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W2008238416 abstract "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 (2312) 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 (1970) 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 (182) Google Scholar). In addition, Mg2+ increases 5LO activity in vitro (4Reddy K.V. Hammarberg T. Rådmark O. Biochemistry. 2000; 39: 1840-1848Crossref PubMed Scopus (35) 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 (138) 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 (685) 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 (158) 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 (106) 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 (328) 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.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.DISCUSSIONThe cellular activity and localization of 5LO may depend on its physical interaction with other proteins. To identify interacting proteins, we have used a two-hybrid approach (10Provost P. Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1881-1885Crossref PubMed Scopus (106) Google Scholar). One of the proteins discovered by this approach, CLP, shows homology to coactosin, an actin binding protein from Dictyostelium discoideum (24de Hostos E.L. Bradtke B. Lottspeich F. Gerisch G. Cell Motil. Cytoskeleton. 1993; 26: 181-191Crossref PubMed Scopus (63) Google Scholar). CLP has recently been characterized as a human F-actin-binding protein.2 In the present study, we show that 5LO interacts directly with CLP in vitro and in vivo. In vitro, the interaction resulted in a heterodimeric complex. The formation of this complex proved to be independent of Ca2+, different from the membrane association of 5LO, which is promoted by Ca2+. We have recently shown that binding of Ca2+ to the N-terminal domain of 5LO stimulates 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 (138) Google Scholar). Neither Ca2+-dependent activation nor the membrane association of 5LO seems to involve CLP. In fact, CLP has no direct effect on 5LO activity when purified proteins were mixed in vitro (data not shown).In a search for amino acid residues involved in the actin binding of CLP, we found one mutant, CLP K131A, with a strongly reduced capacity to interact with 5LO. The substitution of lysine 131 by arginine preserved 5LO binding, indicating the requirement of a basic, positively charged amino acid residue at position 131. The fact that the CLP K131A mutant retained its ability to bind actin indicates that its overall secondary structure was not drastically affected. In accord, the replacement of lysine 131 by alanine only slightly changed the circular dichroism spectrum. Lysine 131 thus appears to be part of the surface interacting with 5LO or to be required in particular for maintaining the conformation of CLP necessary for 5LO binding.The regulation of actin polymerization and depolymerization and the localized assembly of filamentous actin into a network within the cortex of polymorphonuclear leukocytes are essential for generating the force necessary for leukocyte locomotion, shape change, phagocytosis, adhesion, and spreading (25Howard T.H. Watts R.G. Curr. Opin. Hematol. 1994; 1: 61-68PubMed Google Scholar, 26Zigmond S.H. Curr. Opin. Cell Biol. 1989; 1: 80-86Crossref PubMed Scopus (27) Google Scholar). A connection between 5LO and actin, as reported previously (9Lepley R.A. Fitzpatrick F.A. J. Biol. Chem. 1994; 269: 24163-24168Abstract Full Text PDF PubMed Google Scholar), is therefore of considerable interest. It raises the possibility that actin is involved in the intracellular translocation of 5LO, which concomitantly with LT biosynthesis follows activation of the cells (for review, see Ref. 27Peters-Golden M. Brock T.G. Am. J. Respir. Crit. Care Med. 2000; 161: S36-S40Crossref PubMed Scopus (73) Google Scholar). There is evidence that the activity of 5LO is influenced by the state of actin in vivo; an up-regulation of formylmethionylleucylphenylalanine-induced leukotriene generation in polymorphonuclear leukocytes is observed when the cells were treated with cytochalasin B (28Ham E.A. Soderman D.D. Zanetti M.E. Dougherty H.W. McCauley E. Kuehl Jr., F.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4349-4353Crossref PubMed Scopus (255) Google Scholar, 29Haurand M. Flohé L. Biochem. Pharmacol. 1989; 38: 2129-2137Crossref PubMed Scopus (35) Google Scholar), which interferes with actin filament formation. In turn, the catalytic products of 5LO appear to affect the actin system. For example, monohydroxy acids, including the 5LO derivate 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), are capable of binding cytosolic actin (30Kang L.-T. Vanderhoek J.Y. J. Lipid Res. 1998; 39: 1476-1482Abstract Full Text Full Text PDF PubMed Google Scholar). LTB4induces oscillatory actin polymerization/depolymerization in polymorphonuclear leukocytes (31Omann G.M. Porasik M.M. Sklar L.A. J. Biol. Chem. 1989; 264: 16355-16358Abstract Full Text PDF PubMed Google Scholar, 32Omann G.M. Rengan R. Hoffman J.F. Linderman J.J. J. Immunol. 1995; 155: 5375-5381PubMed Google Scholar), and LTD4 triggers reorganization of the actin network in intestinal epithelial cells (33Massoumi R. Sjölander A. Eur. J. Cell Biol. 1998; 76: 185-191Crossref PubMed Scopus (20) Google Scholar). Furthermore, leukotrienes have been implicated with the formation of stress fibers in endothelial cells subjected to mechanical stretching (34Wang J.H.-C. Goldschmidt-Clermont P. Moldovan N. Yin F.C.-P. Cell Motil. Cytoskeleton. 2000; 46: 137-145Crossref PubMed Scopus (38) Google Scholar).In our experiments, 5LO inhibited the polymerization of actin in vitro. This inhibitory effect was observed in both cosedimentation and actin polymerization assays. 5LO exerted this effect at submicromolar concentrations, in accord with the estimated concentration of monomeric actin (∼0.1–0.2 μm) in equilibrium with polymerized actin (35Alberts B. Bray D. Lewis J. Raff M. Roberts K. Watson J.D. Molecular Biology of the Cell. 3rd Ed. Garland Publishing, Inc., NY1994Google Scholar, 36Lodish H. Baltimore D. Berk A. Zipurski S.L. Matsudaira P. Darnell J. Molecular Cell Biology. 3rd Ed. Scientific American Books, Inc., W. H. Freeman and Co., NY1995Google Scholar). These data are compatible with the possibility that 5LO inhibits actin polymerization by the sequestration of G-actin. On the other hand, the possibility remains that 5LO interferes with polymerization by the severing or capping of actin filaments, as for instance gelsolin does (37Janmey P.A. Chaponnier C. Lind S.E. Zaner K.S. Stossel T.P. Yin H.L. Biochemistry. 1985; 24: 3714-3723Crossref PubMed Scopus (151) Google Scholar). In our actin polymerization and cosedimentation assays, which provide no evidence for the incorporation of a putative actin-5LO complex into actin, the binding of 5LO to one or the other end of actin filaments would have escaped detection. In fact, the finding that inhibition of actin polymerization by 5LO is not complete and saturates halfway (Fig.7 C), is most easily explained by assuming that 5LO caps the plus ends of actin filaments, thereby raising the critical concentration of G-actin.An indirect relationship of 5LO to the actin system is established by the binding of 5LO to CLP. The question has been addressed whether CLP, 5LO, and actin form a ternary complex. The mutational analysis of CLP indicated that lysine 131 is important for the binding of 5LO but not for the binding of actin. On the contrary, lysine 75 is critical for actin binding but not for the interaction with 5LO.2 Based on these data, the two binding sites on CLP appear to be distinct. However, they may be overlapping, or simultaneous binding of 5LO (78 kDa) and actin (42 kDa) to the small CLP molecule (16 kDa) may suffer from steric hindrance. Our attempts to identify a ternary complex of 5LO, CLP, and filamentous actin were unsuccessful. Rather, data suggest that 5LO prevents CLP from the interaction with actin. This is relevant since preliminary experiments suggest that CLP might antagonize the activity of gelsolin as a capper of the plus ends of actin-filaments (data not shown), a function reported for coactosin, which interfered with the actin-capping activity of fragment S1 of severin, a gelsolin-related protein in Dictyostelium (38Röhrig U. Gerisch G. Morozova L. Schleicher M. Wegner A. FEBS Lett. 1995; 374: 284-286Crossref PubMed Scopus (33) Google Scholar). In the presence of CLP and gelsolin, 5LO may restore the capping activity by sequestering CLP. Thus, one could speculate that 5LO may inhibit the polymerization of actin in two ways: by maintaining the activity of capping proteins in the presence of CLP and by acting itself as an inhibitor of actin polymerization. Considering the role of 5LO in vivo, the caveat has to be taken into account that modification of the proteins involved, such as phosphorylation (3Werz O. Klemm J. Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5261-5266Crossref PubMed Scopus (182) Google Scholar, 39Lepley R.A. Muskardin D.T. Fitzpatrick F.A. J. Biol. Chem. 1996; 271: 6179-6184Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), or the presence of cofactors may turn the inhibitory effect of 5LO into a supporting one.In conclusion, our results demonstrate that 5LO directly interacts with CLP. We also show that 5LO inhibits actin polymerization and interferes with the binding of CLP to F-actin. It may be hypothesized that 5LO, in addition to its key role in leukotriene synthesis, modulates the actin dynamics in inflammatory cells, thus representing a novel regulator of actin function. 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 (2312) 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 (1970) 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 (182) Google Scholar). In addition, Mg2+ increases 5LO activity in vitro (4Reddy K.V. Hammarberg T. Rådmark O. Biochemistry. 2000; 39: 1840-1848Crossref PubMed Scopus (35) 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 (138) 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 (685) 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 (158) 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 (106) 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 (328) 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.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. DISCUSSIONThe cellular activity and localization of 5LO may depend on its physical interaction with other proteins. To identify interacting proteins, we have used a two-hybrid approach (10Provost P. Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1881-1885Crossref PubMed Scopus (106) Google Scholar). One of the proteins discovered by this approach, CLP, shows homology to coactosin, an actin binding protein from Dictyostelium discoideum (24de Hostos E.L. Bradtke B. Lottspeich F. Gerisch G. Cell Motil. Cytoskeleton. 1993; 26: 181-191Crossref PubMed Scopus (63) Google Scholar). CLP has recently been characterized as a human F-actin-binding protein.2 In the present study, we show that 5LO interacts directly with CLP in vitro and in vivo. In vitro, the interaction resulted in a heterodimeric complex. The formation of this complex proved to be independent of Ca2+, different from the membrane association of 5LO, which is promoted by Ca2+. We have recently shown that binding of Ca2+ to the N-terminal domain of 5LO stimulates 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 (138) Google Scholar). Neither Ca2+-dependent activation nor the membrane association of 5LO seems to involve CLP. In fact, CLP has no direct effect on 5LO activity when purified proteins were mixed in vitro (data not shown).In a search for amino acid residues involved in the actin binding of CLP, we found one mutant, CLP K131A, with a strongly reduced capacity to interact with 5LO. The substitution of lysine 131 by arginine preserved 5LO binding, indicating the requirement of a basic, positively charged amino acid residue at position 131. The fact that the CLP K131A mutant retained its ability to bind actin indicates that its overall secondary structure was not drastically affected. In accord, the replacement of lysine 131 by alanine only slightly changed the circular dichroism spectrum. Lysine 131 thus appears to be part of the surface interacting with 5LO or to be required in particular for maintaining the conformation of CLP necessary for 5LO binding.The regulation of actin polymerization and depolymerization and the localized assembly of filamentous actin into a network within the cortex of polymorphonuclear leukocytes are essential for generating the force necessary for leukocyte locomotion, shape change, phagocytosis, adhesion, and spreading (25Howard T.H. Watts R.G. Curr. Opin. Hematol. 