BAK1 Forms a Heterodimer with BRI1 and Enhances BR Signaling

BAK1 is a member of the LRRII RLK subfamily and was identified independently by activation tagging for suppressors of bril-5 (Li et al. 2002) and by a yeast two-hybrid screen for BRI1 interacting proteins using the BRI1 cyto-plasmic domain as bait (Nam and Li 2002). The extracellular domain of BAK1 lacks the embedded island domain and has only five LRR motifs, differing markedly from BRI1 (Fig. 1). Therefore, it is highly unlikely that BAK1 binds BL directly and it has also been shown that BRI1 can bind BL in a BAK1 mutant background (Kinoshita et al. 2005). Genetic analysis has demonstrated a role for BAK1 in BR signaling, and overexpression of a kinase-inactive mutant form of BAK1 in bril-5 led to a dominant-negative effect, most likely arising by the disruption of a heterodimeric complex between BRI1 and BAK1 (Li et al. 2002). Direct physical interaction between BRI1 and BAK1 has been demonstrated repeatedly (Li et al. 2002, Nam and Li 2002, Russinova et al. 2004), and co-immunoprecipitation experiments in transgenic plants expressing both BRIl-Flag and BAK1-GFP showed that this association was BR-dependent (Wang et al. 2005b). Using the same experimental system as described above for BRI1, it was also demonstrated that the in vivo phospho-rylation of BAK1 on Thr residues was BL-dependent (Fig. 3).

The finding that BRI1 forms a heterodimer with BAK1 in a ligand-dependent manner supports the hypothesis that BR signaling shares some tt> a.

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BRI1-Flag/BAK1-GFP Double Transgenic

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(anti-Flag IP Flag Western} (anti-Flag IP GFP Western) "BAK1-GFP

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Fig. 3 BL-dependence of early events in BR signaling. Transgenic Arabidopsis plants expressing both BRIl-Flag and BAK1-GFP were grown in shaking liquid culture in the light for 6 days. Half of the flasks were then treated with the BR biosynthesis inhibitor brassi-nazole (+BRZ), for 5 additional days to reduce endogenous BR levels. Plants were then treated with 100 nM brassinolide (+BL) or solvent (-BL) for 90 min. Total membrane protein was purified from each sample and BRI-Flag or BAK1-GFP were immunoprecipitated from the solubilized membranes. Equal amounts of protein were separated by SDS-PAGE and Western blot analysis was performed as indicated. A-D Approximately equal amounts of protein were present in each treatment. E Association of BRI1 and BAK1 in vivo, as determined by co-immunoprecipitation is BL-dependent. (F, G) Phosphorylation of Thr residues in BRI1-FLAG and BAK1-GFP is BL-dependent. Adapted with permission from Fig. 1 of Wang et al. 2005b mechanistic similarities to mammalian receptor tyrosine kinase and TGF-P receptor kinase signaling. In mammals, the TGF-P family of polypeptides regulate multiple aspects of development and are perceived at the cell surface by a complex of Type I (RI) and Type II (RII) TGF-P receptor serine/threonine kinases. TGF-P RII homodimerizes in a ligand-independent manner and exhibits constitutive kinase activity. TGF-P binding by RII induces formation of the heterotetramer with RI and results in phosphorylation of RI by RII on specific Thr and Ser resides. Phosphorylated RI then propagates the signal by phosphorylating substrates, termed Smads, which translocate to the nucleus where they associate with transcription factors to regulate the expression of TGF-^-responsive genes (Massague 1998). The RII receptor also phosphorylates specific cytoplasmic substrates, including TGF-P receptor interacting protein 1 (TRIP-1), a WD-40 domain protein that has dual functions as a modulator of TGF-P regulated gene expression and as an essential subunit of the eukaryotic translation initiation factor, eIF3 (Chen et al. 1995; Choy and Derynck 1998). The recent demonstration of several BR signaling features, including BRI1 homodimerization (Russinova et al. 2004; Wang et al. 2005a), the binding of BL directly to BRI1 but not BAK1, the ligand-dependent in vivo association of BRI1 and BAK1 (Wang et al. 2005b), and, as discussed below, the fact that the Arabidopsis ortholog of mammalian TRIP-1 is a putative in vivo substrate of BRI1 (Ehsan et al. 2005), suggest intriguing parallels between BR and TGF-P signaling. However, there are numerous features that are not consistent, including phosphorylation of BRI1 by BAK1, lack of Smad orthologs as BAK1 substrates, and significantly different lig-and and extracellular domain structures. Thus, BR signaling shares some of the signaling logic of the TGF-P pathway without a direct evolutionary relationship.

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