Epidermal Growth Factor Inhibits Gap Junctional Communication and Stimulates Serine-Phosphorylation of Connexin43 in WB Cells by a Protein Kinase C-Independent Mechanism

Epidermal growth factor (EGF) stimulated the phosphorylation of connexin43 (Cx43) in WB cells as evidenced by the formation of multiple irnmunoreactive Cx43 proteins of higher molecular mass which were abolished by treatment with alkaline phosphatase. Phosphorylation of Cx43 occurred within 10 min of EGF stimulation, was sustained for 1 h, and was associated with almost complete inhibition of gap junctional communication in these cells. EGF-induced phosphorylation and communication inhibition were retained in cells pretreated with phorbol 12-myristate 13-acetate (PMA) to deplete protein kinase C. These results show that the EGF inhibition of communication is tightly linked to protein kinase C-independent phosphorylation of Cx43. Further, Cx43 phosphorylated in the presence of EGF did not react with phosphotyrosine antibodies and in ³²P_i incorporation experiments was shown to contain only phosphoserine indicating that the tyrosine kinase activity of the EGF receptor was not directly involved.     

A potential role of connexin 43 in epidermal growth factor-induced proliferation of mouse embryonic stem cells: involvement of Ca2+/PKC, p44/42 and p38 MAPKs pathways

Abstract. Objectives: The gap junction protein, connexin (Cx), plays an important role in maintaining cellular homeostasis and cell proliferation by allowing communication between adjacent cells. Therefore, this study has examined the effect of epidermal growth factor (EGF) on Cx43 and its relationship to proliferation of mouse embryonic stem cells. Materials and methods: Expressions of Cx43, mitogen‐activated protein kinases (MAPKs) and cell cycle regulatory proteins were assessed by Western blot analysis. Cell proliferation was assayed with [³H]thymidine incorporation. Intercellular communication level was measured by a scrape loading/dye transfer method. Results: The results showed that EGF increased the level of Cx43 phosphorylation in a time‐ (≥5 min) and dose‐ (≥10 ng/mL) dependent manner. Indeed, EGF‐induced increase in phospho‐Cx43 level was significantly blocked by either AG 1478 or herbimycin A (tyrosine kinase inhibitors). EGF increased Ca²⁺ influx and protein kinase C (PKC) translocation from the cytosolic compartment to the membrane compartment. Moreover, pre‐treatment with BAPTA‐AM (an intracellular Ca²⁺ chelator), EGTA (an extracellular Ca²⁺ chelator), bisindolylmaleimide I or staurosporine (PKC inhibitors) inhibited the EGF‐induced phosphorylation of Cx43. EGF induced phosphorylation of p38 and p44/42 MAPKs, and this was blocked by SB 203580 (a p38 MAPK inhibitor) and PD 98059 (a p44/42 MAPK inhibitor), respectively. EGF or 18α‐glycyrrhetinic acid (GA; a gap junction inhibitor) increased expression levels of the protooncogenes (c‐fos, c‐jun and c‐myc), cell cycle regulatory proteins [cyclin D1, cyclin E, cyclin‐dependent kinase 2 (CDK2), CDK4 and p‐Rb], [³H]thymidine incorporation and cell number, but decreased expression levels of the p21^WAF1/Cip1 and p27^Kip1, CDK inhibitory proteins. Transfection of Cx43 siRNA also increased the level of [³H]thymidine incorporation and cell number. EGF, 18α‐GA or transfection of Cx43 siRNA increased 2‐DG uptake and GLUT‐1 protein expression. Conclusions: EGF‐induced phosphorylation of Cx43, which was mediated by the Ca²⁺/PKC, p44/42 and p38 MAPKs pathways, partially contributed to regulation of mouse embryonic stem cell proliferation.     

Role of Cx43 Phosphorylation and MAP Kinase Activation in EGF Induced Enhancement of Cell Communication in Human Kidney Epithelial Cells

