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. 1994 Mar 1;124(5):717–727. doi: 10.1083/jcb.124.5.717

Phorbol myristate acetate-mediated stimulation of transcytosis and apical recycling in MDCK cells

PMCID: PMC2119954  PMID: 8120094

Abstract

We observed that phorbol myristate acetate (PMA) stimulates transcytosis of the polymeric immunoglobulin receptor (pIgR) in MDCK cells. Apical release of pre-endocytosed ligand (dimeric IgA) bound to the pIgR can be stimulated twofold within 7 min of addition of PMA while recycling of the ligand from the basal surface is not affected. In addition, apical surface delivery of pIgR and cleavage of its ectodomain to secretory component (SC) is also stimulated by PMA. The recycling of apically internalized ligand back to the apical surface is similarly stimulated. These results suggest that the stimulation of apical delivery is from an apical recycling compartment. The effect of PMA suggests that protein kinase C (PKC) is involved in the regulation of pIgR trafficking in MDCK cells. To test this we down regulated PKC activity by pre-treating cells with PMA for 16 h and observed that transcytosis could no longer be stimulated by PMA. Western blots show that the PKC isozymes alpha and to a lesser extent epsilon, are depleted from MDCK cells which have been pre-treated with PMA for 16 h and that treatment of MDCK cells with PMA for 5 min causes a dramatic translocation of the PKC alpha isozyme and a partial translocation of the epsilon isozyme from the cytosol to the membrane fraction of cell homogenates. This translocation suggests that the alpha and/or epsilon isozymes may be involved in PMA mediated stimulation of transcytosis. A mutant pIgR in which serines 664 and 726, the major sites of phosphorylation, are replaced by alanine is stimulated to transcytose by PMA, suggesting that phosphorylation of pIgR at these sites is not required for the effect of PMA. These results suggest that PMA-mediated stimulation of pIgR transcytosis may involve the activation of PKC alpha and/or epsilon, and that this stimulation occurs independently of the major phosphorylation sites on the pIgR. Finally, PMA stimulates transcytosis of basolaterally internalized transferrin, suggesting that PMA acts to generally stimulate delivery of endocytosed proteins to the apical surface.

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Selected References

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  1. Ahmed S., Lee J., Kozma R., Best A., Monfries C., Lim L. A novel functional target for tumor-promoting phorbol esters and lysophosphatidic acid. The p21rac-GTPase activating protein n-chimaerin. J Biol Chem. 1993 May 25;268(15):10709–10712. [PubMed] [Google Scholar]
  2. Aroeti B., Casanova J., Okamoto C., Cardone M., Pollack A., Tang K., Mostov K. Polymeric immunoglobulin receptor. Int Rev Cytol. 1992;137B:157–168. doi: 10.1016/s0074-7696(08)62603-0. [DOI] [PubMed] [Google Scholar]
  3. Bacallao R., Antony C., Dotti C., Karsenti E., Stelzer E. H., Simons K. The subcellular organization of Madin-Darby canine kidney cells during the formation of a polarized epithelium. J Cell Biol. 1989 Dec;109(6 Pt 1):2817–2832. doi: 10.1083/jcb.109.6.2817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Backer J. M., King G. L. Regulation of receptor-mediated endocytosis by phorbol esters. Biochem Pharmacol. 1991 May 1;41(9):1267–1277. doi: 10.1016/0006-2952(91)90097-o. [DOI] [PubMed] [Google Scholar]
  5. Barroso M., Sztul E. S. Basolateral to apical transcytosis in polarized cells is indirect and involves BFA and trimeric G protein sensitive passage through the apical endosome. J Cell Biol. 1994 Jan;124(1-2):83–100. doi: 10.1083/jcb.124.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bell R. M., Burns D. J. Lipid activation of protein kinase C. J Biol Chem. 1991 Mar 15;266(8):4661–4664. [PubMed] [Google Scholar]
  7. Bomsel M., Mostov K. E. Possible role of both the alpha and beta gamma subunits of the heterotrimeric G protein, Gs, in transcytosis of the polymeric immunoglobulin receptor. J Biol Chem. 1993 Dec 5;268(34):25824–25835. [PubMed] [Google Scholar]
  8. Breitfeld P. P., Casanova J. E., Harris J. M., Simister N. E., Mostov K. E. Expression and analysis of the polymeric immunoglobulin receptor in Madin-Darby canine kidney cells using retroviral vectors. Methods Cell Biol. 1989;32:329–337. doi: 10.1016/s0091-679x(08)61178-4. [DOI] [PubMed] [Google Scholar]
  9. Breitfeld P. P., Casanova J. E., McKinnon W. C., Mostov K. E. Deletions in the cytoplasmic domain of the polymeric immunoglobulin receptor differentially affect endocytotic rate and postendocytotic traffic. J Biol Chem. 1990 Aug 15;265(23):13750–13757. [PubMed] [Google Scholar]
  10. Breitfeld P. P., Harris J. M., Mostov K. E. Postendocytotic sorting of the ligand for the polymeric immunoglobulin receptor in Madin-Darby canine kidney cells. J Cell Biol. 1989 Aug;109(2):475–486. doi: 10.1083/jcb.109.2.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Casanova J. E., Breitfeld P. P., Ross S. A., Mostov K. E. Phosphorylation of the polymeric immunoglobulin receptor required for its efficient transcytosis. Science. 1990 May 11;248(4956):742–745. doi: 10.1126/science.2110383. [DOI] [PubMed] [Google Scholar]
  12. Coleman R. A., Wade J. B. Role of nonacidic endosomes in recycling of ADH-sensitive water channel structures. Eur J Cell Biol. 1992 Jun;58(1):44–56. [PubMed] [Google Scholar]
  13. De Matteis M. A., Santini G., Kahn R. A., Di Tullio G., Luini A. Receptor and protein kinase C-mediated regulation of ARF binding to the Golgi complex. Nature. 1993 Aug 26;364(6440):818–821. doi: 10.1038/364818a0. [DOI] [PubMed] [Google Scholar]
  14. Freier S., Eran M., Faber J. Effect of cholecystokinin and of its antagonist, of atropine, and of food on the release of immunoglobulin A and immunoglobulin G specific antibodies in the rat intestine. Gastroenterology. 1987 Dec;93(6):1242–1246. doi: 10.1016/0016-5085(87)90251-4. [DOI] [PubMed] [Google Scholar]
  15. Geuze H. J., Slot J. W., Strous G. J., Peppard J., von Figura K., Hasilik A., Schwartz A. L. Intracellular receptor sorting during endocytosis: comparative immunoelectron microscopy of multiple receptors in rat liver. Cell. 1984 May;37(1):195–204. doi: 10.1016/0092-8674(84)90315-5. [DOI] [PubMed] [Google Scholar]
  16. Godson C., Weiss B. A., Insel P. A. Differential activation of protein kinase C alpha is associated with arachidonate release in Madin-Darby canine kidney cells. J Biol Chem. 1990 May 25;265(15):8369–8372. [PubMed] [Google Scholar]
  17. Hartwig J. H., Thelen M., Rosen A., Janmey P. A., Nairn A. C., Aderem A. MARCKS is an actin filament crosslinking protein regulated by protein kinase C and calcium-calmodulin. Nature. 1992 Apr 16;356(6370):618–622. doi: 10.1038/356618a0. [DOI] [PubMed] [Google Scholar]
  18. Hirt R. P., Hughes G. J., Frutiger S., Michetti P., Perregaux C., Poulain-Godefroy O., Jeanguenat N., Neutra M. R., Kraehenbuhl J. P. Transcytosis of the polymeric Ig receptor requires phosphorylation of serine 664 in the absence but not the presence of dimeric IgA. Cell. 1993 Jul 30;74(2):245–255. doi: 10.1016/0092-8674(93)90416-n. [DOI] [PubMed] [Google Scholar]
  19. Hopkins C. R. Polarity signals. Cell. 1991 Sep 6;66(5):827–829. doi: 10.1016/0092-8674(91)90427-z. [DOI] [PubMed] [Google Scholar]
  20. Hoppe C. A., Connolly T. P., Hubbard A. L. Transcellular transport of polymeric IgA in the rat hepatocyte: biochemical and morphological characterization of the transport pathway. J Cell Biol. 1985 Dec;101(6):2113–2123. doi: 10.1083/jcb.101.6.2113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hug H., Sarre T. F. Protein kinase C isoenzymes: divergence in signal transduction? Biochem J. 1993 Apr 15;291(Pt 2):329–343. doi: 10.1042/bj2910329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hunziker W., Mâle P., Mellman I. Differential microtubule requirements for transcytosis in MDCK cells. EMBO J. 1990 Nov;9(11):3515–3525. doi: 10.1002/j.1460-2075.1990.tb07560.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hunziker W., Whitney J. A., Mellman I. Selective inhibition of transcytosis by brefeldin A in MDCK cells. Cell. 1991 Nov 1;67(3):617–627. doi: 10.1016/0092-8674(91)90535-7. [DOI] [PubMed] [Google Scholar]
  24. Huotari V., Sormunen R., Lehto V. P., Eskelinen S. Different organizational states of fodrin in cultured MDCK cells are induced by treatment with low pH, calmodulin antagonist TFP, and tumor promoter PMA. J Cell Physiol. 1992 Nov;153(2):340–352. doi: 10.1002/jcp.1041530214. [DOI] [PubMed] [Google Scholar]
  25. Jin G. F., Guo Y. S., Houston C. W. Bombesin: an activator of specific Aeromonas antibody secretion in rat intestine. Dig Dis Sci. 1989 Nov;34(11):1708–1712. doi: 10.1007/BF01540048. [DOI] [PubMed] [Google Scholar]
  26. Kelly R. B. Secretion. A question of endosomes. Nature. 1993 Aug 5;364(6437):487–488. doi: 10.1038/364487a0. [DOI] [PubMed] [Google Scholar]
  27. Kelly R. B. Storage and release of neurotransmitters. Cell. 1993 Jan;72 (Suppl):43–53. doi: 10.1016/s0092-8674(05)80027-3. [DOI] [PubMed] [Google Scholar]
  28. Kennelly P. J., Krebs E. G. Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases. J Biol Chem. 1991 Aug 25;266(24):15555–15558. [PubMed] [Google Scholar]
  29. Kraft A. S., Anderson W. B. Phorbol esters increase the amount of Ca2+, phospholipid-dependent protein kinase associated with plasma membrane. Nature. 1983 Feb 17;301(5901):621–623. doi: 10.1038/301621a0. [DOI] [PubMed] [Google Scholar]
  30. Luna E. J., Hitt A. L. Cytoskeleton--plasma membrane interactions. Science. 1992 Nov 6;258(5084):955–964. doi: 10.1126/science.1439807. [DOI] [PubMed] [Google Scholar]
  31. McGraw T. E., Dunn K. W., Maxfield F. R. Phorbol ester treatment increases the exocytic rate of the transferrin receptor recycling pathway independent of serine-24 phosphorylation. J Cell Biol. 1988 Apr;106(4):1061–1066. doi: 10.1083/jcb.106.4.1061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. McGraw T. E., Dunn K. W., Maxfield F. R. Phorbol ester treatment increases the exocytic rate of the transferrin receptor recycling pathway independent of serine-24 phosphorylation. J Cell Biol. 1988 Apr;106(4):1061–1066. doi: 10.1083/jcb.106.4.1061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Meier K. E., Sperling D. M., Insel P. A. Agonist-mediated regulation of alpha 1- and beta 2-adrenergic receptors in cloned MDCK cells. Am J Physiol. 1985 Jul;249(1 Pt 1):C69–C77. doi: 10.1152/ajpcell.1985.249.1.C69. [DOI] [PubMed] [Google Scholar]
  34. Mochly-Rosen D., Henrich C. J., Cheever L., Khaner H., Simpson P. C. A protein kinase C isozyme is translocated to cytoskeletal elements on activation. Cell Regul. 1990 Aug;1(9):693–706. doi: 10.1091/mbc.1.9.693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Mochly-Rosen D., Khaner H., Lopez J. Identification of intracellular receptor proteins for activated protein kinase C. Proc Natl Acad Sci U S A. 1991 May 1;88(9):3997–4000. doi: 10.1073/pnas.88.9.3997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mostov K. E., de Bruyn Kops A., Deitcher D. L. Deletion of the cytoplasmic domain of the polymeric immunoglobulin receptor prevents basolateral localization and endocytosis. Cell. 1986 Nov 7;47(3):359–364. doi: 10.1016/0092-8674(86)90592-1. [DOI] [PubMed] [Google Scholar]
  37. Mostov K., Apodaca G., Aroeti B., Okamoto C. Plasma membrane protein sorting in polarized epithelial cells. J Cell Biol. 1992 Feb;116(3):577–583. doi: 10.1083/jcb.116.3.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science. 1992 Oct 23;258(5082):607–614. doi: 10.1126/science.1411571. [DOI] [PubMed] [Google Scholar]
  39. Ojakian G. K. Tumor promoter-induced changes in the permeability of epithelial cell tight junctions. Cell. 1981 Jan;23(1):95–103. doi: 10.1016/0092-8674(81)90274-9. [DOI] [PubMed] [Google Scholar]
  40. Ono Y., Fujii T., Ogita K., Kikkawa U., Igarashi K., Nishizuka Y. Protein kinase C zeta subspecies from rat brain: its structure, expression, and properties. Proc Natl Acad Sci U S A. 1989 May;86(9):3099–3103. doi: 10.1073/pnas.86.9.3099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Podbilewicz B., Mellman I. ATP and cytosol requirements for transferrin recycling in intact and disrupted MDCK cells. EMBO J. 1990 Nov;9(11):3477–3487. doi: 10.1002/j.1460-2075.1990.tb07556.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Quintart J., Baudhuin P., Courtoy P. J. Marker enzymes in rat liver vesicles involved in transcellular transport. Eur J Biochem. 1989 Oct 1;184(3):567–574. doi: 10.1111/j.1432-1033.1989.tb15051.x. [DOI] [PubMed] [Google Scholar]
  43. Raphael G. D., Druce H. M., Baraniuk J. N., Kaliner M. A. Pathophysiology of rhinitis. 1. Assessment of the sources of protein in methacholine-induced nasal secretions. Am Rev Respir Dis. 1988 Aug;138(2):413–420. doi: 10.1164/ajrccm/138.2.413. [DOI] [PubMed] [Google Scholar]
  44. Rodriguez-Boulan E., Powell S. K. Polarity of epithelial and neuronal cells. Annu Rev Cell Biol. 1992;8:395–427. doi: 10.1146/annurev.cb.08.110192.002143. [DOI] [PubMed] [Google Scholar]
  45. Rothenberger S., Iacopetta B. J., Kühn L. C. Endocytosis of the transferrin receptor requires the cytoplasmic domain but not its phosphorylation site. Cell. 1987 May 8;49(3):423–431. doi: 10.1016/0092-8674(87)90295-9. [DOI] [PubMed] [Google Scholar]
  46. Song W., Bomsel M., Casanova J., Vaerman J. P., Mostov K. Stimulation of transcytosis of the polymeric immunoglobulin receptor by dimeric IgA. Proc Natl Acad Sci U S A. 1994 Jan 4;91(1):163–166. doi: 10.1073/pnas.91.1.163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wilson I. D., Soltis R. D., Olson R. E., Erlandsen S. L. Cholinergic stimulation of immunoglobulin A secretion in rat intestine. Gastroenterology. 1982 Oct;83(4):881–888. [PubMed] [Google Scholar]
  48. Young S., Parker P. J., Ullrich A., Stabel S. Down-regulation of protein kinase C is due to an increased rate of degradation. Biochem J. 1987 Jun 15;244(3):775–779. doi: 10.1042/bj2440775. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zerial M., Suomalainen M., Zanetti-Schneider M., Schneider C., Garoff H. Phosphorylation of the human transferrin receptor by protein kinase C is not required for endocytosis and recycling in mouse 3T3 cells. EMBO J. 1987 Sep;6(9):2661–2667. doi: 10.1002/j.1460-2075.1987.tb02557.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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