Phosphorylation-dependent regulation of phospholipase A2 by G-proteins and Ca2+ in HL60 granulocytes.
We studied the regulation of arachidonic acid (AA) release by guanosine 5'-O-(3-thiotriphosphate (GTP gamma S) and Ca2+ in electropermeabilized HL60 granulocytes. Stimulation of AA release by GTP gamma S and Ca2+ was mediated by phospholipase A2 (PLA2) and required the presence of MgATP (EC50: 100-250 microM). The nucleotide effects were Ca(2+)-dependent (maximal effects detected at 1 microM free cation). UTP and ATP gamma S, which stimulate AA release in intact HL60 granulocytes with potencies and efficacies similar to those of ATP, were ineffective in supporting the effects of GTP gamma S in electropermeabilized cells. Pretreatment with pertussis toxin affected stimulation of AA release by ATP in intact cell, without altering the nucleotide effects in permeabilized cells. We observed the protein kinase C-dependent phosphorylation of PLA2 in permeabilized HL60 granulocytes, together with a correlation between the effects of phorbol esters and staurosporine on this reaction and on AA release. ATP-independent activation of PLA2 by GTP gamma S and/or Ca2+ was measured in subcellular fractions prepared from HL60 granulocytes. These data appear consistent with a model in which PLA2 activity in resting HL60 granulocytes is subjected to an inhibitory constraint that prevents its activation by Ca2+ and G-proteins. Removal of this constraint, either by the protein kinase C-dependent phosphorylation of the enzyme in vivo or physical disruption of the regulatory assembly (e.g. by N2 cavitation), allows its activation by Ca2+ and G-proteins.
Xing
M
M
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Mattera
R
R
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D016718:Arachidonic Acid / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006098:Granulocytes / Q000187:drug effects / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D006706:Homeostasis; D006801:Humans; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D015473:Leukemia, Promyelocytic, Acute; D008954:Models, Biological; D037342:Pertussis Toxin; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D012265:Ribonucleotides / Q000494:pharmacology; D019311:Staurosporine; D014407:Tumor Cells, Cultured; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000470:Alkaloids; D012265:Ribonucleotides; D010566:Virulence Factors, Bordetella; D007531:Isoflurophate; D000266:Adenylyl Imidodiphosphate; D016718:Arachidonic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D037342:Pertussis Toxin; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D019311:Staurosporine; D002118:Calcium