REGULATION OF MEMBRANE-BOUND PHOSPHOLIPASE-D BY PROTEIN-KINASE-C IN HL-60 CELLS - SYNERGISTIC ACTION OF SMALL GTP-BINDING PROTEIN RHOA

In HL60 cells, the membrane-bound phospholipase D (PLD) was stimulated by 4 beta-phorbol 12-myristate 13-acetate (PMA) in the presence of th e cytosolic fraction from HL60 cells or rat brain. The cytosolic facto r for this PMA-induced PLD activation was subjected to purification fr om rat brain by sequential chromatographies. The PLD stimulating activ ity was found in protein kinase C (PKC) fraction containing alpha, bet a I, beta II, and gamma isozymes. PKC isozymes were further separated by hydroxylapatite chromatography. PKC alpha and -beta, but not gamma, isozymes were found to activate membrane-bound PLD. PKC alpha was muc h more effective than PKC beta for PLD activation. Millimolar concentr ations of MgATP were required for the PKC-mediated PLD activation in H L60 membranes. MgATP is utilized to maintain the levels of phosphatidy linositol 4,5-bisphosphate (PIP2) under these assay conditions. The PK C-mediated PLD activation was completely inhibited by neomycin, a high affinity ligand for PIP2, and this suppression was recovered by the a ddition of exogenous PIP2(.) Thus, these results suggest that PIP2 is supposed to play a key role in PKC-mediated PLD activity in HL60 membr anes. Furthermore, PKC alpha-mediated PLD activation was potentiated b y the addition of recombinant RhoA protein in the presence of guanosin e 5'-O-(3-thiotriphosphate) (GTP gamma S). The results obtained here i ndicate that PKC alpha and RhoA (GTP form) exert a synergistic action in the membrane-bound PLD activation in HL60 cells.