From the Department of Pharmacology, Kyoto University
Faculty of Medicine, ¶ PRESTO-Japan Science and
Technology Corporation, Sakyo-ku, Kyoto 606-8315, and
** Department of Neurochemistry, University of Tokyo Graduate
School of Medicine, Bunkyo-ku, Tokyo 113-0033, Japan
Received for publication, January 17, 2003, and in revised form, February 26, 2003
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ABSTRACT |
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During a screen for novel putative
Ca2+/calmodulin-dependent protein kinase
(CaMK)-like CREB kinases (CLICKs), we have cloned a full-length
cDNA for CLICK-III/CaMKI Neuronal Ca2+ is known to play a critical role as an
intracellular second messenger, linking neuronal excitability with many kinds of cellular biological events including synaptic plasticity and
neuronal cell survival/apoptosis (1-4). One of the unique features of
Ca2+ is that its concentration can be dynamically regulated
both temporally and spatially (5). Despite a growing knowledge about
the critical molecules involved in neuronal Ca2+ influx and
mobilization (e.g.
N-methyl-D-aspartic acid receptors, voltage-gated Ca2+ channels, inositol 1,4,5-trisphosphate
receptors, and ryanodine receptors), how these are converted to the
specific cellular events remains largely unknown.
A significant part of signaling downstream Ca2+ is thought
to be mediated by calmodulin
(CaM),1 a ubiquitous and
evolutionary well conserved intracellular Ca2+ receptor (6,
7). Although a large number of molecules have been shown to be targeted
and activated by the Ca2+/CaM complex, one subgroup of
multifunctional kinases,
Ca2+/calmodulin-dependent protein kinases
(CaMKs), has been ascribed a prominent role. This is because several
unique characteristics of this group of kinases, such as rapid
activation by Ca2+, steep Ca2+/CaM dependence,
and induction of an autonomous kinase activity following activation,
render its members good candidates as molecular devices able to convert
a transient burst of synaptic activity into a longer lasting covalent
modification of substrate proteins (8-15). Indeed, among the CaMK
family members, CaMKII In this study, we report the molecular cloning of mouse full-length
CLICK-III/CaMKI The kinase activity of CLICK-III was similar in many respects to
CaMKI Cloning and Plasmid Constructions--
Human hippocampal
cDNA was prepared from poly(A)+ RNA
(Clontech) using Omniscript reverse transcriptase
(Qiagen) and oligo(dT) primers. An hCLICK-III cDNA was obtained by
nested PCR using two primer pairs (F1, 5'-CCA CTC CCT GCA ATA AAG CAT
CCT C-3', and R1, 5'-CTG CCT ATG AGT GGG AGA GGC CTT T-3' as a first
primer set; F2, 5'-TGG AGG CAA TGG GTC GAA AGG AAG AA-3', and R2,
5'-TGT CCA TTT CTT TCA GTC CTG TTG A-3' as a nested primer set) and
subcloned into pCR-Blunt vector (Invitrogen). ICR mouse hippocampal
poly(A)+ RNA was purified using Trizol (Invitrogen) and
µMACS mRNA isolation kit (Miltenyl Biotec), and mCLICK-III was
subsequently obtained by the 3'-RACE procedure, using SMART RACE
cDNA amplification kit (Clontech) following the
manufacturer's instructions. A gene-specific 5'-primer for 3'-RACE was
designed based on the cDNA sequence of hCLICK-III and that of rat
CaMKI Northern Blotting--
For Northern blot analysis, a total RNA
blot filter was purchased from Seegene (Mouse Brain Aging Blot), and
poly(A)+ RNA blot filters were from
Clontech (all other blots). Double-stranded probe
templates, corresponding to the unique sequence of CLICK-III at the
C-terminal region, were generated by PCR using primers (F, 5'-AAG CCT
CAG AAA CCT CTA GAC CCA G-3', and R, 5'-TTC AGA CCC AAG CTG GGG CTC CAT
CT-3' for hCLICK-III; F, 5'-ATG AAC CTG CAC AGC CCC AGT G-3', and R,
5'-TTA TTG GCC TTT CTG AAG AGG-3' for mCLICK-III). The probes were
labeled with [ In Situ Hybridization--
Brain cryosections (10 µm) were
obtained from 2-month-old female ICR mice and processed for in
situ hybridization as described previously (34). N-terminal (231 bp) and C-terminal (374 bp) fragments were amplified by PCR (5'-GCA GCT
TCA ACT CTG GAG G-3' and 5'-TAG GCT GCT GTC CCG GAA GG-3' for
N-terminal fragment, 5'-ATG AAC CTG CAC AGC CCC AGT G-3' and 5'-TTA TTG
GCC TTT CTG AAG AGG-3' for C-terminal fragment) and subcloned into
pBluescriptII KS(+) vector at the SacII site.
[35S]-Labeled riboprobes were generated using T7 RNA
polymerase (Stratagene) and [ CaM-Sepharose Binding Assay--
COS-7 cells were maintained in
Dulbecco's modified Eagle medium containing 10% fetal calf serum.
Cells were subcultured in 6-cm dishes 12 h before transfection.
pcDNA3-HAmCL3wt (2 µg) or empty vector (2 µg) were transfected
using 4 µl of LipofectAMINE 2000 reagent (Invitrogen). After 24 h, the cells were washed twice with ice-cold PBS( Immunoprecipitate Kinase Assay--
COS-7 cells were plated onto
6-well plates at a density of 2 × 105 per well, and
12 h later were transiently transfected with pcDNA3-HAmCL3wt (0.3 µg), pcDNA3- HAmCL3dC (0.3 µg), or empty vector (0.3 µg) and pd2EGFPN1-CaMKKactiveMyc (0.6 µg), or empty vector (0.6 µg) using LipofectAMINE 2000 reagent. For immunoprecipitate kinase assay, cells were washed twice with ice-cold PBS( Luciferase Assay--
COS-7 cells were plated onto 24-well
plates at a density of 5 × 104 per well, and 12 h later were transfected with pFR-Luc, pFA-CREB, pRL-CMV (200 ng, 50 ng, 4 ng each; Stratagene), and pIRES-HAmCL3dC (100 ng). 24 h
after transfection, luciferase activity was measured using
Dual-Luciferase Reporter Assay System (Promega) according to the
manufacturer's protocol. The firefly and the Renilla
luciferase activities were both measured using SIRIUS luminometer
(Berthold). All results were normalized using Renilla
luciferase (pRL-CMV), which was co-transfected with the other reporter
genes (pFR-Luc and pFA-CREB).
Culture and Transient Transfection of CA1/CA3
Hippocampal Neurons--
Culture of mouse CA1/CA3 hippocampal neurons
was carried out as described previously for rat (16). A cDNA
transfection was carried out at 7 days in vitro using a
modification of the calcium phosphate
method.3
BODIPY-TR-ceramide Labeling and Fluorescence Microscopy--
For
BODIPY-TR-ceramide (Molecular Probes) and green fluorescent
protein (GFP) imaging, COS-7 were plated on Lab-Tek chambered coverglass (4 well, Nunc) and transfected with expression vectors (0.4 µg/well), pEGFP-rCaMKI Subcellular Fractionation--
COS-7 cells were grown to 70%
confluency on a 10 cm-dish and transfected with each expression vector
(4 µg per dish) using LipofectAMINE 2000 reagent. 48 h after
transfection, the cells were washed twice in ice-cold PBS( Western Blot Analysis--
After SDS-PAGE, the proteins were
transferred onto a nitrocellulose membrane (Optitran BAS-85, Schleicher
& Schuell), and immunoreactive proteins were detected using ECL-Plus
(Amersham Biosciences) with the following concentration of primary
antibodies: anti-HA tag monoclonal antibody (1:2500, 12CA5; Roche
Applied Science), anti-GFP monoclonal antibody (1:1000, 3E6; Molecular Probes), and anti-phospho-CREB (Ser-133) polyclonal antibody (1:1000; Cell Signaling). Horseradish peroxidase-linked anti-mouse or
anti-rabbit IgG (1:2000; Amersham Biosciences) were used as secondary
antibodies. The chemiluminescence image was acquired using a FAS-1000
system (Toyobo) equipped with a 16-bit cooled CCD camera. For
quantification of signal intensity of detected immunoreactive bands, a
rectangular window, which had a sufficient area to completely surround
each band, was defined, and the total pixel intensity of each area was
calculated using Photoshop 6.0 (Adobe). The signal intensity was
normalized as a percentage of the total intensity, calculated as the
sum of the band intensities detected in cytosolic and membrane fractions (S or P or P'/(S + P)). Statistical analyses were carried out
using Prism 3.0 (GraphPad software). Statistical significance (p < 0.05) was determined between two groups using
unpaired Student's t test and between three groups using
one-way analysis of variance (followed by post hoc Bonferroni and
Neuman-Keuls tests). All data are given as means ± S.E.
Molecular Cloning of CLICK-III, a Novel Brain-enriched
CaMK--
During the course of studying two novel putative CaMK-like
CREB kinase-I and -II (CLICK-I and -II) cloned by degenerate PCR strategies,2 BLAST search revealed the presence of a
putative human gene product with high homology to both CLICKs. We named
this novel kinase human CLICK-III. A putative open reading frame of
human CLICK-III had been deposited as a novel rat
Ca2+/calmodulin-dependent protein kinase-like
gene, an assembly of presumed exon sequences obtained from the draft
Human Genome Sequence (GenBankTM accession number
AL023754). We cloned a human CLICK-III (hCLICK-III) cDNA
(GenBankTM accession number AY212935) by PCR from a human
hippocampal cDNA pool, using primer sequences corresponding to the
deposited sequence, and we confirmed the authenticity of this gene
product. We next used a 3'-RACE strategy to obtain a full-length mouse hippocampal CLICK-III cDNA (GenBankTM accession number
AY212936). Mouse CLICK-III (mCLICK-III) showed a high degree of amino
acid identities with CKLiK, a recently reported CaMK-like kinase (18),
as well as with rat CaMKI
The open reading frame of CLICK-III cDNAs contained an N-terminal
kinase domain, a conserved threonine at position 178 in the activation
loop, and an overlapping autoinhibitory/Ca2+/CaM-binding
domain in its middle portion; these domains showed high homology across
the CaMKI-related kinases (Fig. 1B). The C-terminal end of
CLICK-III was unusually extended in comparison with CKLiK, CaMKI
As shown in Fig. 2A, Northern
blot analyses suggested that in adult tissues, mCLICK-III was highly
expressed in the brain, although heart, testis, and kidney also showed
detectable amounts of hybridization signals. The mCLICK-III transcript
first became detected at embryonic day 11 (E11), in parallel with the
onset of the development of the central nervous system (CNS). Its
expression level remained constant from E11 onward, throughout
development and during adulthood (Fig. 2A, upper
panels). In human mRNA blots, CLICK-III expression was almost
confined to the brain, with small amount of signals in the skeletal
muscles, kidney, spleen, and liver (Fig. 2A, lower
panels). Within the CNS, the strongest signal was detected in the
forebrain neocortex (cerebral cortex, occipital pole, frontal lobe, and
temporal lobe), the striatum (putamen and caudate nucleus), and the
limbic system (amygdala and hippocampus) (Fig. 2A,
lower panels).
CLICK-III Is Neuronally Expressed and Is Abundant in the CeA and
the VMH--
We next examined CNS expression of CLICK-III mRNA by
in situ hybridization using 35S-labeled cRNA
riboprobes. X-ray film autoradiography of hybridized parasagittal and
coronal sections showed specific signals in the neuronal cell layers of
the cerebral cortex, olfactory bulb, and hippocampus (Fig.
2B, I and III). Most remarkably,
intense hybridization signals were shown in the CeA and the VMH (Fig.
2B, III). We obtained identical results using two
independent antisense probes, either from the N-terminal or from
C-terminal regions (data not shown), whereas sense probes only
generated background signals (Fig. 2B, II and
IV). Hybridization in the presence of excess cold riboprobes also completely abolished the antisense probe signals (data not shown),
confirming the specificity of these obtained signals.
Emulsion autoradiography of individual sections revealed that CLICK-III
was particularly expressed in neurons, but not found in glia, in any
areas that we examined. Heavy amounts of silver grains associated with
most neuronal cell bodies present in the CeA (Fig.
3, A and B) and the
VMH (Fig. 3, C and D). Pyramidal cells in the
cerebral cortex, especially in the layer V, and those in the area CA1
of the hippocampus were also positive in CLICK-III mRNA signals
(Fig. 3E). In contrast, in the dentate gyrus of the hippocampus, only scattered non-principal cells, presumably hilar interneurons, had significant amount of silver grains (Fig.
3F).
Taken together, these sets of data established that CLICK-III was a
CaMKI family member specifically expressed in neurons, with a
remarkable abundance in the CeA and VMH. Such specificity in its
expression profile particularly stands out among all CaMKs presently
identified (16-21, 35-37).
Enzymatic Properties of CLICK-III--
We next tested whether
CLICK-III was indeed a Ca2+/CaM-dependent
protein kinase, as suggested from its domain structure (Fig. 1B). To this end, an HA-tagged CLICK-III was constructed and
transfected to COS-7 cells. Crude lysates of transfected COS-7 cells
were collected and incubated with CaM-Sepharose beads in the presence of 2 mM Ca2+. The beads were centrifuged and
separated from the supernatant and then washed several times in the
presence or absence of Ca2+. SDS-PAGE analyses showed that
HA-immunoreactive bands were mostly recovered with the beads in the
presence of Ca2+, whereas they were largely washed out in 0 Ca2+, 2 EGTA (Fig.
4A). Thus, CLICK-III was
clearly able to bind CaM in a Ca2+-dependent
manner.
Ca2+/CaM-dependent regulation of the protein
kinase activity of CLICK-III was investigated using an
immunoprecipitate kinase assay, with MBP and CREM as kinase substrates
(Fig. 4B). Both Ca2+/CaM and CaMKK
cotransfection were required to reach maximal levels of activation of
the wild type CLICK-III (HAmCL3wt). In contrast, a C-terminal deletion
mutant in which the entire autoinhibitory/Ca2+/CaM binding
domain was removed (HAmCL3dC) showed a constitutive, Ca2+-independent kinase activity even in the absence of
Ca2+, when active CaMKK was cotransfected (Fig.
4B). CLICK-III activity monitored with
[
Taken together, these data indicated that the enzymatic properties of
CLICK-III recapitulated all major features of CaMKI isoforms, such as
dual regulation by Ca2+/CaM and CaMKK, and activation of
CREB pathway in vitro and in a heterologous system.
Membrane Localization of CLICK-III in Hippocampal Neurons and in
COS-7 Cells--
As we failed to raise high titer antibodies against
CLICK-III, we expressed GFP-tagged CLICK-III in hippocampal pyramidal neurons 7 days in vitro, and we monitored its subcellular
distribution in fixed (Fig. 5) or live
samples (data not shown). Under either condition, GFP alone was
diffusely distributed throughout the cytoplasm and the nucleus, whereas
GFP-rCaMKI
To better analyze in detail the potential mechanisms that might
underlie the distinct localization of CLICK-III, we overexpressed CLICK-III in COS-7 cells. Ectopic expression of GFP-mCL3 in COS-7 cells
confirmed that subcellular distribution of CLICK-III was indeed
distinct from that of GFP or GFP-rCaMKI A Critical Role for a CAAX Motif in the Localization of CLICK-III
to the Golgi Complex and Plasma Membranes--
What might be the cause
of such distinct subcellular localization between rCaMKI
To confirm this point further, mCL3-overexpressing cells were
pretreated with compactin, an HMG-CoA reductase inhibitor. As blockade
of this rate-limiting step of cholesterol synthesis should also
significantly reduce prenylation of the CAAX box cysteine (33), we expected that compactin treatment should mimic the effect of
Cys-to-Ser mutation at position 474. Consistently, although vehicle
(Me2SO) treatment had no effect on localization of
GFP-mCL3, which was strongly detected in association with Golgi and
plasma membranes (Fig.
8A, left
panels, arrow), an overnight compactin treatment
dramatically abolished this membrane anchoring (Fig. 8A, right panels). Prevention of membrane
recruitment of GFP-mCL3 and its retention to the soluble fractions by
compactin treatment was also verified by using biochemical
fractionation as well (Fig. 8, B and C).
Together, these experiments demonstrated that CLICK-III, a neuronally
expressed CaMKI isoform, was able to be localized to the Golgi
apparatus and plasma membranes, at least in part, via its C-terminal
CAAX box in a prenylation-dependent manner.
CaMK represents a class of the multifunctional protein kinase
family, with a particular significance for the physiology of excitable
cells such as neurons or smooth muscles (1-10). A better understanding
of the role of neuronal CaMKs has been of urgency because CaMK activity
has been suggested to be crucial for linking neuronal activity with
various types of neuronal plasticity (2-4, 8-11, 39-42). Recent
experiments have further revealed that, in fact, various CaMK isoforms
play a critical role in establishing distinct types of memory in
mammals (9, 12, 43-46). So far, however, most analyses have focused on
the role of CaMKII In this study, we have identified a neuronally enriched CaMKI isoform
that has the potential to be membrane-anchored. This CaMKI isoform,
CLICK-III/CaMKI First, we confirmed that the activation of CLICK-III required not only
Ca2+/CaM but also a phosphorylation by other kinases,
presumably by a CaMKK. Such strong CaMKK dependence for its enzymatic
activation was qualitatively similar to what was reported previously
for CaMKI Second, unlike CaMKI Third, our studies have clarified for the first time a mechanism by
which CaMK may be anchored to the Golgi and plasma membranes. A
CAAX motif that we identified in the unusually extended
C-terminal end of CLICK-III played a critical role in determining the
targeting of CLICK-III to the membrane compartments in a heterologous
expression system. The high degree of sequence similarity of the
CAAX motif of CLICK-III with that of Ras (Table I) and the
disruption of membrane anchoring by either a point mutation at the
putative prenylation site or by pretreatment with an HMG-CoA reductase inhibitor have suggested that prenylation of the CAAX box
may constitute a mechanism through which CLICK-III may be actively sorted to the membranes. The prenyl moiety is derived from the mevalonate/cholesterol biosynthetic pathway. It is interesting to note
that this pathway has been shown recently (50, 51) to affect various
neuronal phenotypes. Furthermore, it is also notable that many neuronal
signaling proteins (e.g. Ras, Rho, paralemmin, and PSD-95)
have been modified by the addition of long fatty acid chains including
prenylation and palmitoylation (52, 53). These post-translational
modifications are suggested to play a critical role for precise
membrane localization especially in highly polarized neuronal cells.