Epidermal growth factor (EGF) has been found to induce enhanced gap junctional intercellular communication (GJIC) in the human kidney epithelial cell line K7. This is in contrast to what is reported for other cell types, which all show decreased GJIC in response to EGF. In the present study it is shown that 12-O-tetradecanoylphorbol-13-acetate (TPA) and EGF induce similar phosphorylation pattern of the gap junction protein connexin43 (Cx43) in K7 cells, although their effects on GJIC are opposite. Tyrosine phosphorylation of a 42 kD protein was observed to be induced concomitantly with phosphorylation of Cx43. EGF was however found to induce only serine phosphorylation of Cx43, indicating that the tyrosine kinase activity of the EGF receptor was not directly affecting the gap junction protein. The 42 kD protein phosphorylated on tyrosine was identified to be a mitogen activated protein (MAP) kinase. Both EGF and TPA was found to activate MAP kinase in these cells. Phosphorylation of Cx43 and enhancement of GJIC in response to EGF occurred with difference in time course. Phosphorylation of Cx43 was completed within 15 min, while the enhanced GJIC appeared 2-3 h later. It is therefore possible that regulation of synthesis or transport of Cx43 is responsible for the increase in GJIC, rather than direct involvement of Cx43 phosphorylation. This is in support of our previous finding that protein synthesis is necessary for EGF induced upregulation of GJIC in K7 cells.     

Epidermal growth factor stimulates the disruption of gap junctional communication and connexin43 phosphorylation independent of 12-0-tetradecanoylphorbol 13-acetate-sensitive protein kinase C: the possible involvement of mitogen-activated protein kinase.

We previously reported that epidermal growth factor (EGF) induced the disruption of gap junctional communication (gjc) and serine phosphorylation of connexin43 (Cx43) in T51B rat liver epithelial cells. However, the cascade of events linking EGF receptor activation to these particular responses have not been fully characterized. Furthermore, the serine kinase(s) acting directly on Cx43 remain unidentified. In the current study, we demonstrate that downmodulation of 12-0-tetradecanoylphorbol 13-acetate (TPA)-sensitive protein kinase C (PKC) activity does not affect EGF's ability to reduce junctional permeability or phosphorylate Cx43 in T51B cells. EGF in the presence or absence of chronic TPA treatment stimulated marked increases in Cx43 phosphorylation on numerous sites as determined by two-dimensional tryptic phosphopeptide mapping. Computer-assisted sequence analysis of Cx43 identified several protein kinase phosphorylation consensus sites including two sites for mitogen-activated protein (MAP) kinase. EGF stimulated activation of MAP kinase in a time- and dose-dependent manner where the kinetics of kinase activity corroborated its possible involvement in mediating EGF's effects. Moreover, purified MAP kinase directly phosphorylated Cx43 on serine residues in vitro. Two-dimensional tryptic and chymotryptic phosphopeptide mapping demonstrated that the in vitro phosphopeptides represented a specific subset of the in vivo phosphopeptides produced in response to EGF after chronic TPA treatment. Therefore, EGF-induced disruption of gjc and phosphorylation of Cx43 may be mediated in part by MAP kinase in vivo.     

A Mitosis-specific Phosphorylation of the Gap Junction Protein Connexin43 in Human Vascular Cells: Biochemical Characterization and Localization

Western blotting studies revealed that connexin43 (Cx43), one of the major gap junction proteins in human vascular endothelial cells, is posttranslationally modified during mitosis. This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43_m. Cx43_m was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all ³²P_i from Cx43_m by PP2A. Immunofluorescent confocal microscopy of mitotic cells revealed that Cx43 is intracellularly located, while in nonmitotic cells Cx43 is located at regions of cell–cell contact. Dye coupling studies revealed that mitotic endothelial cells were uncoupled from each other and from nonmitotic cells. After cytokinesis, sister cells resumed cell coupling independent of de novo protein synthesis. The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.     

Mitogen-activated protein kinase and phosphorylation of connexin43 are not sufficient for the disruption of gap junctional communication by platelet-derived growth factor and tetradecanoylphorbol acetate

Disruption of gap junctional communication (GJC) by various compounds, including growth factors and tumor promoters, is believed to be modulated by the phosphorylation of a gap junctional protein, connexin43 (Cx43). We have previously demonstrated a platelet-derived growth factor (PDGF)-induced blockade of GJC and phosphorylation of Cx43 in T51B rat liver epithelial cells expressing wild-type PDGF receptor beta (PDGFr beta). Both of these actions of PDGF required participation of protein kinase C (PKC) and mitogen-activated protein kinase (MAPK). Similar requirements of MAPK were suggested in the modulation of GJC by other agents, including epidermal growth factor (EGF) and lysophosphatidic acid (LPA). Since many of these agents activate additional protein kinases, our present study examined whether activation of MAPK was sufficient for Cx43 phosphorylation and GJC blockade. By utilizing a variety of MAPK activators, we now show that activation of MAPK is not always associated with either Cx43 phosphorylation or disruption of GJC, which suggests a requirement for additional factors. Furthermore, pretreatment with hydrogen peroxide (H2O2), a potent MAPK activator but inefficient GJC/Cx43 modulator, abrogated PDGF- or TPA-induced disruption of GJC. While a 5 min H2O2 pretreatment abolished both PDGF- and TPA-induced Cx43 phosphorylation and GJC blockade, a simultaneous H2O2 treatment interfered only with GJC closure but not with the phosphorylation of Cx43 induced by PDGF and TPA. This finding indicates that, in addition to the Cx43 phosphorylation step, inhibition of GJC requires interaction with other components. H2O2-mediated abrogation of PDGF/TPA signaling can be neutralized by the antioxidant N-acetylcysteine (NAC) or by the tyrosine kinase inhibitor genistein. Taken together, our results suggest that disruption of GJC is not solely mediated by either activated MAPK or Cx43 phosphorylation but requires the participation of additional kinases and regulatory components. This complex mode of regulation is perhaps essential for the proposed functional role of GJC.     

