From the Division of Cellular Biochemistry, The Netherlands Cancer Institute and Centre for Biomedical Genetics, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands
Received for publication, July 5, 2000, and in revised form, December 19, 2000
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ABSTRACT |
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Cell-cell communication via connexin-43
(Cx43)-based gap junctions is transiently inhibited by certain
mitogens, but the underlying regulatory mechanisms are incompletely
understood. Our previous studies have implicated the c-Src tyrosine
kinase in mediating transient closure of Cx43-based gap junctions in
normal fibroblasts. Here we show that activated c-Src
(c-SrcK+) phosphorylates the COOH-terminal tail of
Cx43, both in vitro and in intact cells.
Coimmunoprecipitation experiments reveal that Cx43 associates with
c-SrcK+ and, to a lesser extent, with wild-type c-Src, but
not with kinase-dead c-Src. Mutation of residue Cx43
Tyr265 (Cx43-Y265F mutant) abolishes both tyrosine
phosphorylation of Cx43 and its coprecipitation with c-Src. Expression
of c-SrcK+ in Rat-1 cells disrupts gap junctional
communication. Strikingly, the communication-defective phenotype is
bypassed after coexpression of the Cx43-Y265F mutant or a
COOH-terminally truncated version of Cx43 (Cx43 Gap junctions mediate communication between adjacent cells in
almost all tissues. Gap junctions are composed of arrays of channel-forming integral membrane proteins, termed connexins (for review, see Refs. 1 and 2). The connexin proteins contain four
transmembrane domains with the amino- and COOH-terminal regions located
intracellularly. Connexin-43
(Cx43)1 is the most
widespread and best studied member of the connexin family, with much
attention being focused on its regulation by physiological and
pathophysiological stimuli. Cx43-based gap junctional communication
(GJC) is transiently inhibited by certain growth factors (1, 3-6) and
permanently reduced or disrupted in most cancer cells and in
v-Src-transformed fibroblasts (1, 7-11). The COOH-terminal tail of
Cx43 contains several serine/threonine and tyrosine phosphorylation
sites that are thought to be important for regulating the opening and
closure ("gating") of Cx43-based channels (1), but little is still
known about how phosphorylation of Cx43 may be linked to the
regulation of GJC.
We have reported that Cx43-based GJC in normal fibroblasts is rapidly
inhibited after activation of certain G protein-coupled receptors, such
as those for lysophosphatidic acid (LPA), endothelin, and thrombin (5).
We found that agonist-induced gap junction closure is independent of
classic second messengers, such as calcium and cyclic nucleotides, and
does not require the mitogen-activated protein kinase cascade. Instead,
gap junction closure was prevented by tyrosine kinase inhibitors, by
dominant-negative c-Src, and in c-Src-deficient cells (5). It thus
appears that the c-Src tyrosine kinase is required for disruption of
Cx43-based GJC by these G protein-coupled receptor agonists. Although
agonist-induced tyrosine phosphorylation of Cx43 could not be detected
(5), the possibility remains that active c-Src may directly interact with Cx43 by analogy of what has been reported for oncogenic v-Src, whose effects on Cx43 and Cx43-based GJC have been investigated extensively (1, 7-13). Residue Tyr265 in the COOH-terminal
tail of Cx43 has been implicated as a major target for the v-Src
kinase, but uncertainty exists as to the relevance of
Tyr265 phosphorylation in channel gating (12, 13). In
Xenopus oocyte pairs, v-Src-mediated closure of Cx43-based
gap junctions was shown to be critically dependent on residue
Tyr265, since mutation of Tyr265 in Cx43
prevented gap junction closure (12). In contrast, more recent
experiments using the Xenopus system have suggested that v-Src-induced inhibition of Cx43-based GJC occurs via a pathway that is
independent of the Tyr265 site but seems to require serine
phosphorylation sites for mitogen-activated protein kinase (13). The
reason for the discrepancy between both studies is unclear. While the
action of oncogenic v-Src on Cx43 has been examined in considerable
detail, it remains unknown whether endogenous wild-type c-Src can act
similarly on Cx43. Overexpressed c-Src has long been known to mimic
v-Src in inhibiting GJC in 3T3 cells (14). However, where tested, an
association between wild-type c-Src and Cx43 in vivo could
not be detected (11).
In the present study we have examined the possible interaction between
c-Src and Cx43, both in vitro and in transfected cells. We
find that residue Tyr265 in the COOH-terminal tail of Cx43
is phosphorylated by active c-Src and, furthermore, that Cx43 and
active c-Src can be coprecipitated, whereas kinase-dead c-Src fails to
stably interact with Cx43. Tyrosine phosphorylation and coprecipitation
of Cx43 are abolished after mutation of residue Cx43
Tyr265. The communication-defective phenotype can be
bypassed after introduction of mutant versions of Cx43 that cannot be
tyrosine-phosphorylated on Tyr265. We conclude that
activated c-Src phosphorylates the C-terminal tail of Cx43 on residue
Tyr265, resulting in a stable interaction between both
proteins that contributes to the inhibition of GJC by extracellular agonists.
Cell Culture--
Rat-1 cells, COS-7, A431, and HeLa cells were
cultured in Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum. Prior to experimentation, cells were
serum-starved for 16 h.
Construction of Plasmids--
Chicken c-SrcY527A
(SrcK+), c-SrcK295M (SrcK cDNA Transfection--
Cells were transfected with
pCDNA3 plasmid encoding Myc-tagged Cx43 and/or versions of c-Src
using standard calcium phosphate precipitation. COS cells were
transfected by the standard DEAE-dextran transfection method, using 1 µg of plasmid DNA per 105 cells.
Antibodies--
Mouse anti-Cx43 and anti-phosphotyrosine (PY20)
monoclonal antibodies were from Transduction Laboratories (Lexington,
KY). Mouse anti-Myc was applied as culture supernatant from the 9E10 hybridoma (ATCC). Rabbit anti-human c-Src (SRC2) was from Santa Cruz
Biotechnology (Santa Cruz, CA), and mouse anti v-Src monoclonal antibody was from Calbiochem. Secondary antibodies (rabbit anti-mouse and swine anti-rabbit) conjugated to horseradish peroxidase were obtained from Dako (Glostrup, Denmark), and antibodies conjungated to
Texas Red were from Molecular Probes (Leiden, The Netherlands).
Immunoprecipitation--
Cells were lysed in 1% Nonidet P-40,
0.25% sodium deoxycholate lysis buffer as described previously
(5). Lysates were clarified and precipitated with antibody precoupled
to protein A-Sepharose Cl-4B (Amersham Pharmacia Biotech,
Uppsala, Sweden). Incubation was for 1 h to overnight at 4 °C.
Precipitates were washed, and proteins were eluted in Laemmli sample
buffer (LSB: 50 mM Tris-Cl, pH 6.8, 2% SDS, 10% glycerol,
5% In Vitro Kinase Assay--
Src was expressed in COS-7
cells as described above. Cells were lysed 40 h post-transfection
in Triton X-100 buffer (150 mM NaCl, 50 mM Tris-Cl, pH 7.4, 2 mM EGTA, and 0.1% Triton
X-100). Src immunoprecipitates were washed in in
vitro kinase buffer (IVKB: 1 mM dithiothreitol,
10 mM MgCl2, 50 mM Tris-Cl, pH
7.4). Substrate (10 µg of GST and GST-Cx43CT) and 0.37 Mbq of
[ SDS-PAGE and Immunoblotting--
Samples in LSB were boiled for
5 min, subjected to SDS-PAGE (12.5% gel), and transferred to
nitrocellulose, as described previously (5). The blots were
blocked and subsequently probed with primary and secondary antibodies.
Immunostained proteins were visualized using enhanced chemoluminescence
(Amersham Pharmacia Biotech).
Gap Junctional Communication and Immunofluorescence
Assays--
Scrape-loading of lucifer yellow, microinjections,
electrophysiological measurements, and immunofluorescence assays were
carried out as described previously (5).
The COOH-terminal Tail of Cx43 Is a Substrate for Activated c-Src
in Vitro--
Previous studies have shown that Cx43 is
tyrosine-phoshorylated by oncogenic v-Src, which correlates with
permanent disruption of GJC in v-Src-transformed cells (1, 8-12),
while endogenous c-Src has been implicated in the transient inhibition
of Cx43-based GJC in normal cells (5). We set out to examine whether
c-Src can phosphorylate Cx43 on its COOH-terminal tail in
vitro. To this end, we used the Cx43 tail (residues 227-382; Fig.
1, upper panel) fused to GST
(GST-Cx43CT) and three distinct versions of c-Src: (i) wild-type (wt)
c-Src; (ii) constitutively active c-Src (c-SrcK+), in which the inhibitory
intramolecular interaction between phosphotyrosine 527 and the SH2
domain is disrupted (Y527A mutation); and (iii) kinase-dead c-Src
(c-SrcK Active c-Src Phosphorylates Cx43 on Residue Tyr265 in
Transfected Cells--
Swenson et al. (12) reported that
residue Tyr265 in the Cx43 tail is critical for
v-Src-induced tyrosine phosphorylation of Cx43 and inhibition of GJC in
paired Xenopus oocytes. Coexpression experiments in HEK293
cells have confirmed that oncogenic v-Src can phosphorylate Cx43 on
residue Tyr265 (11). However, since v-Src and c-Src show
differential association to cellular substrates (16, 17), it remains
unclear whether c-Src may act similarly to v-Src in phosphorylating
Cx43. To examine the importance of Cx43 residue Tyr265 in
the action of c-Src, we generated mutant versions of Cx43 in which
tyrosine residues 265 and 267 were substituted by phenylalanine (Cx43-Y265F and Cx43-Y267F, respectively; Fig. 1, upper
panel). COS-7 cells were transfected with Myc-tagged Cx43 (wt,
Y265F, and Y267F) with or without the various versions of c-Src.
Immunoblot analysis of total cell lysates reveals that the three
distinct Cx43 proteins are properly expressed to comparable levels
(Fig. 3A, middle panels). No
tyrosine phosphorylation of any of the Cx43 proteins is detected in
control transfectants, nor in cells expressing kinase-dead c-Src (Fig.
3). When c-Src or c-SrcK+ is cotransfected, however,
phosphotyrosine is detected in wt Cx43 and Cx43-Y267F, but not in the
Cx43-Y265F mutant (Fig. 3A). We also analyzed Cx43
immunoprecipitates for the presence of phosphotyrosine. As shown in
Fig. 3B, wt Cx43 and mutant Cx43Y267F are
tyrosine-phosphorylated in c-Src-overexpressing cells, whereas
Cx43Y265F (precipitated at comparable amounts) is not. From these
results we conclude that active c-Src phosphorylates Cx43 on residue
Tyr265 and that residue Tyr267 is not a Src
phosphorylation site.
We note that, at least in some experiments, a faint phosphotyrosine
signal was still detectable in the Cx43-Y265F mutant (Fig. 3B). This may represent endogenous Cx43 that is
coprecipitated with transfected Myc-tagged Cx43. An alternative or
additional possibility is that c-Src may phosphorylate Cx43 on another
tyrosine residue with very low stoichiometry. The latter possibility
was not investigated in further detail.
Association between Active c-Src and Cx43--
We next examined
whether c-Src can stably interact with Cx43 in intact cells. COS-7
cells were transfected with Myc-tagged versions of Cx43 (wt, Y265F, and
Y267F mutants) together with the various c-Src constructs. We then
assayed for the presence of Cx43 in Src precipitates. In
SrcK+ precipitates, both wt Cx43 and the Cx43-Y267F mutant
are readily detected, whereas the Cx43-Y265F mutant is not (Fig.
