Residual Cx45 and its relationship to Cx43 in murine ventricular myocardium

Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin(Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre+;Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts. We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/μg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre+;Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates 8 Ser/Thr residues in Cx45 in vitro. Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation.     

Residual Cx45 and its relationship to Cx43 in murine ventricular myocardium

Gap junction channels in ventricular myocardium are required for electrical and metabolic coupling between cardiac myocytes and for normal cardiac pump function. Although much is known about expression patterns and remodeling of cardiac connexin(Cx)43, little is known about the less abundant Cx45, which is required for embryonic development and viability, is downregulated in adult hearts, and is pathophysiologically upregulated in human end-stage heart failure. We applied quantitative immunoblotting and immunoprecipitation to native myocardial extracts, immunogold electron microscopy to cardiac tissue and membrane sections, electrophysiological recordings to whole hearts, and high-resolution tandem mass spectrometry to Cx45 fusion protein, and developed two new tools, anti-Cx45 antisera and Cre(+);Cx45 floxed mice, to facilitate characterization of Cx45 in adult mammalian hearts. We found that Cx45 represents 0.3% of total Cx protein (predominantly 200 fmol Cx43 protein/μg ventricular protein) and colocalizes with Cx43 in native ventricular gap junctions, particularly in the apex and septum. Cre(+);Cx45 floxed mice express 85% less Cx45, but do not exhibit overt electrophysiologic abnormalities. Although the basal phosphorylation status of native Cx45 remains unknown, CaMKII phosphorylates 8 Ser/Thr residues in Cx45 in vitro. Thus, although downregulation of Cx45 does not produce notable deficits in electrical conduction in adult, disease-free hearts, Cx45 is a target of the multifunctional kinase CaMKII, and the phosphorylation status of Cx45 and the role of Cx43/Cx45 heteromeric gap junction channels in both normal and diseased hearts merits further investigation.     

Temporal regulation of connexin phosphorylation in embryonic and adult tissues

Gap junctions, composed of proteins from the connexin family, allow for intercellular communication between cells in tissues and are important in development, tissue/cellular homeostasis, and carcinogenesis. Genome databases indicate that there are at least 20 connexins in the mouse and human. Connexin phosphorylation has been implicated in connexin assembly into gap junctions, gap junction turnover, and cell signaling events that occur in response to tumor promoters and oncogenes. Connexin43 (Cx43), the most widely expressed and abundant gap junction protein, can be phosphorylated at several different serine and tyrosine residues. Here, we focus on the dynamic regulation of Cx43 phosphorylation in tissue and how these regulatory events are affected during development, wound healing, and carcinogenesis. The activation of several kinases, including protein kinase A, protein kinase C, p34cdc2/cyclin B kinase, casein kinase 1, mitogen-activated protein kinase, and pp60src kinase, can lead to the phosphorylation of different residues in the C-terminal region of Cx43. The use of antibodies specific for phosphorylation at defined residues has allowed the examination of specific phosphorylation events both in tissue culture and in vivo. These new antibody tools and those under development will allow us to correlate specific phosphorylation events with changes in connexin function.     

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.     

Regulation of Synaptotagmin I Phosphorylation by Multiple Protein Kinases

Synaptotagmin I has been suggested to function as a low-affinity calcium sensor for calcium-triggered exocytosis from neurons and neuroendocrine cells. We have studied the phosphorylation of synaptotagmin I by a variety of protein kinases in vitro and in intact preparations. SyntagI, the purified, recombinant, cytoplasmic domain of rat synaptotagmin I, was an effective substrate in vitro for Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC), and casein kinase II (caskII). Sequencing of tryptic phosphopeptides from syntagI revealed that CaMKII and PKC phosphorylated the same residue, corresponding to Thr112, whereas caskII phosphorylated two residues, corresponding to Thr125 and Thr128. Endogenous synaptotagmin I was phosphorylated on purified synaptic vesicles by all three kinases. In contrast, no phosphorylation was observed on clathrin-coated vesicles, suggesting that phosphorylation of synaptotagmin I in vivo occurs only at specific stage(s) of the synaptic vesicle life cycle. In rat brain synaptosomes and PC12 cells, K+-evoked depolarization or treatment with phorbol ester caused an increase in the phosphorylation state of synaptotagmin I at Thr112. The results suggest the possibility that the phosphorylation of synaptotagmin I by CaMKII and PKC contributes to the mechanism(s) by which these two kinases regulate neurotransmitter release.     

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.     

