Internalized Group V Secretory Phospholipase A2 Acts on the Perinuclear Membranes

Abstract

Mammalian secretory phospholipases A2 (sPLA2) have been implicated in cellular eicosanoid biosynthesis but the mechanism of their cellular action remains unknown. To elucidate the spatiotemporal dynamics of sPLA2 mobilization and determine the site of its lipolytic action, we performed time-lapse confocal microscopic imaging of fluorescently labeled sPLA2 acting on human embryonic kidney (HEK) 293 cells the membranes of which are labeled with a fluorogenic phospholipid,N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine. The Western blotting analysis of HEK293 cells treated with exogenous sPLA2s showed that not only the affinity for heparan sulfate proteoglycan but also other factors, such as sPLA2hydrolysis products or cytokines, are necessary for the internalization of sPLA2 into HEK293 cells. Live cell imaging showed that the hydrolysis of fluorogenic phospholipids incorporated into HEK293 cell membranes was synchronized with the spatiotemporal dynamics of sPLA2 internalization, detectable initially at the plasma membrane and then at the perinuclear region. Also, immunocytostaining showed that human group V sPLA2 induced the translocation of 5-lipoxygenase to the nuclear envelope at which they were co-localized. Together, these studies provide the first experimental evidence that the internalized sPLA2 acts on the nuclear envelope to provide arachidonate for other enzymes involved in the eicosanoid biosynthesis. Mammalian secretory phospholipases A2 (sPLA2) have been implicated in cellular eicosanoid biosynthesis but the mechanism of their cellular action remains unknown. To elucidate the spatiotemporal dynamics of sPLA2 mobilization and determine the site of its lipolytic action, we performed time-lapse confocal microscopic imaging of fluorescently labeled sPLA2 acting on human embryonic kidney (HEK) 293 cells the membranes of which are labeled with a fluorogenic phospholipid,N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine. The Western blotting analysis of HEK293 cells treated with exogenous sPLA2s showed that not only the affinity for heparan sulfate proteoglycan but also other factors, such as sPLA2hydrolysis products or cytokines, are necessary for the internalization of sPLA2 into HEK293 cells. Live cell imaging showed that the hydrolysis of fluorogenic phospholipids incorporated into HEK293 cell membranes was synchronized with the spatiotemporal dynamics of sPLA2 internalization, detectable initially at the plasma membrane and then at the perinuclear region. Also, immunocytostaining showed that human group V sPLA2 induced the translocation of 5-lipoxygenase to the nuclear envelope at which they were co-localized. Together, these studies provide the first experimental evidence that the internalized sPLA2 acts on the nuclear envelope to provide arachidonate for other enzymes involved in the eicosanoid biosynthesis. phospholipase A2 arachidonic acid bovine serum albumin group VI cytosolic PLA2 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate Dulbecco's modified Eagle's medium human embryonic kidney human group IIa PLA2 heparan sulfate proteoglycan human group V PLA2 5-lipoxygenase phosphate-buffered saline phosphatidylcholine N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine triethylammonium salt 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine secretory PLA2 interleukin Phospholipases A2 (PLA2)1catalyze the hydrolysis of membrane phospholipids, the products of which can be transformed into potent inflammatory lipid mediators, platelet activating factor and eicosanoids that include prostaglandins, thromboxanes, leukotrienes, and lipoxins. Multiple forms of secretory PLA2s (sPLA2) and intracellular PLA2s have been found in mammalian tissues (1Six D.A. Dennis E.A. Biochim. Biophys. Acta. 2000; 1488: 1-19Crossref PubMed Scopus (1220) Google Scholar). Recent cell studies have indicated that some sPLA2 isoforms work in concert with group IV cytosolic PLA2 (cPLA2) to induce immediate and delayed eicosanoid formation (2Balsinde J. Balboa M.A. Dennis E.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7951-7956Crossref PubMed Scopus (171) Google Scholar, 3Murakami 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 (339) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar). At present, the identity of proinflammatory sPLA2, the spatiotemporal dynamics of sPLA2 mobilization, and the signaling mechanism that links sPLA2, cPLA2, and other enzymes involved in eicosanoid biosynthesis are not fully understood. It has been reported that the heparan sulfate proteoglycan (HSPG)-mediated internalization of sPLA2 is an important step in sPLA2 actions on mammalian cells (3Murakami 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 (339) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 5Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 6Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar); however, functional consequences of sPLA2 internalization remain controversial. In agonist-induced human embryonic kidney 293 (HEK293) cells transfected with various sPLA2s, the sPLA2 internalization resulted in arachidonic acid (AA) release and prostaglandin synthesis (3Murakami 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 (339) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 5Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 6Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), whereas in human neutrophils (7Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar) and mast cells (8Enomoto A. Murakami M. Kudo I. Biochem. Biophys. Res. Commun. 2000; 276: 667-672Crossref PubMed Scopus (23) Google Scholar) the sPLA2internalization led to protein degradation. This study was undertaken to clarify the effect of sPLA2 internalization on the cellular eicosanoid biosynthesis and determine the location of sPLA2 lipolytic actions. Results described herein provide the first experimental evidence that the internalized sPLA2liberates fatty acids from the phospholipids in the nuclear envelope at which other eicosanoid-producing enzymes are localized during the cellular eicosanoid biosynthesis. 1,1′-Didodecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiIC12),N-((6-(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphoethanolamine triethylammonium salt (PED6), and Texas RedTMC2 maleimide were purchased from Molecular Probes, Inc. (Eugene, OR). Cholesterol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) were from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. Phospholipid concentrations were determined by phosphate analysis (9Kates M. Techniques of Lipidology.2nd Ed. Elsevier, Amsterdam1986: 114-115Google Scholar). Dublecco's modified Eagle's medium (DMEM) and inactivated fetal bovine serum were from Invitrogen (Grand Island, NY). HEK293 cells and Zeocin were from Invitrogen (San Diego, CA). Fatty acid-free bovine serum albumin (BSA) was from Bayer Inc. (Kankakee, IL). Arachidonyl trifluoromethyl ketone was from Calbiochem(San Diego, CA). Recombinant human group V PLA2(hVPLA2) (10Han S.-K. Yoon E.T. Cho W. Biochem. J. 1998; 331: 353-357Crossref PubMed Scopus (53) Google Scholar), its mutants (7Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 11Han S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar), and human group IIa PLA2 (hIIaPLA2) (12Snitko Y. Koduri R. Han S.-K. Othman R. Baker S.F. Molini B.J. Wilton D.C. Gelb M.H. Cho W. Biochemistry. 1997; 36: 14325-14333Crossref PubMed Scopus (110) Google Scholar) were expressed and purified as described previously. HEK293 cells were treated with 100 nm of hVPLA2-W79A, W79A/W31A, W79A/R100E/K101E, and hIIaPLA2 for the indicated period, and the incubation was quenched by adding a solution of ice-cold 0.6 m NaCl in DMEM. After washing with the same solution, the was by and then in of and After on the cell were at for to the cell The were then with of and the were for The were to The of from the to membrane was a The membrane was with for then with of the N.M. Kim K. Han S.-K. E. H. Cho W. Leff A.R. 2000; PubMed Scopus Google Scholar) or a hIIaPLA2 in saline The membranes were for with with was in and with membrane for The membrane was with and with an of human neutrophils cells with was performed as described (7Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). of HEK293 cells was by the cells with for at was by washing the cells with The was quenched by adding of ice-cold and the cell and the medium were by then the of and was by To a for the was performed as described S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). was and purified to the used for S.-K. Yoon E.T. Cho W. Biochem. J. 1998; 331: 353-357Crossref PubMed Scopus (53) Google Scholar). was in of m and treated with of Texas RedTMC2 maleimide for at The labeled protein was with sulfate on by at and at for and in of m The labeled protein was purified a that was to and in the same protein was with the of NaCl to m in the same The to a protein were for at and then at The of cell membranes by was performed as described J.D. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) with some of in in was and in by the of The solution was the was to to that of was of was to the and were by of the on HEK293 cells were into of on a with the with fetal bovine serum and and at with for The solution was then to of and with HEK293 cells for at HEK293 cells were with phosphate-buffered saline in of and nm sPLA2 and were was with a confocal with the to the The from the Texas to was with a and a whereas the from the was with a and a was used for were the analysis in the these of in the and the membranes were and the in a was as a of To HEK293 cell membranes with of of solution in was to of HEK293 cells that were for in with fetal bovine serum and in of on a was by washing with HEK293 cells were in of without The was on the of a confocal with a and a nm were and the imaging was performed as described HEK293 cells were a and at with The HEK293 cell 5-lipoxygenase was by the cells with by of in the of for The cells were treated with nm of in for and in a At the cells were with and then were at with in for After the cells were with and in a solution serum and 100 in at for The cells were then with and for at with and with the N.M. Kim K. Han S.-K. E. H. Cho W. Leff A.R. 2000; PubMed Scopus Google Scholar) and human in the of After incubation at the were and cells were with in was for at by washing and incubation with in for at After washing with the was with was with a confocal The hydrolysis of in the of was at in of m The of hydrolysis was as an in at nm a with the at was at nm for and of were determined from the of The internalization of sPLA2 to mammalian cells has been the cells were treated with sPLA2 (7Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, A. Murakami M. Kudo I. Biochem. Biophys. Res. Commun. 2000; 276: 667-672Crossref PubMed Scopus (23) Google Scholar) or the cells sPLA2s were with such as (3Murakami 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 (339) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 5Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 6Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). To and determine the for sPLA2 internalization, we the exogenous that the of the protein labeled with a for The was to the of a protein the or affinity not we the internalization of its mutants and and hIIaPLA2 to HEK293 cells by Western blotting analysis of cell sPLA2 the is used in of is fully and a of S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). showed that has on mammalian cells hIIaPLA2 of its to and S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). also showed that of has on membranes S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar) and that has but has affinity for (7Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). in only showed a of internalization at and hIIaPLA2 not detectable internalization not was internalized at a a at which only to that of at HEK293 cells were treated with in the of PLA2 that was to have M. S. Cho W. Biochemistry. 1999; PubMed Scopus Google Scholar), was internalized as as The was not to PLA2 not with N.M. Kim K. Han S.-K. E. H. Cho W. Leff A.R. 2000; PubMed Scopus Google Scholar) and to its affinity was not internalized experimental affinity and the to the plasma membrane are for a sPLA2 to HEK293 cells. In W79A/W31A, and hIIaPLA2 but not W79A/R100E/K101E, were internalized HEK293 cells were with This that affinity is necessary and for the internalization of sPLA2 into HEK293 cells. The internalized sPLA2 in HEK293 which is in to the of internalized sPLA2 in neutrophils (7Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). these that affinity is a factor for the internalization of sPLA2 into HEK293 other factors, such as PLA2 hydrolysis products and cytokines, are also necessary for the have that sPLA2s they are localized J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar) or (3Murakami 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 (339) Google Scholar, 4Murakami M. Kambe T. Shimbara S. Kudo I. J. Biol. Chem. 1999; 274: 3103-3115Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 5Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 6Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), experimental evidence for the has been To determine the the internalization of sPLA2 and its intracellular lipolytic we treated human neutrophils and HEK293 cells with and and the of we reported (7Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), the of from human neutrophils by a whereas the release by to The of the release by is to its internalization into neutrophils and (7Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). the release from HEK293 cells by and showed and the for an In of the of the internalized in neutrophils and HEK293 cells these that the of release from HEK293 cells is to the action of internalized on intracellular To the that the internalized sPLA2 is on intracellular membranes and to determine the intracellular location of sPLA2 lipolytic action, we labeled HEK293 cells with a fluorogenic which has been used for in PLA2 Biochem. 1999; 276: PubMed Scopus Google M. R. 2001; PubMed Scopus Google Scholar). In the in is quenched by the group in the which is the hydrolysis the fatty in sPLA2s used in these studies showed on in the in however, of the in is not the cellular of can be cells were with a in was localized in the plasma membrane the first of incubation but various cellular membranes to of incubation experimental of PLA2 on for was was determined as an of The was in The for was in a was determined as an of The was in first treated HEK293 cells with hVPLA2-W79A, and in the of to the cells resulted in the of first at the plasma membrane and then with a the The of the of that the at the plasma membrane at and the at the nuclear envelope the at The that is nuclear envelope initially to to to the of the fatty acid from the nuclear with Western blotting hIIaPLA2 not induce Also, showed the at the plasma membrane intracellular with the to be to the of internalized the not the and This also the of imaging in with the Western To the that the intracellular lipid in are to the intracellular of fatty acid from the plasma we performed the in which the is labeled in the plasma membrane of HEK293 cells and the at the plasma membranes and in the medium in the the intracellular was the that the intracellular with HEK293 cells membranes are labeled with from the intracellular membrane It is also that the intracellular from or group VI HEK293 cells of these intracellular PLA2s (3Murakami 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 (339) Google Scholar) and the of cells with trifluoromethyl ketone effect on lipid then with HEK293 cells. acts on HEK293 cells as as at the plasma membrane and the perinuclear not In the of the at the plasma membrane was not as from its but the intracellular was hIIaPLA2 not a at the nuclear envelope but a that have a site of lipolytic these in with Western blotting that sPLA2s, are internalized into the HEK293 cells and on intracellular microscopic imaging of on plasma HEK293 cells and are the same as described for that is labeled in the plasma membrane of to with a of fatty were determined at the nuclear envelope and plasma membrane then treated the HEK293 cells with the Texas to the internalization and the lipid hydrolysis by is to a Texas to a by the was on that the is fully and a of S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). The of by Texas maleimide and the the modified protein that is from the and Texas were as as by a Texas was to HEK293 the labeled and then was localized in the perinuclear The release of fatty acid was synchronized and with the internalization, initially at the plasma membrane and then the perinuclear region. we the in membrane during sPLA2 internalization membrane in the of exogenous to HEK293 cells led to the formation of the plasma The of lipid was not a cellular hIIaPLA2 not induce formation the same of HEK293 cells with or at also induced the that products of hydrolysis at the plasma membrane protein internalization To the of internalized sPLA2 and its with other involved in eicosanoid we treated HEK293 cells with and their cellular location by It has been that is in the and the in cells and to the nuclear envelope cell M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). in is in the and the to the cells. of to the nuclear as indicated by a the also to the nuclear at a After and are at the nuclear In of the these that sPLA2 has a in cellular eicosanoid the of at the nuclear envelope and the translocation of other to the nuclear has been reported on the and of sPLA2 isoforms inflammatory is the spatiotemporal dynamics of In the mechanism and the functional consequences of sPLA2 internalization remain The into these important HEK293 cells for these studies sPLA2 internalization studies have been performed with these cells transfected with various sPLA2 isoforms (3Murakami 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 (339) Google Scholar, 5Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (157) Google M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Western blotting analysis that at for sPLA2 isoforms used in these the affinity is an but not factor for the internalization of sPLA2 into HEK293 other factors, such as PLA2 hydrolysis products and cytokines, are also necessary for the This also for the reported the on human neutrophils and on HEK293 cells. In the only that has and affinity was internalized (7Kim K.P. Rafter J.D. Bittova L. Han S.K. Snitko Y. Munoz N.M. Leff A.R. Cho W. J. Biol. Chem. 2001; 276: 11126-11134Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar), whereas in the sPLA2s were internalized (3Murakami 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 (339) Google Scholar, 5Murakami M. Kambe T. Shimbara S. Yamamoto S. Kuwata H. Kudo I. J. Biol. Chem. 1999; 274: 29927-29936Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 6Murakami M. Koduri R.S. Enomoto A. Shimbara S. Seki M. Yoshihara K. Singer A. Valentin E. Ghomashchi F. Lambeau G. Gelb M.H. Kudo I. J. Biol. Chem. 2001; 276: 10083-10096Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). the mechanism of sPLA2 internalization is at present, is to the of the in sPLA2 The imaging of HEK293 cells a membrane that products the formation of intracellular It is however, they the by the of plasma membrane or by a further studies are to of the mechanism of internalization, with HEK293 cells that the internalized is on intracellular the cell imaging sPLA2s, Texas and HEK293 cells the of the fatty acid the cells. with HEK293 the plasma membrane of which is labeled with the that the intracellular fatty acid from the internalization of fatty acid from the plasma membrane by fatty acid or The time-lapse that the nuclear envelope is the site of action for the internalized The from the of fatty acid from the perinuclear membranes or the hydrolysis of incorporated in internalized The in which the the nuclear envelope the In the that the the at the nuclear the cell imaging of Texas and immunocytostaining of and that the internalized is with at the nuclear The translocation of to the nuclear envelope is induced by the in intracellular M. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), which has been to be induced by PLA2 and fatty at the plasma J. Kim and W. Together, these that the nuclear envelope is the site of action at for internalized This is also with the that the nuclear envelope is in D.A. J. Res. 2000; Full Text Full Text PDF PubMed Google Scholar) and has on membranes (10Han S.-K. Yoon E.T. Cho W. Biochem. J. 1998; 331: 353-357Crossref PubMed Scopus (53) Google Scholar, 11Han S.K. Kim K.P. Koduri R. Bittova L. Munoz N.M. Leff A.R. Wilton D.C. Gelb M.H. Cho W. J. Biol. Chem. 1999; 274: 11881-11888Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). sPLA2s are in the of and for is not internalized can on the nuclear the in the the nuclear envelope is to and the to the perinuclear membrane is not and by cellular the group of sPLA2s and the lipid of various intracellular membranes Biol. PubMed Scopus Google Scholar, G. Biochim. Biophys. Acta. PubMed Scopus Google Scholar), is that other sPLA2s on intracellular hIIaPLA2 that has not a at the nuclear envelope a studies are necessary to determine the intracellular site of action for other sPLA2 isoforms and these enzymes are to their membrane study in HEK293 cells that the of to the of fatty acid at the nuclear envelope is experimental This is further by the that that by effect on fatty acid release and Kim and W. in It be however, that HEK293 cells a of sPLA2 in an or on other mammalian cells that sPLA2 work in concert with as reported W. Biochim. Biophys. Acta. 2000; 1488: PubMed Scopus Google Scholar, A. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar, Biochem. Biophys. Res. Commun. 2001; PubMed Scopus Google Scholar). In provide the first experimental evidence that the internalized sPLA2 acts on the nuclear envelope other enzymes in the eicosanoid cPLA2, and are localized during eicosanoid biosynthesis. The experimental used in these studies as a for further studies on the by which sPLA2 isoforms are internalized and to a and in various mammalian cells with