From the Ben May Institute for Cancer Research,
§ Department of Pharmacological and Physiological
Sciences, and ¶ Department of Pediatrics, University of
Chicago, Chicago, Illinois 60637
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ABSTRACT |
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ERK7, a member of the mitogen-activated protein
kinase family, has a carboxyl-terminal tail that is required for ERK7
activation, cellular localization, and its ability to inhibit DNA
synthesis. To identify proteins that interact with ERK7, we utilized a
yeast two-hybrid screen with the COOH-terminal tail of ERK7 as bait and
isolated the cDNA for a novel protein termed CLIC3. The interaction between CLIC3 and ERK7 in mammalian cells was confirmed by
co-immunoprecipitation. CLIC3 has significant homology to human
intracellular chloride channels 1 (NCC27/CLIC1) and 2 and bovine kidney
chloride channel p64. Like NCC27/CLIC1, CLIC3 is predominantly
localized in the nucleus and stimulates chloride conductance when
expressed in cells. Taken together, these results suggest that CLIC3 is
a new member of the human CLIC family. The observed interaction between CLIC3 and ERK7 is the first demonstration of a stable complex between a
protein that activates chloride ion transport and a member of the
mitogen-activated protein kinase family of signal transducers. The
specific association of CLIC3 with the COOH-terminal tail of ERK7
suggests that CLIC3 may play a role in the regulation of cell growth.
Chloride channels are a diverse group of proteins that regulate
fundamental cellular processes including stabilization of cell membrane
potential (1, 2), transepithelial transport, maintenance of
intracellular pH (3), and regulation of cell volume (4, 5). Various
chloride channels have also been implicated in human hereditary
diseases such as the dominant and recessive forms of myotonia (6),
X-linked hereditary nephrolithiasis (7), and cystic fibrosis (8).
Although the majority of chloride channels characterized to date have
been localized to the plasma membrane (9), a new family of proteins
that activate intracellular chloride permeability has been identified
within the past few years.
Termed the "chloride intracellular channel" or
CLIC1 family (10), at least
two members have been described. The CLIC family has homology to p64, a
bovine kidney microsomal chloride ion transporter characterized by
Landry and colleagues (11-13). A rat homologue of p64 (p64G1) has also
been cloned and shown to play a role in intracellular chloride ion
transport within the endoplasmic reticulum (14). The highly related but
distinct CLIC class of chloride ion transporters shares homology with
the COOH-terminal half of p64 (10, 15). The prototype for this family
is NCC27/CLIC1 (15), a small protein of 241 amino acids that is
expressed primarily in the nucleus and exhibits both nuclear and plasma
membrane chloride ion channel activity. CLIC2, a 243-amino acid protein
that shares a putative nuclear localization signal, has not yet been
characterized but has been mapped to a region of the chromosome that
codes for proteins important in a number of diseases. Although the
specific function of these proteins is not yet known, their high degree of conservation and unusual cellular localization suggest that they
play a central role in cellular function. Since the CLIC proteins lack
the membrane-spanning domains characteristic of channel proteins
and are largely intracellular, it is possible that they are not
themselves chloride channels but instead function as activators of
chloride channels.
There is increasing evidence indicating that cellular signaling
cascades play a fundamental role as regulators of ion channels. For
example, the high voltage-activated Ca2+ channel and
Ca2+-activated K+ channels can be modulated by
growth factors (16, 17). Both NMDA receptors and the human Kv1.5
K+ channel were shown to be regulated by the tyrosine
kinase Src (18, 19). In lymphocytes, the tyrosine kinase p561ck
mediates activation of a swelling-activated chloride channel (20) and
also activates an outwardly rectifying chloride channel during
Fas-mediated apoptosis (21). However, little is known about how
signaling pathways modulate chloride channels.