What could be the biological significance of such membrane targeting of
a CaMK? In the case of Ras, CAAX box-dependent
membrane anchoring was absolutely required to allow proper membrane
recruitment of its downstream kinases, c-Raf and B-Raf (23). Similarly, CAAX-mediated localization of CLICK-III to the membranes
might represent an advantageous mechanism to help tightly couple
upstream signals to local downstream phosphorylation targets. In
principle, a direct anchoring of CaMK activity to various membrane
compartments is likely to facilitate the synaptic activity-induced
local protein phosphorylation at or very close to the site of
Ca2+ entry/mobilization. Whether such membrane-delimited
excitation-phosphorylation coupling can indeed be triggered by
electrical activity of the neurons, either postsynaptically or
presynaptically, remains to be studied. Alternatively, or additionally,
the presence of a CaMK in the Golgi apparatus might enable an efficient
coupling of synaptic activity with intracellular trafficking of
neuronal proteins. Experiments are underway to provide answers to these issues.
, an isoform of the CaMKI family with an
extended C-terminal domain ending with CAAX motif (where
AA is aliphatic acid). As expected from the similarity of
its kinase domain with the other CaMKI isoforms, full activation of
CLICK-III/CaMKI
required both Ca2+/CaM and
phosphorylation by CaMKK. We also found that
Ca2+/cAMP-response element-binding protein (CREB) was
a good substrate for CLICK-III/CaMKI
, at least in vitro.
Interestingly enough, CLICK-III/CaMKI
transcripts were most abundant
in neurons, with the highest levels in limited nuclei such as the
central nucleus of the amygdala (CeA) and the ventromedial
hypothalamus. Consistent with the presence of the CAAX
motif, CLICK-III/CaMKI
was found to be anchored to various membrane
compartments, especially to Golgi and plasma membranes. Both point
mutation in the CAAX motif and treatment with compactin, a
3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, disrupted
such membrane localization, suggesting that membrane localization of
CLICK-III/CaMKI
occurred in a prenylation-dependent way.
These findings provide a novel mechanism by which neuronal CaMK
activity could be targeted to specific membrane compartments.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
isoforms, which are present
postsynaptically and presynaptically, have been implicated in various
kinds of synaptic plasticity and homeostasis (8-11), whereas a
CaMKK/CaMKIV cascade has been shown to couple synaptic stimuli with
CREB-dependent gene expression (4, 12, 16). The ability of
the CaMKI isoforms and a related kinase, CKLiK, to phosphorylate
neuronal substrates, such as synapsin I and CREB in vitro,
has been demonstrated so far (13, 17, 18). However, little is yet known
about the physiological role of CaMKI, although CaMKI
and CaMKI
have been shown to be expressed both in neural as well as non-neural
peripheral tissues (13, 17-21).
(occasionally abbreviated to CLICK-III in this
paper), an isoform of CaMKI family, that has a longer C-terminal region
terminating with a CAAX motif (where AA is
aliphatic acid). This motif has been shown to be conjugated with
isoprenoid lipids, thereby allowing proper targeting of various
signaling molecules to cellular membrane compartments (22, 23).
CLICK-III was identified during a screen for novel putative CaMK-like
CREB kinases (CLICKs), as a kinase homologous to CLICK-I and CLICK-II (the cloning and characterization of CLICK-I and -II will be described elsewhere).2
and related kinases. Thus, full activation of CLICK-III required both Ca2+/CaM and phosphorylation by a CaMKK
(24-31). In addition, Ca2+/cAMP-response element-binding
protein (CREB) (4, 32) was a good substrate for CLICK-III, at least
in vitro. Interestingly, CLICK-III transcripts were most
abundant in neurons, with the highest levels in limited nuclei such as
the central nucleus of the amygdala (CeA) and the ventromedial
hypothalamus (VMH). Furthermore, CLICK-III was found to be anchored to
membrane compartments, consistent with the presence of the
CAAX motif at its C-terminal end. A point mutation in the
CAAX motif and treatment with compactin, a
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor
(33), disrupted such membrane localization, suggesting that membrane
localization of CLICK-III occurred in a
prenylation-dependent way. These findings provide a novel
mechanism by which neuronal CaMK activity could be targeted to specific
membrane compartments.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(19) at the 5'-untranslated region adjacent to the first
methionine (5'-GCA GCT TCA ACT CTG GAG G-3'). A single RACE-amplified
fragment was obtained and subcloned into pCR-Blunt vector, and its
nucleotide sequence was determined. Two independent clones were
recovered and yielded an identical sequence. To construct
pIRES-HAmCL3wt and pIRES-HAmCL3dC, cDNA fragments were amplified by
PCR using primers F and R1 or R2 (F, 5'-GGG GGA ATT CTG GCG GCC GCT ATG
GGG CGT AAG GAG GAG GAG-3', and R1 for wt, 5'-GGG GGA ATT CGT TTG GCG
GCC GCC TCC TGG GAT CAC ATA ACG AG-3', or R2 for dC, 5'-GGG GGA ATT CGT
TTG GCG GCC GCA AAG TTC TTC TAA ATC TGG AG-3') and inserted in-frame
downstream to an HA tag cassette at the NotI site of
pIRES-S-Tag-EGFP vector (a kind gift from Dr. Hirohide
Takebayashi). To construct pcDNA3-HAmCL3wt and
pcDNA3- HAmCL3dC, EcoRV/EcoRI fragments of
each pIRES-S-Tag-EGFP construct were blunted and subcloned into the
EcoRV site of pcDNA3 (Invitrogen). To construct
pEGFP-mCL3 and pEGFP-mCL3C474S, cDNA fragments amplified with
primers F and R1 or R2 (F, 5'-GCT TCG AAT TCA GGC TTC AAC TC-3', and R1
for wt, 5'-CCC TCC CGC GGT CAC ATA ACG AGA GAC ACC CCA GTC-3', or R2
for C474S, 5'-CCC TCC CGC GGT CAC ATA ACG AGA CAC ACC CCA GTC-3') were
inserted into a pEGFP-C3 vector (Clontech) cut at
EcoRI and SacII sites. To construct pEGFP-CLVM, two oligonucleotide pairs (5'-CTG TCT CGT TAT GTG AGT GCA CA-3' and
5'-GAT CTG TGC ACT CAC ATA ACG AGA CAG GGC C-3') were annealed, phosphorylated, and inserted to a pEGFP-C2 vector
(Clontech) at the ApaI and
BamHI sites. To construct pd2EGFPN1-CaMKKactiveMyc, a 1.3-kb
cDNA fragment encoding the N-terminal kinase domain of rat CaMKK
was PCR-cloned from a Sprague-Dawley rat hippocampal cDNA pool,
fused to a Myc epitope tag using a pair of primers (F, 5'-ACG GTA CCA
TGG AGC GCA GTC CAG CCG, and R, 5'-ATT GGA TCC CTA CAG GTC CTC CTC GCT
GAT CAG CTT CTG CTC TCC ATG CTT GGT CAC CCA-3'), and inserted into a
pd2EGFPN1 vector (Clontech) cut at KpnI
and BamHI site. To construct pEGFP-rCaMKI
, a cDNA
fragment corresponding to the full-length coding region of rat CaMKI
was obtained by PCR using primers (F, 5'-AAC TCGAGT GGG CCA TGC CAG GGG
CAG TG-3', and R, 5'-ATT GGA TCC TAG TCC TAG TCC ATG GCC CTA GAG
CT-3'), subcloned into a pBluescriptII KS(+) vector cut at XhoI and BamHI sites, and thereafter transferred
in-frame to pEGFP-C1 vector (Clontech) at
EcoRI and BamHI sites. All inserts in the expression vectors were verified by sequencing.
-32P]dCTP using Ready-to-Go DNA labeling
beads (Amersham Biosciences) and hybridized with the filters in
ExpressHyb Hybridization Solution (Clontech) at
68 °C. The filters were washed four times in 0.05% SDS, 2× SSC for
10 min at 50 °C, washed once in 0.1% SDS, 0.1× SSC for 40 min at
50 °C, and subjected to x-ray film autoradiography.
-35S]CTP.
) and lysed in lysis
buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM CaCl2, 1% Triton
X-100, 0.5% deoxycholate, and protease inhibitors (Complete tablet,
Roche Applied Science). After the protein concentrations were
determined using a DC protein assay kit (Bio-Rad), 250 µg of the
lysates were incubated with 15 µl of CaM-Sepharose (Amersham
Bioscience) in the lysis buffer for 2 h at 4 °C. Beads were
washed for six times in the lysis buffer or Ca2+-free lysis
buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EGTA, 1% Triton X-100, 0.5%
deoxycholate, and protease inhibitors. CaM-bound mCLICK-III was
detected by Western blot with an anti-HA tag monoclonal antibody
(1:2500, 12CA5; Roche Diagnostics).
) and lysed in lysis
buffer containing 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM MgCl2, 1% Nonidet
P-40, 1 mM dithiothreitol, 25 mM NaF, 10 mM
-glycerophosphate, 5 mM sodium
pyrophosphate, 0.1 µM calyculin A, and protease
inhibitors. Lysates were immunoprecipitated with an anti-HA antibody
(12CA5) and protein-G-Sepharose (Amersham Biosciences).
Immunoprecipitates were washed three times in the lysis buffer and
washed twice in kinase buffer containing 50 mM HEPES-NaOH
(pH 7.5), 10 mM MgCl2, 1 mM
Ca2+, 20 mM
-glycerophosphate, 0.02%
Nonidet P-40, 1 mM dithiothreitol, and protease inhibitors.
Kinase assay was performed in the presence of 1 µM CaM,
50 µM ATP, 0.5 µCi of [
-32P]ATP in the
kinase buffer using 2.5 µg of CREM (cAMP-response element
modulator) (Santa Cruz Biotechnology) or 5 µg of MBP (Calbiochem) as
substrates for 10 min at 30 °C. For kinase assay without
Ca2+/CaM, 1 µM CaM was omitted, and 1 mM EGTA was substituted for 1 mM
Ca2+ in the kinase buffer.
, pEGFP-mCL3, pEGFP-mCL3C474S, and pEGFP-CLVM using LipofectAMINE 2000 reagent. After 24 h, the cells were rinsed twice in HBSS/HEPES, incubated for 20 min at room temperature with 2.5 µM BODIPY-TR ceramide/bovine serum
albumin in HBSS/HEPES. The cells were washed twice with HBSS/HEPES and replaced with pre-warmed fresh medium, followed by a 1-h incubation in
a CO2 incubator. To simultaneously acquire GFP and
BODIPY-TR-ceramide images under live conditions, a Carl Zeiss LSM 510 system equipped with a Carl Zeiss Axiovert 100TV inverted microscope
and a ×63 Plan-Apochromat (NA 1.4, oil) objective (Carl Zeiss) was
used. For all images, stacks of multiple Z-scan sections were obtained, and projected images were calculated off-line using a projection software on the LSM 510 system. All pseudocolor representations were
assembled using Photoshop version 5.5 (Adobe). For compactin pretreatment, the normal medium was replaced with a growth medium containing 40 µM compactin (Wako Pure Chemicals) for
12 h. No apparent phototoxicity was observed under our
experimental conditions.
),
suspended in 250 µl (per dish) of homogenizing buffer containing 10 mM HEPES-NaOH (pH 7.5), 10 mM KCl, 2 mM MgCl2, protease inhibitors, disrupted in a
Potter-Elvehjem homogenizer with 40 strokes, and adjusted to 0.25 M sucrose. To remove unbroken cells and nuclei, the
homogenate was centrifuged at 600 × g for 10 min. The
supernatant was separated by centrifugation at 100,000 × g for 60 min. After collecting the resulting supernatant (cytosolic fraction, S), the membrane pellet was washed once in the
homogenizing buffer containing 0.25 M sucrose and again
centrifuged at 100,000 × g for 60 min. For the salt
wash experiment, the 600 × g supernatant was divided
into two microtubes and separated by ultracentrifugation at
100,000 × g for 60 min. The membrane pellet was
resuspended in homogenization buffer containing 10 mM
HEPES-NaOH (pH 7.5), 10 mM KCl, 2 mM
MgCl2, 0.25 M sucrose or high salt
homogenization buffer containing 1 M NaCl, followed by
incubation on ice for 1 h, and ultracentrifuged again at
100,000 × g. For all analyses, the membrane pellets
were finally extracted with the elution buffer containing 25 mM HEPES-NaOH (pH 7.5), 2% SDS, 150 mM NaCl,
protease inhibitors for 1 h at room temperature and centrifuged
for 15 min at 15,000 rpm. This supernatant was collected and designated
as crude membrane fraction (P or P'). The volume of the elution buffer
was adjusted to be equal to the volume of input (identical to the
600 × g supernatant). For Western blot analyses,
one-third volume of 4× Laemmli buffer was added and boiled for 4 min,
and 20 µl of each fractions were subjected to SDS-PAGE. For compactin
treatment, the medium was replaced with growth medium containing 40 µM compactin for 16 h.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(13) and CaMKI
(19) (Fig.
1, A and B). A
partial sequence of a rat ortholog of CLICK-III was previously reported
as rCaMKI
(19) (asterisk, Fig. 1C).
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Fig. 1.
CLICK-III/CaMKI is an isoform of the
CaMKI family with an unusually extended C-terminal domain.
A, the deduced amino acid sequence of mCLICK-III was aligned
with CKLiK, rCaMKI
, and rCaMKI
by using the ClustalW Multiple
Sequence Alignment Program version 1.8. The asterisks
indicate positions of identical amino acid residues. The
colons and dots indicate stronger and weaker
degrees of residue conservation, respectively. B, comparison
of amino acid identities between kinase domain sequences of known
CaMKI-related kinases. The kinase domain of mCLICK-III shows high
identity with all known CaMKI-related kinases, CKLiK (74.8%),
CaMKI
(71.1%), and rCaMKI
(65.3%), respectively, in the
order of identity (left column). The exact position of amino
acid (aa) residues used for comparison are shown in
parentheses. C, similarity and difference between
CLICK-III and other CaMKI-related kinases. CLICK-III shares a high
homology in its catalytic domain at the N terminus followed by a
regulatory domain consisting of an autoinhibitory domain
(AID) and a Ca2+/CaM binding domain
(CBD). Unlike the other CaMKI-related kinases, however,
mCLICK-III has a distinctively longer C-terminal region ending with a
putative CAAX motif (CLVM). A 309-amino
acid-long partial sequence of rat of CLICK-III has been reported
before as a rat CaMKI
(asterisk) (19).
GenBankTM accession numbers are as follows:
mCLICK-III, AY212936; CKLiK, AF286366; rCaMKI
, L24907 and L26288;
rCaMKI
, D86556; and rCaMKI
(partial cDNA), D86557.
,
and CaMKI
, suggesting a unique role for CLICK-III (Fig.
1C). The very C-terminal end of hCLICK-III and mCLICK-III were, however, divergent, indicating the existence of multiple C-terminal variants (data not shown).
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Fig. 2.
Abundant expression of CLICK-III transcripts
in the CNS. A, Northern blotting using specific probes
for CLICK-III showed that both mCLICK-III and hCLICK-III are most
strongly expressed in the brain. Note that mCLICK-III transcripts are
expressed from embryonic day (E) 11, and hCLICK-III
transcripts are expressed in a forebrain-specific manner in the CNS.
Sk, skeletal. B, in situ hybridization
was performed with 35S-labeled riboprobes. Film
autoradiographs of mouse brain parasagittal (I and
II) and coronal sections (III and IV)
showed specific hybridization signals for mCLICK-III antisense probes
in restricted brain areas such as the olfactory bulbs (OB),
the cerebral cortex (Cx), the hippocampus
(Hippo.), the central nucleus of amygdala (CeA),
and the ventromedial nucleus of hypothalamus (VMH). These
signals disappeared when sense probes were used (II and
IV).
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Fig. 3.
CLICK-III transcripts are expressed in
neurons. Dark- (A, C, and E) and
bright-field (B, D, and F)
photomicrographs showed strong signals in the central nucleus of
amygdala (A, CeA) and the ventromedial
hypothalamic nucleus (C, VMH) (also see Fig.
2B, III). Silver grains were present only on
neuronal cell bodies (arrows, B and
D). Moderate signals were detected on the layer V
pyramidal neurons in the cerebral cortex and the CA1
pyramidal neurons in the hippocampus (E). In the
hippocampus, non-principal neurons were also labeled with silver grains
(arrows, F). CA1, CA1 area for
Ammon's horn; hi, the hilus of the dentate gyrus;
Gr, the granule cell layer of the dentate gyrus. Scale
bars, A, C, and E, 0.5 mm;
B, D, and F, 50 µm.
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Fig. 4.
Activation of CLICK-III by
Ca2+/CaM and CaMKK. A, mCLICK-III expressed
in COS-7 cells is bound to CaM-Sepharose beads in the presence of
Ca2+. Lysates from COS-7 cells transfected with either an
empty vector (mock) or a mCLICK-III cDNA
(HAmCL3wt) were incubated with CaM-Sepharose beads.
mCLICK-III remained bound to CaM-Sepharose after washing in the
presence of 2 mM Ca2+ (Beads,
Ca2+), whereas most CLICK-III was washed out in 0 mM Ca2+, 2 mM EGTA (Beads,
EGTA). WB, Western blot. B,
immunoprecipitate kinase assays of CLICK-III. COS-7 cells were
transfected with an empty vector (mock), a wild type
mCLICK-III cDNA (HAmCL3wt), or mCLICK-III C-terminal
deletion mutant (HAmCL3dC) with (+) or without ( ) a
cDNA encoding a constitutive active form of rat CaMKK
(CaMKK). After immunoprecipitation with an anti-HA antibody,
kinase assays were performed in the presence (+) or the absence (
) of
Ca2+/CaM using MBP (5 µg) or CREM (2.5 µg) as
substrates. Note that both mCL3wt and mCL3dC require CaMKK activity for
their full activation. C-terminal deletion mutant of mCLICK-III showed
a clear constitutive kinase activity in the presence of CaMKK,
independent of the presence of Ca2+/CaM, whereas the wild
type kinase was only active in the presence of Ca2+/CaM.
C, CREM phosphorylation was also detected in a manner
similar to 32P incorporation, using an anti-phospho-CREB
(Ser-133) antibody. D, the C-terminal deletion mutant of
CLICK-III can activate CREB-dependent transcription in
COS-7. Cells were transfected with CLICK-III C-terminal deletion mutant
(pIRES-HAmCL3dC) and reporter genes coding Gal4-CREB and
UAS-luciferase and collected, and lysates were extracted to measure the
luciferase activity 24 h later. The bars represent
luciferase activities relative to that of mock-transfected lysates
(pIRES) and are shown as means ± S.E.
(n = 3). *** denotes p < 0.001.
-32P]ATP incorporation into CREM closely paralleled
the amount of phospho-CREB Ser-133 immunoreactivity, suggesting that
wild type CLICK-III is able to directly phosphorylate CREB at Ser-133,
in a Ca2+/CaM- and CaMKK-dependent manner, at
least in vitro (Fig. 4C). We further confirmed
that CLICK-III may work as CREB kinase in vivo by using a
Gal4-CREB/UAS-luciferase reporter system. Transfection of a
constitutively active CLICK-III (pIRES-HAmCL3dC) significantly augmented CREB-dependent gene expression in COS-7 cells
(Fig. 4D). The result was normalized using
Renilla luciferase (pRL-CMV), which was co-transfected with
the other reporter genes (pFR-Luc and pFA-CREB).
remained largely excluded from the nucleus, in keeping
with prior reports (Fig. 5) (15, 16). A striking difference was noted
in the subcellular localization of GFP-tagged CLICK-III (GFP-mCL3);
GFP-mCL3 distribution was not diffuse but seemed rather associated with
intracellular compartments reminiscent of endomembrane systems such as
the Golgi complexes or the endoplasmic reticulum (Fig. 5,
arrowheads). Furthermore, CLICK-III was also found to be
enriched at the tips of thin processes that were most likely filopodia
(Fig. 5, arrows); these structures have been proposed to
constitute intermediate membrane protrusions that precede the formation
of stable dendritic spines during synaptogenesis (38).
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Fig. 5.
GFP-rCaMKI and
GFP-mCL3 show distinct subcellular localization in hippocampal
neurons. Mouse CA1/CA3 hippocampal neurons were transfected with
GFP, GFP-rCaMKI
, and GFP-mCL3 for 7 days in vitro, fixed
48 h later, and examined by confocal microscopy. Three
representative neurons were shown for each construct
(#1-#3). GFP showed diffuse distribution across the cell,
whereas GFP-rCaMKI
was diffusely expressed mainly in the cytoplasm.
In contrast, GFP-mCL3 expression was more localized in specific
intracellular compartments (arrowheads) and concentrated at
the tips of filopodia-like processes (arrows).
Bar, 5 µm.
. Unlike the diffuse distribution of GFP (Fig.
6A, left panels) or
the largely cytoplasmic distribution GFP-rCaMKI
(Fig.
6A, middle panels), GFP-mCL3 localization overlapped with the perinuclear Golgi complex, as demonstrated by a
high degree of spatial superimposition between GFP fluorescence of
GFP-mCL3 and a Golgi-specific vital dye, BODIPY TR-ceramide (Fig.
6A, right panels, arrows). Such a
co-localization was not detected between GFP-rCaMKI
, or GFP alone,
and the BODIPY TR-ceramide stain (Fig. 6A, upper
panels). In addition, a significant amount of GFP-mCL3 signals was
seen at the plasma membranes (Fig. 6A, right
panels, arrowheads). Consistent with such significant
localization of GFP-mCL3 with the Golgi complex and the plasma
membranes, a sizable pool of GFP-mCL3 protein was recovered in the
membranes fraction (P), after 100,000 × g
ultracentrifugation (Fig. 6, B and C). In
contrast, GFP-rCaMKI
was predominantly present in the supernatants
(S) and little in the membrane pellet fraction (P) (Fig. 6,
B and C).
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Fig. 6.
Localization of GFP-mCL3 with the Golgi
complex and plasma membranes in COS-7 cells. A, COS-7
cells transfected with GFP, GFP-rCaMKI , and GFP-mCL3 were stained
with a Golgi-specific vital dye (BODIPY TR-ceramide) and examined under
live conditions. Projected images from stacks of z-planes
(Merge and TR-ceramide) and single z-plane images
taken near the bottom, middle, and top
of the cells showed localization of CLICK-III to the Golgi complex
(arrows) and to the plasma membranes
(arrowheads). Green, GFP image; red,
BODIPY TR-ceramide. Bar, 10 µm. B, lysates of
COS-7 cells transfected with the indicated constructs were fractionated
by ultracentrifugation at 100,000 × g. The
supernatants (S) were collected as cytosolic fractions, and
the pellets (P) were washed once and centrifuged again. The
resulting pellet was recovered as crude membrane fraction
(P). Each fraction was blotted and visualized using an anti-GFP antibody.
C, chemiluminescent signal intensities, quantified by a CCD
camera-based imaging system, were presented in percentages relative to
the total signal intensities detected in both fractions (S + P). The bars represent means ± S.E.
(n = 4). ***, p < 0.001.
and
CLICK-III? Sequence search identified a putative CAAX
membrane-anchoring motif in the very C-terminal end of mCLICK-III (Fig.
1 and Table I). We thus examined whether the CAAX box in mCLICK-III was necessary for its membrane
localization. A single amino acid substitution at the putative
prenylation site (GFP-mCL3C474S) completely abolished recruitment of
mCL3 to the perinuclear Golgi (Fig.
7A, left
panels, arrows) and plasma membranes (Fig.
7A, left panels, arrowheads), and this
mutant was now diffusely expressed in the cytoplasm (Fig.
7A, middle panels). Conversely, attachment of the
CAAX box (CVLM) of the mCL3 to the C terminus of GFP
(GFP-CLVM) was sufficient to confer perinuclear Golgi localization to
GFP (Fig. 7A, right panels, arrows).
We found, however, that GFP-CLVM did not appear to localize well to the
plasma membranes, consistent with the notion that residues upstream of
the CAAX motif may also play a role in the correct
localization and the proper sorting to the plasma membranes (Fig.
7A, right panels) (22, 23). In accordance with
these observations, biochemical fractionation experiments showed a
significant reduction in the amount of membrane-bound GFP-mCL3C474S
especially after high salt wash (P'), whereas GFP-CVLM still remained
heavily associated with the membranes (Fig. 7, B and
C). Together, these data suggested that the CAAX
box of CLICK-III is necessary to localize CLICK-III to the Golgi
membranes.
C-terminal sequences of Ras proteins and mCLICK-III
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Fig. 7.
A critical role of the C-terminal
CAAX motif in determining mCLICK-III
localization. A, COS-7 cells transfected with either
wild type CLICK-III (GFP-mCL3), a mutant that has a point
mutation at the prenylation site of CAAX motif
(GFP-mCL3C474S), or GFP-CLVM were stained with
BODIPY TR-ceramide and examined alive. The arrows and
arrowheads indicate the localization to the Golgi complex
and to the plasma membranes, respectively. Merge, projected
images; top, middle, and bottom,
single z-plane images. Green, GFP image; red,
BODIPY TR-ceramide. Bar, 10 µm. B, COS-7
lysates were fractionated by ultracentrifugation at 100,000 × g. The supernatants were collected (S), and the
pellets (P) were washed in either normal homogenization
buffer or high salt homogenization buffer, followed by
ultracentrifugation at 100,000 × g. The cytosolic
fractions (S), the membrane fractions (P), and
the high salt-washed membrane fractions (P') were subjected
to Western blotting with an anti-GFP antibody. C,
quantification of signal intensities detected in B. The
bars represent means ± S.E. (wt, n = 4; C474S, n = 4; CLVM, n = 3). *,
p < 0.05; **, p < 0.01; ***,
p < 0.001.
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Fig. 8.
Compactin, an HMG-CoA reductase inhibitor,
prevents GFP-mCL3 localization to the Golgi and plasma membranes.
A, COS-7 cells transfected with GFP-mCL3 were pretreated
with vehicle alone (DMSO) or 40 µM compactin
for 12 h, stained with BODIPY TR-ceramide, and examined under live
conditions. The co-localization of GFP-mCL3 and BODIPY TR-ceramide
(arrow) was abolished in compactin-treated cells.
Green, GFP image; red, BODIPY TR-ceramide.
Bar, 10 µm. B, COS-7 cells transfected with
GFP-mCL3 were treated with 40 µM compactin for 16 h.
The cytosolic fractions (S), the membrane fractions
(P), the high salt-washed membrane fractions (P')
were collected and subjected to Western blotting using an anti-GFP
antibody. C, quantification of signal intensities detected
in B. The bars represent means ± S.E.
(n = 5). * denotes p < 0.05.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
, and CaMKIV, because these are the most well
characterized CaMKs widely expressed in forebrain neurons (35, 36). In
contrast, studies concerning CaMKI have lagged behind, in part because
the originally isolated CaMKI
has been expressed ubiquitously and little has been shown so far about its physiological role or substrates in neurons (13, 15, 17).
, possessed an extended C-terminal domain distinct
from known CaMKs. We found that CLICK-III had three particular
characteristics that were noteworthy.
(27-29), indicating that the catalytic core of CLICK-III may resemble CaMKI
and may be regulated in a manner identical to
CaMKI
. Thus, maximal activation of CLICK-III might only be triggered
in close vicinity to an upstream kinase such as CaMKK.
, however, CLICK-III expression was strongly
enriched in neurons. More interestingly, examination of its mRNA
distribution, by in situ hybridization analyses, revealed a
very strong expression in the CeA and the VMH. Such peculiar distribution stands out among all known CaMKs and indicates the possibility that CLICK-III may play a role in the proper function of
these nuclei. The CeA has been shown to be a relay for most autonomic
outputs and, furthermore, has been associated with the expression of
the stimulus-specific state of fear (47). The VMH, on the other hand,
has been linked with control of the homeostasis of feeding and sexual
behaviors (48, 49). Future studies are needed to determine whether some
of these functions mediated through either CeA or VMH might indeed be
controlled by the kinase activity of CLICK-III.
![]() |
ACKNOWLEDGEMENTS |
---|
We are indebted to Drs. Masaaki Tsuda and Akiko Tabuchi (Toyama Medical and Pharmaceutical University), Dr. Hiroshi Tokumitsu (Kagawa Medical University), and Dr. Ryuichi Shigemoto (National Institute for Physiological Sciences) for comments during the course of the study. We are also grateful to Dr. Hirohide Takebayashi (National Institute for Physiological Sciences) for providing pIRES-S-Tag-EGFP and Dr. Hiroyuki Sakagami (Tohoku University) for sharing unpublished results. We thank Kimiko Nonomura for technical assistance and Tae Arai and Hiroko Nose for secretarial help.
![]() |
FOOTNOTES |
---|
* This work was supported by grants-in-aid from the Ministry of Education, Science, Sports, Culture and Technology, the Ministry of Health, Labor, and Welfare of Japan, a PRESTO investigatorship from the Japan Science and Technology Corp., and grants from the Asahi Glass Foundation, the Human Frontier Science Program, the Narishige Neuroscience Research Foundation, the Tokyo Biochemical Research Foundation, the Ube Research Foundation, and the Yamanouchi Foundation for Research on Metabolic Disorders.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.
§ Predoctoral fellows from the Japan Society for Promotion of Science.
Postdoctoral fellows from the Japan Society for Promotion of Science.
To whom correspondence should be addressed. Tel.:
81-3-5841-3559; Fax: 81-3-3814-8154; E-mail:
hbito@m.u-tokyo.ac.jp.
Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.M300578200
2 S. Ohmae, S. Takemoto-Kimura, and H. Bito, manuscript in preparation.
3 S. Takemoto-Kimura and H. Bito, manuscript in preparation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CaM, calmodulin; CaMK, Ca2+/calmodulin-dependent protein kinase; CREB, Ca2+/cAMP-response element-binding protein; CLICKs, CaMK-like CREB kinases; VMH, ventromedial hypothalamus; HMG-CoA, 3-hydroxy-3-methylglutaryl-coenzyme A; RACE, rapid amplification of cDNA ends; HA, hemagglutinin; GFP, green fluorescent protein; EGFP, enhanced GFP; wt, wild type; PBS, phosphate-buffered saline; MBP, myelin basic protein; HBSS, Hanks' balanced salt solution; CNS, central nervous system; CeA, central nucleus of amygdala; CMV, cytomegalovirus.
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REFERENCES |
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