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

Jean-Paul Mira,Thierry Dubois,Jean-Paul Oudinet,Sandra Lukowski,Françoise Russo-Marie,Blandine Geny

doi:10.1074/jbc.272.16.10474

Jean-Paul Mira, Thierry Dubois + Show 4 more

Open Access

https://doi.org/10.1074/jbc.272.16.10474

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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.

Highlights

Annexin V belongs to a family of proteins that interact with phospholipids in a Ca2؉-dependent manner
Using GF109203x, a specific protein kinase C (PKC) inhibitor, we demonstrated that this pathway is of minor importance in our model. cytosolic PLA2 (cPLA2) inhibition by annexin V is not linked to PKC inhibition
We demonstrate that the Ca2؉ site located in domain I is necessary for the inhibitory effect of annexin V on cPLA2 activity

Summary

EXPERIMENTAL PROCEDURES

Reagents—HL-60 cells were given by Dr T. Cells were resuspended at a concentration of 2 ϫ 107 cells/ml in buffer A and labeled at 37 °C for 1 h with 1 ␮Ci/ml [3H]arachidonic acid Under these conditions, more than 70% of total radioactive molecules were incorporated into cellular phospholipids. At the end of labeling, cells were washed three times and resuspended at a concentration of 2 ϫ 107 cells/ml in buffer B containing 137 mM NaCl, 2.7 mM KCl, 20 mM PIPES, and 0.2% protease and fatty acid-free bovine serum albumin, pH 6.8. The mutants Glu-72 3 Gln (M1), Asp-144 3 Asn (M2), and Asp-303 3 Asn (M4) were produced with the synthetic oligonucleotides 5Ј-PCCTGAAATCACAAGCTGACTGGAAAATTTG-3Ј, 5Ј-PGGAAGATGACGTCGTGGGTAACACTTCAGG-3Ј, and 5Ј-PGATTTAAGGGAAATACTAGTGGCGACTATAAG-3Ј respectively Another calcium-binding site, related to the presence of Trp-187 and located in domain III, appears to be essential [7]. All data shown are means Ϯ standard error of the means

RESULTS

Phospholipase Activity

DISCUSSION