Downstream Components of BR Signaling

Four components of the downstream segment of BR signaling have been characterized in some detail, including a kinase, a phosphatase and two novel transcription factors (Vert et al. 2005; Li and Jin 2007). The brassinosteroid insensitive 2 (bin2) mutant is a semi-dominant gain-of-function allele that in the homozygous state closely resembles the dwarf bri1 phenotype (Li and Nam 2002). BIN2 encodes a Ser/Thr kinase with sequence similarity to the Drosophila shaggy kinase and mammalian glycogen synthase kinase 3 (GSK-3), which often function as negative regulators of signaling pathways controlling metabolism, cell fate determination, and pattern formation (Kim and Kimmel 2000). Loss-of-function alleles of bin2 show no phenotype, but when combined as a triple mutant with null alleles of the two most closely related GSKs in Arabidopsis, a constitutive BR phenotype is observed similar to the overexpression of BRI1 (Vert and Chory 2006). Thus, BIN2 is a negative regulator of BR signaling and considerable evidence suggests that it functions by phosphorylating two transcription factors that are required for the expression of BR-regulated genes.

The semidominant or dominant mutants, bri1-ems-suppressor1 (bes1-D) and brassinazole-resistant1 (bzr1-D), show constitutive brassinosteroid responses and the BES1 and BZR1 proteins share 88% amino acid identity (He et al. 2002; Wang et al. 2002; Yin et al. 2002). Recombinant BIN2 phosphory-lates BES1 and BZR1 in vitro, and the in planta expression level of BIN2 is correlated with BES1 and BZR1 phosphorylation levels in vivo. BR treatment leads to rapid dephosphorylation of BES1 and BZR1, most likely by inactivating BIN2, allowing the BRI1 SUPPRESSOR 1 (BSU1) phosphatase (Mora-Garcia et al. 2004) to increase the levels of hypophosphorylated BES1 and BZR1 in the nucleus. Recent studies have shown that BES1 and BZR1 are novel transcription factors that bind to specific BR response elements in the promoters of BR-regulated genes, either as homodimers or heterordimers with other transcription factors (He et al. 2005; Yin et al. 2005). Recent microarray analyses have cataloged hundreds of BR-regulated genes in functional categories ranging from wall-modifying proteins to transcription factors (Goda et al. 2002, 2004; Mussig et al. 2002; Nemhauser et al. 2004, 2006; Vert et al. 2005, Nakamura et al. 2006a).

GSK-3/shaggy kinases play an important negative regulatory role in the animal Wingless/wnt signaling pathways by phosphorylating P-catenin, promoting its proteasome-dependent degradation. Ligand binding to a transmembrane receptor leads to GSK-3/shaggy kinase inactivation resulting in an accumulation of unphosphorylated P-catenin, which escapes degradation by the proteasome and translocates to the nucleus. There, it interacts with transcription factors to regulate the expression of genes essential for developmental pattern formation (Kim and Kimmel 2000). There is evidence that the phosphorylated forms of BES1/BZR1 are subject to proteasome-mediated degradation. A parallel mechanism for BR signaling was proposed in which BR binding to BRI1 initiated a signaling cascade that inactivated BIN2 kinase by an unknown mechanism (Wang et al. 2002; Yin et al. 2002; He et al. 2005). This allowed the accumulation of hypophosphorylated BES1/BZR1, which then translocated to the nucleus to activate BR-regulated gene transcription.

Recent studies, however, have suggested an alternative mechanism. In this model, BES1 is constitutively nuclear-localized and is phosphorylated by BIN2 and dephosphorylated by BSU1 in the nucleus. Proteasome-mediated degradation of the hyperphosphorylated form is proposed to be important for protein turnover but not for BR signaling. Instead, the hyperphosphory-lated form of BES1 is inactive because of its inability to bind to BR response elements in the promoters of BR-regulated genes. The rapid loss of phosphorylation on BES1/BZR1 after BR binding to BRI1, allows DNA binding and the activation of BR-regulated gene expression (Vert and Chory 2006).

0 0

Post a comment