Identification of a cellular protein that functionally interacts with the C2 domain of cytosolic phospholipase A2α

Abstract

Cytosolic phospholipase A2 (cPLA2) α plays critical roles in lipid mediator synthesis. We performed far-Western analysis and identified a 60-kDa protein (P60) that interacted with cPLA2α in a Ca2+-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 cPLA2α and the head domain of vimentin. Immunofluorescence microscopic analysis demonstrated that cPLA2α and vimentin colocalized around the perinuclear area in cPLA2α-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 cPLA2α to function properly during the eicosanoid-biosynthetic process.