4, upper right panel). Fig. 4
further shows that a very weak association is detected between wt Cx43
and wt c-Src and that Cx43 fails to interact with kinase-dead c-Src
(SrcK
Our efforts to detect an interaction between endogenous c-Src and Cx43
in agonist-stimulated Rat-1 cells were not successful. This negative
result is not entirely unexpected, however, given that: (i)
agonist-induced c-Src activation and disruption of GJC are transient
events (5, 18, 19); (ii) c-Src and Cx43 are expressed at relatively low
levels in normal fibroblasts; and (iii) the high stringency lysis
conditions required to solubilize Cx43 from junctional plaques is
likely to disrupt protein-protein interactions, particularly when such
interactions are labile and of transient nature.
Cx43-Y265F Rescues the Communication-defective Phenotype in
SrcK+-expressing Rat-1 Cells--
We next examined the
importance of Cx43 residue Tyr265 in c-Src-mediated closure
of gap junctions in fibroblasts. Rat-1 cells are ideally suited for
these studies, because they express Cx43 as the sole gap junction
protein and Cx43-based communication is tightly regulated (5). Rat-1
cells were transiently transfected with cDNAs encoding
c-SrcK+ together with different versions of Cx43. In all
cases, GFP cDNA was cotransfected to identify successfully
transfected cells. At about 40 h after transfection functional
cell-cell contacts were formed and GFP-expressing cells were
micro-injected with Lucifer Yellow (LY) together with ethidium
bromide (EtBr) as a marker to detect injected cells. Cell-to-cell
diffusion of LY was measured at 3 min after LY/EtBr injection. LY
diffusion from the transfected cell (GFP- and EtBr-positive) to its
direct neighbor indicates the open state of Cx43-based gap junctions.
When two or more adjacent cells showed LY fluorescence, communication
was scored positive. Of note, this assay measures single-cell
"all-or-none" responses, since GJC is subject to regulation only in
the transfected cell (GFP-positive) in a cell-autonomous manner.
Fig. 5A shows representative
results of the LY diffusion experiments, while quantitation of all
experiments is summarized in Fig. 5B. In nontransfected
Rat-1 cells ("control"), 90% of the micro-injected cells show LY
diffusion to their neighbors, indicative of normal gap junctional
communication. When active c-SrcK+ is introduced, however,
communication is strongly inhibited: in about 80% of the transfected
cells gap junctional communication is completely lost (Fig.
5B). Cotransfection of wt Cx43 does not restore normal
communication, nor does expression of Cx43-Y267F, which both serve as
Src substrates (Fig. 5A, middle left). Strikingly, when the
Src phosphorylation site Tyr265 in Cx43 is removed either
by point mutation (Cx43Y265F) or by COOH-terminal tail truncation
(Cx43
Our findings support a model in which G protein-coupled receptor
agonists, such as LPA, endothelin, and thrombin, transiently disrupt
GJC through a pathway that involves c-Src-mediated phoshorylation of
Cx43 residue Tyr265 as a critical step (5). In an attempt
to test this model directly, we stably expressed either wt Cx43 or
mutant Cx43-Y265F in communication-defective A431 and HeLa carcinoma
cells, and subsequently examined GJC in response to LPA and other
growth factors. Unexpectedly, however, while expression of wt Cx43 or
Cx43-Y265F did confer GJC to these cells (as determined by dye
diffusion and electrophysiological measurements), coupling in the
transfectants could not be inhibited by LPA, thrombin, epidermal growth
factor, or phorbol ester.2 It
thus appears that, although transfected Cx43 does restore GJC, its
regulation by extracellular agonists is impaired, as if an essential
signaling component or Cx43-interacting partner is lacking or
nonfunctional in the Cx43 transfectants.
Concluding Remarks--
We have shown that Tyr265 in
Cx43 is a critical site for an interaction between Cx43 and active
c-Src and inhibition of GJC. Our results support a model in which
activated c-Src phosphorylates the Cx43 tail on residue
Tyr265, resulting in a stable interaction between both
proteins, most likely via a Src SH2-domain interaction, which then
leads to inhibition of GJC. A similar model emphasizing the importance
of Cx43 residue Tyr265 has been proposed for oncogenic
v-Src in closing Cx43-based gap junctions (Ref. 10; but see also Ref.
13 for a model in which the Cx43-Tyr265 has no major role).
Yet, it should be noted that there are various important differences
between v-Src and c-Src. Not only is v-Src constitutively active
because it lacks the auto-inhibitory Tyr527 residue, c-Src
and v-Src also show differential association to cellular substrates
(16, 17). Of particular relevance is the finding that the isolated SH3
domain of v-Src binds to proline-rich motifs in Cx43 in
vitro, whereas the c-Src SH3 domain mediates only weak binding to
Cx43 under the same conditions (11). Hence, it will be very difficult
to detect a pre-existing SH3-mediated association between endogenous
c-Src and Cx43 in vivo. Although tyrosine phoshorylation of
Cx43 could not be detected in Rat-1 cells following receptor
stimulation (for likely reasons outlined above), a recent study showed
tyrosine phosphorylation of Cx43 in cardiomyopathic heart tissue (20).
This correlated with increased c-Src activity and decreased GJC (20),
consistent with a model in which active c-Src interacts with Cx43 and
thereby mediates inhibition of GJC.
In conclusion, our results provide further evidence for the
direct involvement of c-Src in inhibiting Cx43-based GJC following stimulation of G protein-coupled receptors (5), and they emphasize the
importance of residue Tyr265 as a c-Src target in this
process. Obviously, a full understanding of how Cx43-based GJC is
regulated by physiological and/or pathophysiological stimuli can only
be obtained if all Cx43-interacting partners are known. In addition to
c-Src, we have identified the ZO-1 protein as an interacting partner of
the Cx43 COOH-terminal tail (a PDZ-domain interaction; Ref. 15). One
challenge for future studies is to elucidate if and how c-Src and ZO-1
may interact to regulate the function of the Cx43 multiprotein complex
and channel gating. Future studies should also elucidate the G
protein-effector pathway that is responsible for
Src-dependent inhibition of Cx43-based GJC in normal cells.
263) that lacks
residue Tyr265. Our results support a model in which
activated c-Src phosphorylates the COOH-terminal tail of Cx43 on
residue Tyr265, resulting in a stable interaction between
both proteins leading to inhibition of gap junctional communication.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
), and
c-Src in pMT2 were kindly provided by S. Courtneidge (Sugen, Inc.,
Redwood City, CA). Myc epitope-tagged full-length Cx43 and Cx43
263
in pCDNA3 (Invitrogen) were constructed as described previously
(15). The Cx43Y265F and Cx43Y267F mutations were introduced by
site-directed mutagenesis using 5' oligonucleotides (5' TGC GGA
TCC CCA AAA TTC GCC TAC TTC AAT GGC T 3' and 5' TGC GGA
TCC CCA AAA TAC GCC TTC TTC AAT GGC TGC TCC T 3', respectively)
and the 3' oligonucleotide (5' GGG GCGGCCGC C AAT CTC CAG
GTC ATC AG 3'). Polymerase chain reaction was performed according to
standard procedures, using rat Cx43 cDNA as template. Polymerase
chain reaction products were isolated from gel and
BamHI/NotI ligated into Cx43
263Myc, in which a
BamHI site was introduced in the nucleotides encoding amino
acids 261 and 262 (silent mutation). Mutations were verified by
sequencing. To construct GST-Cx43CT, the COOH-terminal tail of Cx43 was
isolated from pMD4Cx43CT (15) with SalI/EcoRI and ligated, in frame with glutathione S-transferase (GST) cDNA, into pGEX-1n (XhoI/EcoRI). GST and GSTCxCT proteins
were isolated from DH5
bacteria induced with
isopropyl-
-D-thioglactopyranoside according
standard procedures. GFP in pMT2 was obtained from G. Zondag (The
Netherlands Cancer Institute).
