An Essential Role for 14-3-3 Proteins in Brassinosteroid Signal Transduction in Arabidopsis

Abstract

Brassinosteroids (BRs) are essential hormones for plant growth and development. BRs regulate gene expression by inducing dephosphorylation of two key transcription factors, BZR1 and BZR2/BES1, through a signal transduction pathway that involves cell-surface receptors (BRI1 and BAK1) and a GSK3 kinase (BIN2). How BR-regulated phosphorylation controls the activities of BZR1/BZR2 is not fully understood. Here, we show that BIN2-catalyzed phosphorylation of BZR1/BZR2 not only inhibits DNA binding, but also promotes binding to the 14-3-3 proteins. Mutations of a BIN2-phosphorylation site in BZR1 abolish 14-3-3 binding and lead to increased nuclear localization of BZR1 protein and enhanced BR responses in transgenic plants. Further, BR deficiency increases cytoplasmic localization, and BR treatment induces rapid nuclear localization of BZR1/BZR2. Thus, 14-3-3 binding is required for efficient inhibition of phosphorylated BR transcription factors, largely through cytoplasmic retention. This study demonstrates that multiple mechanisms are required for BR regulation of gene expression and plant growth.

A large-scale protein phosphorylation analysis reveals novel phosphorylation motifs and phosphoregulatory networks in Arabidopsis

“…In this study, the kinase motif [sxP, A1] also contains one of two types of 14-3-3 protein binding motifs, the mode II motif [RxxxsxP] [60]. Totally 14 phosphoproteins (15.7%) in subgroup A1 were identified with the mode II motif, no mode I motif [RxxsxP] was found.…”

“…Some of the motifs in subgroup A contain either a proline residue [sxP, A1], or acidic aspartic acid residues [tDD, A5] and an acidic protein C-terminus [sxxxx-, A4; tx-, A6], and thus may mediate the recognition by proline-directed (GSK-3, CDK, and MAPK) or acidophilic (CKII) protein kinases. As an example, the BZR1 protein, a key transcription factor activated by brassiosteroid signaling in Arabidopsis, contains a [sxP, A1] motif at Ser-173 (pSCP) and has been found to be phosphorylated by the proline-directed GSK3-like kinase BIN2 [60]. Some other motifs in subgroup A contain a phenyl group in the phenylalanine residue such as [sF, A2], which may attract kinases with hydrophobic amino acids around the binding site.…”

Structural and Functional Characterization of Arabidopsis GSK3-like Kinase AtSK12

“…Agrobacterium-mediated transient expression in tobacco was performed as described previously (Gampala et al, 2007). Fluorescence of YFP was analyzed by SP5 confocal microscope (Leica Microsystems, Germany).…”

“…The ratio between nuclear and cytoplasmic fluorescent signals of YFP-tagged AtSK12 variants was quantified as previously described (Gampala et al, 2007). Briefly, a fixed size (18 pixels) was drawn from representative areas within the nucleus, cytoplasm, and background of each cell, and then integrated densities were measured.…”

“…Since the bin2-1 mutant carrying E263K mutation was isolated as a BR-insensitive mutant (Choe et al, 2002;Li and Nam, 2002;Perez-Perez et al, 2002), genetic and biochemical analyses have illustrated that BIN2, a GSK3-like kinase, negatively regulates BR-responsive transcription factors, BZR1 and BZR2, leading to the inhibition of plant growth when BR levels are low. BIN2 phosphorylation of BZR1/2 causes the inhibition of BZR1/2 activity through multiple mechanisms such as cytoplasmic retention mediated by 14-3-3 protein, loss of DNA binding activity, and degradation by 26S proteasome (Gampala et al, 2007;He et al, 2002;Ryu et al, 2007;Vert and Chory, 2006;Zhao et al, 2002). Of ten GSK3-like kinases in Arabidopsis, recent studies suggest that seven members including BIN2 are involved in BR signaling (Kim et al, 2009;Rozhon et al, 2010).…”