Specific Binding of a 14-3-3 Protein to Autophosphorylated WPK4, an SNF1-related Wheat Protein Kinase, and to WPK4-phosphorylated Nitrate Reductase*

WPK4 is a wheat protein kinase related to the yeast protein kinase SNF1, which plays a role in catabolite repression. To identify proteins involved in signal transduction through WPK4, we performed yeast two-hybrid screens and isolated two cDNA clones designated as TaWIN1 and TaWIN2. Both encode 14-3-3 proteins that, upon autophosphorylation, bind the C-terminal regulatory domain of WPK4. Mutational analysis through amino acid substitution revealed that TaWIN1 and TaWIN2 primarily bind WPK4 through phosphoserines at the positions 388 and 418, both located in the C-terminal region. Mutations in the conserved residues of the TaWIN1 amphipathic groove impaired the ability of TaWIN1 to bind to WPK4. A screen for in vitro phosphorylation of proteins involved in nutrient metabolism revealed a putative WPK4 substrate, nitrate reductase; its hinge 1 region was efficiently phosphorylated by WPK4. Subsequent far Western blots showed that it specifically bound TaWIN1. Since nitrate reductase has been shown to be inactivated by phosphorylation upon 14-3-3 binding, the present findings strongly suggest that WPK4 is the protein kinase responsible for controlling the nitrogen metabolic pathway, assembling the nitrate reductase and 14-3-3 complex through its phosphorylation specificity.

WPK4 is a wheat protein kinase related to the yeast protein kinase SNF1, which plays a role in catabolite repression. To identify proteins involved in signal transduction through WPK4, we performed yeast two-hybrid screens and isolated two cDNA clones designated as TaWIN1 and TaWIN2. Both encode 14-3-3 proteins that, upon autophosphorylation, bind the C-terminal regulatory domain of WPK4. Mutational analysis through amino acid substitution revealed that TaWIN1 and TaWIN2 primarily bind WPK4 through phosphoserines at the positions 388 and 418, both located in the C-terminal region. Mutations in the conserved residues of the TaWIN1 amphipathic groove impaired the ability of TaWIN1 to bind to WPK4. A screen for in vitro phosphorylation of proteins involved in nutrient metabolism revealed a putative WPK4 substrate, nitrate reductase; its hinge 1 region was efficiently phosphorylated by WPK4. Subsequent far Western blots showed that it specifically bound TaWIN1. Since nitrate reductase has been shown to be inactivated by phosphorylation upon 14-3-3 binding, the present findings strongly suggest that WPK4 is the protein kinase responsible for controlling the nitrogen metabolic pathway, assembling the nitrate reductase and 14-3-3 complex through its phosphorylation specificity.
Members of the SNF1-related protein kinases play a central role in phosphorylation cascades involved in carbon assimilation in animals, fungi, and plants (1). In Saccharomyces cerevisiae, the SNF1 complex is activated in response to glucose deprivation and inhibits the repression of many glucose-repressed genes, including genes involved in alternate carbon source utilization, respiration, and gluconeogenesis (1).
In plants, more than 20 genes encoding the SNF1-related protein kinases (SnRKs) 1 have been identified (2). Based on sequence similarity, they are classified into three distinct subgroups (SnRK1-SnRK3) of which SnRK1 has been the best characterized (2). For example, SnRK1s from rye (RKIN1), tobacco (NPK5), and Arabidopsis thaliana (AKIN10, AKIN11) were shown to complement yeast snf1 mutant cells (2). Antisense expression of a potato SNF1 homolog resulted in a loss of sugar-inducible expression of sucrose synthase (3). These observations suggest that members of the SnRK1 subgroup are SNF1 orthologs that activate sugar-inducible gene expression in sugar signaling pathways (2). However, the physiological functions of the other two subgroups, SnRK2 and SnRK3, remain relatively poorly characterized.
