From the Department of Internal Medicine and
Therapeutics and the ¶ Department of Pathology and
Pathophysiology, Osaka University Graduate School of Medicine, Suita,
Osaka 565-0871, Japan
Received for publication, July 3, 2000, and in revised form, October 10, 2000
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ABSTRACT |
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Connexin-43 is known to interact directly with
ZO-1 in cardiac myocytes, but little is known about the role of ZO-1 in
connexin-43 function. In cardiac myocytes, constitutively active c-Src
inhibited endogenous interaction between connexin-43 and ZO-1 by
binding to connexin-43. In HEK293 cells, by contrast, a connexin-43
mutant lacking the Src phosphorylation site (Tyr265)
interacted with ZO-1 despite cotransfection of a constitutively active
c-Src. Moreover, in vitro binding assays using recombinant proteins synthesized from regions of connexin-43 and ZO-1 showed that
the tyrosine-phosphorylated C terminus of connexin-43 interacts with
the c-Src SH2 domain in parallel with the loss of its interaction with
ZO-1. Cell surface biotinylation revealed that, by phosphorylating Tyr265, constitutively active c-Src reduces total and cell
surface connexin-43 down to the levels seen in cells expressing a
mutant connexin-43 lacking the ZO-1 binding domain. Finally,
electrophysiological analysis showed that both the tyrosine
phosphorylation site and the ZO-1-binding domain of connexin-43 were
involved in the regulation of gap junctional function. We therefore
conclude that c-Src regulates the interaction between connexin-43 and
ZO-1 through tyrosine phosphorylation and through the binding of its
SH2 domain to connexin-43.
Gap junctional communication occurs in almost all tissues and has
been implicated in the control of cell proliferation, embryonic development, and tumor suppression (1, 2). The channels of gap
junctions are permeable to small (<1 kDa) molecules, including such
second messengers as Ca2+, inositol phosphates, and cyclic
nucleotides, and in excitable tissues, such as cardiac myocytes and
neurons, they permit rapid and synchronous propagation of action
potentials. Gap junctions are assembled from connexins, specialized
proteins encoded by a multigene family (1, 2), and the principal member
in cardiac myocytes is connexin-43
(Cx43).1
Communication through gap junctions is sensitive to a variety of
physiological stimuli, including changes in the level of intracellular
Ca2+ (3), pH (4), and transjunctional applied voltage (5). Of particular interest is the finding that several protein kinases can
influence junctional permeability (1). For example, epidermal growth
factor-induced activation of protein kinase C or mitogen-activating protein kinase (MAPK), which correlates with enhanced phosphorylation of Cx43 on serines within a region spanning amino acids 365-382 (6) or
on Ser255, Ser279, and Ser282 (7,
8), abolishes gap junctional coupling, whereas v-Src, which
phosphorylates Cx43 on Tyr265 (9) and possibly
Tyr247 (10), reduces junctional coupling.
The phosphorylation of Tyr265 has been shown to be
important for the binding of v-Src to Cx43, as has the second of two
proline-rich putative SH3 binding domains in the C terminus of Cx43
(11). The phosphorylation of Tyr265 may thus be the key
element in the regulation of gap junctions, although recent studies
indicate that this issue is still controversial. Zhou et
al. (12) found that in oocytes v-Src phosphorylates Cx43 at
Ser255, Ser279, and Ser282 by
activating MAPK and that eliminating the effect of MAPK blocked v-Src-mediated gap junctional closure. On the contrary, Postma et
al. (13) found that c-Src, which is activated via G
protein-coupled receptors in mammalian cells, closes gap junctions by
phosphorylating Tyr265.
The phosphorylation of gap junctional proteins appears to regulate
channel function and the rates of channel assembly and turnover
(14-16). In addition, channel assembly also appears to be regulated by
the interaction of Cx43 and ZO-1 (17, 18). The aim of the present study
was to determine whether c-Src-mediated closure of gap junctions is
related to a change in the interaction of Cx43 and ZO-1. Indirect
evidence supporting that hypothesis includes the observations that gap
junctions in cardiomyopathic heart cells appear disparate rather than
as dense particles as they do in normal cardiac myocytes (19), which
suggests improper incorporation of Cx43 into the gap junctional plaque
in the cardiomyopathic heart, and that cardiomyopathic heart cells
reduce gap junctional communication through c-Src-mediated tyrosine
phosphorylation of Cx43 (20). By using immunoprecipitation-immunoblot
assays, we show here that constitutively active c-Src disrupts the
interaction between Cx43 and ZO-1. Our electrophysiological analysis,
moreover, confirms that the inhibitory effect of c-Src on gap
junctional conductance is due in part to disruption of this
interaction. This molecular mechanism may thus explain how Src kinase
depresses gap junctional communication in the diseased heart.
Reagents--
Mammalian expression vectors, pcDNA3 and
pZeoSV, the expression in eukaryotic cells is under control of the
cytomegalovirus immediate-early promoter, and Zeocin were obtained from
Invitrogen Corp. (San Diego, CA). Expression vector pPET28a,
Escherichia coli (BL21(DE3)pLysS, and a His-bind column were
from Novagen (Madison, WI). G418 was from Sigma. Expression vector
pGEX-3X, Protein A-Sepharose CL-4B, and glutathione-Sepharose 4B were
from Amersham Pharmacia Biotech. Mouse monoclonal anti-connexin-43 IgG
antibody (Ab) was from Transduction Laboratories (Lexington, KY).
Rabbit polyclonal anti-ZO-1 Ab was from Zymed Laboratories Inc. (San Francisco, CA). Rabbit polyclonal anti-c-Src Ab was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). A centriprep 30 microconcentrator was from Amicon (Beverly, MA). An enhanced chemiluminescence assay kit (ECL) and [ Constructs--
cDNAs encoding the full-length of Cx43,
wild-type c-Src (c-Src), and constitutively active c-Src (c-Src(Y527F))
have been described previously (18, 20). Other mutant forms of Cx43 and
c-Src were constructed by PCR-based mutagenesis (18, 21). After
verifying the nucleotide sequences, the respective mutated fragments
were ligated into their original positions in the cDNA. The Cx43
mutants included the following: Cx43(Y265F), containing a
Tyr265 to Phe substitution; Cx43(S255/279/282F), containing
Ser255, Ser279, and Ser282 to Phe
substitutions; Cx43(P277/280/283/284A), containing Pro277,
Pro280, Pro283, and Pro284 to Ala
substitutions; Cx43( Construction and Purification of Fusion Proteins--
cDNA
encoding Cx43-C, the C-terminal domain of Cx43 (amino acid residues
227-382), was synthesized by PCR. The amplified fragment was excised
with EcoRI and XhoI and ligated, in-frame, into a PET28a vector, after which the resultant vector encoding fusion proteins carrying His tag and T7 tag at the N terminus was transfected into E. coli (BL21(DE3)pLysS). Overnight cultures were
diluted 1:10, incubated for 2 h, and then induced for 3 h
with 1 mM
isopropyl-
ZO-P2, the PDZ2 domain of ZO-1 (amino residues 150-312), and
c-Src-SH2, the SH2 domain of c-Src (amino residues 143-245), were
synthesized by PCR. They were excised using EcoRI and
ligated, in-frame, into pGEX-3X vector and encoded a fusion protein
carrying GST at the N terminus. After expression in E. coli
(DH5 Cell Culture and Transfection Procedure--
Rat neonatal
cardiac myocytes were prepared as described previously (22). Briefly,
hearts were isolated from 1-day-old HLA-Wistar rats. The ventricles
were minced, and the cells were dispersed by digestion with 0.1%
collagenase at 37 °C. The dispersed cells were resuspended in high
glucose DMEM supplemented with 10% fetal calf serum and 10 µg/ml
bromodeoxyuridine and pre-plated onto culture dishes for 30 min to
remove fibroblasts. The cells were then plated at an initial density of
105 cells/ml and maintained at 37 °C under an atmosphere
of 5% CO2, 95% air.
