PKC-dependent phosphorylation may regulate the ability of connexin43 to inhibit DNA synthesis

Phosphorylation affects several biological functions of connexin43 (Cx43), although its role on Cx43-mediated inhibition of DNA synthesis is not known. Previous studies showed increased Cx43 phosphorylation on serine in response to growth factor stimulation of cardiomyocytes, mediated by protein kinase C-epsilon (PKCepsilon). Here we report that activation of PKCepsilon is also necessary for stimulation of cardiomyocyte DNA synthesis and mitosis. We have investigated the participation of specific serine residues that are putative PKC targets in producing phosphorylated Cx43 species and also in regulating DNA synthesis in cardiomyocytes. Interference with the PKC signaling system and/or the phosphorylation of specific amino-acids of Cx43 may allow regulation of the mitogenic response.     

PKC-Dependent Phosphorylation May Regulate the Ability of Connexin43 to Inhibit DNA Synthesis

Phosphorylation affects several biological functions of connexin43 (Cx43), although its role on Cx43-mediated inhibition of DNA synthesis is not known. Previous studies showed increased Cx43 phosphorylation on serine in response to growth factor stimulation of cardiomyocytes, mediated by protein kinase C-epsilon (PKCε). Here we report that activation of PKCε is also necessary for stimulation of cardiomyocyte DNA synthesis and mitosis. We have investigated the participation of specific serine residues that are putative PKC targets in producing phosphorylated Cx43 species and also in regulating DNA synthesis in cardiomyocytes. Interference with the PKC signaling system and/or the phosphorylation of specific amino-acids of Cx43 may allow regulation of the mitogenic response.     

Regulation of Connexin-43-Mediated Growth Inhibition by a Phosphorylatable Amino-Acid is Independent of Gap Junction-Forming Ability

The ability of the gap junction phosphoprotein connexin-43 (Cx43) to inhibit DNA synthesis in primary cardiomyocytes is regulated by serine (S) 262, a protein kinase C phosphorylation site that also affects metabolic coupling. We have now examined if the S262-regulated growth suppression is operating in transformed cells and if so whether it depends on gap junction channel forming ability. Serine 262 became phosphorylated in response to protein kinase C stimulation in HEK293 cells transiently expressing either Cx43 or the non-channel-forming carboxy-terminal tail of Cx43 (Cx43CT). Expression of either wild type Cx43 or Cx43CT inhibited DNA synthesis, as did their mutated versions simulating lack of phosphorylation by carrying an S262-to-alanine substitution. The ability to inhibit DNA synthesis was eliminated when expressing mutated versions of either Cx43 or Cx43CT simulating constitutive phosphorylation by carrying an S262-to-aspartate substitution. We conclude that S262 phosphorylation cancels growth inhibition by Cx43 independently of channel-forming ability.     

Connexin 43 in LA-25 cells with active v-src is phosphorylated on Y247, Y265, S262, S279/282, and S368 via multiple signaling pathways

Modulation of gap junction structures and gap junctional communication is important in maintaining tissue homeostasis and can be controlled via phosphorylation of connexin 43 (Cx43) through several different signaling pathways. Transformation of cells by v-src has been shown to down-regulate gap junction communication coincident with an increase in tyrosine phosphorylation on Cx43. Activation of mitogen-activated protein kinase (MAPK) and protein kinase C (PKC) also lead to down-regulation via phosphorylation on specific serine residues. Using phosphospecific anti-Cx43 antibodies generated by the authors' laboratory to specific tyrosines (src substrates) and serine residues (MAPK and PKC substrates) to probe LA-25 cells (which express temperature-sensitive v-src), the authors show that distinct tyrosine and serines residues are phosphorylated in response to v-src activity. They show that tyrosine phosphorylation appears to occur predominantly in gap junction plaques when src is active. In addition, src activation led to increased phosphorylation of apparent MAPK and PKC sites in Cx43. These results indicate all three signaling pathways could contribute to gap junction down-regulation during src transformation in LA-25 cells.     

