Abstract
Signal-activated phospholipase-catalyzed hydrolysis of phosphatidylcholin involves three distinct enzymes: phospholipase A2, phospholipase C (PLC), and phospholipase D (PLD) (1). PLC-catalyzed hydrolysis generates sn-1,2-diacylglycerol (DAG) and choline phosphate, whereas PLD stimulates the generation of phosphatidate (PA) and choline. Choline and choline phosphate are probably not messengers, although there have been some claims of a signaling role for the latter. DAG is the physiological activator of protein kinase C, whereas PA has an incompletely defined messenger function but has been demonstrated to activate a number of serine/threonine kinases and to play a role in secretion and rho-dependent actin stress-fiber formation (2). PA and DAG are apparently interconvertable through the action of phosphatidate phosphohydrolase and diacylglycerol kinase. However, work from this laboratory has recently demonstrated that the acyl structure of PLD-derived PA and PLC-derived DAG is distinct, the latter being polyunsaturated, whereas the former is more saturated/monounsaturated suggesting a specificity between the two signaling pathways (3,4). Indeed, the DAG generated from PLD-derived PA does not activate protein kinase C in vivo. It is thus of importance to be clear of the source of the DAG and PA when attempting to define the signaling of an agonist-stimulated cell. Polyunsaturated DAG is generally derived from phospholipase C-catalyzed phosphatidylinositol 4,5-bis-phosphate hydrolysis; however, there are examples of agonist-stimulated phospholipase C-catalyzed phosphatidylcholine hydrolysis (5–7).
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Wakelam, M. J. O. (1994) An overview of the regulation of phospholipase-linked cell signalling, in New Targets for Cancer Chemotherapy (Workman, P. and Kerr, D. J., eds.), CRC, pp. 31–44.
Exton, J. H. (1994) Phosphatidylcholine breakdown and signal transduction. Biochim. Biophys. Acta 1212, 26–42.
Pettitt, T. R. and Wakelam, M. J. O. (1993) Bombesin stimulates distinct time-dependent changes in the sn-1,2-diradylglycerol molecular species profile from Swiss 3T3 fibroblasts as analysed by 3,5-dinitrobenzoyl derivitisation and HPLC separation. Biochem. J. 289, 487–495.
Pettitt, T. R., Martin, A., Horton, T., Liossiss, C., Lord, J. M., and Wakelam, M. J. O. (1997) Diacylglycerol and phosphatidate generated by phospholipases C and D respectively have distinct fatty acid compositions and functions: phospholipase D-derived diacylglycerol does not activate protein kinase C in PAE cells. J. Biol. Chem. 272, 17,354–17,359.
Cook, S. J. and Wakelam, M. J. O. (1992) Epidermal growth factor increases sn-1,2-diacylglycerol levels and activates phospholipase D-catalysed phosphatidylcholine breakdown in Swiss 3T3 cells in the absence of inositol-lipid hydrolysis. Biochem. J. 284, 247–253.
Pettitt, T. R., Zaqqa, M., and Wakelam, M. J. O. (1994) Epidermal growth factor stimulates distinct diradylglycerol species generation in Swiss 3T3 fibroblasts: evidence for a potential phosphatidylcholine-phospholipase C-catalysed pathway. Biochem J. 298, 655–660.
Pyne, S. and Pyne, N. J. (1995) Bradykinin-stimulated phosphatidylcholine hydrolysis in airway smooth muscle—the role of calcium and protein kinase C. Biochem. J. 311, 637–642.
Wakelam, M. J. O., Hodgkin, M., and Martin, A. (1995) The measurement of phospholipase D linked signalling in cells, in Receptor Transduction Protocols (Kendall, D. A and Hill, S. J., eds.), Humana, New Jersey, pp. 271–278.
Cook, S. J. and Wakelam, M. J. O. (1989) Analysis of the water soluble products of phosphatidylcholine breakdown by ion exchange chromatography: bombesin and c-kinase stimulated choline generation in Swiss 3T3 cells. Biochem. J. 263, 581–587.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1998 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Wakelam, M.J.O., Pettitt, T.R. (1998). Determination of Phospholipase C-or Phospholipase D-Catalyzed Phosphatidylcholine Hydrolysis. In: Bird, I.M. (eds) Phospholipid Signaling Protocols. Methods in Molecular Biology™, vol 105. Humana Press. https://doi.org/10.1385/0-89603-491-7:141
Download citation
DOI: https://doi.org/10.1385/0-89603-491-7:141
Publisher Name: Humana Press
Print ISBN: 978-0-89603-491-4
Online ISBN: 978-1-59259-255-5
eBook Packages: Springer Protocols