Regulation of gap junctions by protein phosphorylation

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Phosphorylation of gap junctionsBrazilian Journal of Medical and Biological Research (1998) 31: 593-600ISSN 0100-879X

Regulation of gap junctions byprotein phosphorylation

Departamento de Ciencias Fisiológicas,Pontificia Universidad Católica de Chile, Santiago, Chile

J.C. Sáez,A.D. Martínez,

M.C. Brañes andH.E. González

Abstract

Gap junctions are constituted by intercellular channels and provide apathway for transfer of ions and small molecules between adjacentcells of most tissues. The degree of intercellular coupling mediated bygap junctions depends on the number of gap junction channels andtheir activity may be a function of the state of phosphorylation ofconnexins, the structural subunit of gap junction channels. Proteinphosphorylation has been proposed to control intercellular gap junc-tional communication at several steps from gene expression to proteindegradation, including translational and post-translational modifica-tion of connexins (i.e., phosphorylation of the assembled channelacting as a gating mechanism) and assembly into and removal from theplasma membrane. Several connexins contain sites for phosphoryla-tion for more than one protein kinase. These consensus sites varybetween connexins and have been preferentially identified in theC-terminus. Changes in intercellular communication mediated byprotein phosphorylation are believed to control various physiologicaltissue and cell functions as well as to be altered under pathologicalconditions.

CorrespondenceJ.C. SáezDepartamento de CienciasFisiológicas

Pontificia Universidad Católicade ChileAlameda 340SantiagoChile

Fax: (562) 222-5515E-mail: [email protected]

Presented at the XII Annual Meeting

of the Federação de Sociedades deBiologia Experimental, Caxambu,MG, Brasil, August 27-30, 1997.

J.C. Sáez, A.D. Martínez and

M.C. Brañes are recipients ofFONDECYT grants (Nos. 1960-559,2960-001 and 2960-002, respectively).

Received September 10, 1997Accepted September 22, 1997

Key words• Gap junctions• Connexins• Protein phosphorylation• Sites of phosphorylation• Protein kinases• Cell-to-cell communication

Introduction

Most cells, with the exception of fewtypes, such as spermatozoids, red blood cells,and skeletal muscle of adult vertebrates, cancommunicate to adjacent cells by gap junc-tions. These membrane specializations, alsoreferred to as nexuses or macula communi-cans, contain intercellular channels whichmediate movement of ions and small mol-ecules (<1.2 kDa) between contacting cells.Each channel is formed by two hemichan-nels or connexons and each one of them is

contributed by one of the two adjacent cells.A connexon is an oligomeric assembly of sixpolypeptide subunits termed connexins (Cxs)which are highly homologous and are en-coded by a gene family (1,2). While a par-ticular Cx (e.g., Cx43) can be expressed by awide spectrum of tissues and cell types (3),the expression of some other Cxs (e.g., Cx33,Cx50 and Cx30.1, found in testis, lenses andskin, respectively) is apparently much morerestricted (4,5).

Although Cx33 is unable to form func-tional homotypic channels when its tran-

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script is translated in Xenopus oocytes, usu-ally the expression of a single Cx type issufficient to establish intercellular gap junc-tional communication (2). Frequently, asingle cell can express more than one Cx,which can be localized in the same (6,7) or indifferent gap junction plaques (8). More-over, functional gap junctions can be estab-lished in an exogenous expression system(4,5,9-17) where the role of protein phos-phorylation in channels formed by a particu-lar Cx can be analyzed at the functionallevel.

The extent to which cells are functionallycoupled by gap junction channels dependson a multiplicity of control mechanisms,including gene transcription, message stabil-ity, translational and post-translational modi-fication of the protein, and assembly into andremoval from the membrane. In addition, anumber of factors affect gating of assembledchannels (1). Analysis of mechanism andregulation of each of these steps has becomea key element in the study of gap junctions.

Gap junction regulatory mechanisms canlead to an increase or reduction of intercellu-lar coupling with a wide spectrum of timecourses (from milliseconds to hours) (18).The turnover of Cx26, Cx32 and Cx43 isbetween 2 and 5 h (7,19-25). Hence, changesin intercellular coupling that occur within atime course of a few hours in cell typesexpressing these Cxs could involve any ofthe steps in Cx biosynthesis from transcrip-tion to degradation. It has been shown thatchanges in Cx mRNA transcription rate andmRNA stability can be affected by the acti-vation of intracellular second messenger path-ways and affect intercellular gap junctionalcommunication within a few hours. Althoughthese changes could involve protein phos-phorylation they will not be presented in thisarticle and readers are referred to reviewspublished elsewhere (1,18).

Formation of gap junctions also requiresappropriate cell adhesion mediated by eitherCa2+-dependent (NCAMs) or Ca2+- independ-

ent (cadherins) cell adhesion molecules(23,26-30). Cell lines deficient in cell adhe-sion molecules do not assemble gap junc-tions and Cx43 localizes in a perinuclearcytoplasmic compartment (23,29), where itis found preferentially in its unphosphory-lated form (23). Transfection with cDNAencoding a cell adhesion molecule inducesat least two changes in Cx43: it promotes i)the insertion of Cx43 into the plasma mem-brane and assembly into gap junctions (23,29)and ii) phosphorylation of Cx43 (23). Al-though phosphorylation might play a roleregulating the insertion and/or assembly of aphospho-Cx into the plasma membrane (23),this might not be an absolute requirement.For example, in exogenous expression sys-tems the expression of mRNAs encodingCx43 or Cx32 mutants with a shortenedcarboxyl terminal (devoid of all phosphory-latable seryl residues) is able to induce inter-cellular coupling (12,17). In addition, it hasbeen reported that in an MDCK cell line,while formation of gap junctions depends oncell contact, phosphorylation of Cx43 by aprotein kinase C-dependent pathway canoccur in the absence of Ca2+-dependent celladhesion activity (30) and does not correlatewith expression of intercellular coupling.

Phosphorylation of Cxs and possiblefunctional roles

Studies on the membrane topologyof Cxs have shown that both the aminoand carboxyl termini of the Cxs are locatedinside the cell (31-33). The carboxy terminalregion is one of the most divergent regionsamong Cxs and is where most of thephosphorylation sites have been identified(34-36). Thus, this region is likely toaccount for many Cx-specific properties, in-cluding responses to phosphorylation.Nonetheless, phosphorylation of aminoacid residues located in the cytoplasmic loopof Cx56 has been recently shown to occur(36).

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Cx26 has been reported not to be phos-phorylated in rat hepatocytes (7) or in iso-lated mouse gap junctions treated withcAMP-dependent kinase (cAMP-dPK) (7,34),protein kinase C (PKC) or Ca2+/calmodulin-dependent kinase II (Ca2+-CM-dPK II) (34).Presently, nine Cxs are known to be phos-phoproteins and their state of phosphoryla-tion can be affected by different proteinkinases (Table 1). Nonetheless, only fewphosphorylation sites and protein kinasesthat phosphorylate them have been identi-fied.

Various phospho-forms of Cx43 can beresolved by immunoblotting, making it auseful technique to evaluate its state of phos-phorylation (13-15,20,25,29,30,33,35,37,41,42,45-56). Nonetheless, not all changes inthe state of phosphorylation of Cx43 alterthe electrophoretic mobility of the protein(42). In addition, immunoblots do not allowfurther analysis of the different Cx43phospho-forms (i.e., amino acid analysis andtwo-dimensional tryptic maps). Therefore,analysis of the state of phosphorylation ofCxs, including Cx43, requires complemen-tary studies with metabolic radiolabellingfollowed by immunoprecipitation.

Cyclic nucleotides, diacylglycerol, lyso-phosphatidic acid, tumor promoter phorbolesters, growth factors, and oncogeneproducts (pp60v-src, p130gag-fps and rasoncogen) alter intercellular gap junctionalcommunication and effect of some theseagents depends on cell and Cx type (13,16,20,30,34-36,38,41-47,51-71). It has also beensuggested that phosphorylation of Cx43 inseryl residues might be involved in a numberof different processes including its insertioninto the plasma membrane (56), increase inits degradation, changes in unitary conduc-tance of single gap junction channels andclosure of gap junction channels (13,16,45,57,61,62). In addition, phosphorylationof tyrosine residues has been associatedwith reduction in intercellular coupling(10,20).

Effect of Cx32 phosphorylationon intercellular gap junctionalcommunication

Cx32 was one of the first channel-formingproteins shown to be phosphorylated inintact cells (63). Activation of eithercAMP-dPK or PKC increases the state ofphosphorylation of Cx32 (34,63,70,72) andat least the effect of cAMP-dPK is tempo-rally correlated with an increased junctionalconductance (gj). Moreover, Chanson et al.(71) reported that an increase in intracellular[cAMP] in a human colonic T84 cell lineinduces a rapid (<20 min) increase in inter-cellular gap junctional communication me-diated by Cx32 gap junctions, that is directlyrelated to an increase in fluid secretion.

Table 1 - Connexins known to be phosphoproteins.

Phosphorylated amino acid residues identified in the intracellular domains of connex-ins are indicated. Isolated gap junctions, fusion proteins or synthetic peptides wereused as substrates for in vitro phosphorylation assays. Purified protein kinases used inthese assays are also indicated. ND: Not determined; Ser: serine; Thr: threonine; Tyr:tyrosine; PK: protein kinase; EGFR-Tyr K: epidermal growth factor receptor Tyr kinase;cAMP-dPK: cAMP-dependent protein kinase; Ca2+-CM-dPK II: Ca2+/calmodulin-de-pendent protein kinase type II; pp60v-src: Tyr kinase encoded by the oncogene v-src;MAP K: mitogen-activated protein kinase.

Connexin Phosphorylated amino Protein kinase Referenceacid residue

Cx31 ND ND 37

Cx32 Ser 233 cAMP-dPK, PKC 34Thr ND Ca2+-CM-dPK II 34Ser ND cAMP-dPK, PKC 34Tyr ND EGFR-Tyr K 38

Cx40 ND ND 40

Cx43 Ser 266, 279, 282 MAP K 35Ser 368, 372 PKC 42Tyr ND pp60v-src 41

Cx45 ND ND 43

Cx45.6 ND Mg2+-dependent PK 39

Cx46 ND ND 44

Cx50 ND Mg2+-dependent PK 39ND cAMP-dPK

Cx56 Ser 118, 493 PKC, PKA 36

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cAMP increases the amount of metaboli-cally labelled Cx32 in primary cell culturesof fetal hepatocytes (70), implying either anincreased rate of synthesis or a reduced rateof degradation. The stoichiometry of phos-phorylation of Cx32 in vitro is low; by PKCit approaches 1 mol/mol and by cAMP-dPKit is about 0.1 mol of Pi/mol of protein(34,63,72). Nevertheless, the stoichiometryof phosphorylation in vivo and the cell com-partment in which Cx32 phosphorylationoccurs have not been determined. Moreover,in isolated liver gap junctions previouslyphosphorylated by PKC, treatment withcAMP-dPK does not increase the incorpora-tion of 32P into Cx32. On the other hand, ifgap junctions are first phosphorylated bycAMP-dPK, treatment with PKC increasesthe incorporation of 32P into Cx32, suggest-ing that PKC can phosphorylate other aminoacyl residues besides those phosphorylatedby cAMP-dPK (72). Using synthetic pep-tides corresponding to regions of theC-terminus of Cx32, it has been demon-strated that Ser 233 is phosphorylated byboth cAMP-dPK and PKC (34). Other siteshave not yet been identified.

