c-Src Regulates the Interaction between Connexin-43 and ZO-1 in Cardiac Myocytes*

Toshihiko ToyofukuDagger §||, Yoshiki Akamatsu||, Hong Zhang, Tsunehiko Kuzuya, Michihiko Tada, and Masatsugu HoriDagger

From the Dagger  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



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

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 [gamma -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).

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(Delta 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(Delta 83-142), lacking amino acids 83-142; c-Src(Delta 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.

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-beta -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, 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 (DH5alpha ), ZO-P2 and c-Src-SH2 proteins were purified by affinity chromatography using a glutathione-Sepharose 4B column according to the manufacturer's instructions.

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 [gamma -32P]ATP.

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 MOmega ) 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 GOmega ). 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 GOmega , which was more than 100-fold greater than the series resistance (2-5 MOmega ). 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 GOmega ) and the nonjunctional membrane resistance (0.3-0.6 GOmega ), we rarely used series resistance compensation in this study.

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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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(Delta 83-142)) by deleting of SH2 domain, which is required signal transduction through binding to a phosphorylated target residue (c-Src(Delta 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 right-arrow Phe point mutation; c-Src(Delta 83-142) is a deleted c-Src(Y527F) lacking the SH3 domain (aa 83-142); c-Src(Delta 143-245) is a deleted c-Src(Y527F) lacking the SH2 domain (aa 143-245); and c-Src(K295M) contains a Tyr295 right-arrow 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.

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(Delta 143-245), c-Src(Delta 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(Delta 143-245), c-Src(Delta 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.

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(Delta 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 right-arrow Phe point mutation; Cx43(S255/279/282F) contains Ser255, Ser279, and Ser282 right-arrow Phe mutations; Cx43(P277/280/283/284A) contains Pro277, Pro280, Pro283, and Pro284 right-arrow Ala mutations; Cx43(Delta 303-382) lacks the ZO-1-binding site (aa 303-382); Cx43(L380S/I382V) contains Leu380 right-arrow Ser and Ile382 right-arrow 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.

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(Delta 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.

Cx43(Delta 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.

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).



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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.

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).



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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.

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(Delta 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(Delta 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.

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.



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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.

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(Delta 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(Delta 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

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 -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.

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 beta 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 beta 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.


    FOOTNOTES

* 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


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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