HEK293 cells were grown in high glucose DMEM supplemented with 10%
fetal calf serum and penicillin at 37 °C under an atmosphere containing 5% CO2, 95% air.
For stable expression of Cx43 mutants and c-Src constructs, stable cell
lines overexpressing wild-type or mutant Cx43s were established by
transfecting HEK293 cells with the pcDNA3 vector containing Cx43
cDNA and subsequently selected with 800 µg/ml G418 (20, 23).
Clones expressing Cx43 constructs were then transfected with pZeoSV
vectors containing c-Src or c-Src(Y527F) cDNAs, after which the
transfectants were grown for selection in DMEM containing 250 µg/ml
Zeocin and 400 µg/ml G418. Clone selected with Zeocin and G418 were
then subjected to Northern blot and immunoblot analyses.
For transient expression of c-Src constructs in cardiac myocytes, the
cells were cotransfected with vectors Cx43 cDNA and the cDNA
for either c-Src or c-Src(Y527F), respectively, by electroporation using a Gene Pulser Transfection Apparatus (BioRad).
Immunoblot Analysis--
Cardiac myocytes or HEK293 cells were
lysed in lysis buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, 100 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml
leupeptin. The lysates were solubilized in SDS loading buffer, after
which samples were subjected to SDS-PAGE and transferred to
nitrocellulose. The nitrocellulose blots were then incubated with
primary Ab against the target protein, washed three times with TBS
containing 0.1% Tween 20, incubated with peroxidase-labeled affinity
purified Ab against the primary Abs, washed again, and developed using
an enhanced chemiluminescence assay (ECL).
Alkaline Phosphatase Treatment--
By repeated concentration
and re-dilution in a Centriprep 30 microconcentrator, lysates prepared
from cardiac ventricles were equilibrated against phosphatase reaction
buffer (50 mM Tris-HCl (pH 8.0), 10 mM
MgCl2, 150 mM NaCl). The protein was then
incubated for 4 h at 30 °C in the presence of 2 units of
molecular biology grade calf intestinal alkaline phosphatase. Control
reactions were run after the addition of phosphatase inhibitors (100 mM sodium vanadate, 10 mM EDTA and 10 mM PO4) to the reaction mixture.
Immunoprecipitation Analysis--
By repeated concentration and
redilution in a Centriprep 30 microconcentrator, lysates prepared from
cardiac myocytes or HEK293 cells were equilibrated against
immunoprecipitation buffer (50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 150 mM NaCl, 100 mM sodium orthovanadate, and 1% Triton X-100). The lysates
were then incubated with 0.1% albumin-coated protein A-Sepharose for
2 h at 4 °C and clarified by centrifugation for 15 min at
15,000 × g. The supernatants were incubated in a
rotating vessel for 2 h at 4 °C with primary antibodies bound
to protein A-Sepharose. The resultant immunoprecipitates were then
extensively washed with immunoprecipitation buffer, and samples were
subjected to immunoblot analysis using a primary Ab against the target
protein. Primary Ab-antigen complexes were then incubated with
peroxidase-conjugated secondary Ab against mouse or rabbit IgG
antibodies and visualized using ECL.
For the metabolic labeling and immunoprecipitation assay of cell
surface Cx43, cells were first starved in methionine-free minimum
essential medium for 45 min and then labeled with 150 µCi/ml
[35S]methionine (specific activity >1180 Ci/mmol, ICN,
UK) for 2 h at 37 °C. The labeling medium was then removed, and
the cells were washed three times with ice-cold PBS. During the chase
period, normal growth medium supplemented with 5 mM
methionine (Sigma) was added to the cells. Biotinylation of the cell
surface was conducted as described previously (16, 24). Briefly,
metabolically labeled cells were rinsed with ice-cold PBS and incubated
for 30 min at 4 °C with 500 µg/ml NHS-LC-biotin (Pierce) in PBS.
The reaction was terminated by rinsing the cells five times with 15 mM glycine, after which the cells were extracted with lysis
buffer, and each extract was immunoprecipitated with ant-Cx43 Ab. After removing an aliquot for determination of whole-cell levels of Cx43,
avidin-agarose was added to the neutralized supernatant for 1 h to
precipitate the biotinylated proteins (Pierce), which were then
subjected to SDS-PAGE. Once dried, the gels were analyzed by
autoradiography, or they were exposed to a PhotoImaging cassette (model
425E using ImageQuant version 4.2 software; Molecular Dynamics, Inc.)
for several hours, after which the bands read with a PhosphorImager and quantified.
In Vitro Phosphorylation of the C Terminus of Cx43 by Purified
c-Src--
T7-tagged Cx43 protein was purified on His-Bind columns,
concentrated using an Amicon concentrator 10, and re-equilibrated to a
concentration of ~1 µg/µl. Samples containing 1 µg of purified protein were incubated for 15 min at room temperature with 10 units of
purified c-Src (Upstate Biotechnology Inc.) in kinase buffer (20 mM HEPES (pH 7.4), 12 mM MgCl2, 1 mM dithiothreitol, 100 µM
Na2VO4) also containing 1 mM ATP in
a total volume of 40 µl. The reaction was stopped with addition of 10 volumes of binding buffer containing 100 µM
Na2VO4 and then purified using a His-Bind column. The reaction was confirmed in a parallel experiment, in which
purified Cx43 protein was incubated with purified c-Src in kinase
buffer and 10 mCi of [ Affinity Binding Assay--
ZO-P2 proteins bound to
glutathione-Sepharose 4B beads were extensively washed, first with PBS
and then with binding buffer (10 mM Tris (pH 7.5), 150 mM NaCl, 5% bovine serum albumin, proteinase Inhibitor
Complete), after which they were incubated in a rotating vessel with
purified Cx43-C proteins for 2 h at 4 °C. The molar ratio of
Cx43-C to ZO-P2 was 1:10, a condition under which all of the Cx43-C was
bound to ZO-P2. After incubation, the beads were extensively washed
with binding buffer, which was collected as the
glutathione-Sepharose-unbound fraction for later experimentation. Proteins associated with the beads (glutathione Sepharose-bound fraction) were eluted with SDS sample buffer and subjected to SDS-PAGE.
Proteins in the glutathione-Sepharose-unbound fraction were purified on
a His-Bind column, eluted with SDS sample buffer, and subjected to
SDS-PAGE. After transfer to nitrocellulose, immunoblot analysis was
performed with anti-T7 IgG Ab. Primary Ab-antigen complexes were
visualized using ECL and peroxidase-conjugated secondary Ab against
mouse IgG. In addition, T7-fused Cx43-C protein extracted from the
glutathione-Sepharose-unbound fraction, to which ATP and c-Src had been
added, were also immunoprecipitated with anti-T7 Ab; the immobilized
complexes were then incubated with or without purified GST or GST-fused
c-Src-SH2 proteins before immunoblotting.