A Cx32 mutant, with serine residues 233and 240 replaced by asparagine residues,forms gap junctions in Xenopus oocytes withmacroscopic gating properties (voltage de-pendence and pH sensitivity) that are indis-tinguishable from those formed by thewild-type Cx (12). These findings suggestthat phosphorylation of those serine residuesis not required for channel opening or clos-ing by these conditions. Nevertheless, modu-lation of gj due to changes in the assembly orretrieval of channels into or from the plasmamembrane has not been studied. Phospho-rylated Cx32 by PKC but not by cAMP-dPKis less sensitive to degradation by m andmµ-calpains (73), suggesting an alternativemechanism for the regulation of intercellularcoupling. Thus, the extent to which intercel-lular coupling between Cx32-containing cellsis modulated by phosphorylation requires a

more exhaustive exploration.Although Cx32 in isolated rat liver gap

junctions is also a moderate substrate forCa2+-CM-dPK II, resulting in serine andthreonine phosphorylation (34), the state ofphosphorylation of Cx32 has not been ex-haustively studied in cells treated with agentsthat selectively activate this kinase. Cx32 isnot phosphorylated by pp60v-src in isolatedrat liver gap junctions (Sáez JC, Nairn ACand Hertzberg EL, unpublished observation)or in Xenopus oocytes (10). Nevertheless,tyrosyl phosphorylation of Cx32 can occurin isolated liver gap junctions treated withthe epidermal growth factor receptor tyrosinekinase (38). The functional consequence ofCx32 tyrosyl phosphorylation remains un-raveled.

Effect of Cx43 phosphorylation onintercellular gap junctionalcommunication

In cell lines and in neonatal (25,42) cardiacmyocytes (33,50), two phosphorylated formsof Cx43 with slower electrophoretic mobili-ties (43-47 kDa) than the unphosphorylatedform (41 kDa) can be identified either byimmunoblotting or in immunoprecipitatedCx43 from 32P-labelled cells. Immunoblot a-nalysis of Cx43 in various rat tissues showsdifferent, although tissue-specific ratios of thedifferent forms of Cx43 (49). Variations inlevels of the different Cx43 phospho-formshave also been detected during the ontogenyof the pineal gland (46). In primary culture ofneonatal rat cardiocytes, Cx43 is predomi-nantly phosphorylated in seryl residues and toa lesser extent in threonyl residues (25,42,50).Pulse-chase studies indicate that phosphoryla-tion occurs soon after synthesis (20,23-25)and, at least in fibroblasts, dephosphorylationoccurs thereafter (20). Inhibition of proteintrafficking with monensin or brefeldin A re-veals that Cx43 is partially phosphorylatedbefore its exit from the Golgi apparatus(21).

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In Rous sarcoma virus-transformed fi-broblasts, where coupling is low, all formsof Cx43 are phosphorylated in both seryl andtyrosyl residues, and threonine phosphoryla-tion of the least mobile species is also noted(20). A mutation replacing tyrosine 265 ofCx43 with phenylalanine does not preventgap junction formation in Xenopus oocytesbut it completely abolishes the inhibition ofintercellular gap junctional communicationand the tyrosyl phosphorylation induced bypp60v-src; gap junctions formed by wild-typeCx43 and then exposed to src are phospho-rylated in tyrosine residues and intercellulargap junctional communication is completelyinhibited (10).

Activation of cAMP-dPK or PKC leadsto various effects on cells of different types,even when cells expressing the same junc-tional proteins are compared (18). Frequently,in cells where the unphosphorylated form ofCx43 (Cx43-NP) predominates under basalconditions, stimulation of PKC with a tumorpromoter phorbol ester (e.g., TPA) leads torapid cell uncoupling and shifts the electro-phoretic mobility of Cx43 forms (30,45,52,53,55). By contrast, in cells where the phos-phorylated forms of Cx43 predominate (e.g.,rat cardiocytes) TPA promotes intercellularcommunication with no detectable changesin the state of phosphorylation, as evaluatedby Western blots (42). Nevertheless, thereare exceptions: in rat leptomeningeal cells(8), and in a rat liver epithelial cell line (IRA20) (54) the phosphorylated forms of Cx43predominate and TPA induces uncouplingwithout detectable changes in the state ofCx43 phosphorylation detected by Westernblots. In IRA 20 cells TPA did not change thelevels of Cx43 or of its mRNA but did resultin the loss of Cx43 immunoreactivity byindirect immunofluorescence (54), suggest-ing that the inhibition in intercellular gapjunctional communication might involve apost-translational modification that perhapscannot be detected in denaturing gels. There-fore, Western blot analysis might not be the