Effects of 18-glycyrrhetinic acid on serine 368 phosphorylation of connexin43 in rat neonatal cardiomyocytes

18Beta-glycyrrhetinic acid (18beta-GA) regulates serine/threonine dephosphorylation of connexin43 (Cx43). Phospho-specific antibodies were used here to determine the effect of 18beta-GA on serine 368-phosphorylated Cx43 (pSer368Cx43) in cultured rat neonatal cardiomyocytes by immunofluorescence microscopy and immunoblot analyses. 18beta-GA caused a time-dependent increase in pSer368Cx43 levels and induced gap junction disassembly, shown by a change in pSer368Cx43 immunostaining from large aggregates to dispersed punctates at cell-cell contact areas. 18beta-GA also induced a time-dependent increase in the levels of serine 729-phosphorylated PKCepsilon, the active form of PKCepsilon. The 18beta-GA-induced increase in pSer368Cx43 levels and changes in pSer368Cx43 staining pattern were abolished by the PKC inhibitor, chelerythrine. Furthermore, 18beta-GA increased the co-immunoprecipitation of Cx43 with PKCepsilon. However, the 18beta-GA-induced increase in pSer368Cx43 levels and increased association of Cx43 with PKCepsilon were inhibited by co-treatment with the protein phosphatase type 1 and type 2A inhibitor, calyculin A. We conclude that 18beta-GA induces Ser368 phosphorylation of Cx43 via PKCepsilon.     

Protein Kinase C-Epsilon Mediates Phorbol Ester-Induced Phosphorylation of Connexin-43

We have used adenoviral vectors to express dominant negative variants of protein kinase C epsilon (PKC?) or mitogen kinase kinase 1 (MKK1) to investigate their involvement in phorbol ester-induced connexin-43 (Cx43) phosphorylation in cardiomyocytes. Stimulation of cardiomyocytes with phorbol 12-myristate 13-acetate (PMA) increased the fraction of the slower migrating (≥45 kDa) and more extensively phosphorylated Cx43 species. Expression of dominant negative MKKI did not prevent the effect of PMA on Cx43 phosphorylation. Selective inhibition of PKC? significantly decreased baseline levels of Cx43 phosphorylation and the PMA-induced accumulation of ≥45 kDa Cx43. Thus, production of the more extensively phosphorylated species of Cx43 in cardiomyocytes by PMA requires activation of PKC?.     

Enhanced PKCε mediated phosphorylation of connexin43 at serine 368 by a carboxyl-terminal mimetic peptide is dependent on injury

The gap junction (GJ) protein connexin (Cx43) is important for organized action potential propagation between mammalian cardiomyocytes. Disruption of the highly ordered distribution of Cx43 GJs is characteristic of cardiac tissue after ischemic injury. We recently demonstrated that epicardial administration of a peptide mimetic of the Cx43 carboxyl-terminus reduced pathologic remodeling of Cx43 GJs and protected against induced arrhythmias following ventricular injury. Treatment of injuries with the carboxyl-terminal peptide was associated with an increase in phosphorylation at serine 368 of the Cx43 carboxyl-terminus. Here, we report that Cx43 peptide treatment of uninjured hearts does not prompt a similar increase in phosphorylation. Moreover, we show that peptide treatment of undisturbed cultured HeLa cells expressing a Cx43 construct also exhibit no changes in Cx43 phosphorylation at serine 368. However, in parallel with the results in vivo, a trend of increasing phosphorylation at serine 368 was observed in Cx43-expressing HeLa cells following scratch wounding of cultured monolayers. These results suggest that peptide-enhanced phosphorylation of the Cx43 carboxyl-terminus is dependent on injury-mediated cellular responses.     

Enhanced PKCε mediated phosphorylation of connexin43 at serine 368 by a carboxyl-terminal mimetic peptide is dependent on injury

The gap junction (GJ) protein connexin (Cx43) is important for organized action potential propagation between mammalian cardiomyocytes. Disruption of the highly ordered distribution of Cx43 GJs is characteristic of cardiac tissue after ischemic injury. We recently demonstrated that epicardial administration of a peptide mimetic of the Cx43 carboxyl-terminus reduced pathologic remodeling of Cx43 GJs and protected against induced arrhythmias following ventricular injury. Treatment of injuries with the carboxyl-terminal peptide was associated with an increase in phosphorylation at serine 368 of the Cx43 carboxyl-terminus. Here, we report that Cx43 peptide treatment of uninjured hearts does not prompt a similar increase in phosphorylation. Moreover, we show that peptide treatment of undisturbed cultured HeLa cells expressing a Cx43 construct also exhibit no changes in Cx43 phosphorylation at serine 368. However, in parallel with the results in vivo, a trend of increasing phosphorylation at serine 368 was observed in Cx43-expressing HeLa cells following scratch wounding of cultured monolayers. These results suggest that peptide-enhanced phosphorylation of the Cx43 carboxyl-terminus is dependent on injury-mediated cellular responses.     

Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication

Phorbol esters (e.g., TPA) activate protein kinase C (PKC), increase connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. We asked whether PKC directly phosphorylates and regulates Cx43. Rat epithelial T51B cells metabolically labeled with (32)P(i) yielded two-dimensional phosphotryptic maps of Cx43 with several phosphopeptides that increased in intensity upon TPA treatment. One of these peptides comigrated with the major phosphopeptide observed after PKC phosphorylation of immunoaffinity-purified Cx43. Purification of this comigrating peptide and subsequent sequencing indicated that the phosphorylated serine was residue 368. To pursue the functional importance of phosphorylation at this site, fibroblasts from Cx43(-/-) mice were transfected with either wild-type (Cx43wt) or mutant Cx43 (Cx43-S368A). Intercellular dye transfer studies revealed different responses to TPA and were followed by single channel analyses. TPA stimulation of T51B cells or Cx43wt-transfected fibroblasts caused a large increase in the relative frequency of approximately 50-pS channel events and a concomitant loss of approximately 100-pS channel events. This change to approximately 50-pS events was absent when cells transfected with Cx43-S368A were treated with TPA. These data strongly suggest that PKC directly phosphorylates Cx43 on S368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication.     

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.     

18beta-glycyrrhetinic acid promotes src interaction with connexin43 in rat cardiomyocytes

The mechanism by which 18beta-glycyrrhetinic acid regulates gap junction intercellular communication (GJIC) remains poorly understood. In this study, treatment of cultured rat neonatal cardiomyocytes with 18beta-glycyrrhetinic acid resulted in dose-dependent inhibition of GJIC as assessed by fluorescent dye transfer analysis. 18beta-Glycyrrhetinic acid induced time-dependent serine/threonine dephosphorylation and redistribution of connexin43 (Cx43) in cardiomyocytes and the induced Cx43 dephosphorylation was prevented by the protein phosphatase inhibitor, calyculin A. However, functional analyses showed that the inhibitory effect of 18beta-glycyrrhetinic acid on dye spreading among cardiomyocytes was not blocked by calyculin A, but was blocked by the Src-selective tyrosine kinase inhibitor, PP2. 18beta-Glycyrrhetinic acid also induced an increase in the levels of phosphorylated Src, and this effect was prevented by PP2. Immunoprecipitation using anti-Cx43 and anti-p-Src antibodies showed that 18beta-glycyrrhetinic acid increased the association between p-Src and Cx43 and induced tyrosine phosphorylation of Cx43. We conclude that the inhibitory effect of 18beta-glycyrrhetinic acid on GJIC in cardiomyocytes involves Src-mediated tyrosine phosphorylation of Cx43.     

Administration of FGF-2 to the Heart Stimulates Connexin-43 Phosphorylation at Protein Kinase C Target Sites

Fibroblast growth factor-2 (FGF-2) confers acute, preconditioning-like cardiac resistance to ischemic injury in a protein kinase C (PKC)-dependent fashion. One of the downstream targets of PKC is the gap junction protein connexin-43 (Cx43). We thus examined the effects of FGF-2 on Cx43 phosphorylation at specific PKC sites in the adult heart. Rat hearts perfused ex vivo for 20 min with an FGF-2-containing solution displayed increased levels of phosphorylated 44-45 kDa Cx43, assessed by western blotting. In addition, FGF-2 significantly upregulated phosphorylation of the PKC target serines 262 and 368 on Cx43 at intercalated disks, assessed using phosphospecific antibodies in immunolocalization and western blotting assays. Our data show that FGF-2, administered by perfusion, can alter the phosphorylation status of Cx43 at cardiomyocyte intercalated disks, and suggest a link between phosphorylation of Cx43 at specific PKC sites and FGF-2 cardioprotection.     

Administration of FGF-2 to the heart stimulates connexin-43 phosphorylation at protein kinase C target sites

Fibroblast growth factor-2 (FGF-2) confers acute, preconditioning-like cardiac resistance to ischemic injury in a protein kinase C (PKC)-dependent fashion. One of the downstream targets of PKC is the gap junction protein connexin-43 (Cx43). We thus examined the effects of FGF-2 on Cx43 phosphorylation at specific PKC sites in the adult heart. Rat hearts perfused ex vivo for 20 min with an FGF-2-containing solution displayed increased levels of phosphorylated 44-45 kDa Cx43, assessed by western blotting. In addition, FGF-2 significantly upregulated phosphorylation of the PKC target serines 262 and 368 on Cx43 at intercalated disks, assessed using phosphospecific antibodies in immunolocalization and western blotting assays. Our data show that FGF-2, administered by perfusion, can alter the phosphorylation status of Cx43 at cardiomyocyte intercalated disks, and suggest a link between phosphorylation of Cx43 at specific PKC sites and FGF-2 cardioprotection.     

Protein Kinase Cδ-mediated Phosphorylation of Connexin43 Gap Junction Channels Causes Movement within Gap Junctions followed by Vesicle Internalization and Protein Degradation

Phosphorylation of gap junction proteins, connexins, plays a role in global signaling events involving kinases. Connexin43 (Cx43), a ubiquitous and important connexin, has several phosphorylation sites for specific kinases. We appended an imaging reporter tag for the activity of the δ isoform of protein kinase C (PKCδ) to the carboxyl terminus of Cx43. The FRET signal of this reporter is inversely related to the phosphorylation of serine 368 of Cx43. By activating PKC with the phorbol ester phorbol 12,13-dibutyrate (PDBu) or a natural stimulant, UTP, time lapse live cell imaging movies indicated phosphorylated Ser-368 Cx43 separated into discrete domains within gap junctions and was internalized in small vesicles, after which it was degraded by lysosomes and proteasomes. Mutation of Ser-368 to an Ala eliminated the response to PDBu and changes in phosphorylation of the reporter. A phosphatase inhibitor, calyculin A, does not change this pattern, indicating PKC phosphorylation causes degradation of Cx43 without dephosphorylation, which is in accordance with current hypotheses that cells control their intercellular communication by a fast and constant turnover of connexins, using phosphorylation as part of this mechanism.     