1994; 1: 61-68PubMed Google Scholar, 26Zigmond S.H. Curr. Opin. Cell Biol. 1989; 1: 80-86Crossref PubMed Scopus (27) Google Scholar). A connection between 5LO and actin, as reported previously (9Lepley R.A. Fitzpatrick F.A. J. Biol. Chem. 1994; 269: 24163-24168Abstract Full Text PDF PubMed Google Scholar), is therefore of considerable interest. It raises the possibility that actin is involved in the intracellular translocation of 5LO, which concomitantly with LT biosynthesis follows activation of the cells (for review, see Ref. 27Peters-Golden M. Brock T.G. Am. J. Respir. Crit. Care Med. 2000; 161: S36-S40Crossref PubMed Scopus (73) Google Scholar). There is evidence that the activity of 5LO is influenced by the state of actin in vivo; an up-regulation of formylmethionylleucylphenylalanine-induced leukotriene generation in polymorphonuclear leukocytes is observed when the cells were treated with cytochalasin B (28Ham E.A. Soderman D.D. Zanetti M.E. Dougherty H.W. McCauley E. Kuehl Jr., F.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4349-4353Crossref PubMed Scopus (255) Google Scholar, 29Haurand M. Flohé L. Biochem. Pharmacol. 1989; 38: 2129-2137Crossref PubMed Scopus (35) Google Scholar), which interferes with actin filament formation. In turn, the catalytic products of 5LO appear to affect the actin system. For example, monohydroxy acids, including the 5LO derivate 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), are capable of binding cytosolic actin (30Kang L.-T. Vanderhoek J.Y. J. Lipid Res. 1998; 39: 1476-1482Abstract Full Text Full Text PDF PubMed Google Scholar). LTB4induces oscillatory actin polymerization/depolymerization in polymorphonuclear leukocytes (31Omann G.M. Porasik M.M. Sklar L.A. J. Biol. Chem. 1989; 264: 16355-16358Abstract Full Text PDF PubMed Google Scholar, 32Omann G.M. Rengan R. Hoffman J.F. Linderman J.J. J. Immunol. 1995; 155: 5375-5381PubMed Google Scholar), and LTD4 triggers reorganization of the actin network in intestinal epithelial cells (33Massoumi R. Sjölander A. Eur. J. Cell Biol. 1998; 76: 185-191Crossref PubMed Scopus (20) Google Scholar). Furthermore, leukotrienes have been implicated with the formation of stress fibers in endothelial cells subjected to mechanical stretching (34Wang J.H.-C. Goldschmidt-Clermont P. Moldovan N. Yin F.C.-P. Cell Motil. Cytoskeleton. 2000; 46: 137-145Crossref PubMed Scopus (38) Google Scholar).In our experiments, 5LO inhibited the polymerization of actin in vitro. This inhibitory effect was observed in both cosedimentation and actin polymerization assays. 5LO exerted this effect at submicromolar concentrations, in accord with the estimated concentration of monomeric actin (∼0.1–0.2 μm) in equilibrium with polymerized actin (35Alberts B. Bray D. Lewis J. Raff M. Roberts K. Watson J.D. Molecular Biology of the Cell. 3rd Ed. Garland Publishing, Inc., NY1994Google Scholar, 36Lodish H. Baltimore D. Berk A. Zipurski S.L. Matsudaira P. Darnell J. Molecular Cell Biology. 3rd Ed. Scientific American Books, Inc., W. H. Freeman and Co., NY1995Google Scholar). These data are compatible with the possibility that 5LO inhibits actin polymerization by the sequestration of G-actin. On the other hand, the possibility remains that 5LO interferes with polymerization by the severing or capping of actin filaments, as for instance gelsolin does (37Janmey P.A. Chaponnier C. Lind S.E. Zaner K.S. Stossel T.P. Yin H.L. Biochemistry. 1985; 24: 3714-3723Crossref PubMed Scopus (151) Google Scholar). In our actin polymerization and cosedimentation assays, which provide no evidence for the incorporation of a putative actin-5LO complex into actin, the binding of 5LO to one or the other end of actin filaments would have escaped detection. In fact, the finding that inhibition of actin polymerization by 5LO is not complete and saturates halfway (Fig.7 C), is most easily explained by assuming that 5LO caps the plus ends of actin filaments, thereby raising the critical concentration of G-actin.An indirect relationship of 5LO to the actin system is established by the binding of 5LO to CLP. The question has been addressed whether CLP, 5LO, and actin form a ternary complex. The mutational analysis of CLP indicated that lysine 131 is important for the binding of 5LO but not for the binding of actin. On the contrary, lysine 75 is critical for actin binding but not for the interaction with 5LO.2 Based on these data, the two binding sites on CLP appear to be distinct. However, they may be overlapping, or simultaneous binding of 5LO (78 kDa) and actin (42 kDa) to the