title: Inhibition of cytosolic phospholipase A2 by annexin V in differentiated permeabilized HL-60 cells. Evidence of crucial importance of domain I type II Ca2+-binding site in the mechanism of inhibition.
abstract: Annexin V belongs to a family of proteins that interact with phospholipids in a Ca2+-dependent manner. This protein has been demonstrated to have anti-phospholipase A2 activity. However, this effect has never yet been reported with the 85-kDa cytosolic PLA2 (cPLA2). We studied, in a model of differentiated and streptolysin O-permeabilized HL-60 cells, the effect of annexin V on cPLA2 activity after stimulation by calcium, GTPgammaS (guanosine 5'-O-(3-thiotriphosphate)), formyl-Met-Leu-Phe, or phorbol 12-myristate 13-acetate. Both recombinant and human placental purified annexin V inhibit cPLA2 activity whatever the stimulus used. The decrease of arachidonic acid release is of 40 and 50%, respectively, at [Ca2+] of 3 and 10 microM. The mechanism of inhibition was also analyzed. cPLA2 requires calcium and protein kinase C (PKC) or mitogen-activated protein kinase phosphorylation for its activation. As annexin V was shown to be an endogenous inhibitor of PKC, PKC-stimulated cPLA2 activity was analyzed. Using GF109203x, a specific PKC inhibitor, we demonstrated that this pathway is of minor importance in our model. cPLA2 inhibition by annexin V is not linked to PKC inhibition. To test the hypothesis of phospholipid depletion, mutants of annexin V were constructed using mutagenesis directed to Ca2+ site. We demonstrate that the Ca2+ site located in domain I is necessary for the inhibitory effect of annexin V on cPLA2 activity. The site in domain IV is also involved but with less efficiency. In contrast, mutations in site II and III do not modify this effect. Moreover, annexin V mutated on all sites does not inhibit cPLA2. Thus, we propose a predominant role of module (I/IV) in the biological action of annexin V, which, in physiological conditions, may control cPLA2 activity by depletion of the phospholipid substrate.
authors.affiliation: Unité 332, Institut Cochin de Génétique Moleculaire, INSERM, 22 rue Mechain, 75014 Paris, France.
authors.forename: J P
authors.initials: JP
authors.lastname: Mira
authors.forename: T
authors.initials: T
authors.lastname: Dubois
authors.forename: J P
authors.initials: JP
authors.lastname: Oudinet
authors.forename: S
authors.initials: S
authors.lastname: Lukowski
authors.forename: F
authors.initials: F
authors.lastname: Russo-Marie
authors.forename: B
authors.initials: B
authors.lastname: Geny
mesh_terms: D017304:Annexin A5 / Q000494:pharmacology*; D016718:Arachidonic Acid / Q000378:metabolism; D001426:Bacterial Proteins; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D002454:Cell Differentiation; D002463:Cell Membrane Permeability; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2; D010920:Placenta; D011247:Pregnancy; D011493:Protein Kinase C / Q000378:metabolism; D011994:Recombinant Proteins / Q000494:pharmacology*; D013301:Streptolysins; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D017304:Annexin A5; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D011994:Recombinant Proteins; D013301:Streptolysins; C025964:streptolysin O; D016718:Arachidonic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium