5-Lipoxygenase Interacts with Coactosin-like Protein

We have recently identified coactosin-like protein (CLP) in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait. In this report, we demonstrate a direct interaction between 5LO and CLP. 5LO associated with CLP, which was expressed as a glutathione S-transferase fusion protein, in a dose-dependent manner. Coimmunoprecipitation experiments using epitope-tagged 5LO and CLP proteins transiently expressed in human embryonic kidney 293 cells revealed the presence of CLP in 5LO immunoprecipitates. In reciprocal experiments, 5LO was detected in CLP immunoprecipitates. Non-denaturing polyacrylamide gel electrophoresis and cross-linking experiments showed that 5LO binds CLP in a 1:1 molar stoichiometry in a Ca2+-independent manner. Site-directed mutagenesis suggested an important role for lysine 131 of CLP in mediating 5LO binding. In view of the ability of CLP to bind 5LO and filamentous actin (F-actin), we determined whether CLP could physically link 5LO to actin filaments. However, no F-actin-CLP·5LO ternary complex was observed. In contrast, 5LO appeared to compete with F-actin for the binding of CLP. Moreover, 5LO was found to interfere with actin polymerization. Our results indicate that the 5LO-CLP and CLP-F-actin interactions are mutually exclusive and suggest a modulatory role for 5LO in actin dynamics. We have recently identified coactosin-like protein (CLP) in a yeast two-hybrid screen using 5-lipoxygenase (5LO) as a bait. In this report, we demonstrate a direct interaction between 5LO and CLP. 5LO associated with CLP, which was expressed as a glutathione S-transferase fusion protein, in a dose-dependent manner. Coimmunoprecipitation experiments using epitope-tagged 5LO and CLP proteins transiently expressed in human embryonic kidney 293 cells revealed the presence of CLP in 5LO immunoprecipitates. In reciprocal experiments, 5LO was detected in CLP immunoprecipitates. Non-denaturing polyacrylamide gel electrophoresis and cross-linking experiments showed that 5LO binds CLP in a 1:1 molar stoichiometry in a Ca2+-independent manner. Site-directed mutagenesis suggested an important role for lysine 131 of CLP in mediating 5LO binding. In view of the ability of CLP to bind 5LO and filamentous actin (F-actin), we determined whether CLP could physically link 5LO to actin filaments. However, no F-actin-CLP·5LO ternary complex was observed. In contrast, 5LO appeared to compete with F-actin for the binding of CLP. Moreover, 5LO was found to interfere with actin polymerization. Our results indicate that the 5LO-CLP and CLP-F-actin interactions are mutually exclusive and suggest a modulatory role for 5LO in actin dynamics. 5-lipoxygenase bovine serum albumin coactosin-like protein 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride filamentous actin globular actin glutathioneS-transferase human embryonic kidney 293 cells leukotriene 3-(N-morpholino)propanesulfonic acid N-hydroxysulfosuccinimide polyacrylamide gel electrophoresis 5-Lipoxygenase (5LO)1 is of central importance in cellular leukotriene (LT) synthesis. This enzyme converts arachidonic acid released from the membranes by the cytosolic phospholipase A2 into 5(S)-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE) and subsequently into the epoxide intermediate LTA4 (1Samuelsson B. Science. 1983; 220: 568-575Crossref PubMed Scopus (2385) Google Scholar). LTA4 is further metabolized into LTB4 by the LTA4 hydrolase or into LTC4 through the action of the LTC4 synthase. LTC4 is then sequentially degraded into LTD4 and LTE4. Whereas LTB4 exerts potent stimulatory effects on various leukocyte functions, including chemotaxis, adhesion, degranulation, and aggregation, the cysteinyl-LTs (LTC4, LTD4, and LTE4) are known to contract airway smooth muscle, increase vascular permeability, and promote mucus secretion (2Samuelsson B. Dahlén S.-E. Lindgren J.A. Rouzer C.A. Serhan C.N. Science. 1987; 237: 1171-1176Crossref PubMed Scopus (2056) Google Scholar). 