One method of determining the physiological role of SnRKs is to identify proteins that interact with them. For example, SNF1 and its mammalian homolog AMPK, a master switch that regulates carbohydrate and fat metabolism (4), combine with other proteins to form a heterotrimer that includes both catalytic subunits, SNF1p and AMPK-␣, respectively, and noncatalytic subunits including SIP1p (SIP2p, GAL83p, AMPK-␤) and SNF4p (AMPK-␥) (4). In the case of SnRKs, a recent survey using yeast two-hybrid system also revealed that the A. thaliana SnRK, AKIN␣1, interacted with noncatalytic subunits AKIN␤1/␤2 and AKIN␥ (1). AKIN10 and AKIN11 were also shown to form complexes with the protein PRL1 (5). CIPK, a member of the SnRK3 subgroup, was found to interact with an EF-hand-type calcium-binding protein similar to yeast calcineurin B (6). These observations clearly indicate that SnRKs function through specific interaction with signaling protein factors involved in nutrient metabolism.
Previously we reported that WPK4, a gene encoding a wheat SnRK3, was up-regulated by light and cytokinins and downregulated by nutrients (7). Primary sequence analysis revealed that C-terminal region of WPK4 is more distantly related than other SnRK subgroups, which suggests that WPK4 may possess another function in nutrient signal transduction pathways (8). To identify target proteins that interact with WPK4, we used the yeast two-hybrid system to identify two cDNA clones. Both encode 14-3-3 proteins that bound WPK4-phosphorylated nitrate reductase.

EXPERIMENTAL PROCEDURES
Yeast Two-Hybrid Screen and Quantitative ␤-Galactosidase Assays-Yeast two-hybrid screening was conducted using a HybriZAP-2.1 twohybrid vector system (Stratagene). The cDNA library was prepared from 7-day-old wheat seedlings grown in water and treated with 100 M N 6 -benzylaminopurine for 24 h using a ZAP-cDNA synthesis kit (Stratagene). A HybriZAP two-hybrid vector was used to construct a GAL4 activation domain fusion cDNA library (2 ϫ 10 7 plaque-forming units) that was converted to a yeast plasmid library by in vivo excision. pBD-WPK4, in which WPK4 cDNA was inserted in-frame with a right orientation into a pBD-GAL4 Cam vector, was used as the bait plasmid. The yeast strain YRG2 (Stratagene), which carries pBD-WPK4, was * This work was supported by Grant-in-aid 09274102 from the Ministry of Education, Science, Sports, and Culture of Japan and Grant for the Future Program JSPS-RFTF97R16001 from the Japan Society for the Promotion of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AB042193 (TaWIN1) and AB042194 (TaWIN2).
subsequently transformed with the library plasmids.
Expression and Purification of Recombinant Proteins-Glutathione S-transferase (GST) and its fusion derivatives were expressed in Escherichia coli DH5␣ cells and purified as described (8). His-tagged proteins were expressed in the E. coli strain BL21 (DE3) and purified by Ni-NTA superflow resin (Qiagen). Purified proteins were dialyzed and stored in 30% glycerol until use.
Gel Filtration Analysis-GST-TaWIN1 fusion proteins were retained in a glutathione-Sepharose 4B resin. Beads were then incubated with T buffer containing 100 units of thrombin (Amersham Pharmacia Biotech) at 4°C for 24 h. TaWIN1, purified with a DEAE-Toyopearl column, was put through high resolution gel filtration chromatography and equilibrated with 2ϫ phosphate-buffered saline.
In Vitro Binding Assays-For GST pull-down assays, His-tagged TaWIN1 protein was dissolved in a protein binding buffer (20 mM Tris-HCl (pH 7.5), 140 mM KCl, 10 mM 2-mercaptoethanol, 0.5 mM EDTA (pH 8.0), 5 mM MgCl 2 , 0.5% Tween 20) to a final concentration of 0.05%. Glutathione-Sepharose beads bound to GST, GST-WPK4, or GST-K75D were incubated in a kinase buffer (20 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), 20 mM MgCl 2 , and 20 M ATP) at room temperature for 1 h and washed with the protein binding buffer. 20 l of each sample of glutathione-Sepharose beads was added to 50 l of the Histagged TaWIN1 protein solution, then incubated at 4°C for 1 h. Proteins were released from the resins by boiling in Laemmli's sample buffer and separated by 12.5% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. His-TaWIN1 was detected using an anti-(His) 5 monoclonal antibody (Novagen) and chemilluminescence (ECL, Amersham Pharmacia Biotech).
