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 Calpha (PKCalpha), all cofactors for PLD activation. Whereas fMLP recruited all three proteins to membranes, V4+-OOH only elicited RhoA translocation and PMA induced ARF and PKCalpha 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 GTPgammaS-stimulated PLD activity in membranes and recruitment of ARF, RhoA, and PKCalpha to the membrane fraction. The data suggest that a tyrosine phosphorylation event is located upstream of the translocation of ARF, RhoA, and PKCalpha 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 PKCalpha 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 PKCalpha translocation was increased. We propose that RhoA translocation may be regulated by PKC in an ATP-independent manner. Furthermore, increased fMLP-induced PKCalpha 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.
Houle
M G
MG
Centre de Recherche en Rhumatologie et Immunologie, Centre de Recherche du CHUL, Ste-Foy, Québec, Canada.
Naccache
P H
PH
Bourgoin
S
S
D020727:ADP-Ribosylation Factors / Q000378:metabolism; D002934:Cinnamates / Q000494:pharmacology; D004357:Drug Synergism; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D018922:HL-60 Cells / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D010739:Phospholipase D / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D051571:Protein Kinase C-alpha; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D013440:Sulfides / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014443:Tyrosine / Q000378:metabolism; D017968:Vanadium Compounds / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D002934:Cinnamates; D020536:Enzyme Activators; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D013440:Sulfides; D017968:Vanadium Compounds; C060747:ST 638; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014443:Tyrosine; D009240:N-Formylmethionine Leucyl-Phenylalanine; D011505:Protein-Tyrosine Kinases; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D010739:Phospholipase D; D020558:GTP Phosphohydrolases; D020727:ADP-Ribosylation Factors; D020742:rhoA GTP-Binding Protein; D013755:Tetradecanoylphorbol Acetate