BRI1 is the BR Receptor

The BRI receptor kinase has been shown by mutational analysis in Arabidop-sis, rice, tomato and pea to be absolutely required for normal BR perception and plant growth (Clouse et al. 1996; Kauschmann et al. 1996; Li and Chory 1997; Noguchi et al. 1999; Friedrichsen et al. 2000; Koka et al. 2000; Yamamuro et al. 2000; Montoya et al. 2002; Nomura et al. 2003). Arabidopsis BRI (a member of LRR RLK subfamily X) is an 1196 amino acid protein with a 791 amino acid extracellular domain containing more than 20 LRR motifs, followed by a predicted hydrophobic transmembrane domain spanning amino acids 792814 (Fig. 1). The cytoplasmic portion of BRI1 consists of a juxtamembrane region (amino acids 815-882), followed by a Ser/Thr kinase domain (amino acids 883-1155) with a final short sequence of 41 amino acids comprising the carboxy-terminal domain. The large number of bril mutant alleles available

Fig. 1 Structure of two LRR RLKs involved in BR signaling. BRI1 and BAK1 form a ligand-dependent heterodimer in cell membranes and are both involved in BR signaling. All segments of the protein N-terminal to the transmembrane domain are extracellular, while those portions C-terminal of the transmembrane domain lie in the cytoplasm. Annotation of BRI1 is based on Vert et al. 2005; and BAK1 on Nam and Li 2002, and Li et al. 2002

Fig. 1 Structure of two LRR RLKs involved in BR signaling. BRI1 and BAK1 form a ligand-dependent heterodimer in cell membranes and are both involved in BR signaling. All segments of the protein N-terminal to the transmembrane domain are extracellular, while those portions C-terminal of the transmembrane domain lie in the cytoplasm. Annotation of BRI1 is based on Vert et al. 2005; and BAK1 on Nam and Li 2002, and Li et al. 2002

clearly shows the biological significance of both the extracellular and kinase domains. The original annotation of Arabidopsis BRI1 called for 25 tandem LRRs with an average length of 24 amino acids, with a 70 amino acid "island domain" embedded between LRR 21 and 22 (Li and Chory 1997). A recent re-annotation suggests there are only 24 LRRs with the island domain lying between LRR 20 and 21 (Vert et al. 2005). BRI1 has been cloned in several crop species and the number of LRR motifs varies between 22 and 25 with an island domain of 68-70 amino acids inserted four LRRs upstream of the membrane-spanning region in all BRI1 sequences examined (Bishop 2003).

Direct biochemical approaches using radiolabeled BL and transgenic plants overexpressing BRI1-GFP fusions demonstrated the role of BRI1 as at least one component of the BR receptor. The BL-binding activity was precipitated by antibodies to GFP, was competitively inhibited by active, but not by inactive, BRs, and was of an affinity (Kd = 7.4 ± 0.9 nM) consistent with known physiological concentrations of BR required to regulate physiological responses in planta (Wang et al. 2001). Subsequent experiments using a photo-affinity labeled BR analog showed that BR binds directly to BRI1 without an intermediate accessory protein (Kinoshita et al. 2005). LRRs are involved in protein-protein interaction and are generally not known for binding small molecules such as steroids, suggesting that BR binding to BRI1 might be through the island domain embedded in the LRR region. This was experimentally confirmed by using radiolabeled BL and recombinant proteins consisting of the island domain with various combinations of adjacent LRRs. The 70 amino acid island domain in conjunction with the adjacent C-terminal LRR (LRR 21 or 22, depending on annotation) was necessary and sufficient for BL binding and thus defines a novel 94 amino acid steroid-binding motif distinctly different in structure to steroid-binding sequences in animals (Kinoshita et al. 2005). The experiments described above clearly show that BRI1 is a BR receptor. Interestingly, based on biochemical evidence tomato BRI1 has been proposed to have a dual function, binding both BR and the peptide hormone systemin, involved in wound response signaling (Montoya et al. 2002; Szekeres 2003; Boller 2005). Systemin is not found in Arabidop-sis, although several other Arabidopsis LRR RLKs are known to have small peptides as ligands (Matsubayashi and Sakagami 2006).

