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Brassinosteroid Perception and Signaling: Heterodimerization and Phosphorylation of Receptor-Like Kinases BRI1 and BAK1

neckardt{at}aspb.org

Brassinosteroids (BRs) are plant growth hormones that are structurally similar to insect and animal steroids. Since the discovery of BRs in Brassica napus pollen, more than 40 BR-like compounds have been identified in numerous plant species (Altmann, 1999). BRs show a strong growth-promoting effect when applied to plants in nanomolar concentrations because of positive effects on both cell division and cell elongation. BR mutants typically show dwarfism, dark green coloration, reduced fertility, delayed senescence, and altered defense responses, suggesting roles in diverse developmental processes (Clouse and Sasse, 1998).

	BR PERCEPTION AND EARLY SIGNALING EVENTS

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Since the initial discovery of the brassinosteroid-insensitive1 (bri1) mutant (Clouse et al., 1996), many bri mutant alleles (presumably affected in BR perception and signal transduction) have been mapped to the BRI1 locus in Arabidopsis and several other species. Arabidopsis BRI1 encodes a membrane-localized Leu-rich repeat (LRR) receptor-like kinase (RLK) that binds BR in an extracellular domain (Li and Chory, 1997; Wang et al., 2001; Kinoshita et al., 2005). BRI1 has been found to interact with another LRR RLK called BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1), which is also essential for BR signal transduction (Li et al., 2002; Nam and Li, 2002). Mammalian receptor kinases are known to undergo ligand-induced homo- or hetero-oligomerization followed by autophosphorylation of the cytoplasmic domain and subsequent signal transduction to other downstream components (Schlessinger, 2000, 2002). In this issue of The Plant Cell, Wang et al. (2005a; pages 1685–1703), working with Steve Clouse and colleagues, show that the in vivo association of BRI1 and BAK1 is enhanced by BR, and phosphorylation of Thr residues on both proteins is BR dependent. These data provide strong support for the notion that ligand binding induces hetero-oligomerization of BRI1 and BAK1 and autophosphorylation of numerous Thr residues to initiate BR signal transduction.

In addition, the work provides valuable information on receptor kinase mechanism of action. Typical receptor kinases comprise a putative extracellular ligand binding domain, a single-pass transmembrane domain, and an intracellular kinase domain that is regulated in response to a stimulus (i.e., ligand binding) (reviewed in Becraft, 2002). The two major classes of receptor kinases in animals are receptor Tyr kinases (which phosphorylate on Tyr residues) and receptor Ser/Thr kinases (which phosphorylate on Ser and Thr residues). Plant genomes contain a large number of genes encoding proteins having the basic receptor Ser/Thr kinase structure, which have been termed RLKs because in most cases receptor function (i.e., specific ligand binding) has not been demonstrated. The Arabidopsis genome contains more than 600 RLKs, which account for almost all of the transmembrane kinases present in this species (Shiu and Bleecker, 2003). Thus, RLKs likely play a major role in signal perception and transduction, controlling a wide range of physiological responses in plants. One model of receptor kinase function in animals is that ligand binding induces dimerization of inactive kinase monomers, which brings kinase domains close together and allows them to transphosphorylate and activate each other. In many cases, such as the epidermal growth factor receptor Tyr kinase in animals, the kinase undergoes hetero-oligomerization with other protein kinases (Schlessinger, 2002). While BRI1 and BAK1 have previously been shown to form heterodimers in planta (Li et al., 2002; Nam and Li, 2002; Russinova et al., 2004), Wang et al. (2005a) now show that this hetero-dimerization is most likely directly associated with BR signaling.