One of the major signaling cascades activated by growth and
differentiating factors in cells is the mitogen-activated protein (MAP)
kinase cascade. A superfamily of highly homologous proline-directed serine/threonine kinases, MAP kinases are regulated through distinct signaling cascades involving upstream protein kinases that
phosphorylate both tyrosine and threonine residues in a
Thr-X-Tyr (TXY) motif positioned within the
activation loop of MAP kinases (reviewed in Ref. 22). Recently, a novel
60-kDa member of the MAP kinase family, termed extracellular
signal-regulated kinase 7 (ERK7), has been cloned and characterized
(23). Although it has the signature TEY activation motif of ERK1 and
ERK2, ERK7 is significantly different from previously identified ERKs.
First, ERK7 does not appear to be activated either by extracellular
stimuli that typically activate ERK1 or ERK2 or by common activators of
the JNK or p38 pathways. Instead, this novel MAP kinase has appreciable
constitutive activity in serum-starved cells, and this activity
requires the presence of a COOH-terminal tail. Second, the enzyme is
expressed in the nucleus in a COOH-terminal tail-dependent
fashion. Finally, ERK7 can function as a negative regulator of cell
growth, and this activity is dependent on the presence of the
COOH-terminal tail but independent of its kinase activity. Taken
together, these results describe a new type of MAP kinase family member
whereby interactions via its COOH-terminal tail, rather than
extracellular signal-mediated activation cascades, regulate its
activity, its localization, and its function.
In order to elucidate the mechanism by which ERK7 is regulated, we
identified proteins that interact with the COOH-terminal tail of ERK7
using a two-hybrid screen. In the present study, we describe the
isolation and characterization of a new member of the CLIC family of
intracellular chloride channels termed CLIC3 that specifically
associates with ERK7. Like NCC27/CLIC1, CLIC3 is a small protein that
is localized primarily in the nucleus and stimulates chloride ion
channel activity. The observed association of CLIC3 with a member of
the MAP kinase family, in conjunction with the nuclear localization of
both of these proteins, raises the possibility that CLIC3 may
participate in cellular growth control.
Plasmids and Antibodies--
Peroxidase-conjugated goat anti-rat
IgG and peroxidase-conjugated goat anti-mouse IgG were purchased from
Sigma. Monoclonal antibody (12CA5) against the hemagglutinin (HA)
epitope was purchased from Babco (Emeryville, CA). High affinity rat
anti-HA monoclonal antibody (3F10) was purchased from Boehringer
Mannheim. Antibody against the FLAG-epitope M5 was purchased from IBI
(Eastman Kodak). Enhanced Chemiluminescence Reagents were purchased
from NEN Life Science Products. The pCMV- Yeast Two-hybrid Screen--
The
BamHI-NotI fragment of ERK7 comprising the
COOH-terminal tail (23) was subcloned into a Bluescript II KS(+) vector
to construct plasmid pB-ERK7. The EcoRI-NotI
fragment with ERK7 from pB-ERK7 was fused into a LexA DNA binding
domain in pEG202, resulting in the pEG202-ERK7 construct. The human
fetal brain library, kindly provided by Dr. Roger Brent, was made from
a 22-week-old human fetal frontal cortex and was used to search for
novel proteins that interact with ERK7. In a screen of
~1×106 primary transformants, two clones (S9 and S46)
showing strong interaction with ERK7 were obtained. Yeast
transformation and routine yeast work were performed as described
(25).
Tissue Northern Blot--
The EcoRI fragment from
pCLIC3 plasmid was isolated and radiolabeled with 32P using
the Megaprime DNA labeling system (Amersham Pharmacia Biotech) and
hybridized to a poly(A)+ RNA human multiple tissue Northern
blot (CLONTECH). Hybridization was performed
according to the user manual (CLONTECH).
Epitope Tagging--
Primer A
(5'-ATGACTACAAGGACGACGATGACAAGGTCCTGCTCCTCAAGGGCGTACCTTTCACCCTCACCACGGTG-3')
and primer B (5'-GCTCGAGGACAAAGATGCCTTTATTGG-3') were used to clone the
CLIC3 from S9 cDNA by PCR with ID Proof Taq (ID Lab).
The PCR products were ligated into the pCR3.1 TA vector (Invitrogen) to
construct pCLIC3. Primer A included nucleotides coding for the FLAG
epitope right after the ATG codon and the 23 nucleotides missing in the
5' end of S9 cDNA. The EcoRI fragment containing CLIC3
was subcloned into a pGEX-2T vector to express the GST-CLIC3 fusion
protein in bacterial BL21 cells. GST fusion proteins were prepared
using a GST purification system (Amersham Pharmacia Biotech). An HA
tag, YPYDVPDY, was inserted right after the initiating methionine of
ERK7 by PCR (23). The HA-K43R ERK7 mutant was generated by unique site
elimination mutagenesis (26).
Cell Culture and Transient Transfections--
COS, CV-1, and LTK
cells were grown in Dulbecco's modified Eagle's medium supplemented
with antibiotics (50 units/ml penicillin and 50 µg/ml streptomycin)
and 10% fetal bovine serum in a 95% air, 5% CO2
incubator at 37 °C. COS and CV-1 cells were transfected with a total
of 10 µg of plasmid DNA, and transfection was performed with the
TransITTM polyamine transfection reagent according to the
manufacturer's instructions (Panvera, Madison, WI). The
LipofectAMINETM Reagent (Life Technologies, Inc.) was used
for transfection of LTK cells. The pGreen Lantern-1 (Life
Technologies, Inc.) green fluorescent protein (GFP) or the
pCMV- Cell Lysis and Western Blot--
Cultured cells were washed
twice with ice-cold phosphate-buffered saline and lysed in 1%
Triton-based lysis buffer containing 50 mM Tris-HCl, pH
7.5, 100 mM NaCl, 50 mM NaF, 1% Triton X-100, 40 mM Immunoprecipitation and in Vitro Kinase
Assays--
Immunoprecipitation was performed as described previously
(28). In order to assay ERK7 kinase activity, COS cells were
transfected with HA-ERK7 or HA-ERK7-K43R. After 24-48 h, cells were
lysed in 0.5% Triton X-100 buffer, and ERK7 and ERK7-K43R were
immunoprecipitated using the anti-HA antibody. The immune complex was
washed three times with lysis buffer and twice with kinase buffer (20 mM Hepes, pH 7.4, 10 mM MgCl2, 1 mM DTT, 0.2 mM sodium vanadate, 10 mM Immunocytochemistry--
The CV-1 cells were plated on
coverslips and incubated overnight. Cells were transfected with
FLAG-CLIC3 and pCMV- Electrophysiological Experiments--
LTK cells were transfected
with both FLAG-CLIC3 and GFP or with GFP alone. Electrophysiological
experiments were performed 48-72 h after transfection. Transfected
cells were identified by GFP fluorescence. Membrane voltage was
controlled with a voltage clamp in the whole-cell, tight-seal
configuration (29). The resistance was 2-3 M Isolation of the Clone Encoding CLIC3, a Protein That Interacts
with the COOH-terminal Tail of ERK7--
In order to elucidate the
mechanism by which the COOH-terminal tail of ERK7 regulates ERK7
activity and function, we screened for interacting proteins using the
LexA-based yeast two-hybrid system (25). A restriction fragment of the
ERK7 cDNA containing the COOH-terminal tail was used as bait for
screening a human fetal brain library. Two cDNA clones (S9 and S46)
out of ~1 × 106 transformants interacted strongly
with the tail of ERK7 but not with other unrelated control proteins
(data not shown).
Sequence analysis indicated that both of these clones contained the
same partial-length cDNA and were missing 23 nucleotides at the 5'
end of the full-length cDNA. Searching the human EST Data Base
revealed an overlapping cDNA clone (gb/N41765) that was missing the
3' end but had the complete 5' end of the gene. Together, the cDNA
sequences from S9 and the EST clones encoded a polypeptide of 208 amino
acids with a predicted Mr of 23,569. The
potential initiating methionine codon is right after a stop codon in
the same reading frame and occurs in a consensus region favorable for
initiation of translation in higher eukaryotes (31). Fig.
1A shows the nucleotide and
predicted amino acid sequences of the protein encoded by the S9 clone.
Comparison of the deduced amino acid sequence to other sequences in the
Swiss Protein Data Base revealed a significant homology to human
chloride intracellular channel 1 (NCC27/CLIC1) (15) and chloride
intracellular channel 2 (CLIC2) (10). The protein encoded by S9 had
48-49% identity and 60-61% similarity to NCC27/CLIC1 and CLIC2.
Furthermore, the S9 protein shared 47% identity and 60% similarity
with the COOH-terminal half of the bovine chloride channel p64
(11-13). Due to the high homology among these proteins, the protein
encoded by the S9 clone was termed CLIC3 for chloride intracellular
channel 3.
The multiple alignment of CLIC3 with NCC27/CLIC1 and CLIC2 is shown in
Fig. 1B. A strongly hydrophobic region in CLIC3, from amino
acids 137-155 (Fig. 1A), was found by Kyte-Doolittle
hydrophobicity analysis (32). This region represents a potential
transmembrane domain that is very conserved among the CLIC genes.
Searching for other possible motifs in CLIC3 revealed two potential
casein kinase II phosphorylation sites at Thr-13 and Thr-141, two
potential protein kinase C phosphorylation sites at Thr-17 and Ser-130, and one potential N-myristoylation site at Gly-7.
In Vivo Interaction between CLIC3 and ERK7--
We generated a
full-length clone of CLIC3 by PCR extension of the S9 sequence and then
determined whether the protein could be expressed in mammalian cells.
The full-length CLIC3 cDNA was tagged with a sequence encoding the
FLAG epitope at the 5' end, and then transiently transfected into COS
cells. The transfected cell lysates were resolved by SDS-PAGE and
immunoblotted with anti-FLAG antibody. A protein of 24 kDa, consistent
with the predicted molecular mass of CLIC3, was recognized specifically
by the anti-FLAG antibody in the cells transfected with CLIC3, but not
in the cells transfected with the control vector (Fig.
2). To determine whether ERK7 and CLIC3
associate in vivo, COS cells were transiently co-transfected with HA-tagged ERK7 and either FLAG-tagged CLIC3 or the control vector.
After immunoprecipitation of the cell lysates with anti-HA antibody,
the immunoprecipitates were resolved by SDS-PAGE and probed for
expression of both the HA-ERK7 and FLAG-CLIC3 proteins by
immunoblotting with anti-HA antibody and anti-FLAG antibody, respectively. As shown in Fig. 2B, the FLAG-CLIC3 protein
co-precipitated with ERK7, indicating that these two proteins do
associate in vivo. Surprisingly, when FLAG-CLIC3 was
immunoprecipitated first from cells transfected with both CLIC3 and
ERK7, no ERK7 was detected in the immunoprecipitates (data not shown).
This result may be due to steric hindrance by the anti-FLAG antibody
bound to protein A beads preventing interaction with ERK7.
Tissue Distribution of CLIC3--
A Northern blot from
CLONTECH containing purified poly(A)+
RNA from different human tissues was used to analyze the mRNA
expression of CLIC3. As shown in Fig. 3,
CLIC3 is expressed as a single transcript of approximately 1.15 kilobase in several human tissues, indicating that CLCI3 is widely
expressed. However, the level of CLIC3 expression varied, depending
upon the particular tissue type. CLIC3 has a very high expression level
in placenta and is abundantly expressed in lung and heart, suggesting
that it may play an important role in these tissues. CLIC3 is also
expressed at a much lower level in skeletal muscle, kidney, and
pancreas, and could not be detected in brain.
Localization of CLIC3--
If CLIC3 associates with ERK7 in
vivo, it should co-localize with ERK7 in the cell. Since the
intracellular location of ERK7 is predominantly in the cell nucleus
(23), we determined whether CLIC3 is also localized in the nucleus.
CV-1 cells were transfected with FLAG-CLIC3 as well as
CMV- CLIC3 Is Not a Direct Substrate of ERK7--
To determine whether
CLIC3 could potentially be a direct substrate of ERK7, we performed an
in vitro kinase assay using a GST-CLIC3 fusion protein as a
substrate. A kinase-inactive mutant of ERK7, ERK7-K43R, was used as a
negative control (23). Cell lysates extracted from COS cells
transfected with either HA-ERK7 or HA-ERK7-K43R were immunoprecipitated
and then incubated with either GST-CLIC3 or GST-Fos in kinase assay
buffer. In contrast to GST-Fos, no significant phosphorylation of CLIC3
by ERK7 was observed (Fig. 5). Although
we cannot rule out the possibility that a phosphorylation site on CLIC3
was unavailable due to the addition of the GST, these results suggest
that CLIC3 associates with, but is not a direct substrate of, ERK7.
Electrophysiological Properties of CLIC3 in LTK Cells--
When
FLAG-CLIC3 is overexpressed in LTK cells, some of the protein is
expressed in the plasma membrane as well as the cytoplasm. A similar
protein distribution was observed upon overexpression of NCC27/CLIC1
(15). Therefore, we measured the current-voltage properties of CLIC3
expressed in the plasma membrane of LTK cells, which have a very low
background of chloride channel activity. We first measured the
current-voltage properties of untransfected LTK cells or cells
transfected with GFP alone using the voltage clamp technique in the
whole cell configuration. As illustrated in Fig.
6A, the current-voltage
relation measured in the normal extra cellular saline solution was
nearly linear between In an effort to identify proteins that associate with the
COOH-terminal tail of ERK7, we isolated the intracellular chloride channel 3 (CLIC3) cDNA by a yeast two-hybrid screen. The
interaction between CLIC3 and ERK7 was confirmed by
co-immunoprecipitation studies in COS cells. CLIC3 is a novel protein
that is highly related to human CLIC1 (NCC27/CLIC1), CLIC2, and bovine
kidney chloride channel p64. A strongly hydrophobic region representing a transmembrane domain is very conserved among NCC27/CLIC1, CLIC2, p64,
and CLIC3. Like NCC27/CLICl, CLIC3 localized predominantly in the
nucleus and induced chloride conductance when expressed in cells.
Overall, these results implicate CLIC3 as a new member of the human
CLIC family.
To date, only one of the CLIC family members has been shown to have
nuclear chloride ion transport activity. NCC27/CLIC1 can promote
chloride transport across both the nuclear and plasma membranes (15).
By analogy based upon sequence homology, cellular localization, and
plasma membrane channel activity, it is likely that CLIC3 also has
nuclear as well as plasma membrane ion transport activity. Since it is
very difficult to detect nuclear ion channel activity, we focused on
demonstrating the function of CLIC3 as an inducer of ion transport by
probing the plasma membrane in transfected cells. Furthermore, the
electrophysiological properties of NCC27/CLIC1 in the nuclear
versus the plasma membranes were similar. The role of
nuclear ion transport is not clear at the present time, but the
existence of three discrete family members suggests that the role is an
important one.
Based on the current-voltage relationship of the LTK cells transfected
with CLIC3, we conclude that CLIC3 stimulates a chloride/anion ion
conductance. For example, reducing the extracellular chloride concentration from 152.5 mM to 22.5 mM reduced
the outward current observed at positive potentials. The change is
easily explained if cells expressing the CLIC3 protein, unlike cells
transfected with GFP only, have a relatively large membrane conductance
to Cl The CLIC family members are much smaller in length than the
characterized chloride ion channels and lack the characteristic transmembrane chloride channel (10). Thus, CLIC3, which has a limited
hydrophobic domain, must either multimerize or associate with other
subunits if it indeed is part of a membrane channel. Despite the
electrophysiological experimental results supporting the hypothesis
that CLIC3 is a component of a Cl Although CLIC3 is principally localized in the nucleus, no nuclear
localization sequence was found. A conserved KKYR has been proposed as
a nuclear localization signal motif in the 3' ends of NCC27/CLIC1,
CLIC2, and p64 (15), but this motif is not present in CLIC3. Therefore,
it is possible that CLIC3 is transported into the nucleus via
association with other proteins that are translocated to the nucleus.
The observation that CLIC3 is not completely nuclear is consistent with
the possibility. Since CLIC3 co-localizes with ERK7 in the nucleus, one
potential role of ERK7 could be to bind CLIC3 and transport it to the nucleus.
