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INTRODUCTION |
Tyrosine phosphorylation plays an important role in the regulation
of many cellular processes. The activity of the enzymes governing this
modification, protein-tyrosine kinases and phosphatases, needs to be
tightly controlled for tyrosine phosphorylation to proceed in an
orderly manner. Improper phosphorylation due to malfunction of
protein-tyrosine kinases and phosphatases can lead to diseases in
humans and animals, including immunodeficiencies and cancer (1). In
higher plants, several serine/threonine kinases have been identified,
but the presence of protein-tyrosine kinases remains controversial. No
tyrosine kinase has hitherto been cloned from plants. However, tyrosine
phosphorylation in plants has been shown to be involved in various
physiological processes (2-4). Dual specificity protein kinases have
been reported in Arabidopsis (5, 6) and soybean (7), but the
functions for these kinases remain unknown. We recently reported that
serine/threonine/tyrosine (STY)1 protein kinase from
Arachis hypogaea is developmentally regulated and induced by
abiotic stress (8). The site of tyrosine phosphorylation has not been
identified for any dual specificity protein kinases in plants. However,
MAPKs have been shown to be activated by dual phosphorylation of
threonine and tyrosine in the Thr-Glu-Tyr (TEY) motif in the activation
loop (9). Recently, the role of threonines in the phosphorylation of
receptor-like kinase has been identified in plants (10, 11).
Most protein kinases retain their kinase domain conserved sequence
motifs, which are separated into subdomains I-XI. The region between
the conserved DFG sequence motif in subdomain VII and the APE
sequence motif in subdomain VIII is referred to as the activation loop.
Several protein kinases such as cAMP-dependent protein
kinase (12), a MAPK family (13), a MAPK kinase (MEK) family (14, 15), a
cyclin-dependent protein kinase family (16), and
c-Src tyrosine kinase (17) are activated by phosphorylation of
residues within the activation loop (18). Phosphorylation negatively
regulates a substantial number of protein kinases. For example,
phosphorylation of cyclin-dependent protein kinase at
threonine and tyrosine residues in the ATP-binding loop by wee1-related kinases leads to kinase inactivation (16), and phosphorylation of the Src tyrosine kinase at a tyrosine residue in the
C terminus maintains the kinase in an inactive conformation (18).
Here we report a novel site of autoinhibition of STY protein kinase. In
contrast to the above-mentioned kinases, we demonstrate here that the
phosphorylation of Tyr148 in the ATP-binding domain and of
Tyr317 in the C-terminal domain activates STY protein
kinase. The replacement of Tyr297 with Phe in the
activation loop resulted in a drastic reduction in the phosphorylation
of peanut STY protein kinase with ATP and histone. This study provides
direct evidence for the tyrosine phosphorylation of peanut STY protein kinase.
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EXPERIMENTAL PROCEDURES |
Materials--
Histone H1 (type IIIS), monoclonal
anti-phosphotyrosine antibody, phosphoamino acids, and calf intestinal
alkaline phosphatase were purchased from Sigma.
[
-32P]ATP (3000 Ci/mmol) was obtained from PerkinElmer
Life Sciences. Restriction endonucleases were from MBI Fermentas (St.
Leon-Rot, Germany). Pfu polymerase was from New England
Biolabs Inc. (Beverly, MA). Nickel-nitrilotriacetic acid-agarose was
obtained from QIAGEN Inc. (Chatsworth, CA). Oligonucleotides were
synthesized by Microsynth (Balgach, Switzerland).
Bacterial Strains, Growth Conditions, and DNA
Manipulations--
Escherichia coli strain DH5
(Invitrogen) was the recipient for all plasmids used in subcloning. The
BL21(DE3) pLysS strain (19) was used for bacterial expression of
pRSET-C-STY and its protein kinase tyrosine mutants. LB medium with 50 µg/ml ampicillin was used for growing E. coli cells
containing the plasmids. Plasmids were prepared by the alkaline lysis
method (20). The DNA fragments were eluted from the agarose gel by the
low melting agarose gel method. The preparation of competent cells and
transformation were carried out as described (20).
