Role of rho proteins in agonist regulation of phospholipase D in HL-60 cells.
Rho family GTP-binding proteins have been demonstrated to play a role in the regulation of phospholipase D (PLD) activity. In the present study, we examined the role of Rho proteins in PLD activation in differentiated HL-60 cells using C3 exoenzyme from Clostridium botulinum, which ADP-ribosylates and inactivates Rho proteins. Introduction of C3 exoenzyme into differentiated HL-60 cells by electroporation resulted in complete inhibition of PLD activity stimulated by formyl methionine-leucine-phenylalanine (fMLP) and ATP, two receptor agonists. Phorbol myristate acetate-induced PLD activation was also inhibited in C3 exoenzyme-treated cells, but the inhibition was only partial. GTPgammaS-dependent activation of PLD, measured in the absence or presence of ATP in permeabilized cells, was also partially affected by C3 exoenzyme treatment. Thus, these results indicate that Rho proteins play a key role in receptor-mediated PLD regulation in differentiated HL-60 cells, but play a partial role in the in vivo action of PMA and in vitro action of GTPgammaS on PLD. ATP produced a significant enhancement of the in vitro effect of GTPgammaS on PLD activity, but the effect of ATP was not altered by inhibitors of serine/threonine and tyrosine kinases. However, it was markedly reduced by neomycin and accompanied by an increase in phosphatidylinositol 4,5-bisphosphate (PtdInsP2) synthesis. These data indicate that in permeabilized HL-60 cells, the stimulatory effect of ATP on PLD does not involve protein phosphorylation but is due to an increase in PtdInsP2.
Guillemain
I
I
Howard Hughes Medical Institute and Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232, USA.
Exton
J H
JH
D036002:ADP Ribose Transferases / Q000378:metabolism / Q000494:pharmacology; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D001905:Botulinum Toxins; D002454:Cell Differentiation; D003014:Clostridium botulinum / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D007700:Kinetics; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010739:Phospholipase D / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation
D019269:Phosphatidylinositol 4,5-Diphosphate; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; D000255:Adenosine Triphosphate; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D010739:Phospholipase D; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins