Regulation of membrane-bound phospholipase D by protein kinase C in HL60 cells. Synergistic action of small GTP-binding protein RhoA.
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.
Ohguchi
K
K
Department of Biochemistry, Gifu University School of Medicine, Tsukasamachi-40, Gifu 500, Japan.
Banno
Y
Y
Nakashima
S
S
Nozawa
Y
Y
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D017886:Durapatite; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D010739:Phospholipase D / Q000378:metabolism*; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D007527:Isoenzymes; D009829:Oleic Acids; D019301:Oleic Acid; D017886:Durapatite; D011493:Protein Kinase C; D010739:Phospholipase D; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate