From the Departments of Physiology,
§ Biology, and ¶ Pharmaco-Bio-Informatics, Faculty of
Medicine, Kagawa Medical University, 1750-1 Ikenobe, Miki-cho,
Kita-gun, Kagawa 761-0793, Japan
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ABSTRACT |
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A cDNA clone that encodes a novel
Ca2+-binding protein was isolated from a human brain
cDNA library. The gene for this clone, termed calbrain, encodes a
70-amino acid polypeptide with a predicted molecular mass of 8.06 kDa.
The analysis of deduced amino acid sequence revealed that calbrain
contains two putative EF-hand motifs that show significantly high
homology to those of the calmodulin (CaM) family rather than two
EF-hand protein families. By Northern hybridization analysis, an
approximate 1.5-kilobase pair transcript of calbrain was detected
exclusively in the brain, and in situ hybridization study
revealed its abundant expression in the hippocampus, habenular area in
the epithalamus, and in the cerebellum. A recombinant calbrain protein
showed a Ca2+ binding capacity, suggesting the functional
potency as a regulator of Ca2+-mediated cellular processes.
Ca2+/calmodulin-dependent kinase II, the most
abundant protein kinase in the hippocampus and strongly implicated in
the basic neuronal functions, was used to evaluate the physiological
roles of calbrain. Studies in vitro revealed that calbrain
competitively inhibited CaM binding to
Ca2+/calmodulin-dependent kinase II
(Ki = 129 nM) and reduced its kinase
activity and autophosphorylation.
Calcium ion (Ca2+) is a universally employed cytosolic
messenger in eukaryotic cells. It is involved in many cellular
processes such as signal transduction, contraction, secretion, and cell proliferation (1, 2). In the central nervous system, Ca2+
plays a major role in the activities and functions of neuronal cells
(3-5). One of the most widely recognized roles of Ca2+ in
synaptic function is its action in neurotransmission. Studies on the
effects of Ca2+ on neurotransmitter release, synaptic
protein phosphorylation, synaptic vesicles, and synaptic membrane
interactions have provided experimental evidence that Ca2+
regulates several biochemical and morphological events in synaptic preparations (6, 7).
In many cases, the effects of Ca2+ are mediated by the
Ca2+-binding proteins (8). One superfamily of these
proteins is the EF-hand protein family. The EF-hand proteins are
characterized by single or multiple pairs of a common helix-loop-helix
motif that coordinates Ca2+ (9, 10). For instance,
CaM,1 troponin C, and myosin
light chain have four EF-hand motifs/molecule, whereas S100 proteins
have only two motifs per molecule. In addition to the role as a
Ca2+-buffering system, the binding of Ca2+
causes a conformational change of EF-hand proteins and enables them to
interact with their target proteins. Most of the EF-hand proteins
except CaM show specific tissue distribution, suggesting their
particular functions in each tissue. CaM has broad distribution within
the cell and throughout different tissues and is a multifunctional regulatory protein that, in a Ca2+-dependent
manner, activates a number of enzymes that are involved in a variety of
physiological processes (11). Among these enzymes, CaM-kinase II is one
of the most abundant Ca2+-activated protein kinases in the
brain, and it plays important roles in a variety of neural functions
including receptor function, neurotransmitter release, and synaptic
plasticity (12).
CaM-kinase II is activated by binding to the Ca2+-bound
form of CaM, which dramatically increases the affinity of the enzyme for Mg2+/ATP, thus leading to substrate phosphorylation and
autophosphorylation (13-15). This self-regulation system coupled to
Ca2+/CaM-dependent autophosphorylation may be
involved in important physiological roles responding to transient
elevation of intracellular Ca2+ (16). During
autophosphorylation (of Thr286), trapping of CaM in
CaM-kinase II occurs, resulting in prolongation of the activation
period of CaM-kinase II. This process is understood as a good model of
memory formation (15, 17).
In the present study, we cloned and characterized a novel two EF-hand
Ca2+-binding protein, termed calbrain, that is
brain-specific and highly expressed in the hippocampus. To characterize
the physiological function of this protein in the hippocampus, the
effects of calbrain on CaM-kinase II were examined. The results
revealed that calbrain competitively inhibited the activity of
CaM-kinase II, suggesting that calbrain may be involved in neuronal
signal transduction and memory.
Polymerase Chain Reaction and Human cDNA Library
Screening--
Degenerate primers were originally designed from
transmembrane regions of several mammalian G-protein-coupled receptors.
