Tyrosine kinase-regulated small GTPase translocation and the activation of phospholipase D in HL60 granulocytes

We focus on the mechanisms of regulation of phospholipase D (PLD) activity. Three agonists known to stimulate PLD activity, fMet‐Leu‐Phe (fMLP), phorbol 12‐myristate 13‐acetate (PMA) and V4+‐OOH, induced a differential translocation of ADP‐ribosylation factor (ARF), RhoA, and protein kinase Cα (PKCα), all cofactors for PLD activation. Whereas fMLP recruited all three proteins to membranes, V4+‐OOH only elicited RhoA translocation and PMA induced ARF and PKCα translocation. Three tyrosine kinases inhibitors, ST‐638, methyl 2,5‐dihydroxycinnamate, and genistein reduced fMLP‐stimulated PLD activity by up to 80%. Furthermore, tyrosine kinase inhibitors reduced the fMLP‐induced increase of GTPγS‐stimulated PLD activity in membranes and recruitment of ARF, RhoA, and PKCα to the membrane fraction. The data suggest that a tyrosine phosphorylation event is located upstream of the translocation of ARF, RhoA, and PKCα in the signaling pathway leading to PLD activation by fMLP. RO 31‐8220, a specific inhibitor of PKC, reduced PMA‐induced PLD activity by 80% in intact HL60 granulocytes but enhanced fMLP‐stimulated PLD activity by 60%. Although PMA alone had no effect on RhoA recruitment to the membrane fraction, in the presence of RO 31‐8220 the levels of membrane‐bound RhoA were increased. The levels of membrane‐bound ARF and PKCα were unaffected by RO 31‐8220 during PMA stimulation. In contrast, fMLP‐induced recruitment of ARF and RhoA was insensitive to RO 31‐8220 but PKCα translocation was increased. We propose that RhoA translocation may be regulated by PKC in an ATP‐independent manner. Furthermore, increased fMLP‐induced PKCα translocation in the presence of RO 31‐8220 may partially account for the synergistic activation of PLD observed when both fMLP and RO 31‐8220 are used together in intact HL60 cells. J. Leukoc. Biol. 66: 1021–1030; 1999.