Two Putative BIN2 Substrates Are Nuclear Components of Brassinosteroid Signaling

Abstract

GSK3 is a highly conserved kinase that negatively regulates many cellular processes by phosphorylating a variety of protein substrates. BIN2 is a GSK3-like kinase in Arabidopsis that functions as a negative regulator of brassinosteroid (BR) signaling. It was proposed that BR signals, perceived by a membrane BR receptor complex that contains the leucine (Leu)-rich repeat receptor-like kinase BRI1, inactivate BIN2 to relieve its inhibitory effect on unknown downstream BR-signaling components. Using a yeast (Saccharomyces cerevisiae) two-hybrid approach, we discovered a potential BIN2 substrate that is identical to a recently identified BR-signaling protein, BES1. BES1 and its closest homolog, BZR1, which was also uncovered as a potential BR-signaling protein, display specific interactions with BIN2 in yeast. Both BES1 and BZR1 contain many copies of a conserved GSK3 phosphorylation site and can be phosphorylated by BIN2 in vitro via a novel GSK3 phosphorylation mechanism that is independent of a priming phosphorylation or a scaffold protein. Five independent bes1 alleles containing the same proline-233-Leu mutation were identified as semidominant suppressors of two different bri1 mutations. Overexpression of the wild-type BZR1 gene partially complemented bin2/ϩ mutants and resulted in a BRI1 overexpression phenotype in a BIN2 ؉ background, whereas overexpression of a mutated BZR1 gene containing the corresponding proline-234-Leu mutation rescued a weak bri1 mutation and led to a bes1-like phenotype. Confocal microscopic analysis indicated that both BES1 and BZR1 proteins were mainly localized in the nucleus. We propose that BES1/BZR1 are two nuclear components of BR signaling that are negatively regulated by BIN2 through a phosphorylation-initiated process.BRs are a special class of plant polyhydroxysteroids that have wide distribution throughout the plant kingdom and play many important roles throughout plant development that include seed germination, stem elongation, pollen tube growth, vascular differentiation, skotomorphogenesis, and stress resistance (Clouse and Sasse, 1998;Steber and McCourt, 2001). It was well documented that gene regulation is critical for many BR-elicited physiological responses (Clouse and Feldmann, 1999), however, the signaling mechanism from BR perception to gene regulation remains largely unknown. Extensive genetic screens for BR-insensitive-signaling mutants in Arabidopsis have so far identified only two genes, BRI1 and BIN2 (Clouse et al., 1996;Kauschmann et al., 1996;Li and Chory, 1997;Noguchi et al., 1999;Li et al., 2001;Li and Nam, 2002).BRI1 encodes a Leu-rich repeat receptor-like kinase that is composed of an extracellular domain, a singlepass transmembrane segment, and an intracellular kinase domain (Li and Chory, 1997). BRI1 is a plasma membrane-localized protein and can function as a Ser/Thr kinase when expressed in Escherichia coli or animal cell culture (Friedrichsen et al., 2000;Oh et al., 2000). The extracellular domain of BRI1 can confer BR responsiveness to the kinase ...

Regulation ofArabidopsisBrassinosteroid Signaling by Atypical Basic Helix-Loop-Helix Proteins

“…To test ATBS1-AIF4 interaction, the ORF of At3g06590 was PCR amplified and cloned into pACT2 vector (Clontech). The pAS2-BIN2 and pACT2-BES1/BZR1 constructs used as controls were described previously (Zhao et al, 2002).…”

“…Unlike animal steroids that move into cells to bind intracellular receptors, BRs are perceived at the cell surface by the leucine-rich repeat receptor-like kinase BRASSINOSTEROID-INSENSITIVE1 (BRI1) that initiates a phosphorylation-mediated signaling cascade (Li and Chory, 1997;Li, 2005). In the absence of BRs, BRI1 and its coreceptor BRI1-Associated receptor Kinase (BAK1) are inactive kinases Nam and Li, 2002;Wang et al, 2005a), while a GSK3-like kinase BRASSINOSTEROID-INSENSITIVE2 (BIN2) is constitutively active to phosphorylate two highly similar transcription factors, bri1-EMS-Suppressor 1 (BES1) and BrassinazoleResistant1 (BZR1) Wang et al, 2002;Yin et al, 2002;Zhao et al, 2002), thus promoting their degradation, retaining them in cytosol, and/or inhibiting their DNA binding activity (Vert and Chory, 2006;Bai et al, 2007;Gampala et al, 2007;Ryu et al, 2007). BR binding to BRI1 triggers a rapid dissociation of BRI1 Kinase Inhibitor1 from BRI1 and promotes heterotetramerization and transphosphorylation of BRI1 and BAK1 (Wang et al, 2005bWang and Chory, 2006), leading to phosphorylation of BR-signaling kinases and inhibition of BIN2 (Peng et al, 2008;Tang et al, 2008).…”

Membrane steroid-binding protein 1 (MSBP1) negatively regulates brassinosteroid signaling by enhancing the endocytosis of BAK1

“…On the opposite, another BRI1-interacting protein, BKI1, negatively regulates BR signaling by limiting the association of BRI1 and BAK1 [17]. BIN2 is a GSK3-like protein and negatively controls BR signaling by phosphorylating two nuclear components BES1 and BZR1 [18,19]. As nuclear transcription factors, the unphosphorylated BES1 and BZR1 can activate the expressions of BRresponsive genes to promote plant growth (BZR1 also inhibits the expression of BR biosynthetic genes), while the phosphorylated BES1 loses its DNA-binding activity to the BR-responsive genes, and phosphorylated BZR1 is rapidly degraded by proteasome to relieve its feedback inhibition of several BR biosynthetic genes [20][21][22][23][24].…”

Functions of OsBZR1 and 14-3-3 proteins in brassinosteroid signaling in rice

“…Activation of BRI1 leads to interaction with, and activation of, another receptor kinase, BAK1, which has been proposed to act as BRI1's coreceptor (9,10). Downstream of the receptor kinases, the BIN2 kinase and BSU1 phosphatase regulate the phosphorylation status of BZR1 and BZR2/BES1 (11)(12)(13)(14). BR activate BZR1 and BZR2/BES1 by inducing their dephosphorylation, possibly by inhibiting BIN2 or activating BSU1.…”