Matrix-assisted Laser Desorption Ionization (MALDI) Mass
Spectrometry--
Two micrograms of STY protein kinase or
autophosphorylated STY protein kinase was electrophoresed on
SDS-polyacrylamide gel and visualized by Coomassie Blue staining. STY
protein kinase bands were then excised from the gel, transferred to an
acid-washed tube, rehydrated with water, crushed, washed three times
for 20 min with 50 mM Tris-HCl (pH 8.0) and 50%
acetonitrile, and dried. The sample was incubated for 6 h at
32 °C with 0.80 ng/ml trypsin in 25 mM Tris-HCl (pH 8.5)
to digest the protein. The tryptic fragments were then extracted with
50% acetonitrile and 0.1% trifluoroacetic acid; dried; suspended in
10 mg/ml 4-hydroxy-
-cyanocinnamic acid in 50% acetonitrile and
0.1% trifluoroacetic acid containing angiotensin as an internal
standard; and applied to a MALDI sample plate, which was dried and
washed with water to remove excess buffer salts. MALDI mass
spectrometry analysis was performed on a Kratos PCKompact Seq 1.2.2 mass spectrometer in the linear mode. The masses obtained in these
experiments were searched against predicted tryptic fragments of STY
protein kinase using the program PeptideMass (21).
Site-directed Mutagenesis--
Wild-type STY protein kinase
(pRSET-C-STY) template (80 ng) and sense and antisense primers (25 pmol) were added to PCR tubes containing 0.2 mM dNTPs, 1 mM MgSO4, 2.5 units of Pfu
polymerase, and 1× reaction buffer. Amplification was carried out
under the following conditions: denaturation of the template at
95 °C for 4 min, followed by 20 cycles at 94 °C for 45 s
(denaturation), 52 °C for 1 min (annealing), and 72 °C for 9 min
(extension). The reaction was continued for 20 min at 72 °C to
complete the extension. The primers for K160R (sense,
5'-ATAATGGTGAGGATGTAGCTA-3'; and antisense,
5'-TAGCTACATCCTCACCATTAT-3'), Y148F (sense,
5'-GGAAAACTCTTTAGGGGAACTT-3'; and antisense,
5'-AAGTTCCCCTAAAGAGTTTTCC-3'), Y213F (sense,
5'-GTAACGGAATTTGCCAAAGGG-3'; and antisense,
5'-CCCTTTGGCAAATTCCGTTAC-3'), Y297F (sense,
5'-ACTGGAACATTCCGTTGGATG-3'; and antisense,
5'-CATCCAACGGAATGTTCCAGT-3'), and Y317F (sense, 5'-GGTGGATGTGTTTAGCTTTGGG-3'; and antisense,
5'-CCCAAAGCTAAACACATCCACC-3') were used on recombinant pRSET-C-STY
protein kinase to construct site-directed mutants. The PCR-amplified
mixture was treated with DpnI (10 units) at 37 °C for
1 h to digest the methylated template and transformed into DH5
competent cells. The presence of mutations was confirmed by sequencing
the plasmid DNA.
Expression and Purification of Wild-type STY Protein Kinase and
Its Mutants--
The cDNA spanning the coding region of STY
protein kinase (lacking 11 amino acids from the N terminus) was
subcloned into the histidine-tagged fusion protein expression vector
pRSET-C at BglI and KpnI restriction sites. The
resultant construct was expressed in E. coli
BL21(DE3) pLysS. The recombinant fusion protein was induced with
0.4 mM isopropyl-1-thio-
-D-galactopyranoside for 4 h. The recombinant protein was induced in large-scale (500 ml) and purified by nickel-nitrilotriacetic acid-agarose
chromatography. Protein concentrations were determined by the method of
Bradford (22). Purified fractions containing the eluted protein were analyzed by 12% SDS-PAGE, followed by Coomassie Blue staining (23).
Mutant proteins were purified similarly to wild-type STY protein kinase.
Assay of Tyrosine Phosphorylation--
Samples of the
His6-tagged STY protein kinase and mutant fusion proteins
were separated on 12% SDS-polyacrylamide gel, transferred onto a
nitrocellulose membrane, and probed with monoclonal
anti-phosphotyrosine antibody.
In Vitro Kinase Assay--
ATP dependence assays were performed
by incubating the STY protein kinase and mutant fusion proteins (1 µg) with 0.1-500 µM ATP containing 1.5 µCi of
[
-32P]ATP in a total volume of 20 µl of kinase
buffer and stopped at 20 min by the addition of 10 µl of 3× gel
loading buffer. Histone phosphorylation assays were performed by
incubating the STY protein kinase and mutant fusion proteins with
2.5-40 µM histone H1 containing 50 µM
unlabeled ATP and 1.5 µCi of [
-32P]ATP in a total
volume of 30 µl of kinase buffer and stopped at 20 min by the
addition of 15 µl of 3× gel loading buffer. The reaction products
were separated by 12% SDS-PAGE. The Coomassie Blue-stained protein
bands were recovered and then measured using a
-scintillation
counter. Km and Vmax values
were estimated according to the double-reciprocal plot and linear
regression using SigmaPlot 2000 Version 6 software (SPSS Inc., Chicago, IL).
