Identification of a cellular protein that functionally interacts with the C2 domain of cytosolic phospholipase A(2)alpha.
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.
Nakatani
Y
Y
Department of Health Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.
Tanioka
T
T
Sunaga
S
S
Murakami
M
M
Kudo
I
I
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001665:Binding Sites; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D002478:Cells, Cultured; D003600:Cytosol / Q000201:enzymology; D005347:Fibroblasts; D054513:Group IV Phospholipases A2; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007668:Kidney; D017737:Macrophages, Peritoneal / Q000201:enzymology*; D008297:Male; D016297:Mutagenesis, Site-Directed; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D014162:Transfection; D014746:Vimentin / Q000737:chemistry* / Q000378:metabolism*
D007527:Isoenzymes; D011994:Recombinant Proteins; D014746:Vimentin; D016718:Arachidonic Acid; D000001:Calcimycin; D010741:Phospholipases A; D054513:Group IV Phospholipases A2