Search tips
Search criteria 


Logo of jcellbiolHomeEditorsContactInstructions for Authors
J Cell Biol. 1990 November 1; 111(5): 2077–2088.
PMCID: PMC2116332

Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines


Connexin43 is a member of the highly homologous connexin family of gap junction proteins. We have studied how connexin monomers are assembled into functional gap junction plaques by examining the biosynthesis of connexin43 in cell types that differ greatly in their ability to form functional gap junctions. Using a combination of metabolic radiolabeling and immunoprecipitation, we have shown that connexin43 is synthesized in gap junctional communication-competent cells as a 42-kD protein that is efficiently converted to a approximately 46-kD species (connexin43-P2) by the posttranslational addition of phosphate. Surprisingly, certain cell lines severely deficient in gap junctional communication and known cell-cell adhesion molecules (S180 and L929 cells) also expressed 42-kD connexin43. Connexin43 in these communication-deficient cell lines was not, however, phosphorylated to the P2 form. Conversion of S180 cells to a communication-competent phenotype by transfection with a cDNA encoding the cell-cell adhesion molecule L-CAM induced phosphorylation of connexin43 to the P2 form; conversely, blocking junctional communication in ordinarily communication-competent cells inhibited connexin43-P2 formation. Immunohistochemical localization studies indicated that only communication-competent cells accumulated connexin43 in visible gap junction plaques. Together, these results establish a strong correlation between the ability of cells to process connexin43 to the P2 form and to produce functional gap junctions. Connexin43 phosphorylation may therefore play a functional role in gap junction assembly and/or activity.

