Unique targeting of cytosolic phospholipase A2 to plasma membranes mediated by the NADPH oxidase in phagocytes

Abstract

Cytosolic phospholipase A2 (cPLA2)–generated arachidonic acid (AA) has been shown to be an essential requirement for the activation of NADPH oxidase, in addition to its being the major enzyme involved in the formation of eicosanoid at the nuclear membranes. The mechanism by which cPLA2 regulates NADPH oxidase activity is not known, particularly since the NADPH oxidase complex is localized in the plasma membranes of stimulated cells. The present study is the first to demonstrate that upon stimulation cPLA2 is transiently recruited to the plasma membranes by a functional NADPH oxidase in neutrophils and in granulocyte-like PLB-985 cells. Coimmunoprecipitation experiments and double labeling immunofluorescence analysis demonstrated the unique colocalization of cPLA2 and the NADPH oxidase in plasma membranes of stimulated cells, in correlation with the kinetic burst of superoxide production. A specific affinity in vitro binding was detected between GST-p47phox or GST-p67phox and cPLA2 in lysates of stimulated cells. The association between these two enzymes provides the molecular basis for AA released by cPLA2 to activate the assembled NADPH oxidase. The ability of cPLA2 to regulate two different functions in the same cells (superoxide generation and eicosanoid production) is achieved by a novel dual subcellular localization of cPLA2 to different targets.

A Regulated Adaptor Function of p40phox: Distinct p67phoxMembrane Targeting by p40phoxand by p47phox

“…Interestingly, both cPLA 2 and secretory PLA 2 accumulate on newly formed phagosomes (Shmelzer et al, 2003;Girotti et al, 2004;Balestrieri et al, 2006). Furthermore, cells lacking Nox2 show no translocation of cPLA 2 to membranes, and it seems that human cPLA 2 associates directly with the cytoplasmic phox proteins and the membrane-bound Nox2 complex after stimulation by several agonists that activate the oxidase (Shmelzer et al, 2003).…”

“…However, macrophages or neutrophils from cPLA 2 -deficient mice exhibited normal stimulated ROS release (Gijon et al, 2000;Rubin et al, 2005), suggesting the murine systems are less dependent on AA. Surprisingly, human cPLA 2 -deficient cells show normal translocation of phox proteins (Dana et al, 1994;Dana et al, 1998;Shmelzer et al, 2003), suggesting cPLA 2 serves other roles in oxidase activation during Nox2 assembly. Interestingly, both cPLA 2 and secretory PLA 2 accumulate on newly formed phagosomes (Shmelzer et al, 2003;Girotti et al, 2004;Balestrieri et al, 2006).…”

“…Surprisingly, human cPLA 2 -deficient cells show normal translocation of phox proteins (Dana et al, 1994;Dana et al, 1998;Shmelzer et al, 2003), suggesting cPLA 2 serves other roles in oxidase activation during Nox2 assembly. Interestingly, both cPLA 2 and secretory PLA 2 accumulate on newly formed phagosomes (Shmelzer et al, 2003;Girotti et al, 2004;Balestrieri et al, 2006). Furthermore, cells lacking Nox2 show no translocation of cPLA 2 to membranes, and it seems that human cPLA 2 associates directly with the cytoplasmic phox proteins and the membrane-bound Nox2 complex after stimulation by several agonists that activate the oxidase (Shmelzer et al, 2003).…”

“…Although relatively high concentrations of AA (50 -200 M) are required for activation of Nox2 both in vitro and in vivo, functional roles for cytosolic phospholipase A 2 (cPLA 2 ), which produces AA, in Nox2 activation have been demonstrated at the cellular level (Dana et al, 1998;Zhao et al, 2002;Shmelzer et al, 2003). Furthermore, recent studies have shown that the orchestration of low concentrations of AA produced by PLA 2 together with protein kinase C (PKC)-dependent phosphorylation promotes translocation of p47 phox and enhances ROS production by Nox2 (Shiose and Sumimoto, 2000;Peng et al, 2003).…”

Polymorphonuclear leukocyte mediated oxidative inactivation of alpha-1-proteinase inhibitor: Modulation by nitric oxide

“…PMA ( a non physiological stimulus) activates cells by stimulating extracellular signal regulated kinases (ERKs) through a PKC dependent pathway, whereas OZ involves the tyrosine kinase, Pyk2 dependent activation of cytosolic phospholipase A2 and the stimulation is further mediated by FC~" receptor and not by complement receptor C3b protein (2,3,34). The precise mechanism for AA mediated activation of NADPH oxidase is still elusive, however several studies suggest that AA induces structural changes in NADPH oxidase components that may promote productive interactions between different oxidase subunits to provide a fully active oxidase or directly affecting the function of flavorcytochrome b (35,36,37). FMLP, a chemoattractant bacterial peptide, is a potent activator of neutrophil degranulation (38) and the activation occurs via diverse pathways including phosphorylation/inactivation of the FO• subfamily of fork-head transcription factors (FKHR, FKHR-L1 and AFX) through the phosphatidylinositol-3-kinase/Akt (protein kinase B) and the RAS mitogen-activated protein kinase pathways (39,40,41,42).…”