Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2001 Dec 1;360(Pt 2):295–304. doi: 10.1042/0264-6021:3600295

Tight-junction protein zonula occludens 2 is a target of phosphorylation by protein kinase C.

A Avila-Flores 1, E Rendón-Huerta 1, J Moreno 1, S Islas 1, A Betanzos 1, M Robles-Flores 1, L González-Mariscal 1
PMCID: PMC1222229  PMID: 11716757

Abstract

Zonula occludens 2 (ZO-2) protein is a tight-junction phos phorylated protein that belongs to the membrane-associated guanylate kinase ('MAGUK') family. Here we study the interaction between ZO-2 and protein kinase C (PKC). We have constructed two ZO-2 fusion proteins of the middle (3PSG) and C-terminal (AP) regions of the molecule and demonstrate that they are phosphorylated by PKC isoenzymes beta, epsilon, lambda and zeta. To understand the physiological significance of the interaction between ZO-2 and PKC, we analysed the phosphorylation state of ZO-2 immunoprecipitated from monolayers with mature tight junctions or from cells that either lack them or have them disassembled through Ca(2+) chelation. We found that in the latter condition the phosphorylation level of ZO-2 is significantly higher and is due to the action of both PKC and cAMP-dependent protein kinase. These results therefore suggest that the phosphorylated state of ZO-2 restrains its capacity to operate at the junctional complex.

Full Text

The Full Text of this article is available as a PDF (417.8 KB).

Selected References

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

  1. Balda M. S., Anderson J. M., Matter K. The SH3 domain of the tight junction protein ZO-1 binds to a serine protein kinase that phosphorylates a region C-terminal to this domain. FEBS Lett. 1996 Dec 16;399(3):326–332. doi: 10.1016/s0014-5793(96)01352-x. [DOI] [PubMed] [Google Scholar]
  2. Balda M. S., Gonzalez-Mariscal L., Matter K., Cereijido M., Anderson J. M. Assembly of the tight junction: the role of diacylglycerol. J Cell Biol. 1993 Oct;123(2):293–302. doi: 10.1083/jcb.123.2.293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Balda M. S., González-Mariscal L., Contreras R. G., Macias-Silva M., Torres-Marquez M. E., García-Sáinz J. A., Cereijido M. Assembly and sealing of tight junctions: possible participation of G-proteins, phospholipase C, protein kinase C and calmodulin. J Membr Biol. 1991 Jun;122(3):193–202. doi: 10.1007/BF01871420. [DOI] [PubMed] [Google Scholar]
  4. Beatch M., Jesaitis L. A., Gallin W. J., Goodenough D. A., Stevenson B. R. The tight junction protein ZO-2 contains three PDZ (PSD-95/Discs-Large/ZO-1) domains and an alternatively spliced region. J Biol Chem. 1996 Oct 18;271(42):25723–25726. doi: 10.1074/jbc.271.42.25723. [DOI] [PubMed] [Google Scholar]
  5. Boyle W. J., van der Geer P., Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. doi: 10.1016/0076-6879(91)01013-r. [DOI] [PubMed] [Google Scholar]
  6. Cereijido M., Robbins E. S., Dolan W. J., Rotunno C. A., Sabatini D. D. Polarized monolayers formed by epithelial cells on a permeable and translucent support. J Cell Biol. 1978 Jun;77(3):853–880. doi: 10.1083/jcb.77.3.853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Citi S. Protein kinase inhibitors prevent junction dissociation induced by low extracellular calcium in MDCK epithelial cells. J Cell Biol. 1992 Apr;117(1):169–178. doi: 10.1083/jcb.117.1.169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Denisenko N., Burighel P., Citi S. Different effects of protein kinase inhibitors on the localization of junctional proteins at cell-cell contact sites. J Cell Sci. 1994 Apr;107(Pt 4):969–981. doi: 10.1242/jcs.107.4.969. [DOI] [PubMed] [Google Scholar]
  9. Dodane V., Kachar B. Identification of isoforms of G proteins and PKC that colocalize with tight junctions. J Membr Biol. 1996 Feb;149(3):199–209. doi: 10.1007/s002329900020. [DOI] [PubMed] [Google Scholar]
  10. Fanning A. S., Jameson B. J., Jesaitis L. A., Anderson J. M. The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. J Biol Chem. 1998 Nov 6;273(45):29745–29753. doi: 10.1074/jbc.273.45.29745. [DOI] [PubMed] [Google Scholar]
  11. Gonzalez-Mariscal L., Chávez de Ramírez B., Cereijido M. Tight junction formation in cultured epithelial cells (MDCK). J Membr Biol. 1985;86(2):113–125. doi: 10.1007/BF01870778. [DOI] [PubMed] [Google Scholar]
  12. Gonzalez-Mariscal L., Contreras R. G., Bolívar J. J., Ponce A., Chávez De Ramirez B., Cereijido M. Role of calcium in tight junction formation between epithelial cells. Am J Physiol. 1990 Dec;259(6 Pt 1):C978–C986. doi: 10.1152/ajpcell.1990.259.6.C978. [DOI] [PubMed] [Google Scholar]
  13. González-Mariscal L., Betanzos A., Avila-Flores A. MAGUK proteins: structure and role in the tight junction. Semin Cell Dev Biol. 2000 Aug;11(4):315–324. doi: 10.1006/scdb.2000.0178. [DOI] [PubMed] [Google Scholar]
  14. Gopalakrishnan S., Raman N., Atkinson S. J., Marrs J. A. Rho GTPase signaling regulates tight junction assembly and protects tight junctions during ATP depletion. Am J Physiol. 1998 Sep;275(3 Pt 1):C798–C809. doi: 10.1152/ajpcell.1998.275.3.C798. [DOI] [PubMed] [Google Scholar]
  15. Itoh M., Furuse M., Morita K., Kubota K., Saitou M., Tsukita S. Direct binding of three tight junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins. J Cell Biol. 1999 Dec 13;147(6):1351–1363. doi: 10.1083/jcb.147.6.1351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Itoh M., Morita K., Tsukita S. Characterization of ZO-2 as a MAGUK family member associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin. J Biol Chem. 1999 Feb 26;274(9):5981–5986. doi: 10.1074/jbc.274.9.5981. [DOI] [PubMed] [Google Scholar]
  17. Itoh M., Morita K., Tsukita S. Characterization of ZO-2 as a MAGUK family member associated with tight as well as adherens junctions with a binding affinity to occludin and alpha catenin. J Biol Chem. 1999 Feb 26;274(9):5981–5986. doi: 10.1074/jbc.274.9.5981. [DOI] [PubMed] [Google Scholar]
