Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1996 Nov 15;320(Pt 1):221–226. doi: 10.1042/bj3200221

Phosphorylation and activation of p42 and p44 mitogen-activated protein kinase are required for the P2 purinoceptor stimulation of endothelial prostacyclin production.

V Patel 1, C Brown 1, A Goodwin 1, N Wilkie 1, M R Boarder 1
PMCID: PMC1217921  PMID: 8947491

Abstract

Extracellular ATP and ADP, released from platelets and other sites stimulate the endothelial production of prostacyclin (PGI2) by acting on G-protein-coupled P2Y2 and P2Y2 purinoceptors, contributing to the maintenance of a non-thrombogenic surface. The mechanism, widely described as being dependent on elevated cytosolic [Ca2+], also requires protein tyrosine phosphorylation. Here we show that activation of both these P2 receptor types leads to the tyrosine phosphorylation and activation of both the p42 and p44 forms of mitogen-activated protein kinase (MAPK). 2-Methylthio-ATP and UTP, selectively activating P2Y1 and P2Y2 purinoceptors respectively, and ATP, a non-selective agonist at these two receptors, stimulate the tyrosine phosphorylation of both p42mapk and p44mapk, as revealed by Western blots with an antiserum specific for the tyrosine-phosphorylated forms of the enzymes. By using separation on Resource Q columns, peptide kinase activity associated with the phosphorylated MAPK enzymes distributes into two peaks, one mainly p42mapk and one mainly p44mapk, both of which are stimulated by ATP with respect to kinase activity and phospho-MAPK immunoreactivity. Stimulation of P2Y1 or P2Y2 purinoceptors leads to a severalfold increase in PGI2 efflux; this was blocked in a dose-dependent manner by the selective MAPK kinase inhibitor PD98059. This drug also blocked the agonist-stimulated increase in phospho-MAPK immunoreactivity for both p42mapk and p44mapk but left the phospholipase C response to P2 agonists essentially unchanged. Olomoucine has been reported to inhibit p44mapk activity. Here we show that in the same concentration range olomoucine inhibits activity in both peaks from the Resource Q column and also the agonist stimulation of 6-keto-PGF1, but has no effect on agonist-stimulated phospho-MAPK immunoreactivity. These results provide direct evidence for the involvement of p42 and p44 MAPK in the PGI2 response of intact endothelial cells: we have shown that both the endothelial P2Y purinoceptors are linked to activation of MAPK, and that activation of this pathway is a requirement for the stimulation by ATP/ADP of endothelial PGI2 production.

Full Text

The Full Text of this article is available as a PDF (377.5 KB).

Selected References

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

  1. Alessi D. R., Cuenda A., Cohen P., Dudley D. T., Saltiel A. R. PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem. 1995 Nov 17;270(46):27489–27494. doi: 10.1074/jbc.270.46.27489. [DOI] [PubMed] [Google Scholar]
  2. Boarder M. R., Weisman G. A., Turner J. T., Wilkinson G. F. G protein-coupled P2 purinoceptors: from molecular biology to functional responses. Trends Pharmacol Sci. 1995 Apr;16(4):133–139. doi: 10.1016/s0165-6147(00)89001-x. [DOI] [PubMed] [Google Scholar]
  3. Booyse F. M., Sedlak B. J., Rafelson M. E., Jr Culture of arterial endothelial cells: characterization and growth of bovine aortic cells. Thromb Diath Haemorrh. 1975 Dec 15;34(3):825–839. [PubMed] [Google Scholar]
  4. Bourne H. R. Signal transduction. Team blue sees red. Nature. 1995 Aug 31;376(6543):727–729. doi: 10.1038/376727a0. [DOI] [PubMed] [Google Scholar]
  5. Bowden A., Patel V., Brown C., Boarder M. R. Evidence for requirement of tyrosine phosphorylation in endothelial P2Y- and P2U- purinoceptor stimulation of prostacyclin release. Br J Pharmacol. 1995 Nov;116(6):2563–2568. doi: 10.1111/j.1476-5381.1995.tb17208.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Carter T. D., Hallam T. J., Cusack N. J., Pearson J. D. Regulation of P2y-purinoceptor-mediated prostacyclin release from human endothelial cells by cytoplasmic calcium concentration. Br J Pharmacol. 1988 Dec;95(4):1181–1190. doi: 10.1111/j.1476-5381.1988.tb11754.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carter T. D., Hallam T. J., Pearson J. D. Protein kinase C activation alters the sensitivity of agonist-stimulated endothelial-cell prostacyclin production to intracellular Ca2+. Biochem J. 1989 Sep 1;262(2):431–437. doi: 10.1042/bj2620431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clark-Lewis I., Sanghera J. S., Pelech S. L. Definition of a consensus sequence for peptide substrate recognition by p44mpk, the meiosis-activated myelin basic protein kinase. J Biol Chem. 1991 Aug 15;266(23):15180–15184. [PubMed] [Google Scholar]
  9. Crespo P., Cachero T. G., Xu N., Gutkind J. S. Dual effect of beta-adrenergic receptors on mitogen-activated protein kinase. Evidence for a beta gamma-dependent activation and a G alpha s-cAMP-mediated inhibition. J Biol Chem. 1995 Oct 20;270(42):25259–25265. doi: 10.1074/jbc.270.42.25259. [DOI] [PubMed] [Google Scholar]
  10. Crespo P., Xu N., Simonds W. F., Gutkind J. S. Ras-dependent activation of MAP kinase pathway mediated by G-protein beta gamma subunits. Nature. 1994 Jun 2;369(6479):418–420. doi: 10.1038/369418a0. [DOI] [PubMed] [Google Scholar]
  11. Crews C. M., Alessandrini A., Erikson R. L. The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. Science. 1992 Oct 16;258(5081):478–480. doi: 10.1126/science.1411546. [DOI] [PubMed] [Google Scholar]
  12. Force T., Kyriakis J. M., Avruch J., Bonventre J. V. Endothelin, vasopressin, and angiotensin II enhance tyrosine phosphorylation by protein kinase C-dependent and -independent pathways in glomerular mesangial cells. J Biol Chem. 1991 Apr 5;266(10):6650–6656. [PubMed] [Google Scholar]
  13. Gould G. W., Cuenda A., Thomson F. J., Cohen P. The activation of distinct mitogen-activated protein kinase cascades is required for the stimulation of 2-deoxyglucose uptake by interleukin-1 and insulin-like growth factor-1 in KB cells. Biochem J. 1995 Nov 1;311(Pt 3):735–738. doi: 10.1042/bj3110735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Graham A., McLees A., Kennedy C., Gould G. W., Plevin R. Stimulation by the nucleotides, ATP and UTP of mitogen-activated protein kinase in EAhy 926 endothelial cells. Br J Pharmacol. 1996 Mar;117(6):1341–1347. doi: 10.1111/j.1476-5381.1996.tb16734.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Huwiler A., Pfeilschifter J. Stimulation by extracellular ATP and UTP of the mitogen-activated protein kinase cascade and proliferation of rat renal mesangial cells. Br J Pharmacol. 1994 Dec;113(4):1455–1463. doi: 10.1111/j.1476-5381.1994.tb17160.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kosako H., Gotoh Y., Matsuda S., Ishikawa M., Nishida E. Xenopus MAP kinase activator is a serine/threonine/tyrosine kinase activated by threonine phosphorylation. EMBO J. 1992 Aug;11(8):2903–2908. doi: 10.1002/j.1460-2075.1992.tb05359.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lev S., Moreno H., Martinez R., Canoll P., Peles E., Musacchio J. M., Plowman G. D., Rudy B., Schlessinger J. Protein tyrosine kinase PYK2 involved in Ca(2+)-induced regulation of ion channel and MAP kinase functions. Nature. 1995 Aug 31;376(6543):737–745. doi: 10.1038/376737a0. [DOI] [PubMed] [Google Scholar]
  18. Lin L. L., Lin A. Y., Knopf J. L. Cytosolic phospholipase A2 is coupled to hormonally regulated release of arachidonic acid. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6147–6151. doi: 10.1073/pnas.89.13.6147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lin L. L., Wartmann M., Lin A. Y., Knopf J. L., Seth A., Davis R. J. cPLA2 is phosphorylated and activated by MAP kinase. Cell. 1993 Jan 29;72(2):269–278. doi: 10.1016/0092-8674(93)90666-e. [DOI] [PubMed] [Google Scholar]
  20. Lustig K. D., Sportiello M. G., Erb L., Weisman G. A. A nucleotide receptor in vascular endothelial cells is specifically activated by the fully ionized forms of ATP and UTP. Biochem J. 1992 Jun 15;284(Pt 3):733–739. doi: 10.1042/bj2840733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Luttrell L. M., van Biesen T., Hawes B. E., Koch W. J., Touhara K., Lefkowitz R. J. G beta gamma subunits mediate mitogen-activated protein kinase activation by the tyrosine kinase insulin-like growth factor 1 receptor. J Biol Chem. 1995 Jul 14;270(28):16495–16498. doi: 10.1074/jbc.270.28.16495. [DOI] [PubMed] [Google Scholar]
  22. Marrero M. B., Schieffer B., Paxton W. G., Heerdt L., Berk B. C., Delafontaine P., Bernstein K. E. Direct stimulation of Jak/STAT pathway by the angiotensin II AT1 receptor. Nature. 1995 May 18;375(6528):247–250. doi: 10.1038/375247a0. [DOI] [PubMed] [Google Scholar]
  23. Martin T. W., Wysolmerski R. B. Ca2+-dependent and Ca2+-independent pathways for release of arachidonic acid from phosphatidylinositol in endothelial cells. J Biol Chem. 1987 Sep 25;262(27):13086–13092. [PubMed] [Google Scholar]
  24. Motte S., Pirotton S., Boeynaems J. M. Heterogeneity of ATP receptors in aortic endothelial cells. Involvement of P2y and P2u receptors in inositol phosphate response. Circ Res. 1993 Mar;72(3):504–510. doi: 10.1161/01.res.72.3.504. [DOI] [PubMed] [Google Scholar]
  25. Needham L., Cusack N. J., Pearson J. D., Gordon J. L. Characteristics of the P2 purinoceptor that mediates prostacyclin production by pig aortic endothelial cells. Eur J Pharmacol. 1987 Feb 10;134(2):199–209. doi: 10.1016/0014-2999(87)90166-x. [DOI] [PubMed] [Google Scholar]
  26. Nemenoff R. A., Winitz S., Qian N. X., Van Putten V., Johnson G. L., Heasley L. E. Phosphorylation and activation of a high molecular weight form of phospholipase A2 by p42 microtubule-associated protein 2 kinase and protein kinase C. J Biol Chem. 1993 Jan 25;268(3):1960–1964. [PubMed] [Google Scholar]
  27. Pang L., Sawada T., Decker S. J., Saltiel A. R. Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J Biol Chem. 1995 Jun 9;270(23):13585–13588. doi: 10.1074/jbc.270.23.13585. [DOI] [PubMed] [Google Scholar]
  28. Patel V., Brown C., Boarder M. R. Protein kinase C isoforms in bovine aortic endothelial cells: role in regulation of P2Y- and P2U-purinoceptor-stimulated prostacyclin release. Br J Pharmacol. 1996 May;118(1):123–130. doi: 10.1111/j.1476-5381.1996.tb15374.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pearson J. D., Slakey L. L., Gordon J. L. Stimulation of prostaglandin production through purinoceptors on cultured porcine endothelial cells. Biochem J. 1983 Jul 15;214(1):273–276. doi: 10.1042/bj2140273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Purkiss J. R., Wilkinson G. F., Boarder M. R. Differential regulation of inositol 1,4,5-trisphosphate by co-existing P2Y-purinoceptors and nucleotide receptors on bovine aortic endothelial cells. Br J Pharmacol. 1994 Mar;111(3):723–728. doi: 10.1111/j.1476-5381.1994.tb14797.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Sa G., Murugesan G., Jaye M., Ivashchenko Y., Fox P. L. Activation of cytosolic phospholipase A2 by basic fibroblast growth factor via a p42 mitogen-activated protein kinase-dependent phosphorylation pathway in endothelial cells. J Biol Chem. 1995 Feb 3;270(5):2360–2366. doi: 10.1074/jbc.270.5.2360. [DOI] [PubMed] [Google Scholar]
  32. Schalkwijk C. G., van der Heijden M. A., Bunt G., Maas R., Tertoolen L. G., van Bergen en Henegouwen P. M., Verkleij A. J., van den Bosch H., Boonstra J. Maximal epidermal growth-factor-induced cytosolic phospholipase A2 activation in vivo requires phosphorylation followed by an increased intracellular calcium concentration. Biochem J. 1996 Jan 1;313(Pt 1):91–96. doi: 10.1042/bj3130091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schievella A. R., Regier M. K., Smith W. L., Lin L. L. Calcium-mediated translocation of cytosolic phospholipase A2 to the nuclear envelope and endoplasmic reticulum. J Biol Chem. 1995 Dec 22;270(51):30749–30754. doi: 10.1074/jbc.270.51.30749. [DOI] [PubMed] [Google Scholar]
  34. Veselý J., Havlicek L., Strnad M., Blow J. J., Donella-Deana A., Pinna L., Letham D. S., Kato J., Detivaud L., Leclerc S. Inhibition of cyclin-dependent kinases by purine analogues. Eur J Biochem. 1994 Sep 1;224(2):771–786. doi: 10.1111/j.1432-1033.1994.00771.x. [DOI] [PubMed] [Google Scholar]
  35. Wheeler-Jones C. P., May M. J., Houliston R. A., Pearson J. D. Inhibition of MAP kinase kinase (MEK) blocks endothelial PGI2 release but has no effect on von Willebrand factor secretion or E-selectin expression. FEBS Lett. 1996 Jun 17;388(2-3):180–184. doi: 10.1016/0014-5793(96)00547-9. [DOI] [PubMed] [Google Scholar]
  36. Wilkinson G. F., McKechnie K., Dainty I. A., Boarder M. R. P2Y purinoceptor and nucleotide receptor-induced relaxation of precontracted bovine aortic collateral artery rings: differential sensitivity to suramin and indomethacin. J Pharmacol Exp Ther. 1994 Feb;268(2):881–887. [PubMed] [Google Scholar]
  37. Wilkinson G. F., Purkiss J. R., Boarder M. R. Differential heterologous and homologous desensitization of two receptors for ATP (P2y purinoceptors and nucleotide receptors) coexisting on endothelial cells. Mol Pharmacol. 1994 Apr;45(4):731–736. [PubMed] [Google Scholar]
  38. Wilkinson G. F., Purkiss J. R., Boarder M. R. The regulation of aortic endothelial cells by purines and pyrimidines involves co-existing P2y-purinoceptors and nucleotide receptors linked to phospholipase C. Br J Pharmacol. 1993 Mar;108(3):689–693. doi: 10.1111/j.1476-5381.1993.tb12862.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Winitz S., Gupta S. K., Qian N. X., Heasley L. E., Nemenoff R. A., Johnson G. L. Expression of a mutant Gi2 alpha subunit inhibits ATP and thrombin stimulation of cytoplasmic phospholipase A2-mediated arachidonic acid release independent of Ca2+ and mitogen-activated protein kinase regulation. J Biol Chem. 1994 Jan 21;269(3):1889–1895. [PubMed] [Google Scholar]
  40. Zavoico G. B., Hrbolich J. K., Gimbrone M. A., Jr, Schafer A. I. Enhancement of thrombin- and ionomycin-stimulated prostacyclin and platelet-activating factor production in cultured endothelial cells by a tumor-promoting phorbol ester. J Cell Physiol. 1990 Jun;143(3):596–605. doi: 10.1002/jcp.1041430326. [DOI] [PubMed] [Google Scholar]
  41. van Biesen T., Hawes B. E., Luttrell D. K., Krueger K. M., Touhara K., Porfiri E., Sakaue M., Luttrell L. M., Lefkowitz R. J. Receptor-tyrosine-kinase- and G beta gamma-mediated MAP kinase activation by a common signalling pathway. Nature. 1995 Aug 31;376(6543):781–784. doi: 10.1038/376781a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES