Functional Insights of Plant GSK3-like Kinases: Multi-Taskers in Diverse Cellular Signal Transduction Pathways

Accepted Manuscript Functional Insights of Plant GSK3-Like Kinases: Multi-taskers in Diverse Cellular Signal Transduction Pathways Ji-Hyun Youn, Tae-Wuk Kim PII:

S1674-2052(14)00035-5

DOI:

10.1016/j.molp.2014.12.006

Reference:

MOLP 34

To appear in:

MOLECULAR PLANT

Received Date: 2 September 2014 Revised Date:

15 October 2014

Accepted Date: 2 December 2014

Please cite this article as: Youn J.-H., and Kim T.-W. (2014). Functional Insights of Plant GSK3-Like Kinases: Multi-taskers in Diverse Cellular Signal Transduction Pathways. Mol. Plant. doi: 10.1016/ j.molp.2014.12.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Functional Insights of Plant GSK3-Like Kinases: Multi-taskers in Diverse Cellular Signal Transduction Pathways

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Ji-Hyun Youna and Tae-Wuk Kima,b,1

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b

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Department of Life Science, College of Natural Sciences, Hanyang University, Seoul, 133791 Korea Natural Science Institute, Hanyang University, Seoul, 133-791 Korea

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To whom correspondence should be addressed. E-mail [email protected], tel. 82-22220-2547, fax. 82-2-2299-3495.

Short summary

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Running title: Plant GSK3-like kinase-mediated cellular signaling

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Recent progress indicates that GSK3-like kinases have versatile functions in growth and development of plants. Various cellular signaling events mediated by GSK3-like kinases were reviewed based on newly identified substrates of GSK3-like kinases in Arabidopsis and rice.

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Keywords: GSK3-like kinases; brassinosteroids; signal transduction pathway; Arabidopsis; rice

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ACCEPTED MANUSCRIPT ABSTRACT The physiological importance of GSK3-like kinases in plants emerged when the functional role of plant GSK3-like kinases represented by BIN2 was first elucidated in the brassinosteroid (BR)-regulated signal transduction pathway. While early studies focused

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more on understanding how GSK3-like kinases regulate BR signaling, recent studies have implicated many novel substrates of GSK3-like kinases that are involved in a variety of cellular processes as well as BR signaling. Plant GSK3-like kinases play diverse roles in physiological and developmental processes such as cell growth, root and stomatal cell

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development, flower development, xylem differentiation, light response, and stress responses. Here, we have reviewed the progress made over recent years in understanding the

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versatile functions of plant GSK3-like kinases. Based on the relationship between GSK3-like kinases and their newly identified substrates, we will discuss the physiological and

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biochemical relevance of various cellular signaling mediated by GSK3-like kinases in plants.

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ACCEPTED MANUSCRIPT INTRODUCTION GLYCOGEN SYNTHASE KINASE 3 (GSK3), also known as a Shaggy in Drosophila (Ruel et al., 1993), is a versatile kinase that is highly conserved in all eukaryotes (Ali et al., 2001; Saidi et al., 2012). Since the function of GSK3 was first reported as an enzyme inactivating

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glycogen synthase in rabbit skeletal muscle (Embi et al., 1980; Woodgett and Cohen, 1984), many more dynamic roles of GSK3s in numerous cellular signal transduction pathways have been characterized over the past three decades (Frame and Cohen, 2001; Kaidanovich-Beilin and Woodgett, 2011). In mammals, about 80 different proteins have been reported as

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substrates of two GSK3 isoforms (GSK3α and GSK3β), indicating that GSK3s control a wide range of cellular responses (Sutherland, 2011). To date, our understanding is that mammal GSK3s regulate diverse physiological and developmental processes, such as metabolic

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homeostasis, cell fate determination, embryo development, neuronal differentiation, and circadian rhythms (Doble and Woodgett, 2003; Hur and Zhou, 2010; Kim and Kimmel, 2006). More specifically, GSK3s function as key regulators in essential signaling events including Wnt, insulin, notch, and hedgehog signaling (Doble and Woodgett, 2003; Wu and Pan, 2010). Plants also contain GSK3-like kinase in their genome. In contrast to mammal GSK3s,

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plant GSK3 isoforms are more divergent (10 in Arabidopsis, 9 in rice) (Figure 1) (Yoo et al., 2006). BR-INSENSITIVE 2 (BIN2) was the first plant GSK3-like kinase to be characterized from genetic screening (Li and Nam, 2002; Li et al., 2001). Biochemical and genetic analyses have established that BIN2 plays a negative role in BR signaling, regulating cell growth.

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Since two BR-responsive transcription factors (BRASSINAZOLE RESISTANT 1, BZR1 and bri1 EMS SUPPRESSOR1, BES1/BZR2) were identified as substrates of GSK3-like kinase

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(He et al., 2002; Wang et al., 2002; Yin et al., 2002; Zhao et al., 2002), only a few other substrates have been characterized. However, notably, in recent years, many studies have indicated that plant GSK3-like kinases conduct diverse physiological and developmental programs through the phosphorylation of different kinds of substrates. In this review, we describe the structural properties and regulatory mechanism of plant GSK3-like kinases in comparison to those of mammals. We also discuss an overview of the multiple functions of Arabidopsis and rice GSK3-like kinases based on the dynamic relationship between GSK3like kinases and their substrates. 3

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NOMENCLATURE OF SHAGGY/GSK3-LIKE KINASES IN ARABIDOPSIS AND RICE Kinase domains of GSK3-like kinases found in Arabidopsis and rice share 65~72% sequence

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similarity with human GSK3β. From early studies, homologous genes of Shaggy/GSK3 of Arabidopsis and rice, which are divided into four subgroups in a phylogenetic tree, have been given different names and have been mixed up, as described in Figure 1. In order to avoid confusion and overlap with protein names widely used in other fields, we propose here to

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unify their official names as AtSKs (Arabidopsis thaliana Shaggy/GSK3-like Kinases) or OsSKs (Oryza sativa Shaggy/GSK3-like Kinases) unless it is identified by genetic screening

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and has a specific name for an allele (Figure 1).

STRUCTURE AND REGULATION OF GSK3

In animals, the majority of known GSK3 substrates contain typical phosphorylation motif

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(Ser/Thr-X-X-X-Ser/Thr, X is any amino acid). The catalytic function of animal GSK3 is facilitated by the priming phosphorylation or interaction with scaffold proteins including Axin and FRAT (Behrens et al., 1998; Hart et al., 1998; Ikeda et al., 1998). GSK3s’ activities phosphorylating substrates are greatly increased up to ~1,000 fold when the C-terminal

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Ser/Thr residues are phosphorylated by another kinase (Thomas et al., 1999). All GSK3s that have identified to date have a ‘priming phosphate-binding pocket’ created by three basic

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residues (Arg96, Arg180, and Lys205 in human GSK3β) (Dajani et al., 2001; ter Haar et al., 2001), suggesting that the priming phosphorylation might be crucial for the catalytic activities of all GSK3s. It seems, however, that the priming phosphorylation and scaffold protein are not essential for the catalytic activity of plant GSK3-like kinases as plant GSK3like kinases were shown to directly interact with and phosphorylate their substrates in vitro (He et al., 2002; Zhao et al., 2002). AtSK21/BIN2 phosphorylated the calf intestine phosphatase-treated BZR1 and BES1 in vitro (Zhao et al., 2002). In addition, a substitution of Arg to Ala at the 80th amino acid of AtSK21/BIN2, which corresponds with Arg96 of human GSK3β, did not result in a decrease in in vivo activity (Peng et al., 2010). In contrast, it was 4

ACCEPTED MANUSCRIPT reported that a mutation on Arg178 of AtSK32, corresponding with Arg96 of human GSK3β, greatly reduces the kinase activity towards primed substrates, but not non-primed substrates in vitro (Claisse et al., 2007). Interestingly, overexpression of AtSK32 R178A caused more severe defects in growth and flower morphogenesis than did overexpression of wild-type AtSK32 (Claisse et al., 2007). Thus, it is possible that the priming phosphorylation for plant

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GSK3-like kinases might not be essential, but might have functional roles depending on the substrates. Nevertheless, so far, it is unclear whether the priming phosphorylation or the aid of scaffold proteins significantly increases the kinase activity of plant GSK3-like kinase in

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plant cells.

Mammal GSK3s share an almost identical kinase domain but have variations in Nterminal and C-terminal extension. Two major phosphorylation events are known as

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regulatory mechanisms of GSK3s. First, the N-terminal serine phosphorylation (Ser9/21 of human GSK3β/α), which provides a pseudo-primed phosphate occupying the priming phosphate-binding pocket, inhibits GSK3 activity (Dajani et al., 2001; Frame and Cohen, 2001; ter Haar et al., 2001). Several kinases suppress the catalytic function of GSK3s through N-terminal serine phosphorylation while protein phosphatase 1 activates GSK3s by

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eliminating a phosphate from N-terminal phosphoserine (Cross et al., 1995; Eldar-Finkelman et al., 1995; Sutherland et al., 1993; Szatmari et al., 2005; Zhang et al., 2003). However, nonmammalian GSK3s, such as those of higher plants, yeast, Dictyostelium, and Caenorhabditis elegans, do not contain a conserved N-terminal region as do mammal GSK3s (Forde and

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Dale, 2007), implying that inhibitory N-terminal serine phosphorylation might only operate in mammals. Second, the auto-phosphorylation of tyrosine in the T-loop of the kinase domain

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(at Tyr216/Tyr279 in human GSK3β/α), is essential for full kinase activity of GSK3s (Cole et al., 2004; Hughes et al., 1993; Lochhead et al., 2006). Unlike N-terminal serine phosphorylation, tyrosine phosphorylation is believed to occur in all GSK3s because the tyrosine residue in the T-loop of GSK3s is absolutely conserved in all identified GSK3s thus far (Kaidanovich-Beilin and Woodgett, 2011). In

Arabidopsis,

the

phospho-Tyr200

residue

(corresponding

to

phospho-

Tyr216/Tyr279 of human GSK3β/α) of AtSK21/BIN2 is dephosphorylated by bri1 SUPPRESSOR 1 (BSU1) phosphatase (Kim et al., 2009), leading to the inhibition of kinase 5

ACCEPTED MANUSCRIPT activity and promotion of AtSK21/BIN2 degradation (see details in next section). The kinase domains and C-terminal regions of all AtSK members are also highly conserved, while Nterminal regions are usually different. Whereas the C-terminal tail of AtSKs seems to be critical for their interaction with substrates, the N-terminal variation of plant GSK3-like kinases may affect their subcellular localization. The deletion of the C-terminal region of

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AtSK12 abolished AtSK12 binding to BZR1 in vivo while deletion of the N-terminal region of AtSK12 induced the nuclear accumulation of AtSK12 (Kim et al., 2009; Youn et al., 2013). Interestingly, plant GSK3-like kinases appear to localize in a broad range of subcellular compartments. AtSK21/BIN2 and AtSK12 are observed in both the nucleus and cytoplasm

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(Kim et al., 2009; Ryu et al., 2010b; Vert and Chory, 2006). The nuclear localization of AtSK21/BIN2 and AtSK12, in particular, was demonstrated to be critical for their inhibitory

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function in BR signaling (Ryu et al., 2010b; Vert and Chory, 2006; Youn et al., 2013). In contrast, AtSK41 localizes to the plasma membrane and the cytoplasm (Bayer et al., 2012) while Medicago GSK3-like kinase MsK4 is detected as a plastid-localized protein associated with starch granules (Kempa et al., 2007).

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ARABIDOPSIS GSK3-LIKE KINASES AND THEIR VERSATILE FUNCTIONS Growth Regulation Mediated by AtSKs

Substrates of AtSKs identified thus far are summarized in Table 1. Of these, many of AtSKs

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substrates are involved in BR signaling. BRs are polyhydroxylated steroid hormones that regulate diverse cellular responses for plant growth and development, such as cell expansion,

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photomorphogenesis, seed germination, vascular differentiation, male fertility, stress tolerance, senescence, and stomatal development (Clouse, 2011; Wang et al., 2012). Perception of BR by BR INSENSITIVE 1 (BRI1) receptor kinase on the cell surface triggers a sequential signaling cascade, ultimately leading to the regulation of gene expression, which is mediated by BR-responsive transcription factors, namely BZR1 and BES1 (Kim and Wang, 2010). A dominant mutant bin2-1 (BR Insensitive 2-1) was identified by a genetic screening for BR-insensitive mutants (Li et al., 2001). The bin2-1 mutant displays pleiotropic defects, 6

ACCEPTED MANUSCRIPT such as extreme dwarfism, curled and dark-green leaves, abnormal skotomorphogenesis, and male sterility, which are very similar characteristics to those of the bri1 null mutant (Li et al., 2001). Fine mapping of a bin2-1 allele revealed that the BIN2 gene encodes for a member of AtSKs (AtSK21), implicating that plant GSK3-like kinase is crucial for steroid hormone signaling (Li and Nam, 2002). The bin2-1 mutant contains an E263K mutation in the catalytic

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domain of AtSK21/BIN2. Among eight gain-of-function bin2 alleles (also named as dwf12 or ucu1) isolated so far as BR-insensitive dominant mutants, seven alleles including bin2-1 have mutations within the TREE motif (from 261th to 264th amino acid of AtSK21/BIN2) (Choe et al., 2002; Li and Nam, 2002; Li et al., 2001; Perez-Perez et al., 2002). In addition, transgenic

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plants that overexpress AtSKs (AtSK12, AtSK22/BIL2, or AtSK23/BIL1) carrying a mutation (corresponding to an E263K mutation of bin2-1) show dwarf phenotype similar to the bin2-1

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mutant, indicating that the TREE motif is critical for the suppression of AtSKs (Kim et al., 2009; Yan et al., 2009; Youn et al., 2013).

Further studies demonstrated that AtSK21/BIN2 acts as a key negative regulator of BR signaling. AtSK21/BIN2 directly interacts with and phosphorylates BZR1 and BES1 in vitro and in vivo (Table 1) (He et al., 2002; Zhao et al., 2002). Phosphorylated BZR1 and

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BES1 are rapidly dephosphorylated upon BR treatment in wild-type, but not in the bin2-1 mutant (He et al., 2002; Vert and Chory, 2006). When BR levels are low, AtSK21/BIN2 is constitutively active and phosphorylates BZR1 and BES1, resulting in cytoplasmic retention, inhibition of DNA binding activity, and proteasomal degradation of BZR1 and BES1

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(Gampala et al., 2007; He et al., 2002; Vert and Chory, 2006). Both BZR1 and BES1 have 25 putative GSK3 phosphorylation motifs. Of these, ten and nine amino acids of BZR1 and

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BES1, respectively, were experimentally confirmed as in vitro or in vivo phosphorylation sites (Gampala et al., 2007; Ryu et al., 2010a; Ryu et al., 2007; Tang et al., 2008b). In particular, AtSK21/BIN2 phosphorylation of the Ser173 residue of BZR1 (Ser171 of BES1) creates a binding site for phosphopeptide binding protein 14-3-3, which induces cytoplasmic retention of phosphorylated BZR transcription factors (Gampala et al., 2007; Ryu et al., 2010a; Ryu et al., 2007). Recently, a 12-amino acid motif (VKPWEGERIHDV) in the Cterminal region of BZR1 was identified as a minimal AtSK21/BIN2-docking motif. In this study, AtSK21/BIN2 failed to interact with and phosphorylate BZR1 without the AtSK21/BIN2-docking motif both in vitro and in vivo (Peng et al., 2010). Given that the 127

ACCEPTED MANUSCRIPT amino acid motif of BZR1 is not conserved in the other AtSK21/BIN2 substrates identified so far, it seems that AtSK21/BIN2 recognizes various binding motifs with a broad range of specificities. Whereas the bin2 loss-of-function mutant shows a subtle phenotype, the triple

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knockout mutant (bin2-3bil1bil2) of subgroup II members displays the phenotype of constitutively activated BR signaling, including characteristics such as curved hypocotyls in the dark, elongated petioles, and BRZ-resistant phenotypes, suggesting that AtSKs (AtSK21/BIN2, AtSK22/BIL2, and AtSK23/BIL1) belonging to subgroup II have redundant

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roles in BR signaling (Vert and Chory, 2006; Yan et al., 2009). Further studies have confirmed that seven (subgroup I, II, and AtSK32 of subgroup III) of ten AtSKs members regulate BR signaling (Kim et al., 2009; Rozhon et al., 2010; Youn et al., 2013). This is

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further supported by the specificity of BIN2 KINASE INHIBITOR (bikinin), which acts through competition with ATP, as identified by a chemical screening that causes constitutive BR response. Of ten members, bikinin only inhibits the kinase activity of seven AtSKs (De Rybel et al., 2009). The structures of those seven members seem to be somewhat similar. On the other hand, the BZR family is composed of six members (BZR1, BES1, BEH1, BEH2,

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BEH3, and BEH4). BR has been shown to regulate the phosphorylation status of BEHs as well as BZR1 and BES1, suggesting that BZR family members function redundantly in BR signaling (Yin et al., 2005). Thereby, it is quite possible that seven members of AtSKs bind to each BZR family member with different binding specificities, specifying their physiological

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roles.

Aside from the BZR family, recent studies have revealed many more substrates of

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AtSKs that modulate BR signaling (Table 1). The cesta-D mutant isolated from activation tagging mutant pools shows a typical BR mutant phenotype overproducing BRs. Transcript levels of BR biosynthetic genes such as CPD, DWF4, and ROT3 are elevated in the cesta-D mutant. CESTA encodes a bHLH transcription factor similar to the BR EARLY RESPONSIVE GENEs (BEEs), acting as positive regulators of BR response. CESTA binds to the G-box motif (CATGTG) of CPD promoter, which is known as a BZR1 target site. The BiFC and yeast two-hybrid experiment demonstrated that CESTA directly interacts with BEE1. Like BEE1, both BR and bikinin treatment induce a speckled localization of CESTA8

ACCEPTED MANUSCRIPT YFP in the nucleus. Together, a complex of CESTA and BEE1 may serve as a functional unit that positively regulates the expression of BR biosynthetic genes (Poppenberger et al., 2011). Although AtSK21/BIN2 phosphorylates CESTA in vitro, the functional significance of CESTA phosphorylation by AtSK21/BIN2 is not yet evident. Unlike the BZR family,

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CESTA’s DNA binding activity is not altered by AtSK21/BIN2 phosphorylation. The direct connection between auxin and BR signaling at molecular level was demonstrated by a physical interaction between AUXIN RESPONSE FACTOR 2 (ARF2) and AtSK21/BIN2 (Vert et al., 2008). In agreement with the observation that auxin-induced

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hypocotyl elongation was not only enhanced by BR, but also dependent on BR, ARF2 acting as a transcriptional repressor in auxin pathway was isolated as a AtSK21/BIN2-interacting protein in yeast two-hybrid screening. AtSK21/BIN2 phosphorylation of ARF2 alleviates

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DNA binding and repressor activity of ARF2, implying that BR signaling facilitates an auxin response by release of AtSK21/BIN2’s inhibition of ARF2 (Vert et al., 2008). Previously, PHYTOCHROME-INTERACTING FACTOR 4 (PIF4) was shown to interact with BZR1, which integrates BR signaling and environmental responses (Bai et al., 2012; Oh et al., 2012). Most recently, it was demonstrated that a light-regulated transcription

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factor, PIF4, is destabilized by BRZ, but stabilized by BR treatment. AtSK21/BIN2 appeared to interact with PIF4 in vitro and in vivo, and three amino acid residues (Thr160, Ser164, and Ser168) were defined as major AtSK21/BIN2 phosphorylation sites. AtSK21/BIN2 phosphorylation dramatically changes the stability of PIF4 in vivo. PIF4 protein possessing

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mutations on AtSK21/BIN2 target sites (Thr160, Ser164, and Ser168) accumulates in both light and dark, and its overexpression induces extremely early flowering and petiole

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elongation. Interestingly, it was shown that AtSK21/BIN2 regulation of PIF4 mainly occurred at dawn, which correlates with rhythmic hypocotyl growth in diurnal time (Bernardo-Garcia et al., 2014). It would be interesting to investigate whether AtSK21/BIN2 activities are altered in by light and dark, or the diurnal time scale. In animals and plants, GSK3s usually negatively regulate their substrates through direct phosphorylation. Recent studies proposed several examples of positive regulation of substrates by AtSKs. MYLEOBLASTOSIS FAMILY TRANSCRIPTION FACTOR-LIKE 2 (MYBL2) was originally identified as a BR-repressed BES1 target at the transcription level 9

ACCEPTED MANUSCRIPT (Ye et al., 2012). Interestingly, the MYBL2 protein down-regulates BR-repressed gene expression by direct interaction with BES1. The expression of BES1-repressed genes is increased in the bri1-5mybl2 double mutant than in the bri1-5 mutant. In the presence of MYBL2, BES1 further suppressed DWF4 expression in transient luciferase assay. In vitro analysis indicated that AtSK21/BIN2 directly interacts with and phosphorylates MYBL2.

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Importantly, AtSK21/BIN2 phosphorylation of MYBL2 induces the stabilization of MYBL2. Both BR and bikinin treatments destabilize the MYBL2 protein in vivo (Ye et al., 2012). This is the first example of positive regulation of a substrate by plant GSK3-like kinases. Considering that upstream BR signaling inhibits the MYBL2 co-repressor that cooperates

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with BES1, MYBL2 might be involved in the feedback regulation mechanism buffering excessive BR signaling output. The detailed balancing mechanism of BES1 and MYBL2 by

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AtSK21/BIN2 remains elusive.

Arabidopsis thaliana HOMEODOMAIN-LEUCINE ZIPPER PROTEIN 1 (HAT1) and HAT3 have also been proposed as co-repressors of BR-repressed gene expression mediated by BES1 (Zhang et al., 2014). BR-repressed gene expression is increased in the hat1hat3 double mutant, and reduced in the plants overexpressing HAT1. Genetic studies

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demonstrated that HAT1 positively regulates BR-mediated growth. Similarly to MYBL2, HAT1 not only interacts with BES1, but also AtSK21/BIN2 in vitro and in vivo. Moreover, AtSK21/BIN2 phosphorylates HAT1, resulting in the stabilization of HAT1. Phosphorylated HAT1 level is decreased in response to BR or bikinin treatment. Interestingly, studies also

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that some BR-repressed genes, such as DWF4, contain the HOMEOBOX BINDING (HB)binding site (TAATAATTA). BES1 and HAT1 bind to BRRE (BR Response Element,

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CACACG) and the HB-binding site on the DWF4 promoter, respectively. In transient luciferase assays, the expression of DWF4pro::LUC was further repressed when both BES1 and HAT1 were co-expressed (Zhang et al., 2014). Although AtSK21/BIN2 phosphorylation appeared to stabilize the HAT1 protein, it is unclear how it affects HAT1 activity in DNA binding or BES1 interaction. Plasma membrane-localized BSKs classified into the RLCK XII subfamily transduce the BR signal from BRI1 to BSU1 phosphatase (Kim et al., 2009; Tang et al., 2008a). Sreeramulu et al. reported that several BSKs physically interact with not only BRI1 but also 10

ACCEPTED MANUSCRIPT AtSK21/BIN2 and AtSK23/BIL2 in a yeast two-hybrid and a BiFC assay. In addition, they showed that both AtSK21/BIN2 and AtSK23/BIL2 phosphorylate many BSK members such as BSK1, BSK3, BSK5, BSK6, BSK8 and BSK11 in vitro (Sreeramulu et al., 2013). However, their in vivo interaction and specificity in plant cells, and the biological relevance

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of AtSK21/BIN2 phosphorylation of BSKs in BR signaling will require further investigation. An atypical basic helix-loop-helix (bHLH) protein lacking critical amino acid residues in DNA binding domain was also identified as a AtSK21/BIN2 substrate involved in BR signaling (Wang et al., 2009). A dominant mutation of ACTIVATION-TAGGED bri1-301

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SUPPRESSOR 1 (ATBS1) bHLH protein suppressed the growth defects of weak alleles of BR mutants. Another bHLH protein, ATBS1-INTERACTING FACTOR 1 (AIF1), was isolated by yeast two-hybrid assay using ATBS1 as a bait (Wang et al., 2009). Overexpression

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of AIF1 abolished the phenotype of bri1-301 suppressed by the Atbs1-D dominant mutation, suggesting that interaction between AIF1 and ATBS1 inhibits ATBS1’s activity, thereby promoting BR signaling. Although AtSK21/BIN2 was shown to interact with and phosphorylates AIF1, it is still unclear how two atypical bHLH proteins modulate BR signaling. The effect of AtSK21/BIN2 phosphorylation on the interaction of AIF1 and

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ATBS1 or the relationship with BZR1/BES1 will require further investigation.

AtSK-Mediated Stomatal Development

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MAP kinase pathways, which are evolutionarily conserved in eukaryotes, transduce various upstream signals to nuclear transcription factors in a wide range of cellular responses (Hamel

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et al., 2006; Rodriguez et al., 2010). In plants, MAP kinase pathways mediate various responses for abiotic and biotic stress, and regulate the development of stomatal cells and embryos (Rodriguez et al., 2010). Although MAP kinase pathways are thought to be regulated by signal-triggered receptor kinases, their regulation mechanism is poorly understood. Importantly, recent works demonstrated that plant GSK3-like kinases control MAP kinases-dependent stomatal development pathway (Khan et al., 2013; Kim et al., 2012). In Arabidopsis, stomatal development is regulated by the ERECTA (ER) receptor kinasetriggered signal transduction pathway. Genetic studies have confirmed that downstream 11

ACCEPTED MANUSCRIPT signaling components of ER include MAP kinase modules and bHLH transcription factors (Dong and Bergmann, 2010). In this pathway, the MAP kinase module is composed of YODA (YDA) MAPKKK, MKK4/5/7/9 MAPKK, and MPK3/6 MAPK. MPK3/6 negatively regulates SPEECHLESS (SPCH) transcription factor through direct phosphorylation, leading

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to the inhibition of SPCH-mediated stomatal formation. Recent studies show that AtSKs control at least three different signaling components downstream of the ER pathway, which provides opposite regulation of the stomatal development in leaves and hypocotyls (Gudesblat et al., 2012; Khan et al., 2013; Kim et al.,

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2012). In leaves, BR negatively regulates stomatal formation. The BR-deficient det2 mutant displays stomatal clusters and increased numbers of stomatal cells, which are suppressed by BL or bikinin but enhanced by BRZ treatment. Particularly, AtSK21/BIN2 gain-of-function

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mutant, such as bin2-1 and AtSK21/BIN2-overexpressing plants, and the bsu-q mutant possess severe abnormalities in stomatal cell development. However, the bzr1-1D mutant showed normal stomatal patterns and failed to suppress the stomatal clusters of bin2-1 or bsuq, suggesting that BR-regulated stomatal development is BZR1-independent process. Genetic studies confirmed that the canonical stomatal pathway mediated by ER is directly connected

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to BRI1-mediated signaling. Bikinin suppressed stomatal clusters of er triple and tmm mutants, but not yda, mpk3/6 (HOPAI1 overexpression mutant), and Scrm-D mutants, indicating that GSK3-like kinase functions downstream of ER/TMM and upstream of the YDA pathway. YDA contains 84 putative GSK3 phosphorylation sites. Biochemical analyses

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supported that AtSK21/BIN2 interacts with and strongly phosphorylates the auto-regulatory domain of YDA MAPKKK, leading to the inactivation of YDA phosphorylating MKK4.

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Moreover, YDA phosphorylation status is dependent on AtSK21/BIN2 activity in vivo. In Arabidopsis seedlings, YDA was phosphorylated and dephosphorylated by BRZ and bikinin treatment, respectively (Kim et al., 2012). Similarly, Khan et al. proposed a negative role of BR signaling in stomatal development of leaves (Khan et al., 2013). Stomatal clusters were generated by not only BRZ treatment but also bin2-1 mutation or overexpression of AtSK32. They found that three AtSK members (AtSK11, AtSK21/BIN2, and AtSK32) phosphorylate MKK4 MAPKK, resulting in the alleviation of its catalytic activity against MPK6. Mass spectrometry analysis suggested that Ser230 and Thr234 of MKK4 are the main AtSK21/BIN2 phosphorylation sites. In transgenic Arabidopsis, overexpression of MKK4 12

ACCEPTED MANUSCRIPT suppressed the stomatal clusters caused by BR deficiency. Taken together, two studies indicate that BR regulates stomatal production through GSK3-like kinases-mediated inhibitory phosphorylation of a MAP kinase module (Khan et al., 2013; Kim et al., 2012). In contrast, a positive role of BR in stomatal development of hypocotyls has been

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proposed by Gudesblat et al (Gudesblat et al., 2012). BR-deficient and BR-insensitive mutants possess reduced numbers of stomatal cells while plants overproducing BR displayed increased numbers of stomatal cells in hypocotyls. Stomatal formation in hypocotyls was increased and decreased by BL and BRZ, respectively. The key mechanism is that

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AtSK21/BIN2 directly regulates SPCH bHLH transcription factor initiating stomatal cell formation. SPCH protein is stabilized by BR, and AtSK21/BIN2 phosphorylation promotes 26S proteasome-mediated degradation of SPCH. Mass spectrometry analysis identified ten in

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vitro AtSK21/BIN2 phosphorylation sites (Ser38, Thr40, Ser43, Thr44, Ser65, Ser171, Ser177, Ser181, Ser193, and Thr214) and five in vivo (Ser65, Ser171, Ser186, Ser193, and Ser219) phosphorylation sites of SPCH. Of these, phosphorylation of three residues (Ser65, Ser171, and Ser186) was reduced by BR. However, no in vivo phosphorylation site matches to the typical GSK3 phosphorylation motif. It is likely that seven amino acid residues (Ser38,

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Thr40, Ser43, Thr44, Ser65, Ser171, and Ser181) of SPCH are AtSK21/BIN2-specific phosphorylation sites because five phospho-residues (Ser177, Ser186, Ser193, Thr214, and Ser219) overlap with target sites phosphorylated by MPK3/6 (Lampard et al., 2008). Although those studies provide strong evidence that AtSK21/BIN2 is a key player

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integrating BR signaling and the stomatal development pathway, there is some conflict between the studies reported by three independent groups (Gudesblat et al., 2012; Khan et al.,

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2013; Kim et al., 2012). Interestingly, BR-regulated AtSK21/BIN2 appeared to have opposite function in leaves and hypocotyls. This finding is not surprising because similar observation in stomatal development has been reported. As the tmm mutant shows stomatal cluster in leaves but no stomata in hypocotyls, stomatal formation is believed to be inversely regulated by TMM in cotyledons and hypocotyls (Bhave et al., 2009). Of seven AtSKs involved in BR signaling, three AtSKs belonging to subgroup II (AtSK21/BIN2, AtSK22/BIL2, and AtSK23/BIL1) strongly bound to YDA in yeast cells. Consistently, overexpression of AtSK12 of subgroup I caused dwarf phenotypes but no stomatal cluster while that of AtSK21/BIN2 13

ACCEPTED MANUSCRIPT induced both growth defects and stomatal clusters, suggesting that AtSK members have overlapping but non-identical function depending on their substrate specificity (Youn et al., 2013). It would be interesting to investigate the binding affinity of SPCH with all AtSK members. It is possible that each AtSK member may differently or selectively regulate three substrates (YDA, MKK4, and SPCH) in different tissues, developmental stages or

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environment-specific manners. Regulation by AtSKs at multiple crosstalk points may orchestrate stomatal development in response to dynamic developmental and environmental

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signals.

AtSK-Mediated Root Development and Xylem Cell Differentiation

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BR signaling negatively regulates root hair development independently of BES1 activity. Whereas other BR-deficient or BR-insensitive mutants show increased numbers and different patterning of root hairs, the BES1 RNAi mutant develops normal root hairs. AtSK21/BIN2 specifies root epidermal cell fate via direct regulation of transcriptional complex composed of WEREWOLF (WER), ENHANCER OF GLABRA 3 (EGL3), and TRANSPARENT TESTA

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GLABRA1 (TTG1). AtSK21/BIN2 interacts with three key transcription factors in vitro and in vivo, and EGL3 and TTG1 are phosphorylated by AtSK21/BIN2 in vitro. Four amino acids (Thr209, Thr213, Thr399, and Thr403) of EGL3 are identified as in vitro AtSK21/BIN2 phosphorylation sites. Genetic analysis using wild-type EGL3 and its mutated forms

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confirmed that both cytoplasmic localization of EGL3 and its movement from hair cells to non-hair cells are controlled by AtSK21/BIN2 phosphorylation. In addition, AtSK21/BIN2

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phosphorylation of TTG1 inhibited the transcriptional activity of the WER-EGL3-TTG1 complex (Cheng et al., 2014). Cell fate determination is tightly regulated in a spatio-temporal manner. Based on the distinct expression pattern of AtSKs in roots, it would be interesting to investigate the functional involvement and substrate specificity of other AtSKs in the regulation of the WER-EGL3-TTG1 complex. Auxin-mediated lateral root formation is precisely controlled by ARF7 and ARF19. Recently, Cho et al. demonstrated that AtSK21/BIN2 promotes lateral root development by activating ARF7 and ARF19 (Cho et al., 2014). Several lines of evidences support that 14

ACCEPTED MANUSCRIPT AtSK21/BIN2 phosphorylation of ARF7 and ARF19 increases the auxin response in lateral root development. The bin2-3bil1bil2 triple mutant showed reduced lateral root development while bin2 gain-of function mutant possessing increased numbers of lateral roots was hypersensitive to

auxin-induced

lateral

root

development.

AtSK21/BIN2

directly

phosphorylates two amino acid residues (Ser698 and Ser707) on the C-terminus of ARF7,

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which leads to the attenuation of ARF7 binding to IAA19 in vitro. In addition, ARF7 inhibition by IAA19 was released by AtSK21/BIN2 phosphorylation of ARF7 in a reporter gene assay and ChIP analysis, suggesting that AtSK21/BIN2 phosphorylation of ARF7 enhances ARF7’s transcriptional activity. However, a mutant ARF7 carrying alanine

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substitution of serine residues phosphorylated by AtSK21/BIN2 still partly rescued the lateral root development arf7-1. Thus, BIN2 phosphorylation of ARF7 contributes to the

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potentiation of the auxin response in lateral root development. Furthermore, Cho et al. showed direct regulation of AtSK21/BIN2 by a receptor-like kinase, TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR RECEPTOR (TDR). TDR protein, which belongs to LEUCINE-RICH REPEAT RECEPTOR-LIKE KINASE (LRR-RLK) family, was identified as a AtSK21/BIN2 interacting partner in yeast two-hybrid assay (Cho et al., 2014). TDR acting as a receptor of the TDIF peptide is known to promote procambial

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cell proliferation and inhibit xylem differentiation. Importantly, TDIF peptide-induced lateral root formation was abolished by bikinin treatment. In the presence of TDR and TDIF, AtSK21/BIN2-medated ARF7 activation was greatly increased in protoplasts, implying that the TDIF-TDR module activates AtSK21/BIN2 phosphorylation of ARF7 during lateral root

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development. In this scenario, BR seems to have a minor role in AtSK21/BIN2-mediated lateral root development, although previous studies showed that BR promotes lateral root

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development. It remains to be determined whether AtSK21/BIN2 activated by TDIF-TDR has an effect on the transcriptional activity of the BZR family and how it releases distinct signaling output independently of downstream BR signaling.

Emerging Roles of AtSKs in Response to Abiotic and Biotic Stress Very early studies suggested that plant GSK3-like kinases might function in response to abiotic stress. RT-PCR analysis showed that three AtSK members (AtSK13, AtSK31, and 15

ACCEPTED MANUSCRIPT AtSK42) are transcriptionally up-regulated by salt stress (Charrier et al., 2002). AtSK22/BIL2 is specifically induced by NaCl and ABA (Piao et al., 2001), and heterologous expression of AtSK22/BIL2 rescued salt stress-sensitive yeast mutant (Piao et al., 1999). Transgenic Arabidopsis overexpressing AtSK22/BIL2 shows increased expression of salt stressresponsive genes, results in enhanced resistance to salt stress (Piao et al., 2001). The

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functional involvement of AtSK11 in abiotic stress responses has been recently elucidated (Dal Santo et al., 2012). GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G6PD), which regulates the cellular redox state, is activated by AtSK11 phosphorylation, leading to the tolerance for salt in Arabidopsis. High salinity increases the kinase activity of AtSK11 and

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impaired salt resistance is observed in the atsk11 knockout mutant. Salt-induced G6PD activity is increased in the atsk11 knockout and decreased in the AtSK11-overexpressing

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mutant. In particular, it was revealed that AtSK11 activates cytosolic G6PD6. Combinatory analysis of mass spectrometry and mutational studies confirmed that the Thr467 residue of G6PD6 is a AtSK11 phosphorylation site. Whereas a T467A mutation of G6PD6 diminished AtSK11-mediated activation, G6PD6 carrying a phospho-mimic T467E mutation showed elevated catalytic activity in vitro and in vivo, suggesting that AtSK11 activates G6PD6

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through phosphorylation of the Thr467 residue of G6PD6 (Dal Santo et al., 2012). Although the bin2-1 gain-of-function mutant was originally isolated as a BRinsensitive mutant, the ABA-hypersensitive phenotype of the bin2-1 mutant in primary root inhibition assay was also reported (Li et al., 2001), implicating AtSK21/BIN2 as a mediator

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of ABA signaling. Cai et al. recently further confirmed that the triple knockout mutant of AtSK subgroup II (bin2-3bil1bil2) is hyposensitive to ABA while bin2-1 is hypersensitive to

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ABA both in primary root inhibition assay and ABA-induced gene expression (Cai et al., 2014). They found that two SNF1-RELATED KINASE 2s (SnRK2s), SnRK2.2 and SnRK2.3, are phosphorylated by AtSK21/BIN2 in vitro and in vivo. AtSK21/BIN2 phosphorylation enhanced the ability of SnRK2.3 to phosphorylate its downstream substrate ABA RESPONSE ELEMENTS-BINDING FACTOR 2 (ABF2). Of four AtSK21/BIN2 phosphorylation sites (Ser172, Ser176, Ser177, and Thr180) of SnRK2.3 identified in vitro, the Thr180 residue of SnRK2.3 that is crucial for its catalytic activity was defined as a major AtSK21/BIN2 phosphorylation site. Thr180 phosphorylation of SnRK2.3 in Arabidopsis was greatly reduced by bikinin treatment and transgenic plants expressing SnRK2.3 T180A was 16

ACCEPTED MANUSCRIPT less sensitive in ABA-induced gene expression. This study revealed a novel mechanism by which AtSK21/BIN2 positively regulates ABA signaling through direct targeting of Arabidopsis SnRK2 proteins (Cai et al., 2014). It remains to be elucidated whether ABA signaling directly regulates AtSK21/BIN2 activity, and the mechanism by which

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AtSK21/BIN2 is activated by abiotic stress will also requires further investigation. Plant GSK3-like kinases seem to be involved in pathogen responses regulated by the MAP kinase pathway. Treatment of the elicitor cellulose rapidly decreased protein level as well as the kinase activity of Medicago GSK3-like kinase MsK1. Transgenic Arabidopsis

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overexpressing MsK1 showed both enhanced susceptibility to virulent Pseudomonas syringae and reduced MPK3/6 activation, indicating that MsK1 might negatively regulate plant immune response (Wrzaczek et al., 2007). However, specific substrate of plant GSK3-

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like kinases that mediates immune responses has not yet been determined. Interestingly, AtSK21/BIN2 phosphorylates geminivirus pathogenicity protein C4 (Piroux et al., 2007). Although biological relevance remains to be determined, this implies that AtSKs might

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modulate the interaction between pathogen and host cell.

