Effects of endoplasmic reticulum stress on group VIA phospholipase A ₂in beta cells include tyrosine phosphorylation and increased association with calnexin

The Group VIA phospholipase A₂ (iPLA₂β) hydrolyzes glycerophospholipids at the sn-2-position to yield a free fatty acid and a 2-lysophospholipid, and iPLA₂β has been reported to participate in apoptosis, phospholipid remodeling, insulin secretion, transcriptional regulation, and other processes. Induction of endoplasmic reticulum (ER) stress in β-cells and vascular myocytes with SERCA inhibitors activates iPLA₂β, resulting in hydrolysis of arachidonic acid from membrane phospholipids, by a mechanism that is not well understood. Regulatory proteins interact with iPLA₂β, including the Ca²⁺/calmodulin-dependent protein kinase IIβ, and we have characterized the iPLA₂β interactome further using affinity capture and LC/electrospray ionization/MS/MS. An iPLA₂β-FLAG fusion protein was expressed in an INS-1 insulinoma cell line and then adsorbed to an anti-FLAG matrix after cell lysis. iPLA₂β and any associated proteins were then displaced with FLAG peptide and analyzed by SDS-PAGE. Gel sections were digested with trypsin, and the resultant peptide mixtures were analyzed by LC/MS/MS with database searching. This identified 37 proteins that associate with iPLA₂β, and nearly half of them reside in ER or mitochondria. They include the ER chaperone calnexin, whose association with iPLA₂β increases upon induction of ER stress. Phosphorylation of iPLA₂β at Tyr⁶¹⁶ also occurs upon induction of ER stress, and the phosphoprotein associates with calnexin. The activity of iPLA₂β in vitro increases upon co-incubation with calnexin, and overexpression of calnexin in INS-1 cells results in augmentation of ER stress-induced, iPLA₂β-catalyzed hydrolysis of arachidonic acid from membrane phospholipids, reflecting the functional significance of the interaction. Similar results were obtained with mouse pancreatic islets.