Phosphoamino Acid Analysis--
Purified STY protein kinase and
the mutant proteins were labeled in vitro with
[
-32P]ATP as described above and electroblotted onto a
polyvinylidene difluoride membrane. After autoradiography, radioactive
bands of interest were excised and hydrolyzed in 200 µl of 6 M HCl for 2 h at 110 °C. The hydrolysate was dried
in a SpeedVac concentrator and resuspended in 20 µl of water
containing 1 mg/ml each of the phosphoamino acid markers such as
phosphoserine, phosphothreonine, and phosphotyrosine. Two microliters
of the hydrolysate was analyzed by ascending silica thin-layer
chromatography (Merck) using a solvent system containing a mixture of
ethanol and ammonia (3.5:1.6, v/v) (24). The positions of phosphoamino
acid markers were detected by ninhydrin staining of the silica
thin-layer plate (0.25% ninhydrin in acetone). The plate was then
exposed for autoradiography to locate the positions of the
32P-labeled amino acids.
Calf Intestinal Alkaline Phosphatase Treatment of the Recombinant
Kinase--
The histidine fusion protein of STY protein kinase was
incubated with 1 unit of calf intestinal alkaline phosphatase for 30 min at 37 °C, and the phosphorylated and dephosphorylated kinases were resolved by 15% SDS-PAGE.
Circular Dichroism--
CD studies were performed using a Jasco
J-720 spectropolarimeter with a thermostatically controlled 10-mm
cylindrical CD cell at 4 µM protein. For near-UV and
UV-visible wavelengths, protein concentrations ranged from 0.8 to 1 mg/ml using a 1-cm path length. The CD spectra are the averages of five
independent experiments on different samples of the same enzyme
preparation recorded at a scan speed of 50 nm/min with a bandwidth of 2 nm and averaged automatically. All spectra were corrected for the
appropriate blank solutions, recorded in the absence of enzyme. The
values are expressed in terms of molar ellipticity.
 |
RESULTS |
Sequence Comparison of the STY Protein Kinase Activation Loop with
Tyrosine Kinases and Dual Specificity Protein Kinases Regulated
by Tyrosine Phosphorylation--
A schematic representation of the STY
protein kinase catalytic domain with all 11 subdomains representing the
site-directed mutations generated in this study is shown in Fig.
1A. STY protein kinase has
four conserved tyrosine residues that are present in the ATP-binding
domain (subdomain I), the TEY sequence motif (subdomain V), the
activation loop (subdomain VIII), and toward the C terminus (subdomain
IX). Short sequences between the conserved DFG motif in subdomain VII
and the APE sequence motif in subdomain VIII of the protein kinase
domains are referred to as the activation loop. STY protein kinase
contains a single tyrosine (Tyr297) in the activation loop
(Fig. 1B). The site of tyrosine phosphorylation has not been
identified in plant kinases. Therefore, we compared STY protein kinase
with the dual specificity protein kinases from all the species (Fig.
1B). The protein sequence of STY protein kinase has homology
to the DYRK family of kinases from mammals, and DYRK is an
intracellular dual specificity protein kinase that is regulated by
tyrosine phosphorylation of the activation loop (25). Based on the
assumption that the kinase activity of STY protein kinase may be
regulated by the phosphorylation of the tyrosine residue in the
activation loop, we constructed an STY protein kinase mutant with
Tyr297 replaced with Phe by site-directed mutagenesis.
Alignment of Src tyrosine kinases and STY protein kinase revealed the
presence of a conserved tyrosine in the TEY motif subdomain V (Fig.
1C). To elucidate the role of Tyr213 in the TEY
motif, site-directed substitution of tyrosine with phenylalanine
was performed.

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Fig. 1.