	BR-DEPENDENT INTERACTION AND PHOSPHORYLATION OF BRI1 AND BAK1 IN VIVO

TOP BR PERCEPTION AND EARLY... BR-DEPENDENT INTERACTION AND... SITE-DIRECTED MUTAGENESIS... REFERENCES

 
Wang et al. (2005a) made use of double transgenic Arabidopsis plants expressing BRI1 tagged with the Flag epitope (BRI1-Flag) and BAK1 labeled with green fluorescent protein (BAK1-GFP). Plants grown in liquid culture were divided into four treatment groups: treated or untreated with the BR synthesis inhibitor brassinazole to reduce endogenous levels of BR and each of these groups subsequently treated or untreated with the BR brassinolide. The total amount of BRI1-Flag and BAK1-GFP present in the solubilized membrane fractions was unaffected by the treatments, but the interaction of the two proteins was found to be significantly enhanced by endogenously or exogenously applied BR.

The authors also examined phosphorylation of BRI1 and BAK1 in vivo using the double transgenic plants and an antiphosphothreonine antibody that specifically recognizes phosphorylated forms of the proteins. These experiments showed that in vivo phosphorylation of BRI1-Flag and BAK1-GFP in the transgenic plants was dramatically enhanced by treatment with brassinolide and greatly reduced when endogenous BR was reduced by brassinazole treatment. Further experiments using immunoprecipitation followed by mass spectrometry showed that BRI1 is phosphorylated on multiple Thr and Ser residues in vivo.

Recent work from Joanne Chory's group (Wang et al., 2005b) provides additional information on the activation of BRI1. This group showed that removal of the BRI1 C terminus leads to hypersensitive receptor activity, characterized by enhanced phosphorylation of BRI1 and enhanced brassinosteroid signaling activity. Their results further suggest that BRI1 monomers interact and show that BAK1 is not required for BR binding to BRI1. The authors suggest that BR binding might activate BRI1 by inducing a conformational change in preformed BRI1 homodimers and that activated BRI1 homodimers subsequently interact with BAK1 via the kinase domain (Wang et al., 2005b).

Taken together, the results of both groups (Wang et al., 2005a, 2005b) suggest that BR signaling in plants may be analogous to that of Transforming Growth Factor-ß (TGF-ß) signaling in animals, which involves formation of heterotetramers of Ser/Thr receptor kinase pairs Tß-RI and Tß-RII. In this system, Tß-RII homodimerizes in the absence of ligand (Massague, 1998), and binding of the ligand TGF-ß by Tß-RII induces formation of a heterotetramer with Tß-RI and results in phosphorylation of Tß-RI by Tß-RII on specific Thr and Ser resides (Huse et al., 2001). Additional evidence of similarities between TGF-ß and BR signaling was provided recently by the Clouse group (Ehsan et al., 2005), who showed that the plant homolog of a mammalian Tß-RII substrate is a putative BRI1 substrate in Arabidopsis. However, it also remains possible that BR binding to the inactive BRI1 homodimer induces dissociation of the BRI1 monomers and formation of activated BRI1-BAK1 heterodimers, a mechanism that is known to occur in the case of some animal receptor Tyr kinases (Schlessinger, 2000).

	SITE-DIRECTED MUTAGENESIS IDENTIFIES KEY PHOSPHORYLATION SITES

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Many protein kinases have a highly conserved initial mechanism of action involving phosphorylation of one to three residues in an activation loop located in a specific kinase subdomain. Most kinases also have an invariant Asp residue that is required for catalytic activity, and those kinases requiring activation loop phosphorylation are usually RD kinases (referring to adjacent Arg and Asp residues, respectively) that have an Arg residue immediately upstream of the invariant Asp. BRI1, BAK1, and numerous other plant RLKs appear to be of the RD-type based on the presence of these two residues. Wang et al. (2005a) examined sequence conservation among all 610 members of the Arabidopsis RLK family, of which at least 213 are LRR RLKs and 121 are RD-type LRR RLKs. They found that a number of Ser and Thr residues corresponding to BRI1 phosphorylation sites are highly conserved specifically among the RD-type LRR RLKs.