-mercaptoethanol, 0.1% bromphenol blue).
-32P]ATP (Amersham Pharmacia Biotech) were added in
IVKB. Reactions carried out at 30 °C were stopped after 1 h
with LSB. Samples were subjected to SDS-PAGE and analyzed by
autoradiography and immunoblotting.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
, K295M mutation) (Fig. 1, lower
panel). Src proteins were immunoprecipitated from transfected
COS-7 cells and incubated with GST-Cx43CT. [
-32P]ATP
was then added for in vitro kinase assays. As shown in Fig. 2, the Cx43CT fusion protein is
phosphorylated by c-SrcK+ (although radiolabeling is low
relative to the in vitro autophosphorylation of c-Src), but
not by kinase-dead SrcK
, and only very weakly by wt
c-Src. Thus, the COOH-terminal tail of Cx43 can serve as a substrate
for active c-Src. For further analysis we turned to intact cell
systems.
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Fig. 1.
Schematic representation of Cx43 and
c-Src. Upper panel, Cx43 has four transmembrane domains
(TM) and a relatively large COOH-terminal cytoplasmic tail.
Residue 227 is the first amino acid in the GST-Cx43CT fusion protein.
Residue 263 represents the new COOH-terminal amino acid in the
Cx43 263 truncation mutant. Residues Tyr265 and
Tyr267 were mutated to yield point mutants Cx43-Y265F and
Cx43-Y267F, respectively. Lower panel, c-Src with its SH2,
SH3, and kinase domains. The K295M point mutation abolishes ATP binding
and, hence, kinase activity (SrcK
). The Y527A mutation
renders c-Src constitutively active (SrcK+).
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Fig. 2.
In vitro phosphorylation of the
COOH-terminal of Cx43 tail by c-Src. Different versions of c-Src
(c-SrcK+, wt c-Src, and c-SrcK ) were
expressed in COS-7 cells. Src proteins were immunoprecipitated, probed
with anti-Src antibody (lower panel), and used for in
vitro kinase assays (upper panel), as described under
"Experimental Procedures." The Cx43 COOH-terminal tail (amino acids
227-382) fused to GST was used as a substrate (GST-Cx43CT)
(right lane, Ponceau S staining).
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Fig. 3.
Tyrosine 265 of Cx43 is phosphorylated by
c-Src. COS-7 cells were cotransfected with c-Src and Myc-tagged
versions of Cx43 (wt, Cx43Y265F, or Cx43-Y267F). A, cell
lysates were subjected to SDS-PAGE followed by immunoblotting using
antibodies to phosphotyrosine (PY20), Myc tag
(9E10), and Src, as indicated. Overexpressed c-Src or
SrcK+ phosphorylates wt Cx43 and the Cx43-Y267F mutant, but
not Cx43-Y265F (upper lanes). Lower panels show
Cx43 and Src expression controls. B, Cx43 was
immunoprecipitated at equal amounts (second panel, 9E10
blot). Cx43 immunoprecipitates were probed with anti-phosphotyrosine
mAb PY20 (upper panel). When c-Src is overexpressed, wt Cx43
and the Y267F mutant Cx43 are phosphorylated, whereas the Y265F mutant
shows little or no tyrosine phosphorylation. Lower panel
shows Src expression controls.
). These results emphasize the importance of Cx43
residue Tyr265, and they support a model in which
phosphorylation of Cx43-Tyr265 is essential for stable
interaction between Cx43 and active c-Src.
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Fig. 4.
Cx43 residue Tyr265 and c-Src
kinase activity are essential for c-Src-Cx43 association. COS-7
cells were transfected with wt c-Src, SrcK , or
SrcK+ together with Myc-tagged versions of Cx43 (wt, Y265F,
or Y267F). Src was precipitated from cell lysates, subjected to
SDS-PAGE, blotted to nitrocellulose, and probed with antibodies to Myc
tag (9E10), Cx43, and Src, as indicated. It is seen (upper
panels) that wt Cx43 coprecipitates with c-SrcK+ and, to a much
lesser extent, c-Src, but not with c-SrcK
.
Coprecipitation is abolished with mutant Cx43-Y265F, but not with
Cx43-Y267F.
63), gap junctional communication is restored (Fig. 5,
A and B). Immunofluorescence analysis of transfected cells did not reveal gross differences in expression levels
of the Cx43 mutants (Fig. 5C), although we cannot rule out
the formal possibility that activated Src may somehow affect Cx43
localization and/or gap junction assembly. Nevertheless, our results
suggest that c-Src-mediated phosphorylation of Cx43 on residue
Tyr265 is a key event in closure of Cx43-based gap
junctions.
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Fig. 5.
Importance of Cx43 residue Tyr265
in c-Src-induced inhibition of GJC in Rat-1 cells. A,
phase-contrast and fluorescence photomicrographs of Rat-1 cells in
monolayer. SrcK+ and Cx43 (wt or mutants) were transiently
cotransfected as indicated. Successfully transfected cells were
identified by GFP coexpression. GFP-positive cells were micro-injected
with a mixture of LY and EtBr as a marker. Cell-to-cell diffusion of LY
was measured at 3 min after micro-injection. It is seen that
SrcK+ expression blocks GJC. Strikingly, communication is
restored after coexpression of either COOH-terminally truncated
Cx43- 263 or Cx43-Y265F, whereas expression of wt Cx43 or Cx43-Y267F
fails to bypass SrcK+ action. B, quantitation of
the LY micro-injection experiments illustrated in A. The
degree of intercellular LY diffusion was determined at 3 min after dye
injection into GFP-positive cells (as illustrated in A). GJC
was scored as either "positive" or "negative." In control
cells, 90% of gap junctions are open (left bar).
SrcK+ expression largely blocks GJC. This effect is rescued
by cotransfection of Cx43-Y265F or Cx43-
263, but not by wt Cx43 or
Cx43-Y267F. That rescue is not 100% complete may be due to less than
100% triple-transfection efficiency per cell. C, expression
and localization of Cx43 in SrcK+-transfected Rat-1 cells.
Rat-1 cells were transiently transfected with the indicated Cx43 and
SrcK+ constructs, using the same procedures as in
A. GFP was cotransfected to identify transfected cells.
Cells were stained for Cx43-Myc, using 9E10 anti-Myc and Texas
Red-conjugated goat-anti-mouse antibodies. It is seen that transfected
Cx43-Myc is located both at the cell periphery and in intracellular
vesicles. Bar: 20 µm.
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ACKNOWLEDGEMENTS |
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We thank Sara Courtneidge and Gerben Zondag for plasmids and Ingrid Verlaan for assistance with the biochemical assays.
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FOOTNOTES |
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* This work was supported by the Dutch Cancer Society.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 31-20-512-1974;
Fax: 31-20-512-1989; E-mail: wmoolen@nki.nl.
Published, JBC Papers in Press, December 20, 2000, DOI 10.1074/jbc.M005847200
2 B. Giepmans, T. Hengeveld, F. Postma, and W. Moolenaar, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: Cx43, connexin-43; EtBr, ethidium bromide; GFP, green fluorescent protein; GJC, gap junctional communication; GST, glutathione S-transferase; LPA, lysophosphatidic acid; LY, Lucifer Yellow; PAGE, polyacrylamide gel electrophoresis; wt, wild-type.
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REFERENCES |
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