Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1993 Nov 1;295(Pt 3):735–742. doi: 10.1042/bj2950735

Dynamics of connexin43 phosphorylation in pp60v-src-transformed cells.

G S Goldberg 1, A F Lau 1
PMCID: PMC1134622  PMID: 7694570

Abstract

Connexin43 phosphorylation was analysed in non-transformed and pp60v-src-transformed Rat-1 fibroblasts. Connexin43 appeared to be the primary connexin expressed in these cells. Although gap-junctional communication was disrupted in pp60v-src-transformed cells, they contained more connexin43 protein and RNA than their non-transformed counterpart. Connexin43 was phosphorylated within minutes of its synthesis in both cell types and appeared to be degraded while in the phosphorylated state. Phosphopeptide and phosphoamino acid analyses suggested that connexin43 in both cell types contained at least five fragments with serine phosphorylation. The major difference in connexin43 phosphorylation between the pp60v-src-transformed and non-transformed cells was that, whereas approx. 70% of the phosphorylated connexin43 in the former contained phosphotyrosine, this phosphoamino acid was not detected in connexin43 isolated from the latter cells. These data support the hypothesis that phosphorylation of connexin43 on tyrosine is critical for the blockade of gap-junctional communication which occurs concomitantly with transformation by the pp60v-src oncogene.

Full text

PDF
735

Images in this article

737Figure 1
on p.737
738Figure 2
on p.738
738Figure 3
on p.738
739Figure 4
on p.739
739Figure 5
on p.739
740Figure 6
on p.740
740Figure 7
on p.740

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Azarnia R., Loewenstein W. R. Intercellular communication and the control of growth: XII. Alteration of junctional permeability by simian virus 40. Roles of the large and small T antigens. J Membr Biol. 1984;82(3):213–220. doi: 10.1007/BF01871631. [DOI] [PubMed] [Google Scholar]
  2. Azarnia R., Loewenstein W. R. Polyomavirus middle T antigen downregulates junctional cell-to-cell communication. Mol Cell Biol. 1987 Feb;7(2):946–950. doi: 10.1128/mcb.7.2.946. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Beyer E. C., Kistler J., Paul D. L., Goodenough D. A. 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. doi: 10.1083/jcb.108.2.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beyer E. C., Paul D. L., Goodenough D. A. 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. doi: 10.1083/jcb.105.6.2621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brissette J. L., Kumar N. M., Gilula N. B., Dotto G. P. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate and the ras oncogene modulate expression and phosphorylation of gap junction proteins. Mol Cell Biol. 1991 Oct;11(10):5364–5371. doi: 10.1128/mcb.11.10.5364. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Cooper J. A., Hunter T. Major substrate for growth factor-activated protein-tyrosine kinases is a low-abundance protein. Mol Cell Biol. 1985 Nov;5(11):3304–3309. doi: 10.1128/mcb.5.11.3304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Crow D. S., Beyer E. C., Paul D. L., Kobe S. S., Lau A. F. Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts. Mol Cell Biol. 1990 Apr;10(4):1754–1763. doi: 10.1128/mcb.10.4.1754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Crow D. S., Kurata W. E., Lau A. F. Phosphorylation of connexin43 in cells containing mutant src oncogenes. Oncogene. 1992 May;7(5):999–1003. [PubMed] [Google Scholar]
  10. Davis S., Lu M. L., Lo S. H., Lin S., Butler J. A., Druker B. J., Roberts T. M., An Q., Chen L. B. Presence of an SH2 domain in the actin-binding protein tensin. Science. 1991 May 3;252(5006):712–715. doi: 10.1126/science.1708917. [DOI] [PubMed] [Google Scholar]
  11. Eldridge S. R., Martens T. W., Sattler C. A., Gould M. N. Association of decreased intercellular communication with the immortal but not the tumorigenic phenotype in human mammary epithelial cells. Cancer Res. 1989 Aug 1;49(15):4326–4331. [PubMed] [Google Scholar]
  12. Ellis C., Moran M., McCormick F., Pawson T. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature. 1990 Jan 25;343(6256):377–381. doi: 10.1038/343377a0. [DOI] [PubMed] [Google Scholar]
  13. Filson A. J., Azarnia R., Beyer E. C., Loewenstein W. R., Brugge J. S. Tyrosine phosphorylation of a gap junction protein correlates with inhibition of cell-to-cell communication. Cell Growth Differ. 1990 Dec;1(12):661–668. [PubMed] [Google Scholar]
  14. Glenney J. R., Jr Tyrosine-phosphorylated proteins: mediators of signal transduction from the tyrosine kinases. Biochim Biophys Acta. 1992 Mar 16;1134(2):113–127. doi: 10.1016/0167-4889(92)90034-9. [DOI] [PubMed] [Google Scholar]
  15. Glenney J. R., Jr, Zokas L. Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton. J Cell Biol. 1989 Jun;108(6):2401–2408. doi: 10.1083/jcb.108.6.2401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Heussen G. A., Alink G. M. Inhibition of gap-junctional intercellular communication by TPA and airborne particulate matter in primary cultures of rat alveolar type II cells. Carcinogenesis. 1992 Apr;13(4):719–722. doi: 10.1093/carcin/13.4.719. [DOI] [PubMed] [Google Scholar]
  17. Johnson R. M., Wasilenko W. J., Mattingly R. R., Weber M. J., Garrison J. C. Fibroblasts transformed with v-src show enhanced formation of an inositol tetrakisphosphate. Science. 1989 Oct 6;246(4926):121–124. doi: 10.1126/science.2506643. [DOI] [PubMed] [Google Scholar]
  18. Kadle R., Zhang J. T., Nicholson B. J. Tissue-specific distribution of differentially phosphorylated forms of Cx43. Mol Cell Biol. 1991 Jan;11(1):363–369. doi: 10.1128/mcb.11.1.363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kamps M. P., Sefton B. M. Acid and base hydrolysis of phosphoproteins bound to immobilon facilitates analysis of phosphoamino acids in gel-fractionated proteins. Anal Biochem. 1989 Jan;176(1):22–27. doi: 10.1016/0003-2697(89)90266-2. [DOI] [PubMed] [Google Scholar]
