connexins, gap junctional intercellular communication and kinases

Review

Connexins, gap junctional intercellular communication and kinases

Véronique Cruciani, Svein-Ole Mikalsen *

Department of Environmental and Occupational Cancer, Institute for Cancer Research, The Norwegian Radium Hospital, N-0310 Oslo, Norway

Received 7 March 2002; accepted 13 September 2002

Abstract

A number of kinases and signal transduction pathways are known to affect gap junctional intercellular communication and/or phosphory-lation of connexins. Most of the information is available for protein kinase A, protein kinase C, mitogen-activated protein kinase, and thetyrosine kinase Src. Much less is known for protein kinase G, Ca2+-calmodulin dependent protein kinase, and casein kinase. However, thepresent lack of knowledge is not necessarily synonymous with lack of importance in the regulation of intercellular communication andphosphorylation of connexins. Kinases and the phosphorylation of connexins may be involved in the regulation of gap junctional intercellularcommunication at all levels ranging from the expression of connexin genes to the degradation of the gap junction channels. The exact role ofthe phosphorylation depends both on the kinase and the connexin involved, as well as the cellular context.

© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Keywords: Connexins; Gap junctions; Intercellular communication; Kinases; Phosphorylation

1. Introduction

The gap junction structure was first observed by electronmicroscopy studies around 45 years ago (Robertson, 1957;Karrer, 1960). In the same period, electrical conductancebetween adjacent cells was observed (Furshpan and Potter,1959; Dewey and Barr, 1962). Some years later, it becameclear that not only small ions, but also molecules of a consid-erable size could diffuse between neighboring cells (Kannoand Loewenstein, 1964; Stoker, 1967; Simpson et al., 1977).Several hormones and second messengers were shown toaffect the size of gap junction plaques or to modulate thepermeability between cells (Hax et al., 1974; Amsterdam etal., 1981; Flagg-Newton et al., 1981; Maldonado et al.,1988). It was also shown that pharmacological agents, espe-cially the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA), could modulate theintercellular communication (Murray and Fitzgerald, 1979;Yotti et al., 1979). The potential of kinases to modulate gapjunctional intercellular communication (GJIC) was furtherunderlined by the finding that introduction of the catalyticsubunit of protein kinase A (PKA) into communication-deficient cells corrected the defect (Wiener and Loewenstein,

1983). Furthermore, the gap junction proteins could be directtargets of kinases as shown by the cAMP-induced stimula-tion of phosphorylation of the main 27 kDa gap junctionprotein (today known as Cx32) from rat hepatocytes (Sáez etal., 1986). Also a temperature-sensitive and oncogenic ty-rosine kinase, v-Src, was shown to strongly influence GJIC(Atkinson et al., 1981).

The first connexin to be cloned was Cx32 (Kumar andGilula, 1986), soon followed by Cx43 (Beyer et al., 1987).Asof today, around 20 connexins have been cloned from hu-mans and rodents. Most of them contain potential phospho-rylation sites, usually in their cytoplasmic C-terminal tail.Many connexins have a basal level of phosphorylation, buttheir phosphorylation can also be induced or changed by theactivation of the proper signal transduction pathways. Suchactivation is often accompanied by changes in the level ofGJIC. Thus, kinases are directly (by phosphorylation of con-nexins) or indirectly involved in the regulation of GJIC. Theaction of kinases is usually balanced by protein phosphatases(see review by Hervé and Sarrouilhe).

The permeability and electrical conductance betweencells are determined by the pore size of the channels, theiropen-state probability, and the number of channels. Thechannels flicker between closed and open states, and evenbetween two or more levels of pore size. The number ofchannels may be affected by synthesis or degradation of

* Corresponding author.E-mail address: [email protected] (S.O. Mikalsen)

Biology of the Cell 94 (2002) 433–443

www.elsevier.com/locate/bicell

© 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.PII: S0248-4900(02)00014-X

connexins, by the kinetics of assembly into connexons, thetransport of connexons to the cell membrane, and the subse-quent assembly of connexons into gap junction plaques. Thepore size may increase or decrease by changing the confor-mation of the protein in a phosphorylation-dependent man-ner (Takens-Kwak and Jongsma, 1992; Moreno et al., 1994).The conformation may also change the kinetics of openingand closing cycles, and thereby affect the open-state prob-ability. In this review, we will mention examples that supportthe involvement of kinases and connexin phosphorylation inmany of these processes.

Recommended early reviews in the field are from Staggand Fletcher (1990) and Sáez et al. (1993a). In the presentreview, “ in vivo” will refer to tissues or intact cells, includingcell cultures, while “ in vitro” will refer to enzymatic assays,reconstituted pathways, etc.

2. The kinases and signal transduction pathways

2.1. Protein kinase A and cyclic AMP

The cAMP/PKA system was early known to affect GJIC(Stagg and Fletcher, 1990; Sáez et al., 1993a). Three types ofresponses are found, depending on the system studied: (i) aslow increase in GJIC; (ii) a rapid decrease in GJIC; or (iii) arapid increase in GJIC. The first of these responses is mainlydependent on increased gene expression and/or protein syn-thesis. Little is known about the second type of response, butit will be discussed at the end of this section. We will firstfocus on the third type of response.

A very interesting early work used CHO cells lackingeither PKAI or PKAII (Wiener and Loewenstein, 1983). Thecategorization of PKA into isoforms I and II is based on twomain types of regulatory subunits, and the catalytic subunitscan freely exchange between them. The PKAI-lacking cellsshowed low GJIC, while the PKAII-lacking cells had a be-havior more similar to wild-type cells. When the catalyticsubunit of PKA was introduced into PKAI-lacking cells,GJIC was normalized. This not only clearly suggests theimportance of the catalytic subunit, but may also suggestdifferences in the functions of the regulatory subunits ofPKA. However, regulatory subunits of type II injected intowell-coupled pig granulosa cells had no effect on GJIC, incontrast to a PKA inhibitory peptide (Godwin et al., 1993).

One possible explanation of the rapid responses to cAMPis PKA-mediated phosphorylation of connexins. Rat hepato-cytes increased their conductance within minutes in responseto cAMP analogs (Sáez et al., 1986). PKA phosphorylatedCx32 (Sáez et al., 1986), but to a considerably lower extentthan PKC (see Section 2.4). The major phosphorylation oc-curred at S233 in an in vitro system (Sáez et al., 1990). Thesame residue is phosphorylated by PKC. Also Cx40 becamephosphorylated in response to cAMP (Traub et al., 1994;van Rijen et al., 2000), and the conductance and dye transfer

between Cx40-transfected SKHep1 cells increased(van Rijen et al., 2000).

There is some controversy on whether PKA phosphory-lates Cx43 in vitro (Dasgupta et al., 2001) or not (Sáez et al.,1993b; TenBroek et al., 2001). Some cell types do not changethe phosphorylation status of Cx43 in response to cAMPelevating agents (Berthoud et al., 1992; Bånrud et al., 1994;Paulson et al., 2000; TenBroek et al., 2001), while others do(Granot and Dekel, 1994; Atkinson et al., 1995). In a recentpaper, it was concluded that PKA did not phosphorylateCx43 directly, but the enhanced assembly of Cx43-containing gap junctions was dependent on the basal phos-phorylation of S364 by an unknown kinase (TenBroek et al.,2001; see also Section 2.9).

cAMP/PKA may affect GJIC by changing the transportkinetics through the Golgi system. It was observed that a24-h exposure to 8-Br-cAMP decreased the amount of intra-cellular Cx43 (Atkinson et al., 1995), probably located in theGolgi, suggesting a recruitment of Cx43 from the Golgi tothe cell membrane. Also considerably shorter exposures mayrecruit Cx43 from the Golgi, as pre-treatment with monensin(which blocks protein transport at the level of the transGolginetwork) or brefeldin A (which blocks protein transport bycollapsing the Golgi into the endoplasmic reticulum) inhib-ited the rapid increase in GJIC by 8-Br-cAMP (Burghardt etal., 1995; Paulson et al., 2000). Thus, cAMP-induced in-crease in GJIC in Cx43-containing cells may occur by cell-specific mechanisms with increased open-state probability,increased single channel conductance, enhanced assembly ofCx43 connexons into functional channels, or recruitment ofCx43 from the Golgi-located subpopulation as the majoralternatives. We also note that other agents may affect GJICby the latter mechanism as a high concentration of WY-14,643, a peroxisome proliferator, induced a rapid increaseof GJIC in V79 hamster fibroblasts, which was fully counter-acted by brefeldin A (Cruciani and Mikalsen, 2002).

PKA appeared to phosphorylate chick Cx56 in vitro atsome of the same sites as PKC, but 8-Br-cAMP-stimulatedlens cells did not show any changes in phosphopeptide pat-tern compared with controls (Berthoud et al., 1997). Func-tional consequences are therefore uncertain.

