DOI QR코드

DOI QR Code

EP2 Induces p38 Phosphorylation via the Activation of Src in HEK 293 Cells

  • Chun, Kyung-Soo (College of Pharmacy, Keimyung University) ;
  • Shim, Minsub (Department of Biological Sciences, University of South Carolina)
  • Received : 2015.04.22
  • Accepted : 2015.08.17
  • Published : 2015.11.01

Abstract

Prostaglandin $E_2$ ($PGE_2$), a major product of cyclooxygenase, binds to four different prostaglandin $E_2$ receptors (EP1, EP2, EP3, and EP4) which are G-protein coupled transmembrane receptors (GPCRs). Although GPCRs including EP receptors have been shown to be associated with their specific G proteins, recent evidences suggest that GPCRs can regulate MAPK signaling via non-G protein coupled pathways including Src. EP2 is differentially expressed in various tissues and the expression of EP2 is induced by extracellular stimuli. We hypothesized that an increased level of EP2 expression may affect MAPK signaling. The overexpression of EP2 in HEK 293 cells resulted in significant increase in intracellular cAMP levels response to treatment with butaprost, a specific EP2 agonist, while overexpression of EP2 alone did not increase intracellular cAMP levels. However, EP2 overexpression in the absence of $PGE_2$ induced an increase in the level of p38 phosphorylation as well as the kinase activity of p38, suggesting that up-regulation of EP2 may promote p38 activation via non-G protein coupled pathway. Inhibition of Src completely blocked EP2-induced p38 phosphorylation and overexpression of Src increased the level of p38 phosphorylation, indicating that Src is upstream kinase for EP2-induced p38 phosphorylation. EP2 overexpression also increased the Src activity and EP2 protein was co-immunoprecipitated with Src. Furthermore, sequential co-immunoprecipitation studies showed that EP2, Src, and ${\beta}$-arrestin can form a complex. Our study found a novel pathway in which EP2 is associated with Src, regulating p38 pathway.

Keywords

References

  1. Abrahamsen, H., Vang, T. and Tasken, K. (2003) Protein kinase A intersects SRC signaling in membrane microdomains. J. Biol. Chem. 278, 17170-17177. https://doi.org/10.1074/jbc.M211426200
  2. Arinsburg, S. S., Cohen, I. S. and Yu, H. G. (2006) Constitutively active Src tyrosine kinase changes gating of HCN4 channels through direct binding to the channel proteins. J. Cardiovasc. Pharmacol. 47, 578-586. https://doi.org/10.1097/01.fjc.0000211740.47960.8b
  3. Biscardi, J. S., Maa, M. C., Tice, D. A., Cox, M. E., Leu, T. H. and Parsons, S. J. (1999) c-Src-mediated phosphorylation of the epidermal growth factor receptor on Tyr845 and Tyr1101 is associated with modulation of receptor function. J. Biol. Chem. 274, 8335-8343. https://doi.org/10.1074/jbc.274.12.8335
  4. Bos, C. L., Richel, D. J., Ritsema, T., Peppelenbosch, M. P. and Versteeg, H. H. (2004) Prostanoids and prostanoid receptors in signal transduction. Int. J. Biochem. Cell Biol. 36, 1187-1205. https://doi.org/10.1016/j.biocel.2003.08.006
  5. Buchanan, F. G., Gorden, D. L., Matta, P., Shi, Q., Matrisian, L. M. and DuBois, R. N. (2006) Role of beta-arrestin 1 in the metastatic progression of colorectal cancer. Proc Natl. Acad. Sci. U.S.A. 103, 1492-1497. https://doi.org/10.1073/pnas.0510562103
  6. Cao, W., Luttrell, L. M., Medvedev, A. V., Pierce, K. L., Daniel, K. W., Dixon, T. M., Lefkowitz, R. J. and Collins, S. (2000) Direct binding of activated c-Src to the beta 3-adrenergic receptor is required for MAP kinase activation. J. Biol. Chem. 275, 38131-38134. https://doi.org/10.1074/jbc.C000592200
  7. Castellone, M. D., Teramoto, H., Williams, B. O., Druey, K. M. and Gutkind, J. S. (2005) Prostaglandin E2 promotes colon cancer cell growth through a Gs-axin-beta-catenin signaling axis. Science 310, 1504-1510. https://doi.org/10.1126/science.1116221
  8. Chang, L. and Karin, M. (2001) Mammalian MAP kinase signalling cascades. Nature 410, 37-40. https://doi.org/10.1038/35065000
  9. Chell, S. D., Witherden, I. R., Dobson, R. R., Moorghen, M., Herman, A. A., Qualtrough, D., Williams, A. C. and Paraskeva, C. (2006) Increased EP4 receptor expression in colorectal cancer progression promotes cell growth and anchorage independence. Cancer Res. 66, 3106-3113. https://doi.org/10.1158/0008-5472.CAN-05-3702
  10. DeFea, K. A., Vaughn, Z. D., O'Bryan, E. M., Nishijima, D., Dery, O. and Bunnett, N. W. (2000) The proliferative and antiapoptotic effects of substance P are facilitated by formation of a beta -arrestindependent scaffolding complex. Proc. Natl. Acad. Sci. U.S.A. 97, 11086-11091. https://doi.org/10.1073/pnas.190276697
  11. Derijard, B., Raingeaud, J., Barrett, T., Wu, I. H., Han, J., Ulevitch, R. J. and Davis, R. J. (1995) Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 267, 682-685. https://doi.org/10.1126/science.7839144
  12. Estrella, V. C., Eder, A. M., Liu, S., Pustilnik, T. B., Tabassam, F. H., Claret, F. X., Gallick, G. E., Mills, G. B. and Wiener, J. R. (2007) Lysophosphatidic acid induction of urokinase plasminogen activator secretion requires activation of the p38MAPK pathway. Int. J. Oncol. 31, 441-449.
  13. Faour, W. H., He, Q., Mancini, A., Jovanovic, D., Antoniou, J. and Di Battista, J. A. (2006) Prostaglandin E2 stimulates p53 transactivational activity through specific serine 15 phosphorylation in human synovial fibroblasts. Role in suppression of c/EBP/NF-kappaBmediated MEKK1-induced MMP-1 expression. J. Biol. Chem. 281, 19849-19860. https://doi.org/10.1074/jbc.M601293200
  14. Fujino, H., West, K. A. and Regan, J. W. (2002) Phosphorylation of glycogen synthase kinase-3 and stimulation of T-cell factor signaling following activation of EP2 and EP4 prostanoid receptors by prostaglandin E2. J. Biol. Chem. 277, 2614-2619. https://doi.org/10.1074/jbc.M109440200
  15. Fujino, H., Xu, W. and Regan, J. W. (2003) Prostaglandin E2 induced functional expression of early growth response factor-1 by EP4, but not EP2, prostanoid receptors via the phosphatidylinositol 3-kinase and extracellular signal-regulated kinases. J. Biol. Chem. 278, 12151-12156. https://doi.org/10.1074/jbc.M212665200
  16. Ge, B., Gram, H., Di Padova, F., Huang, B., New, L., Ulevitch, R. J., Luo, Y. and Han, J. (2002) MAPKK-independent activation of p38alpha mediated by TAB1-dependent autophosphorylation of p38alpha. Science 295, 1291-1294. https://doi.org/10.1126/science.1067289
  17. Hanyaloglu, A. C. and Zastrow, M. V. (2008) Regulation of GPCRs by endocytic membrane trafficking and its potential implications. Annu. Rev. Pharmacol. Toxicol. 48, 537-568. https://doi.org/10.1146/annurev.pharmtox.48.113006.094830
  18. Honda, T., Segi-Nishida, E., Miyachi, Y. and Narumiya, S. (2006) Prostacyclin-IP signaling and prostaglandin E2-EP2/EP4 signaling both mediate joint inflammation in mouse collagen-induced arthritis. J. Exp. Med. 203, 325-335. https://doi.org/10.1084/jem.20051310
  19. Hubbard, N. E., Lee, S., Lim, D. and Erickson, K. L. (2001) Differential mRNA expression of prostaglandin receptor subtypes in macrophage activation. Prostaglandins Leukot. Essent. Fatty Acids 65, 287-294. https://doi.org/10.1054/plef.2001.0327
  20. Hunter, T. (1987) A tail of two src's: mutatis mutandis. Cell 49, 1-4. https://doi.org/10.1016/0092-8674(87)90745-8
  21. Ikegami, R., Sugimoto, Y., Segi, E., Katsuyama, M., Karahashi, H., Amano, F., Maruyama, T., Yamane, H., Tsuchiya, S. and Ichikawa, A. (2001) The expression of prostaglandin E receptors EP2 and EP4 and their different regulation by lipopolysaccharide in C3H/HeN peritoneal macrophages. J. Immunol. 166, 4689-4696. https://doi.org/10.4049/jimmunol.166.7.4689
  22. Katsuyama, M., Ikegami, R., Karahashi, H., Amano, F., Sugimoto, Y. and Ichikawa, A. (1998) Characterization of the LPS-stimulated expression of EP2 and EP4 prostaglandin E receptors in mouse macrophage-like cell line, J774.1. Biochem. Biophys. Res. Commun. 251, 727-731. https://doi.org/10.1006/bbrc.1998.9540
  23. Kumar, S., Boehm, J. and Lee, J. C. (2003) p38 MAP kinases: key signalling molecules as therapeutic targets for inflammatory diseases. Nat. Rev. Drug Discov. 2, 717-726. https://doi.org/10.1038/nrd1177
  24. Lefkowitz, R. J. and Whalen, E. J. (2004) beta-arrestins: traffic cops of cell signaling. Curr. Opin. Cell Biol. 16, 162-168. https://doi.org/10.1016/j.ceb.2004.01.001
  25. Luttrell, L. M., Ferguson, S. S., Daaka, Y., Miller, W. E., Maudsley, S., Della Rocca, G. J., Lin, F., Kawakatsu, H., Owada, K., Luttrell, D. K., Caron, M. G. and Lefkowitz, R. J. (1999) Beta-arrestin-dependent formation of beta2 adrenergic receptor-Src protein kinase complexes. Science 283, 655-661. https://doi.org/10.1126/science.283.5402.655
  26. Ma, Y. C. and Huang, X. Y. (2002) Novel signaling pathway through the beta-adrenergic receptor. Trends Cardiovasc. Med. 12, 46-49. https://doi.org/10.1016/S1050-1738(01)00138-4
  27. O'Hayre, M., Degese, M. S. and Gutkind, J. S. (2014) Novel insights into G protein and G protein-coupled receptor signaling in cancer. Curr. Opin. Cell Biol. 27, 126-135. https://doi.org/10.1016/j.ceb.2014.01.005
  28. Raingeaud, J., Whitmarsh, A. J., Barrett, T., Derijard, B. and Davis, R. J. (1996) MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol. Cell Biol. 16, 1247-1255. https://doi.org/10.1128/MCB.16.3.1247
  29. Regan, J. W. (2003) EP2 and EP4 prostanoid receptor signaling. Life Sci. 74, 143-153. https://doi.org/10.1016/j.lfs.2003.09.031
  30. Rouse, J., Cohen, P., Trigon, S., Morange, M., Alonso-Llamazares, A., Zamanillo, D., Hunt, T. and Nebreda, A. R. (1994) A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78, 1027-1037. https://doi.org/10.1016/0092-8674(94)90277-1
  31. Salvador, J. M., Mittelstadt, P. R., Guszczynski, T., Copeland, T. D., Yamaguchi, H., Appella, E., Fornace, A. J., Jr. and Ashwell, J. D. (2005) Alternative p38 activation pathway mediated by T cell receptor-proximal tyrosine kinases. Nat. Immunol. 6, 390-395. https://doi.org/10.1038/ni1177
  32. Shenoy, S. K. and Lefkowitz, R. J. (2005) Seven-transmembrane receptor signaling through beta-arrestin. Sci STKE 2005, cm10.
  33. Summy, J. M., Trevino, J. G., Baker, C. H. and Gallick, G. E. (2005) c-Src regulates constitutive and EGF-mediated VEGF expression in pancreatic tumor cells through activation of phosphatidyl inositol-3 kinase and p38 MAPK. Pancreas 31, 263-274. https://doi.org/10.1097/01.mpa.0000178280.50534.0c
  34. Sun, Y., Huang, J., Xiang, Y., Bastepe, M., Juppner, H., Kobilka, B. K., Zhang, J. J. and Huang, X. Y. (2007a) Dosage-dependent switch from G protein-coupled to G protein-independent signaling by a GPCR. EMBO J. 26, 53-64. https://doi.org/10.1038/sj.emboj.7601502
  35. Sun, Y., McGarrigle, D. and Huang, X. Y. (2007b) When a G proteincoupled receptor does not couple to a G protein. Mol. Biosyst. 3, 849-854. https://doi.org/10.1039/b706343a
  36. Sung, Y. M., He, G., Hwang, D. H. and Fischer, S. M. (2006) Overexpression of the prostaglandin E2 receptor EP2 results in enhanced skin tumor development. Oncogene 25, 5507-5516. https://doi.org/10.1038/sj.onc.1209538
  37. Tice, D. A., Biscardi, J. S., Nickles, A. L. and Parsons, S. J. (1999) Mechanism of biological synergy between cellular Src and epidermal growth factor receptor. Proc. Natl. Acad. Sci. U.S.A. 96, 1415-1420. https://doi.org/10.1073/pnas.96.4.1415
  38. Uddin, S., Lekmine, F., Sharma, N., Majchrzak, B., Mayer, I., Young, P. R., Bokoch, G. M., Fish, E. N. and Platanias, L. C. (2000) The Rac1/p38 mitogen-activated protein kinase pathway is required for interferon alpha-dependent transcriptional activation but not serine phosphorylation of Stat proteins. J. Biol. Chem. 275, 27634-27640.
  39. Williams, J. A. (2001) Intracellular signaling mechanisms activated by cholecystokinin-regulating synthesis and secretion of digestive enzymes in pancreatic acinar cells. Ann. Rev. Physiol. 63, 77-97. https://doi.org/10.1146/annurev.physiol.63.1.77
  40. Xi, X., Han, J. and Zhang, J. Z. (2001) Stimulation of glucose transport by AMP-activated protein kinase via activation of p38 mitogen-actiated protein kinase. J. Biol. Chem. 276, 41029-41034. https://doi.org/10.1074/jbc.M102824200
  41. Yin, G., Yan, C. and Berk, B. C. (2003) Angiotensin II signaling pathways mediated by tyrosine kinases. Int. J. Biochem. Cell Biol. 35, 780-783. https://doi.org/10.1016/S1357-2725(02)00300-X
  42. Yuan, J. and Rozengurt, E. (2008) PKD, PKD2, and p38 MAPK mediate Hsp27 serine-82 phosphorylation induced by neurotensin in pancreatic cancer PANC-1 cells. J. Cell. Biochem. 103, 648-662. https://doi.org/10.1002/jcb.21439
  43. Zhang, L., Jiang, L., Sun, Q., Peng, T., Lou, K., Liu, N. and Leng, J. (2007) Prostaglandin E2 enhances mitogen-activated protein kinase/Erk pathway in human cholangiocarcinoma cells: involvement of EP1 receptor, calcium and EGF receptors signaling. Mol. Cell. Biochem. 305, 19-26. https://doi.org/10.1007/s11010-007-9523-5

Cited by

  1. Reciprocal regulation of β 2 -adrenoceptor-activated cAMP response-element binding protein signalling by arrestin2 and arrestin3 vol.38, 2017, https://doi.org/10.1016/j.cellsig.2017.07.011
  2. Comprehensive Analysis of Non-Synonymous Natural Variants of G Protein-Coupled Receptors vol.26, pp.2, 2018, https://doi.org/10.4062/biomolther.2017.073
  3. Thymoquinone Suppresses Migration of Human Renal Carcinoma Caki-1 Cells through Inhibition of the PGE2-Mediated Activation of the EP2 Receptor Pathway vol.29, pp.1, 2021, https://doi.org/10.4062/biomolther.2020.048