Far Western Blot Analysis-A previously described protocol for far Western blotting was employed, with modifications (9). GST, GST-HMGR, and GST-nitrate reductase (GST-NR) proteins (1 g of each) were phosphorylated by GST-WPK4 in a kinase buffer at 30°C for 30 min, separated by SDS-PAGE, transferred onto a polyvinylidene difluoride membrane, denatured, and renatured with guanidine-HCl. Membranes were kept in a blocking buffer (phosphate-buffered saline, 1% bovine serum albumin, and 0.05% Tween 20) at room temperature for 1 h, then incubated with the 0.5% His-TaWIN1 fusion protein in the same buffer at room temperature for 1 h. Immunological detection was performed as described above.

RESULTS
Isolation of WPK4-interacting Factors-Screening 1 ϫ 10 6 clones by the yeast two-hybrid system, isolated three clones that activated two reporter genes, HIS3 and lacZ. Sequence analysis revealed that two were derived from the same gene and were designated as TaWIN1 (Triticum aestivum WPK4interacting factor 1). The third showed a 70% similarity to TaWIN1 and was designated as TaWIN2. All three encoded 14-3-3 proteins (Fig. 1A). TaWIN1 encoded a polypeptide consisting of 266 amino acid residues with a relative molecular mass of 29.4 kDa. TaWIN1 showed the highest similarity to GF14d, isolated from rice (10). TaWIN2 encoded a polypeptide consisting of 259 amino acid residues with a relative molecular mass of 28.6 kDa. TaWIN2 belonged to the omega-type 14-3-3 proteins but is more distantly related (Fig. 1C). A putative EF-hand motif is also found in both TaWIN1 and TaWIN2 between helices 8 and 9, with the conserved Gly critical for calcium binding (11) (Fig. 1B).
Binding of TaWINs to Autophosphorylated WPK4 -A Thr residue, one of the autophosphorylation sites located in the activation loop, is well conserved in WPK4, SNF1, AMPK, and protein kinase A ( Fig. 2A). Substitution with Ala (T204A) reduced interaction with TaWIN1 and TaWIN2 to 76 and 72% that of wild type, respectively (Fig. 2B). By contrast, substitution with Glu (T204E) strengthened the interaction to 118 and 115%, respectively (Fig. 2B). The mutant lacking phosphorylation activity (K75D) (8) showed essentially no interaction with either TaWIN1 or TaWIN2 (Fig. 2B). The results indicate the necessity of kinase activity for the binding of WPK4 to TaWIN1 and TaWIN2 as well as the crucial role of the Thr in the activation loop. A GST pull-down assay showed that TaWIN1 specifically binds GST-WPK4 but not GST-K75D in vitro, indicating a direct interaction between WPK4 and TaWIN1 Fig. 2C).

Binding of TaWINs to the C-terminal Region of WPK4 -
There are two potential 14-3-3 binding motifs, RPASLN (with S388) and RFISGEP (with S418) in the C-terminal region of WPK4. To determine whether these motifs are indeed binding sites, we constructed mutants in which one or both of the two Ser residues were replaced with Ala (Fig. 3A). A third mutant lacking the C-terminal region was also constructed (⌬C). A quantitative liquid assay showed that the ⌬C mutation completely abolished the binding of WPK4 to TaWIN1 and The protein kinase catalytic domain is represented as a gray box, and the proline-rich region is represented as a hatched box. The upper five constructs were fused to the GAL4 DNA binding domain (GBD) and used as bait for testing TaWIN1 and TaWIN2 binding. Two constructs without the kinase domain were fused to GST; the resulting fusion proteins were purified and used for the binding assay. The positions of Ser to Ala substitutions are shown by solid bars. WT, wild type. B, interaction of wild-type and mutant WPK4 (S388A, S418A, S388/418A) with TaWIN1 and TaWIN2. Y190 cells were cotransformed with the bait and prey combinations shown at the left and quantitatively assayed for ␤-galactosidase activity. Mean values of three independent assays are indicated by bars. C, autophosphorylation and binding of the C-terminal region of WPK4 with TaWIN1 in vitro. 1 g of GST-⌬N or mutated GST-⌬N was incubated with 50 ng of GST-WKP4 fusion protein, separated by 12.5% SDS-PAGE, stained, and dried, followed by exposure to x-ray film (left and middle panels, respectively). Far Western blotting was performed on a separate set, as described under "Experimental Procedures" (right panel). CBB, Coomassie Brilliant Blue.
TaWIN2, thereby demonstrating that the C-terminal region is critical for interaction (Fig. 3B). Binding of the S388A mutant to TaWIN1 and TaWIN2 was reduced to 80 and 76% of the control, respectively; likewise, that of S418A reduced binding to 76 and 59%, respectively (Fig. 3B). The S388A/S418A double mutant had even further reduced binding, with only 25% of the control (Fig. 3B). These results show that the binding of TaWIN1 and TaWIN2 to WPK4 is primarily mediated by the phosphorylated Ser-388 and Ser-418 of WPK4. To determine whether these two Ser residues are sites of autophosphorylation, two N-terminal-truncated mutations (⌬N) containing only the 165-amino acid C-terminal region were constructed (Fig.  3A) and subjected to phosphorylation by GST-WPK4 (Fig. 3C). The results showed no clear difference between ⌬N and Ser-388/418A⌬N in phosphate receptor function, suggesting the presence of another phosphorylation site(s) in the C-terminal region. Far Western blotting analysis showed that phosphorylated Ser-388/418A⌬N was able to bind TaWIN1, although the efficiency was low (Fig. 3C). These results showed that, in addition to two Ser-containing regions described above, there is an additional binding site(s) for TaWIN1 in the C-terminal region of WPK4.
Molecular Properties of TaWIN1-In general, 14-3-3 proteins form dimers and their inner surfaces form amphipathic grooves that serve as the principle binding site for 14-3-3 ligands (12). Site-directed mutagenesis was used to change the conserved basic and hydrophobic residues located in the inner surface of TaWIN1. Basic residues within helix 3 (Lys-60, Arg-67) and hydrophobic residues within helices 7 and 9 (Leu-185, Val-189, Leu-233) were substituted with acidic residues (K60D, R67E, L185D, V189E, L233D) (Fig. 4A). Binding assays showed all of these residues to be critical for interaction between TaWIN1 and WPK4 (Fig. 4A). However, when the Gly in the putative EF-hand motif of TaWIN1 was substituted by Ser (G221S), no loss of binding activity was observed (Fig. 4A). This suggests that, at least in yeast cells, calcium ions are not required for WPK4/TaWIN1 interaction. The capacity for TaWIN1-TaWIN2 dimer formation was then analyzed by a GST pull-down assay, which clearly showed that TaWIN1 was able to bind TaWIN2 (Fig. 4B). This indicates that each are able to form heteromeric dimers. Purified recombinant TaWIN1 protein was subjected to gel filtration followed by SDS-PAGE, whereupon the relative molecular mass of each fraction after each step was estimated to be 64 and 32 kDa, respectively (Fig. 4C).
Binding of TaWIN1 to WPK-phosphorylated Nitrate Reductase-NR is inactivated by phosphorylation followed by 14-3-3 protein binding (13). A peptide from the hinge 1 region of tobacco NR was expressed as a GST fusion protein and subjected to a WPK4 phosphorylation assay (Fig. 5). The results showed that WPK4 phosphorylates GST-NR and GST-HMGR, but not GST (Fig. 5B). TaWIN1 was subsequently examined by far Western blotting for binding phosphorylated NR. It is clear that TaWIN1 efficiently binds phosphorylated NR, but not HMGR in this system (Fig. 5C). TaWIN1 was not found to bind unphosphorylated NR (Fig. 5C).
Ubiquitous Accumulation of TaWIN1 Transcripts-The localization of TaWIN1 in wheat tissues was examined by RNA blot analysis. Although WPK4 transcripts were detected in upper-ground tissues, especially in leaves, those of TaWIN1 were found in all tissues examined (Fig. 6A). The induction profiles of these transcripts were also examined. Although WPK4 transcripts accumulated in response to cytokinins and nutrient deprivation (Fig. 6, B and C) (7), transcripts of TaWIN1 were ubiquitously accumulated regardless of the presence or absence of these factors (Fig. 6, B and C). These results suggest that TaWIN1 is constitutively expressed without being affected by the external factors that regulate WPK4.

DISCUSSION
The present paper provides evidence that TaWINs, different isoforms of 14-3-3 proteins, interact with WPK4, and that TaWIN1 directly binds WPK4-phosphorylated NR. Yeast twohybrid screens were performed to find proteins that interact with WPK4, and distinct clones termed TaWINs were isolated. The primary sequence indicated that they belong to the 14-3-3 protein family. In general, 14-3-3 proteins bind ligands containing phosphorylated consensus motif RSXpSXP (pS is phosphoserine), found in Raf (14), Bad (15), and CDC25 (16), although unphosphorylated peptides have also been shown to be the target of 14-3-3 proteins (17). Our finding that TaWINs bind only autophosphorylated WPK4 suggests that its C-terminal region is only a pseudosubstrate region; this was directly shown for the case of Ser-388 and Ser-418 autophosphorylation, which occurs in the C-terminal region. Plant 14-3-3 proteins are involved in many cellular events, including enzyme regulation, gene expression, and protein translocation (18). Despite the diversity of these physiological functions, few reports have yet been presented on their interaction with protein kinases. One notable study found that they bind calcium-dependent protein kinase, resulting in enzymatic activation in vitro (19). By contrast, the binding of TaWINs to WPK4 did not influence kinase activity under our experimental conditions. 2 Nevertheless, these findings suggest that some protein kinases may generally interact with 14-3-3 proteins, which assist in the transduction of signals that the kinases receive.
The activity of NR, the key enzyme in plant nitrogen assimilation pathways, is both transcriptionally and post-translationally regulated (20). Post-translational regulation is performed by reversible phosphorylation with subsequent binding of inhibitor proteins (20,21). In previous studies, three protein kinases responsible for NR phosphorylation at Ser-543 were FIG. 6. Northern blot analyses of TaWIN1 transcripts. A, tissuespecific accumulation of TaWIN1 transcripts. Total RNA was extracted from spike, leaf sheath, leaf blade, and root, and a 10-g aliquot of each was used for hybridization assays. B, effect of N 6 -benzylaminopurine on TaWIN1 transcript accumulation. Six-day-old green seedlings, grown under continuous light conditions, were treated with 100 M N 6 -benzylaminopurine for the time period indicated; extracted RNAs were used for hybridization assays. C, effect of inorganic salt deprivation on TaWIN1 transcript levels. Six-day-old green seedlings, grown under continuous light in the 1:5 Murashinge and Skoog medium, were transferred to water for the time period indicated. Extracted RNAs were again used for hybridization assays. Hybridization was performed (8) with probes for a 0.3-kilobase 5Ј-untranslated region of TaWIN1 cDNA, a 0.8-kilobase 3Ј region of WPK4 cDNA, and a 1.2-kilobase wheat actin cDNA, respectively. FIG. 7. Proposed pathway for the regulation of NR by WPK4. WPK4 is transcriptionally activated by carbon deprivation (8). As a result, WPK4 autophosphorylates its own C-terminal region, thereby creating the TaWIN1 binding site. At the same time TaWIN1 bind, WPK4-phosphorylated NR, to which TaWIN1 is transferred from WPK4. This transfer forms an enzymatically inactive NR complex. Thus, carbon deficiency can coordinate the suppression of a nitrogen assimilation machinery of a cell. isolated from spinach leaves and designated PKI, PKII, and PKIII (22). PKII was shown to be a calcium-dependent protein kinase, and PKIII was shown to be an SNF1-related kinase (23). NR, which is reversibly phosphorylated at Ser-543, has been shown elsewhere to interact with 14-3-3 proteins, which results in its enzymatic inactivation (13,20). However, mechanisms by which NR kinase is activated and by which 14-3-3 proteins are recruited remain to be determined. In the present study we suggest that one of the NR kinases is WKP4 and propose the following working hypothesis. Upon external stimulation, WPK4 autophosphorylates its regulatory domain, to which the 14-3-3 protein (TaWIN) binds. WPK4 simultaneously phosphorylates NR, to which TaWIN is transferred; this forms the enzymatically inactive NR/TaWIN complex (Fig.  7). Since WPK4 is up-regulated by nutrient deprivation (8), our model may partly explain the complex regulation of NR; the reduction of NR activity by reduced nutrient assimilation (24) could conceivably be mediated through WPK4 system.