Skip to main content
SpringerLink
Log in

Regulation of acetylcholine-induced phosphorylation of PLD1 in porcine tracheal smooth muscle

  • Original Paper
  • Published:
Journal of Biomedical Science

Abstract

The muscarinic agonist, acetylcholine (ACh), stimulates phospholipase D (PLD) activity in tracheal smooth muscle cells. Direct activation of protein kinase C (PKC) by phorbol-12-myristate-13-acetate (PMA) also stimulates PLD in this tissue. Activation of ACh-induced PLD was inhibited by the tyrosine kinase inhibitor genistein in a concentration-dependent manner. Presently known isoforms of PLD, PLD1 and PLD2, were identified in tracheal smooth muscle and their activation-induced phosphorylation status studied. Both ACh and PMA increased phosphorylation of PLD1 that was significantly blocked by genistein or the PKC inhibitor calphostin C. PLD2 phosphorylation was not detected in the present experiments. Western blots probed with an anti-phosphotyrosine antibody indicate that PLD1 in this tissue is phosphorylated on tyrosine residues after ACh or PMA stimulation. Tyrosine phosphorylation of PLD1 was blocked by genistein and calphostin C. No tyrosine residues were phosphorylated on PLD2. Taken together, these results demonstrate that porcine tracheal smooth muscle cells express both isoforms PLD1 and PLD2. However, on muscarinic activation only PLD1 in this tissue is phosphorylated by PKC via a tyrosine-kinase-dependent pathway.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Med Sci 37:911–917;1959.

    Google Scholar 

  2. Bocckino SB, Wilson PB, Exton JH. Phosphatidate-dependent protein phosphorylation. Proc Natl Acad Sci USA 88:6210–6213;1991.

    PubMed  Google Scholar 

  3. Choi WS, Chahdi A, Kim YM, Fraundorfer PF, Beaven MA. Regulation of phospholipase D and secretion in mast cells by protein kinase A and other protein kinases. Ann NY Acad Sci 968:198–212;2002.

    PubMed  Google Scholar 

  4. Colley WC, Sung TC, Roll R, Jenco J, Hammond SM, Altshuller Y, Bar-Sagi D, Morris AJ, Frohman MA. Phospholipase D2, a distinct phospholipase D isoform with novel regulatory properties that provokes cytoskeletal reorganization. Curr Biol 7:191–201;1997.

    Article  PubMed  Google Scholar 

  5. Exton JH. Regulation of phospholipase D. FEBS Lett 53:58–61;2002.

    Article  Google Scholar 

  6. Exton JH. Phospholipase D: Enzymology, mechanisms of regulation, and function. Physiol Rev 77:303–320;1997.

    PubMed  Google Scholar 

  7. Ghelli A, Porcelli AM, Facchini A, Hrelia S, Flamigni F, Rugolo M. Phospholipase D1 is threonine-phosphorylated in human-airway epithelial cells stimulated by sphingosine-1-phosphate by a mechanism involving Src tyrosine kinase and protein kinase C delta. Biochem J 366:187–193;2002.

    PubMed  Google Scholar 

  8. Hammond SM, Jenco JM, Nakashima S, Cadwallader K, Gu Q, Cook S, Nozawa Y, Prestwich GD, Frohman MA, Morris AJ. Characterization of two alternately spliced forms of phospholipase D1. Activation of the purified enzymes by phosphatidylinositol 4,5-bisphosphate, ADP-ribosylation factor, and Rho family monomeric GTP-binding proteins and protein kinase C-alpha. J Biol Chem 272:3860–3868;1997.

    Article  PubMed  Google Scholar 

  9. Han JM, Kim JH, Lee BD, Lee SD, Kim Y, Jung YW, Lee S, Cho W, Ohba M, Kuroki T, Suh PG, Ryu SH. Phosphorylation-dependent regulation of phospholipase D2 by protein kinase C delta in rat Pheochromocytoma PC12 cells. J Biol Chem 277:8290–8297;2002.

    Article  PubMed  Google Scholar 

  10. Han JM, Kim Y, Lee JS, Lee CS, Lee BD, Ohba M, Kuroki T, Suh PG, Ryu SH. Localization of phospholipase D1 to caveolin-enriched membrane via palmitoylation: Implications for epidermal growth factor signaling. Mol Biol Cell 13:3976–3988;2002.

    Article  PubMed  Google Scholar 

  11. Hu T, Exton JH. Mechanisms of regulation of phospholipase D1 by protein kinase Cα. J Biol Chem 278:2348–2355;2003.

    Article  PubMed  Google Scholar 

  12. Kim Y, Han JM, Han BR, Lee KA, Kim JH, Lee BD, Jang IH, Suh PG, Ryu SH. Phospholipase D1 is phosphorylated and activated by protein kinase C in caveolin-enriched microdomains within the plasma membrane. J Biol Chem 275:13621–13627;2000.

    Article  PubMed  Google Scholar 

  13. Krieg T, Qin Q, McIntosh EC, Cohen MV, Downey JM. ACh and adenosine activate PI3-kinase in rabbit hearts through transactivation of receptor tyrosine kinases. Am J Physiol Heart Circ Physiol 283:H2322-H2330;2002.

