β-Cell Calcium-Independent Group VIA Phospholipase A2 (iPLA2β)

Abstract

Evidence that group VIA cytosolic calcium-independent phospholipase A 2 (iPLA 2 ␤) participates in ␤-cell signal transduction includes the observations that inhibition of iPLA 2 ␤ with the bromoenol lactone suicide substrate suppresses glucose-stimulated insulin secretion and that overexpression of iPLA 2 ␤ amplifies insulin secretory responses in INS-1 insulinoma cells. Immunofluorescence analyses also reveal that iPLA 2 2 ) is a diverse group of enzymes that catalyze hydrolysis of the sn-2 substituent from glycerophospholipid substrates to yield a free fatty acid and a 2-lysophospholipid (1,2). Among the PLA 2 s is an 84-kDa cytosolic PLA 2 that does not require Ca 2ϩ for catalysis and is designated group VIA cytosolic calcium-independent phospholipase A 2 (iPLA 2 ␤) (3-5).A role for iPLA 2 ␤ in signal transduction in insulinsecreting ␤-cells is suggested by the observations that inhibition of iPLA 2 ␤ activity with the bromoenol lactone (BEL) suicide substrate of iPLA 2 ␤ suppresses insulin secretion, and overexpression of iPLA 2 ␤ amplifies glucoseand forskolin-stimulated insulin secretion from pancreatic islets and INS-1 insulinoma cells (6,7). Stimulation of iPLA 2 ␤-overexpressing INS-1 cells with cAMP-elevating agents also induces translocation of iPLA 2 ␤ to the perinuclear region (7). This is of interest because glucose promotes both ␤-cell insulin secretion and proliferation, and glucose-induced INS-1 cell mitogenesis is cAMP dependent (8). To characterize iPLA 2 ␤ subcellular movements further, we developed INS-1 cell lines that overexpress iPLA 2 ␤ as a fusion protein with enhanced green fluorescent protein (EGFP) so that green fluorescence associated with EGFP reflects the location of iPLA 2 ␤. Simultaneous monitoring of florescence associated with various tracking molecules allowed us to identify specific subcellular organelles with which iPLA 2 ␤ associates when cells are stimulated. RESEARCH DESIGN AND METHODSPreparation of a construct for expressing iPLA 2 ␤ as a fusion protein with EGFP and selection of stably transfected clones. Full-length iPLA 2 ␤ cDNA was amplified by PCR using the following primer set: sense, 5Ј AGCTTCGAATTCATGCAGTTCTTTGGACGC-3, and antisense, 5Ј-TTC GATATCGGGAGATAGCAGCAGCTGG-3Ј. The amplified full-length iPLA 2 ␤ from the pMSCV-neo-iPLA 2 ␤ constructs were then subcloned into the pEGFP-N2 (Clontech, Palo Alto, CA) after the immediate early promoter of cytomegalovirus major and before EGFP coding sequences in the same code-reading frame with EGFP. The EGFP-N2 control vector and the construct encoding iPLA 2 ␤-EGFP (FPN2) fusion protein were transfected into INS-1 cells with a Gene PORTER transfection system according to the manufacture's instructions (Gene Therapy Systems, San Diego, CA). Stably transfected clones were selected using G418 (0.4 mg/ml This article is based on a presentation at a symposium. The symposium and the publication of this article were made possible by an unrestricted educational grant from Les Laboratoires Servier.BEL, bromoenol lactone; EGFP, enha...

Group VIA Phospholipase A2 Mitigates Palmitate-induced β-Cell Mitochondrial Injury and Apoptosis

“…Phospholipase A 2 Enzymatic Activity-Ca 2ϩ -independent PLA 2 enzymatic activity was assayed as described (33) Immunostaining and Fluorescence Microscopic Analyses-As described (34,35), stably transfected INS-1 cells that expressed an iPLA 2 ␤-GFP construct were cultured on glass coverslips (18-mm diameter) in 6-well plates. After treatment, cells were stained with MitoTracker Red (Invitrogen, M7512) according to the manufacturer's protocol.…”

“…Recombinant His-iPLA 2 ␤-FLAG was purified by immobilized metal affinity chromatography on cobalt-based TALON columns as described (34,38).…”

HL-1 mouse cardiomyocyte injury and death after simulated ischemia and reperfusion: roles of pH, Ca2+-independent phospholipase A2, and Na+/H+ exchange

“…At least five splice variants of iPLA 2 -VIA exist, of which two, iPLA 2 -VIA-1 and iPLA 2 -VIA-2, are catalytically active (27). iPLA 2 -VIA was initially reported to be cytosolic; however, localization of this isoform to endoplasmic reticulum, mitochondria, and nucleus, and association with F-actin has also been reported (4,33,35,53,59,60). The precise mechanisms by which iPLA 2 -VIA contributes to ischemic damage are not understood; however, roles in induction of PCD (53) and in necrotic cell death are likely (43).…”

Characterization of N-terminal processing of group VIA phospholipase A2 and of potential cleavage sites of amyloid precursor protein constructs by automated identification of signature peptides in LC/MS/MS analyses of proteolytic digests

“…Using our optimized parameters, four different isoforms of Figure 9. MS/MS spectrum of the signature peptide 38 YENPTYKFFE 47 from a Glu-C proteolytic digest of APP six myc-tagged C-terminal cleavage products. iPLA 2 ␤ produced by proteolytic processing have been identified among products derived from expression of iPLA 2 ␤ cDNA in cells.…”

“…For expression of iPLA 2 ␤ protein, a 250-ml flask was prepared with a suspension of 50 ml of 10 6 cells/ml and incubated at 27°C for 24 h before infection with baculovirus. For protein expression, cDNA containing the entire coding sequence of native iPLA 2 ␤ [37] or his-tagged iPLA 2 ␤ with the tag at either the C-terminus or at the N-terminus [38], or was cloned into the EcoRI-SalI site of the pFASTBAC tm 1 baculovirus expression vector. The sequence of the insert was verified, and the plasmid was then transformed into DH10Bac cells according to instructions in the Bac-ToBac system manual.…”