small CLP molecule (16 kDa) may suffer from steric hindrance. Our attempts to identify a ternary complex of 5LO, CLP, and filamentous actin were unsuccessful. Rather, data suggest that 5LO prevents CLP from the interaction with actin. This is relevant since preliminary experiments suggest that CLP might antagonize the activity of gelsolin as a capper of the plus ends of actin-filaments (data not shown), a function reported for coactosin, which interfered with the actin-capping activity of fragment S1 of severin, a gelsolin-related protein in Dictyostelium (38Röhrig U. Gerisch G. Morozova L. Schleicher M. Wegner A. FEBS Lett. 1995; 374: 284-286Crossref PubMed Scopus (33) Google Scholar). In the presence of CLP and gelsolin, 5LO may restore the capping activity by sequestering CLP. Thus, one could speculate that 5LO may inhibit the polymerization of actin in two ways: by maintaining the activity of capping proteins in the presence of CLP and by acting itself as an inhibitor of actin polymerization. Considering the role of 5LO in vivo, the caveat has to be taken into account that modification of the proteins involved, such as phosphorylation (3Werz O. Klemm J. Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5261-5266Crossref PubMed Scopus (182) Google Scholar, 39Lepley R.A. Muskardin D.T. Fitzpatrick F.A. J. Biol. Chem. 1996; 271: 6179-6184Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), or the presence of cofactors may turn the inhibitory effect of 5LO into a supporting one.In conclusion, our results demonstrate that 5LO directly interacts with CLP. We also show that 5LO inhibits actin polymerization and interferes with the binding of CLP to F-actin. It may be hypothesized that 5LO, in addition to its key role in leukotriene synthesis, modulates the actin dynamics in inflammatory cells, thus representing a novel regulator of actin function. The cellular activity and localization of 5LO may depend on its physical interaction with other proteins. To identify interacting proteins, we have used a two-hybrid approach (10Provost P. Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1881-1885Crossref PubMed Scopus (106) Google Scholar). One of the proteins discovered by this approach, CLP, shows homology to coactosin, an actin binding protein from Dictyostelium discoideum (24de Hostos E.L. Bradtke B. Lottspeich F. Gerisch G. Cell Motil. Cytoskeleton. 1993; 26: 181-191Crossref PubMed Scopus (63) Google Scholar). CLP has recently been characterized as a human F-actin-binding protein.2 In the present study, we show that 5LO interacts directly with CLP in vitro and in vivo. In vitro, the interaction resulted in a heterodimeric complex. The formation of this complex proved to be independent of Ca2+, different from the membrane association of 5LO, which is promoted by Ca2+. We have recently shown that binding of Ca2+ to the N-terminal domain of 5LO stimulates 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 (138) Google Scholar). Neither Ca2+-dependent activation nor the membrane association of 5LO seems to involve CLP. In fact, CLP has no direct effect on 5LO activity when purified proteins were mixed in vitro (data not shown). In a search for amino acid residues involved in the actin binding of CLP, we found one mutant, CLP K131A, with a strongly reduced capacity to interact with 5LO. The substitution of lysine 131 by arginine preserved 5LO binding, indicating the requirement of a basic, positively charged amino acid residue at position 131. The fact that the CLP K131A mutant retained its ability to bind actin indicates that its overall secondary structure was not drastically affected. In accord, the replacement of lysine 131 by alanine only slightly changed the circular dichroism spectrum. Lysine 131 thus appears to be part of the surface interacting with 5LO or to be required in particular for maintaining the conformation of CLP necessary for 5LO binding. The regulation of actin polymerization and depolymerization and the localized assembly of filamentous actin into a network within the cortex of polymorphonuclear leukocytes are essential for generating the force necessary for leukocyte locomotion, shape change, phagocytosis, adhesion, and spreading (25Howard T.H. Watts R.G. Curr. Opin. Hematol. 