Schematic representation of the STY protein
kinase catalytic domain and identification of site-directed
mutations. A, the locations of
Tyr148, Lys160, Tyr213,
Tyr297, and Tyr317 of STY protein kinase
mutated in this study. B, sequence comparison of protein
kinases that are regulated by phosphorylation of the presumed
activation loop between domains VII and VIII. The conserved DFG
sequence in subdomain VII and the APE sequence in subdomain VIII are
boxed. Asterisks above tyrosine residues indicate
activating phosphorylation sites. The GenBankTM/EBI Data
Bank accession numbers are as follows: STY protein kinase (A. hypogaea), AY037437; ATN1 (Arabidopsis thaliana),
S61766; DYRK (Drosophila melanogaster), P83102; and DYRK
(Homo sapiens), NP_006473. C, sequence alignment
of subdomain V of STY protein kinase with Src tyrosine kinase from
chicken (Protein Data Bank code 2PTK) and human (code 2SRC) and with
c-ABL kinase from mice (code 1IEPA). The TEY motif in subdomain V is
boxed. D, locations of STY protein kinase
autophosphorylation sites (indicated by red circles on the
molecule backbone) in the predicted secondary structure. N
and C indicate the N and C termini of the structure,
respectively. Green arrows indicate boundaries of
the STY protein kinase activation loop, which lies between
Asp277 and Glu303. -Helices and -strands
are colored in magenta and yellow,
respectively.
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Homology Modeling--
The x-ray structures of the mouse c-ABL
kinase domain in complex with the inhibitor STI-571 (Protein Data Bank
code 1IEPA), human protein-tyrosine kinase (code 2SRC), and
chicken protein-tyrosine kinase (code 2PTK) were used as templates for
modeling the STY protein kinase catalytic domain. These templates
produced the best E value when using BLAST against protein
kinase in the Protein Data Bank. A three-dimensional model of the
catalytic domain of STY protein kinase was determined using SWISS-MODEL (26) and visualized by RasMol (27). The consensus tyrosine kinase motif
CWX6RPXF in subdomain XI is conserved
among the proteins. STY protein kinase has the consensus IVTEY motif
that is conserved in Src family kinases in subdomain V. The predicted
STY protein kinase molecule resembles the typical structure of a
protein kinase catalytic domain, consisting of a small lobe, which is
involved in ATP binding and orientation, and a large lobe, which
provides sites for substrate recognition and catalysis (28). The small lobe represents the N terminus of the molecule and consists
predominantly of
-strands, whereas the large lobe represents the C
terminus of the molecule and includes several
-helices. STY protein
kinase autophosphorylation sites are scattered throughout the kinase catalytic domain (Fig. 1D). The activation loop helix that
spans between Asp277 and Glu303 packs between
the N-terminal and C-terminal lobes and sequesters Tyr297.
Phosphorylation Site Mapping by MALDI Time-of-Flight Mass
Spectrometry Analysis--
We carried out mass spectrometry
experiments to identify autophosphorylation sites. Recombinant STY
protein kinase was digested with trypsin before and after an extended
autophosphorylation reaction, and the protein was analyzed by MALDI
mass spectrometry (Fig. 2).
Phosphorylation increased the peptide mass by 80 Da. Tryptic
fragments of STY protein kinase that were found in the unphosphorylated
state and those that were shifted by the mass of a phosphate (+80 Da)
in the autophosphorylated samples were studied. One of the major peaks
(2541.20 Da) fitting these criteria (peak b) has a mass that
corresponds to a peptide from the activation loop of STY protein
kinase. This tryptic peptide contains a single tyrosine
(Tyr297). Two other peaks (peaks a and
c) correspond to Tyr148 (450 Da) in the
ATP-binding domain and Tyr317 (3850.02 Da) in subdomain IX,
respectively. Corresponding unphosphorylated peptides were not
detected in the MALDI time-of-flight mass spectra of the
autophosphorylated kinase. We did not observe any evidence for
phosphorylation of Tyr213 in the TEY motif in STY protein
kinase (Fig. 2). These results suggest the possibility of STY protein
kinase being phosphorylated at multiple sites and that
Tyr148, Tyr297, and Tyr317 are the
autophosphorylation sites.

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Fig. 2.
Identification of STY protein kinase
autophosphorylation sites by MALDI mass spectrometry. Recombinant
kinase or autophosphorylated STY protein kinase (2 µg) was subjected
to 12% SDS-PAGE and visualized by Coomassie Blue staining. The STY
protein kinase bands were excised from the gel and subjected to in-gel
trypsin digestion. Tryptic fragments of native STY protein kinase
(A) and autophosphorylated STY protein kinase (B)
were analyzed by MALDI mass spectrometry. Peaks a-c
are STY protein kinase peptides that were present in their
unphosphorylated state in the native sample, and peaks
a'-c' are STY protein kinase peptides that were present in their
phosphorylated state (+80 Da) in the autophosphorylated sample. There
was no change in the phosphorylation of the tryptic peptide shown by
peak d. The tryptic peptide masses and phosphorylation sites
are summarized at the bottom. Insets show the MALDI
time-of-flight mass spectra of tryptic peptides up to 1000 Da.
