title: Cellular Assays for Evaluating Calcium-Dependent Translocation of cPLA2α to Membrane.
abstract: The group IVA phospholipase A2, commonly called cytosolic phospholipase A2α (cPLA2α), is a widely expressed enzyme that hydrolyzes membrane phospholipid to produce arachidonic acid and lysophospholipids, which are precursors for a number of bioactive lipid mediators. Arachidonic acid is metabolized through the cyclooxygenase and lipoxygenase pathways for production of prostaglandins and leukotrienes that regulate normal physiological processes and contribute to disease pathogenesis. cPLA2α is composed of an N-terminal C2 domain and a C-terminal catalytic domain that contains the Ser-Asp catalytic dyad. The catalytic domain contains phosphorylation sites and basic residues that regulate the catalytic activity of cPLA2α. In response to cell stimulation, cPLA2α is rapidly activated by posttranslational mechanisms including increases in intracellular calcium and phosphorylation by mitogen-activated protein kinases. In resting cells, cPLA2α is localized in the cytosol but translocates to membrane including the Golgi, endoplasmic reticulum, and the peri-nuclear membrane in response to increases in intracellular calcium. Calcium binds to the C2 domain, which promotes the interaction of cPLA2α with membrane through hydrophobic interactions. In this chapter, we describe assays used to study the calcium-dependent translocation of cPLA2α to membrane, a regulatory step necessary for access to phospholipid and release of arachidonic acid.
authors.affiliation: National Jewish Health, Denver, CO, United States.
authors.forename: B
authors.initials: B
authors.lastname: Yun
authors.affiliation: National Jewish Health, Denver, CO, United States; University of Colorado Denver, Aurora, CO, United States. Electronic address: lesliec@njhealth.org.
authors.forename: C C
authors.initials: CC
authors.lastname: Leslie
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001681:Biological Assay; D002118:Calcium / Q000378:metabolism*; D020134:Catalytic Domain; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D054513:Group IV Phospholipases A2 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D051379:Mice; D061848:Optical Imaging / Q000379:methods*; D010766:Phosphorylation; D061251:Primary Cell Culture; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D059008:Time-Lapse Imaging / Q000379:methods*
chemical_list: D001426:Bacterial Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C527701:enhanced cyan fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; D016718:Arachidonic Acid; D054513:Group IV Phospholipases A2; D002118:Calcium
keywords: C2 domain; Calcium; Cytosolic phospholipase A(2); Fluorescent proteins; Golgi; Membrane; Time-lapse imaging; Translocation