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Reconstitution System Based on Cytosol-Depleted Cells to Study the Regulation of Phospholipase D

  • Amanda Fensome-Green
  • Shamshad Cockcroft
Part of the Methods in Molecular Biology™ book series (MIMB, volume 332)

Summary

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to produce the membraneassociated second messenger, phosphatidic acid (PA) and choline. Two phospholipase D enzymes—PLD1 and PLD2—have been identified, although their regulatory mechanisms are yet to be fully understood. To study the regulation of PLD, we established a reconstitution system that allows the study of the PLD enzymes in their native environment while enabling the cytosol to be manipulated. Cells are permeabilized with a bacterial cytolysin (streptolysin O), which produces lesions in the plasma membrane, resulting in the release of cytosolic proteins. With increasing permeabilization times, guanosine 5′-[γ-thio]triphosphate and receptor-activated PLD activity diminishes. Once the conditions for the run-down of the response is established, cellular factors, such as cytosol and purified proteins, can be added to these cells to restore activity. In addition to examining PLD activity, this reconstitution system allows the study of potential cellular targets of PA, such as phosphatidylinositol 4-phosphate (PIP) 5-kinase activity by monitoring PIP2 synthesis, and also functional outputs, such as exocytosis.

Key Words

ARF phosphatidylcholine phosphatidate streptolysin O alcohols permeabilization 

References

  1. 1.
    Cockcroft S. (1997) Phospholipase D: regulation by GTPases and protein kinase C and physiological relevance. Prog. Lipid Res. 35, 345–370.CrossRefGoogle Scholar
  2. 2.
    Cockcroft S. (2001) Signalling roles of mammalian phospholipase D1 and D2. Cell Mol. Life Sci. 58, 1674–1687.PubMedCrossRefGoogle Scholar
  3. 3.
    Exton J. H. (2002) Phospholipase D-structure, regulation and function. Rev. Physiol. Biochem. Pharmacol. 144, 1–94.PubMedCrossRefGoogle Scholar
  4. 4.
    Nakamura S., Akisue T., Jinnai H., et al. (1998) Requirement of GM2 ganglioside activator for phospholipase D activation. Proc. Natl. Acad. Sci. USA 95, 12,249–12,253.PubMedCrossRefGoogle Scholar
  5. 5.
    Sarkar S., Miwa N., Kominami H., et al. (2001) Regulation of mammalian phospholipase D2: interaction with and stimulation by G(M2) activator. Biochem. J. 359, 599–604.PubMedCrossRefGoogle Scholar
  6. 6.
    Lee S., Park J. B., Kim J. H., et al. (2001) Actin directly interacts with phospholipase D, inhibiting its activity. J. Biol. Chem. 276, 28,252–28,260.PubMedCrossRefGoogle Scholar
  7. 7.
    Park J. B., Kim J. H., Kim Y., et al. (2000) Cardiac phospholipase D2 localizes to sarcolemmal membranes and is inhibited by a-actinin in an ADP-ribosylation factor-reversible manner. J. Biol. Chem. 275, 21,295–21,301.PubMedCrossRefGoogle Scholar
  8. 8.
    Jenco J. M., Rawlingson A., Daniels B., and Morris A. J. (1998) Regulation of phospholipase D2-selective inhibition of mammalian phospholipase D isozymes by alpha-and beta-synucleins. Biochemistry 37, 4901–4909.PubMedCrossRefGoogle Scholar
  9. 9.
    Cullen P. J., Cozier G. E., Banting G., and Mellor H. (2001) Modular phosphoinositide-binding domains—their role in signalling and membrane trafficking. Curr. Biol. 11, R882–R893.PubMedCrossRefGoogle Scholar
  10. 10.
