Role of phosphorylation sites and the C2 domain in regulation of cytosolic phospholipase A2.
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.
Gijón
M A
MA
Division of Basic Science, Department of Pediatrics, National Jewish Medical and Research Center, Denver, Colorado 80206, USA.
Spencer
D M
DM
Kaiser
A L
AL
Leslie
C C
CC
D019943:Amino Acid Substitution; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000494:pharmacology; D001427:Bacterial Toxins; D001665:Binding Sites; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D004533:Egtazic Acid / Q000494:pharmacology; D004731:Endotoxins / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D049452:Green Fluorescent Proteins; D006460:Hemolysin Proteins; D007313:Insecta; D008164:Luminescent Proteins; D009685:Nuclear Envelope / Q000187:drug effects / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010741:Phospholipases A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D049452:Green Fluorescent Proteins; D019319:Okadaic Acid; D016718:Arachidonic Acid; D000001:Calcimycin; D004533:Egtazic Acid; D010741:Phospholipases A; D054467:Phospholipases A2; D002118:Calcium