Brassinosteroids signal through two receptor-like kinases

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Brassinosteroids signal through two receptor-like kinases Jianming Li Both animals and plants use steroids to regulate their growth and development, but their mechanisms for steroid perception are different. Animal steroids are mainly recognized by intracellular steroid receptors, whereas plant steroids are perceived by cellsurface receptors that contain a transmembrane receptor serine/ threonine kinase. Recent studies suggest that heterodimerization between two receptor kinases might be a key step in steroid perception and signaling in plants. Addresses Department of Molecular, Cellular, and Developmental Biology, University of Michigan, 830 N University, Ann Arbor, Michigan 48109-1048, USA e-mail: [email protected]

scription factors. Upon steroid binding, these intracellular receptors translocate into the nucleus to regulate gene expression [3]. Alternatively, animal steroids can be perceived by membrane steroid receptors, inducing the socalled ‘non-genomic’ steroid effects [4]. By contrast, plant steroid signaling pathways might be initiated solely by transmembrane receptors that involve two leucine-richrepeat (LRR) receptor-like kinases (RLKs), a cytoplasmic GLYCOGEN SYNTHASE KINASE 3 (GSK3)/SHAGGY kinase, and two nuclear proteins (see [5] for a comprehensive review on BR signaling). The purpose of this review is to summarize recent studies that highlight the important roles of two distinct LRR–RLKs in the perception of plant steroids, and to discuss two competing models for receptor activation in response to BR binding.

Current Opinion in Plant Biology 2003, 6:494–499 This review comes from a themed issue on Cell signalling and gene regulation Edited by Kazuo Shinozaki and Elizabeth Dennis 1369-5266/$ – see front matter ß 2003 Elsevier Ltd. All rights reserved. DOI 10.1016/S1369-5266(03)00088-8

Abbreviations 70AA 70-amino-acid abs1 altered brassinolide sensitivity 1 BAK1 BRI1-ASSOCIATED RECEPTOR KINASE 1 BR brassinosteroid BRI1 BRASSINOSTEROID INSENSITIVE 1 BRL BRI1-LIKE GFP green fluorescent protein LRR leucine-rich repeat RLK receptor-like kinase SR160 160-kDa systemin cell-surface receptor TGFb transforming growth factor b

Introduction Brassinosteroids (BRs) are a unique class of plant polyhydroxysteroids that are structurally related to the animal steroid hormones and elicit a plethora of physiological responses when applied exogenously to plants [1]. The importance of endogenous BRs in plant growth and development was revealed by dramatic morphological changes in BR-biosynthesis mutants in several plant species including Arabidopsis, pea, rice, and tomato [2]. These BR-deficient mutants often share morphological changes that include dwarfed stature in the light and aberrant skotomorphogenesis in the dark [2]. In animals, steroid perception is largely mediated by intracellular steroid receptors that are members of the nuclear receptor superfamily of ligand-dependent tranCurrent Opinion in Plant Biology 2003, 6:494–499

BRI1 is a critical component of a membrane BR receptor The first LRR–RLK to be implicated in BR signaling was BRASSINOSTEROID INSENSITIVE 1 (BRI1), which has been repeatedly identified by several genetic screens for loss-of-function BR-insensitive Arabidopsis mutants [6–9]. Mutations in BRI1 result in a morphological phenotype that is indistinguishable from that of BR-deficient mutants, but the bri1 phenotype cannot be suppressed by exogenous BR application. Interestingly, bri1 mutants accumulate high levels of brassinolide (the presumed end-product of BR biosynthesis) and its biosynthetic intermediates [9], most likely as a result of de-repressing the expression of several key BR biosynthetic genes [10]. This accumulation indicates a critical role for BRI1 in BR homeostasis. Like many other LRR–RLKs, BRI1 consists of an extracellular domain, a single-pass transmembrane segment, and a cytoplasmic serine/threonine kinase domain [8]. The extracellular domain of BRI1 contains several different sequence elements, including a signal peptide, a leucine-zipper motif, two cysteine pairs that flank 25 LRR motifs, and a characteristic 70-amino-acid (70AA) island domain buried between the 21st and 22nd LRRs (Figure 1). Analysis of the sequences of various bri1 mutant alleles revealed the essential role of the kinase domain in transmitting BR signals, and suggested the involvement of the 70AA island in BR binding [8,9]. Consistent with the sequence prediction, BRI1 is a membrane-localized protein, as revealed by confocal microscopic analysis of BRI1::green fluorescent protein (GFP) transgenic plants [11], and functions as a serine/ threonine kinase when expressed in Escherichia coli or in human cell cultures [11,12]. These data suggest that BRI1 could function as a transmembrane BR receptor. BRI1 www.current-opinion.com

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Figure 1

Kinase domain BRI1 Kinase domain BAK1

Signal peptide

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The structures of the two LRR–RLKs that are involved in BR signaling. BRI1 contains 25 LRR motifs and a characteristic 70AA island in its extracellular domain, whereas BAK1 contains only five LRRs and lacks the 70AA island and the second cysteine pair.

might not be able to bind BR directly, however, as its extracellular domain is mainly composed of LRR motifs, which are best known for mediating protein–protein interaction. Instead, it has been proposed that a BRbinding protein might be needed to facilitate BR binding to the 70AA island [8]. A putative secreted serine carboxypeptidase, which was identified by a bri1-suppressor genetic screen as a potential regulator of an early BR signaling event, was postulated to process such a BRbinding protein proteolytically [13]. Although no direct biochemical data are available to demonstrate that purified BRI1 binds brassinolide, several lines of evidence strongly suggest that BRI1 is an essential component of a BR receptor. First, the extracellular domain of BRI1 can confer BR responsiveness to Xa21, a rice LRR-RLK that is involved in disease resistance [14]. Upon BR treatment, a BRI1::Xa21 chimeric receptor — which contains the extracellular domain, the transmembrane a-helix, plus a small intracellular juxtamembrane segment of BRI1 and the Xa21 kinase domain — initiated a Xa21-specific plant defense response in a rice cell culture. A mutation in the 70AA island of BRI1 completely abolished this BR-induced defense response [14], confirming the role of the 70AA island in BR sensing. Second, BRI1 is qualitatively and quantitatively correlated to a BR-binding activity [15]. Transgenic Arabidopsis plants that overexpressed a BRI1::GFP fusion protein not only exhibited greater BR sensitivity but also had a greater BR-binding activity than wildtype plants. Consistent with the BRI1::Xa21 experiment, no binding activity was detected in bri1 mutants that contained mutations in the 70AA island. More importantly, such a BR-binding activity can be co-immunoprecipitated with the BRI1::GFP fusion proteins. Third, treatment of Arabidopsis seedlings with brassinolide stimulated the phosphorylation of BRI1 in wildtype plants but not in a kinasedead bri1 mutant [15]. Thus, binding of BR to the BRI1 extracellular domain, either directly or indirectly via a www.current-opinion.com

BR-binding protein, triggers the activation of the BRI1 kinase and initiates a BR signaling cascade.

BRI1 is a conserved component in BR signaling The importance of BRI1 in BR signaling was further validated by the identification of BRI1 homologs whose mutations also give rise to BR-insensitive dwarf phenotypes in plant species other than Arabidopsis [16,17,18]. Rice contains at least two BRI1 homologs, OsBRI1 and OsBRI2. OsBRI1 is more closely related to BRI1 than OsBRI2. OsBRI1 contains a 70AA-island domain that is similar to that of BRI1 but only 22 LRRs. Mutations in OsBRI1 result in partial BR-insensitivity and a dwarf phenotype with erected leaves [16]. The dwarf phenotype of the two known osbri1 mutants was weaker than that of a rice BR-deficient mutant [19,20], however, most likely because weak OsBRI1 alleles were carried in the two BR-insensitive mutants or because of functional redundancy between OsBRI1 and OsBRI2. Three BR-related dwarf mutants have been described in pea. Two of them, lk and lkb, are defective in BR biosynthesis [21,22]. The third, lka, which exhibited reduced BR sensitivity and accumulated greater levels of endogenous BRs than did wildtype plants, was believed to be a BR-signaling mutant [22]. It was reported recently that the LKA gene does indeed encode a pea homolog (PsBRI1) of the Arabidopsis BRI1 [17]. In tomato, two allelic BR-insensitive dwarf mutants, curl3 and altered brassinolide sensitivity 1 (abs1), were identified on the basis of their similarity to tomato BR-deficient mutants and their reduced BR sensitivity [18,23]. Using a candidate gene approach, two BRI1 homologs were identified from Lycopersicon esculentum [18]. Of these, tBRI1 exhibited higher sequence homology to BRI1 and was mutated in both curl3 and abs1 mutants. Genetic studies revealed a co-segregation pattern for the detected Current Opinion in Plant Biology 2003, 6:494–499

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nucleotide mutations and the BR-insensitive dwarf phenotype, confirming the identity of CURL3 as a tomato ortholog of BRI1. Surprisingly, tBRI1 is almost identical to the 160-kDa systemin cell-surface receptor (SR160) of Lycopersicon peruvianum, which was recently identified by biochemical methods as a putative receptor for systemin [24]. Systemin is the first known plant peptide hormone that is involved in the wounding response [25], raising a tantalizing possibility that tBRI1/SR160 could function as a dual-ligand receptor [26]. Brassinolide failed to inhibit the labeling of SR160 by 125I-systemin in a tomato cell culture, suggesting that tBRI1/SR160 might contain independent binding sites for BR and systemin [24]. Alternatively, BRI1 might heterodimerize with different RLKs to mediate BR and systemin perception in tomato. Biochemical and genetic studies with curl3 and abs1 mutants should determine whether tBRI1 is indeed capable of binding two distinct ligands in tomato. Additional BRI1 homologs have been identified in database searches; these include three BRI1-LIKE (BRL) proteins in Arabidopsis [27], and one BRI1 homolog from Lotus japonicus and from Capsella rubella. Consistent with the molecular genetic studies, phylogenetic analysis of the sequences of 12 proteins revealed that BRI1 and the three BRI1 homologs with known mutations are clustered within a single clade (Figure 2), indicating that they are true orthologs with conserved function. It is interesting to note that all three Arabidopsis BRL proteins are clustered into a separate group that also includes OsBRI2, suggesting that an ancient gene duplication event occurred before the split between monocotyledons and dicotyle-

dons. Some of the BRI1 homologs might retain the original biochemical activity but others might have acquired new functions. For example, both BRL1 and BRL3 could bind brassinolide [27] and rescued a bri1 mutation when driven by the BRI1 promoter (J Li, unpublished data). By contrast, BRL2 failed to bind brassinolide [27] and could not rescue the bri1 mutation (J Li, unpublished data) but was implicated in leaf cell patterning [28].

BAK1 interacts with BRI1 in mediating BR perception A second LRR–RLK that is involved in BR signaling is BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). This protein was independently identified as a bri1 suppressor by an activation-tagging genetic screen [29] and as a BRI1-interacting protein by a yeast two-hybrid screen [30]. BAK1 is a much smaller receptor kinase than BRI1; it contains just five LRR motifs and lacks the second cysteine pair and the 70AA island that are characteristic of BRI1 and its homologs (Figure 1). Sequence comparison indicated that BAK1 is a member of a subfamily of Arabidopsis LRR–RLKs (i.e. LRRII; [31]) that also includes Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR KINASE 1 (AtSERK1), a molecular marker for somatic embryogenesis [32]. BRI1 and BAK1 share similar gene expression and protein localization patterns, and physically interact with each other in yeast and plant cells [29,30]. A role for BAK1 in BR signaling was revealed by both gain-of-function and loss-of-function genetic studies

Figure 2

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Phylogenetic analysis of 12 BRI1/BRI1-homolog sequences. Sequences that were used in the analysis are: BRI1 (At4 g39400), BRL1 (At1 g55610), BRL2 (At2 g01950) and BRL3 (At3 g13380) from Arabidopsis thaliana; CrBRI1 (CAC36390) from Capsella rubella; LjBRI1 (AP004500) from Lotus japonicus; tBRI1 (AAN85409) and LeBRI2 (CAC36401) from Lycopersicon esculentum; SR160 (AAM48285) from Lycopersicon peruvianum; OsBRI1 (BAB68053) and OsBRI2 (AAK52544) from Oryza sativa; and PsBRI1 from Pisum sativum [18]. Alignments were performed with Clustal X by the neighbor-joining method and the tree was analyzed with 100 bootstrap trials. The numbers indicate the bootstrap values for the nodes to the right of the number. Current Opinion in Plant Biology 2003, 6:494–499

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[29,30]. Overexpression of BAK1 suppressed two weak bri1 mutations, one in the extracellular domain and the other in the kinase domain, and gave rise to a BRI1-overexpression phenotype in a wildtype background. By contrast, two null bak1 mutants had a semidwarf stature and reduced BR sensitivity. Given the fact that BAK1 physically interacts with BRI1 in plant cells, these genetic studies led to the hypothesis that BRI1 and BAK1 might function as a heterodimer to mediate plant steroid signaling. This hypothesis is supported by additional genetic data. First, the expression of a kinase-dead BAK1 enhanced the bri1-5 mutation, most likely through a dominant-negative effect on the BRI1-containing BR receptor [29]. Second, BAK1 overexpression could not suppress a null bri1 mutation, implying that BAK1 requires BRI1 for its BR-signaling activity [29]. Third, a null bak1 mutation enhanced a weak bri1 mutation but had no effect on a strong bri1 mutation [30].

Two signaling models have been proposed for the hypothesized BRI1–BAK1 interaction (Figure 3) on the bases of two different sets of biochemical studies with E. coli or yeast-expressed proteins. In E. coli, both BRI1 and BAK1 functioned as active kinases when their kinase domains were expressed as fusion proteins, but BAK1 exhibited a weak phosphorylation activity [30]. When the two kinases were mixed in vitro, BAK1 phosphorylation became much stronger, whereas BRI1 phosphorylation remained the same. As the transphosphorylation level of a kinase-dead BAK1 by BRI1 was very low, the increased BAK1 phosphorylation was likely due to enhanced BAK1 activity as a result of transphosphorylation by BRI1. Because BR treatment was known to activate BRI1 phosphorylation in plants [15], these biochemical data support a two-step activation model in which BR binds BRI1 to activate the BRI1 kinase, which then phosphorylates and activates BAK1 [28]. This model

Figure 3

(a) Sequential phosphorylation model BRI1

Brassinolide and a hypothetical BR-binding protein

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(b) Transphosphorylation model BRI1

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Two signaling models for BRI1–BAK interaction. (a) The sequential phosphorylation model. The binding of brassinolide to BRI1, possibly aided by a putative BR-binding protein, activates BRI1. BR1 can then phosphorylate and activate BAK1 to initiate BR signaling. (b) The transphosphorylation model. Brassinolide binding promotes BRI1–BAK1 dimerization, leading to the activation of both receptor kinases through transphosphorylation. www.current-opinion.com

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of BRI1–BAK1 interaction resembles the transforming growth factor b (TGFb) receptor model, which involves the unidirectional phosphorylation of a homodimeric type-I TGFb receptor by a constitutively active homodimeric type-II receptor [33]. This sequential phosphorylation model suggests that BAK1 is not directly involved in BR binding but functions as a signaling component of a membrane BR receptor. It fails to explain, however, how BRI1 becomes activated upon BR binding. In yeast, neither BRI1 nor BAK1 was an active kinase when expressed alone as a full-length protein; however, both receptor kinases were phosphorylated when coexpressed [29]. Neither BRI1 nor BAK1 was phosphorylated when one of these proteins was inactivated, although the inactivating mutations had no effect on the BRI1–BAK1 dimerization. These results suggest that dimerization is sufficient to activate both receptor kinases but the activation of either receptor kinase requires transphosphorylation by their partner. Given the fact that BR stimulates BRI1 phosphorylation in plants [15], it has been proposed that BR binding would promote or stabilize the BRI1–BAK1 dimerization, leading to the activation of both receptor kinases and subsequent BR signaling events. This model is almost identical to the animal receptor tyrosine kinase model in which ligand binding triggers dimerization, leading to the activation of receptor kinases through transphosphorylation [34]. It predicts that both BRI1 and BAK1 are needed for BR binding on the cell surface.

Conclusions and perspectives Despite the lack of direct evidence that purified BRI1 proteins bind brassinolide, molecular genetic and biochemical studies have now firmly established that BRI1 is a key component of a membrane BR receptor. The recent discovery that BAK1 functions together with BRI1 in mediating BR perception suggests that BRI1–BAK1 heterodimerization is a key step in initiating the BR signal transduction pathway upon BR binding. Further studies are needed to determine whether BAK1 is the second component of the BRI1-containing BR receptor and to reveal the exact biochemical function of BAK1 in BR signaling. Given that Arabidopsis has 13 BAK1-like proteins and that two null bak1 mutations give rise to only a weak bri1-like phenotype, it is possible that some BAK1like proteins could heterodimerize with BRI1 to participate in BR perception and to contribute signaling specificity. Heterodimerization between BAK1 and BRI1 or BRI1 homologs could also increase the diversity of ligands that can be recognized by different receptor dimers to mediate other signaling events, such as leaf cell patterning in Arabidopsis and the wounding response in tomato.

Acknowledgements I would like to thank Dr Kyoung Hee Nam for helpful comments on the manuscript. The work in my laboratory was supported by a grant (GM60519) from the National Institutes of Health. Current Opinion in Plant Biology 2003, 6:494–499

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