Multiple phosphorylation sites in the 165-kilodalton peptide associated with dihydropyridine-sensitive calcium channels

Evidence from electrophysiological and ion flux studies has established that dihydropyridine-sensitive calcium channels are subject to regulation by neurotransmitter-mediated phosphorylation and dephosphorylation reactions. In the present study, we have further characterized the phosphorylation by cAMP-dependent protein kinase and a multifunctional Ca/calmodulin-dependent protein kinase of the membrane-associated form of the 165-kDa polypeptide identified as the skeletal muscle dihydropyridine receptor. The initial rates of phosphorylation of the 165-kDa peptide by both protein kinases were found to be relatively good compared to the rates of phosphorylation of established substrates of the enzymes. Phosphorylation of the 165-kDa peptide by both protein kinases was additive. Prior phosphorylation by either one of the kinases alone did not preclude phosphorylation by the second kinase. The cAMP-dependent protein kinase phosphorylated the 165-kDa peptide preferentially at serine residues, although a small amount of phosphothreonine was also formed. In contrast, after phosphorylation of the 165-kDa peptide by the Ca/calmodulin-dependent protein kinase, slightly more phosphothreonine than phosphoserine was recovered. Phosphopeptide mapping indicated that the two kinases phosphorylated the peptide at distinct as well as similar sites. Notably, one major site phosphorylated by the cAMP-dependent protein kinase was not phosphorylated by the Ca/calmodulin-dependent protein kinase, while other sites were phosphorylated to a high degree by the Ca/calmodulin-dependent protein kinase, but to a much lesser degree by the cAMP-dependent protein kinase. The results show that the 165-kDa dihydropyridine receptor from skeletal muscle can be multiply phosphorylated at distinct sites by the cAMP- and Ca/calmodulin-dependent protein kinases. As the 165-kDa peptide may be the major functional unit of the dihydropyridine-sensitive Ca channel, the results suggest that the phosphorylation-dependent modulation of Ca channel activity by neurotransmitters may involve phosphorylation of the 165-kDa peptide at multiple sites.     

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.     

Spinophilin is phosphorylated by Ca2+/calmodulin-dependent protein kinase II resulting in regulation of its binding to F-actin

Spinophilin is a protein phosphatase-1- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We have recently shown that the interaction of spinophilin with the actin cytoskeleton depends upon phosphorylation by protein kinase A. We have now found that spinophilin is phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in neurons. Ca(2+)/calmodulin-dependent protein kinase II, located within the post-synaptic density of dendritic spines, is known to play a role in synaptic plasticity and is ideally positioned to regulate spinophilin. Using tryptic phosphopeptide mapping, site-directed mutagenesis and microsequencing analysis, we identified two sites of CaMKII phosphorylation (Ser-100 and Ser-116) within the actin-binding domain of spinophilin. Phosphorylation by CaMKII reduced the affinity of spinophilin for F-actin. In neurons, phosphorylation at Ser-100 by CaMKII was Ca(2+) dependent and was associated with an enrichment of spinophilin in the synaptic plasma membrane fraction. These results indicate that spinophilin is phosphorylated by multiple kinases in vivo and that differential phosphorylation may target spinophilin to specific locations within dendritic spines.     

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.     

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.     

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).     

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.     

Key Connexin 43 Phosphorylation Events Regulate the Gap Junction Life Cycle

Connexin 43 (Cx43), the most widely expressed and abundant vertebrate gap junction protein, is phosphorylated at multiple different serine residues during its life cycle. Cx43 is phosphorylated soon after synthesis and phosphorylation changes as it traffics through the endoplasmic reticulum and Golgi to the plasma membrane, ultimately forming a gap junction structure. The electrophoretic mobility of Cx43 changes as the protein proceeds through its life cycle, with prominent bands often labeled P0, P1 and P2. Many reports have indicated changes in “phosphorylation” based on these mobility shifts and others that occur in response to growth factors or other biological effectors. Here, we indicate how phosphospecific and epitope-specific antibodies can be utilized to show when and where certain phosphorylation events occur during the Cx43 life cycle. These reagents show that phosphorylation at S364 and/or S365 is involved in forming the P1 isoform, an event that apparently regulates trafficking to or within the plasma membrane. Phosphorylation at S325, S328 and/or S330 is necessary to form a P2 isoform; and this phosphorylation event is present only in gap junctions. Treatment with protein kinase C activators led to phosphorylation at S368, S279/S282 and S262 with a shift in mobility in CHO, but not MDCK, cells. The shift was dependent on mitogen-activated protein kinase activity but not phosphorylation at S279/S282. However, phosphorylation at S262 could explain the shift. By defining these phosphorylation events, we have begun to sort out the critical signaling pathways that regulate gap junction function.     

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.     

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.     

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.     

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.     

Detailed regulatory mechanism of the interaction between ZO-1 PDZ2 and connexin43 revealed by MD simulations

The gap junction protein connexin43 (Cx43) binds to the second PDZ domain of Zonula occludens-1 (ZO-1) through its C-terminal tail, mediating the regulation of gap junction plaque size and dynamics. Biochemical study demonstrated that the very C-terminal 12 residues of Cx43 are necessary and sufficient for ZO-1 PDZ2 binding and phosphorylation at residues Ser (-9) and Ser (-10) of the peptide can disrupt the association. However, only a crystal structure of ZO-1 PDZ2 in complex with a shorter 9 aa peptide of connexin43 was solved experimentally. Here, the interactions between ZO-1 PDZ2 and the short, long and phosphorylated Cx43 peptides were studied using molecular dynamics (MD) simulations and free energy calculation. The short peptide bound to PDZ2 exhibits large structural variations, while the extension of three upstream residues stabilizes the peptide conformation and enhanced the interaction. Phosphorylation at Ser(-9) significantly weakens the binding and results in conformational flexibility of the peptide. Glu210 of ZO-1 PDZ2 was found to be a key regulatory point in Cx43 binding and phosphorylation induced dissociation.