The authors next analyzed the functional significance of 17 positively identified and predicted BRI1 phosphorylation sites by site-directed mutagenesis of each specific Ser or Thr residue to Ala followed by in vitro biochemical analysis and an in vivo test of the ability of each altered construct to rescue the weak bri1-5 BR-insensitive mutant phenotype. These experiments suggested that phosphorylation of specific Ser and Thr residues controls kinase activation and signal transduction to downstream components of the BR signaling pathway. Future experiments with domain swapping and site-directed mutagenesis should also be instrumental in identifying the precise sequence of events in BRI1 and BAK1 activation as well as downstream interacting components of the BRI1-BAK1 complex.

Mechanisms of receptor activation in plants are poorly understood, and the work of Wang et al. (2005a) presents several major findings that advance our understanding of plant RLKs in general and BRI1 signaling in particular.

	REFERENCES

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Clouse, S.D., Langford, M., and McMorris, T.C. (1996). A brassinosteroid-insensitive mutant in Arabidopsis thaliana exhibits multiple defects in growth and development. Plant Physiol. 111, 671–678.[Abstract]

Ehsan, H., Ray, W.K., Phinney, B., Wang, X., Huber, S.C., and Clouse, S.D. (2005). Interaction of Arabidopsis BRASSINOSTEROID-INSENSITIVE 1 receptor kinase with a homolog of mammalian TGF-ß Receptor Interacting Protein. Plant J., in press.

Huse, M., Muir, T.W., Xu, L., Chen, Y.G., Kuriyan, J., and Massague, J. (2001). The TGF beta receptor activation process: An inhibitor- to substrate-binding switch. Mol. Cell 8, 671–682.[CrossRef][ISI][Medline]

Kinoshita, T., Cano-Delgado, A., Seto, H., Hiranuma, S., Fujioka, S., Yoshida, S., and Chory, J. (2005). Binding of brassinosteroids to the extracellular domain of plant receptor kinase BRI1. Nature 433, 167–171.[CrossRef][Medline]

Li, J., and Chory, J. (1997). A putative leucine-rich repeat receptor kinase involved in brassinsteroid signal transduction. Cell 90, 929–938.[CrossRef][ISI][Medline]

Li, J., Wen, J., Lease, K.A., Doke, J.T., Tax, F.E., and Walker, J.C. (2002). BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling. Cell 110, 213–222.[CrossRef][ISI][Medline]

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Nam, K.H., and Li, J. (2002). BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling. Cell 110, 203–212.[CrossRef][ISI][Medline]

Russinova, E., Borst, J.W., Kwaaitaal, M., Cano-Delgado, A., Yin, Y., Chory, J., and de Vries, S.C. (2004). Heterodimerization and endocytosis of Arabidopsis brassinosteroid receptors BRI1 and AtSERK3 (BAK1). Plant Cell 16, 3216–3229.[Abstract/Free Full Text]

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Schlessinger, J. (2002). Ligand-induced, receptor-mediated dimerization and activation of EGF receptor. Cell 110, 669–672.[CrossRef][ISI][Medline]

Shiu, S.-H., and Bleecker, A.B. (2003). Expansion of the receptor-like kinase/Pelle gene family and receptor-like proteins in Arabidopsis. Plant Physiol. 132, 530–543.[Abstract/Free Full Text]

Wang, X., Goshe, M.B., Soderblom, E.J., Phinney, B.S., Kuchar, J.A., Li, J., Asami, T., Yoshida, S., Huber, S.C., and Clouse, S.D. (2005a). Identification and functional analysis of in vivo phosphorylation sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 receptor kinase. Plant Cell 17, 1685–1703.[Abstract/Free Full Text]

Wang, X., Li, X., Meisenhelder, J., Hunter, T., Yoshida, S., Asami, T., and Chory, J. (2005b). Autoregulation and homodimerization are involved in the activation of the plant steroid receptor BRI1. Dev. Cell, in press.

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Identification and Functional Analysis of in Vivo Phosphorylation Sites of the Arabidopsis BRASSINOSTEROID-INSENSITIVE1 Receptor Kinase

Xiaofeng Wang, Michael B. Goshe, Erik J. Soderblom, Brett S. Phinney, Jason A. Kuchar, Jia Li, Tadao Asami, Shigeo Yoshida, Steven C. Huber, and Steven D. Clouse
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