Phospholipase D activity in phagocytic leucocytes is synergistically regulated by G-protein- and tyrosine kinase-based mechanisms.
The regulation of phospholipase D (PLD)-type effector enzymes by G-proteins and protein kinases/phosphatases was characterized in the U937 human promonocytic leucocyte line. PLD activity was assayed by measuring (in the presence of 1% ethanol) the accumulation of phosphatidylethanol in cells permeabilized with beta-escin, a saponin-like detergent. Basal PLD activity was very low when cells were permeabilized and incubated in cytosol-like medium containing micromolar [Ca2+]. When this medium was supplemented with exogenous MgATP or guanosine 5'-[gamma-thio]triphosphate (GTP[S]), PLD activity increased by 9- and 14-fold respectively. Cells permeabilized in the absence of exogenously added MgATP, but in the presence of 1 microM vanadate/100 microM H2O2, also exhibited a modest 12-fold increase in PLD activity. However, the simultaneous presence of either GTP[S] plus exogenous MgATP or GTP[S] plus vanadate/H2O2 (and endogenous MgATP) induced similar 60-75-fold increases in the rate and extent of phosphatidylethanol accumulation. These latter effects of vanadate/H2O2 were strongly correlated with the very rapid accumulation of multiple tyrosine-phosphorylated proteins. Other studies utilized cells which were permeabilized in the presence of GTP[S] and then washed before assay of PLD. These cells retained approximately 60% of the MgATP-regulatable PLD activity (EC50 approximately = to 100 microM MgATP) observed in freshly permeabilized non-washed cells. In the absence of GTP[S] pre-treatment, washed cells retained minimal PLD activity. Genistein, a tyrosine kinase inhibitor, significantly attenuated the ability of MgATP to stimulate PLD activity and accumulation of tyrosine-phosphorylated proteins in the washed GTP[S]-treated cells. These data suggest that PLD activity in myeloid leucocytes involves co-ordinate regulation by both G-protein(s) and tyrosine phosphorylation.
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, OH 44106.
G R
GR
Dubyak
S J
SJ
Schomisch
D J
DJ
Kusner
M
M
Xie
D000255:Adenosine Triphosphate / Q000494:pharmacology; D002478:Cells, Cultured; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007962:Leukocytes / Q000201:enzymology* / Q000276:immunology; D010587:Phagocytosis; D010739:Phospholipase D / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
C069276:peroxovanadate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014638:Vanadates; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D010739:Phospholipase D; D019204:GTP-Binding Proteins