    Sciorra V. A., Rudge S. A., Prestwich G. D., Frohman M. A., Engebrecht J., and Morris A. J. (1999) Identifcation of a phosphoinositide binding motif that mediates activation of mammalian and yeast phospholipase D isoenzymes. EMBO J. 20, 5911–5921.CrossRefGoogle Scholar
  11. 11.
    Way G., O’Luanaigh N., and Cockcroft S. (2000) Activation of exocytosis by cross-linking of the IgE receptor is dependent on ARF-regulated phospholipase D in RBL-2H3 mast cells: Evidence that the mechanism of activation is via regulation of PIP2 synthesis. Biochem. J. 346, 63–70.PubMedCrossRefGoogle Scholar
  12. 12.
    O’Luanaigh N., Pardo R., Fensome A., et al. (2002) Continual production of phosphatidic acid by phospholipase D is essential for antigen-stimulated membrane ruffling in cultured mast cells. Mol. Biol. Cell 13, 3730–3746.PubMedCrossRefGoogle Scholar
  13. 13.
    Skippen A., Jones D. H., Morgan C. P., Li M., and Cockcroft S. (2002) Mechanism of ADP-ribosylation factor-stimulated phosphatidylinositol 4,5-bisphosphate synthesis in HL60 cells. J. Biol. Chem. 277, 5823–5831.PubMedCrossRefGoogle Scholar
  14. 14.
    Cockcroft S., Thomas G. M. H., Fensome A., et al. (1994) Phospholipase D: A downstream effector of ARF in granulocytes. Science 263, 523–526.PubMedCrossRefGoogle Scholar
  15. 15.
    Fensome A., Cunningham E., Prosser S., et al. (1996) ARF and PITP restore GTPγS-stimulated protein secretion from cytosol-depleted HL60 cells by promoting PIP2 synthesis. Curr. Biol. 6, 730–738.PubMedCrossRefGoogle Scholar
  16. 16.
    Sarri E., Pardo R., Fensome-Green A., and Cockcroft S. (2003) Endogenous phospholipase D2 localizes to the plasma membrane of RBL 2H3 mast cells and can be distinguished from ADP ribosylation factor-stimulated phospholipase D1 activity by its specific sensitivity to oleic acid. Biochem. J. 369, 319–329.PubMedCrossRefGoogle Scholar
  17. 17.
    Fensome A., Whatmore J., Morgan C. P., Jones D., and Cockcroft S. (1998) ADP-ribosylation factor and Rho proteins mediate fMLP-dependent activation of phospholipase D in human neutrophils. J. Biol. Chem. 273, 13,157–13,164.PubMedCrossRefGoogle Scholar
  18. 18.
    Leino L., Forbes L., Segal A., and Cockcroft S. (1999) Reconstitution ofG TPgammaS-induced NADPH oxidase activity in streptolysin-O permeabilised neutrophils by specific cytosol fractions. Biochem. Biophys. Res. Commun. 265, 29–37.PubMedCrossRefGoogle Scholar
  19. 19.
    Tatham P. E. R. and Gomperts B. D. (1990) Cell permeabilisation, in Peptide Hormones— A Practical Approach (Siddle K. and Hutton J. C., eds.). IRL Press, Oxford, pp. 257–269.Google Scholar
  20. 20.
    Geny B. and Cockcroft S. (1992) Synergistic activation of phospholipase D by a protein kinase C-and a G-protein-mediated pathway in streptolysin Opermeabilized HL60 cells. Biochem. J. 284, 531–538.PubMedGoogle Scholar
  21. 21.
    Thomas G. M. H., Cunningham E., Fensome A., et al. (1993) An essential role for phosphatidylinositol transfer protein in phospholipase C-mediated inositol lipid signalling. Cell 74, 919–928.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Amanda Fensome-Green
    • 1
  • Shamshad Cockcroft
    • 2
  1. 1.Department of Physiology, Rockefeller BuildingUniversity College LondonLondonUK
  2. 2.Department of Physiology, Rockefeller BuildingUniversity College LondonLondonUK

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