Polymerase chain reaction was performed with these primers using 100 ng
of human brain cDNA library (CLONTECH) as a
template. The cycling condition was 1 min at 95 °C, 1 min at
55 °C, and 0.5 min at 72 °C for 37 cycles. The polymerase chain
reaction products were end-repaired with T4 DNA polymerase and ligated
into pCR-Script SK(+) vector (Stratagene). Samples were transformed,
and colonies were picked for subsequent sequencing. The sequence data
was obtained using ABI 373 sequencer (Perkin-Elmer) with
dye-terminators by the method of Sanger et al. (18). The
data was searched against SWISSPROT (Ver.30.0) data base by BLAST
algorithm (18). The fragment that showed a similarity to
Ca2+-binding proteins was chosen for further analysis.
About one million recombinants of human brain Northern Hybridization Analysis--
The full-length coding
region of calbrain was labeled with [ In Situ Hybridization Study--
Adult Sprague-Dawley rats (all
male, 8-9 weeks old) were anesthetized, and brains were perfused with
phosphate-buffered saline. Brains were then quickly removed and frozen
at-80 °C. Cryostat sections (10 µm-thickness) were mounted onto
slides. The sections were fixed in 4% paraformaldehyde in
phosphate-buffered saline, incubated with 0.01% proteinase K in
Tris-HCl for 30 min, and then acetylated for 15 min with 0.2 M triethanolamine containing 0.2% acetic acid. After
sequential dehydration through a graded alcohol series, each section
was hybridized either with a digoxigenin-labeled antisense RNA probe or
with a labeled sense probe as a control. The RNA probe was made from a
rat homologue of calbrain gene that is 99.0% identical in nucleotide
level to the human one. After hybridization at 50 °C overnight, the
slides were incubated with RNase A (10 µg/ml) and washed with 2× SSC
and then with 0.2× SSC. Slides were incubated with alkaline
phosphatase-conjugated anti-digoxigenin antibody (Boehringer Mannheim)
for 30 min, washed, and developed by incubating at 4 °C overnight
with 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium.
Expression and Purification of Recombinant
Protein--
Recombinant calbrain protein was expressed using the QIA
expressionist system (QIAGEN). The gene construct encoding calbrain was
subcloned into pQE expression plasmid vector and transformed into
Escherichia coli host strain M15. Transformed M15 cells were cultured in 500 ml of LB medium containing 100 µg/ml ampicillin and
25 µg/ml kanamycin until the A600 of 0.7 at
37 °C. After adding isopropyl-1-thio- Tricine-SDS-PAGE and Electrophoretic
Transfer--
Tricine-SDS-polyacrylamide gel electrophoresis
(Tricine-SDS-PAGE) was carried out as described previously (19).
Protein samples, CaM protein as a control, and molecular weight markers were denatured and applied to 10% Tricine-SDS-PAGE. Separated protein
bands were either stained with Coomassie Brilliant Blue or transferred
to a nitrocellulose membrane by the method of Kyhse-Andersen (20).
Detection of Calcium Binding by
45Ca2+--
The calcium binding study using
45Ca2+ was performed according to the method of
Maruyama et al. (21). Briefly, after the protein transfer,
the membrane was soaked in a solution containing 60 mM KCl,
5 mM MgCl2, and 10 mM imidazole-HCl
(pH 6.8). The membrane was then incubated in the same buffer containing
1 mCi/liter 45CaCl2 for 10 min. Nonspecifically
bound 45Ca2+ was removed by washing with 50%
ethanol for 5 min, and dried membrane was exposed to imaging plates.
The images were analyzed by Fuji phosphor-imaging system as above,
under the Northern hybridization analysis.
Ca2+ binding affinity of recombinant calbrain was
determined by equilibrium dialysis. Calbrain was first dialyzed
overnight against 1000 volumes of a solution containing 150 mM KCl, 10 mM MOPS, pH 7.1, 3 mM
MgCl2, 1 mM dithiothreitol, and 0.1 mM EGTA to remove bound Ca2+ from the protein.
The dialyzed protein was then used for equilibrium dialysis as follows.
A 0.5-ml portion of protein at the concentration of 1 mg/ml was
dialyzed with shaking for 48 h at 4 °C against 100 ml of the
same solution as described above for Ca2+ binding affinity
but containing various amounts of CaCl2 and 45Ca2+ (5 µCi) to achieve the desired
free-Ca2+ concentration. The solutions outside and inside
the dialysis tubing were removed, the absorbance at 278 nm was
determined, and the protein concentration calculated
(A2781% = 0.958). Samples of
these solutions were subjected to liquid scintillation spectrometry.
The association constants for metal and H+ binding to EGTA
were based on values measured by Fabiato (23).
Kinase Activity and Autophosphorylation Assay--
The activity
of CaM-kinase II was assayed by measuring the
Ca2+-dependent phosphorylation (32P
incorporation) of syntide-2 substrate as described previously by
Ochiishi et al. (24). The standard reaction mixture
contained 50 µM[ Inhibition of CaM Binding to CaM-kinase II by Calbrain--
The
effect of calbrain on CaM binding to CaM-kinase II was examined as
described previously (27) with some modifications. The reaction mixture
for the binding studies was the same as that used in the kinase
activity assay without ATP. Various concentrations (0-200
µM) of 125I-CaM and calbrain were incubated
in the reaction mixture at the total volume of 200 µl at 4 °C for
1 h, and they were reacted with 3 µg of polyclonal
anti-CaM-kinase II antibody (Transduction Laboratories) at 4 °C for
16 h. The amount of antibody was proved to be adequate for binding
to entire CaM-kinase II in the reaction mixture. Then, 10 µl of
protein G-Sepharose (Amersham Pharmacia Biotech) was added to each
reaction mixture and incubated at 4 °C for 1 h on a rotating
wheel. After 3 washes with the reaction mixture without CaM-kinase II,
these samples were centrifuged (5000 rpm, 5 min), and the radioactivity
of each pellet was measured by a gamma counter. Double-reciprocal plots
and determination of Ki values were performed as
described by Segel (28).
Calbrain Has Two EF-hand Motifs--
The isolated clone was found
to have a 210-nucleotide open reading frame that encodes a 70-amino
acid polypeptide with a predicted molecular mass of 8.06 kDa (Fig.
1). We named the clone as calbrain. A
motif search suggested the presence of two EF-hands, a motif known to
be involved in calcium binding (29). By a data base search, calbrain
showed significant high homology to CaM and troponin C proteins. The
four EF-hand domains of CaM, troponin C, and two EF-hand domains of
S100 Brain-specific Expression of Calbrain mRNA--
From the
results of Northern hybridization analysis under high stringency
conditions, an approximate 1.5-kilobase single calbrain transcript was
detected exclusively in the brain (Fig.
3). No band was detected in other tissues
examined including heart, placenta, lung, liver, muscle, kidney, and
pancreas.
Localization of Calbrain mRNA in the Brain--
Results of
in situ hybridization study for localization of calbrain
mRNA in rat brain was conducted on rat brain sections showed strong
signals in the pyramidal layers CA1 to CA3 of the hippocampal gyrus and
the granular layer of the dentate gyrus in the hippocampus (Fig.
4). The habenular nucleus in the
epithalamus was also stained strongly. In the cerebellum, the Purkinje
cells were strongly stained. There were also weak and scattering
signals in the cerebral cortex and other areas of the brain. No
staining was observed when a sense probe was used as a control.
Ca2+ Binding Property of Calbrain--
The purified
calbrain and CaM as a control were run on a 10% Tricine-SDS-PAGE gel
with molecular mass markers (Fig.
5A). The molecular mass based
on the amino acid sequence of mammalian CaM is 16.7 kDa, which is
approximately double the size of calbrain. On Tricine-SDS-PAGE gel,
calbrain protein was observed at approximately 8 kDa, and CaM is
observed as the same size of its molecular mass. The control fraction
(sample using the nonrecombinant vector) on the same Tricine-SDS-PAGE
gel did not show any protein bands. The 45Ca2+
binding study of calbrain are shown in Fig. 5A. Both CaM and calbrain showed strong radioactive bands at the expected positions, indicating the binding of 45Ca2+ to these
proteins. Fig. 5B shows a saturation curve for the
Ca2+ binding to calbrain. Scatchard analysis (30) of the
data (Fig. 5B, inset and legend) reveals that, in
the presence of 3.0 mM MgCl2 and 150 mM KCl, calbrain binds 2.0 mol of Ca2+/mol of
protein with an apparent Kd of 0.194 µM.
The Inhibitory Effect of Calbrain on CaM-kinase II Activity,
Autophosphorylation, and CaM Binding--
The effect of calbrain on
CaM-kinase II activity is shown in Fig.
6, A and B. The
activity of CaM-kinase II incubated in the solution containing 0-200
µM calbrain without CaM (Fig. 6A, open circles) revealed that calbrain was not able to activate
CaM-kinase II. On the other hand, Fig. 6B showed that
calbrain competitively inhibited activation of CaM-kinase II by CaM
with a Ki value of 143.5 ± 16.4 nM
(mean ±S.E.). The effect of calbrain on the autophosphorylation of
CaM-kinase II was examined, and the results were shown in Fig.
7. Calbrain also competitively inhibited
CaM-dependent autophosphorylation of CaM-kinase II with a
Ki value of 189.7 ± 12.4 nM (mean
±S.E.), whereas calbrain itself did not induce autophosphorylation of
CaM-kinase II (data not shown). The binding study of CaM and calbrain
to CaM-kinase II revealed that calbrain competitively inhibited CaM
binding to CaM-kinase II, and the Ki value of this
inhibition was 128.6 ± 19.7 nM (mean ±S.E.) (Fig.
8).
In the present study, we have shown that two EF-hand motifs of
calbrain, a novel Ca2+-binding protein, have a significant
homology to those of CaM and other related four EF-hand
Ca2+-binding proteins. The first EF-hand motif of calbrain
is very similar to the first and the third motifs, and the second
EF-hand motif of calbrain very similar to the second and the fourth
motifs of CaM and troponin C. Although calbrain is a two EF-hand
protein, the homology between calbrain and two EF-hand proteins such as S100 The biological functions of EF-hand proteins are strongly related to
the conformational changes of EF-hand domains in response to
Ca2+ binding. EF-hand domains that show large
conformational changes by binding Ca2+ are known to have a
trigger function in the activation of target proteins. The domains that
have regulatory roles are termed regulatory domains (35, 36), and
proteins that have such domain(s) are called Ca2+ sensor
proteins. For example, CaM and troponin C, which are classified in this
category, enable the cell to detect a stimulatory influx of
Ca2+ and thereby transduce this signal into a variety of
cellular processes (37). On the other hand, EF-hand domains that
exhibit small conformational changes are termed structural or buffer
domains (36, 38). These domains are responsible for the structural stability, local Ca2+ transport, and function in buffering
intracellular Ca2+. Proteins possessing these domains, such
as calbindin and parvalbumin, are called Ca2+ buffer
proteins (39, 40). From the amino acid sequence in the present study,
it is difficult to predict whether calbrain has regulatory or
structural domains, and therefore is a Ca2+ sensor or
Ca2+ buffer protein. It has been suggested that the
interhelical angle changes of EF-hand proteins upon Ca2+
binding become a good index for their classification. Another useful
method is to check whether or not the protein can bind to a hydrophobic
column in a Ca2+-dependent manner (36).
Ca2+ sensor proteins that cause large conformational
changes bind to the column by Ca2+-induced exposure of the
hydrophobic surface in the protein formed by a pair of EF-hands. After
that, these proteins can be eluted by removing Ca2+ with
EGTA. This method was successfully applied for the purification of CaM
(41). We have purified the recombinant calbrain protein by this method,
also (data not shown), and the results indicate that calbrain possesses
a regulatory domain that shows a large conformational change with
Ca2+ binding and, therefore, can be classified as a
Ca2+ sensor protein.
The distribution study of calbrain mRNA revealed that calbrain is a
brain-specific Ca2+-binding protein that is expressed
abundantly in the hippocampus, in the habenular nucleus of the
epithalamus and in the Purkinje cell layer of the cerebellum. The
specific tissue distribution of EF-hand proteins has suggested their
particular functions in each tissue (42-44). The localization of
calbrain mRNA in the hippocampus and cerebellum, together with its
functional potency as a Ca2+ sensor protein being involved
in the Ca2+ signal transduction suggest an important role
for this protein in the central nervous system. A number of studies on
hippocampus have shown that this part of the brain is particularly
involved in acquisition and storage of spatial information
(45-47).
