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INTRODUCTION |
We previously isolated the cDNA encoding a novel protein
kinase, TESK11 named after
testis-specific protein kinase 1, by low-stringency hybridization
screening with LIMK1 (LIM motif-containing protein kinase 1) cDNA
as a probe (1). TESK1 contains characteristic structural features
composed of an N-terminal protein kinase domain and a C-terminal
proline-rich region (1). Northern blot and in situ
hybridization analyses revealed that TESK1 mRNA is highly expressed
in testicular germ cells at the stages of pachytene spermatocytes to
round spermatids (1, 2). Immunohistochemical analyses revealed that the
expression of TESK1 protein is almost in parallel with its mRNA
expression in rat spermatogenic cells (2). These findings suggest a
role of TESK1 in spermatogenesis. However, mechanisms of regulation of
TESK1 kinase activity and signaling pathways in which TESK1 is involved
are poorly understood.
Protein kinases are generally classified into serine/threonine kinases
and tyrosine kinases, based on their substrate specificity (3, 4). On
the other hand, several dual-specificity protein kinases have been
identified that are formally categorized into a serine/threonine kinase
family but catalyze phosphorylation on both serine/threonine and
tyrosine residues (reviewed in Refs. 5, 6). The protein kinase domain
of TESK1 is structurally similar to domains of LIMK1 and its relative
LIMK2 (1, 7-9), with about 50% sequence identity. Phylogenetic
analysis of the protein kinase domains revealed that TESK1 forms an
obvious cluster with LIMKs (a LIMK/TESK1 subfamily), and this subfamily
generally belongs to a family of serine/threonine kinases (1). On the other hand, the kinase domain of TESK1 is unique in that it contains an
unusual short sequence motif DLTSKN in the catalytic loop in the
subdomain VIB, and this motif does not match the consensus sequence of
either conventional serine/threonine kinases (DLKXXN) or
tyrosine kinases (DLRXXN or DLXXRN) (3, 4). This
is also the case for LIMK1 and LIMK2, both of which have a sequence
motif DLNSHN in this region (7-9). On the basis of the primary
sequences, it is thus difficult to predict the substrate specificity of
TESK1 and its related kinases, LIMK1 and LIMK2.
We have now obtained evidence that TESK1 is a dual specificity
protein kinase, capable of phosphorylating both serine/ threonine and
tyrosine residues in its sequence and in exogenous substrates. We also
determined the site of autophosphorylation and asked if the
autophosphorylation would play a role in regulating the kinase activity
of TESK1. Most protein kinases retain within their kinase domains
conserved sequence motifs, which are separated into subdomains, termed
I to XI (3). The region between the conserved DFG motif in subdomain
VII and the PE motif in subdomain VIII is referred to as the
"activation loop," because several protein kinases, such as
cAMP-dependent protein kinase (PKA) (10), a
mitogen-activated protein kinase (MAPK) family (11), a MAPK kinase
(MAPKK or MEK) family (12, 13), a cyclin-dependent protein
kinase (CDK) family (14) and c-Src tyrosine kinase (15), are activated
by phosphorylation of residues within this loop (reviewed in Ref. 16).
In site-directed mutagenesis studies where TESK1 replaced serine and
tyrosine residues within the activation loop by alanine, we found the
serine autophosphorylation site of TESK1 to be Ser-215. Using TESK1
mutants with replacement of Ser-215 by alanine or glutamic acid, we
also provide evidence that phosphorylation of Ser-215 is required for
the kinase activity of TESK1.
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EXPERIMENTAL PROCEDURES |
Northern Hybridization--
Total RNA was extracted from various
tissues or cells by the acid guanidine thiocyanate/phenol/chloroform
extraction method (17). Poly(A)+ RNA was purified by two
cycles of Oligotex dT-30 (Rosch) adsorption, according to the
manufacturer instruction. Poly(A)+ RNA (2 µg each) was
denatured with formaldehyde, electrophoresed on 1% agarose gels, and
transferred onto Hybond-N nylon membranes (Amersham Pharmacia Biotech).