ERK7, the MAP kinase family member that associates with CLIC3, is the
newest member of the MAP kinase family. Among the extracellular signal-regulated or ERK family of MAP kinases, only four out of the
seven enzymes designated ERKs have the signature TEY activation motif.
These are ERKs 1, 2, 5, and 7. ERKs 1 and 2, the enzymes that were
originally designated as MAP kinases, are a discrete size between 42 and 44 kDa. In contrast, ERKs 5 and 7 are larger due to the presence of
a carboxyl-terminal tail that modulates the properties of the enzyme.
Although ERK7 has the signature TEY activation motif of ERK1 and ERK2,
this enzyme appears to be significantly different from other ERK family
members (23). In contrast to previously reported ERKs, ERK7 has
significant constitutive activity in serum-starved cells, is targeted
to the nucleus in both active and inactive states, and can function as a negative regulator of growth independent of its kinase activity. In
all cases, these properties are dependent upon the presence of the
COOH-terminal tail. Thus, the interaction of ERK7 with CLIC3 may be
very important in the action of the enzyme.
The functional consequences of the interaction between CLIC3 and ERK7
are not yet clear. One possibility is that ERK7 mediates phosphorylation of CLIC3 and/or chloride ion channel conductance. While
our results suggest CLIC3 is not directly phosphorylated by ERK7, it is
possible that the MAP kinase may facilitate association of CLIC3 with
other key components or enzymes. Since ERK7 can inhibit DNA synthesis,
it is also possible that CLIC3 is linked to a growth regulatory system,
either by competing with growth regulatory proteins for binding to ERK7
or by facilitating chloride ion transport, leading to changes in
intracellular pH and osmolarity.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-galactosidase expression
vector was a gift from V. Sukhatme. Plasmid DNAs were purified using a
Qiagen kit or by CsCl-ethidium bromide gradient centrifugation as
described previously (24). The plasmid DNAs were sequenced by the
Interdisciplinary Center for Biotechnology Research DNA sequencing core
laboratory at the University of Florida or by the University of
Chicago Cancer Research Center DNA sequencing facility.
-galactosidase expression vectors were co-transfected with
other plasmids as markers for visualizing transfected cells or as
indicators of transfection efficiency.
-glycerophosphate, 2 mM EDTA, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl
fluoride, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 20 mM
-nitrophenyl phosphate. Cell extracts were separated on 10% or 12% SDS-polyacrylamide gels. Proteins were transferred to a
nitrocellulose membrane. Western blot analysis was performed as
described previously (27).
-nitrophenyl phosphate). 2 µg of each substrate
were used per reaction in kinase buffer containing 5 µCi of
[
-32P]ATP. The kinase reaction was incubated at
30 °C for 30 min and terminated by boiling the mixtures in sample
buffer for 5 min. The reaction products were separated by 8%
SDS-PAGE.
-galactosidase or HA-ERK7 using the TransIT-LT1
reagent. After 48 h, the cells were fixed in 10% formaldehyde
solution (Fisher) for 15 min, washed with phosphate-buffered saline,
blocked, and incubated simultaneously with mouse anti-FLAG
M5 antibody and rabbit polyclonal anti-
-galactosidase antibody (5 Prime
3 Prime, Boulder, CO) or rat high affinity anti-HA
antibody. After 1 h of incubation at room temperature, the cells
were washed and then incubated with Texas Red-conjugated anti-mouse and
fluorescein isothiocyanate-conjugated anti-rabbit antibodies or
fluorescein isothiocyanate-conjugated anti-rat antibody (Molecular
Probes, Inc., Eugene, OR). The stained cells were analyzed using a
Zeiss Axioplan fluorescent microscope.
. Patch pipettes
contained (in mM): KCl, 135; CaCl2, 1;
MgCl2, 1; K2-EGTA, 10; HEPES, 10; adjusted to
pH 7.2 by the addition of KOH. The standard extracellular saline contained (in mM): NaCl, 140; KCl, 2.5; CaCl2,
3; MgCl2, 2; glucose, 15; HEPES, 10; adjusted to pH 7.4 by
the addition of NaOH. A low chloride saline was made by substituting
NaCl with sodium gluconate. Membrane potential was normally held at
70 mV. A current-voltage relation was measured by recording the
current while the voltage was changed between
100 and +80 mV at a
constant rate of 1.4 V s
1. No correction was made for the
series resistance of the patch pipette. Junction potentials were
corrected as described in Ref. 30. Currents were low pass-filtered
(8-pole with 3 db attenuation at 3 kHz), digitized at 2 KHz, and
subsequently analyzed with custom software written in Axobasic (Axon
Instruments Inc.).
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Fig. 1.
Nucleotide and predicted amino acid sequence
of CLIC3. A, cDNA and deduced protein sequence of
CLIC3. The putative transmembrane domain (137-155) is
underlined. B, sequence comparison of CLIC3 with
other members of the human chloride intracellular channel family. Amino
acids conserved in all proteins are shaded. C,
Kyte-Doolittle hydrophobicity plots of the CLIC3 protein.
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Fig. 2.
In vivo association of CLIC3 and
ERK7. A, expression of the CLIC3 protein. COS cells
were transfected with either an expression vector for FLAG-CLIC3 or
control vector. CLIC3 protein was detected in cell lysates by
immunoblotting with an anti-FLAG antibody. B,
co-immunoprecipitation of CLIC3 and ERK7 in COS cells. Cells were
transiently transfected with expression vectors for either FLAG-CLIC3
(lane 1), HA-ERK7 (lane 2),
or both FLAG-CLIC3 and HA-ERK7 (lane 3). Total
cell lysates were either directly resolved by SDS-PAGE (lane
1) or first immunoprecipitated with anti-HA antibody and
then resolved by SDS-PAGE (lanes 2 and
3). All samples were then immunoblotted with anti-FLAG
antibody or anti-HA antibody. The upper blot was probed with anti-HA
antibody, which detects HA-ERK7, and the lower blot was probed with
anti-FLAG antibody, which detects FLAG-CLIC3.
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Fig. 3.
Northern blot showing tissue distribution of
CLIC3. Poly(A)+ RNA isolated from various human
tissues was probed with a fragment of the CLIC3 cDNA as described
under "Materials and Methods."
-galactosidase to monitor transfected cells. After fixation,
the cells were immunostained with anti-FLAG and anti-
-galactosidase
antibodies as described under "Materials and Methods." As shown in
Fig. 4, the nuclei in the
CLIC3-transfected cells were intensely stained with the anti-FLAG
antibody, but weak staining was also observed in the cytoplasm. In
contrast, the staining by the anti-
-galactosidase antibody was more
evenly distributed between the nucleus and the cytoplasm. These results indicate that the FLAG-CLIC3 is located predominantly in the nucleus. Cells were also co-transfected with plasmids for both FLAG-CLIC3 and
HA-ERK7 to compare the localization of both proteins directly. As shown
in Fig. 4B, HA-ERK7 and CLIC3 co-localized principally in
the nucleus. However, there is significantly more cytoplasmic expression of CLIC3 than ERK7, suggesting that CLIC3 may also function
independently of ERK7. The nuclear localization of CLIC3 was confirmed
using LTK cells (data not shown).
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Fig. 4.
Cellular localization of CLIC3 and ERK7.
A, CV-1 cells were co-transfected with expression vectors
for -galactosidase and FLAG-tagged CLIC3.
-Galactosidase and
CLIC3 were visualized by immunocytochemistry with
anti-
-galactosidase (left panel) or anti-FLAG
(middle panel) antibodies, respectively. The
phase micrograph is shown in the right panel.
B, CV-1 cells were co-transfected with expression vectors
for FLAG-CLIC3 and HA-ERK7. CLIC3 and ERK7 were visualized by
immunocytochemistry with anti-FLAG (left panel)
or anti-HA (right panel) antibodies,
respectively.
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Fig. 5.
