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• W1975287199 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. 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. phospholipase A2 cytosolic PLA2α arachidonic acid prostaglandin cyclooxygenase lipoxygenase glutathioneS-transferase glial fibrillary acidic protein fetal calf serum polyacrylamide gel electrophoresis polymerase chain reaction phosphate-buffered saline enhanced green fluorescent protein Phospholipase A2(PLA2)1hydrolyzes the ester bonds of fatty acids present at thesn-2 positions of phospholipids. PLA2 plays crucial roles in diverse cellular responses, including phospholipid digestion and metabolism, host defense, signal transduction, and probably apoptosis. PLA2 provides precursors for the biosynthesis of eicosanoids, such as prostaglandins (PGs) and leukotrienes, when the hydrolyzed fatty acid is arachidonic acid (AA); platelet-activating factor when the sn-1 position of phosphatidylcholine contains an alkyl ether linkage; and some bioactive lysophospholipids, such as lysophosphatidic acid. As oversynthesis of these lipid mediators causes inflammation and tissue disorders, it is important to elucidate the mechanisms that regulate the functions of PLA2. Mammalian tissues and cells generally contain more than one PLA2, each of which is regulated independently and plays distinct roles. There are three large families of mammalian PLA2s, cytosolic PLA2 (cPLA2), secretory PLA2, and Ca2+-independent PLA2, and the roles of type IV cPLA2α and secretory types IIA and V sPLA2 in lipid mediator synthesis have been studied extensively (1.Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar, 2.Kramer R.M. Sharp J.D. FEBS Lett. 1997; 410: 49-53Crossref PubMed Scopus (235) Google Scholar, 3.Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-283Crossref PubMed Google Scholar).An 85-kDa type IV cPLA2α appears to be one of the most important PLA2 isozymes involved in lipid mediator synthesis following cell activation (1.Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar, 2.Kramer R.M. Sharp J.D. FEBS Lett. 1997; 410: 49-53Crossref PubMed Scopus (235) Google Scholar, 3.Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-283Crossref PubMed Google Scholar). Submicromolar concentrations of Ca2+ appear to be required for cPLA2α to exert its catalytic activity, and this enzyme preferentially hydrolyses phospholipids bearing AA (4.Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1454) Google Scholar, 5.Sharp J.D. White D.L. Chiou X.G. Goodson T. Gamboa G.C. McClure D. Burgett S. Hoskins J. Skatrud P.L. Sportsman J.R. Kang L.H. Roberts E.F. Kramer R.M. J. Biol. Chem. 1991; 266: 14850-14853Abstract Full Text PDF PubMed Google Scholar). cPLA2α is expressed in most mammalian cells, and its activation is regulated by several postreceptor signal transduction events, such as Ca2+ mobilization (6.Qiu Z.H. Gijon M.A. de Carvalho M.S. Spencer D.M. Leslie C.C. J. Biol. Chem. 1998; 273: 8203-8211Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 7.Hirabayashi T. Kume K. Hirose K. Yokomizo T. Iino M. Itoh H. Shimizu T. J. Biol. Chem. 1999; 274: 5163-5169Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), phosphorylation (8.Lin L.-L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1643) Google Scholar, 9.Nakatani Y. Murakami M. Kudo I. Inoue K. J. Immunol. 1994; 153: 796-803PubMed Google Scholar), and gene induction (10.Lin L.-L. Lin A.Y. DeWitt D.L. J. Biol. Chem. 1992; 267: 23451-23454Abstract Full Text PDF PubMed Google Scholar, 11.Hoeck W.G. Ramesha C.S. Chang D.J. Fan N. Heller R.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4475-4479Crossref PubMed Scopus (191) Google Scholar). After stimuli that are accompanied by an increase in the cytoplasmic Ca2+ concentration, cPLA2α translocates rapidly and often transiently to the perinuclear and endoplasmic reticular membranes (12.Glover S. Bayburt T. Jonas M. Chi E. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15367Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 13.Schievella A.R. Regier M.K. Smith W.L. Lin L.