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Role of phosphorylation sites and the C2 domain in regulation of cytosolic phospholipase A2.

ABSTRACT: Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocation of green fluorescent protein (GFP)-cPLA2 to the nuclear envelope. cPLA2 is phosphorylated on multiple sites in Sf9 cells; however, only S505 phosphorylation partially contributes to cPLA2 activation. Although okadaic acid does not increase calcium, mutating the calcium-binding residues D43 and D93 prevents arachidonic acid release and translocation of cPLA2, demonstrating the requirement for a functional C2 domain. However, the D93N mutant is fully functional with A23187, whereas the D43N mutant is nearly inactive. The C2 domain of cPLA2 linked to GFP translocates to the nuclear envelope with calcium-mobilizing agonists but not with okadaic acid. Consequently, the C2 domain is necessary and sufficient for translocation of cPLA2 to the nuclear envelope when calcium is increased; however, it is required but not sufficient with okadaic acid.

SUBMITTER: Gijon MA

PROVIDER: S-EPMC2133140 | biostudies-literature | 1999 Jun

REPOSITORIES: biostudies-literature

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Role of phosphorylation sites and the C2 domain in regulation of cytosolic phospholipase A2.

Gijón M A MA Spencer D M DM Kaiser A L AL Leslie C C CC

The Journal of cell biology 19990601 6

Cytosolic phospholipase A2 (cPLA2) mediates agonist-induced arachidonic acid release, the first step in eicosanoid production. cPLA2 is regulated by phosphorylation and by calcium, which binds to a C2 domain and induces its translocation to membrane. The functional roles of phosphorylation sites and the C2 domain of cPLA2 were investigated. In Sf9 insect cells expressing cPLA2, okadaic acid, and the calcium-mobilizing agonists A23187 and CryIC toxin induce arachidonic acid release and translocat ...[more]

PMID: 10366595

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Project description:BackgroundThe combined effects of glyphosate and hard water on chronic kidney disease of unknown etiology (CDKu) have attracted much interest, but the mechanisms remain unknown. Cytoplasmic phospholipase A2 (cPLA2) plays a key role in the acute and chronic inflammatory reactions. This study explored the effect of glyphosate combined with hard water on renal tubules and the possible targets and mechanisms involved.MethodsIn vivo experiments were conducted to investigate the synergistic effects and potential mechanisms of glyphosate and hard water on renal tubular injury in mice.ResultsAdministration of glyphosate in mice resulted in elevated levels of β2-microglobulin (β2-MG), albumin (ALB), and serum creatinine (SCr) compared to control mice. This increase was more pronounce when glyphosate was combined with hard water. In the glyphosate-treated mice, small areas of the kidney revealed fibroblast proliferation and vacuolar degeneration, particularly at the higher dose of 400 mg/kg glyphosate. However, the combination of glyphosate and hard water induced an even greater degree of pathological changes in the kidney. Immunofluorescence and western blot analyses showed that glyphosate and hard water had a coordinated effect on calcium ions (Ca2+)-activated phospholipase A2 and the activation may play a key role in inflammation and renal tubular injury. Exposure to glyphosate alone or glyphosate plus hard water increased the levels of oxidative stress markers and inflammatory biomarkers, namely, thromboxane A2 (TX-A2), leukotriene B4 (LTB4), prostaglandin E2 (PGE2), nitric oxide synthase (NOS), and nitric oxide (NO). Parameters of oxidative stress, including the levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were decreased. Further analysis showed that the levels of these biomarkers were significantly different between the mice treated with glyphosate plus hard water and the mice treated with glyphosate alone.ConclusionsThese findings suggested that hard water combined with glyphosate can induce renal tubular injury in mice, and this may involve mitogen-activated protein kinases (MAPK)/cytosolic phospholipase A2 (cPLA2)/arachidonic acid (AA) and its downstream factors.

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Role of phosphorylation sites and the C2 domain in regulation of cytosolic phospholipase A2.

Publications

Role of phosphorylation sites and the C2 domain in regulation of cytosolic phospholipase A2.

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