As mentioned in the beginning of this section,cAMP/PKA also causes a decrease of GJIC in some systems.Sometimes, this can be attributed to the connexin expressed.Cx45 forms gap junction channels with low conductance,which are poorly permeable to the commonly used fluores-cent dyes like Lucifer Yellow (Steinberg et al., 1994). cAMPdecreased the electrical coupling between Cx45-transfectedHeLa cells, most likely due to decreased open-state probabil-ity, and increased the phosphorylation of the protein(van Veen et al., 2000). Dopamine and cAMP blocked dyetransfer between horizontal cells in fish retina (Teranishi etal., 1983). Fish retina expresses a number of connexins(Cx27.5, 34.7, 35, 43, 44.1, 55.5) (O’Brien et al., 1998;Wagner et al., 1998; Dermietzel et al., 2000), but it is uncer-tain whether they are regulated in the same manner as their

434 V. Cruciani, S.O. Mikalsen / Biology of the Cell 94 (2002) 433–443

mammalian counterparts. In some cases, connexins involvedin cAMP-induced upregulation of GJIC (see above) may alsobe inhibited by cAMP. For example, cAMP (and cGMP)bound directly to Cx26 and Cx32 before the incorporationinto liposomes, and this completely inhibited the opening ofthe half channels (Bevans and Harris, 1999). Although this isa highly interesting observation, the physiological relevanceis not clear. However, in intact cells also, the dichotomy ofresponse can be found. Increased cAMP levels decreased gapjunctional permeability, although only to a limited extent, inuterine (Cole and Garfield, 1986), vascular (Moore et al.,1991) and corpus cavernosum (Moreno et al., 1993) smoothmuscle cells expressing Cx43. It is not known as to whydifferent cell systems expressing the same connexin responddifferently to cAMP. The most trivial possibility is that thisrapid, but small, response (Moore et al., 1991; Moreno et al.,1993) has been overlooked in studies where long-term effectsof cAMP were investigated. In support of this possibility, theshort-term decrease in GJIC was followed by a long-termincreased synthesis of Cx43 in corpus cavernosum smoothmuscle cells (Moreno et al., 1993). Among the more appeal-ing explanations are the involvement of different subtypes ofPKA, especially of the catalytic subunit (and thereby differ-ent substrate specificities), and different anchoring proteins(causing different localizations of the enzyme). As will bemade clear throughout the present review, the puzzle of“opposite responses” is a recurrent phenomenon in the fieldof GJIC.

2.2. Protein kinase G and cyclic GMP

cGMP decreased conductance between rat cardiomyo-cytes, probably due to the lowering of single channel conduc-tance (Burt and Spray, 1988; Takens-Kwak and Jongsma,1992), but there was no effect in Cx43-expressing rat A7r5cells (Moore et al., 1991). When non-communicatingSKHep1 cells were transfected with rat or human Cx43, onlythe rat Cx43-transfected cells responded to 8-Br-cGMP(Kwak et al., 1995b). 8-Br-cGMP increased the incorpora-tion of phosphate into rat Cx43, but not human Cx43. Thiscould be due to the presence of Ser at position 257 in ratCx43, while in human Cx43, there is Ala at the correspond-ing position (Kwak et al., 1995b). cGMP did not affect theconductance between Cx45-transfected HeLa cells(van Veen et al., 2000).

Nitric oxide (NO) is an important signal molecule, whosesynthesis is induced by many cytokines and by endotoxins,like bacterial lipopolysaccharide (LPS). NO activates guany-late cyclase, thereby increasing the cGMP level. LPS inhib-ited GJIC in rat astrocytes, and GJIC remained low after theremoval of LPS (Bolaños and Medina, 1996). NO may beinvolved in the suppression of Cx43 expression in rat uterus,balancing the stretch-induced expression of Cx43 in thepregnant uterus (Sladek et al., 1999).

2.3. Mitogen-activated protein (MAP) kinases and growthfactors

Growth factors like EGF, PDGF, FGF-2 (previously calledbFGF), and others, have for some time been known to affectGJIC in a number of cell types (Maldonado et al., 1988;Pepper and Meda, 1992). The mentioned growth factorsactivate the ERK pathway (one of several MAP kinase path-ways; two other well-characterized MAP kinase pathwaysare the p38 and the JNK pathways, and there are at least twoMAP kinases in each of the pathways). In rat liver epithelialcells, EGF caused a transient, but strong, decrease in GJICthat was independent of PKC (Kanemitsu and Lau, 1993;Rivedal and Opsahl, 2001). EGF profoundly affected thephosphorylation status of Cx43 in these cell systems. MAPkinase (this term is used when the exact identity is not givenin the paper) was found to phosphorylate purified Cx43, andphosphopeptide mapping showed coincidence with a subsetof phosphopeptides from Cx43 phosphorylated in vivo(Kanemitsu and Lau, 1993). By a combination of site-directed mutagenesis, peptide-based kinase assays, trypticphosphopeptide mapping, and peptide sequencing, the majorMAP kinase phosphorylation sites were determined as S255,S279 and S282 (Warn-Cramer et al., 1996). Phosphorylationof these sites was not necessary for the assembly of Cx43 intogap junction channels or the establishment of GJIC itself(Warn-Cramer et al., 1998). In contrast to the response toEGF described above, a human kidney cell line increasedGJIC after exposure to EGF (Rivedal et al., 1996). This alsoappeared dependent on the ERK pathway, but protein synthe-sis was needed (Rivedal et al., 1996; Vikhamar et al., 1998).The kinetics of Cx43 phosphorylation were considerablymore rapid than the changes in GJIC (Vikhamar et al., 1998).It is presently unknown as to why two cell types stimulatedwith the same growth factor, utilizing the same signal trans-duction pathway, and having the same connexin protein, haveopposite responses with regard to GJIC. However, we notethat the stimulation of ERK in some cases causes an importof ERK into the nucleus, where it presumably affects tran-scription factors, while in other cases, it remains in thecytoplasm (reviewed in Volmat and Pouysségur, 2001). Thelocalization of the MAP kinases has not been studied in anyof these papers.

PDGF induced a strong, but transient, decrease of GJIC infibroblasts (Maldonado et al., 1988; Pelletier and Boynton,1994) and in T51B rat liver epithelial cells transfected with aPDGF receptor (Hossain et al., 1998), but there are differentconclusions on the necessity of PDGF receptor tyrosine ki-nase activity for the response (Pelletier and Boynton, 1994;Hossain et al., 1998). This could suggest that at least twoPDGF modulated pathways might affect GJIC. In T51Bcells, PDGF-induced phosphorylation of Cx43 appeared togive a signal for degradation, followed by resynthesis (Hos-sain et al., 1998). Both ERK1/2 and PKC were necessary, butnot sufficient, to obtain a response in T51B cells, and usingPDGF receptor mutants, it was concluded that several PDGF

435V. Cruciani, S.O. Mikalsen / Biology of the Cell 94 (2002) 433–443

receptor-initiated pathways were involved (Hossain et al.,1999).

FGF-2 increased GJIC in a number of cell systems likemicrovascular endothelial cells (Pepper and Meda, 1992),cardiac fibroblasts (Doble and Kardami, 1995), and chicklens cells (Le and Musil, 2001). In cultured glia cells, bothincreases (Nadarajah et al., 1998) and decreases (Reuss et al.,1998) in GJIC have been found. The increase was generallyrelatively slow (several hours), and could be due to increasedexpression of Cx43, without any considerable change inphosphorylation status. In striking contrast to cardiac fibro-blasts, cardiomyocytes had a rapid decrease in GJIC and anincrease in Cx43 phosphorylation after exposure to FGF-2(Doble et al., 1996). In spite of the activation of the ERK1/2pathway by FGF-2 in cardiomyocytes, the response in GJICwas in this case mediated through PKC (Doble et al., 2000;see Section 2.4). In chick lens cells, ERK mediated theincrease in GJIC (Le and Musil, 2001). Again, the differ-ences between systems are puzzling. It should also be men-tioned that FGF-2 was colocalized with Cx43-containing gapjunctions in the brain (Yamamoto et al., 1991) and the heart(Kardami et al., 1991). The functional importance of thiscolocalization is unclear, especially since the signaling isbelieved to be initiated from the extracellular side of themembrane.

MAP kinases may become activated by agents employingreceptors outside the growth factor systems. TPA activatesMAP kinases (see Section 2.4). Also several ligands usingG-protein coupled receptors activate MAP kinases (Belchevaand Coscia, 2002). While Postma et al. (1998) concluded thatchannel closure by G-protein coupled receptor ligands wasindependent of MAP kinase, ERK1/2 mediated the inhibitionof GJIC in endothelial cells by cannabinoids (Brandes et al.,2002).

2.4. Protein kinase C

Many PKC-mediated effects on GJIC and connexin phos-phorylation have been elucidated by the use of TPA. In many,but not all systems, TPA causes a strong, but transient, de-crease in GJIC, usually measured by dye transfer. However,dye permeability and conductance are differentially affectedby TPA in rat cardiomyocytes (Kwak et al., 1995c). This canbe explained by a smaller pore size restricting dye transfer,while the open-state probability and/or number of channelsare increased. The complexity and differences in responses toTPA is further shown by the fact that TPA enhanced thedegradation of Cx43 in some cell systems (Oh et al., 1991;Matesic et al., 1994), but not in others (Yamasaki et al., 1983;Cruciani et al., 1999). The endogenous PKC activator, dia-cylglycerol, usually gives the same responses as TPA(Enomoto and Yamasaki, 1985; Yada et al., 1985), althoughsome controversy exists on this point (Bastide et al., 1994).

Cx32 was the first connexin found as a PKC substrate invitro (Takeda et al., 1987) and in hepatocyte cultures (Takedaet al., 1989). The PKA and PKC phosphorylation sites are

partially different (Takeda et al., 1989), but the major phos-phorylation site in both cases seems to be S233 (Sáez et al.,1990). TPA does not decrease GJIC in rat hepatocytes (Sáezet al., 1990). The PKC-mediated phosphorylation of Cx32may protect against the degradation of the protein (Elvira etal., 1993). Interestingly, both Cx26-, 32-, 40-, and 43-transfected HeLa cells decreased their GJIC in response toTPA (Mazzoleni et al., 1996). TPA also strongly decreasedGJIC in Cx26-transfected human hepatoma SKHep1 cells(Kwak et al., 1995a). These results are intriguing for tworeasons. First, they show that connexins can deviate in re-sponse depending on their cellular environment. Second,TPA is able to suppress GJIC in cell systems containingCx26, a connexin that presumably is devoid of phosphoryla-tion sites. Thus, TPA may suppress GJIC by mechanisms notdependent on a direct phosphorylation of connexins. Oneexplanation could be that PKC phosphorylates an accessoryprotein that subsequently can bind to Cx26. The concept ofaccessory proteins may also extend to other connexins, in-cluding Cx43 (see review by Thomas et al.).

By far, Cx43 is the most studied connexin in relation toTPA and PKC. Still we do not know why the phosphorylationchanges of Cx43 vary so much between systems, in spite of asimilar response with regard to GJIC (see e.g. Cruciani et al.,1997, 1999). In addition to the cell membrane, there is oftena considerable subpopulation of Cx43 in the Golgi area(Cruciani and Mikalsen, 1999). This subpopulation mainlyconsists of the NP form of Cx43. In cell types where TPAcaused profound changes in phosphorylation status of Cx43,the NP form became phosphorylated, but was still present inthe Golgi (Cruciani and Mikalsen, 1999). However, the phos-phorylation caused a slower migration during electrophore-sis, thus obscuring the (potential) phosphorylation changesoccurring in the plasma membrane-located P1 and P2 forms(Cruciani and Mikalsen, 1999). Ideally, the differently lo-cated subpopulations of Cx43 (and any other connexin)should be separated before analyzing the posttranslationalmodifications.

Where in Cx43 are the sites phosphorylated by PKC? In apeptide-based PKC assay (isoform not given, but may consistof a, b, and c), only the peptide 360–375 became phospho-rylated at the positions corresponding to S368 and S372(Sáez et al., 1993b). The PKC-mediated phosphorylation ofS368 was recently supported by observations in anothersystem (Lampe et al., 2000). This included the transfection ofCx43 knockout cells with mutated (S368A) Cx43. However,phosphopeptide analyses showed that several other PKC-mediated phosphorylation sites exist in vivo (Sáez et al.,1997; Lampe et al., 2000). Some of these phosphorylationsmay be due to an activation of the MAP kinase/ERK pathwayor cdc2 kinase. It was recently shown that ERK1/2 mediateda part of the response to TPA both with regard to Cx43phosphorylation and GJIC in rat liver epithelial cells(Rivedal and Opsahl, 2001; Ruch et al., 2001). Also in V79hamster fibroblasts, a part of the TPA effect on Cx43 phos-phorylation appeared mediated through ERK1/2 (V. Cru-


ciani, S.-O. Mikalsen, unpublished). However, Cx43 phos-phopeptides obtained from neonatal cardiomyocytes underbasal growth conditions did not coincide well with phospho-peptides from MAP kinase or cdc2 kinase in vitro phospho-rylations of the Cx43 cytoplasmic C-terminal tail (Sáez et al.,1997).

PKC is activated by a number of physiological and patho-logical conditions. For example, a short heart ischemia mayactivate PKC (Ytrehus et al., 1994). Gap junctions becomedisordered in the failing heart (reviewed in Severs et al.,2001), uncoupling occurs, and Cx43 changes its phosphory-lation status (Beardslee et al., 2000). PKCe appears as themajor Cx43-interacting isoenzyme in the failing heart, and itmay directly phosphorylate Cx43. On the other hand, PKCamay activate another kinase that phosphorylates Cx43(Bowling et al., 2001). PKCe also appears as the majorisoform involved in FGF-2 induced decrease of GJIC incardiomyocytes (Doble et al., 2000). Curiously, PKCe wasfound involved in TPA-induced decrease of GJIC in earlypassage hamster fibroblasts and in R6 rat fibroblasts, but ithad no visible effect on the phosphorylation status of Cx43 inthese cells (Cruciani et al., 2001, Husøy et al., 2001). PKCewas not involved in responses to TPA in V79 fibroblasts. Inthe latter cells, PKCd appeared as the major mediator ofsuppressed GJIC by TPA, but it had minimal effects on thephosphorylation of Cx43 (Cruciani et al., 2001). Thus, dif-ferent PKC isoenzymes may be of different importance indifferent systems. Interestingly, differences in the total PKCenzyme activity did not determine the sensitivity of fibro-blasts to TPA (Cruciani et al., 2001; Husøy et al., 2001).

PKC (a mixture of a, b, and c isoforms) is able to phos-phorylate chick lens Cx45.6 and Cx56 in vitro (Jiang andGoodenough, 1998). TPA stimulates the phosphorylation ofCx56 in both the intracellular loop (S118) and in theC-terminal tail (S493) (Berthoud et al., 1997). There are alsoprobably other, presently unidentified, phosphorylation sitesin Cx56. The phosphorylation of S118 may act as a signal forincreased degradation of the protein (Berthoud et al., 1997).Cx56 is presently the only connexin where there is goodevidence for phosphorylation of the intracellular loop. PKCcis a candidate for mediating these effects (Berthoud et al.,2000). Additionally, overexpressed PKCc inhibited GJIC inrat lens epithelial cells (Saleh and Takemoto, 2000), and itphosphorylated rat lens Cx46 (Saleh et al., 2001). Thus,PKCc appears to be involved in the regulation of GJIC andconnexin phosphorylation in the lens. PKCc is a neuro-specific isoform of PKC, and these results should thereforenot be directly extended to other tissues.

TPA/PKC and cAMP/PKA often influence GJIC in oppo-site directions. It is therefore not surprising that TPA/PKCcauses increased GJIC in some systems. In Cx45-transfectedHeLa cells, TPA increased electrical conductance by chang-ing the open-state probability, but did not change the phos-phorylation status as judged by western blotting (van Veen etal., 2000). In Cx43-expressing corpus carvernosum smoothmuscle cells, TPA induced an increase in conductance within

15 min, concurrent with a shift to lower single channelunitary conductances (Moreno et al., 1993). Thus, this obser-vation is in line with the results of Kwak et al. (1995c)described above. There are relatively few papers that havecompared dye transfer and electrical conductance, or ana-lyzed the parameters determining GJIC or macroscopic con-ductance (open-state probability, number of channels, singlechannel conductance). Thus, we do not know whether theparadoxical situation with increased conductance concurrentwith decreased pore size or decreased dye transfer is re-stricted to a few cell types and one kinase-activating agent.For retinoic acid, there was a correlation between decreaseddye transfer and decreased conductance in primary humankeratinocytes (Salomon et al., 1992).

2.5. Ca2+-calmodulin dependent protein kinase

There is much literature on Ca2+ and GJIC (which will notbe touched upon here), but Ca2+-calmodulin dependent pro-tein kinase (Ca2+-CDPK) has been little studied. Calmodulinitself can bind to Cx32 at two sites, one in the N-terminal tailand one close to the C-terminal tail (Török et al., 1997). TheN-terminal sequences of the connexins are much conserved,but it is not known whether calmodulin directly interacts withother connexins. It was suggested that calmodulin may act asan integral regulatory subunit of Cx32 connexons (Peracchiaet al., 2000). Calmodulin may enhance the decrease in con-ductance by an increased intracellular Ca2+ level in cardi-omyocytes (Toyama et al., 1994), but it is not known whetherany kinase is involved in the enhancement.

Ca2+-CDPK phosphorylates Cx32, but not at the same siteas PKC or PKA (Sáez et al., 1990). It is uncertain whetherthis phosphorylation has any functional consequences forCx32. In contrast, Ca2+-CDPK may be involved in K+-induced increase of GJIC in astrocytes (De Pina-Benabou etal., 2001), but without any visible change in phosphorylationstatus of Cx43. The increase is probably secondary to theinflux of Ca2+. In the Mauthner neurons of goldfish, Ca2+

may stimulate a Ca2+-CDPK-dependent increase in GJIC(Pereda et al., 1998). Traditionally, Ca2+ is said to decreaseGJIC, but obviously the consequences may depend on thecellular context.

2.6. Casein kinase

Chick lens Cx45.6 was found to be phosphorylated onS363 in vivo (Yin et al., 2000). This site was phosphorylatedby casein kinase II in a peptide-based kinase assay, andphosphopeptide mapping suggested that this is one of severalphosphorylations occurring in vivo (Yin et al., 2000). TheCx45.6 mutant S263A was partly protected against degrada-tion by a caspase-3-like protease (Yin et al., 2000, 2001).Interestingly, human Cx50 (the ortholog of chick Cx45.6)has Ser at the corresponding position, while mouse Cx50 has


Ala. Thus, it is unclear whether the human and mouse Cx50are regulated in the same manner. This is further underlinedby sheep Cx49, which contains Pro at this position. More-over, casein kinase I, but not casein kinase II, phosphorylatedsheep Cx49 (Cheng and Louis, 1999), and it appeared thatthis phosphorylation contributed to lowering of GJIC in thelens cells (Cheng and Louis, 2001). At first sight, this maylook self-contradictory, as Cx50 knockout mice developcataract (White et al., 1998). However, it may be that acertain level of GJIC, not too high, not too low, is needed fordifferentiation and proper homeostasis both in the lens and inother tissues.

There is no clear picture of the cellular functions of caseinkinase I (which is a family of isoforms) and II (reviewed inGross and Anderson, 1998; Faust and Montenarh, 2000).Both are present in most tissues, in many cellular compart-ments, are constitutively active, and have a number of sub-strates. Both kinases normally prefer substrates with numer-ous negative charges N-terminal to the target site. Caseinkinase I may in certain contexts be a phosphate-directedkinase (Gross and Anderson, 1998), and it is involved in Wntsignaling in Xenopus embryos (Peters et al., 1999). How orwhether this relates to the potential role of Wnt in the regu-lation of GJIC and Cx43 expression (Olson et al., 1991;Van der Heyden et al., 1998; Ai et al., 2000), is not known.

2.7. Cdc-2 kinase and cell cycle

We will here concentrate on the phosphorylation changesoccurring in Cx43 during the cell cycle. Whether suchchanges occur in other connexins is presently unknown. Thelarge field of connexin-mediated growth regulation is consid-ered to be outside the scope of the present paper (see reviewby Mesnil).

GJIC is usually strongly decreased during mitosis (Good-all and Maro, 1986; Stein et al., 1992; Xie et al., 1997; Wilsonet al., 2000; but see also O’Lague et al., 1970), although thereis not always a correlation between dye transfer and electricalconductance (Goodall and Maro, 1986; Kwak et al., 1995c).Regardless of whether the cells have been collected by mi-totic shake-off or by chemically induced accumulation ofmitotic cells, the phosphorylation status of Cx43 is muchchanged during mitosis, and the major part of the Cx43population is in a slow migrating form found slightly abovethe P2 position (Xie et al., 1997; Kanemitsu et al., 1998;Lampe et al., 1998). Additionally, Cx43 seems redistributedto an intracellular location (Xie et al., 1997; Lampe et al.,1998), which may explain the decrease in GJIC. It is notknown whether the mitotic version of Cx43 is formed at thelevel of the cell membrane with subsequent internalization,or the protein is internalized before the phosphorylationsoccur. However, the continued presence of an antigenicallymasked form of Cx43 in the cell membrane should not beexcluded. The formation of the mitosis-specific form of Cx43is dependent on cdc2 kinase activity (Kanemitsu et al., 1998;Lampe et al., 1998). While one group found similar Cx43

phosphotryptic peptides from in vitro assays with cdc2 andMAP kinase (Sáez et al., 1997), others identified the majorcandidate phosphorylation site for cdc2 kinase as S255(Kanemitsu et al., 1998; Lampe et al., 1998), potentially withS262 as a minor target (Kanemitsu et al., 1998). There arealso other phosphorylations present in the mitotic form ofCx43 (Kanemitsu et al., 1998; Lampe et al., 1998), whichwere suggested to be caused by unidentified cdc2-activatedmitotic kinases (Lampe et al., 1998). However, as the mitoticform probably is created from NP, P1 and P2 forms present ininterphase cells, it is likely that some of the phosphorylationsites in the mitotic form will correspond to the phosphoryla-tion sites in P1 and P2 forms.

Little work has been done on other parts of the cell cycle.It was reported that quiescent (G0) cells stimulated by serumto enter the cell cycle changed Cx43 phosphorylation statusand lowered GJIC in a reversible and PKC-dependent man-ner (Koo et al., 1997). However, it is not clear whether thesechanges are directly associated with the entry into the cellcycle.

2.8. Src and other tyrosine kinases

Three tyrosine kinases have been found to induce tyrosinephosphorylation of connexins. Little information is availablefor v-Fps, but it seems to affect GJIC and Cx43 in a mannersimilar to v-Src (Kurata and Lau, 1994). The third tyrosinekinase is the EGF receptor, which phosphorylated Cx32 invitro, but only at a low stoichiometry (Diez et al., 1998).Whether this can be extended to the in vivo situation ispresently unclear. In the remaining part, we will concentrateon Src.

A temperature-sensitive variant of the viral and oncogenicversion of the cellular proto-oncogene, Src, was earliershown to have a rapid and strong effect on GJIC (Atkinson etal., 1981). Also overexpressed c-Src can affect GJIC (Azar-nia et al., 1988). Several groups showed that activated ver-sions of Src caused tyrosine phosphorylation of Cx43 (Crowet al., 1990; Filson et al., 1990; Swenson et al., 1990). Thereis some conflict regarding the importance of the tyrosinephosphorylation of Cx43, and the site phosphorylated. Onegroup found the mutation Y265F to abolish v-Src-induceddecrease in GJIC in a Xenopus oocyte system (Swenson etal., 1990), while another group found minimal effects bothfor Y265F and Y247F (Zhou et al., 1999; see also below). Incontrast, site-directed mutation analysis of Cx43 in mamma-lian cell systems suggested thatY265 is a major Src phospho-rylation target, and that this residue is involved in the Src-mediated closure of Cx43 gap junction channels (Giepmanset al., 2001; Lin et al., 2001). The results from Lin et al.(2001) suggested that Y247 is also phosphorylated by v-Src,this phosphorylation being as important as Y265 phosphory-lation. In contrast to these controversies, there is generalagreement that Src interacts with Cx43 through its SH3domain, probably using the second polyproline area (resi-dues 274–284) as target sequence (Kanemitsu et al., 1997;


Zhou et al., 1999; Giepmans et al., 2001). The Cx43–Srcassociation may be further stabilized by the Src SH2 domainbinding to the phosphorylatedY265 (Kanemitsu et al., 1997).

Investigations in Xenopus oocytes suggested that some ofthe effects of v-Src-induced decrease in conductance couldbe mediated through MAP kinases (Zhou et al., 1999), as themutations of Sers or Pros in the area 253–282 counteractedthe effects of v-Src. It should be noted that some of themutations caused decreased binding of v-Src to Cx43, al-though mutations of Y247 and Y265 were more efficient indecreasing the interaction (Zhou et al., 1999). The use of theMEK1 inhibitor PD98059 also counteracted v-Src in Xeno-pus oocytes (Zhou et al., 1999), but not in mammalian cells(Lin et al., 2001).

Constitutively, active versions of Src are well known to beoncogenic in experimental systems, and a part of the onco-genic effect may be mediated through its effect on Cx43 gapjunction channels. Wild-type c-Src is activated by severalphysiological and pathological conditions. Agonists of anumber of G-protein coupled receptors activated c-Src andcaused a decrease in cell–cell coupling, but did not causetyrosine phosphorylation of Cx43 (Postma et al., 1998). Incontrast, c-Src may directly be involved in the reduced elec-trical coupling between myocytes in hypertrophic cardiomy-opathy. In the late stages of cardiomyopathy, Cx43 was foundto be tyrosine phosphorylated concurrent with an increasedc-Src activity (Toyofuku et al., 1999).

The protein tyrosine-phosphatase inhibitors pervanadateand permolybdate had profound effects on both Cx43 phos-phorylation and GJIC in fibroblasts (Husøy et al., 1993;Mikalsen and Kaalhus, 1996, 1997). They induced tyrosinephosphorylation of Cx43 (Mikalsen et al., 1997), but thephosphorylation sites are not known. It is possible that thesecompounds reveal a rapid cycle of tyrosine phosphorylation–dephosphorylation of Cx43, but it is more likely that theyinduce an indirect activation of tyrosine kinases, which thenphosphorylate Cx43 (Mikalsen and Kaalhus, 1998). Thisimplies that under normal conditions, there is no cycle oftyrosine phosphorylation–dephosphorylation of Cx43. Phos-photyrosine in Cx43 is usually not found in control cells(e.g., Crow et al., 1990; Filson et al., 1990; Kurata and Lau,1994; Kanemitsu et al., 1997; Mikalsen et al., 1997), al-though there have been a few such claims (Zhang et al., 1999;Yao et al., 2000). High level of oxidative stress, e.g., experi-mentally induced by H2O2, may also cause tyrosine phospho-rylation of Cx43 (Mikalsen et al., 1997). Ischemia and reoxy-genation processes occurring under pathological conditions(heart attack, brain stroke) cause oxidative stress. This mayexplain the induction of phosphotyrosine in Cx43 found inhuman umbilical vein endothelial cells after a procedure ofhypoxia and reoxygenation (Zhang et al., 1999).

Pervanadate significantly decreased electrical conduc-tance between Cx45-transfected HeLa cells, and it increasedthe amount of phosphorylated Cx45 (van Veen et al., 2000). Itwas not studied if this was due to tyrosine phosphorylation. A

trace of phosphotyrosine in Cx45 was previously found intransfected HeLa cells (Hertlein et al., 1998).

2.9. Unidentified kinases

In spite of the efforts taken to identify kinases involved inphosphorylation of connexins, especially Cx43, some ofthem remain elusive. The kinase(s) involved in the genera-tion of the phospho-connexins under basal cell culture con-ditions or under physiological conditions in the tissues are ingeneral unknown; so are the phosphorylation sites underthese conditions, even for Cx43. It has been suggested thatthere is a hierarchical phosphorylation sequence of Cx43,with the first phosphorylation occurring in the epitope of themonoclonal antibody, 13–8300, as this antibody recognizesonly NP form of Cx43 in rat heart cells and canine smoothmuscle cells (Nagy et al., 1997), but also the minor phospho-rylation form called P' is recognized in non-stimulated V79cells (Cruciani and Mikalsen, 1999). The 13–8300 epitopeencompasses 360–376, and thus includes some potentialPKC phosphorylation sites (see above). However, TPA didnot cause a further diminished recognition of Cx43 in west-ern blots probed by 13–8300 even under conditions whereCx43 clearly gets phosphorylated in response to TPA (Cru-ciani and Mikalsen, 1999). Peptide competition experimentssuggested that the epitope is rather N-terminal in this se-quence, and includes S364 and/or S365 (Cruciani and Mi-kalsen, 1999). The phosphorylation of S364 was recentlyconfirmed by another group (TenBroek et al., 2001). Interest-ingly, several mutations of which S364P and R(362,376)Qwere the most frequent, were detected in Cx43 from patientswith heart heterotaxia and from the hypoplastic heart, respec-tively (Britz-Cunningham et al., 1995; Dasgupta et al., 2001).When the S364P mutant of Cx43 was transfected into non-communicating L929 cells, a low level of GJIC wasachieved, and it was unaffected by PKA in contrast to theincrease in cells transfected with wild-type Cx43 (Britz-Cunningham et al., 1995). Furthermore, PKC caused anincrease in GJIC in mutant cells, but not in cells transfectedwith wild-type Cx43 (Britz-Cunningham et al., 1995). Thus,this area of Cx43 seems involved in the regulation of GJIC. Itis therefore intriguing that Cx43 in chick has Pro, fish hasCys, and Xenopus has Ser (similar to mammals) at position364. The S364P mutant caused a significant fraction of Xeno-pus embryos to develop a symptom similar to the humanheterotaxia (Levin and Mercola, 1998). This could suggest adifferent regulation of Cx43 channels depending on the spe-cies.

Mice Cx45 with site-directed mutations of Sers in thesequence 374-394 were transfected into HeLa cells. Theresults suggested that S374/376/378/381/382 are the major,but not exclusive, phosphorylation sites, and that the phos-phorylations may show cooperativity. The double mutantsS381G/S382A or S384G/S385L showed considerably in-creased degradation rates, suggesting that phospho-Cx45 ismore stable (Hertlein et al., 1998).


3. Concluding remarks

Future aims are to understand the regulation of GJIC, theimportance of connexin phosphorylation, and potential non-GJIC roles of connexins. The bewildering array of connex-ins, of which several are often coexpressed in a cell, theirnumerous phosphorylation sites, their interactions with ac-cessory proteins, and their confusingly different regulationsin different cell types make it difficult to obtain a unifiedpicture of the field. This is underlined by the increasingnumber of diverse pathological conditions where mutationsin connexin genes are found.

References

Ai, Z.W., Fischer, A., Spray, D.C., Brown, A.M.C., Fishman, G.I., 2000.Wnt-1 regulation of connexin 43 in cardiac myocytes. J. Clin. Invest.105, 161–171.

Amsterdam, A., Knecht, M., Catt, K.J., 1981. Hormonal regulation ofcytodifferentiation and intercellular communication in cultured granu-losa cells. Proc. Natl. Acad. Sci. USA 78, 3000–3004.

Atkinson, M.M., Lampe, P.D., Lin, H.H., Kollander, R., Li, X.R.,Kiang, D.T., 1995. Cyclic AMP modifies the cellular distribution ofconnexin 43 and induces a persistent increase in the junctional perme-ability of mouse mammary tumor cells. J. Cell Sci. 108, 3079–3090.

Atkinson, M.M., Menko, A.S., Johnson, R.G., Sheppard, J.R., Sheri-dan, J.D., 1981. Rapid and reversible reduction of junctional permeabil-ity in cells infected with a temperature-sensitive mutant of avian sarcomavirus. J. Cell Biol. 91, 573–578.

Azarnia, R., Reddy, S., Kmiecik, T.E., Shalloway, D., Loewenstein, W.R.,1988. The cellular src gene product regulates junctional cell-to-cellcommunication. Science 239, 398–401.

Bastide, B., Hervé, J.C., Délèze, J., 1994. The uncoupling effect of diacylg-lycerol on gap junctional communication of mammalian heart cells isindependent of protein kinase C. Exp. Cell Res. 214, 519–527.

Beardslee, M.A., Lerner, D.L., Tadros, P.N., Laing, J.G., Beyer, E.C.,Yamada, K.A., Kléber, A.G., Schuessler, R.B., Saffitz, J.E., 2000.Dephosphorylation and intracellular redistribution of ventricular con-nexin 43 during electrical uncoupling induced by ischemia. Circ. Res.87, 656–662.

Belcheva, M.M., Coscia, C.J., 2002. Diversity of G protein-coupled receptorsignaling pathways to ERK/MAP kinase. Neurosignals 11, 34–44.

Berthoud, V.M., Beyer, E.C., Kurata, W.E., Lau, A.F., Lampe, P.D., 1997.The gap-junction protein connexin 56 is phosphorylated in the intracel-lular loop and the carboxy-terminal region. Eur. J. Biochem. 244, 89–97.

Berthoud, V.M., Ledbetter, M.L.S., Hertzberg, E.L., Sáez, J.C., 1992. Con-nexin 43 in MDCK cells: regulation by a tumor-promoting phorbol esterand Ca2+. Eur. J. Cell Biol. 57, 40–50.

Berthoud, V.M., Westphale, E.M., Grigoryeva, A., Beyer, E.C., 2000. PKCisoenzymes in the chicken lens and TPA-induced effects on intercellularcommunication. Invest. Ophthalmol. Vis. Sci. 41, 850–858.

Bevans, C.G., Harris, A.L., 1999. Direct high affinity modulation of con-nexin channel activity by cyclic nucleotides. J. Biol. Chem. 274,3720–3725.

Beyer, E.C., Paul, D.L., Goodenough, D.A., 1987. Connexin 43: a proteinfrom rat heart homologous to a gap junction protein from liver. J. CellBiol. 105, 2621–2629.

Bolaños, J.P., Medina, J.M., 1996. Induction of nitric oxide synthase inhibitsgap junction permeability in cultured rat astrocytes. J. Neurochem. 66,2091–2099.

Bowling, N., Huang, X., Sandusky, G.E., Fouts, R.L., Mintze, K., Ester-man, M., Allen, P.D., Maddi, R., McCall, E., Vlahos, C.J., 2001. Proteinkinase C-a and -e modulate connexin 43 phosphorylation in humanheart. J. Mol. Cell Cardiol. 33, 789–798.

Brandes, R.P., Popp, R., Ott, G., Bredenkötter, D., Wallner, C., Busse, R.,Fleming, I., 2002. The extracellular regulated kinases (ERK) 1/2 mediatecannabinoid-induced inhibition of gap junctional communication inendothelial cells. Br. J. Pharmacol. 136, 709–716.

Britz-Cunningham, S.H., Shah, M.M., Zuppan, C.W., Fletcher, W.H., 1995.Mutations of the connexin 43 gap-junction gene in patients with heartmalformations and defects of laterality. N. Engl. J. Med. 332,1323–1329.

Burghardt, R.C., Barhoumi, R., Sewall, T.C., Bowen, J.A., 1995. CyclicAMP induces rapid increases in gap junction permeability and changesin the cellular distribution of connexin 43. J. Membr. Biol. 148, 243–253.

Burt, J.M., Spray, D.C., 1988. Inotropic agents modulate gap junctionalconductance between cardiac myocytes. Am. J. Physiol. 254,H1206–H1210.

Bånrud, H., Mikalsen, S.O., Berg, K., Moan, J., 1994. Effects of ultravioletradiation on intercellular communication in V79 Chinese hamster fibro-blasts. Carcinogenesis 15, 233–239.

Cheng, H.L., Louis, C.F., 1999. Endogenous casein kinase I catalyzes thephosphorylation of the lens fiber cell connexin 49. Eur. J. Biochem. 263,276–286.

Cheng, H.L., Louis, C.F., 2001. Functional effects of casein kinaseI-catalyzed phosphorylation on lens cell-to-cell coupling. J. Membr.Biol. 181, 21–30.

Cole, W.C., Garfield, R.E., 1986. Evidence for physiological regulation ofmyometrial gap junction permeability. Am. J. Physiol. 251, C411–C420.

Crow, D.S., Beyer, E.C., Paul, D.L., Kobe, S.S., Lau, A.F., 1990. Phospho-rylation of connexin 43 gap junction protein in uninfected and Roussarcoma virus-transformed mammalian fibroblasts. Mol. Cell. Biol. 10,1754–1763.

Cruciani, V., Husøy, T., Mikalsen, S.O., 2001. Pharmacological evidence forsystem-dependent involvement of protein kinase C isoenzymes in phor-bol ester-suppressed gap junctional communication. Exp. Cell Res. 268,150–161.

Cruciani, V., Kaalhus, O., Mikalsen, S.O., 1999. Phosphatases involved inmodulation of gap junctional intercellular communication and dephos-phorylation of connexin 43 in hamster fibroblasts: 2B or not 2B?. Exp.Cell Res. 252, 449–463.

Cruciani, V., Mikalsen, S.O., 1999. Stimulated phosphorylation of intracel-lular connexin 43. Exp. Cell Res. 251, 285–298.

Cruciani, V., Mikalsen, S.O., 2002. Mechanisms involved in responses to theperoxisome proliferator WY-14,643 on gap junctional intercellular com-munication in V79 hamster fibroblasts. Toxicol. Appl. Pharmacol. 181,66–75.

Cruciani, V., Mikalsen, S.O., Vasseur, P., Sanner, T., 1997. Effects of peroxi-some proliferators and 12-O-tetradecanoyl phorbol-13-acetate on inter-cellular communication and connexin 43 in two hamster fibroblast sys-tems. Int. J. Cancer 73, 240–248.

Dasgupta, C., Martinez, A.M., Zuppan, C.W., Shah, M.M., Bailey, L.L.,Fletcher, W.H., 2001. Identification of connexin 43 (a1) gap junctiongene mutations in patients with hypoplastic left heart syndrome bydenaturing gradient gel electrophoresis (DGGE). Mutat. Res. 479,173–186.

