A Single Amino Acid Difference between
and
Ca2+/Calmodulin-dependent Protein Kinase Kinase
Dictates Sensitivity to the Specific Inhibitor, STO-609*
Hiroshi
Tokumitsu
,
Hiroyuki
Inuzuka,
Yumi
Ishikawa, and
Ryoji
Kobayashi
From the Department of Signal Transduction Sciences, Kagawa Medical
University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan
Received for publication, December 26, 2002, and in revised form, January 22, 2003
 |
ABSTRACT |
We recently developed STO-609, a selective
inhibitor of Ca2+/calmodulin-dependent
protein kinase kinase (CaM-KK), and we demonstrated that CaM-KK
is
more sensitive to STO-609 than the CaM-KK
isoform (Tokumitsu
H., Inuzuka H., Ishikawa Y., Ikeda M., Saji I., and Kobayashi R. (2002) J. Biol. Chem. 277, 15813-15818). By using catalytic chimera and point mutants of both isoforms, we demonstrated that Val269 in CaM-KK
/Leu233 in CaM-KK
confers a distinct sensitivity (~10-fold) to STO-609 on CaM-KK
isoforms. Various mutations of Val269 in CaM-KK
indicate
that substitution by hydrophobic residues with bulky side chains
significantly decreases drug sensitivity and that the V269F
mutant is the most effective drug-resistant enzyme (~80-fold higher
IC50 value). These findings are consistent with a result
obtained with a full-length mutant expressed in COS-7 cells.
Furthermore, suppression of CaM-KK-mediated CaM-KIV activation in
transfected HeLa cells by STO-609 treatment was completely abolished by
the co-expression of the CaM-KK
V269F mutant. Based on the results
that the distinct sensitivity of CaM-KK isoforms to STO-609 is because
of a single amino acid substitution (Val/Leu) in the ATP-binding
pocket, we have generated an STO-609-resistant CaM-KK mutant, which
might be useful for validating the pharmacological effects and
specificity of STO-609 in vivo.
 |
INTRODUCTION |
Intracellular Ca2+ is known to be a second messenger
mediating a wide variety of physiological functions such as
contraction, gene expression, and secretion.
Ca2+/calmodulin-dependent protein kinases
(CaM-K)1 constitute a diverse
group of enzymes involved in many cellular responses mediated by an
increase in the concentration of intracellular calcium. Previous
studies have demonstrated that two multifunctional CaM kinases, CaM-KI
and -IV, are activated by the phosphorylation of an activation loop Thr
residue by an upstream CaM kinase kinase (CaM-KK), resulting in a large
increase in catalytic efficiency (1, 2). In mammals, two CaM-KK genes
(CaM-KK
and CaM-KK
) have been cloned, both of which are highly
expressed in the brain and also expressed (to a low degree) in various
peripheral tissues such as the thymus and spleen (3-5). CaM-KK is also
known to be a Ca2+/CaM-regulated enzyme (3, 5-7). Indeed,
Ca2+/CaM binding is absolutely required for the relief of
CaM-KK
autoinhibition, which results in the activation of the enzyme (8-10). In contrast, CaM-KK
exhibits enhanced
Ca2+/CaM-independent activity, because of the suppression
of autoinhibition by the second regulatory segment (residues 129-151)
located at the N terminus of the catalytic domain (5, 11). The CaM-KK gene has been found in Caenorhabditis elegans and
Aspergillus nidulans, and the proteins they encode are
components of the CaM kinase cascade of these organisms (12-15). A
functional CaM kinase cascade leading to the activation of CaM-KIV in
response to Ca2+ mobilization has been demonstrated by
using transfected COS-7 cells (6), Jurkat cells (16), and cultured
hippocampal neurons (17). The cascade has also been shown to be
required for the activation of CaM-KI in PC-12 cells upon membrane
depolarization (18). An important role has been demonstrated for the
CaM-KIV cascade in the regulation of
Ca2+-dependent gene expression by the
phosphorylation of transcription factors such as CREB (19-22). A
recent study of transgenic mice carrying dominant negative CaM-KIV
alleles that confer a defect in the phosphorylation of CREB indicates
that these animals exhibit a disruption of late-phase long term
potentiation, and that they are impaired in the
consolidation/retention phase of hippocampus-dependent memory (23). We have also demonstrated that the CaM kinase cascade regulated CREB-dependent gene expression in neurons of
living nematodes by using transgenic worms (24). Analysis of mice
deficient in CaM-KIV revealed that the CaM-KIV-mediated pathway plays
an important role in the function and development of the cerebellum and
is critical for male and female fertility (25-27). In addition, a
physiological role has been predicted for CaM-KK, with the suggestion that it may act as a regulatory protein kinase in the CaM kinase cascade; however, this has not been demonstrated in vivo. To
evaluate the physiological functions of CaM-KK and those of the CaM
kinase cascade, we recently developed a relatively selective and
cell-permeable inhibitor of CaM-KK, STO-609 (28). However, a recent
search for protein kinase sequences in the human genome revealed that 510 protein kinase genes are encoded in the human genome (29), indicating the practical difficulty of precisely examining the specificity of the protein kinase inhibitor in vivo as well
as in vitro (35). In this study, we further characterized
the inhibitory mechanism of STO-609 on CaM-KK activity; based on the
results, we attempted to generate an inhibitor-resistant CaM-KK mutant that might prove valuable for evaluating the specificity of the pharmacological effect(s) of STO-609 in vivo.
 |
EXPERIMENTAL PROCEDURES |
Materials--
CaM-KK
cDNA (GenBankTM
accession number L42810 (3)) was obtained from a rat brain cDNA
library. Rat CaM-KK
was cloned by reverse transcriptase-PCR, as
described previously (11). Recombinant rat CaM was expressed in the
Escherichia coli strain BL-21(DE3) using pET-CaM (30)
(kindly provided by Dr. Nobuhiro Hayashi, Fujita Health
University, Toyoake, Japan) and was purified by phenyl-Sepharose column
chromatography. Rat CaM-KI-(1-293, K49E) was expressed in E. coli JM-109 as a GST fusion protein and was purified by
glutathione-Sepharose column chromatography (10). STO-609
(7H-benzimidazo[2,1-a]benz[de]isoquinoline-7-one-3-carboxylic acid, MW 314.29) was synthesized as recently described (28).
Construction of GST Fused with CaM-KK Catalytic Domain--
GST
fused with CaM-KK
-(126-434) and CaM-KK
-(162-470) was
constructed by amplification of each fragment by PCR using specific primers (CaM-KK
-(126-434): sense primer,
5'-TCTCTAGAGAACCAGTACAAGCTG-3'; antisense primer,
5'-TTGACTGTCGACTACACCTCCTCCTCAGTC-3'; CaM-KK
-(162-470), sense primer/5'-GCTCTAGAGAATCAGTACACGCTGAAG-3', antisense
primer, 5'-TTGACTGTCGACTAGACCTCCTCTTCGGTC-3') followed by
ligation into an XbaI/SalI site in a pGEX-PreS
vector (11). GST-CaM-KK
-(162-374)/CaM-KK
-(339-434) and
GST-CaM-KK
-(162-303)/CaM-KK
-(268-434) were constructed by replacement of the corresponding fragment using common restriction sites, StuI and ScaI, respectively.
GST-CaM-KK
-(162-271)/CaM-KK
-(236-434) was constructed by PCR
amplification of each cDNA fragment using phosphorylated primers
(sense primer, 5'-GCTCTAGAGAATCAGTACACGCTGAAG-3', antisense
primer, 5'-CCCTTGGTTGACCAGTTCAAACACCATGTA for the amplification of
CaM-KK
-(162-272) and sense primer,
5'-CCAGTCATGGAAGTGCCCTGCGACAAGCCC-3', antisense primer,
5'-TTGACTGTCGACTACACCTCCTCCTCAGTC-3' for the amplification
of CaM-KK
-(237-434)) and pyrobest DNA polymerase, followed by the
insertion of both fragments into an
XbaI/SalI-digested pGEX-PreS vector.
GST-CaM-KK
-(162-250)/CaM-KK
-(215-434) was constructed by PCR
amplification of each cDNA fragment using phosphorylated primers
(sense primer, 5'-GCTCTAGAGAATCAGTACACGCTGAAG-3', antisense primer/5'-TCCACAAGCTTCACCACGTTGGGGTGATCC-3' for the
amplification of CaM-KK
-(162-251) and sense primer,
5'-TAGTCAAGCTTATCGAGGTCCTGGATGATC-3', antisense primer,
5'-TTGACTGTCGACTACACCTCCTCCTCAGTC-3' for the amplification
of CaM-KK
-(212-434)) and pyrobest DNA polymerase, followed by
HindIII digestion and ligation of both fragments into an
XbaI/SalI-digested pGEX-PreS vector.