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Expression and Purification of Wild-type STY Protein Kinase and
Substituted STY Protein Kinase Mutants--
Based on the comparative
sequence analysis, homology modeling, and phosphopeptide mapping, the
conserved Tyr148, Tyr213, Tyr297,
and Tyr317 residues of STY protein kinase were substituted
with Phe by site-directed mutagenesis. All the mutant proteins were
overproduced in E. coli BL21(DE3) pLysS using the T7 RNA
polymerase expression system, induced by
isopropyl-thio-
-D-galactoside, and purified by
nickel-nitrilotriacetic acid affinity chromatography. The expressed
proteins were predominantly present in the soluble fraction, and yields
of the proteins were in the range of 10-15 mg/liter. The expression
levels of and the ability to purify the STY protein kinase mutants
suggested that they were as stable as the wild-type protein.
Tyrosine Phosphorylation of STY Protein Kinase Mutants--
To
determine the degree of autophosphorylation, equal quantities of
histidine fusion proteins of the wild-type and mutant STY protein
kinases were subjected to anti-phosphotyrosine and anti-STY protein
kinase immunoblot analyses. Notably, there was a significant increase
in the reactivity of the anti-phosphotyrosine antibody with the Y213F
mutant protein (Fig. 3A). The
tyrosine mutations at positions 148, 297, and 317 diminished the
reactivity of STY protein kinase with the anti-phosphotyrosine
antibody. When the same blot was subjected to anti-STY protein kinase
Western blot analysis, there was no difference in the reactivities of the wild-type and mutant proteins (Fig. 3B). There was a
slight downshift in the mobility of the Y148F, Y297F, and Y317F mutant proteins with respect to the wild-type protein. When wild-type STY
protein kinase was subjected to phosphatase treatment, we observed
faster mobility of the protein upon SDS-PAGE compared with the
untreated protein (Fig. 3C). The downshift in mobility of
the protein kinase inactive mutants could be due to the phosphorylation status of the protein.

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Fig. 3.
Tyrosine phosphorylation of the STY protein
kinase mutants in which the autophosphorylated tyrosines were replaced
with phenylalanines. A, affinity-purified histidine
fusion proteins of wild-type (WT) STY protein kinase and its
mutants were run on 12% SDS-polyacrylamide gel, transferred onto a
nitrocellulose membrane, and subjected to anti-phosphotyrosine
(Anti-pY) immunoblot analysis. B, shown is an
anti-STY protein kinase immunoblot illustrating equal loading of the
fusion proteins. C, shown is the dephosphorylation of
wild-type STY protein kinase with calf intestinal alkaline phosphatase
(CIAP). Lane 1, 15% SDS-polyacrylamide gel
representing molecular mass standards; lane 2, wild-type STY
protein kinase; lane 3, calf intestinal alkaline
phosphatase-treated STY protein kinase.
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Effect of Tyrosine Mutations on Kinase Activity--
The kinase
activities of all the site-directed mutants were assessed by comparing
their abilities to autophosphorylate and phosphorylate histone in
vitro (Fig. 4). As a negative
control, the conserved lysine in subdomain II involved in ATP binding
was changed to arginine to form a kinase inactive mutant (K160R), and
the mutant did not show autophosphorylation or substrate
phosphorylation. Preincubation of STY protein kinase and the
site-directed mutants with magnesium and unlabeled ATP resulted in a
reduced level of labeled phosphate incorporation (Fig. 4, A
and B). Replacement of Tyr213 with Phe resulted
in a 4-fold increase in autophosphorylation and a 2.8-fold increase in
histone phosphorylation. Elevated tyrosine phosphorylation of the Y213F
mutant protein was accompanied by reduced mobility upon SDS-PAGE. We
analyzed the relative incorporation of 32P label into the
phosphoamino acids. Surprisingly, the Y213F mutant showed an increased
band intensity of phosphoserine, phosphothreonine, and phosphotyrosine
with respect to the wild-type protein, suggesting that serines,
threonines, and tyrosines were phosphorylated (Fig. 5). Substitution of Tyr148 in
the ATP-binding domain with Phe decreased both autophosphorylation and
substrate phosphorylation. There is a single tyrosine
(Tyr297) in the activation loop upstream of the APE
sequence in STY protein kinase, and its replacement with phenylalanine
strongly decreased both autophosphorylation and substrate
phosphorylation. The site-specific mutation of Y317F in the C-terminal
motif also resulted in a drastic reduction in protein kinase activity
(Fig. 4, A-D). Phosphoamino acid analyses of the Y148F,
Y297F, and Y317F mutant proteins revealed a drastic reduction in
phosphorylation of tyrosine with respect to the wild-type protein (Fig.
