EXPRESSION OF IPLA2B AS FUSION PROTEIN W/ EGFP PROVIDES EVIDENCE FOR ITS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Pancreatic islet b-cells and insulinoma cells express a Group VIA Ca2+ -independent phospholipase A2 (iPLA2b) that participates in signal transduction. Inhibition of iPLA2b leads to suppression and overexpression leads to amplification of secretagogue-stimulated insulin secretion. Immunofluorescence analyses have revealed an accumulation of iPLA2b in the perinuclear region of INS-1 cells stimulated with secretagogues. Here, to determine specific subcellular target(s) of iPLA2b, it was expressed in INS-1 cells as a fusion protein (fp) with EGFP, attached at either the N-terminus (fpC2) or C-terminus (fpN2) of iPLA2b. Expression of iPLA2bas a fp allowed iPLA2b tracking in the cell by monitoring EGFP fluorescence and avoids potential non-specific affinities of antibodies. Both fpC2 and fpN2 stably-transfected cells express higher iPLA2b activity than control cells transfected with EGFP cDNA alone. This indicates that adding EGFP to iPLA2b does not interfere with its catalytic activity. Dual fluorescence monitoring of EGFP and organelle Trackers reveals that iPLA2b accumulates in the Golgi and ER of stimulated fpN2, but not, fpC2 expressing cells. These observations suggest that iPLA2b undergoes post-translational modifications, and this is confirmed by immunoaffinity analyses using antibodies against EGFP or iPLA2b. Further, mass spectrometric analyses of purified iPLA2b reveals iPLA2b variants from which variable numbers of N-terminal residues have been removed. These findings suggest that iPLA2b undergoes endogenous proteolytic processing and that stimulation of b-cells causes redistribution of iPLA2b variants to different subcellular compartme