THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 275, No. 41, Issue of October 13, pp. 31695–31700, 2000
Printed in U.S.A.
Specific Binding of a 14-3-3 Protein to Autophosphorylated
WPK4, an SNF1-related Wheat Protein Kinase, and to
WPK4-phosphorylated Nitrate Reductase*
Received for publication, June 6, 2000, and in revised form, July 27, 2000
Published, JBC Papers in Press, July 28, 2000, DOI 10.1074/jbc.M004892200
Yoshihisa Ikeda, Nozomu Koizumi, Tomonobu Kusano, and Hiroshi Sano‡
From the Research and Education Center for Genetic Information, Nara Institute of Science and Technology, Ikoma,
Nara 630-0101, Japan
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
* 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 GenBankTM/EBI Data Bank with accession number(s) AB042193
(TaWIN1) and AB042194 (TaWIN2).
‡ To whom correspondence should be addressed: Tel.: 81-743-725650; Fax: 81-743-72-5659; E-mail: [email protected].
1
The abbreviations used are: SnRK, SNF1-related protein kinase;
GST, glutathione S-transferase; NTA, nickel nitrilotriacetic acid; NR,
nitrate reductase; PAGE, polyacrylamide gel electrophoresis; AMPK,
AMP-activated protein kinase; HMGR, 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
This paper is available on line at http://www.jbc.org
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
N6-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 ⫻ 107 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
31695
31696
Interaction of 14-3-3 with SNF1-related Protein Kinase
subsequently transformed with the library plasmids.
Construction of Plasmids—The K75D-WPK4 (8) and ⌬C-WPK4 genes
were cloned into a pBD-Gal4 Cam vector. A series of WPK4 mutants
(T204A, T204E, S388A, S418A, and S388A/S418A) were constructed
using the Mutan-Express Km kit (TAKARA) and appropriate oligonucleotides. For the S388/418A double mutant, the S338A mutation was
generated on the S418A clone. A series of TaWIN1 mutants (K60D,
R67E, L185D, V189E, G221S, and L233D) were similarly constructed.
All mutagenized inserts were subcloned into the pAD Gal4 vector. The
⌬N-WPK4 gene containing the 3⬘-distal 1-kilobase fragment of WPK4
with the double mutation S388A/S418A was cloned into a pGEX-2T
vector (Amersham Pharmacia Biotech). The entire coding regions of
TaWIN1 and TaWIN2 were cloned in-frame into pGEX-4T-1 (Amersham Pharmacia Biotech), yielding pGEX-TaWIN1 and pGEXTaWIN2, respectively. These were also cloned in-frame into pET32a
(Novagen), yielding pET-TaWIN1 and pET-TaWIN2. A DNA fragment
encoding a peptide corresponding to the residues 469 –560 of tobacco
Nia1 (nitrate reductase) was amplified by polymerase chain reaction
and cloned into pGEX4-T1 (Amersham Pharmacia Biotech). pGEXHMGR was prepared as described (8).
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 2⫻ 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 MgCl2, 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 MgCl2, 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, GSTHMGR, 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 ⫻ 106
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
FIG. 1. Amino acid sequence and phylogenic analysis of
TaWIN1 and TaWIN2. A, sequence alignment of predicted amino acid
sequences of TaWIN1 and TaWIN2 with indication of potential ␣-helical segments. Identical amino acids are shaded, and those invariant
across 84 sequences and 30 species are boxed. Amino acids necessary for
clustering with the phosphorylated consensus motif are shown in reverse contrast. B, amino acid alignment of the putative EF-hand motif
of TaWINs between Glu-207 and Leu-234. Conserved and similar amino
acids are shaded. C, dendrogram of amino acid sequence relationships
among 14-3-3 proteins from A. thaliana, O. sativa, and human. GenBankTM accession numbers are TaWIN1 (AB042193), TaWIN2
(AB042194), At14chi (L09112), At14epsilon (U36446), At14kappa
(U36447), At14lambda (U68545), At14mu (U60444), At14nu (U60445),
At14omega (Q01525), At14phi (L09111), At14psi (L09110), At14RCI1A
(X74141), At14RCI1B (X74141), At14upsilon (L9109), OsGF14b
(U65956), OsGF14c (U65957), OsGF14d (U65968), and OsS94
(D16140).
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
Interaction of 14-3-3 with SNF1-related Protein Kinase
FIG. 2. WPK4 binding assay showing the binding of TaWIN1
and TaWIN2 to the C-terminal region of WPK4. A, amino acid
alignment of the activation loop of WPK4, SNF1, AMPK, and protein
kinase A. DFG and APE motifs are shaded. Thr-204, the phosphorylation site, is shown in reverse contrast. B, interaction of WPK4 and
mutant WPK4 (S389A, S416A, S389/414A) with TaWIN1 and TaWIN2.
Yeast Y190 cells were cotransformed with the bait and prey combinations and quantitatively assayed for ␤-galactosidase activity. Bars indicate the mean of three independent assays. GBD, GAL4 DNA binding
domain. WT, wild type. C, GST pull-down assay for interaction between
TaWIN1 and WPK4. His-tagged TaWIN1 was incubated with immobilized GST, GST-K75D, or GST-WPK4. Proteins bound to the Sepharose
beads were pelleted, washed, subjected to SDS-PAGE, stained with
Coomassie Brilliant Blue (CBB, left), and detected by Western blotting
using an anti-Penta-His antibody (right). Relative molecular masses of
the standard samples are indicated at the left. The arrow indicates
His-TaWIN1.
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).
31697
FIG. 3. Binding of TaWIN1 to the C-terminal region of WPK4. A,
diagrammatic representation of WPK4 constructs used in this study.
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.
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
31698
Interaction of 14-3-3 with SNF1-related Protein Kinase
FIG. 5. Binding of TaWIN1 to a WPK4-phosphorylated NR peptide. A 1-␮g aliquot of GST, GST-HMGR, or GST-NR (lanes 1 through
3) was incubated with 50 ng of GST-WPK4, separated by SDS-PAGE,
stained (A), dried, and exposed to x-ray film (B). As the control, GST-NR
without phosphorylation was used (lane 4). Far Western blotting analysis showing direct interaction of TaWIN1 with phosphorylated NR (C).
Substrate proteins were phosphorylated by GST-WPK4 in the presence
of cold ATP, separated by SDS-PAGE, and electroblotted on polyvinylidene difluoride. The membrane was incubated with His-TaWIN1,
washed thoroughly, and detected using an anti-Penta-His antibody.
Asterisks indicate phosphorylated samples. CBB, Coomassie Brilliant
Blue.
FIG. 4. Effects of amino acid substitution and dimer formation
on TaWIN1 function. A, 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. WT, wild type. B, GST pull-down assay showing the
association of TaWIN1 with TaWIN2. Histidine-tagged TaWIN2 was
incubated with glutathione-Sepharose-immobilized GST or GSTTaWIN1. Proteins bound to the Sepharose beads were pelleted, washed,
subjected to electrophoresis, stained with Coomassie Brilliant Blue
(CBB, left), and detected by Western blotting using an anti-Penta-His
antibody (right). Relative molecular masses of the standard proteins are
indicated at the left. The arrow indicates His-TaWIN1. C, elution profile
of recombinant TaWIN1 proteins by high resolution gel filtration chromatography (left). The elution positions for albumin (67 kDa), ovalbumin (43 kDa), and chymotrypsinogen A (25 kDa) are shown above the
A280 trace for TaWIN1 elution. SDS-PAGE analysis of the peak fraction
is shown in the right panel. Relative molecular masses are indicated in
left.
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 Ser388/418A⌬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
Interaction of 14-3-3 with SNF1-related Protein Kinase
31699
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.
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.
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 N6-benzylaminopurine on
TaWIN1 transcript accumulation. Six-day-old green seedlings, grown
under continuous light conditions, were treated with 100 ␮M N6-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 5⬘-untranslated region of TaWIN1 cDNA,
a 0.8-kilobase 3⬘ region of WPK4 cDNA, and a 1.2-kilobase wheat actin
cDNA, respectively.
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
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
2
Y. Ikeda, N. Koizumi, T. Kusano, and H. Sano, unpublished
observation.
31700
Interaction of 14-3-3 with SNF1-related Protein Kinase
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.
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