-L. J. Biol. Chem. 1995; 270: 30749-30754Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar), is phosphorylated by mitogen-activated protein kinases (8.Lin L.-L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1643) Google Scholar, 9.Nakatani Y. Murakami M. Kudo I. Inoue K. J. Immunol. 1994; 153: 796-803PubMed Google Scholar), and releases AA for immediate conversion to PGs and leukotrienes by the constitutive cyclooxygenase (COX)-1 and 5-lipoxygenase (5-LO), respectively (14.Murakami M. Matsumoto R. Austen K.F. Arm J.P. J. Biol. Chem. 1994; 269: 22269-22275Abstract Full Text PDF PubMed Google Scholar, 15.Morham S.G. Langenbach R. Loftin C.D. Tiano H.F. Vouloumanos N. Jennette J.C. Mahler J.F. Kluckman K.D. Ledford A. Lee C.A. Smithies O. Cell. 1995; 83: 473-482Abstract Full Text PDF PubMed Scopus (1022) Google Scholar, 16.Riendeau D. Guay J. Weech P.K. Laliberte F. Yergey J. Li C. Desmarais S. Perrier H. Liu S. Nicoll-Griffith D. Street I.P. J. Biol. Chem. 1994; 269: 15619-15624Abstract Full Text PDF PubMed Google Scholar, 17.Huang Z. Payette P. Abdullah K. Cromlish W.A. Kennedy B.P. Biochemistry. 1996; 35: 3712-3721Crossref PubMed Scopus (86) Google Scholar). Furthermore, cPLA2α has been implicated in the inducible COX-2-dependent delayed PG generation, which lasts for hours, initiated by proinflammatory stimuli, such as interleukin-1, tumor necrosis factor α, and lipopolysaccharide (18.Reddy S.T. Winstead M.V. Tischfield J.A. Herschman H.R. J. Biol. Chem. 1997; 272: 13591-13596Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 19.Murakami M. Kuwata H. Amakasu Y. Shimbara S. Nakatani Y. Atsumi G. Kudo I. J. Biol. Chem. 1997; 271: 30041-30051Abstract Full Text Full Text PDF Scopus (121) Google Scholar, 20.Marshall L.A. Bolognese B. Winkler J.D. Roshak A. J. Biol. Chem. 1997; 272: 759-765Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 21.Shinohara H. Balboa M.A. Johnson C.A. Balsinde J. Dennis E.A. J. Biol. Chem. 1999; 274: 12263-12268Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). cPLA2α has several functionary distinct regions: an amino-terminal Ca2+-dependent lipid binding domain (amino acids 18–138) called the C2 domain (22.Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (685) Google Scholar, 23.Rizo J. Sudhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar), a carboxyl-terminal region (amino acids 179–749) containing the catalytic domain, a putative pleckstrin homology domain (amino acids 263–354) similar to phospholipase C-δ1 (24.Mosior M. Six D.A. Dennis E.A. J. Biol. Chem. 1998; 273: 2184-2191Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), and two critical serine residues (Ser505 and Ser727), which undergo activation-directed phosphorylation (25.Borsch-Haubold A.G. Bartoli F. Asselin J. Dudler T. Kramer R.M. Apitz-Castro R. Watson S.P. Gelb M.H. J. Biol. Chem. 1998; 273: 4449-4458Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). The C2 domain is responsible for translocation of cPLA2α from the cytosol to the membrane compartment and exhibits significant homology with the C2 domains of several proteins, such as protein kinase C, GTPase-activating protein, synaptotagmin, and phospholipase C, all of which bind to phospholipid membranes in a Ca2+-dependent manner (22.Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (685) Google Scholar, 23.Rizo J. Sudhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (703) Google Scholar). The carboxyl-terminal region mediates the Ca2+-independent enzymatic catalysis, which involves Ser228 at the active site (26.Sharp J.D. Pickard R.T. Chiou X.G. Manetta J.V. Kovacevic S. Miller J.R. Varshavsky A.D. Roberts E.F. Strifler B.A. Brems D.N. Kramer R.M. J. Biol. Chem. 1994; 269: 23250-23254Abstract Full Text PDF PubMed Google Scholar). Interestingly, a number of enzymes involved in AA metabolism, such as COX-1 and -2, 5-LO, and 5-LO-activating protein, are also localized in the nuclear envelope and endoplasmic reticulum (27.Morita I. Schindler M. Reiger M.K. Otto J.C. Hori T. DeWitt D.L. Smith W.L. J. Biol. Chem. 1995; 270: 10902-10908Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar, 28.Spencer A.G. Woods J.W. Arakawa T. Singer I.I. Smith W.L. J. Biol. Chem. 1998; 273: 9886-9893Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 29.Pouliot M. McDonald P.P. Krump E. Mancini J.A. McColl S.R. Weech P.K. Borgeat P. Eur. J. Biochem. 1996; 238: 250-258Crossref PubMed Scopus (98) Google Scholar).Although the C2 domain appears to be responsible for Ca2+-dependent localization of cPLA2α to its membrane substrates, whether additional interactions, such as C2 domain binding to an adaptor protein, also play roles in cPLA2α targeting to particular membrane compartments remains to be elucidated. In this study, we carried out far-Western screening in an attempt to determine how cPLA2α translocates specifically to the perinuclear particles and identified a protein that interacted with cPLA2α. We found that vimentin, a major component protein of intermediate filaments, interacted with the amino-terminal region of cPLA2α in a Ca2+-dependent manner. Further experiments demonstrated that cPLA2α and vimentin colocalized in Ca2+ ionophore-stimulated, but not unstimulated, human embryonic kidney 293 cells stably transfected with cPLA2α. Introduction of vimentin into a vimentin-deficient SW13 cell line restored cPLA2α-dependent AA release in response to a Ca2+ ionophore, whereas introduction of the vimentin head domain, to which cPLA2α bound via its C2 domain, into rat fibroblastic 3Y1 cells reduced it. These results suggest that vimentin is involved in the regulation of cPLA2α in the AA metabolic pathway.DISCUSSIONAs has been shown by a number of studies, there is no doubt that cPLA2α regulates the initial step of AA metabolism in response to stimuli that mobilize intracellular Ca2+ (1.Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar, 2.Kramer R.M. Sharp J.D. FEBS Lett. 1997; 410: 49-53Crossref PubMed Scopus (235) Google Scholar, 3.Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-283Crossref PubMed Google Scholar, 4.Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1454) Google Scholar, 5.Sharp J.D. White D.L. Chiou X.G. Goodson T. Gamboa G.C. McClure D. Burgett S. Hoskins J. Skatrud P.L. Sportsman J.R. Kang L.H. Roberts E.F. Kramer R.M. J. Biol. Chem. 1991; 266: 14850-14853Abstract Full Text PDF PubMed Google Scholar, 6.Qiu Z.H. Gijon M.A. de Carvalho M.S. Spencer D.M. Leslie C.C. J. Biol. Chem. 1998; 273: 8203-8211Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 7.Hirabayashi T. Kume K. Hirose K. Yokomizo T. Iino M. Itoh H. Shimizu T. J. Biol. Chem. 1999; 274: 5163-5169Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 8.Lin L.-L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1643) Google Scholar,12.Glover S. Bayburt T. Jonas M. Chi E. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15367Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 13.Schievella A.R. Regier M.K. Smith W.L. Lin L.-L. J. Biol. Chem. 1995; 270: 30749-30754Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 14.Murakami M. Matsumoto R. Austen K.F. Arm J.P. J. Biol. Chem. 1994; 269: 22269-22275Abstract Full Text PDF PubMed Google Scholar, 15.Morham S.G. Langenbach R. Loftin C.D. Tiano H.F. Vouloumanos N. Jennette J.C. Mahler J.F. Kluckman K.D. Ledford A. Lee C.A. Smithies O. Cell. 1995; 83: 473-482Abstract Full Text PDF PubMed Scopus (1022) Google Scholar, 16.Riendeau D. Guay J. Weech P.K. Laliberte F. Yergey J. Li C. Desmarais S. Perrier H. Liu S. Nicoll-Griffith D. Street I.P. J. Biol. Chem. 1994; 269: 15619-15624Abstract Full Text PDF PubMed Google Scholar, 17.Huang Z. Payette P. Abdullah K. Cromlish W.A. Kennedy B.P. Biochemistry. 1996; 35: 3712-3721Crossref PubMed Scopus (86) Google Scholar, 42.Fujishima H. Mejia R.O.S. Bingham III, C.O. Lam B.K. Sapirstein A. Bonventre J.V. Austen K.F. Arm J.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4803-4807Crossref PubMed Scopus (167) Google Scholar). In vitro studies employing cPLA2α-specific inhibitors (19.Murakami M. Kuwata H. Amakasu Y. Shimbara S. Nakatani Y. Atsumi G. Kudo I. J. Biol. Chem. 1997; 271: 30041-30051Abstract Full Text Full Text PDF Scopus (121) Google Scholar, 34.Kuwata H. Nakatani Y. Murakami M. Kudo I. J. Biol. Chem. 1998; 273: 1733-1740Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 35.Naraba H. Murakami M. Matsumoto H. Shimbara S. Ueno A. Kudo I. Oh-ishi S. J. Immunol. 1998; 160: 2974-2982PubMed Google Scholar), antisense oligonucleotides (20.Marshall L.A. Bolognese B. Winkler J.D. Roshak A. J. Biol. Chem. 1997; 272: 759-765Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 43.Roshak A. Sathe G. Marshall L.A. J. Biol. Chem. 1994; 269: 25999-26005Abstract Full Text PDF PubMed Google Scholar), and cPLA2α overexpression (33.Murakami M. Shimbara S. Kambe T. Kuwata H. Winstead M.V. Tischfield J.A. Kudo I. J. Biol. Chem. 1998; 273: 14411-14423Abstract Full Text Full Text PDF PubMed Scopus (338) Google Scholar,44.Lin L.-L. Lin A.Y. Knopf J.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6147-6151Crossref PubMed Scopus (518) Google Scholar) have shown that this particular PLA2 isozyme, which bears multifunctional domains, represents a rate-limiting step for the initiation of the lipid mediator-biosynthetic pathway. Furthermore, the importance of cPLA2α has been confirmed by in vivo studies on cPLA2α knockout mice (45.Uozumi N. Kume K. Nagase T. Nakatani N. Ishii S. Tashiro F. Komagata Y. Maki K. Ikuta K. Ouchi Y. Miyazaki J. Simizu T. Nature. 1997; 390: 618-622Crossref PubMed Scopus (636) Google Scholar, 46.Bonventre J.V. Huang Z. Taheri M.R. O'Leary E. Li E. Moskowitz M.A. Sapirstein A. Nature. 1997; 390: 622-625Crossref PubMed Scopus (757) Google Scholar). Following agonist-stimulated transmembrane signaling that increases the cytoplasmic Ca2+ level, cPLA2α undergoes translocation from the cytosol to the perinuclear envelope and endoplasmic reticulum (12.Glover S. Bayburt T. Jonas M. Chi E. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15367Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 13.Schievella A.R. Regier M.K. Smith W.L. Lin L.-L. J. Biol. Chem. 1995; 270: 30749-30754Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar), where many of the downstream eicosanoid-biosynthetic enzymes, including COX-1, COX-2, 5-LO, 5-LO-activating protein, and several terminal PG and leukotriene synthases, are located (27.Morita I. Schindler M. Reiger M.K. Otto J.C. Hori T. DeWitt D.L. Smith W.L. J. Biol. Chem. 1995; 270: 10902-10908Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar, 28.Spencer A.G. Woods J.W. Arakawa T. Singer I.I. Smith W.L. J. Biol. Chem. 1998; 273: 9886-9893Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 29.Pouliot M. McDonald P.P. Krump E. Mancini J.A. McColl S.R. Weech P.K. Borgeat P. Eur. J. Biochem. 1996; 238: 250-258Crossref PubMed Scopus (98) Google Scholar). Phosphorylation by mitogen-activated protein kinases increases the intrinsic activity of cPLA2α in vitro (8.Lin L.-L. Wartmann M. Lin A.Y. Knopf J.L. Seth A. Davis R.J. Cell. 1993; 72: 269-278Abstract Full Text PDF PubMed Scopus (1643) Google Scholar) and synergizes with the Ca2+ signaling pathway to provide the optimal AA-releasing response in vivo (6.Qiu Z.H. Gijon M.A. de Carvalho M.S. Spencer D.M. Leslie C.C. J. Biol. Chem. 1998; 273: 8203-8211Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). The C2 domain of cPLA2α is essential for Ca2+-dependent association of cPLA2α with phospholipid vesicles (4.Clark J.D. Lin L.-L. Kriz R.W. Ramesha C.S. Sultzman L.A. Lin A.Y. Milona N. Knopf J.L. Cell. 1991; 65: 1043-1051Abstract Full Text PDF PubMed Scopus (1454) Google Scholar, 47.Nalefski E.A. McDonagh T. Somers W. Seehra J. Falker J.J. Clark J.D. J. Biol. Chem. 1998; 273: 1365-1372Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 48.Davletov B. Perisic O. Williams R.L. J. Biol. Chem. 1998; 273: 19093-19096Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), which is facilitated in the presence of phosphatidylinositol 4,5-bisphosphate and, therefore, consistent with the presence of a putative pleckstrin homology domain in the middle part of the enzyme (24.Mosior M. Six D.A. Dennis E.A. J. Biol. Chem. 1998; 273: 2184-2191Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). However, the molecular mechanism whereby cPLA2α is directed to the intracellular membrane compartments, the perinuclear membranes in particular, in activated cells remains largely obscure.While searching for proteins that physically and functionally interact with cPLA2α, Wu et al. (49.Wu T. Angus W. Yao X.-L. Logun C. Shelhamer J.H. J. Biol. Chem. 1997; 272: 17145-17153Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar) recently identified, by means of the yeast two-hybrid system, a cellular protein, p11, that associated with the carboxyl-terminal portion of cPLA2α. Functional analysis revealed that p11 inhibited cPLA2α activity both in vitro and in vivo, suggesting that it acts as a negative regulator of cPLA2α. In this study, we found that cPLA2α interacted with a major intermediate filament protein, vimentin, which is expressed abundantly in the perinuclear regions of mesenchymal cells and a variety of cultured cells (40.Fuchs E. Weber K. Annu. Rev. Biochem. 1994; 63: 345-382Crossref PubMed Scopus (1272) Google Scholar). This interaction occurred between the C2 domain of cPLA2α and the head domain of vimentin. In agreement with the dependence of their interaction on Ca2+, confocal microscopic analysis revealed that they colocalized around the perinuclear area in A23187-stimulated but not unstimulated cells. Most importantly, overexpression of full-length vimentin augmented cPLA2α-initiated AA metabolism, whereas that of the vimentin head domain exerted a dominant-negative effect. Collectively, these results suggest that vimentin represents a functional adaptor for cPLA2α that determines the intracellular localization and thereby modifies the function of cPLA2α, depending on the changes in the cytoplasmic Ca2+ levels during cellular activation.The C2 domain of cPLA2α, like those of protein kinase C and synaptotagmin, behaves as a Ca2+-dependent lipid-binding domain. Recent crystal structural analysis revealed that the C2 domain of cPLA2α captured two Ca2+ions at one end of the domain between three loops, CBR1, CBR2, and CBR3 (36.Perisic O. Fong S. Lynch D.E. Bycroft M. Williams R.L. J. Biol. Chem. 1998; 273: 1596-1604Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 37.Bittova L. Sumandea M. Cho W. J. Biol. Chem. 1999; 274: 9665-9672Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), and the Ca2+ ions interacted directly with the phosphate moiety of a lipid head group. In this study, we showed that Asp43 (which resides in CBR1) and Asp93 (which resides in CBR3) in the C2 domain are involved in the cPLA2α-vimentin interaction. This is an important finding, because mutation of either of these two aspartates in the native C2 domain reduced Ca2+ binding, eventually leading to reduce phospholipid binding and enzyme activity (37.Bittova L. Sumandea M. Cho W. J. Biol. Chem. 1999; 274: 9665-9672Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The cPLA2α C2 domain consists of eight β-sheet structures, β1–β8 (36.Perisic O. Fong S. Lynch D.E. Bycroft M. Williams R.L. J. Biol. Chem. 1998; 273: 1596-1604Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 63.Dessen A. Tang J. Schmidt H. Stahl M. Clark J.D. Seehra J. Somers W.S. Cell. 1999; 97: 349-360Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar). Our present findings (that cPLA2α (1–81), which contains the first four β-sheets (β1-β4) of the C2 domain, interacted with vimentin, whereas neither cPLA2α (1.Leslie C.C. J. Biol. Chem. 1997; 272: 16709-16712Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar, 2.Kramer R.M. Sharp J.D. FEBS Lett. 1997; 410: 49-53Crossref PubMed Scopus (235) Google Scholar, 3.Murakami M. Nakatani Y. Atsumi G. Inoue K. Kudo I. Crit. Rev. Immunol. 1997; 17: 225-283Crossref PubMed Google Scholar, 4.Clark J.D. 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