Full Text

The Full Text of this article is available as a PDF (2.6M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Atkinson MM, Menko AS, Johnson RG, Sheppard JR, Sheridan JD. Rapid and reversible reduction of junctional permeability in cells infected with a temperature-sensitive mutant of avian sarcoma virus. J Cell Biol. 1981 Nov;91(2 Pt 1):573–578. [PMC free article] [PubMed]
  • Azarnia R, Dahl G, Loewenstein WR. Cell junction and cycle AMP: III. Promotion of junctional membrane permeability and junctional membrane particles in a junction-deficient cell type. J Membr Biol. 1981;63(1-2):133–146. [PubMed]
  • Azarnia R, Reddy S, Kmiecik TE, Shalloway D, Loewenstein WR. The cellular src gene product regulates junctional cell-to-cell communication. Science. 1988 Jan 22;239(4838):398–401. [PubMed]
  • Bennett MV, Goodenough DA. Gap junctions, electrotonic coupling, and intercellular communication. Neurosci Res Program Bull. 1978 Sep;16(3):1–486. [PubMed]
  • Beyer EC, Paul DL, Goodenough DA. Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J Cell Biol. 1987 Dec;105(6 Pt 1):2621–2629. [PMC free article] [PubMed]
  • Beyer EC, Kistler J, Paul DL, Goodenough DA. Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues. J Cell Biol. 1989 Feb;108(2):595–605. [PMC free article] [PubMed]
  • Burt JM, Spray DC. Single-channel events and gating behavior of the cardiac gap junction channel. Proc Natl Acad Sci U S A. 1988 May;85(10):3431–3434. [PubMed]
  • Carr C, McCourt D, Cohen JB. The 43-kilodalton protein of Torpedo nicotinic postsynaptic membranes: purification and determination of primary structure. Biochemistry. 1987 Nov 3;26(22):7090–7102. [PubMed]
  • Chanson M, Bruzzone R, Bosco D, Meda P. Effects of n-alcohols on junctional coupling and amylase secretion of pancreatic acinar cells. J Cell Physiol. 1989 Apr;139(1):147–156. [PubMed]
  • Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. [PubMed]
  • Cooper JA, Sefton BM, Hunter T. Detection and quantification of phosphotyrosine in proteins. Methods Enzymol. 1983;99:387–402. [PubMed]
  • Crow DS, Beyer EC, Paul DL, Kobe SS, Lau AF. Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts. Mol Cell Biol. 1990 Apr;10(4):1754–1763. [PMC free article] [PubMed]
  • Dermietzel R, Traub O, Hwang TK, Beyer E, Bennett MV, Spray DC, Willecke K. Differential expression of three gap junction proteins in developing and mature brain tissues. Proc Natl Acad Sci U S A. 1989 Dec;86(24):10148–10152. [PubMed]
  • Dewey MM, Barr L. Intercellular Connection between Smooth Muscle Cells: the Nexus. Science. 1962 Aug 31;137(3531):670–672. [PubMed]
  • DUNHAM LJ, STEWART HL. A survey of transplantable and transmissible animal tumors. J Natl Cancer Inst. 1953 Apr;13(5):1299–1377. [PubMed]
  • Edelman GM. Morphoregulatory molecules. Biochemistry. 1988 May 17;27(10):3533–3543. [PubMed]
  • el-Fouly MH, Trosko JE, Chang CC. Scrape-loading and dye transfer. A rapid and simple technique to study gap junctional intercellular communication. Exp Cell Res. 1987 Feb;168(2):422–430. [PubMed]
  • Feinberg AP, Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. [PubMed]
  • Fraser SE, Green CR, Bode HR, Gilula NB. Selective disruption of gap junctional communication interferes with a patterning process in hydra. Science. 1987 Jul 3;237(4810):49–55. [PubMed]
  • Furshpan EJ, Potter DD. Low-resistance junctions between cells in embryos and tissue culture. Curr Top Dev Biol. 1968;3:95–127. [PubMed]
  • Gilula NB, Reeves OR, Steinbach A. Metabolic coupling, ionic coupling and cell contacts. Nature. 1972 Feb 4;235(5336):262–265. [PubMed]
  • Goodenough DA, Dick JS, 2nd, Lyons JE. Lens metabolic cooperation: a study of mouse lens transport and permeability visualized with freeze-substitution autoradiography and electron microscopy. J Cell Biol. 1980 Aug;86(2):576–589. [PMC free article] [PubMed]
  • Green CR, Harfst E, Gourdie RG, Severs NJ. Analysis of the rat liver gap junction protein: clarification of anomalies in its molecular size. Proc R Soc Lond B Biol Sci. 1988 Mar 22;233(1271):165–174. [PubMed]
  • Guthrie SC, Gilula NB. Gap junctional communication and development. Trends Neurosci. 1989 Jan;12(1):12–16. [PubMed]
  • Hertzberg EL, Spray DC, Bennett MV. Reduction of gap junctional conductance by microinjection of antibodies against the 27-kDa liver gap junction polypeptide. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2412–2416. [PubMed]
  • Keane RW, Mehta PP, Rose B, Honig LS, Loewenstein WR, Rutishauser U. Neural differentiation, NCAM-mediated adhesion, and gap junctional communication in neuroectoderm. A study in vitro. J Cell Biol. 1988 Apr;106(4):1307–1319. [PMC free article] [PubMed]
  • Krieg PA, Melton DA. Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 1984 Sep 25;12(18):7057–7070. [PMC free article] [PubMed]
  • Kumar NM, Gilula NB. Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein. J Cell Biol. 1986 Sep;103(3):767–776. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Larson DM, Sheridan JD. Junctional transfer in cultured vascular endothelium: II. Dye and nucleotide transfer. J Membr Biol. 1985;83(1-2):157–167. [PubMed]
  • Loewenstein WR. Junctional intercellular communication and the control of growth. Biochim Biophys Acta. 1979 Feb 4;560(1):1–65. [PubMed]
  • Loewenstein WR. Junctional intercellular communication: the cell-to-cell membrane channel. Physiol Rev. 1981 Oct;61(4):829–913. [PubMed]
  • Makowski L, Caspar DL, Phillips WC, Goodenough DA. Gap junction structures. II. Analysis of the x-ray diffraction data. J Cell Biol. 1977 Aug;74(2):629–645. [PMC free article] [PubMed]
  • Mathias RT, Rae JL. Transport properties of the lens. Am J Physiol. 1985 Sep;249(3 Pt 1):C181–C190. [PubMed]
  • Matsuzaki F, Mège RM, Jaffe SH, Friedlander DR, Gallin WJ, Goldberg JI, Cunningham BA, Edelman GM. cDNAs of cell adhesion molecules of different specificity induce changes in cell shape and border formation in cultured S180 cells. J Cell Biol. 1990 Apr;110(4):1239–1252. [PMC free article] [PubMed]
  • Mege RM, Matsuzaki F, Gallin WJ, Goldberg JI, Cunningham BA, Edelman GM. Construction of epithelioid sheets by transfection of mouse sarcoma cells with cDNAs for chicken cell adhesion molecules. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7274–7278. [PubMed]
  • Mehta PP, Bertram JS, Loewenstein WR. Growth inhibition of transformed cells correlates with their junctional communication with normal cells. Cell. 1986 Jan 17;44(1):187–196. [PubMed]
  • Miller TM, Goodenough DA. Gap junction structures after experimental alteration of junctional channel conductance. J Cell Biol. 1985 Nov;101(5 Pt 1):1741–1748. [PMC free article] [PubMed]
  • Musil LS, Beyer EC, Goodenough DA. Expression of the gap junction protein connexin43 in embryonic chick lens: molecular cloning, ultrastructural localization, and post-translational phosphorylation. J Membr Biol. 1990 Jun;116(2):163–175. [PubMed]
  • Nagafuchi A, Shirayoshi Y, Okazaki K, Yasuda K, Takeichi M. Transformation of cell adhesion properties by exogenously introduced E-cadherin cDNA. Nature. 1987 Sep 24;329(6137):341–343. [PubMed]
  • Paul DL. Molecular cloning of cDNA for rat liver gap junction protein. J Cell Biol. 1986 Jul;103(1):123–134. [PMC free article] [PubMed]
  • Rose B, Loewenstein WR. Permeability of cell junction depends on local cytoplasmic calcium activity. Nature. 1975 Mar 20;254(5497):250–252. [PubMed]
  • Saez JC, Spray DC, Nairn AC, Hertzberg E, Greengard P, Bennett MV. cAMP increases junctional conductance and stimulates phosphorylation of the 27-kDa principal gap junction polypeptide. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2473–2477. [PubMed]
  • Schuetze SM, Goodenough DA. Dye transfer between cells of the embryonic chick lens becomes less sensitive to CO2 treatment with development. J Cell Biol. 1982 Mar;92(3):694–705. [PMC free article] [PubMed]
  • Sibley DR, Benovic JL, Caron MG, Lefkowitz RJ. Regulation of transmembrane signaling by receptor phosphorylation. Cell. 1987 Mar 27;48(6):913–922. [PubMed]
  • Spray DC, Harris AL, Bennett MV. Voltage dependence of junctional conductance in early amphibian embryos. Science. 1979 Apr 27;204(4391):432–434. [PubMed]
  • Stevenson BR, Paul DL. The molecular constituents of intercellular junctions. Curr Opin Cell Biol. 1989 Oct;1(5):884–891. [PubMed]
  • Swenson KI, Jordan JR, Beyer EC, Paul DL. Formation of gap junctions by expression of connexins in Xenopus oocyte pairs. Cell. 1989 Apr 7;57(1):145–155. [PubMed]
  • Traub O, Look J, Paul D, Willecke K. Cyclic adenosine monophosphate stimulates biosynthesis and phosphorylation of the 26 kDa gap junction protein in cultured mouse hepatocytes. Eur J Cell Biol. 1987 Feb;43(1):48–54. [PubMed]
  • Traub O, Look J, Dermietzel R, Brümmer F, Hülser D, Willecke K. Comparative characterization of the 21-kD and 26-kD gap junction proteins in murine liver and cultured hepatocytes. J Cell Biol. 1989 Mar;108(3):1039–1051. [PMC free article] [PubMed]
  • Turin L, Warner A. Carbon dioxide reversibly abolishes ionic communication between cells of early amphibian embryo. Nature. 1977 Nov 3;270(5632):56–57. [PubMed]
  • Warner AE, Guthrie SC, Gilula NB. Antibodies to gap-junctional protein selectively disrupt junctional communication in the early amphibian embryo. Nature. 1984 Sep 13;311(5982):127–131. [PubMed]
  • Werner R, Levine E, Rabadan-Diehl C, Dahl G. Formation of hybrid cell-cell channels. Proc Natl Acad Sci U S A. 1989 Jul;86(14):5380–5384. [PubMed]
  • Wiener EC, Loewenstein WR. Correction of cell-cell communication defect by introduction of a protein kinase into mutant cells. Nature. 305(5933):433–435. [PubMed]
  • Willingham MC, Jay G, Pastan I. Localization of the ASV src gene product to the plasma membrane of transformed cells by electron microscopic immunocytochemistry. Cell. 1979 Sep;18(1):125–134. [PubMed]
  • Yancey SB, John SA, Lal R, Austin BJ, Revel JP. The 43-kD polypeptide of heart gap junctions: immunolocalization, topology, and functional domains. J Cell Biol. 1989 Jun;108(6):2241–2254. [PMC free article] [PubMed]
  • Yarden Y, Ullrich A. Growth factor receptor tyrosine kinases. Annu Rev Biochem. 1988;57:443–478. [PubMed]
  • Young JD, Cohn ZA, Gilula NB. Functional assembly of gap junction conductance in lipid bilayers: demonstration that the major 27 kd protein forms the junctional channel. Cell. 1987 Mar 13;48(5):733–743. [PubMed]
  • Zhang JT, Nicholson BJ. Sequence and tissue distribution of a second protein of hepatic gap junctions, Cx26, as deduced from its cDNA. J Cell Biol. 1989 Dec;109(6 Pt 2):3391–3401. [PMC free article] [PubMed]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press