5LO and LTs are, therefore, key components involved in inflammatory disorders, including arthritis, asthma, and allergic reactions. Recently, novel modulatory mechanisms determining cellular 5LO activity were identified. 5LO is phosphorylated by p38 mitogen-activated protein kinase-activated protein (MAPKAP) kinases prepared from stimulated myeloid cells (3Werz O. Klemm J. Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5261-5266Crossref PubMed Scopus (187) Google Scholar). In addition, Mg2+ increases 5LO activity in vitro (4Reddy K.V. Hammarberg T. Rådmark O. Biochemistry. 2000; 39: 1840-1848Crossref PubMed Scopus (36) Google Scholar). Furthermore, a stimulatory Ca2+ binding site has been localized in the N-terminal domain of 5LO, that may function as a C2 domain in the calcium regulation of 5LO catalytic activity (5Hammarberg T. Provost P. Persson B. Rådmark O. J. Biol. Chem. 2000; 275: 38787-38793Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). C2 domains have also been shown to mediate protein-protein interactions (6Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (698) Google Scholar). Additional lines of evidence indicate that cellular 5LO activity and distribution is regulated by interaction with other proteins. For example, the subcellular distribution of 5LO differs among cell types and changes in response to various stimuli. In particular, 5LO translocates to the nuclear membrane from either the cytosol (for polymorphonuclear leukocytes) (7Brock T.G. McNish R.W. Bailie M.B. Peters-Golden M. J. Biol. Chem. 1997; 272: 8276-8280Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar) or from inside the nucleus (for alveolar macrophages) (8Woods J.W. Coffey M.J. Brock T.G. Singer I.I. Peters-Golden M. J. Clin. Invest. 1995; 95: 2035-2046Crossref PubMed Scopus (160) Google Scholar). In that perspective, an association of 5LO with cytoskeletal structures in vivo, which is a reasonable possibility after demonstration of a direct association between 5LO and actin in vitro (9Lepley R.A. Fitzpatrick F.A. J. Biol. Chem. 1994; 269: 24163-24168Abstract Full Text PDF PubMed Google Scholar), could have important implications for translocation and modulation of cellular 5LO activity. In our attempt to determine the protein partners of 5LO using the yeast two-hybrid system, we identified coactosin-like protein (CLP) as a potential 5LO-interacting protein (10Provost P. Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1881-1885Crossref PubMed Scopus (107) Google Scholar). The CLP nucleotide sequence was initially found as a sequence flanking a deletion on chromosome 17 characterizing the Smith-Magenis syndrome (11Chen K.-S. Manian P. Koeuth T. Potocki L. Zhao Q. Chinault A.C. Lee C.C. Lupski J.R. Nat. Genet. 1997; 17: 154-163Crossref PubMed Scopus (329) Google Scholar). In a separate paper, we have characterized CLP as a human filamentous actin (F-actin)-binding protein. 2P. Provost, J. Doucet, A. Stock, G. Gerisch, B. Samuelsson, and O. Rådmark, submitted for publication. Here, we characterize the CLP-5LO interaction and investigate whether 5LO or the CLP-5LO tandem is recruited by F-actin. The human CLP cDNA was obtained by screening of a human lung cDNA library using 5LO as a bait (10Provost P. Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1881-1885Crossref PubMed Scopus (107) Google Scholar). The CLP cDNA was amplified by polymerase chain reaction and cloned in-frame into theBamHI/XhoI sites of pGEX-5X-1 vector (Amersham Pharmacia Biotech). The pGEX-5X-1-CLP construct was also subjected to site-directed mutagenesis, as described in the “site-directed mutagenesis” section, to generate the CLP mutant K131A. The pGEX-5X-1-CLP and pGEX-5X-1-CLP K131A constructs as well as the empty pGEX-5X-1 vector were transformed into the Escherichia coli bacterial strain BL21 (Amersham Pharmacia Biotech) for expression and purification of the GST-CLP fusion proteins or GST only, according to the manufacturer's instructions. In some experiments, the GST-CLP proteins were eluted from the glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) with 4 volumes of glutathione (10 mm) in 50 mm Tris-HCl pH 8.0 (elution buffer). The CLP proteins were then cleaved from the GST moiety with factor Xa (Amersham Pharmacia Biotech) and purified by anion exchange chromatography. The E. coli bacterial strain MV1190 was transformed with the plasmid pT3–5LO and used for expression of 5LO, as described previously (12Hammarberg T. Zhang Y.Y. Lind B. Rådmark O. Samuelsson B. Eur. J. Biochem. 1995; 230: 401-407Crossref PubMed Scopus (57) Google Scholar). The 5LO protein was purified by affinity chromatography using an ATP-agarose column (12Hammarberg T. Zhang Y.Y. Lind B. Rådmark O. Samuelsson B. Eur. J. Biochem. 1995; 230: 401-407Crossref PubMed Scopus (57) Google Scholar). The protein concentrations were determined by the method of Bradford Biochem. PubMed Scopus Google Scholar) using the with bovine serum albumin as For binding in of the GST-CLP or GST-CLP K131A fusion protein or GST to glutathione-Sepharose 4B was with and of purified 5LO and of F-actin in the presence of in of mm mm and mm pH with mm and 50 mm The of on complex was in with 50 mm a the beads were in and the GST-CLP fusion proteins or 5LO F-actin complex was eluted with glutathione (10 mm glutathione in 50 pH The beads were by and the was with and The presence of the 5LO actin protein in the was by gel electrophoresis by embryonic kidney 293 cells were in with bovine and in a For experiments, the 5LO by a sequence M. 1987; PubMed Scopus Google Scholar) and a was cloned into the expression vector the CLP cDNA was cloned into the expression vector which an the were prepared using the plasmid purification with and in The 293 cells in were transiently with of plasmid by the calcium In the of with a the of was to with empty The 293 cells were with and in of mm pH mm mm with were by 4 of cell was for protein and by to 5LO and CLP protein of cell was with either (10 or or for 4 4 (Amersham Pharmacia Biotech) was then and the was for an The were with of and eluted by for in the were by as described between 5LO and CLP was by as described by Biochem. PubMed Scopus Google Scholar). 5LO and CLP were or in the concentrations in mm mm mm and mm pH for LTA4 hydrolase was used as a protein The were then with and 4 were then either with or to membranes for of the of CLP or CLP K131A and 5LO, the was and in for with The was then on of a and the electrophoresis was in the presence of in the The were with CLP and 5LO proteins were and for in (10 mm pH and 50 mm The reaction were by the of 4 mm of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 4 mm N-hydroxysulfosuccinimide the were by the of mm and the were with The presence of was on and either with or by For actin was purified from as described by and M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). The GST-CLP CLP, and 5LO proteins were purified as described the protein for 4 the reaction were prepared and for in in the (10 mm pH and 50 mm In some experiments, the reaction were with concentrations of The were then for 4 in a The and of were and with The proteins in the and were by and the were with was a using a of M. S. J. Biol. Chem. 1983; Full Text PDF PubMed Google Scholar), as described by G. Biochemistry. PubMed Scopus Google Scholar). was used in a molar of with for actin the the of between the and actin G. Biochemistry. PubMed Scopus Google Scholar). The of and in in the presence of 5LO was by the of and concentrations of and 50 In some experiments, was or were in the The of the reaction was for using a The was and the was through a Protein or cell were by using the to membranes (Amersham Pharmacia and as described previously (10Provost P. Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1881-1885Crossref PubMed Scopus (107) Google Scholar). CLP was in of human CLP. was from was from and 5LO was used after proteins were by using and and The binding domain vector the and the domain vector the were obtained from construct the vector the CLP cDNA by polymerase chain reaction as a and used for the pGEX-5X-1-CLP as described was cloned in-frame into the sites of construct the vector a of the 5LO cDNA from pT3–5LO Y.Y. Rådmark O. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) was amplified by polymerase chain reaction and cloned into the sites of the construct by the of the 5LO cDNA from pT3–5LO into the sites of and the 5-lipoxygenase cDNA was as a and then cloned in-frame into the sites of the vector The constructs and were obtained by screening of a human lung cDNA library using human CLP as a The constructs and used as were described previously (10Provost P. Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1881-1885Crossref PubMed Scopus (107) Google Scholar). The yeast strain P. J. E.A. 1996; PubMed Google Scholar) was used to protein-protein as previously described (10Provost P. Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1881-1885Crossref PubMed Scopus (107) Google Scholar). Site-directed mutagenesis of the lysine tandem of CLP, cloned in was by using the site-directed mutagenesis according to the manufacturer's instructions. The were with with the of The A. 1999; PubMed Scopus Google Scholar), that a or a site in The of CLP was subjected to site-directed mutagenesis by polymerase chain reaction J.A. in Scholar). The were with that a site in The presence of the was by and by the CLP cDNA using the (Amersham Pharmacia Biotech) the was used to the of of purified CLP and CLP K131A 50 in mm pH using a cell For were in the from to and the was then in were into and expressed as The of the of CLP and CLP K131A in of and was using the on the P. J.J. Protein PubMed Scopus Google Scholar). the interaction between 5LO and CLP in GST binding experiments were The GST-CLP fusion protein or GST was expressed in to glutathione-Sepharose 4B and with and purified a the beads were and the protein were eluted with The presence of 5LO in the was by by using shown in no binding of 5LO to the beads to GST was However, a dose-dependent association of 5LO to GST-CLP was that CLP and 5LO with For experiments, 293 cells were with expression a epitope-tagged 5LO and an epitope-tagged CLP protein. CLP or 5LO protein was found in cells with empty vector only, and no to or was of the 293 cells the expression of 5LO with activity to that obtained after with 5LO also showed CLP expression in cells with the CLP the presence of the as with CLP. of cells was by and subjected to with the revealed that 5LO was with CLP In the reciprocal in which was used to 5LO, by CLP with 5LO 5LO or CLP was detected in prepared with and indicate that 5LO a complex with CLP in The 5LO-CLP interaction was also using this the of is determined by the of to of a protein. the purified 5LO as a with a and on the of the gel CLP a and as a in the of the LTA4 used as a a further CLP, as a 5LO or CLP was with LTA4 no or changes in the or of the was observed. However, of CLP and 5LO were the CLP and the 5LO was In the presence of the the of 5LO was that the CLP was with 5LO is the of complex was by either or as a of the polyacrylamide with and the presence of CLP in the 5LO shown in of the by a the of CLP in the 5LO The of on the complex was further in GST binding The GST-CLP fusion protein to glutathione-Sepharose 4B beads were with of purified 5LO in the presence of calcium mm) or in the of calcium in the presence of no on 5LO binding to CLP. results indicate an interaction between 5LO and CLP that is The stoichiometry of the complex was determined by binding on In a known of 5LO was with of CLP and and the which CLP appeared or was a of CLP was by of 5LO, the of the CLP was and a molar of 1:1 or a of 5LO was by of CLP, the CLP a molar 1:1 4 In experiments, the of between 5LO and CLP, as or the of the complex was in cross-linking CLP and 5LO were or in in the or presence of the and and the were by and In the of cross-linking CLP and 5LO as was CLP or 5LO was with for However, CLP was with 5LO in the presence of for a of appeared on the showed of this with 4 that the a using and was shown in this on was with and and the for an results indicate that the protein complex is of of 5LO to of CLP. acid of CLP involved in the binding of 5LO, the yeast two-hybrid was The yeast was with 5LO and various CLP mutant The CLP cDNA of CLP construct was to the presence of the and the of and were used as and was used as a protein the CLP were to bind 5LO, the CLP mutant construct This CLP mutant with actin with 5LO of CLP, we found that the mutant could with 5LO, the of lysine 131 5LO binding. results were obtained lysine 131 was by of lysine 131 by the binding of CLP to 5LO, the of a acid 131 of CLP is involved in 5LO binding in the yeast two-hybrid binding domain domain in the of in of yeast strain was with the the fusion and were for the plasmid were for CLP of the was for and 4 or the of yeast In two-hybrid experiments, a protein, and as and CLP with actin for the CLP that the binding domain moiety of the CLP mutant two-hybrid constructs was In addition, CLP in with were that the in CLP of the two-hybrid results were obtained using the and in a The yeast strain was with the the fusion and were for the plasmid were for CLP of the was for and 4 or the of yeast In two-hybrid experiments, a protein, and as and CLP with actin for the CLP that the binding domain moiety of the CLP mutant two-hybrid constructs was In addition, CLP in with were that the in CLP of the two-hybrid results were obtained using the and on the that a acid is involved in 5LO of CLP was by site-directed the in which of the are of the of the by the 5LO binding of CLP. the two-hybrid the importance of lysine 131 of CLP in 5LO binding was in GST binding experiments and in gel For the GST binding the GST-CLP or GST-CLP K131A fusion protein was expressed in to glutathione-Sepharose 4B and with of purified The dose-dependent association of 5LO with CLP was by lysine 131 of CLP by an of the GST the interaction between purified CLP mutant protein and 5LO was by CLP K131A and 5LO were no or other changes in the to the proteins were In particular, no of the 5LO of a protein was of the CLP by a of the proteins the of interaction between the CLP K131A mutant and an important role for lysine 131 in 5LO binding. The of CLP as well as that of the CLP K131A mutant was by and in of and was shown in the of the CLP K131A mutant was from CLP. the P. J.J. Protein PubMed Scopus Google Scholar), CLP was to of and the CLP K131A mutant was found to of and results indicate that of lysine 131 by of the CLP protein a in the that the binding of by and Fitzpatrick (9Lepley R.A. Fitzpatrick F.A. J. Biol. Chem. 1994; 269: 24163-24168Abstract Full Text PDF PubMed Google Scholar) that 5LO with In view of the ability of CLP to bind 5LO as well as was of to determine whether CLP a link between F-actin and This was in GST binding The GST-CLP fusion protein was to glutathione-Sepharose 4B beads and with various concentrations of F-actin Whereas F-actin and 5LO and with CLP, the binding of F-actin to CLP was by concentrations of 5LO the beads could have physically with the of an F-actin-CLP·5LO ternary using purified proteins were In 5LO was concentrations with in the presence or of CLP, and actin was by the of and the and the the actin were for the presence of 5LO and CLP by and shown in of CLP in the mediate 5LO binding to F-actin. as in the GST binding the of an F-actin-CLP·5LO ternary complex in experiments a mutually exclusive of CLP to F-actin was in the or concentrations of This CLP-F-actin association was by of 5LO In a a ternary F-actin-CLP·5LO was detected by cross-linking In in the of CLP, no association of 5LO with F-actin was found 5LO an increase in with a This of 5LO on actin was by a molar of to 5LO and was by the 5LO The that 5LO the of actin in was further in a actin The of with concentrations of 5LO, was by the of and and the of the reaction was shown in in the a and increase in The of 5LO the the of actin the of a 5LO of 5LO no on actin In a subjected to a was in the possibility that the of 5LO is to a interaction with other experiments were in the presence of was in an molar to 5LO, the of 5LO by in the presence and of that this was to the interaction of 5LO with The cellular activity and of 5LO may on interaction with other proteins. we have used a two-hybrid (10Provost P. Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1881-1885Crossref PubMed Scopus (107) Google Scholar). of the proteins by this CLP, to an actin binding protein from B. G. PubMed Scopus Google Scholar). CLP has recently been characterized as a human In the we that 5LO with CLP in vitro and in In the interaction in a The of this complex to of from the membrane association of 5LO, which is by We have recently shown that binding of Ca2+ to the N-terminal domain of 5LO the activity of this enzyme (5Hammarberg T. Provost P. Persson B. Rådmark O. J. Biol. Chem. 2000; 275: 38787-38793Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). the membrane association of 5LO to CLP. In CLP has no direct on 5LO activity purified proteins were in vitro In a for acid involved in the actin binding of CLP, we found CLP with a to with The of lysine 131 by 5LO the of a acid The that the CLP K131A mutant ability to bind actin that was In the of lysine 131 by the 131 to of the with 5LO or to in for the of CLP for 5LO binding. The regulation of actin and and the localized of filamentous actin into a the of polymorphonuclear are for the for leukocyte adhesion, and 1994; Google Biol. PubMed Scopus Google Scholar). between 5LO and as previously (9Lepley R.A. Fitzpatrick F.A. J. Biol. Chem. 1994; 269: 24163-24168Abstract Full Text PDF PubMed Google Scholar), is of the possibility that actin is involved in the translocation of 5LO, which with of the cells (for M. Brock T.G. J. 2000; PubMed Scopus Google Scholar). is evidence that the activity of 5LO is by the of actin in an of leukotriene in polymorphonuclear is the cells were with E.A. E. F.A. Proc. Natl. Acad. Sci. U. S. A. 1983; PubMed Scopus Google M. L. Biochem. PubMed Scopus (36) Google Scholar), which with actin In the catalytic of 5LO to the actin For example, including the 5LO acid are of binding cytosolic actin J. 39: Full Text Full Text PDF PubMed Google Scholar). actin in polymorphonuclear J. Biol. Chem. Full Text PDF PubMed Google J.J. J. 1995; Google Scholar), and LTD4 of the actin in cells A. Eur. J. Biol. PubMed Scopus Google Scholar). Furthermore, have been with the of in cells subjected to P. 2000; PubMed Scopus Google Scholar). In our experiments, 5LO the of actin in This was in and actin 5LO this in with the of actin in with actin B. J. M. of the A. P. J. and Scholar). are with the possibility that 5LO actin by the of the other the possibility that 5LO with by the or of actin as for Lind Biochemistry. PubMed Scopus Google Scholar). In our actin and which no evidence for the of a complex into the binding of 5LO to or the other of actin have In the that of actin by 5LO is and is by that 5LO the of actin the of of 5LO to the actin is by the binding of 5LO to CLP. The has been whether CLP, 5LO, and actin a ternary The of CLP that lysine 131 is important for the binding of 5LO for the binding of the lysine is for actin binding for the interaction with on the binding sites on CLP to However, may or binding of 5LO and actin to the CLP may from Our to a ternary complex of 5LO, CLP, and filamentous actin were suggest that 5LO CLP from the interaction with This is experiments suggest that CLP the activity of as a of the of a function for which with the activity of of a protein in U. G. L. M. A. 1995; PubMed Scopus Google Scholar). In the presence of CLP and 5LO may the activity by CLP. could that 5LO may the of actin in by the activity of proteins in the presence of CLP and by as an of actin polymerization. the role of 5LO in vivo, the has to into that of the proteins as (3Werz O. Klemm J. Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5261-5266Crossref PubMed Scopus (187) Google R.A. Fitzpatrick F.A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar), or the presence of may the of 5LO into a In our results demonstrate that 5LO with CLP. We also that 5LO actin and with the binding of CLP to F-actin. may that 5LO, in to key role in leukotriene the actin in inflammatory a novel of actin We Stock, and for and for We are to for the yeast strain and for purified LTA4