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Effects of endoplasmic reticulum stress on group VIA phospholipase A2 in beta cells include tyrosine phosphorylation and increased association with calnexin.

ABSTRACT: The Group VIA phospholipase A(2) (iPLA(2)?) hydrolyzes glycerophospholipids at the sn-2-position to yield a free fatty acid and a 2-lysophospholipid, and iPLA(2)? has been reported to participate in apoptosis, phospholipid remodeling, insulin secretion, transcriptional regulation, and other processes. Induction of endoplasmic reticulum (ER) stress in ?-cells and vascular myocytes with SERCA inhibitors activates iPLA(2)?, resulting in hydrolysis of arachidonic acid from membrane phospholipids, by a mechanism that is not well understood. Regulatory proteins interact with iPLA(2)?, including the Ca(2+)/calmodulin-dependent protein kinase II?, and we have characterized the iPLA(2)? interactome further using affinity capture and LC/electrospray ionization/MS/MS. An iPLA(2)?-FLAG fusion protein was expressed in an INS-1 insulinoma cell line and then adsorbed to an anti-FLAG matrix after cell lysis. iPLA(2)? 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 iPLA(2)?, and nearly half of them reside in ER or mitochondria. They include the ER chaperone calnexin, whose association with iPLA(2)? increases upon induction of ER stress. Phosphorylation of iPLA(2)? at Tyr(616) also occurs upon induction of ER stress, and the phosphoprotein associates with calnexin. The activity of iPLA(2)? in vitro increases upon co-incubation with calnexin, and overexpression of calnexin in INS-1 cells results in augmentation of ER stress-induced, iPLA(2)?-catalyzed hydrolysis of arachidonic acid from membrane phospholipids, reflecting the functional significance of the interaction. Similar results were obtained with mouse pancreatic islets.

SUBMITTER: Song H

PROVIDER: S-EPMC2962484 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Effects of endoplasmic reticulum stress on group VIA phospholipase A2 in beta cells include tyrosine phosphorylation and increased association with calnexin.

Song Haowei H Rohrs Henry H Tan Min M Wohltmann Mary M Ladenson Jack H JH Turk John J

The Journal of biological chemistry 20100823 44

The Group VIA phospholipase A(2) (iPLA(2)β) hydrolyzes glycerophospholipids at the sn-2-position to yield a free fatty acid and a 2-lysophospholipid, and iPLA(2)β 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 iPLA(2)β, resulting in hydrolysis of arachidonic acid from membrane phospholipids, by ...[more]

PMID: 20732873

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Project description:Cholestasis-induced accumulation of bile acids in the liver leads to farnesoid X receptor (FXR)-mediated transcriptional down-regulation of the bile acid importer Na+-taurocholate cotransporting protein (NTCP) and to induction of endoplasmic reticulum (ER) stress. However, whether ER stress affects bile acid uptake is largely unknown. Here, we investigated the role of ER stress on the regulation and function of the bile acid transporter NTCP. ER stress was induced using thapsigargin or subtilase cytotoxin in human osteosarcoma (U2OS) and human hepatocellular carcinoma (HepG2) cells stably expressing NTCP. Cellular bile acid uptake was determined using radiolabeled taurocholate (TCA). NTCP plasma membrane expression was determined by cell surface biotinylation. Mice received a single injection of thapsigargin, and effects of ER stress on NTCP messenger RNA (mRNA) and protein were measured by reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis. Effects of cholestasis on NTCP and ER stress were assessed in response to 3, 5-diethoxycarbonyl-1, 4-dihydrocollidine (DDC) feeding or bile duct ligation in FXR-/- mice after 7 or 3 days, respectively. Novel NTCP-interacting proteins were identified by mass spectrometry (MS), interaction verified, and assessed by co-immunoprecipitation and TCA uptake for functional relevance in relation to ER stress. ER stress induction strongly reduced NTCP protein expression, plasma membrane abundance, and NTCP-mediated bile acid uptake. This was not controlled by FXR or through a single unfolded protein response (UPR) pathway but mainly depended on the interaction of NTCP with calnexin, an ER chaperone. In mice, expression of both NTCP and calnexin was reduced by thapsigargin or cholestasis-induced ER stress. Calnexin down-regulation in vitro recapitulated the effect of ER stress on NTCP. Conclusion: ER stress-induced down-regulation of calnexin provides an additional mechanism to dampen NTCP-mediated bile acid uptake and protect hepatocytes against bile acid overload during cholestasis.

Dataset Information

Effects of endoplasmic reticulum stress on group VIA phospholipase A2 in beta cells include tyrosine phosphorylation and increased association with calnexin.

Publications

Effects of endoplasmic reticulum stress on group VIA phospholipase A2 in beta cells include tyrosine phosphorylation and increased association with calnexin.

Similar Datasets

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