Electrophysiology--
Gap junctional currents
(Ij) were measured using a Geneclamp 500 amplifier
(Axon Instruments, Inc.) and a double whole-cell patch clamp procedure
(23, 25, 26). HEK293 cells overexpressing mutant Cx43, with or without
c-Src(Y527F), were obtained by freshly dissociating pure populations
from confluent cultures and aliquoting them onto 1-cm diameter glass
coverslips. At 12-36 h after splitting, coverslips with attached cells
were mounted in a chamber, which was placed on the stage of a Nikon Diaphot microscope, and the cells were continuously exchanged with the
bath solution (133 mM NaCl, 3.6 mM KCl, 1.0 mM CaCl2, 0.3 mM MgCl2,
16 mM glucose, 3.0 mM HEPES at pH 7.2) at the
rate of 5 ml/min. For each cell of the pair that was identified by phase-contrast microscopy, access to the cell interior was achieved by
applying gentle suction to the rear of a fire-polished glass pipette
(3-5 M Statistics--
Data are presented as means ± S.D.
Statistical analysis was performed using analysis of variance and
unpaired Student's t tests as appropriate. Values of
p < 0.05 were considered significant.
c-Src Inhibits the Interaction between Cx43 and ZO-1--
The
kinase activity of c-Src is regulated by its phosphorylation state;
activity is initiated by auto-phosphorylation of Tyr419 and
terminated by tyrosine phosphorylation of Tyr527 by Csk
(27). Therefore, to investigate its function, we synthesized a series
of c-Src mutants with differing kinase activities (Fig. 1A). Constitutively active
c-Src was created by substituting a Phe for Tyr527, which
is not inactivated by Csk (c-Src(Y527F)), and nonfunctional c-Srcs were
created by deleting the SH3 domain, which is required for substrate
binding (c-Src(
To determine the extent to which the aforementioned c-Src mutants alter
the levels or the phosphorylation state of Cx43, cardiac myocytes were
transfected with each clone, lysed, and immunoblotted with anti-Cx43
Ab. This analysis showed that the Cx43 band migrated as a doublet with
a major band at 43-45 kDa (Fig. 1B, left panel, P1 and P2, respectively) and a very faint band of
41 kDa (NP). Transfection with c-Src or c-Src(Y527F) did not
alter the mobilities of Cx43. When protein samples were treated with
alkaline phosphatase before immunoblotted with anti-Cx43 Ab, the
43-45-kDa forms collapsed into the 41-kDa form, indicating that the
lower electrophoretic mobilities could be attributed to phosphorylation
of the products. To obtain an indication as to whether
c-Src-phosphorylated Cx43 on a Tyr residue, the lysates were
immunoprecipitated with anti-phospho-Tyr Ab linked to protein
A-Sepharose beads, after which the precipitated proteins were
immunoblotted with anti-Cx43 Ab (Fig. 1B, right panel).
Anti-phospho-Tyr Ab was found to pull down an ~45-kDa form of Cx43
from cardiac myocytes transfected with either c-Src or c-Src(Y527F),
the latter yielding a more intense band indicative of the higher kinase
activity of the constitutively active mutant. In contrast,
anti-phospho-Tyr Ab did not precipitate any form of Cx43 from cells
transfected with the other c-Src mutants, indicating their lack of
kinase activity (data not shown).
We next transfected cardiac myocytes with mutant c-Src and carried out
an immunoprecipitation-immunoblot analysis aimed at detecting any
interaction between Cx43 and ZO-1. The transfectants were solubilized
with 1% Triton X-100, and the soluble cell lysate was
immunoprecipitated with anti-ZO-1 Ab. The precipitates were then
immunoblotted with anti-ZO-1, anti-Cx43, or anti-c-Src Ab (Fig.
1C). All of the samples yielded similar amounts of
endogenous ZO-1 when precipitated with anti-ZO-1 Ab. Cx43 was
precipitated by the anti-ZO-1 Ab in the absence of overexpressed c-Src,
as has been observed previously (18). Overexpression of c-Src or c-Src(Y527F) decreased the amount of precipitated Cx43, although overexpression of c-Src( Tyr265 of Cx43 Is Required for the Inhibitory Effects
of c-Src--
The v-Src regulatory sites in the C terminus of Cx43 are
now known; Tyr265 is a direct target of v-Src (9);
Ser255, Ser279, and Ser282 are
targets of v-Src-activated MAPK (7, 8); and a proline-rich region
(amino acids 274-284) is the site where the SH3 domain of v-Src binds
(11) (Fig. 2A). To determine
which sites are involved in the regulatory effect of c-Src on the
interaction between Cx43 and ZO-1, we synthesized a series of Cx43
mutants as follows: Cx43(Y265F), containing a Tyr265 to Phe
substitution and lacking the Src kinase-targeted tyrosine phosphorylation site; Cx43(S255/279/282F), containing
Ser255, Ser279, and Ser282 to Phe
substitutions and lacking the MAPK-targeted serine phosphorylation sites; Cx43(P277/280/283/284A), containing Pro277,
Pro280, Pro283, and Pro284 to Ala
substitutions and lacking the SH3 domain necessary for binding to Src;
Cx43(
HEK293 cells contain minimal amounts of Cx43 and show minimal
junctional conductance (18, 23). Therefore, to analyze the function of
the Cx43 mutants, we used HEK293 cells to establish stable cell lines
overexpressing respective Cx43 mutants, with or without c-Src(Y527F).
Transfectant cell lysates were immunoprecipitated with anti-Cx43 Ab,
and the precipitates were immunoblotted with anti-ZO-1 or anti-c-Src Ab
(Fig. 2B). All Cx43 mutants except Cx43(
Cx43( Phosphorylation and Binding of the Src-SH2 Domain Prevents Cx43
Binding to ZO-1--
To determine whether the inhibitory effect of
c-Src is direct, we performed an in vitro affinity binding
assay using bacterially expressed Cx43 and ZO-1 domains known to
participate in the interaction between Cx43 and ZO-1 (17, 18). Purified
ZO-P2 protein, coupled to glutathione-Sepharose 4B beads, served as the
substrate for binding purified Cx43-C protein, preincubated with or
without purified c-Src (Fig. 3A,
top panel). In the presence of ATP, Cx43-C was phosphorylated by
the purified c-Src (lane 3). After incubating Cx43-C with
ZO-P2 at a molar ratio of 1:10, the protein associated with ZO-P2 was
collected by extensive washing with binding buffer and resolved by
SDS-PAGE (Fig. 3A, lower left panel). The resultant immunoblots showed that Cx43-C binding to ZO-P2 was dramatically reduced by c-Src-mediated phosphorylation of the former (lane 3).
In a parallel experiment, Cx43-C not associated with ZO-P2
(glutathione-Sepharose-unbound fraction) was collected using a His-Bind
column and was resolved by SDS-PAGE (Fig. 3A, lower right panel). Immunoblots showed that, at a 1:10 molar ratio of Cx43-C to ZO-P2, all of the unphosphorylated Cx43-C was pulled down by ZO-P2
bound to glutathione-Sepharose 4B beads (lanes 1 and
2). In contrast, phosphorylated Cx43-C lost the capacity to
bind ZO-P2 and remained in the wash buffer (lane 3), which
lends further support to the idea that c-Src phosphorylation disrupts a
direct interaction between Cx43 and ZO-1.
To determine whether detachment of Cx43 from ZO-1 coincides with the
association of Cx43 with the SH2 domain of c-Src, we tested the
hypothesis that the association could be accomplished using only free
Src-SH2 domain. Cx43-C-associated proteins in the
glutathione-Sepharose-unbound fraction, also containing ATP and c-Src,
were immunoprecipitated with anti-T7 Ab, and the immobilized complexes
were incubated with or without purified GST or GST-Src-SH2 fusion
proteins before immunoblotting with anti-c-Src or anti-T7 Ab (Fig.
3B). The resultant immunoblots showed that Cx43-C proteins bind to c-Src (lane 1) and that addition of GST alone did
not affect the amount of associated c-Src (lanes 2 and
3). On the other hand, addition of 1 or 2 µg of
GST-Src-SH2 fusion protein completely abolished the association between
Cx43-C and c-Src (lanes 4 and 5), confirming that
phosphorylated Cx43 binds to c-Src via the SH2 domain.
Expression of c-Src(Y527F) Decreases the Stability of Cx43 at the
Cell Surface--
Evidence suggests interaction with ZO-1 is important
for the localization of Cx43 at the intercalated discs of cardiac
myocytes (18). c-Src could thus act by affecting trafficking of Cx43 to
the plasma membrane or by altering connexin-connexin assembly within
the plasma membrane (gap junctional plaque) through regulation of the
Cx43-ZO-1 interaction. We addressed these possibilities using a cell
surface biotinylation technique that enabled us to determine whether
c-Src affects the levels of Cx43 in the plasma membrane.
HEK293 cells expressing mutant Cx43 with or without c-Src(Y527F) were
first starved in methionine-free medium and then labeled with
[35S]methionine for 2 h. Normal growth medium
supplemented with 5 mM methionine was added during the
chase period. At the indicated time points, cell surface proteins were
biotinylated, and cell extracts were prepared, after which protein
equivalent aliquots were immunoprecipitated with anti-Cx43 Ab. After
removal of an aliquot for determination of whole-cell levels of Cx43,
the biotinylated Cx43 fractions were isolated by precipitation with
avidin-agarose and quantified by SDS-PAGE and densitometry (Fig.
4).
Densitometric analysis of several pulse-chase experiments revealed that
one-half of the total Cx43 synthesized during a 2-h pulse was degraded
after 4.1 ± 0.4 h of chase (Figs. 4B and
5B), which is comparable to rates of Cx43 degradation
observed in normal rat kidney, S180, and L929 cells
(t1/2 Cx43 is 2-2.5 h (28)). Cell that had been
biotinylated immediately after the pulse period (0 h) contained
detectable levels of biotinylated Cx43 (Fig. 4A), indicating
that at least a fraction of the Cx43 was situated on the plasma
membrane within 2 h of synthesis. As chase times were prolonged,
levels of cell surface Cx43 declined at a faster rate than total Cx43,
becoming undetectable within 2-4 h. This more rapid disappearance of
biotinylated Cx43 was most likely due to the decreased efficiency with
which Cx43 became biotinylated after incorporation into gap junctional
plaque (16, 28). Indeed, comparison of the whole-cell and cell surface
levels of Cx43 indicated that only several percent of the Cx43
molecules were biotinylated (See Refs. 16 and 28 and this study).
Because of these quantitative limitations of the cell surface
biotinylation assay, we were unable to calculate the
t1/2 of Cx43 transport to the plasma membrane nor
determine the fraction of total Cx43 present in the cell surface at any
given time.
In the presence of c-Src(Y527F), levels of total Cx43 and
Cx43(S255/279/282F) were markedly reduced (t1/2 = 2.3 ± 0.4 h, 1.9 ± 0.3 h, respectively), whereas
less reduction of Cx43(Y265F) or Cx43(P277/280/283/284A) was detected
(t1/2 = 4.3 ± 0.7 h, 4.4 ± 0.5 h, respectively) (Fig. 5). All of the changes in total Cx43 appeared to parallel changes in the cell surface
fraction (biotinylated Cx43) of the same Cx43 mutant, suggesting
reductions in cell surface Cx43 were due to the reductions of total
Cx43, rather than enhanced incorporation of Cx43 into the
biotinylation-inaccessible fraction (gap junctional plaque). Apparently, tyrosine phosphorylation of Tyr265 by c-Src
accelerated the reduction of total Cx43, most likely resulting in the
reduction of cell surface Cx43. Additionally, Cx43( Functional Analysis--
To examine the respective roles of the
phosphorylation sites, the proline-rich region and the ZO-1-binding
site of Cx43 in the function of gap junctions, we measured
Gj between paired HEK293 cells overexpressing
c-Src(Y527F) and mutant Cx43 (Fig. 6). In
addition, we analyzed the involvement of c-Src-mediated tyrosine
phosphorylation using genistein, a Y kinase inhibitor, and
MAPK-mediated serine phosphorylation using PD98059, a MEK inhibitor.
Whereas Gj between control HEK293 cells averaged 6 ± 2 nS, Gj between cells expressing Cx43
alone (control) averaged 53 ± 7 nS, which is comparable to
results from other cell types (29, 30), whereas Gj
between cells coexpressing Cx43 and c-Src(Y527F) were diminished to
10-20% of control. Partial recovery to 30-40 or 70-80% of control
was achieved by application of PD98059 or genistein, respectively.
Gj between cells expressing Cx43 phosphorylation
site mutants showed distinct differences. Coexpression of Cx43(Y265F)
and c-Src(Y527F) reduced Gj to 70-80% of control;
in this case application of PD98059 elicited complete recovery, but
genistein had little effect. Conversely, Gj values
between cells coexpressing Cx43(S255/279/282F) and c-Src(Y527F) were
reduced to 30-40% of control, and while genistein elicited complete
recovery, PD98059 had little effect.
The above results suggest that the inhibitory effect of c-Src(Y527F) on
Gj is mediated by independent mechanisms that
include c-Src-mediated tyrosine phosphorylation and MAPK-mediated serine phosphorylation; apparently 70-80% of c-Src-induced reduction of Gj is due to the former, and 30-40% of the
reduction is due to the latter. When Cx43(P277/280/283/284A) was
coexpressed with c-Src(Y527F), changes in Gj were
similar to those in cells expressing Cx43(Y265F), indicating that the
proline-rich region is required for the c-Src-mediated change in
Gj. Moreover, Gj between cells
expressing Cx43( The results described show that c-Src(Y527F) prevents binding of
Cx43 to the cytoskeletal protein ZO-1 by phosphorylating Tyr265, thereby providing a binding site for the SH2 domain
of c-Src, which directly disrupts the interaction between the C
terminus of Cx43 and ZO-1. Consistent with the finding that the
interaction of Cx43 with ZO-1 is important for the localization of Cx43
(18), c-Src-mediated tyrosine phosphorylation of Cx43 reduced gap
junctional conductance in parallel with decreases in the stability of
Cx43 at the cell surface as well as in the whole cell. We therefore conclude that the function of gap junctions is established by the
interaction of Cx43 with ZO-1 and is down-regulated when that interaction is interfered by c-Src binding to a different site.
The principal mechanisms by which the function of gap junctions is
regulated entail such intramolecular modifications as phosphorylation of connexins by protein kinases, which appear to regulate the gap
junction at several levels, including assembly of the junctions in the
plasma membrane (16, 28, 24) and connexin turnover (31, 32) and by
directly affecting channel gating, revealed as a reduction in single
channel conductance (33, 34). Overexpression of c-Src had a small
effect on Gj, whereas expression of v-Src or
c-Src(Y527F) substantially reduced it (35), with phosphorylation of
Tyr265 mediating the inhibitory effect of v-Src (9). v-Src
associates with Cx43 through the interaction of its SH3 domain with the
proline-rich region of Cx43, which likely brings its catalytic domain
into close proximity with Tyr265 of Cx43 (11).
Phosphorylation of Cx43 at Tyr265 has thus been proposed to
correlate with gap junction closure. However, one recent study using a
Xenopus oocyte expression system showed that neither
tyrosine phosphorylation nor direct interaction of Cx43 with v-Src is
crucial for v-Src-mediated Cx43 channel closure. Instead, it is serine
phosphorylation of Cx43 at positions 255, 279, and 282 that appears to
be essential for gating (12). In its mitogenic pathway, v-Src
associates with and phosphorylates the adaptor protein Shc, which in
turn activates Ras/Raf, eventually activating MAPK. It was therefore
proposed that MAPK acts as a common downstream effector, mediating
uncoupling for both tyrosine kinase growth factor receptors
(e.g. epidermal growth factor and lysophosphatidic acid) and
the v-src oncogene. In contrast to v-Src, G protein-coupled
receptors, including lysophosphatidic acid thrombin and neuropeptides,
use c-Src to decrease transiently the gating of Cx43 independently of
Ca2+, protein kinase C, MAPK, membrane potential, and
Ras/Rho signaling (13). Considered in the context of the finding that
the transforming activity of c-Src(Y527F) is weaker than that of v-Src
in the mammalian cells (36, 37), the discrepancy between the effects of
v-Src and c-Src may be due to differences in their affinity for
cellular substrates. Structural analyses have shown that amino acids 95 and 96 within the SH3 domain are involved in regulating substrate binding (38, 39). This means that v-Src, which contains a Trp95 and an Ile96, has a higher binding
affinity for Cx43 than c-Src, which contains an Arg95 and a
Thr96. Indeed, the binding affinity of c-Src for Cx43 could
be increased by mutating Arg95 and Thr96 to
Trp95 and Ile96, respectively (11).
Another possibility relates to differences in the cellular
compartmentalization of v-Src and c-Src and their proximity to potential regulator and effector molecules. Gap junctional regulation by c-Src should take place in the vicinity of the intercalated discs,
since it requires tyrosine phosphorylation and direct binding to Cx43,
with subsequent detachment of Cx43 from ZO-1 (this study). And in fact
c-Src is enriched at the intercalated discs (40). Gap junctional
regulation by v-Src, by contrast, is probably not limited to any
particular cellular compartment, because it requires neither tyrosine
phosphorylation nor direct binding to Cx43 (12). We therefore speculate
that the difference in cellular compartmentalization of v-Src and c-Src
may determine the involvement of MAPK in the regulation of gap junctions.
Electrophysiological analysis of paired mammalian cells using
phosphorylation site Cx43 mutants and specific inhibitors against each
kinase revealed that c-Src-mediated reduction of Gj is regulated by mechanisms that include c-Src-mediated tyrosine phosphorylation and MAPK-mediated serine phosphorylation, and that the
former had a greater impact on Gj than the latter. Tyrosine phosphorylation of Cx43 decreased Gj by
disrupting the interaction with ZO-1, whereas serine phosphorylation
decreased Gj via an independent mechanism. Since
there have been reports that a truncated Cx43 lacking the C-terminal
phosphorylation sites and the ZO-1-binding site can nonetheless form
channels (41, 42), the interaction with ZO-1 is not essential for the formation of functional channels of Cx43. Rather, we speculated that
this interaction regulates the rates of channel assembly and turnover
and the localization of Cx43. Indeed, the present findings indicated
that Cx43 lacking the ZO-1 binding domain can form channels, whereas
levels of the incorporation into the cell surface and junctional
conductance of this mutant Cx43 are 30-40% of those of wild-type
Cx43. It is therefore likely that interaction with ZO-1 stabilizes Cx43
at the cell surface. ZO-1 was originally identified as an anchoring
protein stabilizing components at cell-cell interfaces, linking to
cadherin in nonepithelial cells or to occludin in epithelial cells
(43). Moreover, the interaction between Cx43 and ZO-1 has been
identified in the cardiac myocytes at the intercalated discs (18),
testis, Rat-1 fibroblasts, and lung epithelial cells (17) and probably
in thyroid epithelial cells (44). Although it remains unclear whether
the localization of Cx43 is determined by binding to ZO-1, localization
of Cx43 along with ZO-1 in the intercalated discs of cardiac myocytes
may serve to synchronize the activity of cells, thus providing an
isochronous front for the wave of excitation.
The PDZ domain of ZO-1 has been proposed to participate in such
protein-protein interactions (45). PDZ domains can be grouped into two
subsets on the basis of their binding properties as follows: PDZ
domains in group 1 select peptides with an E(S/T)X(V/I)
consensus motif, whereas those in group 2 select peptides containing
hydrophobic or aromatic amino acids at positions The properties of gap junctions thus appear to be stabilized by the
interaction between Cx43 and ZO-1, a relationship that is
down-regulated by tyrosine phosphorylation when c-Src is activated under such pathophysiological conditions as end stage cardiomyopathic heart (20). It was recently shown that the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP were
from Amersham Pharmacia Biotech. [35S]Methionine was from
ICN (UK). NHS-LC-biotin and avidin-agarose were from Pierce. A purified
c-Src was from Upstate Biotechnology Inc. (Lake Placid, NY).
303-382), lacking amino acids 303-382; and
Cx43(L380S/I382V), containing Leu380 to Ser and
Ile382 to Val substitutions. c-Src mutants included the
following: c-Src(Y527F), containing a Tyr527 to Phe
substitution; c-Src(
83-142), lacking amino acids 83-142; c-Src(
143-245), lacking amino acids 143-245; and c-Src(K295M), containing a Lys295 to Met substitution. For transfection,
the full-length cDNAs encoding Cx43 and c-Src, respectively, were
ligated into the EcoRI sites of the pcDNA3 and pZeoSV
expression vectors.
-D-thiogalactopyranoside. Expressed proteins
carrying His tag and T7 tag epitopes in the region of the N terminus
were affinity-purified on His-Bind columns according to the
manufacturer's instructions.
), ZO-P2 and c-Src-SH2 proteins were purified by affinity
chromatography using a glutathione-Sepharose 4B column according to the
manufacturer's instructions.
-32P]ATP.
) filled with solution containing 10 nM free
Ca2+ (135 mM CsCl, 0.5 mM
CaCl2, 2 mM MgCl2, 5.5 mM EGTA, 5.0 mM HEPES at pH 7.2) and sealed to
the cell membrane (seal resistance >1 G
). Cells were
voltage-clamped at a holding potential of
40 mV, and voltage pulses
(10 mV, 200-500 ms in duration) were alternately applied to each cell.
Within each cell pair, Ij was measured keeping
constant the membrane potential in one cell and applying the voltage
steps (Vj) to the other cell. Junctional conductances (Gj) were calculated from the equation, Gj = Ij/Vj.
The nonjunctional membrane resistance was usually on the order of
0.3-0.6 G
, which was more than 100-fold greater than the series
resistance (2-5 M
). Consequently, the current flowing through the
junction was approximately equal to the recorded current change
(Ij). Since series resistance of the electrode was
always less than 1% of the parallel sum of the seal resistance (>1
G
) and the nonjunctional membrane resistance (0.3-0.6 G
), we
rarely used series resistance compensation in this study.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
83-142)) by deleting of SH2 domain, which is
required signal transduction through binding to a phosphorylated target
residue (c-Src(
143-245)), or by substituting an Met for Lys295, which is essential for the catalytic activity of
c-Src (c-Src(K295M)).
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Fig. 1.
The effects of mutant c-Src on the Cx43-ZO-1
interaction in the cardiac myocytes. A, schematic
diagram of c-Src showing the domain structure and the regulatory sites;
Lys295 is the ATP-binding site, and Tyr527 is
the Csk phosphorylation (27). c-Src mutants were produced by PCR as
follows: c-Src(Y527F) contains a Tyr527 Phe point
mutation; c-Src(
83-142) is a deleted c-Src(Y527F) lacking the SH3
domain (aa 83-142); c-Src(
143-245) is a deleted c-Src(Y527F)
lacking the SH2 domain (aa 143-245); and c-Src(K295M) contains a
Tyr295
Met point mutation. B, immunoblot
(Blot) analysis for determination of the phosphorylated
forms of Cx43. Lysates were prepared from the cardiac cells in the
absence and presence of c-Src or c-Src(Y527F). Left panel,
lysates were treated with (+) or without (
) alkaline phosphatase
(AP) and then resolved by SDS-PAGE and immunoblotted with
anti-Cx43 Ab. Note that the slower mobility bands of Cx43 were selectively sensitive to alkaline phosphatase.
P (P1 and P2) and NP
indicate phosphorylated and unphosphorylated forms of Cx43,
respectively. Right panel, lysates were immunoprecipitated
(IP) with anti-phospho-Tyr Ab. The immunoprecipitates were
resolved by SDS-PAGE and immunoblotted with anti-Cx43 Ab. Band at 55 kDa (arrow) represents mouse immunoglobulin heavy chains.
Mobility of molecular mass markers is shown on the right. C,
representative results of an immunoprecipitation-immunoblot analysis
for determination of the association among Cx43, ZO-1, and mutant
c-Src. Lysates prepared from cardiac cells expressing with (+) or
without (
) mutant c-Src were immunoprecipitated with anti-ZO-1 Ab,
after which the immunoprecipitates were immunoblotted with anti-ZO-1,
anti-Cx43, or anti-c-Src Ab. Conversely, lysates were
immunoprecipitated with anti-Cx43 Ab, and the immunoprecipitates were
immunoblotted with anti-Cx43, anti-ZO-1, or anti-c-Src Ab. C-Src and
c-Src(Y527F) increased c-Src binding to Cx43 but decreased Cx43 binding
to ZO-1. Other c-Src mutants had little effect. D, summary
of the effects of c-Src mutants on the association between Cx43 and
ZO-1 and on the phosphorylation of Cx43. Each experiment was repeated
at least three times, and each effect was estimated as plus or
minus.
143-245), c-Src(
83-142), or
c-Src(K295M) had no effect. Conversely, when the soluble cell lysates
were immunoprecipitated with anti-Cx43 Ab and the precipitates
immunoblotted with anti-Cx43, anti-ZO-1, or anti-c-Src Ab, c-Src, and
c-Src(Y527F) but not other c-Src mutants reduced the amount of
associated ZO-1 in parallel with their increased binding to Cx43.
c-Src(Y527F) induced a greater reduction in the interaction of Cx43 and
ZO-1 than c-Src and was more extensively bound to Cx43, most likely due
to its increased kinase activity (Fig. 1B). It appears,
therefore, that c-Src disrupts the interaction between Cx43 and ZO-1 by
binding to Cx43. That c-Src(
143-245), c-Src(
83-142), and
c-Src(K295M) lack the ability to phosphorylate Cx43 (data not shown)
suggests the inhibitory effect of c-Src may be due to c-Src-mediated
phosphorylation of Cx43.
303-382), lacking the ZO-1-binding site; and
Cx43(L380S/I382V), containing Leu380 to Ser and
Ile382 to Val substitutions and alteration of the
C-terminal tetrapeptide from DLEI to ESXV, a PDZ domain
consensus motif.
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Fig. 2.
The effects of c-Src(Y527F) on the mutant
Cx43 and ZO-1 interaction in the HEK293 cells. A,
schematic diagram of the C terminus of Cx43 showing the domain
structure and the regulatory sites; Tyr247 and
Tyr265 are the c-Src phosphorylation sites,
Ser255, Ser279, and Ser282 are the
MAPK phosphorylation sites; the proline-rich region (aa 274-284) is
the binding site for the SH3 domain of v-Src, and the C-terminal region
(aa 303-382) is the binding site for the ZO-1. Cx43 mutants were
produced by PCR; Cx43(Y265F) contains a Tyr265 Phe
point mutation; Cx43(S255/279/282F) contains Ser255,
Ser279, and Ser282
Phe mutations;
Cx43(P277/280/283/284A) contains Pro277,
Pro280, Pro283, and Pro284
Ala
mutations; Cx43(
303-382) lacks the ZO-1-binding site (aa 303-382);
Cx43(L380S/I382V) contains Leu380
Ser and
Ile382
Val mutations. Lower panel, schematic
diagrams of mutant Cx43 and summary of the effect of c-Src(Y527F) on
the association between mutant Cx43 and ZO-1. Each experiment was
repeated at least three times, and each effect was estimated as plus or
minus. B, representative results of an immunoprecipitation
(IP)-immunoblot analysis for determination of association
among mutants Cx43, ZO-1, and c-Src. Lysates prepared from cells
expressing mutant Cx43, with (+) or without (
) c-Src(Y527F), were
immunoprecipitated with anti-Cx43 Ab; after that the immunoprecipitates
were immunoblotted with anti-Cx43, anti-ZO-1, or anti-c-Src Ab.
Coexpressed c-Src(Y527F) reduced binding of Cx43, Cx43(S255/279/282F),
and Cx43(L380S/I382V) to ZO-1 but increased their binding to
c-Src.
303-382)
associated with ZO-1 in the absence of c-Src(Y527F). In the presence of
c-Src(Y527F), however, Cx43 and Cx43(S255/279/282F) did not interact
with ZO-1, although Cx43(Y265F) and Cx43(P277/280/283/284A) did. This
result indicates that Tyr265 phosphorylation by c-Src,
which is dependent on the association between the proline-rich region
of Cx43 and the SH3 domain of c-Src, is most likely involved in the
regulation of the interaction of Cx43 with ZO-1 but that
phosphorylation of Ser255, Ser279, and
Ser282 by MAPK is not.
303-382) did not interact with ZO-1 but nonetheless retained
the ability to bind to c-Src(Y527F), indicating the regions that
interact with ZO-1 and c-Src are independent of one another. When the
C-terminal tetrapeptide of Cx43 was exchanged for a PDZ domain
consensus motif, Cx43(L380S/I382V) retained the capacity to bind ZO-1,
which indicates that the Cx43-binding motif for the ZO-1 PDZ2 domain is
not restricted to peptides containing a hydrophobic amino acid at
position 3. Similar results were obtained using anti-ZO-1 Ab for the
immunoprecipitation and anti-Cx43 Ab to probe the immunoblot (data not
shown). The inhibitory effect of c-Src(Y527F) on the interaction
between Cx43 and ZO-1 thus required the phosphorylation of
Tyr265, which likely provides a binding site for the SH2
domain of c-Src.
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Fig. 3.
Phosphorylation and binding with Src-SH2
domain prevent Cx43 from binding to ZO-1. A, top
panel, purified Cx43-C protein, which was preincubated with (+) or
without ( ) purified c-Src, was incubated with ZO-P2 at a molar ratio
of 1:10. Lower panel, the proteins associating with ZO-P2
bound to glutathione-Sepharose 4B beads were collected by extensive
washing with binding buffer and resolved by SDS-PAGE (left
panel). Proteins not associated with ZO-P2 bound to beads were
purified using a His-Bind column and then resolved by SDS-PAGE
(right panel). B, c-Src-associated proteins in
the glutathione-Sepharose-unbound fraction were immunoprecipitated with
anti-T7 Ab, and the immobilized complexes were incubated with or
without purified GST or GST-Src-SH2 fusion proteins before
immunoblotting.
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Fig. 4.
The turnover of total cellular and cell
surface Cx43. A, the autoradiographs representing the
time course of total and cell surface Cx43 in the HEK293 cells in the
absence (left) and presence (right) of
coexpressed c-Src(Y527F). Replicate cell cultures were pulse-labeled
(0-h chase) with [35S]methionine and chased in the
absence of label for the indicated times. At each time point, cell
surface proteins were biotinylated and immunoprecipitated with
anti-Cx43 Ab. One- twentieth of each immunoprecipitate was
removed without the following processes to determine the total cellular
Cx43 (total Cx43). The rest of the immunoprecipitates were
treated with the avidin beads to extract the biotinylated Cx43
(cell surface Cx43). B, plots of the relative
densitometric values (means ± S.D.) for total Cx43 (open
circles) and cell surface Cx43 (closed triangles) in
the absence (left panel) and the presence (right
panel) of coexpressed c-Src(Y527F). At the each time point, the
levels of Cx43 were quantified by PhosphorImager analysis on the
SDS-PAGE gel (normalized to protein level at 0 h). Data from three
independent experiments are shown; especially data of total Cx43 are
shown along with a line derived from a linear fit.
303-382) was
markedly reduced even in the absence of c-Src(Y527F) (t1/2 = 2.8 ± 0.3 h versus
2.7 ± 0.3 h), indicating that interaction with ZO-1 is also
required for stabilization of total Cx43 and that disruption of that
interaction by c-Src may reduce the stability of cell surface Cx43.
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Fig. 5.
The effects of c-Src(Y527F) on the turnover
of total cellular and cell surface Cx43. A, the
autoradiographs representing the total and cell surface Cx43 at 0 h and a 2-h chase in the HEK293 cells with (+) or without ( )
c-Src(Y527F). Note that c-Src(Y527F) markedly reduced both total and
cell surface Cx43 and CX43(S255/279/282F) and that Cx43(
303-382)
exhibited similar reduction in the absence of c-Src(Y527F).
B, the effects of c-Src(Y527F) on the half-life of total
Cx43. Mean normalized densitometric values of the total Cx43 through
the 6-h chase were fit to a single exponential decay so that the
half-life of each Cx43 could be estimated.
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Fig. 6.
Effect of c-Src on gap junctional conductance
(Gj) in cardiac myocytes. Pairs of HEK293
cells overexpressing Cx43 mutants, with (+) or without ( )
c-Src(Y527F), were subjected to whole-cell voltage clamp. Junctional
and nonjunctional conductances were calculated as described under
"Materials and Methods." Data are means ± S.D. obtained from
13 to 16 pairs in each group. *, p < 0.05 versus control.
303-382) were only 30-40% of control, supporting
the notion that the interaction of Cx43 and ZO-1 contributes to gap
junction formation (18). Gj values between cells
expressing Cx43(
303-382) were further reduced to 10% of control by
coexpression with c-Src(Y527F), returning to 30-40% upon application
of PD98059; genistein had little effect. The regulation of
Gj by MAPK-mediated serine phosphorylation does not,
therefore, appear to be affected by the interaction of Cx43 and ZO-1,
whereas regulation of Gj by tyrosine phosphorylation
does. Thus, c-Src-mediated tyrosine phosphorylation apparently
decreases Gj by disrupting the interaction between
Cx43 and ZO-1, whereas serine phosphorylation by c-Src-stimulated MAPK
decreases Gj via an independent mechanism.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1,
2, and
3
(46). Our data indicate that the C-terminal tetrapeptide of Cx43, DLEI, has the capacity to bind to the ZO-1 PDZ2 domain. The Eph-related receptor tyrosine kinase that binds to the PDZ domain of the
Ras-binding protein, AF6, contains a homologous C-terminal core motif,
X(V/I)(E/Q)V (47). Since amino acid mutations at positions
1 and
3 of Cx43, which generates an SXV motif, did not
abolish binding to the ZO-1 PDZ2 domain, it is apparently not
restricted to peptides containing hydrophobic amino acid at position
3. In addition, the interaction of a Cx43 mutant containing the C
terminus SEV motif was inhibited by c-Src(Y527F). Thus, regulation by
c-Src may be due to conformational hindrance; c-Src-mediated tyrosine
phosphorylation and SH2 domain binding may induce a structural change
in the C-terminal region of Cx43, thereby hindering the interaction
between the C-terminal tetrapeptide and the ZO-1 PDZ-2 domain.
2-adrenergic
receptor is sorted to the cell membrane by PDZ domain-mediated protein interactions and that serine phosphorylation by G protein-coupled receptor kinase disrupts this interaction, leading to internalization of
2-adrenergic receptor (48). In that context, the
present findings, together with those of an earlier study showing that in the failing cardiomyopathic heart c-Src tyrosine-phosphorylates Cx43, which decreases gap junctional conductance (20), leads us to
conclude that PDZ domain-containing proteins, including ZO-1, function
as mediators that localize receptors to specific sites, thereby linking
them to defined physiological mechanisms regulating signal transduction.
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FOOTNOTES |
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* This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture of 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.
These authors contributed equally to this work.
§ To whom correspondence should be addressed: Dept. of Internal Medicine and Therapeutics, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-3273; Fax: 81-6-6879-3279; E-mail: toyofuku@mr-path.med.osaka-u.ac.jp.
Published, JBC Papers in Press, October 16, 2000, DOI 10.1074/jbc.M005826200
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ABBREVIATIONS |
---|
The abbreviations used are: Cx43, connexin43; MAPK, mitogen-activating protein kinase; Ab, antibody, ECL, enhanced chemiluminescence assay; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; aa, amino acids; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase.
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---|
1. | Goodenough, D. A., Goliger, J. A., and Paul, D. L. (1996) Annu. Rev. Biochem. 65, 475-502[CrossRef][Medline] [Order article via Infotrieve] |
2. | Kumar, N. M., and Gilula, N. B. (1996) Cell 84, 381-388[Medline] [Order article via Infotrieve] |
3. | Rose, B., Mehta, P. P., and Loewenstein, W. R. (1993) Carcinogenesis 14, 1073-1075[Abstract] |
4. | Spray, D. C. (1994) Biophys. J. 67, 491-492 |
5. | Bennett, M. V., and Verselis, V. K. (1992) Semin. Cell Biol. 3, 29-47[Medline] [Order article via Infotrieve] |
6. | Saez, J. C., Berthoud, V. M., Moreno, A. P., and Spray, D. C. (1993) Adv. Second Messenger Phosphoprotein Res. 27, 163-198[Medline] [Order article via Infotrieve] |
7. | Kanemitsu, M. Y., and Lau, A. F. (1993) Mol. Biol. Cell 4, 837-848[Abstract] |
8. |
Warn-Cramer, B. J.,
Lampe, P. D.,
Kurata, W. E.,
Kanemitsu, M. Y.,
Loo, L. W.,
Eckhart, W.,
and Lau, A. F.
(1996)
J. Biol. Chem.
271,
3779-3786 |
9. | Swenson, K. I., Piwnica Worms, H., McNamee, H., and Paul, D. L. (1990) Cell Regul. 1, 989-1002[Medline] [Order article via Infotrieve] |
10. | Lau, A. F., Kurata, W. E., Kanemitsu, M. Y., Loo, L. W., Warn Cramer, B. J., Eckhart, W., and Lampe, P. D. (1996) J. Bioenerg. Biomembr. 28, 359-368[Medline] [Order article via Infotrieve] |
11. |
Kanemitsu, M. Y.,
Loo, L. W.,
Simon, S.,
Lau, A. F.,
and Eckhart, W.
(1997)
J. Biol. Chem.
272,
22824-22831 |
12. |
Zhou, L.,
Kasperek, E. M.,
and Nicholson, B. J.
(1999)
J. Cell Biol.
144,
1033-1045 |
13. |
Postma, F. R.,
Hengeveld, T.,
Alblas, J.,
Giepmans, B. N.,
Zondag, G. C.,
Jalink, K.,
and Moolenaar, W. H.
(1998)
J. Cell Biol.
140,
1199-1209 |
14. | Brissette, J. L., Kumar, N. M., Gilula, N. B., and Dotto, G. P. (1991) Mol. Cell. Biol. 11, 5364-5371 |
15. | Kwak, B. R., van Veen, T. A., Analbers, L. J., and Jongsma, H. J. (1995) Exp. Cell Res. 220, 456-463[CrossRef][Medline] [Order article via Infotrieve] |
16. | Lampe, P. D. (1994) J. Cell Biol. 127, 1895-1905[Abstract] |
17. | Giepmans, B. N., and Moolenaar, W. H. (1998) Curr. Biol. 8, 931-934[Medline] [Order article via Infotrieve] |
18. |
Toyofuku, T.,
Yabuki, M.,
Otsu, K.,
Kuzuya, T.,
Hori, M.,
and Tada, M.
(1998)
J. Biol. Chem.
273,
12725-12731 |
19. | Luque, E. A., Veenstra, R. D., Beyer, E. C., and Lemanski, L. F. (1994) J. Morphol. 222, 203-213[Medline] [Order article via Infotrieve] |
20. |
Toyofuku, T.,
Yabuki, M.,
Otsu, K.,
Kuzuya, T.,
Tada, M.,
and Hori, M.
(1999)
Circ. Res.
85,
672-681 |
21. | Higuchi, R., Krummel, B., and Saiki, R. K. (1988) Nucleic Acids Res. 16, 7351-7367[Abstract] |
22. | Meidell, R. S., Sen, A., Henderson, S. A., Slahetka, M. F., and Chien, K. R. (1986) Am. J. Physiol. 251, H1076-H1084[Medline] [Order article via Infotrieve] |
23. |
Toyofuku, T.,
Yabuki, M.,
Otsu, K.,
Kuzuya, T.,
Hori, M.,
and Tada, M.
(1998)
J. Biol. Chem.
273,
1519-1528 |
24. | Musil, L. S., and Goodenough, D. A. (1991) J. Cell Biol. 115, 1357-1374[Abstract] |
25. | Neyton, J., and Trautmann, A. (1985) Nature 317, 331-335[Medline] [Order article via Infotrieve] |
26. |
Spray, D. C.,
and Burt, J. M.
(1990)
Am. J. Physiol.
258,
C195-C205 |
27. | Xu, W., Harrison, S. C., and Eck, M. J. (1997) Nature 385, 595-602[CrossRef][Medline] [Order article via Infotrieve] |
28. | Musil, L. S., Cunningham, B. A., Edelman, G. M., and Goodenough, D. A. (1990) J. Cell Biol. 111, 2077-2088[Abstract] |
29. | Elfgang, C., Eckert, R., Lichtenberg Frate, H., Butterweck, A., Traub, O., Klein, R. A., Hulser, D. F., and Willecke, K. (1995) J. Cell Biol. 129, 805-817[Abstract] |
30. | Fishman, G. I., Spray, D. C., and Leinwand, L. A. (1990) J. Cell Biol. 111, 589-598[Abstract] |
31. | Oh, S. Y., Grupen, C. G., and Murray, A. W. (1991) Biochim. Biophys. Acta 1094, 243-245[Medline] [Order article via Infotrieve] |
32. |
Elvira, M.,
Diez, J. A.,
Wang, K. K.,
and Villalobo, A.
(1993)
J. Biol. Chem.
268,
14294-14300 |
33. |
Lampe, P. D.,
TenBroek, E. M.,
Burt, J. M.,
Kurata, W. E.,
Johnson, R. G.,
and Lau, A. F.
(2000)
J. Cell Biol.
149,
1503-1512 |
34. | Moreno, A. P., Saez, J. C., Fishman, G. I., and Spray, D. C. (1994) Circ. Res. 74, 1050-1057[Abstract] |
35. | Azarnia, R., Reddy, S., Kmiecik, T. E., Shalloway, D., and Loewenstein, W. R. (1988) Science 239, 398-401 |
36. | Kmiecik, T. E., and Shalloway, D. (1987) Cell 49, 65-73[Medline] [Order article via Infotrieve] |
37. | Piwnica Worms, H., Saunders, K. B., Roberts, T. M., Smith, A. E., and Cheng, S. H. (1987) Cell 49, 75-82[Medline] [Order article via Infotrieve] |
38. | Kato, J. Y., Takeya, T., Grandori, C., Iba, H., Levy, J. B., and Hanafusa, H. (1986) Mol. Cell. Biol. 6, 4155-4160 |
39. | Potts, W. M., Reynolds, A. B., Lansing, T. J., and Parsons, J. T. (1988) Oncogene Res. 3, 343-355[Medline] [Order article via Infotrieve] |
40. | Tsukita, S., Oishi, K., Akiyama, T., Yamanashi, Y., Yamamoto, T., and Tsukita, S. (1991) J. Cell Biol. 113, 867-879[Abstract] |
41. | Dunham, B., Liu, S., Taffet, S., Trabka Janik, E., Delmar, M., Petryshyn, R., Zheng, S., Perzova, R., and Vallano, M. L. (1992) Circ. Res. 70, 1233-1243[Abstract] |
42. | Fishman, G. I., Moreno, A. P., Spray, D. C., and Leinwand, L. A. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3525-3529[Abstract] |
43. | Itoh, M., Nagafuchi, A., Yonemura, S., Kitani Yasuda, T., Tsukita, S., and Tsukita, S. (1993) J. Cell Biol. 121, 491-502[Abstract] |
44. |
Guerrier, A.,
Fonlupt, P.,
Morand, I.,
Rabilloud, R.,
Audebet, C.,
Krutovskikh, V.,
Gros, D.,
Rousset, B.,
and Munari Silem, Y.
(1995)
J. Cell Sci.
108,
2609-2617 |
45. | Doyle, D. A., Lee, A., Lewis, J., Kim, E., Sheng, M., and MacKinnon, R. (1996) Cell 85, 1067-1076[Medline] [Order article via Infotrieve] |
46. |
Songyang, Z.,
Fanning, A. S.,
Fu, C.,
Xu, J.,
Marfatia, S. M.,
Chishti, A. H.,
Crompton, A.,
Chan, A. C.,
Anderson, J. M.,
and Cantley, L. C.
(1997)
Science
275,
73-77 |
47. |
Hock, B.,
Bohme, B.,
Karn, T.,
Yamamoto, T.,
Kaibuchi, K.,
Holtrich, U.,
Holland, S.,
Pawson, T.,
Rubsamen Waigmann, H.,
and Strebhardt, K.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9779-9784 |
48. | Cao, T. T., Deacon, H. W., Reczek, D., Bretscher, A., and von Zastrow, M. (1999) Nature 401, 286-290[CrossRef][Medline] [Order article via Infotrieve] |