Abstract
Cytosolic phospholipase A(2) (cPLA(2)) alpha plays critical roles in lipid mediator synthesis. We performed far-Western analysis and identified a 60-kDa protein (P60) that interacted with cPLA(2)alpha in a Ca(2+)-dependent manner. Peptide microsequencing revealed that purified P60 was identical to vimentin, a major component of the intermediate filament. The interaction occurred between the C2 domain of cPLA(2)alpha and the head domain of vimentin. Immunofluorescence microscopic analysis demonstrated that cPLA(2)alpha and vimentin colocalized around the perinuclear area in cPLA(2)alpha-overexpressing human embryonic kidney 293 cells following A23187 stimulation. Forcible expression of vimentin in vimentin-deficient SW13 cells augmented A23187-induced arachidonate release. Moreover, overexpression of the vimentin head domain in rat fibroblastic 3Y1 cells exerted a dominant inhibitory effect on arachidonate metabolism, significantly reducing A23187-induced arachidonate release and attendant prostanoid generation. These results suggest that vimentin is an adaptor for cPLA(2)alpha to function properly during the eicosanoid-biosynthetic process.
Highlights
Phospholipase A2 (PLA2)1 hydrolyzes the ester bonds of fatty acids present at the sn-2 positions of phospholipids
Detection of a cytosolic PLA2 (cPLA2)␣-binding Protein—In an attempt to search for a protein that interacts with cPLA2␣, lysates of several cells that generated PGE2 in a cPLA2␣-dependent manner [19, 34, 35] were subjected to far-Western analysis using epitope-tagged cPLA2␣ protein as a probe
To determine whether the C2 domain of cPLA2␣ was involved in the binding of cPLA2␣ to P60, we engineered a truncated cPLA2␣ cDNA construct, which was bacterially expressed as a glutathione Stransferase (GST) fusion protein (GST-cPLA2␣ [1–138])
Summary
Phospholipase A2 (PLA2)1 hydrolyzes the ester bonds of fatty acids present at the sn-2 positions of phospholipids. The head domain of vimentin protein was released from pCR3.1-vimentin by restriction enzyme digestion (EcoRI and XhoI) and subcloned into the pET21a vector, which expressed protein as a fusion protein containing a stretch of 6 consecutive histidine residues (His-tag) at the carboxyl terminus.
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