ATP-INDUCED POTENTIATION OF G-PROTEIN-DEPENDENT PHOSPHOLIPASE-D ACTIVITY IN A CELL-FREE SYSTEM FROM U937 PROMONOCYTIC LEUKOCYTES

Although G-protein- and protein kinase-mediated pathways have been rep orted to activate phospholipase D (PLD) following cell stimulation, th e relation between these activation pathways and the mechanistic detai ls of lipase stimulation remain unknown. We have studied activation of PLD by GTPgammaS (guanosine 5'-O-(thiotriphosphate)), and its potenti ation by ATP, in a cell-free system derived from U937 human promonocyt ic leukocytes. ATP, in the micromolar to millimolar range, significant ly augmented GTPgammaS-stimulated PLD activity (2.6-fold) and the comb ination resulted in a 15-fold increase in PLD activity compared to con trol. ATP alone did not stimulate PLD activity. Measurement of endogen ous cytosolic ATP levels and nucleotide depletion with activated charc oal demonstrated that stimulation of PLD by GTPgammaS proceeds by both ATP-dependent and -independent pathways. Nucleotide specificity data suggested that the ATP-dependent pathway involves kinase activity. The tyrosine phosphatase inhibitor vanadate augmented PLD activity stimul ated by GTPgammaS/ATP by 41% (p < 0.01). Conversely, the tyrosine kina se inhibitors genistein and herbimycin A decreased PLD activity stimul ated by GTPgammaS/ATP by 58 and 35%, respectively (p < 0.001 for each) . Mixing experiments utilizing subcellular fractions from herbimycin A -treated cells suggested that the relevant tyrosine kinase activity is membrane-associated. Despite its role in ATP-induced potentiation, ty rosine kinase activity is neither necessary nor sufficient for activat ion of PLD in this system. Protein kinase C (PKC) is unlikely to play a role in potentiation by ATP as PKC activity is not stimulated under conditions of maximal PLD activation.