  18. Izumi Y., Hirose T., Tamai Y., Hirai S., Nagashima Y., Fujimoto T., Tabuse Y., Kemphues K. J., Ohno S. An atypical PKC directly associates and colocalizes at the epithelial tight junction with ASIP, a mammalian homologue of Caenorhabditis elegans polarity protein PAR-3. J Cell Biol. 1998 Oct 5;143(1):95–106. doi: 10.1083/jcb.143.1.95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Joberty G., Petersen C., Gao L., Macara I. G. The cell-polarity protein Par6 links Par3 and atypical protein kinase C to Cdc42. Nat Cell Biol. 2000 Aug;2(8):531–539. doi: 10.1038/35019573. [DOI] [PubMed] [Google Scholar]
  20. Lin D., Edwards A. S., Fawcett J. P., Mbamalu G., Scott J. D., Pawson T. A mammalian PAR-3-PAR-6 complex implicated in Cdc42/Rac1 and aPKC signalling and cell polarity. Nat Cell Biol. 2000 Aug;2(8):540–547. doi: 10.1038/35019582. [DOI] [PubMed] [Google Scholar]
  21. Martiny-Baron G., Kazanietz M. G., Mischak H., Blumberg P. M., Kochs G., Hug H., Marmé D., Schächtele C. Selective inhibition of protein kinase C isozymes by the indolocarbazole Gö 6976. J Biol Chem. 1993 May 5;268(13):9194–9197. [PubMed] [Google Scholar]
  22. Mattagajasingh S. N., Huang S. C., Hartenstein J. S., Benz E. J., Jr Characterization of the interaction between protein 4.1R and ZO-2. A possible link between the tight junction and the actin cytoskeleton. J Biol Chem. 2000 Sep 29;275(39):30573–30585. doi: 10.1074/jbc.M004578200. [DOI] [PubMed] [Google Scholar]
  23. Nigam S. K., Denisenko N., Rodriguez-Boulan E., Citi S. The role of phosphorylation in development of tight junctions in cultured renal epithelial (MDCK) cells. Biochem Biophys Res Commun. 1991 Dec 16;181(2):548–553. doi: 10.1016/0006-291x(91)91224-z. [DOI] [PubMed] [Google Scholar]
  24. Perez-Moreno M., Avila A., Islas S., Sanchez S., González-Mariscal L. Vinculin but not alpha-actinin is a target of PKC phosphorylation during junctional assembly induced by calcium. J Cell Sci. 1998 Dec;111(Pt 23):3563–3571. doi: 10.1242/jcs.111.23.3563. [DOI] [PubMed] [Google Scholar]
  25. Qiu R. G., Abo A., Steven Martin G. A human homolog of the C. elegans polarity determinant Par-6 links Rac and Cdc42 to PKCzeta signaling and cell transformation. Curr Biol. 2000 Jun 15;10(12):697–707. doi: 10.1016/s0960-9822(00)00535-2. [DOI] [PubMed] [Google Scholar]
  26. Rendón-Huerta E., Mendoza-Hernández G., Robles-Flores M. Characterization of calreticulin as a protein interacting with protein kinase C. Biochem J. 1999 Dec 1;344(Pt 2):469–475. [PMC free article] [PubMed] [Google Scholar]
  27. Robles-Flores M., Alcántara-Hernández R., García-Sáinz J. A. Differences in phorbol ester-induced decrease of the activity of protein kinase C isozymes in rat hepatocytes. Biochim Biophys Acta. 1991 Aug 13;1094(1):77–84. doi: 10.1016/0167-4889(91)90028-v. [DOI] [PubMed] [Google Scholar]
  28. Robles-Flores M., García-Sáinz J. A. Activated protein kinase C binds to intracellular receptors in rat hepatocytes. Biochem J. 1993 Dec 1;296(Pt 2):467–472. doi: 10.1042/bj2960467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Staddon J. M., Herrenknecht K., Smales C., Rubin L. L. Evidence that tyrosine phosphorylation may increase tight junction permeability. J Cell Sci. 1995 Feb;108(Pt 2):609–619. doi: 10.1242/jcs.108.2.609. [DOI] [PubMed] [Google Scholar]
  30. Stuart R. O., Nigam S. K. Regulated assembly of tight junctions by protein kinase C. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):6072–6076. doi: 10.1073/pnas.92.13.6072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Takeda H., Tsukita S. Effects of tyrosine phosphorylation on tight junctions in temperature-sensitive v-src-transfected MDCK cells. Cell Struct Funct. 1995 Oct;20(5):387–393. doi: 10.1247/csf.20.387. [DOI] [PubMed] [Google Scholar]
  32. Wittchen E. S., Haskins J., Stevenson B. R. Protein interactions at the tight junction. Actin has multiple binding partners, and ZO-1 forms independent complexes with ZO-2 and ZO-3. J Biol Chem. 1999 Dec 3;274(49):35179–35185. doi: 10.1074/jbc.274.49.35179. [DOI] [PubMed] [Google Scholar]
  33. Wolf M., Sahyoun N. Protein kinase C and phosphatidylserine bind to Mr 110,000/115,000 polypeptides enriched in cytoskeletal and postsynaptic density preparations. J Biol Chem. 1986 Oct 5;261(28):13327–13332. [PubMed] [Google Scholar]

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

RESOURCES