  20. Kamps M. P., Sefton B. M. Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene. 1988 Apr;2(4):305–315. [PubMed] [Google Scholar]
  21. Klaunig J. E., Ruch R. J. Role of inhibition of intercellular communication in carcinogenesis. Lab Invest. 1990 Feb;62(2):135–146. [PubMed] [Google Scholar]
  22. Kmiecik T. E., Shalloway D. Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation. Cell. 1987 Apr 10;49(1):65–73. doi: 10.1016/0092-8674(87)90756-2. [DOI] [PubMed] [Google Scholar]
  23. Laird D. W., Puranam K. L., Revel J. P. Turnover and phosphorylation dynamics of connexin43 gap junction protein in cultured cardiac myocytes. Biochem J. 1991 Jan 1;273(Pt 1):67–72. doi: 10.1042/bj2730067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Laird D. W., Revel J. P. Biochemical and immunochemical analysis of the arrangement of connexin43 in rat heart gap junction membranes. J Cell Sci. 1990 Sep;97(Pt 1):109–117. doi: 10.1242/jcs.97.1.109. [DOI] [PubMed] [Google Scholar]
  25. Lau A. F., Kanemitsu M. Y., Kurata W. E., Danesh S., Boynton A. L. Epidermal growth factor disrupts gap-junctional communication and induces phosphorylation of connexin43 on serine. Mol Biol Cell. 1992 Aug;3(8):865–874. doi: 10.1091/mbc.3.8.865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lau A. F. Phosphotyrosine-containing 120,000-dalton protein coimmunoprecipitated with pp60v-src from Rous sarcoma virus-transformed mammalian cells. Virology. 1986 May;151(1):86–99. doi: 10.1016/0042-6822(86)90106-6. [DOI] [PubMed] [Google Scholar]
  27. Loewenstein W. R., Rose B. The cell-cell channel in the control of growth. Semin Cell Biol. 1992 Feb;3(1):59–79. doi: 10.1016/s1043-4682(10)80008-x. [DOI] [PubMed] [Google Scholar]
  28. Loewenstein W. R. The cell-to-cell channel of gap junctions. Cell. 1987 Mar 13;48(5):725–726. doi: 10.1016/0092-8674(87)90067-5. [DOI] [PubMed] [Google Scholar]
  29. Luo K., Hurley T. R., Sefton B. M. Transfer of proteins to membranes facilitates both cyanogen bromide cleavage and two-dimensional proteolytic mapping. Oncogene. 1990 Jun;5(6):921–923. [PubMed] [Google Scholar]
  30. Madhukar B. V., Oh S. Y., Chang C. C., Wade M., Trosko J. E. Altered regulation of intercellular communication by epidermal growth factor, transforming growth factor-beta and peptide hormones in normal human keratinocytes. Carcinogenesis. 1989 Jan;10(1):13–20. doi: 10.1093/carcin/10.1.13. [DOI] [PubMed] [Google Scholar]
  31. Maldonado P. E., Rose B., Loewenstein W. R. Growth factors modulate junctional cell-to-cell communication. J Membr Biol. 1988 Dec;106(3):203–210. doi: 10.1007/BF01872158. [DOI] [PubMed] [Google Scholar]
  32. Mehta P. P., Hotz-Wagenblatt A., Rose B., Shalloway D., Loewenstein W. R. Incorporation of the gene for a cell-cell channel protein into transformed cells leads to normalization of growth. J Membr Biol. 1991 Dec;124(3):207–225. doi: 10.1007/BF01994355. [DOI] [PubMed] [Google Scholar]
  33. Musil L. S., Cunningham B. A., Edelman G. M., Goodenough D. A. Differential phosphorylation of the gap junction protein connexin43 in junctional communication-competent and -deficient cell lines. J Cell Biol. 1990 Nov;111(5 Pt 1):2077–2088. doi: 10.1083/jcb.111.5.2077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Musil L. S., Goodenough D. A. Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques. J Cell Biol. 1991 Dec;115(5):1357–1374. doi: 10.1083/jcb.115.5.1357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Nada S., Okada M., MacAuley A., Cooper J. A., Nakagawa H. Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src. Nature. 1991 May 2;351(6321):69–72. doi: 10.1038/351069a0. [DOI] [PubMed] [Google Scholar]
  36. Naus C. C., Zhu D., Todd S. D., Kidder G. M. Characteristics of C6 glioma cells overexpressing a gap junction protein. Cell Mol Neurobiol. 1992 Apr;12(2):163–175. doi: 10.1007/BF00713370. [DOI] [PubMed] [Google Scholar]
  37. Nori M., Shawver L. K., Weber M. J. A Swiss 3T3 variant cell line resistant to the effects of tumor promoters cannot be transformed by src. Mol Cell Biol. 1990 Aug;10(8):4155–4162. doi: 10.1128/mcb.10.8.4155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Oh S. Y., Grupen C. G., Murray A. W. Phorbol ester induces phosphorylation and down-regulation of connexin 43 in WB cells. Biochim Biophys Acta. 1991 Sep 3;1094(2):243–245. doi: 10.1016/0167-4889(91)90016-q. [DOI] [PubMed] [Google Scholar]
  39. Parsons J. T., Weber M. J. Genetics of src: structure and functional organization of a protein tyrosine kinase. Curr Top Microbiol Immunol. 1989;147:79–127. doi: 10.1007/978-3-642-74697-0_3. [DOI] [PubMed] [Google Scholar]
  40. Piwnica-Worms H., Saunders K. B., Roberts T. M., Smith A. E., Cheng S. H. Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src. Cell. 1987 Apr 10;49(1):75–82. doi: 10.1016/0092-8674(87)90757-4. [DOI] [PubMed] [Google Scholar]
  41. Rivedal E., Sanner T. Regulation of gap junctional communication in Syrian hamster embryo cells by retinoic acid and 12-O-tetradecanoylphorbol-13-acetate. Carcinogenesis. 1992 Feb;13(2):199–203. doi: 10.1093/carcin/13.2.199. [DOI] [PubMed] [Google Scholar]
  42. Rogers M., Berestecky J. M., Hossain M. Z., Guo H. M., Kadle R., Nicholson B. J., Bertram J. S. Retinoid-enhanced gap junctional communication is achieved by increased levels of connexin 43 mRNA and protein. Mol Carcinog. 1990;3(6):335–343. doi: 10.1002/mc.2940030605. [DOI] [PubMed] [Google Scholar]
  43. Rose B., Yada T., Loewenstein W. R. Downregulation of cell-to-cell communication by the viral src gene is blocked by TMB-8 and recovery of communication is blocked by vanadate. J Membr Biol. 1986;94(2):129–142. doi: 10.1007/BF01871193. [DOI] [PubMed] [Google Scholar]
  44. Schultz A. M., Henderson L. E., Oroszlan S., Garber E. A., Hanafusa H. Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein. Science. 1985 Jan 25;227(4685):427–429. doi: 10.1126/science.3917576. [DOI] [PubMed] [Google Scholar]
  45. Sefton B. M., Hunter T., Ball E. H., Singer S. J. Vinculin: a cytoskeletal target of the transforming protein of Rous sarcoma virus. Cell. 1981 Apr;24(1):165–174. doi: 10.1016/0092-8674(81)90512-2. [DOI] [PubMed] [Google Scholar]
  46. Seiler-Tuyns A., Eldridge J. D., Paterson B. M. Expression and regulation of chicken actin genes introduced into mouse myogenic and nonmyogenic cells. Proc Natl Acad Sci U S A. 1984 May;81(10):2980–2984. doi: 10.1073/pnas.81.10.2980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Shalloway D., Shenoy S. Oncoprotein kinases in mitosis. Adv Cancer Res. 1991;57:185–225. doi: 10.1016/s0065-230x(08)60999-9. [DOI] [PubMed] [Google Scholar]
  48. Stagg R. B., Fletcher W. H. The hormone-induced regulation of contact-dependent cell-cell communication by phosphorylation. Endocr Rev. 1990 May;11(2):302–325. doi: 10.1210/edrv-11-2-302. [DOI] [PubMed] [Google Scholar]
  49. Stein L. S., Welsh T. H., Jr, Wilson V. G., Burghardt R. C. Cell-to-cell communication competence in simian virus 40-transfected rat ovarian cells is reduced following tumor selection. In Vitro Cell Dev Biol. 1992 Jun;28A(6):436–444. doi: 10.1007/BF02634048. [DOI] [PubMed] [Google Scholar]
  50. Swenson K. I., Piwnica-Worms H., McNamee H., Paul D. L. Tyrosine phosphorylation of the gap junction protein connexin43 is required for the pp60v-src-induced inhibition of communication. Cell Regul. 1990 Dec;1(13):989–1002. doi: 10.1091/mbc.1.13.989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Trosko J. E., Chang C. C., Madhukar B. V., Klaunig J. E. Chemical, oncogene and growth factor inhibition gap junctional intercellular communication: an integrative hypothesis of carcinogenesis. Pathobiology. 1990;58(5):265–278. doi: 10.1159/000163596. [DOI] [PubMed] [Google Scholar]
  52. Udenfriend S., Stein S., Böhlen P., Dairman W., Leimgruber W., Weigele M. Fluorescamine: a reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science. 1972 Nov 24;178(4063):871–872. doi: 10.1126/science.178.4063.871. [DOI] [PubMed] [Google Scholar]
  53. Zhu D., Caveney S., Kidder G. M., Naus C. C. Transfection of C6 glioma cells with connexin 43 cDNA: analysis of expression, intercellular coupling, and cell proliferation. Proc Natl Acad Sci U S A. 1991 Mar 1;88(5):1883–1887. doi: 10.1073/pnas.88.5.1883. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zhu D., Kidder G. M., Caveney S., Naus C. C. Growth retardation in glioma cells cocultured with cells overexpressing a gap junction protein. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10218–10221. doi: 10.1073/pnas.89.21.10218. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES