveals BSU1-mediated pTyr200 dephosphorylation as the primary mechanism for regulating plant GSK3s in the BR signaling pathway. This tyrosine residue is absolutely conserved in all GSK3s identified so far, and its phosphorylation is required for kinase activity in Dictyostelium and mammals^{27, 32, 33}. However, the phosphatase for this regulation has not been identified in these systems^{27, 33–35}. BSU1 represents the first phosphatase that mediates dephosphorylation of this conserved tyrosine residue of GSK3s. BSU1 contains an N-terminal Kelch-repeat domain and a C-terminal phosphatase domain¹⁴, which dephosphorylates both phospho-Ser/Thr and phospho-Tyr residues (Supplementary Information, Fig. S2b, c, S11). The phosphatase domain of BSU1 shares about 45% sequence identity with mammalian protein phosphatase-1 (PP1). Interestingly, PP1 expressed in E. coli exhibits both Tyr and Ser/Thr phosphatase activity, although native PP1 expressed in mammalian cells is inactive on phospho-Tyr due to inhibition by inhibitor-2³⁶. It will be interesting to see if BSU1-related phosphatases mediate tyrosine dephosphorylation of GSK3s in mammals and other species.