5). These results suggest that the mutated sites could play a role in
the regulation of STY protein kinase.

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Fig. 4.
Autophosphorylation and histone
phosphorylation of STY protein kinase and its mutants. Bacterially
produced STY protein kinase and its mutants were purified, and 500 ng
of protein was incubated with [ -32P]ATP to determine
kinase activity. After separation by 12% SDS-PAGE, the resulting gels
were analyzed using a PhosphorImager. A, autoradiographs of
autophosphorylation of wild-type (WT) STY protein kinase and
mutants Y148F, Y213F, Y297F, and Y317F. As indicated, the proteins were
preincubated with or without 0.5 mM unlabeled ATP in the
same buffer for 30 min. B, histogram constructed with data
from A. Black bars, autophosphorylated enzymes
without preincubation with ATP; white bars,
autophosphorylated enzymes preincubated with ATP. The error
bars represent the S.D. of three independent experiments.
C, histone phosphorylation of wild-type and site-directed
mutants. Aliquots of 500 ng of purified STY protein kinase and its
mutants were incubated with 1 µg of histone in the presence of
[ -32P]ATP as described under "Experimental
Procedures." After separation by 12% SDS-PAGE, the resulting gels
were analyzed using a PhosphorImager. Shown are autoradiographs of
histone incubated with wild-type STY protein kinase, K160R, Y148F,
Y213F, Y297F, and Y317F. D, histogram constructed with data
from C. The error bars represent the S.D. of four
experiments.
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Fig. 5.
Phosphoamino acid analyses of the STY protein
kinase mutants in which the autophosphorylated tyrosines were replaced
with phenylalanines. A, phosphoamino acid analysis of
autophosphorylated STY protein kinase and the site-directed mutants.
Recombinant STY protein kinase and the site-directed mutants (1 µg)
were autophosphorylated, resolved by 12% SDS-PAGE, and transferred
onto a polyvinylidene difluoride membrane. The reaction products were
hydrolyzed and separated by silica thin-layer chromatography as
described under "Experimental Procedures." The positions of the
origin (ori), phosphoserine (PS),
phosphothreonine (PT), and phosphotyrosine (PY)
are indicated. WT, wild-type STY protein kinase,
B, histogram constructed with data from A. The
error bars represent the S.D. of four
experiments.
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Kinetics of Autophosphorylation of STY Protein Kinase
Mutants--
STY protein kinase exhibited Michaelis-Menten kinetics
with respect to ATP. The apparent Km value for ATP
was calculated to be 34.4 µM (Table
I). The Y213F mutant showed an increase in phosphorylation activity with respect to the wild-type protein; hence, we investigated whether the kinetic properties of this mutant
deviated significantly from that of the wild-type enzyme. The mutation
decreased the apparent Km from 34.4 to 18.8 µM and increased the kcat value
(Table I). The phosphorylation efficiency of this mutant
(kcat/Km) was 5-fold higher than that of the wild-type enzyme.
The same analyses were performed with mutants Y148F, Y297F, and Y317F
(Table I). There was a significant reduction in the affinity of the
Y148F mutant for ATP, and the Km of the mutant
increased by 3-fold and the kcat was reduced by
5-fold compared with those of the wild-type enzyme. A drastic reduction (17-fold) in the phosphorylation efficiency of this mutant was observed
compared with that of the wild-type protein. The Y297F mutant also
showed a 26-fold reduction in phosphorylation efficiency compared with
the wild-type enzyme. The Km of the Y317F mutant
deviated slightly from that of the wild-type protein, but the
kcat and phosphorylation efficiency of this
mutant protein were drastically reduced by 20- and 38-fold,
respectively, compared with those of the wild-type protein. These
results suggest that the tyrosine mutations reported in this
study significantly altered the phosphorylation activity.
Kinetics of Histone Phosphorylation of STY Kinase
Mutants--
The wild-type enzyme has an apparent
Km of 6.71 µM (Table I). The Y213F
mutation showed no apparent effect on the Km,
but led to an increase (2-fold) in the kcat. The Y148F mutant was substantially less active than the wild-type enzyme,
with 5-fold lower phosphorylation efficiency and a 1.6-fold higher
Km for histone. A decrease in
kcat and histone phosphorylation efficiency was
observed with the Y297F mutant. There was a 3.3-fold reduction in the
histone phosphorylation efficiency of the Y317F mutant protein.
Circular Dichroism of STY Protein Kinase Mutants--
The
secondary structures of these proteins were monitored by CD. The
observed deficiency in enzyme activity for any of these mutant enzymes
could have been caused by alterations in the secondary structures of
the enzyme. The number of secondary structure elements was calculated
from the CD spectra using Antheprot software (29). The CD spectra of
the wild-type and mutant STY protein kinases were similar and were
characterized by a minimum at 204-205 nm and a shoulder in the
222-225 nm range, suggesting the presence of
-sheet and
-helical
structures, respectively. The results from the CD spectral analyses of
STY protein kinase and its mutants are given in Table
II.
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Table II
Fraction of secondary elements as calculated from respective CD spectra
Number of secondary structure elements was calculated from the CD
spectra using Antheprot software (29).
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DISCUSSION |
Regulation of protein kinases is achieved through many different
mechanisms, including protein phosphorylation by other kinases (30),
autophosphorylation (31), and control by regulatory domains or subunits
(32). A key feature of regulation in many protein kinases is thought to
be the phosphorylation of one or more residues within the activation
loop of the catalytic subunit. STY protein kinase activation by
tyrosine phosphorylation within the activation loop is of interest
because it suggests possible involvement of STY protein kinase in
tyrosine kinase-mediated signaling pathways in plants. Most
protein kinases that are activated by phosphorylation of residues in
the activation loop belong to arginine/aspartate kinase family
(18), in which the aspartate residue in the activation loop has
an adjacent arginine. The arginine residue has been hypothesized to
form an ionic bridge with the phosphorylated serine or threonine
residue that stabilizes the catalytically active conformation (34). STY
protein kinase also belongs to the arginine/aspartate kinase
family. In this study, we have shown that a conserved tyrosine
residue within the activation loop regulates STY protein kinase
activity. There is a single tyrosine (Tyr297) in the
activation loop of peanut STY protein kinase, and the substitution of
Tyr297 with Phe led to a significant loss of catalytic
activity in terms of both autophosphorylation and substrate
phosphorylation toward histone. Activation of the mammalian dual
specificity protein kinases is regulated by the tyrosine
phosphorylation of the activation loop. Isoforms of the protein kinase
C subfamily can be activated by tyrosine phosphorylation by Bruton's
tyrosine kinase or Src family tyrosine kinases (35, 36). Tyrosine
phosphorylation of a member of the protein kinase C subfamily is
dependent on the activity of a Bruton's tyrosine kinase that may
directly phosphorylate the protein kinase C (37). All other kinases
that are regulated by tyrosine phosphorylation between subdomains VII
and VIII, e.g. MAPK/ERK, JNK, and GSK3, are components of
signaling pathways that transduce receptor-initiated signals to nuclear
phosphorylation of transcription factors (38). Two requirements have
been thought to be critical for the catalytic activity of protein
kinases. One is the correct juxtaposition of catalytic groups
contributing to the transfer of the
-phosphate group from ATP to a
serine, threonine, or tyrosine side chain of the substrate (34). The other is the accessibility and correct positioning of the
substrate-binding site(s) (30). The mechanism of peanut STY protein
kinase activation by phosphorylation could therefore be that
phosphorylation promotes a conformation of the activation loop in which
the catalytic and substrate-binding sites are correctly formed,
resulting in a significant increase in kinase activity. STY protein
kinase may also be activated in vivo through activation loop
phosphorylation of the tyrosine residue by an upstream kinase(s).
In our studies, mutation of Tyr213 to Phe produced a
recombinant enzyme with a 4-fold increase in autophosphorylation and a
2.8-fold increase in substrate phosphorylation activities. A similar
observation was made earlier in several protein kinases (32, 39). The catalytic activity of many protein kinases is regulated by an autoinhibitory mechanism known as intrasteric regulation (32). Many
protein kinases are maintained in low activity or inactive states
through intermolecular or intramolecular association with inhibitory
molecules or domains. These inhibitory molecules may be disrupted in
response to appropriate stimuli, allowing the protein kinase to adopt
an active conformation. Autoinhibitory sequences have been found in
protein kinase C, calcium/calmodulin-dependent kinase,
myosin light chain kinase, c-Src, the insulin receptor, and twitchin
(32, 39, 40). The affinity of autoinhibitory sequences may also be
regulated by autophosphorylation or other interactions, but the
structural basis for these regulatory mechanisms remains to be
elucidated. Casein kinases are also dual specificity kinases, and
autophosphorylation has an inhibitory effect on protein kinase
activity. Casein kinases are regulated by a C-terminal phosphorylation-dependent autoinhibitory domain (41).
Autophosphorylation inactivates the kinase, and mutagenesis of the
phosphorylation residues of the C-terminal domain reactivates the
kinase. Serine and threonine to alanine mutations that
eliminated these autophosphorylation sites in a recombinant casein
kinase I
protein resulted in 8-fold more activity compared with the
wild-type enzyme. Further supporting the presence of an inhibitory
phosphorylation site in the kinase domain, Kuret and co-workers (42)
described two forms of recombinant yeast casein kinase I
; one form
was autophosphorylated in the kinase domain and had a 4-fold decrease
in activity compared with the unphosphorylated protein kinase.
STY protein kinase has homology to the MLKs from mammals. A point
mutation (Y52A) in the SH3 domain-binding site of MLK3 abolishes binding to the SH3 domain and increases the MLK3 activity (43). The
Y52A mutant shows a 2-fold increase in autophosphorylation and a
2.5-fold increase in histone phosphorylation. SH3 domains are known to
bind to proteins containing PXXP sequences that form polyproline type II helices (44). STY protein kinase also has a similar
sequence motif in the C-terminal region. Apart from the other examples,
substitution of Tyr1162 with Phe in the intact insulin
receptor results in an increase in basal protein kinase activity in the
absence of insulin, consistent with an autoinhibitory role for
Tyr1162 (45). A point mutation in the TEY domain might
disrupt the autoinhibitory domain, leading to a conformational change
that activates the protein kinase, leading to the increased
phosphorylation of all three amino acids. A crystal structure of the
STY protein kinase molecule will be required to understand how
autophosphorylation of Tyr213 might affect the
conformation of STY protein kinase.
STY protein kinase possesses a tyrosine residue within the ATP-binding
motif, whereas the vast majority of kinase family members have a
phenylalanine at this site (1). The protein kinases with a tyrosine in
the ATP-binding domain have been shown to be regulated by tyrosine
phosphorylation. Tyr148 in the ATP-binding domain is
conserved among STY protein kinase-related sequences. The replacement
of Tyr148 with Phe drastically reduced the enzyme activity
of peanut STY protein kinase. The tyrosine in the ATP-binding site of
cyclin-dependent protein kinases and the phosphorylation of
residues in this region are important for the inhibition of kinase
activity (16).
Several protein kinases are regulated by tyrosine phosphorylation of
the C-terminal region. STY protein kinase has structural similarity to
Src tyrosine kinases from chicken and human (46, 47). Src family
kinases have a regulatory tyrosine (Tyr527 in
p60c-src) toward the C terminus and an
autophosphorylation site (Tyr416). Data on the biological
properties of p60c-src have shown that mutation
of its C-terminal sites of tyrosine phosphorylation can serve to
activate or suppress its transforming properties (48-50), indicating
that this process is an important regulatory step. The C-terminal
region of the epidermal growth factor receptor is important in
regulating its biological activity (51). The point mutation of
Tyr317 to Phe in the C-terminal region of STY protein
kinase resulted in the reduction of autophosphorylation activity.
Our results show that STY protein kinase is a multisite-phosphorylated
enzyme and suggest that its phosphorylation may be an intricate process
that regulates its biological functions in very distinct ways.
Multisite phosphorylation is a characteristic of MAPK-activated protein
kinase-2, in which any two of three sites must be phosphorylated to
achieve maximal activation (52). Protein kinase D is also
phosphorylated at multiple sites such as the activation loop,
C-terminal domain, and regulatory domain (33). STY protein kinase is
developmentally regulated and is induced by cold and salt stresses (8).
The distribution of STY protein kinase autophosphorylation sites in the
kinase molecule suggests that different sites may be involved in
distinct mechanisms mediated by STY protein kinase during the onset of
stress response. Src family kinases and MLKs are dual specificity
protein kinases regulated by both autophosphorylation and
autoinhibition. Like Src family tyrosine kinases and mammalian MLKs,
STY protein kinase has a PXXP sequence motif in the
C-terminal region. STY protein kinase might interact through these
proline-rich sites with other kinases that are involved in signal
transduction. Further studies are needed to identify the upstream
kinase(s) that phosphorylate STY protein kinase in its catalytic
segment and to fully elucidate the molecular mechanism of the
regulation of STY protein kinase activity. These mutants provide an
excellent opportunity to dissect in vivo function and
regulation of STY protein kinase.