De Pina-Benabou, M.H., Srinivas, M., Spray, D.C., Scemes, E., 2001.Calmodulin kinase pathway mediates the K+-induced increase in gapjunctional communication between mouse spinal cord astrocytes. J.Neurosci. 21, 6635–6643.

Dermietzel, R., Kremer, M., Paputsoglu, G., Stang, A., Skerrett, I.M.,Gomes, D., Srinivas, M., Janssen-Bienhold, U., Weiler, R., Nichol-son, B.J., Bruzzone, R., Spray, D.C., 2000. Molecular and functionaldiversity of neural connexins in the retina. J. Neurosci. 20, 8331–8343.

Dewey, M.M., Barr, L., 1962. Intercellular connections between smoothmuscle cells: the nexus. Science 137, 670–672.


Diez, J.A., Elvira, M., Villalobo, A., 1998. The epidermal growth factorreceptor tyrosine kinase phoshorylates connexin 32. Mol. Cell. Bio-chem. 187, 201–210.

Doble, B.W., Chen, Y.J., Bose, D.G., Litchfield, D.W., Kardami, E., 1996.Fibroblast growth factor-2 decreases metabolic coupling and stimulatesphosphorylation as well as masking of connexin 43 epitopes in cardiacmyocytes. Circ. Res. 79, 647–658.

Doble, B.W., Kardami, E., 1995. Basic fibroblast growth factor stimulatesconnexin 43 expression and intercellular communication of cardiacfibroblasts. Mol. Cell Biochem. 143, 81–87.

Doble, B.W., Ping, P., Kardami, E., 2000. The e subtype of protein kinase Cis required for cardiomyocyte connexin 43 phosphorylation. Circ. Res.86, 293–301.

Elvira, M., Diez, J.A., Wang, K.K., Villalobo, A., 1993. Phosphorylation ofconnexin 32 by protein kinase C prevents its proteolysis by µ-calpain andm-calpain. J. Biol. Chem. 268, 14294–14300.

Enomoto, T., Yamasaki, H., 1985. Rapid inhibition of intercellular commu-nication between BALB/c 3T3 cells by diacylglycerol, a possible endog-enous functional analogue of phorbol esters. Cancer Res. 45,3706–3710.

Faust, M., Montenarh, M., 2000. Subcellular localization of protein kinaseCK2. A key to its function?. Cell Tissue Res. 301, 329–340.

Filson, A.J., Azarnia, R., Beyer, E.C., Loewenstein, W.R., Brugge, J.S.,1990. Tyrosine phosphorylation of a gap junction protein correlates withinhibition of cell-to-cell communication. Cell Growth Differ. 1,661–668.

Flagg-Newton, J.L., Dahl, G., Loewenstein, W.R., 1981. Cell junction andcyclic AMP: 1. Upregulation of junctional membrane permeability andjunctional membrane particles by administration of cyclic nucleotide orphosphodiesterase inhibitor. J. Membr. Biol. 63, 105–121.

Furshpan, E.J., Potter, D.D., 1959. Transmission at the giant motor synapsesof the crayfish. J. Physiol. 145, 289–325.

Giepmans, B.N., Hengeveld, T., Postma, F.R., Moolenaar, W.H., 2001.Interaction of c-Src with gap junction protein connexin 43: role in theregulation of cell–cell communication. J. Biol. Chem. 276, 8544–8549.

Godwin, A.J., Green, L.M., Walsh, M.P., McDonald, J.R., Walsh, D.A.,Fletcher, W.H., 1993. In situ regulation of cell-cell communication bythe cAMP-dependent protein kinase and protein kinase C. Mol. CellBiochem. 127-128, 293–307.

Goodall, H., Maro, B., 1986. Major loss of junctional coupling duringmitosis in early mouse embryos. J. Cell Biol. 102, 568–575.

Granot, I., Dekel, N., 1994. Phosphorylation and expression of connexin 43ovarian gap junction protein are regulated by luteinizing hormone. J.Biol. Chem. 269, 30502–30509.

Gross, S.D., Anderson, R.A., 1998. Casein kinase I: spatial organization andpositioning of a multifunctional protein kinase family. Cell. Signal. 10,699–711.

Hax, W.M.A., van Venrooij, G.E.P.M., Vossenberg, J.B.J., 1974. Cellcommunication: a cyclic AMP medicated phenomenon. J. Membr. Biol.19, 253–266.

Hertlein, B., Butterweck, A., Haubrich, S., Willecke, K., Traub, O., 1998.Phosphorylated carboxy terminal serine residues stabilize the mouse gapjunction protein connexin 45 against degradation. J. Membr. Biol. 162,247–257.

Hossain, M.Z., Ao, P., Boynton, A.L., 1998. Rapid disruption of gap junc-tional communication and phosphorylation of connexin 43 by platelet-derived growth factor in T51B rat liver epithelial cells expressingplatelet-derived growth factor receptor. J. Cell. Physiol. 174, 66–77.

Hossain, M.Z., Jagdale, A.B., Ao, P., Kazlauskas, A., Boynton, A.L., 1999.Disruption of gap junctional communication by the platelet-derivedgrowth factor is mediated via multiple signaling pathways. J. Biol.Chem. 274, 10489–10496.

Husøy, T., Cruciani, V., Sanner, T., Mikalsen, S.O., 2001. Phosphorylation ofconnexin 43 and inhibition of gap junctional communication in 12-O-tetradecanoylphorbol-13-acetate-exposed R6 fibroblasts: minor role ofprotein kinase CbI and µ. Carcinogenesis 22, 221–231.

Husøy, T., Mikalsen, S.O., Sanner, T., 1993. Phosphatase inhibitors, gapjunctional intercellular communication and [125I]-EGF binding in ham-ster fibroblasts. Carcinogenesis 14, 2257–2265.

Jiang, J.X., Goodenough, D.A., 1998. Phosphorylation of lens-fiber connex-ins in lens organ cultures. Eur. J. Biochem. 255, 37–44.

Kanemitsu, M.Y., Jiang, W., Eckhart, W., 1998. Cdc2-mediated phosphory-lation of the gap junction protein, connexin 43, during mitosis. CellGrowth Differ. 9, 13–21.

Kanemitsu, M.Y., Lau, A.F., 1993. Epidermal growth factor stimulates thedisruption of gap junctional communication and connexin 43 phospho-rylation independent of 12-O-tetradecanoylphorbol 13-acetate-sensitiveprotein kinase C: the possible involvement of mitogen-activated proteinkinase. Mol. Biol. Cell 4, 837–848.

Kanemitsu, M.Y., Loo, L.W.M., Simon, S., Lau, A.F., Eckhart, W., 1997.Tyrosine phosphorylation of connexin 43 by v-Src is mediated by SH2and SH3 domain interactions. J. Biol. Chem. 272, 22824–22831.

Kanno, L., Loewenstein, W.R., 1964. Intercellular diffusion. Science 143,959–960.

Kardami, E., Stoski, R.M., Doble, B.W., Yamamoto, T., Hertzberg, E.L.,Nagy, J.I., 1991. Biochemical and ultrastructural evidence for the asso-ciation of basic fibroblast growth factor with cardiac gap junctions. J.Biol. Chem. 266, 19551–19557.

Karrer, H.E., 1960. Cell interconnections in normal human cervical epithe-lium. J. Biophys. Biochem. Cytol. 7, 181–185.

Koo, S.K., Kim, D.Y., Park, S.D., Kang, K.W., Joe, C.O., 1997. PKCphosphorylation disrupts gap junctional communication at G0/S phase inclone 9 cells. Mol. Cell. Biochem. 167, 41–49.

Kumar, N.M., Gilula, N.B., 1986. Cloning and characterization of humanand rat liver cDNAs coding for a gap junction protein. J. Cell Biol. 103,767–776.

Kurata, W.E., Lau, A.F., 1994. p130gag-fps disrupts gap junctional communi-cation and induces phosphorylation of connexin 43 in a manner similarto that of pp60v-src. Oncogene 9, 329–335.

Kwak, B.R., Hermans, M.M.P., De Jonge, H.R., Lohmann, S.M.,Jongsma, H.J., Chanson, M., 1995a. Differential regulation of distincttypes of gap junction channels by similar phosphorylating conditions.Mol. Biol. Cell 6, 1707–1719.

Kwak, B.R., Sáez, J.C., Wilders, R., Chanson, M., Fishman, G.I.,Hertzberg, E.L., Spray, D.C., Jongsma, H.J., 1995b. Effects of cGMP-dependent phosphorylation on rat and human connexin 43 gap junctionchannels. Pflügers Arch. 430, 770–778.

Kwak, B.R., van Veen, T.A.B., Analbers, L.J.S., Jongsma, H.J., 1995c. TPAincreases conductance but decreases permeability in neonatal rat cardi-omyocyte gap junction channels. Exp. Cell Res 220, 456–463.

Lampe, P.D., Kurata, W.E., Warn-Cramer, B.J., Lau, A.F., 1998. Formationof a distinct connexin 43 phosphoisoform in mitotic cells is dependentupon p34cdc2kinase. J. Cell Sci. 111, 833–841.

Lampe, P.D., TenBroek, E.M., Burt, J.M., Kurata, W.E., Johnson, R.G.,Lau, A.F., 2000. Phosphorylation of connexin 43 on serine368 by proteinkinase C regulates gap junctional communication. J. Cell Biol. 149,1503–1512.

Le, A.C.N., Musil, L.S., 2001. A novel role for FGF and extracellularsignal-regulated kinase in gap junction-mediated intercellular communi-cation in the lens. J. Cell Biol. 154, 197–216.

Levin, M., Mercola, M., 1998. Gap junctions are involved in the earlygeneration of left-right asymmetry. Dev. Biol. 203, 90–105.

Lin, R., Warn-Cramer, B.J., Kurata, W.E., Lau, A.F., 2001. v-Src phospho-rylation of connexin 43 on Tyr247 and Tyr265 disrupts gap junctionalcommunication. J. Cell Biol. 154, 815–828.

Maldonado, P.E., Rose, B., Loewenstein, W.R., 1988. Growth factors modu-late junctional cell-to-cell communication. J. Membr. Biol. 106,203–210.

Matesic, D.F., Rupp, H.L., Bonney, W.J., Ruch, R.J., Trosko, J.E., 1994.Changes in gap-junction permeability, phosphorylation, and numbermediated by phorbol ester and non-phorbol-ester tumor promoters in ratliver epithelial cells. Mol. Carcinogen. 10, 226–236.


Mazzoleni, G., Camplani, A., Telo, P., Pozzi, A., Tanganelli, S., Elfgang, C.,Willecke, K., Ragnotti, G., 1996. Effect of tumor-promoting and anti-promoting chemicals on the viability and junctional coupling of humanHeLa cells transfected with DNAs coding for various murine connexinproteins. Comp. Biochem. Physiol. 113C, 247–256.

Mikalsen, S.O., Husøy, T., Vikhamar, G., Sanner, T., 1997. Induction ofphosphotyrosine in the gap junction protein, connexin 43. FEBS Lett.401, 271–275.

Mikalsen, S.O., Kaalhus, O., 1996. A characterization of pervanadate, aninducer of cellular tyrosine phosphorylation and inhibitor of gap junc-tional intercellular communication. Biochim. Biophys. Acta 1290,308–318.

Mikalsen, S.O., Kaalhus, O., 1997. A characterization of permolybdate andits effect on cellular tyrosine phosphorylation, gap junctional intercellu-lar communication and phosphorylation status of the gap junction pro-tein, connexin 43. Biochim. Biophys. Acta 1356, 207–220.

Mikalsen, S.O., Kaalhus, O., 1998. Properties of pervanadate and permolyb-date. Connexin 43, phosphatase inhibition, and thiol reactivity as modelsystems. J. Biol. Chem. 273, 10036–10045.

Moore, L.K., Beyer, E.C., Burt, J.M., 1991. Characterization of gap junctionchannels in A7r5 vascular smooth muscle cells. Am. J. Physiol. 260,C975–C981.

Moreno, A.P., Campos de Carvalho, A.C., Christ, G., Melman, A.,Spray, D.C., 1993. Gap junctions between human corpus cavernosumsmooth muscle cells: gating properties and unitary conductance. Am. J.Physiol. 264, C80–C92.

Moreno,A.P., Sáez, J.C., Fishman, G.I., Spray, D.C., 1994. Human connexin43 gap junction channels. Regulation of unitary conductances by phos-phorylation. Circ. Res. 74, 1050–1057.

Murray, A.W., Fitzgerald, D.J., 1979. Tumour promoters inhibit metaboliccooperation in cocultures of epidermal and 3T3 cells. Biochem. Bio-phys. Res. Commun. 91, 395–401.

Nadarajah, B., Makarenkova, H., Becker, D.L., Evans, W.H., Parnave-las, J.G., 1998. Basic FGF increases communication between cells of thedeveloping neocortex. J. Neurosci. 18, 7881–7890.

Nagy, J.I., Li, W.E.I., Roy, C., Doble, B.W., Gilchrist, J.S., Kardami, E.,Hertzberg, E.L., 1997. Selective monoclonal antibody recognition andcellular localization of an unphosphorylated form of connexin 43. Exp.Cell Res. 236, 127–136.

O’Brien, J., Bruzzone, R., White, T.W., Al-Ubaidi, M.R., Ripps, H., 1998.Cloning and expression of two related connexins from the perch retinadefine a distinct subgroup of the connexin family. J. Neurosci. 18,7625–7637.

O’Lague, P., Dalen, H., Rubin, H., Tobias, C., 1970. Electrical coupling: lowresistance junctions between mitotic and interphase fibroblasts in tissueculture. Science 170, 464–466.

Oh, S.Y., Grupen, C.G., Murray, A.W., 1991. Phorbol ester induces phos-phorylation and down-regulation of connexin 43 in WB cells. Biochim.Biophys. Acta 1094, 243–245.

Olson, D.J., Christian, J.L., Moon, R.T., 1991. Effect of Wnt-1 and relatedproteins on gap junctional communication in Xenopus embryos. Science252, 1173–1176.

Paulson, A.F., Lampe, P.D., Meyer, R.A., TenBroek, E., Atkinson, M.M.,Walseth, T.F., Johnson, R.G., 2000. Cyclic AMP and LDL trigger a rapidenhancement in gap junction assembly through a stimulation of con-nexin trafficking. J. Cell Sci. 113, 3037–3049.

Pelletier, D.B., Boynton, A.L., 1994. Dissociation of PDGF receptortyrosine kinase activity from PDGF-mediated inhibition of gap junc-tional communication. J. Cell. Physiol. 158, 427–434.

Pepper, M.S., Meda, P., 1992. Basic fibroblast growth factor increasesjunctional communication and connexin 43 expression in microvascularendothelial cells. J. Cell. Physiol. 153, 196–205.

Peracchia, C., Sotkis, A., Wang, X.G., Peracchia, L.L., Persechini, A., 2000.Calmodulin directly gates gap junction channels. J. Biol. Chem. 275,26220–26224.

Pereda, A.E., Bell, T.D., Chang, B.H., Czernik, A.J., Nairn, A.C., Soder-ling, T.R., Faber, D.S., 1998. Ca2+/calmodulin-dependent kinase II medi-ates simultaneous enhancement of gap-junctional conductance andglutamatergic transmission. Proc. Natl. Acad. Sci. USA 95,13272–13277.

Peters, J.M., McKay, R.M., McKay, J.P., Graff, J.M., 1999. Casein kinase Itransduces Wnt signals. Nature 401, 345–350.

Postma, F.R., Hengeveld, T., Alblas, J., Giepmans, B.N.G., Zondag, G.C.M.,Jalink, K., Moolenaar, W.H., 1998. Acute loss of cell-cell communica-tion caused by G protein-coupled receptors: A critical role for c-Src. J.Cell Biol. 140, 1199–1209.

Reuss, B., Dermietzel, R., Unsicker, K., 1998. Fibroblast growth factor 2(FGF-2) differentially regulates connexin (cx) 43 expression and func-tion in astroglial cells from distinct brain regions. Glia 22, 19–30.

Rivedal, E., Mollerup, S., Haugen, A., Vikhamar, G., 1996. Modulation ofgap junctional intercellular communication by EGF in human kidneyepithelial cells. Carcinogenesis 17, 2321–2328.

Rivedal, E., Opsahl, H., 2001. Role of PKC and MAP kinase in EGF- andTPA-induced connexin 43 phosphorylation and inhibition of gap junc-tion intercellular communication in rat liver epithelial cells. Carcinogen-esis 22, 1543–1550.

Robertson, J.D., 1957. New observations on the ultrastructure of the mem-branes of frog peripheral nerves fibers. J. Biophys. Biochem. Cytol. 3,1043–1051.

Ruch, R.J., Trosko, J.E., Madhukar, B.V., 2001. Inhibition of connexin 43gap junctional intercellular communication by TPA requires ERK acti-vation. J. Cell. Biochem. 83, 163–169.

Saleh, S.M., Takemoto, D.J., 2000. Overexpression of protein kinase Ccinhibits gap junctional intercellular communication in the lens epithelialcells. Exp. Eye Res. 71, 99–102.

Saleh, S.M., Takemoto, L.J., Zoukhri, D., Takemoto, D.J., 2001. PKC-cphosphorylation of connexin 46 in the lens cortex. Mol. Vis. 7, 240–246.

Salomon, D., Chanson, M., Vischer, S., Masgrau, E., Vozzi, C., Saurat, J.H.,Spray, D.C., Meda, P., 1992. Gap junctional communication of primaryhuman keratinocytes: characterization by dual voltage clamp and dyetransfer. Exp. Cell Res. 201, 452–461.

Sáez, J.C., Berthoud, V.M., Moreno, A.P., Spray, D.C., 1993a. Gapjunctions: multiplicity of controls in differentiated and undifferentiatedcells and possible functional implications. Adv. Second MessengerPhosphoprotein Res. 27, 163–198.

Sáez, J.C., Nairn, A.C., Czernik, A.J., Fishman, G.I., Spray, D.C.,Hertzberg, E.L., 1997. Phosphorylation of connexin 43 and the regula-tion of neonatal rat cardiac myocyte gap junctions. J. Mol. Cell. Cardiol.29, 2131–2145.

Sáez, J.C., Nairn, A.C., Czernik, A.J., Spray, D.C., Hertzberg, E.L., 1993b.Rat connexin 43: regulation by phosphorylation in heart. Prog. Cell Res.3, 275–281.

Sáez, J.C., Nairn, A.C., Czernik, A.J., Spray, D.C., Hertzberg, E.L., Green-gard, P., Bennett, M.V.L., 1990. Phosphorylation of connexin 32, ahepatocyte gap-junction protein, by cAMP-dependent protein kinase,protein kinase C and Ca2+/calmodulin-dependent protein kinase II. Eur.J. Biochem. 192, 263–273.

Sáez, J.C., Spray, D.C., Nairn, A.C., Hertzberg, E., Greengard, P., Ben-nett, M.V., 1986. cAMP increases junctional conductance and stimulatesphosphorylation of the 27-kDa principal gap junction polypeptide. Proc.Natl. Acad. Sci. USA 83, 2473–2477.

Severs, N.J., Rothery, S., Dupont, E., Coppen, S.R., Yeh, H.I., Ko, Y.S.,Matsushita, T., Kaba, R., Halliday, D., 2001. Immunocytochemicalanalysis of connexin expression in the healthy and diseased cardiovas-cular system. Microsc. Res. Tech. 52, 301–322.

Simpson, I., Rose, B., Loewenstein, W.R., 1977. Size limit of moleculespermeating the junctional membrane channels. Science 195, 294–296.

Sladek, S.M., Westerhausen-Larson, A., Roberts, J.M., 1999. Endogenousnitric oxide suppresses rat myometrial connexin 43 gap junction proteinexpression during pregnancy. Biol. Reprod. 61, 8–13.


Stagg, R.B., Fletcher, W.H., 1990. The hormone-induced regulation ofcontact-dependent cell–cell communication by phosphorylation.Endocr. Rev. 11, 302–325.

Stein, L.S., Boonstra, J., Burghardt, R.C., 1992. Reduced cell–cell commu-nication between mitotic and nonmitotic coupled cells. Exp. Cell Res.198, 1–7.

Steinberg, T.H., Civitelli, R., Geist, S.T., Robertson, A.J., Hick, E., Veen-stra, R.D., Wang, H.Z., Warlow, P.M., Westphale, E.M., Laing, J.G.,Beyer, E.C., 1994. Connexin 43 and connexin 45 form gap junctions withdifferent molecular permeabilities in osteoblastic cells. EMBO J. 13,744–750.

Stoker, M.G.P., 1967. Transfer of growth inhibition between normal andvirus-transformed cells: autoradiographic studies using marked cells. J.Cell Sci. 2, 293–304.

Swenson, K.I., Piwnica-Worms, H., McNamee, H., Paul, D.L., 1990.Tyrosine phosphorylation of the gap junction protein connexin 43 isrequired for the pp60v-src-induced inhibition of communication. CellRegul. 1, 989–1002.

Takeda, A., Hashimoto, E.,Yamamura, H., Shimazu, T., 1987. Phosphoryla-tion of liver gap junction protein by protein kinase C. FEBS Lett. 210,169–172.

Takeda, A., Saheki, S., Shimazu, T., Takeuchi, N., 1989. Phosphorylation ofthe 27-kDa gap junction protein by protein kinase C in vitro and in rathepatocytes. J. Biochem. 106, 723–727.

Takens-Kwak, B.R., Jongsma, H.J., 1992. Cardiac gap junctions: threedistinct single channel conductances and their modulation by phospho-rylating treatments. Pflügers Arch. 422, 198–200.

TenBroek, E.M., Lampe, P.D., Solan, J.L., Reynhout, J.K., Johnson, R.G.,2001. Ser364 of connexin 43 and the upregulation of gap junctionassembly by cAMP. J. Cell Biol. 155, 1307–1318.

Teranishi, T., Negishi, K., Kato, S., 1983. Dopamine modulates S-potentialamplitude and dye-coupling between external horizontal cells in carpretina. Nature 301, 243–246.

Toyama, J., Sugiura, H., Kamiya, K., Kodama, I., Terasawa, M., Hidaka, H.,1994. Ca2+-calmodulin mediated modulation of the electrical couplingof ventricular myocytes isolated from guinea pig heart. J. Mol. Cell.Cardiol. 26, 1007–1015.

Toyofuku, T., Yabuki, M., Otsu, K., Kuzuya, T., Tada, M., Hori, M., 1999.Functional role of c-Src in gap junctions of the cardiomyopathic heart.Circ. Res. 85, 672–681.

Traub, O., Eckert, R., Lichtenberg-Fraté, H., Elfgang, C., Bastide, B.,Scheidtmann, K.H., Hülser, D.F., Willecke, K., 1994. Immunochemicaland electrophysiological characterization of murine connexin 40 and 43in mouse tissues and transfected human cells. Eur. J. Cell Biol. 64,101–112.

Török, K., Stauffer, K., Evans, W.H., 1997. Connexin 32 of gap junctionscontains two cytoplasmic calmodulin-binding domains. Biochem. J.326, 479–483.

Van der Heyden, M.A.G., Rook, M.B., Hermans, M.M.P., Rijksen, G.,Boonstra, J., Defize, L.H.K., Destrée, O.H.J., 1998. Identification ofconnexin 43 as a functional target for Wnt signalling. J. Cell Sci. 111,1741–1749.

van Rijen, H.V.M., van Veen, T.A.B., Hermans, M.M.P., Jongsma, H.J.,2000. Human connexin 40 gap junction channels are modulated bycAMP. Cardiovasc. Res. 45, 941–951.

van Veen, T.A., van Rijen, H.V., Jongsma, H.J., 2000. Electrical conductanceof mouse connexin 45 gap junction channels is modulated by phospho-rylation. Cardiovasc. Res. 46, 496–510.

Vikhamar, G., Rivedal, E., Mollerup, S., Sanner, T., 1998. Role of Cx43phosphorylation and MAP kinase activation in EGF induced enhance-ment of cell communication in human kidney epithelial cells. CellAdhes. Commun. 5, 451–460.

Volmat, V., Pouysségur, J., 2001. Spatiotemporal regulation of the p42/p44MAPK pathway. Biol. Cell 93, 71–79.

Wagner, T.L.E., Beyer, E.C., McMahon, D.G., 1998. Cloning and functionalexpression of a novel gap junction channel from the retina of Danioaquipinnatus. Visual Neurosci. 15, 1137–1144.

Warn-Cramer, B.J., Cottrell, G.T., Burt, J.M., Lau, A.F., 1998. Regulation ofconnexin 43 gap junctional intercellular communication by mitogen-activated protein kinase. J. Biol. Chem. 273, 9188–9196.

Warn-Cramer, B.J., Lampe, P.D., Kurata, W.E., Kanemitsu, M.Y.,Loo, L.W.M., Eckhart, W., Lau, A.F., 1996. Characterization of themitogen-activated protein kinase phosphorylation sites on the connexin43 gap junction protein. J. Biol. Chem. 271, 3779–3786.

White, T.W., Goodenough, D.A., Paul, D.L., 1998. Targeted ablation ofconnexin 50 in mice results in microphthalmia and zonular pulverulentcataracts. J. Cell Biol. 143, 815–825.

Wiener, E.C., Loewenstein, W.R., 1983. Correction of cell–cell communi-cation defect by introduction of a protein kinase into mutant cells. Nature305, 433–435.

Wilson, M.R., Close, T.W., Trosko, J.E., 2000. Cell population dynamics(apoptosis, mitosis, and cell–cell communication) during disruption ofhomeostasis. Exp. Cell Res. 254, 257–268.

Xie, H.Q., Laird, D.W., Chang, T.H., Hu, V.W., 1997. A mitosis-specificphosphorylation of the gap junction protein connexin 43 in humanvascular cells: biochemical characterization and localization. J. CellBiol. 137, 203–210.

Yada, T., Rose, B., Loewenstein, W.R., 1985. Diacylglycerol downregulatesjunctional membrane permeability. TMB-8 blocks this effect. J. Membr.Biol. 88, 217–232.

Yamamoto, T., Kardami, E., Nagy, J.I., 1991. Basic fibroblast growth factorin rat brain: localization to glial gap junctions correlates with connexin43 distribution. Brain Res. 554, 336–343.

Yamasaki, H., Enomoto, T., Martel, N., Shiba, Y., Kanno, Y., 1983. Tumourpromoter-mediated reversible inhibition of cell–cell communication(electrical coupling). Relationship with phorbol ester binding and denovo macromolecule synthesis. Exp. Cell Res. 146, 297–308.

Yao, J., Morioka, T., Oite, T., 2000. PDGF regulates gap junction commu-nication and connexin 43 phosphorylation by PI 3-kinase in mesangialcells. Kidney Int. 57, 1915–1926.

Yin, X., Gu, S., Jiang, J.X., 2001. The development-associated cleavage oflens connexin 45.6 by caspase-3-like protease is regulated by caseinkinase II-mediated phosphorylation. J. Biol. Chem. 276, 34567–34572.

Yin, X.Y., Jedrzejewski, P.T., Jiang, J.X., 2000. Casein kinase II phosphory-lates lens connexin 45.6 and is involved in its degradation. J. Biol. Chem.275, 6850–6856.

Yotti, L.P., Chang, C., Trosko, J.E., 1979. Elimination of metabolic coopera-tion in Chinese hamster cells by a tumor promoter. Science 206,1089–1091.

Ytrehus, K., Liu,Y., Downey, J.M., 1994. Preconditioning protects ischemicrabbit heart by protein kinase C activation. Am. J. Physiol. 266,H1145–H1152.

Zhang, Y.W., Morita, I., Nishida, M., Murota, S.I., 1999. Involvement oftyrosine kinase in the hypoxia/reoxygenation-induced gap junctionalintercellular communication abnormality in cultured human umbilicalvein endothelial cells. J. Cell. Physiol. 180, 305–313.

Zhou, L., Kasperek, E.M., Nicholson, B.J., 1999. Dissection of the molecu-lar basis of pp60v-src induced gating of connexin 43 gap junction chan-nels. J. Cell Biol. 144, 1033–1045.


connexins, gap junctional intercellular communication and kinases

Documents