AtSKs and Flower Development

AtSK11 and AtSK12 are highly expressed in floral organs. Consistently, genetic manipulation of AtSK11 and AtSK12 through antisense approach causes abnormal flower development

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carrying increased number of petal and sepals, and alteration of gynoecium patterning (Dornelas et al., 2000). However, because their specific substrates, the proteins specifying

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flower development are still unknown, action mechanism of AtSKs in floral organ development remains to be determined. Interestingly, overexpression of AtSK32 carrying a mutation, which impairs priming phosphate-binding pocket, caused development of smaller floral organs, while that of wild-type AtSK32 did not alter flower morphogenesis. Primed substrate may be required for AtSK32-mediated flower development (Claisse et al., 2007).

RICE GSK3-LIKE KINASES AND BR SIGNALING 17

ACCEPTED MANUSCRIPT The rice genome contains nine GSK3-like kinases that also classified into four subgroups (Figure 1). Of these, the two rice GSK3-like kinases that have the highest homology with Arabidopsis AtSK21/BIN2 were analyzed by genetic studies (Figure 1). The OsSK21 T-DNA insertional knockout mutant showed enhanced sensitivity to BL in coleoptile elongation and BR-regulated gene expression. While ectopic overexpression of OsSK21 in Arabidopsis

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caused stunted growth phenotype, the OsSK21 knockout mutant showed a more obvious phenotype in response to abiotic stress. OsSK21 is likely to act as a negative regulator in responses to abiotic stress, such as drought, heat and cold (Koh et al., 2007). However, by contrast, AtSK11 and AtSK22/BIL2 exert positive roles to stress tolerance (Dal Santo et al.,

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2012; Piao et al., 2001). It might be possible that the function of GSK3-like kinases in stress responses has evolved oppositely depending on species specificity or their habitats.

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On the other hand, recently, the functional role of OsSK22 was elucidated in detail. Overexpression of OsSK22 possessing a gain-of-function mutation corresponding with Arabidopsis bin2-1 or bin2-2 caused phenotypes typical of BR-insensitive rice mutants, such as dwarf, dark green and curly leaves, and the insensitivity to BL. Furthermore, phenotypes induced by RNAi suppression of OsSK22 were very similar to those of the m107 mutant that

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overproduces endogenous BR, but opposite to those of gain-of-function mutant of OsSK22. The phosphorylated forms of a rice ortholog of Arabidopsis BZR1 (OsBZR1) were increased by overexpression of OsSK22, suggesting that OsSK22 mediates BR signaling through OsBZR1 phosphorylation in rice (Tong et al., 2012).

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In addition to OsBZR1, two substrates of OsSKs have recently been characterized as BR signaling components (Tong et al., 2012; Zhang et al., 2012). DWARF AND LOW-

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TILLERING (DLT), which encodes a member of the GRAS family, mediates BR responses in rice. Phenotypic and genomic analysis of DLT knockout and overexpression mutants suggested that DLT positively regulates BR signaling in rice. Genetic studies further confirmed that DLT functions downstream of OsSK22. The function of DLT in rice BR signaling has high similarities to OsBZR1. DLT is directly regulated by OsSK22 phosphorylation. DLT was detected with two bands in immunoblots, which corresponded to phosphorylated and dephosphorylated forms of DLT. BR treatment induced the accumulation of dephosphorylated DLT protein whereas BRZ reduced DLT protein level (Tong et al., 2012). 18

ACCEPTED MANUSCRIPT It is possible that DLT cooperatively regulates the downstream events of BR signaling together with OsBZR1. How DLT regulates BR-regulated gene expression as a transcription factor remains unknown. In contrast to DLT, LEAF AND TILLER ANGLE INCREASED CONTROLLER

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(LIC) plays a negative role in BR signaling (Zhang et al., 2012). In BR-induced rice lamina inclination assay, the lic-1 gain-of-function and LIC overexpression mutant showed greatly reduced lamina joint angles, whereas the LIC antisense line is hypersensitive to BR. BR induces dephosphorylation of LIC, leading to the nuclear localization of LIC.

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Dephosphorylated LIC accumulates in the nucleus. It was revealed that LIC interacts with OsSK21, OsSK24, and even Arabidopsis AtSK21/BIN2. OsSK21 phosphorylation of LIC was demonstrated by an in vitro kinase assay. LIC phosphorylation by OsSK21 reduces

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nuclear localization of LIC. Co-transformation of OsSK21 with LIC in tobacco leaf cells reduced the nuclear localization of LIC. A delicate feedback loop has been suggested, in which LIC and BZR1 act as a pair of antagonistic partners in their transcriptional regulation (Zhang et al., 2012). Expression of BZR1 was increased by low concentration of BR while LIC was only induced by high concentration of BR. LIC repressed the expression of BZR1

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and a BZR1 target gene, ILI1, through direct binding on their promoter. Inversely, BZR1 bound to the promoter of LIC, and BZR1 RNAi caused up-regulation of LIC. Thus, LIC is likely to control the negative feedback loop of BR signaling, which alleviates BZR1 activity. These studies provide an interesting “seesaw” mechanism of dynamic transcriptional

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regulation, in which OsSKs play a pivotal role in determination of the subcellular localization

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of BZR1 and LIC (Zhang et al., 2012).

REGULATIONS OF PLANT GSK3-LIKE KINASES BR SIGNALING

Regulation mechanism of plant GSK3-like kinases has been first demonstrated in BR signaling. In order to activate BR-responsive BZR transcription factors, upstream BR signaling inhibits the catalytic activity of AtSK21/BIN2 phosphorylating BZR transcription factors. In the presence BR, upon BR binding to BRI1, BRI1 forms a receptor complex with 19

ACCEPTED MANUSCRIPT BRI1-ASSOCIATED KINASE 1 (BAK1), leading to the sequential transphosphorylation and full activation of BRI1 (Wang et al., 2008). Activated BRI1 then phosphorylates RECEPTOR-LIKE CYTOPLASMIC KINASES (RLCKs). BR SIGNALING KINASE 1 (BSK1), or CONSTITUTIVE DIFFERENTIAL GROWTH 1 (CDG1), is phosphorylated by BRI1, resulting in an increase in binding affinity to BSU1 phosphatase (Kim et al., 2011; Kim

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et al., 2009; Tang et al., 2008a). BSU1 is activated by CDG1 phosphorylation or unknown mechanism via BSK1 interaction (Kim et al., 2011). BSU1 then inhibits AtSK21/BIN2, but not bin2-1, through dephosphorylation of the phosho-Tyr200 residue. A phospho-peptide of AtSK21/BIN2 containing phospho-Tyr200 residue was identified both in vitro and in vivo. In

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addition, a mutation of Tyr200 to Phe200 (Y200F) in AtSK21/BIN2 greatly reduces phosphorylation of BZR1 as well as autophosphorylation of AtSK21/BIN2 in vitro,

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suggesting that a phospho-Tyr200 residue of AtSK21/BIN2 is essential for full kinase activity of AtSK21/BIN2 (Kim et al., 2009). Because the phospho-Tyr residue of AtSKs is the only in vivo phosphorylation site detected by proteomic studies so far, change in the phosphorylation status of Tyr residue of AtSKs is believed to be the primary mechanism that regulates GSK3like kinases in Arabidopsis. Nevertheless, it is possible that AtSKs might also be regulated by another phosphorylation event or protein-protein interaction similar to those observed in

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mammal GSK3s.

Notably, mutant bin2-1 protein accumulates in greater quantities than wild-type AtSK21/BIN2 in plant cells (Peng et al., 2008). The BR biosynthetic inhibitor

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BRASSINAZOLE (BRZ) induces the accumulation of AtSK21/BIN2 in plant cells. In contrast, the protein level of AtSK21/BIN2, but not of bin2-1, is rapidly reduced by

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BRASSINOLIDE (BL, the most active BR) treatment, which can be suppressed by treatment with MG132, an inhibitor of 26S proteasome (Peng et al., 2008). Dephosphorylation of AtSK21/BIN2 by BSU1 is thought to induce AtSK21/BIN2 degradation because overexpression of BSU1 decreases the protein level of AtSK21/BIN2 (Kim et al., 2009). However, the detailed mechanism that regulates AtSK21/BIN2 degradation requires further study for elucidation.

TDIF/TDR Pathway 20

ACCEPTED MANUSCRIPT More recently, Kondo et al. demonstrated that a TDIF-TDR module activates AtSK members in procambial cells (Kondo et al., 2014). Six AtSK members that belong to subgroups I and II strongly interact with the TDR kinase domain in yeast cells. FRET analysis revealed that physical interaction between TDR and AtSK21/BIN2 is decreased by overexpression or treatment with TDIF. In contrast, AtSK21/BIN2 binding to kinase-inactive TDR was not

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inhibited by TDIF, suggesting that TDIF-induced dissociation of AtSK21/BIN2 from TDR is dependent to kinase activity of TDR. Cytoplasmic retention of BZR1-CFP caused by AtSK21/BIN2 phosphorylation was increased by TDIF treatment in wild-type procambial cells, but not in those of the tdr-1 mutant. The effect of TDIF blocking xylem differentiation

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was diminished in the AtSKs quadruple mutant (bin2-3bil1bil2/AtSK13RNAi). Genetic and anatomical analyses showed that AtSK21/BIN2 regulates xylem differentiation through the

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inhibition of BES1, but not BZR1, in procambial cells. This study provides a novel concept in which another LRR-RLK regulates BZR family members through AtSKs activation independently of BRI1 (Kondo et al., 2014). Further study is needed to better understand BRI1-independent AtSK21/BIN2 regulation, and the mechanism by which the TDIF-TDR

HSP90

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module activates AtSK21/BIN2.

Nuclear localization of AtSKs is crucial for inhibition of BZR1 and BES1 in BR signaling.

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Nuclear accumulation of AtSK21/BIN2 and AtSK12 caused by NLS tagging and deletion of N-terminal domain, respectively, induced severe growth defects, suggesting that AtSKs more

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effectively inhibit BR signaling in the nucleus (Ryu et al., 2010b; Youn et al., 2013). A recent study showed that the subcellular localization of AtSK21/BIN2 is regulated by Arabidopsis HEAT SHOCK PROTEIN 90s (HSP90s) (Samakovli et al., 2014). Whereas BR induced the cytoplasmic localization of HSP90s, nuclear localization of both HSP90s and AtSK21/BIN2 was blocked by long-term treatment with the HSP90 inhibitor geldanamycin. Two members (HSP90.1 and HSP90.3) of the HSP90 family physically interact with AtSK21/BIN2, which their interaction is predominantly observed in the nucleus. In contrast, BR treatment promoted the cytoplasmic localization of HSP90-AtSK21/BIN2 complex (Samakovli et al., 2014). It has not been confirmed whether BR promotes the cytoplasmic accumulation of 21

ACCEPTED MANUSCRIPT AtSK21/BIN2. The subcellular localization of HSP90, but not of AtSK21/BIN2, might affect their interaction. Examination of the subcellular localization of AtSK21/BIN2 in the hsp90s knockout mutant will aid in the elucidation of whether HSP90s are essential to determine the

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subcellular localization of AtSK21/BIN2 in BR signaling.

AGB1

It has been proposed that HETEROTRIMERIC GTP-BINDING PROTEINS (G-protein) may

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modulate BR signaling because the phenotypes and physiological responses of G proteinknockout mutants are similar to those of BR-deficient or BR-insensitive mutants (Chen et al., 2004; Gao et al., 2008; Ullah et al., 2002). However, how G proteins participate in the BR

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signaling has remained unknown. Recently, it was demonstrated that Arabidopsis G protein β subunit, AGB1, interacts with AtSK21/BIN2 without phosphorylation, resulting in the activation of BR responses independently of BZR1 (Tsugama et al., 2013). This could be an example of a scaffold protein that modulates the activity of AtSK21/BIN2 in plant cells. However, the physiological relevance of AGB1 binding to AtSK21/BIN2 has not yet been

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determined.

CONCLUSIONS AND PERSPECTIVES

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Versatile roles for GSK3-like kinases in a wide range of physiological and developmental programs are emerging with identification of their novel substrates. So far, it has been

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revealed that plant GSK3-like kinases regulate growth, root and vascular development, flower and stomatal development, and environmental responses such as light, and biotic and abiotic stress (Figure 2). Considering that plants contain divergent GSK3-like kinases, many more GSK3-like kinases substrates may be further identified. This will allow us to understand how plants coordinate complicate physiological processes in response to diverse environmental stimuli. In particular, plant GSK3-like kinases play pivotal roles in the crosstalk between multiple signal transduction pathways. Therefore, deciphering detailed relationships between GSK3-like kinases and their substrates in various signal transduction 22

ACCEPTED MANUSCRIPT pathways will shed light on how internal and external signals are integrated and branched in plant cells. Our knowledge of GSK3-like kinases and their substrates is rapidly expanding. However, it is important to validate whether identified substrates are true substrates in

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physiological conditions. Plant GSK3-like kinases tend to show a broad range of substrate specificity in vitro, yielding non-specific phosphorylation in in vitro kinase assays. It should be further supported by combined experiments including in vivo interaction, identification of in vivo phosphorylation site(s), co-expression and co-localization in same cells or tissues, and

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their relevance in physiological or developmental regulation.

One major issue facing researchers is uncovering the detailed and distinctive

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regulation mechanism for GSK3-like kinases in different cellular responses. Given that the BR-independent GSK3-mediated pathway is operational, the existence of novel mechanism(s) to regulate the activity of plant GSK3-like kinases is speculated. In addition, identification of the ubiquitin E3 ligase for GSK3-like kinases is important in the process of understanding how GSK3-like kinases are degraded by 26S proteasome in plant cells.

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Unlike animals, in which most of substrates are negatively regulated by GSK3 phosphorylation, the phosphorylation by plant GSK3-like kinases gives rise to positive regulations as well as negative regulations toward substrates. It will be interesting to understand how plant GSK3-like kinases have different effects on their different substrates

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through phosphorylation. For example, transcriptional activities of two different transcription factors, BZR1 and MYBL2, are oppositely regulated by AtSK21/BIN2 phosphorylation. One

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simple explanation is that the catalytic function of substrates might be differently affected by the location or number of phosphorylated amino acids according to their structural characteristics. Nevertheless, further detailed investigation is required for a better understanding the biochemical regulatory mechanism by plant GSK3-like kinases. Although plant GSK3-like kinases are mainly detected in the nucleus and cytoplasm,

some evidence indicates that GSK3-like kinases might also function in other subcellular organelles such as the plasma membrane and plastids. Together with the substrate specificity of GSK3-like kinases, elucidation of cell-type or organelle-specific regulation of substrates 23

ACCEPTED MANUSCRIPT by GSK3-like kinases will be essential for understanding the crosstalk network of GSK3-like kinase-mediated cellular signaling.

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FUNDING This work was supported by grants from the Next-Generation BioGreen 21 Program (SSAC, PJ009026), Rural Development Administration, and Basic Science Research Program (2012R1A1A1011986) through the National Research Foundation of Korea (NRF) funded by

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the Ministry of Science, ICT & Future Planning, Republic of Korea.

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ACKNOWLEDGEMENT

We apologize to our colleagues whose work could not be included due to space limitation.

FIGURE LEGEND

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Figure 1. Classification and phylogenetic tree of Arabidopsis and rice GSK3-like kinases. (A) GSK3-like kinase family of Arabidopsis thaliana and Oryza sativa is classified into four subgroups (I~IV). We propose to unify their official name as AtSKs (Arabidopsis thaliana Shaggy/GSK3-Like Kinases) or OsSKs (Oryza sativa Shaggy/GSK3-Like Kinases). (B)

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Phylogenetic analysis of Arabidopsis and rice GSK3-like kinases. Amino acid sequences of GSK3-like kinases, which were obtained from TAIR and TIGR resources, were aligned with

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ClustalW. The evolutionary history was inferred using the Maximum Parsimony method (Nei and Kumar, 2000). Evolutionary analyses were conducted in MEGA6 (Tamura et al., 2013). The scale bar indicates 10 amino acid substitutions.

Figure 2. Signaling network mediated by GSK3-like kinases in Arabidopsis and rice. Arrows and bar ends indicate activation and inhibitory action, respectively, while dotted lines indicate hypothetical regulation. Substrates phosphorylated by GSK3-like kinases are marked with red 24

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circles containing the letter P.

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ACCEPTED MANUSCRIPT Table 1. Substrates phosphorylated by GSK3-like kinases in Arabidopsis and rice. Name

P site(s) a

Effect toward substrate(s)

G6PD6

ND b T467*

Inhibition Activation

Promotion of stomatal development (leaves) Enhancement of tolerance for salt stress

BZR1

ND

Inhibition

Suppression of BR signaling and cell growth

BSKs

ND S102, S130, S134, S171, S173, S177, S181, S185, S220*, S224* S113, S117, S121, S125, S129, S133, S137, S171, S175

ND

Substrate

Function of GSK3-like kinase through substrate phosphorylation

Reference(s)

< Arabidopsis thaliana >

BZR1

BES1

Suppression of BR signaling and cell growth

Inhibition

Suppression of BR signaling and cell growth

Ryu et al., 2010a

Feedback regulation of BR signaling Feedback regulation of BR signaling ND Promotion of stomatal development (leaves) Promotion of stomatal development (leaves)

Poppenberger et al., 2011 Vert et al., 2008 Bernando-Garcia et al., 2014 Ye et al., 2012 Zhang et al., 2014 Wang et al., 2009 Kim et al., 2012 Khan et al., 2013

Inhibition of stomatal development (hypocotyls)

Gudesblat et al., 2012

Promotion of root hair development Promotion of root hair development Potentiation of lateral root development Potentiation of lateral root development Enhancement of ABA signaling Enhancement of ABA signaling

Cheng et al., 2014 Cheng et al., 2014

ND

ND

ARF2

ND

Inhibition

PIF4

T160, S164, S168

Destabilization

MYBL2 HAT1 AIF1 YDA MKK4

Stabilization Stabilization ND Inhibition Inhibition

EGL3 TTG1 ARF7 ARF19 SnRK2.2 SnRK2.3

ND ND ND 185-322 aa S230, T234* S38, T40, S43, T44, S65*, S171*, S177, S181, S186*, S193, T214, S219* T209, T213, T399, T403 ND S698, S707 ND ND S172, S176, S177, T180

BSKs

ND

ND

ND

Inhibition

Suppression of BR signaling and cell growth

ND

Inhibition

Suppression of BR signaling and cell growth

ND

Inhibition

Suppression of BR signaling and cell growth

Rozhon et al., 2011

ND

Inhibition

Promotion of stomatal development (leaves)

Khan et al., 2013

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BZR1/BES1

AtSK23/BIL1

BZR1/BES1

AtSK32

BZR1/BES1 /BEH2 MKK4

< Oryza sativa >

a

Inhibition

CESTA

ND

Inhibition of Auxin signaling

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SPCH

AtSK22/BIL2

ND

Inhibition

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AtSK21/BIN2

Khan et al., 2013 Dal Santo et al., 2012 Kim et al., 2009 Youn et al.,2013 Sreeramulu et al., 2013 Gampala et al., 2007 Ryu et al., 2007, Tang et al., 2008b

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AtSK12

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MKK4

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AtSK11

Inhibition Inhibition Activation Activation Activation Activation

Regulation of rhythmic hypocotyls growth

ND

Cho et al., 2014 Cai et al., 2014 Sreeramulu et al., 2013 Ryu et al., 2007 Yan et al., 2009 Ryu et al., 2007 Yan et al., 2009

OsSK21

OsBZR1 LIC

ND 188-255 aa

Inhibition Inhibition

Suppression of BR signaling Enhancement of BR signaling

Bai et al., 2007 Zhang et al, 2012

OsSK22

OsBZR1 DLT

ND ND

Inhibition Inhibition

Suppression of BR signaling Suppression of BR signaling

Tong et al., 2012

OsSK24

LIC

188-255 aa

Inhibition

Suppression of BR signaling

Zhang et al., 2012

b

Phosphorylation site, Not determined, * in vivo phosphorylation site. 26

ACCEPTED MANUSCRIPT REFERENCES

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31

ACCEPTED MANUSCRIPT

ASK5, ASK

At5g14640

AtSK21/BIN2

ASK7, ASK , BIN2, UCU1, DWF12

At4g18710

AtSK22./BIL2

ASK9, ASK , BIL2, AtGSK1

At1g06390

AtSK23/BIL1

ASK6, ASK , BIL1

At2g30980

AtSK31

ASK2, ASK

At3g61160

AtSK32

ASK8, ASK

At4g00720

AtSK41

ASK10, ASK , AtK-1

At1g09840

AtSK42

ASK4, ASK

At1g57870

OsGSK3

OsSK13

OsGSK6, OsGSK3, GSK1

Os05g04340

OsSK21

OsGSK1, OSKζ

Os01g10840

OsSK22

OsGSK7, GSK2

Os05g11730

OsSK23

OsGSK4, GSK3

Os02g14130

OsSK24

OsGSK8, GSK4, OSK , SK

Os06g35530

III

OsSK31

OsGSK9

Os10g37740

IV

OsSK41

OsGSK5

Os03g62500

Os01g19150

10

OsSK41

AC C

IV

AtS K4 1

II

EP

OsSK12

Os01g14860

4 K2

OsGSK2, OSK

2 K3

OsSK11 I

Locus

A tS

Other names

1 K2

Os SK 23

3 K1 tS

1 OsSK3

Proposed name

12

Clade

AtSK11

A

S At

21 SK Os OsSK 22

K

Oryza sativa

II 2 AtSK2 At SK 23

AtSK13

SC

At3g05840

M AN U

ASK3, ASK

S Os

AtSK12

I

Os SK 13

At5g26751

11

ASK1, ASK

tS

IV

AtSK11

A

III

Locus

K OsS

II

Other names

OsSK12

I

Proposed name

TE D

Clade

RI PT

B

Arabidopsis thaliana

K4 2 AtS

A

At S

K3 1

III

Figure 1

ACCEPTED MANUSCRIPT

Tolerance for abiotic stress

P

P

P

G6PD6

P

P

Xylem differentiation

EGL3, TTG1

TDR

P

LIC

P

ARF7/19

GSK3GSK3-like kinases

P

P

BSKs

CESTA

P

P P P P

BZRs

ARF2

MYBL2

HAT1

AIF1

PIF4

AC C

BSUs

EP

TE D

DLT

P

Hypocotyl growth

P

Auxin signaling

M AN U

OsBZR1

AGB1

SnRK2s

SC

SPCH

Root hair development

Lateral root development

BR signaling & growth regulation (rice)

YDA

HSP90s

P

MKK4/5

P

ABA signaling

RI PT

Stomatal development

BR signaling & growth regulation

Figure 2