Construction of Site-directed Mutants of GST-CaM-KK
and
--
Site-directed mutagenesis was performed by
GenEditorTM (Promega) using GST-CaM-KK
-(162-470)
cDNA as a template and the following mutagenic primers: V251I,
5'-ACGTGGTGAAGCTTATCGAGGTCCTGGATG-3'; N258A,
5'-CTGGATGACCCTGCTGAGGACCATCTGTAC-3'; H261N,
5'-GACCCTAACGAGGACAATCTGTACATGGTG-3'; M264L,
5'-AGGACCATCTGTACTTGGTGTTTGAACTGG-3'; E267D,
5'-ACATGGTGTTTGACCTAGTCAACCAAGGGC-3'; V269L,
5'-TGGTGTTTGAACTGCTCAACCAAGGGCCTG-3'; N270R,
5'-TTTGAACTGGTCAGACAAGGGCCTGTGATG-3'; Q271K,
5'-TTGAACTGGTCAACAAGGGGCCTGTGATGG-3'; V269A,
5'-TGGTGTTTGAACTGGCCAACCAAGGGCCTG-3'; V269I,
5'-TGGTGTTTGAACTGATCAACCAAGGGCCTG-3'; V269F,
5'-TGGTGTTTGAACTGTTCAACCAAGGGCCTG-3'; V269H,
5'-TGGTGTTTGAACTGCACAACCAAGGGCCTG-3'; V269M,
5'-TGGTGTTTGAACTGATGAACCAAGGGCCTG-3'; V269P
5'-TGGTGTTTGAACTGCCCAACCAAGGGCCTG-3'.
GST-CaM-KK
-(126-434) L233V mutant was constructed by site-directed
mutagenesis as described above using GST-CaM-KK
-(126-434) cDNA
as a template and 5'-TTTGACCTCGTGAGAAAGGGCCCAGTCATG-3' as a
mutagenic primer. The nucleotide sequences of each mutant were
confirmed by automated sequencing using an Applied Biosystems 377 DNA
sequencer. GST-fused CaM-KK mutants were expressed in E. coli JM-109 and purified by glutathione-Sepharose (Amersham Biosciences) as described previously (11).
In Vitro Assay for CaM-KK Activity--
Either purified
recombinant CaM-KKs (20-30 ng of GST-fusion enzyme) or CaM-KK
expressed in COS-7 cells (4 ng enzyme) were incubated with 10 µg of
GST-CaM-KI-(1-293, K49E) at 30 °C for 5 min in a solution (25 µl)
containing 50 mM HEPES (pH 7.5), 10 mM
Mg(Ac)2, 1 mM dithiothreitol, 50 µM [
-32P]ATP (3000-7000 cpm/pmol) with
various concentrations of STO-609 (0-1 µg/ml in Me2SO at
a final concentration of 4%) in the presence of either 1 mM EGTA for GST fusion enzymes or 1 mM
CaCl2, 2 µM CaM for CaM-KK
expressed
in COS-7 cells. The reaction was initiated by the addition of
[
-32P]ATP and terminated by spotting aliquots (15 µl) onto phosphocellulose paper (Whatman P-81) followed by several
washes with 75 mM phosphoric acid (31). Phosphate
incorporation into GST-CaM-KI-(1-293, K49E) was determined by liquid
scintillation counting of the filters. A 5-min reaction was chosen to
determine CaM-KK activity based on a recently described time course
experiment (11).
Transient Expression of HA-CaM-KIV with CaM-KK
and
Immunoprecipitation--
HeLa cells were maintained in Dulbecco's
modified Eagle's medium containing 10% fetal bovine serum. Cells were
subcultured in 6-cm dishes 12 h before transfection. The cells
were then transferred to serum-free medium and treated with a mixture
of either 2.7 µg of pME18s plasmid DNA (DNAX Research Institute,
Inc.) or 2.7 µg of HA (hemagglutinin-tagged)-CaM-KIV with or without
40 ng of CaM-KK
and 20 µg of LipofectAMINE reagent
(Invitrogen) in 2.5 ml of medium. After 40 h of culture,
the cells were further cultured in serum-free medium for 6 h in
either the absence or presence of various concentrations of STO-609 (1 and 10 µg/ml in Me2SO at a final concentration of 0.5%)
and then treated with or without 1 µM ionomycin for 5 min. Stimulation was terminated by the addition of 1 ml of lysis buffer
(150 mM NaCl, 20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 2 mM EGTA, 1% Nonidet P-40, 10%
glycerol, 0.2 mM phenylmethylsulfonyl fluoride, 10 mg/liter
leupeptin, 10 mg/liter trypsin inhibitor, and 1 µM
mycrocistin-LR), and the cells were lysed for 30 min on ice. The cell
extract was collected and centrifuged at 15,000 × g
for 15 min and the supernatant was precleared with 40 µl of Protein
G-Sepharose (50% slurry, Amersham Biosciences) for 2 h at
4 °C. The supernatant was then mixed with 4 µg of anti-HA antibody
(clone 12CA5, Roche Molecular Biochemicals) for 3 h. In addition,
40 µl of Protein G-Sepharose was applied to the extract and incubated
overnight. The immunoprecipitated resin was washed three times with 1 ml of the lysis buffer, as described above, and then the resin was
washed with 1 ml of kinase buffer (50 mM HEPES, pH 7.5, 10 mM Mg(Ac)2, 1 mM EGTA, and 1 µM mycrocistin-LR). Protein G-Sepharose with
immunoprecipitated HA-CaM-KIV was incubated with 40 µM
syntide-2 at 30 °C for 10 min in a solution (40 µl) containing 50 mM HEPES (pH 7.5), 10 mM Mg(Ac)2, 1 mM dithiothreitol, 1 µM mycrocistin-LR, 100 µM [
-32P]ATP (10000 cpm/pmol) in the
presence of 1 mM EGTA. To estimate the amount of
immunoprecipitated HA-CaM-KIV, SDS-PAGE sample buffer (50 µl) was
added to immunoprecipitated samples and then heated at 95 °C for 10 min. After centrifugation, 5 µl of the sample was subjected to
SDS-7.5% PAGE followed by Western blotting using anti-CaM-KIV antibody
(1:2000, Transduction Laboratories). To confirm the expression level of
co-expressed CaM-KK
(wild-type and V269F mutant), transfected HeLa
cells were lysed with 160 µl of SDS-PAGE sample buffer after
stimulation with or without ionomycin as described above, and then the
sample was heated at 95 °C for 10 min. After centrifugation, 20 µl
of the sample was subjected to SDS-7.5% PAGE followed by Western
blotting using anti-CaM-KK antibody (1:2000, Transduction Laboratories).
Expression of CaM-KK
in COS-7 Cells--
Expression of
full-length CaM-KK
in COS-7 cells was carried out essentially as
described above, using 3 µg of either pME-CaM-KK
(wild-type or
V269F mutant) or empty vector (pME18s, mock) and 20 µg of
LipofectAMINE reagent (Invitrogen). After 36 h culture, cells in
6-cm dishes were lysed with 250 µl of lysis buffer, as described
above, without EGTA. After centrifugation at 15,000 × g for 15 min, the supernatant was collected and stored at
30 °C until used for the CaM-KK activity assay. The amount of
CaM-KK
in the cell extract was estimated by Western blot analysis
with anti-CaM-KK antibody (1:2000, Transduction Laboratories) using purified recombinant wild-type CaM-KK
(11) as a standard. The antibody was capable of recognizing both
and
isoforms of CaM-KK (11).
Others--
Western blot analysis was performed using
horseradish peroxidase-conjugated anti-mouse IgG antibody (Amersham
Biosciences) as a secondary antibody and chemiluminescence reagent
(PerkinElmer Life Sciences) for detection. Protein
concentrations for E. coli expressing GST-fused CaM-KK
mutants were estimated by Coomassie dye binding (Bio-Rad) using bovine
serum albumin as a standard.
 |
RESULTS AND DISCUSSION |
We have recently developed a potent and relatively selective
inhibitor of CaM-KK, STO-609, which can permeate cells and is a
competitive inhibitor of ATP (28). According to previous results, the
sensitivity of CaM-KK
to the compound is >5-fold higher than that
of the
isoform whereas the apparent Km values of
both isoforms for ATP (~33 µM) are indistinguishable
(28). To elucidate the mechanism of the differential sensitivity of CaM-KK isoforms to STO-609, we examined the inhibitory effect of
STO-609 on various catalytic domain chimera mutants of both isoforms,
because the inhibitor has been shown to target the catalytic domain of
CaM-KK (28). Catalytic domain mutants, including the wild-type enzyme,
lack both an N-terminal extension domain (residues 1-125 in the
isoform and residues 1-161 in the
isoform) and a C-terminal domain
containing an autoinhibitory segment and a CaM-binding segment (8, 11).
These mutants were expressed in E. coli and purified as GST
fusion proteins. Thus, all of the recombinant enzyme phosphorylates the
protein substrate (GST-CaM-KI-(1-293), K49E) in a complete
Ca2+/CaM-independent manner. Despite the 70% amino acid
identity of the catalytic domain between rat CaM-KK
and -
(Fig.
1A), STO-609 suppressed
CaM-KK
-(162-470) activity more efficiently (IC50 = ~10 ng/ml) than did the
isoform (126-434, IC50 = ~130 ng/ml) in the presence of 50 µM ATP (Fig.
1B). When we compared the inhibitory potency of STO-609
against various chimera mutants,
CaM-KK
-(162-374)/CaM-KK
-(339-434), CaM-KK
-(162-303)/CaM-KK
-(268-434), and
CaM-KK
-(162-271)/CaM-KK
-(236-434) mutants showed no significant
difference in their IC50 value (~10 ng/ml) from that of
CaM-KK
-(162-470). Only the
CaM-KK
-(162-250)/CaM-KK
-(215-434) mutant drastically decreased
the sensitivity to STO-609 (IC50 = ~130 ng/ml), whose
IC50 value is comparable with that of the CaM-KK
isoform
(126-434). This result suggests that residues 251-271 in CaM-KK
that correspond to residues 215-235 in the
isoform (Fig.
1A) appear to be involved in the sensitivity to inhibition
by STO-609. These residues of both CaM-KK isoforms are located between
subdomains IV and V in the protein kinase catalytic domain (32),
containing an ATP-binding hydrophobic pocket.

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Fig. 1.
Inhibition of CaM-KK catalytic domain chimera
mutants by STO-609. A, amino acid sequences of the
catalytic domain of rat CaM-KK (residues 126-434 (3)) and (residues 162-470 (4)) are aligned. Residues identical in both CaM-KK
isoforms are indicated by colons. The subdomain location in
the catalytic domain was determined by the numbering system of Hanks
et al. (32) and is indicated by dashed lines. The
RP domain was previously identified as a region involved in substrate
recognition of CaM-KK (13). The region involved in STO-609 sensitivity
of each CaM-KK (residues 215-235 in CaM-KK , and residues 251-271
in CaM-KK ) is indicated by a shaded box. B,
schematic representation of catalytic domain chimera mutants of CaM-KK
isoforms is shown. CaM-KK catalytic domain chimera mutants, including
the wild-type enzyme, were constructed and expressed in E. coli JM-109 as a GST fusion protein and purified as described
under "Experimental Procedures." Purified CaM-KK mutants (20-30
ng) were incubated with 10 µg of GST-CaM-KI-(1-293, K49E) at
30 °C for 5 min in a solution (25 µl) containing 50 mM
HEPES (pH 7.5), 10 mM Mg(Ac)2, 1 mM
dithiothreitol, and 50 µM [ -32P]ATP with
various concentrations of STO-609 (0-1 µg/ml in Me2SO at
a final concentration of 4%) in the presence of 1 mM EGTA.
The CaM-KK activities of recombinant enzymes measured in the absence of
STO-609 are shown and the IC50 values were calculated, as
described under "Experimental Procedures." Results represent the
mean ± S.E. of three experiments.
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According to the amino acid sequence alignment of the region involved
in the distinct sensitivity of CaM-KK isoforms to the inhibitor
described above, eight amino acid residues were shown to differ (Fig.
2). Then, we replaced each of the
differing amino acids among residues 251-271 of CaM-KK
-(162-470)
by the corresponding residues in the
isoform and measured the
inhibitory effects of STO-609 on the activities of the mutants (Fig.
2). The IC50 values of seven of eight substitution mutants
(V251I, N258A, H261N, M264L, E267D, N270R, and Q271L) for inhibition by
STO-609 were indistinguishable to that of
CaM-KK
-(162-470)(IC50 = ~10 ng/ml). Only the V269L
mutation significantly reduced the sensitivity of the enzyme to STO-609
(IC50 = ~140 ng/ml) without any effects on the catalytic
efficiency. This IC50 value reflects a similar inhibitory
potency to CaM-KK
-(126-434). To confirm whether Leu233
(i.e. the equivalent of Val269 in the
isoform) in CaM-KK
is indeed involved in lowered drug sensitivity,
as compared with the
isoform, we replaced Leu233 in
CaM-KK
-(126-434) with a Val residue and measured the inhibitory effect of STO-609 on the mutant. As was expected, the IC50
value of the L233V mutant of CaM-KK
-(126-434) for inhibition by
STO-609 was ~8-fold lower than that of the wild-type enzyme. The
catalytic activity of the L233V mutant in the absence of STO-609 was
comparable with that of the wild-type CaM-KK
-(126-434). These
results strongly indicate that a single amino acid difference (Val/Leu)
in the catalytic domain of CaM-KK isoforms dictates sensitivity to the inhibitor. Val residue at position 269 in the rat CaM-KK
is
conserved in human CaM-KK
(Val270) (5) and C. elegans CaM-KK (Val125) (13). According to the crystal
structure of PKA, Val269 in CaM-KK
is also conserved in
subdomain V in PKA as Val123, which helps to anchor Mg-ATP
by hydrogen bonding, and also contributes to a hydrophobic pocket that
surrounds the adenine ring (33, 34).

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Fig. 2.
Involvement of Val269 in
CaM-KK /Leu233 in
CaM-KK in the sensitivity of the inhibition of
CaM-KK isoforms by STO-609. Amino acid sequences of rat CaM-KK
(residues 215-235) and (residues 251-271) are aligned. Residues
identical between both CaM-KK isoforms are indicated by
colons. Schematic representation of point mutants of
GST-fused CaM-KK -(162-470) and CaM-KK -(126-434) is shown. Point
mutants of CaM-KK isoforms, including the wild-type enzyme, were
expressed and purified as described under "Experimental Procedures"
and 20-30 ng of each mutant was assayed to measure the inhibitory
effect of STO-609, as described in the legend to Fig. 1. The CaM-KK
activities of recombinant enzymes measured in the absence of STO-609
are shown and the IC50 values were calculated, as described
under "Experimental Procedures." Results represent the mean ± S.E. of three experiments.
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Next, we mutated Val269 in CaM-KK
-(162-470) with
various amino acid residues, and in particular, hydrophobic residues,
to maintain the hydrophobic ATP-binding pocket. We then examined the
inhibitory potency of the inhibitor against these mutants (Fig.
3). Although the Ala mutation had little
or no significant effect on the drug sensitivity of the enzyme, the
mutations with hydrophobic residues with relatively bulky side chains
(Leu, His, and Met) resulted in a significant increase (>10-fold) in
the IC50 value for inhibition by STO-609 of the CaM-KK
mutants. We found that the mutation of Val269 by Phe caused
the most dramatic effect (i.e. ~80-fold increase in the
IC50 value) on the drug sensitivity of CaM-KK
. It is of note that a Pro mutation results in an inactive enzyme, probably because of the destruction of the structure of the ATP-binding pocket
necessary for efficient catalysis. Because the V269A mutant was
inhibited by STO-609 with the same potency as was the wild-type enzyme,
an amino acid residue at Val269 in
CaM-KK
/Leu233 in CaM-KK
may not be involved in the
direct binding of the compound to CaM-KK. However, mutation of
Val269 by hydrophobic residues with relatively larger side
chains (e.g. Leu, Met, His, and Phe) can efficiently block
the STO-609/CaM-KK interaction resulting in decreased drug sensitivity
of CaM-KK.

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Fig. 3.
Effect of various mutations at
Val269 in CaM-KK on STO-609
sensitivity. Schematic representation of point mutants at
Val269 of GST-fused CaM-KK -(162-470) is shown. Point
mutants of GST-fused CaM-KK -(162-470), including the wild-type
enzyme, were expressed and purified as described under "Experimental
Procedures" and 20-30 ng of each mutant was assayed to measure the
inhibitory effect of STO-609, as described in the legend to Fig. 1. The
CaM-KK activities of recombinant enzymes measured in the absence of
STO-609 are shown and the IC50 values were calculated as
described under "Experimental Procedures." The results represent
the mean ± S.E. of three experiments. ND, not
detected.
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Identification of Val269/Leu233 in the CaM-KK
isoforms involved in the STO-609 sensitivity described above was
performed by using E. coli expressing GST fusion proteins
with the catalytic domain of each CaM-KK. We attempted to express the
V269F mutant of full-length CaM-KK
in mammalian cells to confirm the
drug sensitivity of the mutant enzyme. As shown in Fig.
4, the protein kinase activity of
wild-type CaM-KK
expressed in COS-7 cells was inhibited by ~80%
in the presence of 0.1 µg/ml STO-609 with an IC50 value
of 20-30 ng/ml. This result is consistent with that of a previous report using E. coli expressing full-length CaM-KK
(28).
In contrast, although the CaM-KK activities of COS-7 cells expressing the wild-type CaM-KK
(504 ± 15 nmol/min/mg) and the V269F
mutant (656 ± 22 nmol/min/mg) in the absence of STO-609 were
comparable, 1 µg/ml STO-609 suppressed only 40% of the V269F mutant
activity. And the IC50 value for inhibition by STO-609 of
the mutant was approximately 2 orders of magnitude higher than that of
the wild-type enzyme. This finding is in good agreement with the
results obtained with catalytic domain mutants, as shown in Fig. 3.

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Fig. 4.
Effect of STO-609 on the activities of
full-length CaM-KK wild-type and V269F mutant
expressed in COS-7 cells. Wild-type CaM-KK (CaM-KK WT,
closed circle) and V269F mutant (CaM-KK V269F, open
circle) cDNA was transfected into COS-7 cells and the cell
extracts (4 ng of CaM-KK) were assayed to measure the inhibitory
effects of STO-609 in the presence of 1 mM
CaCl2, 2 µM CaM, as described under
"Experimental Procedures." The CaM-KK activity of each enzyme,
including the extract from cells transfected with an empty vector that
was negligible, was measured as described under "Experimental
Procedures." The results are expressed as a percentage of the value
in the absence of STO-609 (CaM-KK wild-type, 504 ± 15 nmol/min/mg; CaM-KK V269F, 656 ± 22 nmol/min/mg). The results
represent the mean ± S.E. of three experiments. Each extract,
including the extract from the cells transfected with an empty vector
(Mock), was subjected to SDS-10% PAGE followed by Western
blot analysis using anti-CaM-KK antibody (inset). The
arrow indicates CaM-KK .
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Finally, we examined whether or not ectopical expression of the
CaM-KK
V269F mutant induces STO-609 resistance in the
kinase-expressing cells. After we co-transfected HA-tagged CaM-KIV with
an empty vector, wild-type CaM-KK
, or V269F mutant cDNA into
HeLa cells, the cells were treated with (1 and 10 µg/ml) or without
STO-609, and then the cells were stimulated with ionomycin for 5 min,
thereby increasing the intracellular Ca2+ concentration.
Then, HA-CaM-KIV was immunoprecipitated with anti-HA antibody followed
by measurement of its Ca2+/CaM-independent activity using
syntide-2 as a substrate (Fig. 5).
Western blot analysis of the cell extract using anti-CaM-KK antibody
revealed that HeLa cells contain endogenous CaM-KK
and the
expression levels of overexpressed wild-type CaM-KK
and V269F mutant
were indistinguishable (Fig. 5, upper inset). The amount of
immunoprecipitated HA-CaM-KIV was also confirmed by Western blot
analysis (Fig. 5, lower inset). The protein kinase activity of immunoprecipitated CaM-KIV from cells co-expressed with or without
wild-type CaM-KK
or V269F mutant was largely activated by
Ca2+ mobilization. Maximum activity of the
immunoprecipitated CaM-KIV induced by ionomycin was not affected by
co-expression of either wild-type CaM-KK
or V269F mutant.
Suppression of Ca2+-dependent induction of
CaM-KIV activity by STO-609 treatment indicates an endogenous
CaM-KK-mediated CaM-K cascade in HeLa cells. STO-609 is an
ATP-competitive inhibitor (28), and the ATP-concentration in cells is
in the millimolar range. Therefore, a much higher concentration of
STO-609 appears to be required to suppress CaM-KK activity in these
cells, as compared with the concentration required for the inhibition
of CaM-KK activity in vitro. Although the overexpression of
wild-type CaM-KK
significantly reduced the inhibitory potency of
STO-609 for the activation of CaM-KIV in these cells, when compared
with the inhibition of endogenous CaM-KK activity in HeLa cells, it was
nonetheless shown that 50-60% of the ionomycin-induced CaM-KIV
activation was suppressed by treatment with 10 µg/ml STO-609 in the
presence of overexpressed wild-type CaM-KK
. In contrast, activation
of CaM-KIV from the cells co-expressed with the V269F mutant was
completely resistant to the inhibitor at concentrations up to 10 µg/ml. This result establishes that the suppression of
ionomycin-induced CaM-KIV activity by STO-609 was because of direct
inhibition of endogenous CaM-KK in these cells, as effected by the
inhibitor. We have observed a similar result with the same transfection
experiment using COS-7 cells (data not shown). Taken together, these
results support the idea that the V269F mutant of CaM-KK
functions
as an STO-609-resistant mutant in intact cells as well as in
vitro.

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|
Fig. 5.
CaM-KK V269F mutant
induces STO-609 resistance in HeLa cells. After transfection of
either HA-CaM-KIV cDNA (2.7 µg, +) or empty vector (left
lane, ) without ( ) or with either 40 ng of wild-type CaM-KK
(WT) or V269F mutant cDNA (V269F) into HeLa cells, the
cells were either untreated ( ) or treated with various concentrations
of STO-609 (1 and 10 µg/ml), as indicated, for 6 h in serum-free
medium. The cells were then stimulated with 1 µM
ionomycin for 5 min (+), or not stimulated ( ). HA-CaM-KIV was
immunoprecipitated and its Ca2+/CaM-independent activity
was measured at 30 °C for 10 min in the presence of 1 mM
EGTA, as described under "Experimental Procedures." The results
represent the mean of two independent transfections. Similar results
were obtained in three separate experiments. Transfected HeLa cell
extracts (1/8 volume) and immunoprecipitated samples (1/10 volume),
including a sample prepared from cells transfected with an empty
vector, were subjected to Western blot analysis with an anti-CaM-KK
antibody (upper inset) and an anti-CaM-KIV antibody
(lower inset), respectively, as described under
"Experimental Procedures." The upper and lower
arrows in the upper inset indicate overexpressed
CaM-KK and endogenous CaM-KK , respectively. The upper
and lower arrows in the lower inset indicate
HA-CaM-KIV and the anti-HA antibody (IgG), respectively.
|
|
In summary, we have characterized the inhibitory mechanism of a
recently developed inhibitor of CaM-KK, STO-609, with regard to the
distinct sensitivity of CaM-KK isoforms to the inhibitor. We have shown
in this report that a single amino acid difference (Val269
in CaM-KK
/Leu233 in CaM-KK
) in the ATP-binding
hydrophobic pocket of the catalytic domain of CaM-KK isoforms is
directly involved in the distinct sensitivity to the inhibitor. This
conclusion is well correlated with findings from the kinetic analysis
of STO-609 inhibition, which revealed that the compound is
ATP-competitive (28). Although Val269 is conserved in PKA
(Val123) as an important residue that contributes to a
hydrophobic pocket and also helps to anchor Mg-ATP, STO-609 has a less
effective inhibitory potency (<100-fold) against PKA than does CaM-KK
(28). This suggests that the reduced inhibitory potency of this
compound against PKA appears to proceed in a totally different manner
from that of CaM-KK. According to the present results with various point mutants of Val269 in CaM-KK
, substitution of
Val269 by residues with bulky side chains resulted in
effectively increasing IC50 values of the mutants. This
result is probably because of interference with the binding of STO-609
to CaM-KK by bulky side chains, because the substitution of
Val269 by a residue with a relatively small side chain such
as Ala had no impact on inhibition by STO-609. Based on these results,
we generated an STO-609-resistant CaM-KK
mutant (V269F), which was able to induce STO-609 resistance in transfected HeLa cells in terms of
the activation of CaM-KK-mediated CaM-KIV activation. Because 510 protein kinases are likely encoded in the human genome (29), it is very
difficult to evaluate the specificity of a protein kinase inhibitor,
in vitro as well as in vivo. Therefore, the
availability of an STO-609-resistant CaM-KK mutant could be useful to
validate the pharmacological effect(s) and the specificity of a
compound in vivo with regard to elucidation of the
physiological function(s) mediated by the CaM kinase cascade.
 |
ACKNOWLEDGEMENTS |
We thank Sumitomo Pharmaceuticals for the
synthesis of STO-609 and N. Ishikawa (Kagawa Medical University) for
excellent technical assistance. We are also grateful to Y. Watanabe
(Kagawa Medical University) for helpful discussions.
 |
FOOTNOTES |
*
This work was supported in part by Grant-in-aid for
Scientific Research 14580649 from the Ministry of Education, Culture, Sports, and Technology of Japan (to H. T.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Signal
Transduction Sciences, Kagawa Medical University, 1750-1 Miki-cho, Kita-gun, Kagawa 761-0793, Japan. Tel./Fax: 81-87-891-2368; E-mail: tokumit@kms.ac.jp.
Published, JBC Papers in Press, January 22, 2003, DOI 10.1074/jbc.M213183200
 |
ABBREVIATIONS |
The abbreviations used are:
CaM-K, Ca2+/CaM-dependent protein kinase;
CaM, calmodulin;
PKA, cAMP-dependent protein kinase;
CREB, cAMP-response element binding protein;
HA, hemagglutinin;
GST, glutathione S-transferase.
 |
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