Protein kinase C (PKC) mediated interaction between conexin43 (Cx43) and K+(ATP) channel subunit (Kir6.1) in cardiomyocyte mitochondria: Implications in cytoprotection against hypoxia induced cell apoptosis

Purpose of researchWe have recently shown that adenosine-triphosphate-sensitive potassium [K⁺(ATP)] channel protein subunit, Kir6.1 is a phosphospecific interaction partner of the gap-junction protein connexin43 (Cx43). Since, both Cx43 and K⁺(ATP) are known to be involved in cell survival during hypoxia, we addressed the question, whether the interaction between Cx43 and K⁺(ATP) has a role in protecting cell against hypoxia-induced cell death.Principle resultsWe report here that the Kir6.1 protein interacts, in a phosphospecific manner with Cx43 in the mitochondria of cardiomyocytic cell line H9C2. The hypoxia for 12-h resulted in the appreciable increase in the phosphorylation at the serine 262 (S262) of the Cx43 with the concomitant increase in the Cx43 and Kir6.1 interaction. Moreover, the increased interaction was mediated by a signaling pathway involving PKC and more specifically by PKC epsilon. Functional implications of the association between the Cx43 and Kir6.1 were found to prevent mitochondria mediated hypoxia induced cell apoptosis.Major conclusionsOur results demonstrate that PKC epsilon regulates the interaction between Cx43 and Kir6.1 in the cardiomyocyte mitochondria and this interaction prevents hypoxia induced cell death. Our results provide an interesting lead in developing effective strategies to protect cardiomyocytes from hypoxia/ischemia induced cell death.     

Connexin43 phosphorylation by PKC and MAPK signals VEGF-mediated gap junction internalization

Gap junctions (GJs) exhibit a complex modus of assembly and degradation to maintain balanced intercellular communication (GJIC). Several growth factors, including vascular endothelial growth factor (VEGF), have been reported to disrupt cell–cell junctions and abolish GJIC. VEGF directly stimulates VEGF-receptor tyrosine kinases on endothelial cell surfaces. Exposing primary porcine pulmonary artery endothelial cells (PAECs) to VEGF for 15 min resulted in a rapid and almost complete loss of connexin43 (Cx43) GJs at cell–cell appositions and a concomitant increase in cytoplasmic, vesicular Cx43. After prolonged incubation periods (60 min), Cx43 GJs reformed and intracellular Cx43 were restored to levels observed before treatment. GJ internalization correlated with efficient inhibition of GJIC, up to 2.8-fold increased phosphorylation of Cx43 serine residues 255, 262, 279/282, and 368, and appeared to be clathrin driven. Phosphorylation of serines 255, 262, and 279/282 was mediated by MAPK, whereas serine 368 phosphorylation was mediated by PKC. Pharmacological inhibition of both signaling pathways significantly reduced Cx43 phosphorylation and GJ internalization. Together, our results indicate that growth factors such as VEGF activate a hierarchical kinase program—including PKC and MAPK—that induces GJ internalization via phosphorylation of well-known regulatory amino acid residues located in the Cx43 C-terminal tail.     

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.     

Mechanism of regulation of the gap junction protein connexin 43 by protein kinase C-mediated phosphorylation

Phosphorylation of the gap junction protein connexin 43 (Cx43) by protein kinase C (PKC) decreases dye coupling in many cell types. We report an investigation of the regulation by PKC of Cx43 gap junctional hemichannels (GJH) expressed in Xenopus laevis oocytes. The activity of GJH was assessed from the uptake of hydrophilic fluorescent probes. PKC inhibitors increased probe uptake in isolated oocytes expressing recombinant Cx43, indicating that the regulatory effect occurs at the hemichannel level. We identified by mutational analysis the carboxy-terminal (CT) domain sequences involved in this response. We found that 1) Ser368 is responsible for the regulation of Cx43 GJH solute permeability by PKC-mediated phosphorylation, 2) CT domain residues 253-270 and 288-359 are not necessary for the effect of PKC, and 3) the prolinerich CT region is not involved in the effect of phosphorylation by PKC. Our results demonstrate that Ser368 (but not Ser372) is involved in the regulation of Cx43 solute permeability by PKC-mediated phosphorylation, and we conclude that different molecular mechanisms underlie the regulation of Cx43 by intracellular pH and PKC-mediated phosphorylation. protein kinase C blocker; dye loading; hemichannel