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 C alpha (PKC alpha), all cofactors for PLD activation. Whereas fMLP recruited all three proteins to membranes, V4+-OOH only elicited RhoA translocation and PMA induced ARF and PKC alpha 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 GTP gamma S-stimulated PLD activity in membranes and recruitment of ARF, RhoA, and PKC alpha to the membrane fraction. The data suggest that a tyrosine phosphorylation event is located upstream of the translocation of ARF, RhoA, and PKC alpha 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 PKC alpha 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 PKC alpha translocation was increased, We propose that RhoA translocation may be regulated by PKC in an ATP-independent manner. Furthermore, increased fMLP-induced PKC alpha 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.