Rapid disruption of gap junctional communication and phosphorylation of connexin43 by platelet-derived growth factor in T51B rat liver epithelial cells expressing platelet-derived growth factor receptor

Gap junctional communication (GJC) between contacting cells has been postulated to be involved in the regulation of cell proliferation. This suggestion stems from numerous studies showing modulation of GJC by agents that influence cellular proliferation. Platelet-derived growth factor (PDGF), a strong mitogen, inhibits GJC in many cell types. To understand the molecular nature of the signal transduction pathway responsible for the GJC blockade, T51B rat liver epithelial cells, which lack endogenous PDGF receptor (PDGFr), were infected with a retrovirus containing either wild-type full-length cDNA of human PDGFrβ (Kin⁺) or a mutant PDGFrβ lacking receptor tyrosine kinase activity (Kin⁻). PDGF caused a complete but transient interruption of cell communication in Kin⁺ cells within 15–20 min of addition. This interruption of GJC was not associated with a gross destabilization of gap junction plaques but with the phosphorylation of connexin43 (Cx43), the only known gap junction protein expressed in these cells. These effects were not exhibited in either control T51B cells or in Kin⁻ cells, indicating a requirement of the receptor tyrosine kinase activity. Further examination revealed that the newly phosphorylated Cx43 then undergoes a rapid degradation utilizing the lysosomal pathway resulting in a decreased total Cx43 protein level. The re-establishment of GJC following PDGF treatment was dependent on protein synthesis. This report describes a suitable cell system which is currently being utilized for the characterization of the PDGF signaling pathway responsible for the inhibition of GJC. J. Cell. Physiol. 174:66–77, 1998. © 1998 Wiley-Liss, Inc.     

Cyclic-GMP-Dependent Protein Kinase Inhibits the Ras/Mitogen-Activated Protein Kinase Pathway

Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3′,5′-cyclic monophosphate and 8-bromoguanosine-3′,5′-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Iβ expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser⁴³, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser⁴³ uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser⁴³ was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser⁴³ in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser⁴³ phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by phosphorylating c-Raf kinase on Ser⁴³ and thereby inhibiting its activation and (ii) by inducing MAP kinase phosphatase 1 expression.     

Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication.

Gap junction communication in some cells has been shown to be inhibited by pp60v-src, a protein tyrosine kinase encoded by the viral oncogene v-src. The gap junction protein connexin43 (Cx43) has been shown to be phosphorylated on serine in the absence of pp60v-src and on both serine and tyrosine in cells expressing pp60v-src. However, it is not known if the effect of v-src expression on communication results directly from tyrosine phosphorylation of the Cx43 or indirectly, for example, by activation of other second-messenger systems. In addition, the effect of v-src expression on communication based on other connexins has not been examined. We have used a functional expression system consisting of paired Xenopus oocytes to examine the effect of v-src expression on the regulation of communication by gap junctions comprised of different connexins. Expression of pp60v-src completely blocked the communication induced by Cx43 but had only a modest effect on communication induced by connexin32 (Cx32). Phosphoamino acid analysis showed that pp60v-src induced tyrosine phosphorylation of Cx43, but not Cx32. A mutation replacing tyrosine 265 of Cx43 with phenylalanine abolished both the inhibition of communication and the tyrosine phosphorylation induced by pp60v-src without affecting the ability of this protein to form gap junctions. These data show that the effect of pp60v-src on gap junctional communication is connexin specific and that the inhibition of Cx43-mediated junctional communication by pp60v-src requires tyrosine phosphorylation of Cx43.     

Interplay between PKC and the MAP kinase pathway in Connexin43 phosphorylation and inhibition of gap junction intercellular communication

Gap junction channels are made of a family proteins called connexins. The best-studied type of connexin, Connexin43 (Cx43), is phosphorylated at several sites in its C-terminus. The tumor-promoting phorbol ester TPA strongly inhibits Cx43 gap junction channels. In this study we have investigated mechanisms involved in TPA-induced phosphorylation of Cx43 and inhibition of gap junction channels. The data show that TPA-induced inhibition of gap junction intercellular communication (GJIC) is dependent on both PKC and the MAP kinase pathway. The data suggest that PKC-induced activation of MAP kinase partly involves Src-independent trans-activation of the EGF receptor, and that TPA-induced shift in SDS-PAGE gel mobility of Cx43 is caused by MAP kinase phosphorylation, whereas phosphorylation of S368 by PKC does not alter gel migration of Cx43. We also show that TPA, in addition to phosphorylation of S368, also induces phosphorylation of S255 and S262, in a MAP kinase-dependent manner. The data add to our understanding of the molecular mechanisms involved in the interplay between signaling pathways in regulation of GJIC.     

Regulation of epidermal growth factor-induced connexin 43 gap junction communication by big mitogen-activated protein kinase1/ERK5 but not ERK1/2 kinase activation

The gap junction protein, Cx43, plays a pivotal role in coupling cells electrically and metabolically, and the putative phosphorylation sites that modulate its function are reflected as changes in gap junction communication. Growth factor stimulation has been correlated with a decrease in gap junction communication and a parallel activation of ERK1/2; the inhibition of epidermal growth factor (EGF)-induced Cx43 gap junction uncoupling was observed by using the MEK1/2 inhibitor, PD98059. Because 1) BMK1/ERK5, another MAPK family member also activated by growth factors, possesses a phosphorylation motif similar to ERK1/2, and 2) it has been reported that PD98059 can inhibit not only MEK1/2-ERK1/2 but also MEK5-BMK1 activation, we investigated whether BMK1 can regulate EGF-induced Cx43 gap junction uncoupling and phosphorylation, comparing this to the role of ERK1/2 on Cx43 function and phosphorylation induced by EGF. Selective activation or inactivation of ERK1/2 by using a constitutively active form or a dominant negative form of MEK1 did not regulate Cx43 gap junction coupling. In contrast, we found that BMK1, selectively activated by constitutively active MEK5alpha, induced gap junction uncoupling, and the inhibition of BMK1 activation by transfection of dominant negative BMK1 prevented EGF-induced gap junction uncoupling. Activated BMK1 selectively phosphorylates Cx43 on Ser-255 in vitro and in vivo, but not on S279/S282, which are reported as the consensus phosphorylation sites for MAPK. Furthermore, by co-immunoprecipitation, we found that BMK1 directly associates with Cx43 in vivo. These data indicate that BMK1 is more important than ERK1/2 in EGF-mediated Cx43 gap junction uncoupling by association and Cx43 Ser- 255 phosphorylation.     

Connexin phosphorylation as a regulatory event linked to gap junction channel assembly

Gap junctions, composed of proteins from the connexin family, allow for intercellular communication between cells and are important in development and maintenance of cell homeostasis. Phosphorylation has been implicated in the regulation of gap junctional communication at several stages of the cell cycle and the connexin “lifecycle”, such as trafficking, assembly/disassembly, degradation, as well as in the gating of “hemi” channels or intact gap junction channels. This review focuses on how phosphorylation can regulate the early stages of the connexin life cycle through assembly of functional gap junctional channels. The availability of sequences from the human genome databases has indicated that the number of connexins in the gene family is approximately 20, but we know mostly about how connexin43 (Cx43) is regulated. Recent technologies and investigations of interacting proteins have shown that activation of several kinases including protein kinase A, protein kinase C (PKC), p34^cdc2/cyclin B kinase, casein kinase 1 (CK1), mitogen-activated protein kinase (MAPK) and pp60^src kinase can lead to phosphorylation of the majority of the 21 serine and two of the tyrosine residues in the C-terminal region of Cx43. While many studies have correlated changes in kinase activity with changes in gap junctional communication, further research is needed to directly link specific phosphorylation events with changes in connexin oligomerization and gap junction assembly.     