The blots were hybridized with 32P-labeled 3.6-kb
full-length rat TESK1 cDNA or its 0.7-kb EcoRI fragment
(nucleotide residues 1-664) used as a probe and analyzed using a
BAS1000 Bio-Image Analyzer (Fuji Film), as described (1).
Plasmid Construction--
The full-length 3.6-kb rat TESK1
cDNA (1) was inserted into pBluescript II SK
(Stratagene) at EcoRI site (pBS-TESK1). To generate the
plasmid encoding TESK1-PK, composed of the N-terminal protein kinase
domain (amino acid residues 1-311) and the C-terminal antigenic
peptide sequence (residues 619-628, PSLQLPGARS) of rat TESK1, the
cDNA fragment was amplified by polymerase chain reaction, using
primers, 5'-GGAATTCCAGAGGTGTTGCGGGGAG-3' (containing EcoRI
site) and
5'-GGAATTCCCTAAGAGCGTGCCCCAGGCAGCTGCAGGCTGGGCTCCAGGATTTGTTCCAGGTGCTGG-3' (containing codons for a C-terminal peptide, a stop codon and EcoRI site). The polymerase chain reaction-amplified
fragment was inserted into pBluescript II SK
at
EcoRI site, digested with XbaI and
CbiI, and ligated with XbaI-CbiI
fragment (nucleotides 1-1936) of pBS-TESK1 to generate pBS-TESK1-PK.
To construct expression plasmids pUC-SR
-TESK1, pBS-TESK1 were
digested with NcoI and EcoRV, ligated with
NotI linker, and subcloned into the NotI site of
pUC-SR
expression vector (18). The plasmid coding for glutathione
S-transferase (GST) fused with the protein kinase domain of
TESK1 (GST-PK) was constructed by inserting NcoI,
EcoRV-digested pBS-TESK1-PK into the SmaI site of
pGEX-2T (Amersham Pharmacia Biotech). Point mutation in the kinase
domain of TESK1 was introduced, using mutated oligonucleotides and an
in vitro site-directed mutagenesis kit
(CLONTECH). The authenticity of expression plasmids
was confirmed by nucleotide sequence analysis.
Purification of GST-fusion Proteins--
GST-fusion proteins
were expressed in Escherichia coli and purified on
glutathione-Sepharose (Amersham Pharmacia Biotech), as described
(19).
Cell Culture and Transfection--
COS-7 and HeLa cells were
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum. For transfection, 5 × 105
cells were grown in 100-mm culture dish and transfected with 15 µg of
plasmid DNA/100-mm dish, following the modified calcium phosphate
method (20), then the cells were cultured for 36 h.
Immunoprecipitation and Immunoblotting--
Immunoprecipitation
and immunoblotting were carried out as described (1, 2), using rabbit
anti-TESK1 antibody (TK-C21) raised against the C-terminal 21-amino
acids peptide of rat TESK1 (2).
In Vitro Kinase Assay--
The glutathione-Sepharose beads that
bound GST-fusion protein or immunoprecipitates were washed twice with
kinase reaction buffer (50 mM HEPES, pH 7.2, 150 mM NaCl, 1 mM dithiothreitol, 1 mM
NaF, 0.1 mM sodium vanadate, 5 mM
MnCl2, 5 mM MgCl2, 0.1% Triton
X-100, and 5% glycerol) and incubated for 30 min at 30 °C in 40 µl of kinase reaction buffer containing 10 µM ATP and 10 µCi of [
-32P]ATP (3000 Ci/mmol, Amersham
Pharmacia Biotech) in the presence or absence of 0.5 mg/ml histone H3
or myelin basic protein (MBP). To detect the autophosphorylation, the
reaction mixture was centrifuged, and the pellets were washed twice
with kinase reaction buffer and subjected to SDS-polyacrylamide
gel electrophoresis (PAGE). To detect the phosphorylation of
exogenous substrates, the supernatants of the kinase reaction mixture
were resolved by SDS-PAGE. Proteins were transferred onto
polyvinylidene difluoride membranes (Bio-Rad), and
32P-labeled proteins were visualized by autoradiography.
Phosphoamino Acid Analysis--
Phosphoamino acid analysis was
performed as described (21). The region of the membrane containing the
32P-labeled protein was excised and incubated with 6 N HCl
for 2 h at 105 °C. After removal of the membrane, the
hydrolysates were separated by two-dimensional electrophoresis, and
32P-labeled phosphoamino acids were detected by
autoradiography by comparing with the elution positions of internal
phosphoamino acid standards stained with ninhydrin.
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RESULTS |
Expression of TESK1 mRNA in Rat Tissues and Various Cell
Lines--
We previously showed that TESK1 mRNA was predominantly
expressed in mouse and rat testicular germ cells (1, 2). To further investigate the expression of TESK1 mRNA in other tissues, we performed Northern blot analysis of poly(A)+ RNAs from
various tissues of adult rats and several cell lines of various
origins, using the 3.6-kb full-length rat TESK1 cDNA as a probe. In
addition to the predominant expression of a 3.6-kb TESK1 mRNA in
the testis, a faint hybridizing band of about 2.5-kb was detected in
all other tissues examined (Fig.
1A). When the blot was probed
with the 0.7-kb fragment of rat TESK1 cDNA corresponding to the
5'-untranslated region (nucleotide residues 1-664), the major 3.6-kb
mRNA species in the testis was detected, but the minor 2.5-kb
mRNA species in other tissues was not detectable even after longer
exposure (data not shown). These findings suggest that the two mRNA
species differ in the length of 5'-untranslated region and are probably
generated by an alternative initiation of a single TESK1 gene (see
"Discussion"). Expression of a similar 2.5-kb TESK1 mRNA was
also detected in various cell lines, including COS-7 kidney transformed
cells, HeLa epithelial carcinoma, HepG2 hepatoma, KB epidermoid
carcinoma, and NIH3T3 and Swiss3T3 fibroblasts (Fig. 1B).
Radiophotometric analyses revealed that the level of expression of
3.6-kb mRNA in the testis was about 100-fold higher than that of
2.5-kb mRNA in other tissues or cell lines. Even though the
expression level was low, the ubiquitous expression of 2.5-kb TESK1
mRNA in various tissues and cell lines suggests general cellular
functions of TESK1 other than spermatogenesis.

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Fig. 1.
Expression of TESK1 mRNA in rat tissues
and various cell lines. Poly(A)+ RNAs (2 µg each)
from adult rat tissues (A) and various cell lines
(B) were subjected to Northern blot analysis, using the
3.6-kb rat TESK1 full-length cDNA as a probe. The blots were also
hybridized with rat glyceraldehyde phosphate dehydrogenase
(GAPDH) cDNA to confirm the integrity of the RNA samples
(lower panels). Positions of molecular size markers are
indicated at the left.
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Dual Specificity Kinase Activity of TESK1--
To characterize
autophosphorylation and substrate phosphorylation activities of TESK1,
GST fusion protein of the N-terminal protein kinase domain of TESK1
(GST-PK) was expressed in E. coli and purified by
glutathione-Sepharose (see Fig.
2A). The C-terminal peptide
was conjugated to detect the expressed protein immunologically using
anti-TESK1 antibody. As a control, its kinase-defective mutant,
GST-PK-D170A, in which the presumptive catalytic residue Asp-170 in
subdomain VIB was replaced by alanine, was also expressed and purified.
Attempts to obtain the GST-fusion protein of full-length TESK1 from
E. coli were unsuccessful because it was unstable and obtained only as variously degraded products. Immunoblot analysis confirmed the expression of GST-PK and GST-PK-D170A as major
immunoreactive products with the expected molecular masses of around
61-kDa (Fig. 2B). When these fusion proteins were subjected
to autophosphorylation reaction in the presence of
[
-32P]ATP, GST-PK but not GST-PK-D170A was
radiolabeled (Fig. 2C), which indicates that
32P-incorporation into GST-PK was caused by the
autophosphorylation related to the intrinsic activity of GST-PK and not
by phosphorylation by contaminating bacterial protein kinases. Thrombin
cleavage of 32P-labeled GST-PK at the site between GST and
the kinase domain of TESK1 generated a 35-kDa phosphorylated protein,
which corresponds to the protein kinase domain of TESK1, thereby
indicating that autophosphorylation occurred within the kinase domain
(data not shown). Phosphoamino acid analysis of 32P-labeled
GST-PK revealed that GST-PK autophosphorylated primarily on serine and
tyrosine residues (Fig. 2D).

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Fig. 2.
Autophosphorylation of GST-PK on serine and
tyrosine residues. A, schematic diagrams showing
structures of rat TESK1 and GST-PK fusion proteins. The amino acid
residue numbers of TESK1 are indicated at the top.
PK, protein kinase domain; Pro-rich, proline-rich
domain. B, GST-PK and GST-PK-D170A were expressed in
E. coli, purified with glutathione-Sepharose, run on
SDS-PAGE, and immunoblotted with an anti-TESK1 antibody. C,
GST-PK and GST-PK-D170A bound on glutathione beads were subjected to
in vitro kinase reaction with [ -32P]ATP.
The reaction mixture was run on SDS-PAGE, transferred onto the
polyvinylidene difluoride membrane, and visualized by autoradiography.
B and C, arrowheads indicate the
positions of GST-PK. D, two-dimensional phosphoamino acid
analysis of the hydrolysates of 32P-labeled GST-PK obtained
from the membrane prepared as in panel C. The
positions of standard phosphoamino acids are indicated.
pSer, phosphoserine; pThr, phosphothreonine;
pTyr, phosphotyrosine.
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To characterize the protein kinase activity of TESK1 toward exogenous
substrates, we examined the potential of GST-PK to phosphorylate histone H3 and MBP. When GST-PK and GST-PK-D170A were subjected to
in vitro kinase reaction in the presence of
[
-32P]ATP and substrate proteins, GST-PK but not
GST-PK-D170A phosphorylated histone H3 and MBP (Fig.
3A). Phosphoamino acid
analysis revealed that histone H3 was phosphorylated primarily on
tyrosine in addition to serine and threonine, whereas MBP was
phosphorylated primarily on serine in addition to threonine and
tyrosine (Fig. 3B). Taken together these results indicate
that TESK1 has dual specificity kinase activity, which is capable of
phosphorylating both serine/threonine and tyrosine residues in its own
sequence (autophosphorylation) and in exogenous substrates.

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Fig. 3.
Dual specificity kinase activity of GST-PK
toward exogenous substrates. A, GST-PK and GST-PK-D170A
were subjected to in vitro kinase reaction in the presence
of histone H3 or MBP as the exogenous substrate. The reaction mixture
was separated on SDS-PAGE and visualized by autoradiography
(upper panels). Expression of GST-PK and GST-PK-D170A was
analyzed by immunoblotting using an anti-TESK1 antibody (lower
panels). B, two-dimensional phosphoamino acid analyses
of the hydrolysates of 32P-labeled histone H3 and MBP
obtained from the membranes prepared as in A. The positions
of standard phosphoamino acids are indicated, as in Fig. 2.
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Identification of the Serine Autophosphorylation Site--
The
short sequence between the conserved DFG motif in subdomain VII and the
PE motif in subdomain VIII of protein kinase domains has been referred
to as the "activation loop" (16). As shown in Fig.