    PubMed  Google Scholar 

  14. Liscovitch M. Signal-dependent activation of phosphatidylcholine hydrolysis: Role of phospholipase D. Biochem Soc Trans 19:402–407;1991.

    PubMed  Google Scholar 

  15. Liscovitch M, Czarny M, Fiucci G, Tang X. Phospholipase D: Molecular and cell biology of a novel gene family. Biochem J 345:401–415;2000.

    PubMed  Google Scholar 

  16. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275;1951.

    PubMed  Google Scholar 

  17. Mamoon AM, Baker RC, Farley JM. Activation of phospholipase D in porcine tracheal smooth muscle: role of phosphatidylinositol 3-kinase and RhoA activation. Eur Pharmacol 433:7–16;2001.

    Article  Google Scholar 

  18. Mamoon AM, Smith J, Baker RC, Farley JM. Activation of protein kinase A increases phospholipase D activity and inhibits phospholipase D activation by acetylcholine in tracheal smooth muscle. J Pharmacol Exp Ther 291:1188–1195;1999.

    PubMed  Google Scholar 

  19. Marcil J, Harbour D, Naccache PH, Bourgoin S. Human phospholipase D1 can be tyrosine-phosphorylated in HL-60 granulocytes. J Biol Chem 272:20660–20664;1997.

    Article  PubMed  Google Scholar 

  20. Mehta S, Maglio J, Kobayashi MS, Sipple AM, Horwitz J. Activation of phospholipase D is not mediated by direct phosphorylation on tyrosine residues. Biochim Biophys Acta 1631:246–254;2003.

    PubMed  Google Scholar 

  21. Meier KE, Gibbs TC, Knoepp SM, Ella KM. Expression of phospholipase D isoforms in mammalian cells. Biochim Biophys Acta 1439:199–213;1999.

    PubMed  Google Scholar 

  22. Min DS, Kim EG, Exton JH. Involvement of tyrosine phosphorylation and protein kinase C in the activation of phospholipase D by H2O2 in Swiss 3T3 fibroblasts. J Biol Chem 273:29986–29994;1998.

    Article  PubMed  Google Scholar 

  23. Moehren G, Gustavsson L, Hoek JB. Activation and desensitization of phospholipase D in intact rat hepatocytes. J Biol Chem 269:838–848;1994.

    PubMed  Google Scholar 

  24. Morris AJ, Frohman MA, Engebrecht J. Measurement of phospholipase D activity. Anal Biochem 252:1–9;1997.

    Article  PubMed  Google Scholar 

  25. Natarajan V, Scribner WM, Vepa S. Regulation of phospholipase D by tyrosine kinases. Chem Phys Lipids 80:103–116;1996.

    Article  PubMed  Google Scholar 

  26. Oka M, Hitomi T, Okada T, Nakamura SiS, Nagai H, Ohba M, Kuroki T, Kikkawa U, Ichihashi M. Dual regulation of phospholipase D1 by protein kinase C alpha in vivo. Biochem Biophys Res Commun 294:1109–1113;2002.

    Article  PubMed  Google Scholar 

  27. Oka M, Okada T, Nakamura S, Ohba M, Kuroki T, Kikkawa U, Nagai H, Ichihashi M, Nishigori C. PKCdelta inhibits PKCalpha-mediated activation of phospholipase D1 in a manner independent of its protein kinase activity. FEBS Lett 554:179–183;2003.

    Article  PubMed  Google Scholar 

  28. Pochet S, Metioui M, Grosfils K, Gomez-Munoz A, Marino A, Dehaye JP. Regulation of phospholipase D by muscarinic receptors in rat submandibular ductal cells. Cell Signal 15:103–113;2003.

    Article  PubMed  Google Scholar 

  29. Simons K, Ikonen E. Functional rafts in cell membranes. Nature 387:569–572;1997.

    Article  PubMed  Google Scholar 

  30. Slaaby R, Jensen T, Hansen HS, Frohman MA, Seedorf K. PLD2 complexes with the EGF receptor and undergoes tyrosine phosphorylation at a single site upon agonist stimulation. J Biol Chem 273:33722–33727;1998.

    Article  PubMed  Google Scholar 

  31. Tomic S, Greiser U, Lammers R, Kharitonenkov A, Imyanitov E, Ullrich A, Bohmer FD. Association of SH2 domain protein tyrosine phosphatases with the epidermal growth factor receptor in human tumor cells. Phosphatidic acid activates receptor dephosphorylation by PTP1C. J Biol Chem 270:21277–21284;1995.

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmacology and Toxicology, University of Mississippi Medical Center, 2500 North State Street, 39216-4505, Jackson, MS, USA

    Abulkhair M. Mamoon, Rodney C. Baker & Jerry M. Farley PhD

Authors
  1. Abulkhair M. Mamoon
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rodney C. Baker
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jerry M. Farley PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Mamoon, A.M., Baker, R.C. & Farley, J.M. Regulation of acetylcholine-induced phosphorylation of PLD1 in porcine tracheal smooth muscle. J Biomed Sci 11, 810–817 (2004). https://doi.org/10.1007/BF02254366

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02254366

Key Words

Search

Navigation