EGF induces efficient Cx43 gap junction endocytosis in mouse embryonic stem cell colonies via phosphorylation of Ser262, Ser279/282, and Ser368

Gap junctions (GJs) traverse apposing membranes of neighboring cells to mediate intercellular communication by passive diffusion of signaling molecules. We have shown previously that cells endocytose GJs utilizing the clathrin machinery. Endocytosis generates cytoplasmic double-membrane vesicles termed annular gap junctions or connexosomes. However, the signaling pathways and protein modifications that trigger GJ endocytosis are largely unknown. Treating mouse embryonic stem cell colonies – endogenously expressing the GJ protein connexin43 (Cx43) – with epidermal growth factor (EGF) inhibited intercellular communication by 64% and activated both, MAPK and PKC signaling cascades to phosphorylate Cx43 on serines 262, 279/282, and 368. Upon EGF treatment Cx43 phosphorylation transiently increased up to 4-fold and induced efficient (66.4%) GJ endocytosis as evidenced by a 5.9-fold increase in Cx43/clathrin co-precipitation.     

The detergent resistance of Connexin43 is lost upon TPA or EGF treatment and is an early step in gap junction endocytosis

Gap junctions are plasma membrane domains containing channels that directly connect the cytosols of neighbouring cells. Gap junction channels are made of a family of transmembrane proteins called connexins, of which the best studied is Connexin43 (Cx43). MAP kinase-induced phosphorylation of Cx43 has previously been shown to cause inhibition of gap junction channel permeability and increased Cx43 endocytosis. As Cx43 assembles into gap junction plaques, Cx43 acquires detergent resistance. Here we report that the detergent resistance is lost after activation of MAP kinase. Treatment of IAR20 rat liver epithelial cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) or epidermal growth factor (EGF) caused a rapid increase in the solubility of Cx43 in Triton X-100. This process was mediated by MAP kinase and was initiated at the plasma membrane. The data suggest that loss of the detergent resistance of Cx43 is an early step in TPA- and EGF-induced endocytosis of gap junctions.     

A 14‐3‐3 Mode‐1 Binding Motif Initiates Gap Junction Internalization During Acute Cardiac Ischemia

Altered phosphorylation and trafficking of connexin 43 (Cx43) during acute ischemia contributes to arrhythmogenic gap junction remodeling, yet the critical sequence and accessory proteins necessary for Cx43 internalization remain unresolved. 14‐3‐3 proteins can regulate protein trafficking, and a 14‐3‐3 mode‐1 binding motif is activated upon phosphorylation of Ser373 of the Cx43 C‐terminus. We hypothesized that Cx43^Ser373 phosphorylation is important to pathological gap junction remodeling. Immunofluorescence in human heart reveals the enrichment of 14‐3‐3 proteins at intercalated discs, suggesting interaction with gap junctions. Knockdown of 14‐3‐3τ in cell lines increases gap junction plaque size at cell–cell borders. Cx43^S373A mutation prevents Cx43/14‐3‐3 complexing and stabilizes Cx43 at the cell surface, indicating avoidance of degradation. Using Langendorff‐perfused mouse hearts, we detect phosphorylation of newly internalized Cx43 at Ser373 and Ser368 within 30 min of no‐flow ischemia. Phosphorylation of Cx43 at Ser368 by protein kinase C and Ser255 by mitogen‐activated protein kinase has previously been implicated in Cx43 internalization. The Cx43^S373A mutant is resistant to phosphorylation at both these residues and does not undergo ubiquitination, revealing Ser373 phosphorylation as an upstream gatekeeper of a posttranslational modification cascade necessary for Cx43 internalization. Cx43^Ser373 phosphorylation is a potent target for therapeutic interventions to preserve gap junction coupling in the stressed myocardium.      

Doxorubicin induces EGF receptor-dependent downregulation of gap junctional intercellular communication in rat liver epithelial cells

Exposure of rat liver epithelial cells to doxorubicin, an anthraquinone derivative widely employed in cancer chemotherapy, led to a dose-dependent decrease in gap junctional intercellular communication (GJC). Gap junctions are clusters of inter-cellular channels consisting of connexins, the major connexin in the cells used being connexin-43 (Cx43). Doxorubicin-induced loss of GJC was mediated by activation of extracellular signal-regulated kinase (ERK)-1 and ERK-2, as demonstrated using inhibitors of ERK activation. Furthermore, activation of the epidermal growth factor (EGF) receptor by doxorubicin was responsible for ERK activation and the subsequent attenuation of GJC. Inhibition of GJC, however, was not by direct phosphorylation of Cx43 by ERK-1/2, whereas menadione, a 1,4-naphthoquinone derivative that was previously demonstrated to activate the same EGF receptor-dependent pathway as doxorubicin, resulting in downregulation of GJC, caused strong phos-phorylation of Cx43 at serines 279 and 282. Thus, ERK-dependent downregulation of GJC upon exposure to quinones may occur both by direct phosphorylation of Cx43 and in a phosphorylation-independent manner.     

The regulatory role of the C-terminal domain of connexin43

The C-terminal (CT) domain of connexin43 (Cx43) is thought to be important in the control of gap junction function via: a.) CT phosphorylation-dependent control of gap junction assembly and gating, b.) interactions of CT with key regulatory binding partners. To more closely examine CT-dependent regulation, we have expressed a hemagglutinin-Cx43CT (amino acids 235-382) fusion protein in Normal Rat Kidney (NRK) cells under a tetracycline-responsive inducible promoter. Western blot analysis shows that Cx43CT expression is markedly induced by at least 48 h oftreatment with the tetracycline analogue, doxycycline. Furthermore, Cx43CT is modified within the cell, as several treatments/conditions that increase endogenous Cx43 phosphorylation induced a mobility shift in Cx43CT. Treatment with kinase activators, including epidermal growth factor (EGF) and the tumor promoting phorbol ester 12-O-tetradecanylphorbol-13-acetate (TPA), caused a shift in the mobility of the Cx43CT in a manner consistent with the mobility shift observed upon increased phosphorylation of endogenous Cx43. Similarly, Cx43CT in mitotic cells is extensively shifted, consistent with reports which show that Cx43 is phosphorylated to a unique phosphoisoform in mitotic cells. These results indicate that the Cx43CT can interact with at least some of the kinases that phosphorylate endogenous Cx43 in cells and possibly modulate the effects of kinase activation on gap junctional communication.     

v-Src tyrosine phosphorylation of connexin43: regulation of gap junction communication and effects on cell transformation

The oncogenic tyrosine kinase, v-Src, phosphorylates connexin43 (Cx43) on Y247 and Y265 and inhibits Cx43 gap junctional communication (GJC), the process of intercellular exchange of ions and metabolites. To test the role of a negative charge on Cx43 induced by tyrosine phosphorylation, we expressed Cx43 with glutamic acid substitutions at Y247 or Y265. The Cx43Y247E or Cx43Y265E channels were functional in Cx43 knockout fibroblasts, indicating that introducing a negative charge on Cx43 was not likely the mechanism for v-Src disruption of GJC. Cells coexpressing v-Src and the triple serine to alanine mutant, Cx43S255/279/282A, confirmed that mitogen-activated protein (MAP) kinase phosphorylation of Cx43 was not required for v-Src-induced disruption of GJC and that tyrosine phosphorylation was sufficient. In addition, v-Src cells containing v-Src-resistant gap junctions, Cx43Y247/265F, displayed properties of cell migration, adhesion, and proliferation similar to Cx43wt/v-Src cells, suggesting that Cx43 tyrosine phosphorylation and disruption of GJC are not involved in these transformed cell properties.     

Dissection of the molecular basis of pp60(v-src) induced gating of connexin 43 gap junction channels

Suppression of gap-junctional communication by various protein kinases, growth factors, and oncogenes frequently correlates with enhanced mitogenesis. The oncogene v-src appears to cause acute closure of gap junction channels. Tyr265 in the COOH-terminal tail of connexin 43 (Cx43) has been implicated as a potential target of v-src, although v-src action has also been associated with changes in serine phosphorylation. We have investigated the mechanism of this acute regulation through mutagenesis of Cx43 expressed in Xenopus laevis oocyte pairs. Truncations of the COOH-terminal domain led to an almost complete loss of response of Cx43 to v-src, but this was restored by coexpression of the independent COOH-terminal polypeptide. This suggests a ball and chain gating mechanism, similar to the mechanism proposed for pH gating of Cx43, and K+ channel inactivation. Surprisingly, we found that v-src mediated gating of Cx43 did not require the tyrosine site, but did seem to depend on the presence of two potential SH3 binding domains and the mitogen-activated protein (MAP) kinase phosphorylation sites within them. Further point mutagenesis and pharmacological studies in normal rat kidney (NRK) cells implicated MAP kinase in the gating response to v-src, while the stable binding of v-src to Cx43 (in part mediated by SH3 domains) did not correlate with its ability to mediate channel closure. This suggests a common link between closure of gap junctions by v-src and other mitogens, such as EGF and lysophosphatidic acid (LPA).