4, various protein kinases are activated
by autophosphorylation or phosphorylation by other kinases on
serine/threonine or tyrosine residues within this region (10-15).
TESK1 contains one serine (Ser-215) and two tyrosine (Tyr-201 and
Tyr-217) residues within the activation loop. Based on the assumption
that the kinase activity of TESK1 may be regulated by phosphorylation
of residues within the activation loop, we constructed GST-PK mutants
with replacement of each of these residues by alanine, by site-directed
mutagenesis, and we then examined the related autophosphorylation
activity. As shown in Fig. 5A,
GST-PK-S215A, with replacement of Ser-215 by alanine, was significantly
reduced regarding its autophosphorylation activity. On the other hand,
GST-PK-Y201A and GST-PK-Y217A, with replacement of Tyr-201 and Tyr-217
by alanine, respectively, exhibited autophosphorylation activity at a
level similar to that seen with wild-type GST-PK. Phosphoamino acid
analyses revealed that GST-PK-S215A mutant autophosphorylated faintly
on tyrosine residue, but no phosphoserine was detected (Fig.
5B). In contrast, GST-PK-Y201A and GST-PK-Y217A mutants
autophosphorylated on both serine and tyrosine residues in the manner
similar to that for the wild-type GST-PK. These results suggest that
Ser-215 is the site of serine autophosphorylation of GST-PK, while
neither Tyr-201 nor Tyr-217 is the site of tyrosine
autophosphorylation. The tyrosine autophosphorylation site of GST-PK
was not further examined.

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Fig. 4.
Sequence alignments of the activation loops
of several protein kinases. Shaded amino acids represent conserved
DFG residues in subdomain VII and PE residues in subdomain VIII (3).
Phosphorylation sites involved in activation of the kinase activity are
shown in boxes for MEK1 (13), MAPK (12),
cAMP-dependent protein kinase (11), and CDC2 (15). The
locations of Ser-215, Tyr-201, and Tyr-217 residues of TESK1 mutated in
this study are indicated by asterisks.
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Fig. 5.
Autophosphorylation of site-directed mutants
of GST-PK. A, GST-PK and its mutants (S215A, Y201A, and
Y217A) were expressed in E. coli, purified with
glutathione-Sepharose, and subjected to in vitro kinase
reaction with [ -32P]ATP. The reaction mixture was
separated on SDS-PAGE and visualized by autoradiography (upper
panel). Expression of GST fusion proteins was analyzed by
immunoblotting with an anti-TESK1 antibody (lower panel).
Arrowheads indicate the positions of GST-PK. B,
two-dimensional phosphoamino acid analysis of the hydrolysates of
32P-labeled GST-PK mutants obtained from the membrane
prepared as in panel A. The positions of standard
phosphoamino acids are indicated as in Fig. 2.
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Kinase Activity of GST-PK Mutants with Replacement of Ser-215 by
Alanine and Glutamic Acid--
To examine the role of phosphorylation
of Ser-215 for the kinase activity of GST-PK, we constructed the
GST-PK-S215E mutant, in which Ser-215 of GST-PK was replaced by an
acidic glutamate residue, which was expected to mimic the
phosphorylated state of serine. As in the case of GST-PK-S215A
described above, GST-PK-S215E autophosphorylated only faintly, compared
with the wild-type GST-PK, probably due to the replacement of Ser-215
by a nonphosphorylatable glutamate residue (Fig.
6A, middle panel).
When we examined the kinase activity of GST-PK-S215A and
GST-PK-S215E toward histone H3, the former failed to phosphorylate
histone H3 but the latter did phosphorylate it at a level similar to
that for the wild-type GST-PK (Fig. 6A, upper
panel). Thus, replacement of Ser-215 by alanine, which is expected
to mimic the nonphosphorylated state, lost the kinase activity toward
histone H3, whereas replacement of Ser-215 by glutamic acid, an event
which mimics the phosphorylated state, retained the almost full kinase
activity. We further examined the time course of histone H3
phosphorylation by GST-PK and its mutants. Phosphorylation of histone
H3 by wild-type GST-PK and GST-PK-S215E increased almost in parallel;
the increase was linear and was dependent on the incubation time, until
10 min, and reached a maximum level after 60 min (Fig. 6, B
and C). No 32P-incorporation into histone H3 was
detectable during a 90-min incubation with GST-PK-D170A or
GST-PK-S215A. Taken together these findings suggest that Ser-215 in the
activation loop of TESK1 is the site of autophosphorylation, and this
phosphorylation is important to regulate the kinase activity of
TESK1.

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Fig. 6.
Histone H3 kinase activity of site-directed
mutants of GST-PK. A, GST-PK and its mutants (D170A,
S215E, and S215A) were subjected to in vitro kinase reaction
with [ -32P]ATP in the presence of histone H3 as an
exogenous substrate. The reaction mixture was separated on SDS-PAGE,
and 32P-incorporation into histone H3 (upper
panel) or GST-PK fusion proteins (middle panel) was
analyzed by autoradiography. Expression of GST-PK fusion proteins was
analyzed by immunoblotting with an anti-TESK1 antibody (lower
panel). B, time courses of histone H3 phosphorylation
reaction by GST-PK and its mutant proteins. The reaction mixtures at
indicated times were separated on SDS-PAGE and histone H3
phosphorylation was visualized by autoradiography. C,
relative amounts of 32P-incorporation into histone H3 by
GST-PK fusion proteins, calculated by dividing the radioactivity
incorporated into histone H3 by the amount of GST-PK fusion protein
estimated by densitometer, were plotted against the incubation time.
Histone H3 phosphorylating activity of GST-PK at 90-min incubation was
taken as 100%. Each value represents the mean of duplicate
measurements.
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Kinase Activity of Full-length TESK1 and Its Mutants Expressed in
COS Cells--
To examine the autophosphorylation and histone H3
kinase activity of the full-length TESK1, we expressed in COS-7 cells
the full-length wild-type TESK1 and its mutants with replacement of Ser-215 by alanine (TESK1-S215A) or glutamic acid (TESK1-S215E) or with
replacement of catalytic Asp-170 by alanine (TESK1-D170A). Immunoblot
analysis revealed the expression of these proteins as the major
products with estimated molecular masses of about 68 kDa (Fig.
7A, upper panel). When
immunoprecipitates were subjected to in vitro kinase
reaction, weak autophosphorylation of wild-type TESK1 was detected, but
autophosphorylation of TESK1-D170A, TESK1-S215A, and TESK1-S215E
mutants was not detected over the background level seen in the
immunoprecipitates from mock-transfected cells (Fig. 7A, lower
panel). When immunoprecipitates were subjected to in vitro kinase reaction with histone H3, wild-type TESK1 and its S215E mutant phosphorylated histone H3, to a similar extent, but TESK1-D170A and TESK1-S215A mutants did not do so (Fig. 7B).
These results suggest that autophosphorylation of Ser-215 is important for the kinase activity of full-length TESK1.

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Fig. 7.
Autophosphorylation and histone H3 kinase
activity of full-length TESK1 and its mutants. A, cell
lysates of COS-7 cells transfected with the expression plasmid for
full-length TESK1 (wt) or its site-directed mutant (D170A,
S215E, or S215A) or vector alone (mock) were
immunoprecipitated with an anti-TESK1 antibody and subjected to
in vitro kinase reaction with [ -32P]ATP and
histone H3. The pellet fractions were run on SDS-PAGE and analyzed by
immunoblotting using an anti-TESK1 antibody (upper panel) or
autoradiography to detect autophosphorylation (lower panel).
B, the supernatants of kinase reaction mixtures were run on
SDS-PAGE and analyzed by autoradiography to detect histone H3
phosphorylation (upper panel) or amido black staining
(lower panel). C, lysates of HeLa cells were
immunoprecipitated with anti-TESK1 antibody in the absence or presence
of excess amounts of antigenic peptide, run on SDS-PAGE, and
immunoblotted with the same antibody (upper panel).
Immunoprecipitates were subjected to in vitro kinase
reaction and analyzed in the same way as in B. The position
of molecular weight marker proteins were indicated on the left.
IgH, immunoglobulin heavy chain. Arrowheads indicate
the positions of TESK1 and histone H3, respectively.
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Expression and Kinase Activity of Endogenous TESK1 Protein--
We
also examined the kinase activity of TESK1 endogenously expressed in
HeLa cells. When lysates of HeLa cells were immunoprecipitated and
immunoblotted with anti-TESK1 antibody, one major immunoreactive band
migrating at around 68 kDa was detected (Fig. 7C). The size was similar to the molecular mass (67,709 Da) predicted from human TESK1 sequence, and this band was not detected when lysates were immunoprecipitated with anti-TESK1 antibody preincubated with excess
amounts of antigenic peptide, indicating that this band corresponds to
the endogenous TESK1. In vitro kinase reaction revealed that
endogenous TESK1 has activity to phosphorylate histone H3 (Fig.
7C). Thus, it is likely that the 2.5-kb TESK1 mRNAs
expressed in various tissues and cell lines yield a kinase-active
translation product with the size similar to that of testicular TESK1 protein.
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DISCUSSION |
We reported that the 3.6-kb TESK1 mRNA was predominantly
expressed in rat testicular germ cells at stages of pachytene
spermatocytes to round spermatids (1, 2). In the present work, we
obtained evidence for the ubiquitous expression of minor 2.5-kb TESK1
mRNA in various tissues and cell lines, a finding which suggests
more general functions of TESK1 for various cells other than the
function in spermatogenesis in the testis. The 2.5-kb mRNA species
was not hybridized with the probe of the 5'-untranslated region of the
3.6-kb TESK1 cDNA, suggesting that the former is the 5'-shortened form of the latter. The mouse TESK1 gene exists as a single copy gene,
which spans 6.1 kb and is composed of ten exons and nine introns (22).
Exon 1 contains the 1.0-kb 5'-untranslated region and the coding region
corresponding to the N-terminal 68-amino acid residues. We earlier
isolated the 2.5-kb TESK1 cDNA from a cDNA library of HepG2
human hepatoma cells. This cDNA contained a short 5'-untranslated
region, the sequence of which was similar to that found in exon 1 of
the mouse TESK1 gene (1, 22). Based on these observations, we suggest
that the 2.5- and 3.6-kb TESK1 mRNAs are derived from a single
TESK1 gene, and the 2.5-kb mRNA is generated by the use of an
alternative transcription initiation site, which locates in the exon 1 of the TESK1 gene. We also showed in this study the expression and
kinase activity of 68-kDa TESK1 protein endogenously expressed in HeLa
cells. This finding further suggests that the 2.5-kb TESK1 transcript
yields the kinase-active translation product similar to the one derived
from the testicular 3.6-kb transcript (2). Several Sp1-binding GC-box
elements, but no TATAA element, exist near the putative transcription
initiation site of the 2.5-kb TESK1 mRNA (22). Because Sp1
recognizes the GC-box element, which is often found singly or multiply
in TATAA-deficient promoter regions of genes expressed ubiquitously in
many tissues and at different stages of development (23), the
ubiquitous expression of the 2.5-kb TESK1 mRNA in various tissues
and cell lines may be under the control of Sp1.
The results obtained here clearly demonstrate that TESK1 has dual
specificity protein kinase activity catalyzing autophosphorylation and
phosphorylation of exogenous substrates on both serine/threonine and
tyrosine residues. It phosphorylates histone H3 mainly on tyrosine,
while it phosphorylates MBP mainly on serine. Thus, the preference for
serine/threonine versus tyrosine residue in the substrate
specificity of TESK1 seems to depend on the substrate used, probably
due to the sequence around the phosphorylatable residue and/or the
accessibility of the enzyme dependent on the substrate conformation.
Whether serine/threonine kinase activity or tyrosine kinase activity
(or both) is relevant to physiological functions of TESK1 is unknown
because cellular targets of TESK1 have yet to be identified. Except for
the cases for MAPK kinases (e.g. MEK1), the
well-characterized dual specificity kinases, which are known to
activate MAPKs by phosphorylation of both threonine and tyrosine
residues in the TxY motifs in the activation loop of MAPKs (24), the
biological significance of dual specificity kinase activity remains to
be determined for most dual-specificity kinases so far identified,
including STY/Clk1 (25, 26), ERK2 (27), Esk (28), GSK3
(29), Dyrk
(30), TIP kinase (31), and type II transforming growth factor-
receptor (32). Searches for their cellular targets will be important in
understanding the physiological significance of dual specificity for
these kinases. It could be that they have their own cellular targets
with strict substrate specificity, as in the cases of MAPK kinases.
Dual specificity kinases, including TESK1, generally belong to a
serine/threonine kinase family on a phylogenetic tree when categorized
based on primary sequences. They have no obviously recognizable
structural elements in common other than the highly conserved residues
common for all serine/threonine kinases (5). Thus, at present it is
difficult to predict dual specificity based on the primary sequence
data of protein kinases. In contrast to other protein kinases, the
kinase domain of TESK1 contains an unusual short sequence motif,
DLTSKN, in the catalytic loop of subdomain VIB, which does not
correspond to the diagnostic sequence motifs conserved in
serine/threonine kinases (DLKXXN) or tyrosine kinases
(DLRXXN or DLXXRN). This sequence may contribute
to the dual specificity kinase activity of TESK1. In this respect, it is interesting to note that LIMK1, which has a DLNSHN sequence in the
catalytic loop, was found to exhibit dual specificity kinase activity
to undergo autophosphorylation on serine/threonine and tyrosine
residues (33). Biochemical and tertiary structure analyses on the
respective kinases are needed to understand differences in substrate
specificity between the serine/threonine, tyrosine, and dual
specificity kinases.
Site-specific mutation analyses indicate that TESK1 autophosphorylates
Ser-215, which lies in the activation loop within the linkage region
between subdomain VII and VIII. The TESK1 mutant with replacement of
Ser-215 by alanine has no activity to phosphorylate histone H3, which
suggests that autophosphorylation of Ser-215 is required to exhibit the
kinase activity of TESK1. Several protein kinases are phosphorylated on
residues located in the activation loop, as shown in Fig. 4 (10-16).
Crystal structures of cAMP-dependent protein kinase (34)
and MAPK, also called ERK (extracellular signal-related protein
kinase), in phosphorylated and nonphosphorylated forms (35, 36),
revealed that phosphorylation of the residue in the activation loop
significantly changes conformation of the loop as well as its overall
structure outside the loop, an event which leads to active conformation
with the correct disposition of substrate binding and catalytic groups
and relief of steric hindrance, which provides access of substrates to
the catalytic site. In contrast to the S215A mutant of TESK1, the S215E
mutant with replacement of Ser-215 by glutamic acid exhibited histone H3 kinase activity to the level of wild-type TESK1. This finding suggests that the S215E mutant mimics the conformation similar to that
of TESK1 autophosphorylated at Ser-215. Taken together these findings
suggest that autophosphorylation of Ser-215 is an important regulatory
mechanism for the kinase activity of TESK1. As described for MEK1 (37,
38) and protein kinase C (39), the S215E mutant of TESK1 may serve as
the constitutively active form to examine the role of TESK1 in
signal transduction pathways.