appropriate approach to study the correla-tion between all changes in phosphorylation.For example, the state of Cx43 phosphoryla-tion studied by Western blot in rat heartmyocytes does not change in response toagents that affect the activity of protein ki-nases or phosphoprotein phosphatases, al-though treatment with some of these agentsdoes affect the incorporation of 32P intoCx43 (42). The stimulation of cAMP- orcGMP-dPK or PKC does not significantlyincrease the incorporation of 32P presumablybecause Cx43 is maximally phosphorylatedunder basal conditions. However, the incor-poration of 32 P is greatly reduced after inhi-bition of protein kinase activities with stau-rosporine, and can then be stimulated byTPA (42). In neonatal rat cardiac myocytesCx43 is predominantly phosphorylated(25,42,50) and it is localized in the plasmamembrane (33). Agents that affect the incor-poration of 32P into Cx43 do not affect thedistribution of the protein tested immunocy-tochemically in rat myocytes, suggesting thatchanges in the rate of phosphorylation de-tected with 32Pi occur within the plasma mem-brane compartment. In all the systems men-tioned above it seems that PKC mediates theeffects of TPA. In support of this, after PKCis down-regulated cells are coupled and bothcoupling and state of phosphorylation ofCx43 become insensitive to TPA and phos-phorylated forms of Cx43 are still detectedas major components (45). Using syntheticpeptides corresponding to the deduced se-quences in the C-terminal region or recom-binant fusion protein of the C-terminus ofCx43 it has been shown that residues 368and 372 are phosphorylated by PKC but notby cAMP- or cGMP-dPK or Ca2+-CM-dPKII (42) (Table 1). Both sites have been foundmutated in visceroatrial heterotaxia and there-fore implicated in the pathogenesis of thisdisease (74).

Cx43 can be phosphorylated bymitogen-activated protein (MAP) kinase inseryl residues 266, 279 and 282 (35) which

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presumably are the same residues phospho-rylated by cdc2 kinase, since a fusion proteinof the carboxy terminal of Cx43 phosphoryl-ated either by MAP or cdc2 kinase showsidentical tryptic fingerprints (42) (Table 1).Furthermore, EGF-induced cell uncoupling ismediated by MAP kinase Cx43 phosphoryla-tion in seryl residues of proline rich regions(35). A similar mechanism may operate in thePDGF-induced cell uncoupling which is PKCindependent (68).

A membrane permeable derivative ofcGMP reduces gj in neonatal rat myocytes(61) and in SKHep1 cells transfected withrat Cx43 but not in SKHep1 cells transfectedwith human Cx43 (14). The state of phos-phorylation of Cx43 expressed by SKHep1cells stably transfected with rat Cx43 cDNAis increased by 8Br-cGMP but the site ofphosphorylation is unknown as also is its

interaction with other sites of phosphoryla-tion by other protein kinases. In rat but not inhuman Cx43 the seryl residue 257 located inthe carboxy terminal is flanked by prolineand lysine, making it a possible site for phos-phorylation by cGMP-dependent protein ki-nase. Although Cx43 does not present anobvious consensus site for cAMP-dPK phos-phorylation, an increase in intracellular[cAMP] increases gj in neonatal rat myo-cytes (57) and induces phosphorylation ofCx43 in the ovary (47).

Finally, indications that phosphoproteinphosphatases 1 and 2A participate in de-phosphorylation of Cx43 have been obtained.In MDCK cells, treatment with okadaic acid,an inhibitor of these phosphatases, potenti-ates the increase in the relative amount ofphosphorylated Cx43 induced by activationof a PKC-dependent pathway (30).

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