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. 1993 May 15;292(Pt 1):121–128. doi: 10.1042/bj2920121

Phospholipase D activity in phagocytic leucocytes is synergistically regulated by G-protein- and tyrosine kinase-based mechanisms.

G R Dubyak 1, S J Schomisch 1, D J Kusner 1, M Xie 1
PMCID: PMC1134277  PMID: 8503838

Abstract

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.

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Selected References

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