Recent evidence suggests that BL may bind to a preformed ligand-independent BRI1 homodimer in planta. Russinova et al. (2004) found that BRI1-CFP and BRI1-YFP interacted in plant protoplasts using fluorescence lifetime imaging microscopy (FLIM) and Forster resonance energy transfer (FRET) analysis. Moreover, Wang et al. (2005a) found that BRI-CFP and BRI-Flag co-immunoprecipitated in transgenic Arabidopsis plants both in the presence and absence of BR. The addition of BR appeared to stabilize the preformed BRI1 homodimer. Expression of a kinase-inactive BRI1 mutant in either bri1-5 or wild-type plants leads to a dominant negative phenotype, further suggesting BRI1-BRI1 interaction in vivo.

In animal receptor kinases, activation of the cytoplasmic kinase domain by phosphorylation of specific residues is a consequence of ligand binding to the extracellular domain. The activation of many protein kinases occurs by autophosphorylation of one to three residues within the activation loop of subdomain VII/VIII, which is thought to allow substrate access to the catalytic site in subdomain VIb (Johnson et al. 1996). Further phosphorylation of multiple residues in the juxtamembrane and carboxy-terminal domains generates docking sites for binding downstream kinase substrates in the specific signaling pathway. Immunoprecipitation of BRI-Flag from 11-day old light-grown Arabidopsis plants treated with or without BL, followed by im-munoblot analysis with anti-phosphoThr antibody, demonstrated that BRI1 phosphorylation (at least on Thr residues) was BL-dependent in planta (Wang et al. 2005b). The use of a more general phosphorylation stain also suggested that at least one Ser residue was constitutively phosphorylated. Using the same experimental system, immunoprecipitation of BRI1-Flag followed by liquid chromatography tandem mass spectrometry (LC/MS/MS) showed that at least 11 Ser or Thr residues were phosphorylated in vivo in BL-treated Ara-bidopsis plants, including six sites in the juxtamembrane region, one in the carboxy terminal domain and four in the kinase catalytic domain (Wang et al. 2005b). At least three residues in the subdomain VII/VIII activation loop were phosphorylated in vivo. Most of the BRI1 in vivo phosphorylation sites had been previously identified in vitro using recombinant BRI1 cytoplasmic domain expressed in bacteria (Oh et al. 2000). Therefore, at least in the case of BRI1, the in vitro autophosphorylation sites were highly predictive of in vivo phosphorylation.

The functional significance of each of the identified and predicted phos-phorylation sites in Arabidopsis BRI1 was assessed by site-directed muta-genesis of each specific Ser or Thr to Ala followed by biochemical analysis in vitro and testing for the ability of the altered construct to rescue the weak bri1-5 BR-insensitive mutant in planta. The mutations T-1049-A and S-1044-A in the kinase domain activation loop nearly abolished kinase activity, with respect to both autophosphorylation and peptide substrate phos-phorylation. Moreover, these constructs failed to rescue bri1-5 and resulted in a dominant negative effect (Fig. 2), similar to transformation with a kinase inactive mutant (Wang et al. 2005b). Thus, BRI1 appears to share the activation mechanism of many animal kinases by requiring phosphorylation within the activation loop for kinase function. Autophosphorylation within this region is also likely to occur in other plant RLKs. Sequence alignment of RLKs reveals that the activation loop is highly conserved including several Ser and Thr residues that are routinely present. In fact, the residue corresponding to T-1049 in the BRI1 activation loop is nearly invariant, being either a Ser or Thr in 100 of the most closely related Arabidopsis LRR RLKs. Juxtamembrane substitutions did not result in appreciable differences in autophosphorylation compared with wild-type constructs, but resulted in

Ws-2 bril-S

Ws-2 bril-S

bri1-5:\NT BRI1 -Flag fcr/i-5:(T1049A)BRI1-Flag bri1-5:\NT BRI1 -Flag fcr/i-5:(T1049A)BRI1-Flag

Fig. 2 Effect of mutating BRI1 kinase domain activation loop residue Thr-1049 on rescue of the bril-5 mutant. Transgenic constructs contained 1699 bp of 5' upstream BRI1 sequence (covering the BRI1 promoter and 5' UTR), the entire coding region, and an in-frame C-terminal epitope tag (WT BRI1-Flag). Three independent transgenic lines for each construct are shown. All lines were grown under the same conditions and are the same age (65 days). Adapted with permission from Fig. 8 of Wang et al. 2005b

Fig. 2 Effect of mutating BRI1 kinase domain activation loop residue Thr-1049 on rescue of the bril-5 mutant. Transgenic constructs contained 1699 bp of 5' upstream BRI1 sequence (covering the BRI1 promoter and 5' UTR), the entire coding region, and an in-frame C-terminal epitope tag (WT BRI1-Flag). Three independent transgenic lines for each construct are shown. All lines were grown under the same conditions and are the same age (65 days). Adapted with permission from Fig. 8 of Wang et al. 2005b

74 -88% reduction in phosphorylation of a synthetic peptide substrate when compared to the non-mutated kinase control (Wang et al. 2005b). These findings are consistent with the model that autophosphorylation of the activation loop is required for general kinase activity, while autophosphorylation of juxtamembrane and carboxy terminal residues either generates docking sites for specific downstream substrates or affects catalytic activity towards those substrates.

Besides generating docking sites, phosphorylation of juxtamembrane and carboxy terminal domains may also lead to a general activation of the kinase catalytic domain by a variety of mechanisms (Pawson 2002). An inhibitory affect of the C-terminal domain on BRI1 kinase activity was demonstrated both in vitro and in planta (Wang et al. 2005a). Deletion of the C-terminal domain in BRI1-Flag constructs in transgenic plants led to a hypersensitive BR phenotype including elongated hypocotyls, expanded leaves, and elongated petioles. Deletion of the C-terminus also enhanced BRI1 kinase activity in vitro and in planta as did substitution of specific Ser and Thr residues in the C-terminus with Asp, which can mimic constitutive phosphorylation. This data suggests that BL binding to BRI1 results in phosphorylation of the C-terminal region of the cytoplasmic domain, leading to a release of inhibition and subsequent BRI1 kinase activation, most likely via phosphorylation in the activation loop (Wang et al. 2005a). However, another study in which specific Ser and Thr residues in the BRI1 C-terminal domain were substituted with Ala, which prevents phosphorylation at those residues, showed very little effect on general kinase activity or in planta function (Wang et al. 2005b). Thus, the precise mechanism by which the C-terminus regulates BRI1 function requires further examination.

BRI1 activity plays a critical role in plant development and the biochemical mechanisms of its ligand-dependent activation are beginning to be clarified, as described above. Several novel reports on the impact of BRI1 on overall plant development and physiology also recently appeared. Limiting expression of BRI1 to the epidermal layer by transforming a bril null mutant in Arabidopsis with a full-length construct driven by an Ll-specific promoter, resulted in nearly complete rescue of several developmental defects including hypocotyl length and leaf and petiole size (Savaldi-Goldstein et al. 2007). However, defects in vascular organization could only be fully rescued by expression of BRI1 in ground layers. Thus, the role of BRI1 in growth can be uncoupled from vascular organization and differentiation. Several new mutant alleles of the rice BRI1 ortholog, OsBRIl, were identified and functional characterization suggested that OsBRIl is critical for organ development by controlling cell expansion and division, but that it is not essential for organ initiation or pattern development in rice (Nakamura et al. 2006b). A second study in rice revealed potential practical agricultural applications of regulating BRI1 expression. A partial suppression of OsBRI1 expression in trans-genic rice gave plants with an erect-leaf phenotype that led to 30% increases in yield compared to control when planted at high densities (Morinaka et al. 2006).

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