ADP-RIBOSYLATION FACTOR TRANSLOCATION CORRELATES WITH POTENTIATION OF GTP-GAMMA-S-STIMULATED PHOSPHOLIPASE-D ACTIVITY IN MEMBRANE-FRACTIONS OF HL-60 CELLS

Phospholipase D (PLD) activation by guanine nucleotides requires protein cofactors from both the membrane and the cytosol. The small GTP-binding protein ADP-ribosylation factor (ARF) has been established as one important component of PLD activation. By stimulating HL-60 cells with various agonists and then isolating the membrane fraction and assaying PLD activity in the presence and absence of GTP gamma S, we observed that fMet-Leu-Phe (fMLP) and phorbol esters induced a potentiation of GTP gamma S-stimulated PLD activity in the membrane fractions of these cells. Inactive phorbol esters induced no such potentiation. Both fMLP and active phorbol esters induced a 2-3-fold increase in GTP gamma S-stimulated PLD in HL-60 membranes, Membranes derived from stimulated HL-60 cells contained 60-70% more ARF as compared with membranes derived from control cells. Membrane contents of ARF were assessed by Western blotting with the anti-ARF monoclonal antibody 1D9 followed by densitometric evaluation. Therefore, ARF translocation correlates with the potentiation of the GTP gamma S-stimulated PLD activity. The effect on PLD activity and ARF membrane content achieved through fMLP stimulation was greatly enhanced by prior treatment of the cells with cytochalasin B. Membranes derived from control and fMLP-stimulated cells were assayed for PLD activity in the presence of exogenous ARF and a 50-kDa fraction known to contain elements implicated in PLD activation. The ability of ARF and the 50-kDa fraction to enhance GTP gamma S-sensitive PLD activity was significantly reduced when the membranes were derived from fMLP-stimulated cells. The data indicate that, in addition to ARF, elements of the 50-kDa PLD-inducing factors were likely already translocated to the membranes upon stimulation. We propose that ARF, upon stimulation with agonists such as fMLP or phorbol esters, is translocated to the membrane and in concert with other protein components of the 50-kDa fraction enhances PLD activity.