Determination of Phospholipase C-or Phospholipase D-Catalyzed Phosphatidylcholine Hydrolysis

Signal-activated phospholipase-catalyzed hydrolysis of phosphatidylcholin involves three distinct enzymes: phospholipase A
2
, 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
).