CaM-kinase II is a multifunctional serine/threonine protein kinase
capable of phosphorylating several endogenous proteins in the brain
(48, 49) and is highly expressed in the mammalian central nervous
system (50-52). This enzyme is a major component of postsynaptic
density (24, 53, 54) and plays important roles in the regulation of the
neurotransmitter synthesis, receptor function, axonal transport, gene
expression, and especially in the long-term potentiation, which is an
established model of neural plasticity (14, 55, 56). Many biochemical
studies have indicated that phosphorylation induced by CaM-kinase II
can act as a molecular switch, conferring properties that are
advantageous for long-lasting storage of changes initiated by brief
Ca2+ signals (57-59). The binding study of CaM and
calbrain to CaM-kinase II indicated that under nonautophosphorylated
conditions, calbrain inhibited CaM binding to CaM-kinase II
competitively (Fig. 8). Although calbrain could not activate CaM-kinase
II, Fig. 6B showed that calbrain competitively inhibited the
activation of CaM-kinase II by CaM. Because Ki
values of binding and activity inhibition were similar, it was supposed
that inhibition of CaM binding by calbrain may caused the reduction of
kinase activity. CaM-kinase II activity is regulated by
Ca2+/CaM and autophosphorylation (60). When CaM binds to
the CaM binding domain, a conformational change is induced in the
regulatory region, and the interaction of inhibitory domain with the
active site is disrupted. It allows the active site to become
accessible to exogenous substrate. When Ca2+/CaM is bound,
CaM-kinase II is rapidly autophosphorylated on Thr286, and
autophosphorylation increases CaM binding affinity to the kinase
dramatically by decreasing of CaM-releasing time (61). Under both
experimental conditions used for the kinase activity assay (incubation
at 30 °C for 1 min) and autophosphorylation assay (at 0 °C for 10 min), autophosphorylation on Thr286 of CaM-bound kinase
occurs (24-26). Therefore, the activity detected in Fig. 6B
may be influenced by a change of CaM affinity caused by
autophosphorylation. Ki values of kinase activity (143.5 ± 16.4 nM) and autophosphorylation (189.7 ± 12.4 nM) did not significantly differ from that of CaM
binding (128.6 ± 19.7 nM) under nonautophosphorylated condition.
This is the first report of a novel Ca2+-binding protein
that can inhibit CaM-dependent CaM-kinase II activity.
Although calbrain can reduce autophosphorylation, it is supposed that
this protein is involved in the physiological regulation of CaM-kinase
II. As CaM-kinase II has multiple functions and essential roles in the
brain, calbrain may also play important roles in the central nerve system.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
gt11 cDNA library
(CLONTECH) were grown and transferred onto nylon
membranes. The polymerase chain reaction fragment was labeled with
[
-32P]dCTP and used as a probe for the screening.
Hybridization-positive plaques were picked and grown to purify the DNA.
Inserts from those positive clones were subcloned into EcoRI
cloning site of pBluescript KS (+) vector (Stratagene). The sequence
was determined, and data was searched as above.
-32P]dCTP and
used as a probe for Northern hybridization analysis. A human multiple
tissue Northern blot filter (CLONTECH) containing 2 µg of poly(A)+RNA in each lane was prehybridized in a solution containing 5× saline/sodium phosphate/EDTA, 10× Denhardt's, 100 µg/ml salmon sperm DNA, 50% formamide, and 2% SDS for 3 h at
42 °C. The blot was then hybridized in the same solution with the labeled probe at 42 °C for 20 h. The blot was washed several
times with a mixture of 2× SSC (1× SSC = 0.15 M NaCl
and 0.015 M sodium citrate) and 0.05% SDS for 30 min at
room temperature and then once with a mixture of 0.1× SSC and 0.1%
SDS for 40 min at 50 °C. The filter was exposed to Fuji BASIII
imaging plates (Fuji Biomedical, Japan), and the image was analyzed by
the BAS 1000 phosphor-imaging system (Fuji Biomedical, Japan).
-D-galactropyanoside to a final
concentration of 1 mM, the cells were incubated for another
5 h. As a negative control, a plasmid vector with no insert was
transformed and expressed in the same way. The cells were harvested and
lysed with 10 ml of lysis buffer containing 1 mg/ml lysozyme before
sonication. After removal of cellular debris, the supernatant was
collected, and histidine-tagged calbrain protein was purified on nickel
nitrilotriacetic acid resin in a batch procedure. The protein-resin
complex was set into a column and washed with washing buffer containing
50 mM sodium phosphate, 300 mM NaCl, 10%
glycerol, pH 5.7. After washing, the protein was eluted with 0.1-0.5
M imidazole gradient in washing buffer.
-32P]ATP, 8 mM Mg(CH3COO)2, 20 µM
syntide-2, 0.25 mM CaCl2, 0.1 mM EGTA, 50 mM HEPES buffer, pH 8.0, and 0.3 µg/ml
CaM-kinase II (purified from bovine brain, kindly provided by Dr.
Yamauchi (25)). A range of CaM or recombinant calbrain (0-200
nM) was employed in the assay mixture at the total volume
of 20 µl. The reaction was carried out at 30 °C for 1 min and
stopped by spotting onto P81 filter paper. The filter papers were
washed several times with 75 mM phosphoric acid, and
radioactivity was measured by a liquid scintillation counter. An
inhibition assay of Ca2+-dependent activity of
CaM-kinase II was performed in the standard mixture as described above
in the presence of various concentrations of CaM. Autophosphorylation
of CaM-kinase II was assayed in the standard mixture at the total
volume of 60 µl. The reactions were carried out at 0 °C for 10 min
as described previously (26), then samples were boiled for 3 min and
were subjected to SDS-PAGE. After electrophoresis, the gel was stained
with Coomassie Brilliant Blue, and the band of CaM-kinase II protein
was excised from the gel. Radioactivity of the gel was determined by a
liquid scintillation counter.
RESULTS
protein and calbrain were aligned, and the amino acid sequences
were compared as shown in Fig. 2. The first domain of calbrain is highly (50.0% each) homologous to the
first and the third domain of CaM. The second domain of calbrain is
homologous to the second and the fourth domains of CaM (48.1 and
51.9%, respectively) and those of troponin C (51.9% each). The first
and the second domains of calbrain showed only 29.6% homology.
Although calbrain has only two EF-hand motifs, the similarity between
calbrain and two EF-hand protein (S100
) is very low (Fig. 2).
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Fig. 1.
Nucleotide sequence and deduced amino acid
sequence of human calbrain. The sequence was derived from human
brain cDNA clones. Two EF-hand domains are indicated by
lines; the in-frame methionine and stop codon are shown in
bold face type.
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Fig. 2.
Alignment of the amino acid sequences of
EF-hand domains. The first and second EF-hand domains of calbrain
(CALBR1 and CALBR2) and S100 (S100B1 and S100B2) and the first,
second, third, and fourth domain of CaM and troponin C (CALM1 to CALM4,
TROPO1 to TROPO4, respectively) were aligned. The conserved amino acid
residues are shown in shaded boxes. The lines
with arrows indicate the helix and loop region of EF-hand.
The numbers on the right indicate the percentages of
similarity between either CALBR1 or CALBR2 and EF-domains in each
protein.
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Fig. 3.
Northern blot analysis of the tissue
expression pattern of calbrain. A Northern blot filter containing
2 µg of human poly(A)+ RNA (CLONTECH)
was hybridized with 32P-labeled calbrain probe and washed
following the protocol of Church-Gilbert. Numbers on the
left refer to the size of RNA standard run in parallel.
32P-Labeled -actin was hybridized on the same Northern
blot filter as a quantitative control. A single transcript was
visualized in the brain sample lane (approximately 1.5 kilobases). No signal was detected in other tissues examined.
kb, kilobases.
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Fig. 4.
Localization of calbrain mRNA in rat
brain. In situ hybridization was performed. A rat
homologue of calbrain probe (99.0% identical to human one) was labeled
and hybridized to a coronal section (A) or a sagittal
section (B) of adult rat brain. The hippocampal area
(C) and the cerebellar cortex (D) are shown in
higher magnification. The strong signals were detected in the CA1 to
CA3 of the hippocampal gyrus and granular layer of the dentate gyrus
(DG) in the hippocampus (C). In the area of
cerebellum, the Purkinje cell layer was stained intensively
(D).
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Fig. 5.
Purification of recombinant calbrain protein
and its calcium binding property. Panel A, the recombinant
calbrain protein from plasmid expression system was purified by a
nickel nitrilotriacetic acid resin column. 4 µg of pure CaM
(lane 1), 3 µg of recombinant calbrain protein (lane
2), and control sample (proteins obtained from E. coli
with nonrecombinant plasmid and purified by the same procedure)
(lane 3) were run on a 10% Tricine SDS-PAGE gel. They were
electrophoresed and stained with Coomassie Brilliant Blue. The
duplicate of the electrophoresed gel was transferred to a
nitrocellulose membrane and incubated with
45Ca2+ and washed, then the membrane was
exposed and analyzed by Fuji imaging system. The negative control
sample (proteins obtained from E. coli with nonrecombinant
plasmid) showed no signal. M, molecular mass markers.
Panel B, the data was carried out by equilibrium dialysis.
Conditions: 10 mM MOPS, pH 7.1, 150 mM KCl, 1.0 mM dithiothreitol, 0.1 mM EGTA, and 3 mM MgCl2. Inset, Scatchard plot of
data. V, mol of Ca2+ bound/mol of calbrain;
C, free [Ca2+].
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Fig. 6.
Comparison of in vitro activation
of CaM-kinase II by calbrain and CaM. Panel A, the activity
of purified bovine CaM-kinase II in the presence of various
concentrations of calbrain or CaM was assessed using syntide-2 as a
substrate for phosphorylation. The kinase activity in the presence of
100 nM CaM was measured and designated as 100%. The
relative ratio (%) of activities in the presence of various
concentrations of calbrain (open circles) and CaM
(filled circles) were then calculated. Data are represented
as the means of triplicate determinations (±S.E.). Panel B,
activation assays were performed in the presence of indicated
concentrations of calbrain and varying concentrations of CaM.
1/v represents 1/activity of CaM-kinase II (%). Note that
calbrain competitively inhibits CaM-kinase II activation by CaM. The
results are the mean (±S.E.) for two successive experiments performed
in duplicate.
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Fig. 7.
Inhibition of CaM-kinase II
autophosphorylation by calbrain. The autophosphorylation of
purified bovine CaM-kinase II in the presence of indicated
concentrations of calbrain and varying concentrations of CaM was
assessed by measuring [ -32P]ATP radioactivity of
CaM-kinase II extracted from electrophoresed gels. Autophosphorylation
assays were performed as described under "Experimental Procedures."
1/v represents 1/autophosphorylation of CaM-kinase II (%).
Note that calbrain competitively inhibits CaM-dependent
CaM-kinase II autophosphorylation. The results are the mean (±S.E.)
for two successive experiments performed in duplicate.
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Fig. 8.
Competitive inhibition of CaM binding to
CaM-kinase II by calbrain. The binding of CaM to CaM-kinase II in
the presence of indicated concentrations of calbrain was studied.
125I-CaM and calbrain were mixed with CaM-kinase II and
Ca2+ under nonautophosphorylated conditions (in ATP-free
reaction mixture). CaM-kinase II was immunoprecipitated by
anti-CaM-kinase II antibody after the reaction, and the radioactivity
of each sample was measured. The radioactivity of the bound
125I-CaM in the presence of 500 nM CaM and 0 nM calbrain was measured and designated as 100%. The
relative ratio (%) of radioactivities of the samples were then
calculated. 1/v represents 1/CaM binding rate to CaM-kinase
II (%). The results are the mean (±S.E.) for two successive
experiments performed in duplicate.
DISCUSSION
was found to be very low. From the high sequence homology between EF-hand motifs, it has been proposed that CaM, troponin C, and
myosin light chain gene evolved from a common four-domain molecule (29,
31, 32). In each of these proteins, the first and the third and the
second and fourth EF-hand domains show high homology, supporting the
hypothesis that these proteins evolved from a two-domain precursor by
gene duplication (33, 34). The significant homology between the EF-hand
motifs of calbrain and CaM family suggests that although calbrain is a
two EF-hand protein, evolutionally it is related closely to the CaM
family rather than two EF-hand protein families. As calbrain is the
first two EF-hand protein whose domains appear to be very similar to those of four EF-hand proteins, our findings may be interesting from
the aspect of the evolution of EF-hand proteins.
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ACKNOWLEDGEMENTS |
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We thank Dr. T. Yamauchi for providing CaM-kinase II. We are also grateful to Dr. T. Yamauchi, Dr. S. Kimura, and Dr. N. A. Janjua for preparation of the manuscript and the figures.
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FOOTNOTES |
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* This research was financially supported by Grant-in-aid for Scientific Research (A) of the Ministry of Education, Science, and Culture, Japan.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X94700.
To whom correspondence should be addressed. Tel.:
81-878-91-2095; Fax: 81-878-91-2096; E-mail: tokuda{at}kms.ac.jp.
The abbreviations used are: CaM, calmodulin; CaM-kinase, Ca2+/calmodulin-dependent protein kinase; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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