Phosphorylation of GST-Fos but not GST-CLIC3
by ERK7. COS cells were transfected with an expression vector for
either HA-ERK7 or HA-K43R (the kinase-deficient mutant). GST-CLIC3 or
GST-Fos were isolated from bacteria and incubated with HA-ERK7 or
HA-K43R immunoprecipitated from the transfected COS cells. Kinase
buffer with [32P]ATP was added, and the samples were
assayed for kinase activity as described under "Materials and
Methods." Following incubation, the samples were resolved by SDS-PAGE
and analyzed by autoradiography. The positions of the
32P-labeled GST-Fos and GST-CLIC3 bands are
indicated.
100 and +80 mV with a slope of 5 G
(Fig.
6A, traces 1 and 3). A
similar relation was observed when cells were superfused with a saline solution containing a low Cl
concentration
(trace 2). In contrast, cells co-transfected with GFP and CLIC3 had two different behaviors. A minority of the cells behaved exactly like cells transfected with GFP alone; these cells had
an input resistance of 4.3 ± 4.8 G
(range: 0.630-13.6 G
; n = 7) and a linear current-voltage relation, which was
not changed by superfusing with a saline solution containing a low
Cl
concentration. These cells probably express GFP but
not CLIC3 at the plasma membrane. A majority of the cells exhibited an
altered behavior; the input resistance of 646 ± 242 M
(range:
140-1169 M
; n = 8) was lower, and exposure to a
saline solution containing a low Cl
concentration altered
the current-voltage relation (Fig. 6B). Reducing the
extracellular Cl
concentration from 152.5 mM
to 22.5 mM reduced the outward current observed at positive
potentials. These results indicate that CLIC3 mediates chloride ion
transport across the membrane.
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Fig. 6.
Current-voltage plots of the plasma membrane
of LTK cells transfected with CLIC3. Current-voltage plots were
obtained before (trace 1), during
(trace 2), and after (trace
3) superfusion of the cell with a reduced-chloride solution
(see "Materials and Methods"). A, current-voltage plot
from a control LTK cell transfected with an expression vector for GFP.
B, current-voltage plot from a cell transfected with
expression vectors for CLIC3 and GFP.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
. When Cl
is removed from the
extracellular saline solution, fewer anions are available to enter
resulting in a reduction of outward current. Since intracellular
Cl
ions are still present and able to permeate, the
current-voltage relation observed at negative potentials is unchanged.
Each curve crosses the voltage axes at a different point. In this case
the shift in crossing point is +20 mV. Because this is less than the 48 mV predicted by the Nernst equation for a perfect Cl- channel, the
membrane must also be permeable to other ions. For example, gluconate
used to replace Cl
may be able to carry current with
reduced efficiency. The Goldman-Hodgkin-Katz equation predicts a
gluconate:Cl
permeability ratio of 0.173. Based on this
ratio, the pore formed by CLIC3 appears to be relatively large.
channel, it is also
possible that CLIC3 may act as a regulator of a channel. One example of
such a protein is pICln, which was cloned and initially identified as a
swelling-induced chloride channel (33). Subsequently, Krapivinsky and
co-workers (34) showed that anti-pICln monoclonal antibodies can block
the native swelling-induced chloride channel in Xenopus
oocytes, and that pICln was abundant in the cytoplasm and bound to
actin. pICln was also shown to bind to a human homolog of yeast SKb1
protein (IBP72), which is associated with Shk1 kinase (35). Taken
together, these results suggest that pICln may play an indirect role in Cl
channel conductance, possibly via interactions with
the cytoskeletal network. CLIC3, which is also associated with a
kinase, may function in a similar manner.
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ACKNOWLEDGEMENTS |
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We thank J. Booker for assistance with the manuscript, V. Sukhatme for generously providing reagents, and Wen Kuo for helpful discussions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants NS33858 (to M. R. R.), HL56399 (to M. R. R.), and HL03867 (to M. K. A.), and a grant from the Cornelius Crane Trust Fund for Eczema Research (to M. R. R.).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) AF102166.
To whom correspondence should be addressed: Dept. of
Pharmacological and Physiological Sciences, University of Chicago,
Chicago, IL 60637.
The abbreviations used are:
CLIC, chloride
intracellular channel; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; HA, hemagglutinin; , ohm(s); PCR, polymerase chain reaction; GST, glutathione
S-transferase; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis.
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REFERENCES |
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