Mutation of Human Connexin43 Amino Acids S279/S282 Increases Protein Stability Upon Treatment with Epidermal Growth Factor

Connexins are the structural units of gap junctions, structures allowing interchanging of information between the adjacent cells. Connexin43 (Cx43) is the most abundant gap junction protein. Cx43 can be degraded by lysosome- and proteasome-mediated processes upon internalisation of the entire structure. Only little is known about the role of phosphorylation during the gap junction degradation. In Cx43, a protein containing 14 amino acids susceptible to be phosphorylated, amino acids S279 and S282 are phosphorylated upon epidermal growth factor (EGF) treatment by erk1/2 MAP kinases. Here, we show that the wild-type Cx43 protein, as well as HeLa cells expressing the mutated Cx43 proteins S279A, S282A, and S279A/S282A, is mainly located at the plasma membrane. However, the protein stability is not altered in the isolated single mutants, whereas the double mutant S279A/S282A is strongly degradation impaired upon EGF treatment. This effect is not due to the decreased Cx43 internalisation, but seems to be related to a reduced ubiquitination.     

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.     

Phosphorylation of Serine Residues in the C-terminal Cytoplasmic Tail of Connexin43 Regulates Proliferation of Ovarian Granulosa Cells

Connexin43 (Cx43) forms gap junctions that couple the granulosa cells of ovarian follicles. In Cx43 knockout mice, follicle growth is restricted as a result of impaired granulosa cell proliferation. We have used these mice to examine the importance of specific Cx43 phosphorylation sites in follicle growth. Serines at residues 255, 262, 279, and 282 are MAP kinase substrates that, when phosphorylated, reduce junctional conductance. Mutant forms of Cx43 were constructed with these serines replaced with amino acids that cannot be phosphorylated. These mutants were transduced into Cx43 knockout ovarian somatic cells that were combined with wild-type oocytes and grafted into immunocompromised female mice permitting follicle growth in vivo. Despite residues 255 or 262 being mutated to prevent their being phosphorylated, recombinant ovaries constructed with these mutants were able to rescue the null phenotype, restoring complete folliculogenesis. In contrast, Cx43 with serine to alanine mutations at both residues 279 and 282 or at all four residues failed to rescue folliculogenesis; the mutant molecules were largely confined to intracellular sites, with few gap junctions. Using an in vitro proliferation assay, we confirmed a decrease in proliferation of granulosa cells expressing the double mutant construct. These results indicate that Cx43 phosphorylation by MAP kinase at serines 279 and 282 occurs in granulosa cells of early follicles and that this is involved in regulating follicle development.     

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.     

Phosphorylation and subcellular distribution of connexin43 in normal and stressed cells

We have developed polyclonal antibodies (SA226P) to a peptide of the human connexin43 (Cx43) protein between amino acids 271 and 288 containing phosphorylated S279 and S282. Antibodies specific for the phosphorylated form of the peptide were isolated by double immunoaffinity chromatography and were characterised using proteins of the cell line WB-F344, known to contain large amounts of Cx43. SA226P recognises specifically the slowest migrating Cx43 band in immunoblots of proteins isolated from untreated cells. In immunofluorescence experiments SA226P scarcely stains the plasma membrane in untreated cells in contrast to a commercial antibody recognising all isoforms of the Cx43 protein. EGF or stress treatment of the cells results in a rapid increase in the phosphorylated forms of Cx43 as revealed by immunoblotting. Immunofluorescence experiments reveal that both phosphorylated and non-phosphorylated Cx43 could be found at the plasma membrane. Whether phosphorylation of S279/S282 takes place before or after incorporation of Cx43 into the membranes is so far unknown. More interestingly, confocal microscopy using our antibodies and a commercial antibody recognising all isoforms of Cx43 shows the coexistence of differentially phosphorylated forms of the protein at the plasma membrane. Our results indicate that MAP kinases erk1/2 are mainly responsible for this phosphorylation, as already published. Nevertheless, treatment of the cells with anisomycin, known to activate stress kinase p38 but not erk1/2, also results in a weak but reproducible Cx43 phosphorylation.     

Phosphorylation at S365 is a gatekeeper event that changes the structure of Cx43 and prevents down-regulation by PKC

Phosphorylation at unspecified sites is known to regulate the life cycle (assembly, gating, and turnover) of the gap junction protein, Cx43. In this paper, we show that Cx43 is phosphorylated on S365 in cultured cells and heart tissue. Nuclear magnetic resonance structural studies of the C-terminal region of Cx43 with an S365D mutation indicate that it forms a different stable conformation than unphosphorylated wild-type Cx43. Immunolabeling with an antibody specific for Cx43 phosphorylated at S365 shows staining on gap junction structures in heart tissue that is lost upon hypoxia when Cx43 is no longer specifically localized to the intercalated disk. Efficient phosphorylation at S368, an important Cx43 channel regulatory event that increases during ischemia or PKC activation, depends on S365 being unphosphorylated. Thus, phosphorylation at S365 can serve a “gatekeeper” function that may represent a mechanism to protect cells from ischemia and phorbol ester-induced down-regulation of channel conductance.     

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.     

Phosphorylation on Ser-279 and Ser-282 of connexin43 regulates endocytosis and gap junction assembly in pancreatic cancer cells

The molecular mechanisms regulating the assembly of connexins (Cxs) into gap junctions are poorly understood. Using human pancreatic tumor cell lines BxPC3 and Capan-1, which express Cx26 and Cx43, we show that, upon arrival at the cell surface, the assembly of Cx43 is impaired. Connexin43 fails to assemble, because it is internalized by clathrin-mediated endocytosis. Assembly is restored upon expressing a sorting-motif mutant of Cx43, which does not interact with the AP2 complex, and by expressing mutants that cannot be phosphorylated on Ser-279 and Ser-282. The mutants restore assembly by preventing clathrin-mediated endocytosis of Cx43. Our results also document that the sorting-motif mutant is assembled into gap junctions in cells in which the expression of endogenous Cx43 has been knocked down. Remarkably, Cx43 mutants that cannot be phosphorylated on Ser-279 or Ser-282 are assembled into gap junctions only when connexons are composed of Cx43 forms that can be phosphorylated on these serines and forms in which phosphorylation on these serines is abolished. Based on the subcellular fate of Cx43 in single and contacting cells, our results document that the endocytic itinerary of Cx43 is altered upon cell–cell contact, which causes Cx43 to traffic by EEA1-negative endosomes en route to lysosomes. Our results further show that gap-junctional plaques formed of a sorting motif–deficient mutant of Cx43, which is unable to be internalized by the clathrin-mediated pathway, are predominantly endocytosed in the form of annular junctions. Thus the differential phosphorylation of Cx43 on Ser-279 and Ser-282 is fine-tuned to control Cx43’s endocytosis and assembly into gap junctions.     

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.     

Heteromeric connexin 43/connexin 33 complex endocytosis: A connexin phosphorylation independent mechanism

The role of gap junctions in proliferation, differentiation and apoptosis has been recently highlighted. Nevertheless, the molecular mechanisms that control these physiological events by acting on gap junction channels are still unknown. We have recently demonstrated that heteromeric gap junction plaques composed by Cx43 and Cx33 are unstable at the cell boundary and are rapidly internalized by endocytosis. In the present study, we analyze the phosphorylation status of Cx43 in homomeric (Cx43/Cx43) and heteromeric (Cx33/Cx43) complexes and their association with the tyrosine kinase c-Src. Our data show that c-Src interaction and P2 phosphorylation of Cx43, which are essential for homomeric Cx43 complex endocytosis, were altered in the heteromeric Cx33/Cx43 complex: lack of association between Cx33 and activated c-Src and disappearance of the P2 phosphorylated Cx43 isoform. The present findings demonstrate that the interaction of Cx33 with Cx43 within a same heteromeric complex may conduce to channel instability through alteration of the phosphorylation status of Cx43 independently of the control of the c-Src kinase. The data described here emphasize a new mechanism of Cx43 internalization Src kinase-independent.     

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.     

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.     

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.     

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.     

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.     

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.     

Connexin43 in MDCK cells: regulation by a tumor-promoting phorbol ester and Ca2+.

Prior to confluence, cultures of Madin Darby canine kidney (MDCK) cells expressed gap junctional communication, as assessed by fluorescent dye transfer, as well as relatively high levels of an anti-connexin43 immunoreactive component referred to as connexin43 (Cx43). After confluence, dye coupling and levels of Cx43 were dramatically reduced. Immunofluorescence analysis of the distribution of Cx43 in subconfluent cultures showed punctate labeling on the plasma membrane at regions of cell apposition and a more diffuse labeling in perinuclear regions. Western blots of total cell homogenates showed that the dephosphorylated form of Cx43 was more abundant than the phosphorylated forms. Phosphorylation of Cx43 was not significantly affected by 8-Bromo-cAMP or 8-Bromo-cGMP. However, 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited dye coupling and induced an increase in the amount of phosphorylated forms of Cx43 at the expense of the dephosphorylated form. This effect occurred as rapidly as 5 min after TPA treatment without apparent changes in distribution of Cx43 or cell morphology. These results suggest that second messenger pathways involving protein kinase C, but not cAMP- or cGMP-dependent protein kinase, led to changes in electrophoretic mobility of Cx43, revealed by Western blot, consistent with an alteration in the state of phosphorylation of the gap junction protein. Treatments with staurosporine, a protein kinase inhibitor, or okadaic acid, a protein phosphatase inhibitor, either alone or in combination with TPA, indicated that the abundance of the dephosphorylated form of Cx43 in MDCK cells was due to low kinase activity. It was also found that lowering the concentration of extracellular Ca2+, which reduced cell contact, did not affect the abundance, the state of phosphorylation, or the TPA-induced phosphorylation of Cx43. These results suggest that neither extracellular Ca2+ nor cell contact is required for basal or TPA-induced phosphorylation of Cx43.     

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.      

Casein kinase 1 regulates connexin-43 gap junction assembly

Phosphorylation of members of the connexin family of gap junction proteins has been correlated with gap junction assembly, but the mechanisms involved remain unclear. We have examined the role of casein kinase 1 (CK1) in connexin-43 (Cx43) gap junction assembly. Cellular co-immunoprecipitation experiments and in vitro CK1 phosphorylation reactions indicate that CK1 interacted with and phosphorylated Cx43, initially on serine(s) 325, 328, or 330. (32)P(i)-Metabolically labeled cells treated with CKI-7, a specific CK1 inhibitor, showed a reduction in Cx43 phosphorylation on site(s) that can be phosphorylated by CK1 in vitro. To examine CK1 function, normal rat kidney cells were treated with CKI-7, and Cx43 content was analyzed by Triton X-100 extraction, cell-surface biotinylation, and immunofluorescence. Western blot analysis indicated a slight increase in total Cx43, whereas gap junctional (Triton-insoluble) Cx43 decreased, and non-junctional plasma membrane Cx43 increased (as detected by cell surface biotinylation). Immunofluorescence experiments in the presence of CK1 inhibitor showed increases in Cx43 plasma membrane localization but not necessarily accumulation at cell-cell interfaces. Decreased gap junctional and phosphorylated Cx43 was also detected when cells were treated with IC261, a CK1 inhibitor specific for delta or epsilon isoforms. These data suggest CK1delta could regulate Cx43 gap junction assembly by directly phosphorylating Cx43.     

Relationship of gap junction formation to phosphorylation of connexin43 in mouse preimplantation embryos

To clarify the relationship of gap junction formation to phosphorylation of connexin43 (Cx43) in mouse preimplantation embryos, immunofluorescence and Western blot analysis were conducted. Immunofluorescence showed Cx43 positive spots first at the mid-eight-cell stage (6 hr postdivision to the eight-cell stage). The number of spots increased from 6 to 15 hr postdivision to the eight-cell stage. Western blot analysis suggested Cx43 to possibly be present in the nonphosphorylated form at the mid-four-cell stage (6 hr postdivision to the four-cell stage), and phosphorylated Cx43 to increase from the mid-eight-cell stage (6 hr post-division to the eight-cell stage) onward. Dibutyryl cAMP (dbcAMP), a protein kinase A (PKA) activator, added to the culture medium increased the phosphorylation of Cx43 and Cx43 positive spots. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator, increased the phosphorylation of Cx43, but decreased Cx43 positive spots. These results suggest that the phosphorylation of Cx43, induced by different protein kinase, leads to a different effect on gap junction formation in mouse preimplantation embryos.