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 reported to activate phospholipase D (PLD) following cell stimulation, the relation between these activation pathways and the mechanistic details of lipase stimulation remain unknown. We have studied activation of PLD by GTP gamma S (guanosine 5'-O-(thiotriphosphate)), and its potentiation by ATP, in a cell-free system derived from U937 human promonocytic leukocytes. ATP, in the micromolar to millimolar range, significantly augmented GTP gamma S-stimulated PLD activity (2.6-fold) and the combination resulted in a 15-fold increase in PLD activity compared to control. ATP alone did not stimulate PLD activity. Measurement of endogenous cytosolic ATP levels and nucleotide depletion with activated charcoal demonstrated that stimulation of PLD by GTP gamma S 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 stimulated by GTP gamma S/ATP by 41% (p < 0.01). Conversely, the tyrosine kinase inhibitors genistein and herbimycin A decreased PLD activity stimulated by GTP gamma S/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, tyrosine kinase activity is neither necessary nor sufficient for activation 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.
Kusner
D J
DJ
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106.
Schomisch
S J
SJ
Dubyak
G R
GR
D000255:Adenosine Triphosphate / Q000378:metabolism* / Q000494:pharmacology*; D002474:Cell-Free System; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D009000:Monocytes; D010739:Phospholipase D / Q000378:metabolism*; D012265:Ribonucleotides / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured; D014638:Vanadates / Q000494:pharmacology
D012265:Ribonucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014638:Vanadates; D000255:Adenosine Triphosphate; D010739:Phospholipase D; D019204:GTP-Binding Proteins