GSK3s and brassinosteroid signalling in flowering plants

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Steroid hormone signalling is found in plants, animals and fungi and, therefore, might be predicted to have an ancient evolutionary origin [12]. However, the proteins involved in plant steroid signalling belong to plant-specific protein families, thus indicating a separate origin for steroid signalling in the plant lineage. Brassinosteroid (BR) signalling modulates a range of physiological responses in flowering plants, including cell expansion, greening, flowering time, fertility and differentiation of vascular tissue (reviewed in [13-16]).

In Arabidopsis, BRs are perceived by a plasma membrane-localized receptor kinase, BRI1 [13-15]. The downstream signal transduction involves a gene isolated by forward genetic studies, BRASSINOSTEROID-INSENSITIVE 2 (BIN2). Dominant mutations in bin2 result in brassinosteroid-insensitive dwarf plants. BIN2 encodes a GSK3 with a catalytic domain sharing ~70% identity with animal GSK3β [17-20] (Figure 1). Gain-of-function bin2 point mutations and over-expression of BIN2 all cause the same BR insensitivity phenotype, confirming BIN2 as an inhibitor of BR signalling [17-20].

In the absence of a BR signal, active BIN2 negatively regulates BR-specific transcription factors, BRASSINAZOLE RESISTANT 1 (BZR1) and bri1-EMS-SUPPRESSOR 1 (BES1/BZR2) (Figure 2). BIN2 phosphorylates BZR1/BES1 on many serine and threonine residues, which leads to their proteasomal degradation [21-23], reminiscent of the role of animal GSK3 during both Wnt- and Hedgehog signalling [4, 24]. BIN2 can also promote BZR1/BES1 nuclear export, thus inhibiting BZR1/BES1 binding to DNA [25-26]. Again, this is a role also demonstrated by activated (phosphorylated (Figure 1)) animal and amoebal GSK3, which control the nuclear export of the transcription factors NF-ATc and StatA, respectively [27-28]. Interaction between BIN2 and BZR1/BES1 is via a specific docking mechanism not seen in other GSK3 substrates [29], and independent of prime phosphorylation or a scaffold protein [21]. In the presence of BR, BIN2 activity towards BES1/BZR1 is inhibited and, consequently, unphosphorylated BZR1 and BES1 are no longer degraded and can direct transcription of BR-responsive genes (Figure 2) [13-15].

The exact mechanism by which the BR signal is transduced from the plasma membrane to BIN2 has now been elucidated [13-15,30-32] and it demonstrates novel modes of inhibition of GSK3 activity. BR is perceived by the BRI1 brassinosteroid receptor and BAK1 (BRI1-associated receptor kinase) co-receptor (Figure 2). BR binding to BRI1 enables transphosphorylation of BRI1 and BAK1, and activation of BRI1. BRI1 then phosphorylates BR-signalling kinases (BSKs) at the plasma membrane. Phosphorylated BSK binds and activates BSU1 phosphatases, which dephosphorylate tyrosine 200 (Y200) in BIN2, inactivating its kinase activity [31] (Figure 2). Interestingly, the dominant bin2-1 (E263K) mutation (Figure 1) renders BIN2 hyperactive by blocking its dephosphorylation by BSU1 [31]. In response to BR, BIN2 is also degraded by the proteasome, whereas the bin2-1 mutant protein is considerably stabilized [30]. Whether it is the Y200-dephosphorylated form of BIN2 that is specifically targeted for degradation has yet to be determined. Y200 in BIN2 is homologous to Y216 in human GSK3β (Figure 1), a tyrosine residue absolutely required for GSK3 activity. The homologous tyrosine residue in other AtGSK3s is also phosphorylated in vivo, and this phosphorylation is required for kinase activity [10]. In GSK3β, this residue is autophosphorylated by GSK3 itself just after the protein is synthesized, with the help of the Hsp90 chaperone protein [33]. BSU1-mediated dephosphorylation of Y200 in BIN2 is the first example of direct dephosphorylation of the conserved tyrosine residue of GSK3 as a means of negatively regulating kinase activity. Furthermore, most plant GSK3s (all except clade III kinases, Table 1) do not contain the N-terminal serine 9 residue found in GSK3β that is key to regulating GSK3β inhibition (Figure 1) (reviewed in [2-3]). It is, therefore, possible that a different mechanism exists in plants for GSK3 inactivation.

The mechanism for brassinosteroid perception and transduction involves GSK3 homologues functioning redundantly for the negative regulation of BR responses [26,34-35]. Experiments using loss- and gain-of-function mutants showed that two other GSK3 homologues, AtSK22/BIL1 and AtSK23/BIL2 (Table 1), function in the same way as BIN2 during BR signalling [35]. Together these genes make up sub-clade II of the Arabidopsis GSK3s in the flowering plant phylogeny [9,36] (Table 1). Although the triple mutant lines of BIN2, BIL1 and BIL2 show a BR-enhanced phenotype, analysis of the levels of phosphorylated BZR1 showed that despite knockouts of all clade II GSK3s there was still substantial phosphorylation of BZR1 [35]. This indicated a role for additional GSK3(s) in the BR signalling pathway, which have been identified as AtSK31/ASKθ [37], a member of sub-clade III, and possibly all three of the subclade I GSK3s [31]. Therefore, at least seven out of the ten Arabidopsis GSK3 proteins are implicated in BR signalling. Importantly, a chemical activator of BR signalling, bikinin, specifically inhibits the same subset of seven Arabidopsis GSK3 proteins [38]. BIN2 orthologues from cotton (Gossypium hirsutum) and rice (Oryza sativa) have been isolated and their overexpression in Arabidopsis has been shown to cause severe growth defects similar to bin2 gain-of-function (BR-insensitive) mutants [39-40]. This suggests that BIN2 orthologues encode isoforms that share a conserved function in BR signalling.