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  • Title: Regulation of membrane-bound phospholipase D by protein kinase C in HL60 cells. Synergistic action of small GTP-binding protein RhoA.
    Author: Ohguchi K, Banno Y, Nakashima S, Nozawa Y.
    Journal: J Biol Chem; 1996 Feb 23; 271(8):4366-72. PubMed ID: 8626786.
    Abstract:
    In HL60 cells, the membrane-bound phospholipase D (PLD) was stimulated by 4beta-phorbol 12-myristate 13-acetate (PMA) in the presence of the cytosolic fraction from HL60 cells or rat brain. The cytosolic factor for this PMA-induced PLD activation was subjected to purification from rat brain by sequential chromatographies. The PLD stimulating activity was found in protein kinase C (PKC) fraction containing alpha, betaI, betaII, and gamma isozymes. PKC isozymes were further separated by hydroxylapatite chromatography. PKCalpha and - beta, but not gamma, isozymes were found to activate membrane-bound PLD. PKCalpha was much more effective than PKCbeta for PLD activation. Millimolar concentrations of MgATP were required for the PKC-mediated PLD activation in HL60 membranes. MgATP is utilized to maintain the levels of phosphatidylinositol 4,5-bisphosphate (PIP2) under these assay conditions. The PKC-mediated PLD activation was completely inhibited by neomycin, a high affinity ligand for PIP2, and this suppression was recovered by the addition of exogenous PIP2. Thus, these results suggest that PIP2 is supposed to play a key role in PKC-mediated PLD activity in HL60 membranes. Furthermore, PKCalpha-mediated PLD activation was potentiated by the addition of recombinant RhoA protein in the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The results obtained here indicate that PKCalpha and RhoA (GTP form) exert a synergistic action in the membrane-bound PLD activation in HL60 cells.
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