1994; 1: 61-68PubMed Google Scholar, 26Zigmond S.H. Curr. Opin. Cell Biol. 1989; 1: 80-86Crossref PubMed Scopus (27) Google Scholar). A connection between 5LO and actin, as reported previously (9Lepley R.A. Fitzpatrick F.A. J. Biol. Chem. 1994; 269: 24163-24168Abstract Full Text PDF PubMed Google Scholar), is therefore of considerable interest. It raises the possibility that actin is involved in the intracellular translocation of 5LO, which concomitantly with LT biosynthesis follows activation of the cells (for review, see Ref. 27Peters-Golden M. Brock T.G. Am. J. Respir. Crit. Care Med. 2000; 161: S36-S40Crossref PubMed Scopus (73) Google Scholar). There is evidence that the activity of 5LO is influenced by the state of actin in vivo; an up-regulation of formylmethionylleucylphenylalanine-induced leukotriene generation in polymorphonuclear leukocytes is observed when the cells were treated with cytochalasin B (28Ham E.A. Soderman D.D. Zanetti M.E. Dougherty H.W. McCauley E. Kuehl Jr., F.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4349-4353Crossref PubMed Scopus (255) Google Scholar, 29Haurand M. Flohé L. Biochem. Pharmacol. 1989; 38: 2129-2137Crossref PubMed Scopus (35) Google Scholar), which interferes with actin filament formation. In turn, the catalytic products of 5LO appear to affect the actin system. For example, monohydroxy acids, including the 5LO derivate 5(S)-hydroxy-6,8,11,14-eicosatetraenoic acid (5-HETE), are capable of binding cytosolic actin (30Kang L.-T. Vanderhoek J.Y. J. Lipid Res. 1998; 39: 1476-1482Abstract Full Text Full Text PDF PubMed Google Scholar). LTB4induces oscillatory actin polymerization/depolymerization in polymorphonuclear leukocytes (31Omann G.M. Porasik M.M. Sklar L.A. J. Biol. Chem. 1989; 264: 16355-16358Abstract Full Text PDF PubMed Google Scholar, 32Omann G.M. Rengan R. Hoffman J.F. Linderman J.J. J. Immunol. 1995; 155: 5375-5381PubMed Google Scholar), and LTD4 triggers reorganization of the actin network in intestinal epithelial cells (33Massoumi R. Sjölander A. Eur. J. Cell Biol. 1998; 76: 185-191Crossref PubMed Scopus (20) Google Scholar). Furthermore, leukotrienes have been implicated with the formation of stress fibers in endothelial cells subjected to mechanical stretching (34Wang J.H.-C. Goldschmidt-Clermont P. Moldovan N. Yin F.C.-P. Cell Motil. Cytoskeleton. 2000; 46: 137-145Crossref PubMed Scopus (38) Google Scholar). In our experiments, 5LO inhibited the polymerization of actin in vitro. This inhibitory effect was observed in both cosedimentation and actin polymerization assays. 5LO exerted this effect at submicromolar concentrations, in accord with the estimated concentration of monomeric actin (∼0.1–0.2 μm) in equilibrium with polymerized actin (35Alberts B. Bray D. Lewis J. Raff M. Roberts K. Watson J.D. Molecular Biology of the Cell. 3rd Ed. Garland Publishing, Inc., NY1994Google Scholar, 36Lodish H. Baltimore D. Berk A. Zipurski S.L. Matsudaira P. Darnell J. Molecular Cell Biology. 3rd Ed. Scientific American Books, Inc., W. H. Freeman and Co., NY1995Google Scholar). These data are compatible with the possibility that 5LO inhibits actin polymerization by the sequestration of G-actin. On the other hand, the possibility remains that 5LO interferes with polymerization by the severing or capping of actin filaments, as for instance gelsolin does (37Janmey P.A. Chaponnier C. Lind S.E. Zaner K.S. Stossel T.P. Yin H.L. Biochemistry. 1985; 24: 3714-3723Crossref PubMed Scopus (151) Google Scholar). In our actin polymerization and cosedimentation assays, which provide no evidence for the incorporation of a putative actin-5LO complex into actin, the binding of 5LO to one or the other end of actin filaments would have escaped detection. In fact, the finding that inhibition of actin polymerization by 5LO is not complete and saturates halfway (Fig.7 C), is most easily explained by assuming that 5LO caps the plus ends of actin filaments, thereby raising the critical concentration of G-actin. An indirect relationship of 5LO to the actin system is established by the binding of 5LO to CLP. The question has been addressed whether CLP, 5LO, and actin form a ternary complex. The mutational analysis of CLP indicated that lysine 131 is important for the binding of 5LO but not for the binding of actin. On the contrary, lysine 75 is critical for actin binding but not for the interaction with 5LO.2 Based on these data, the two binding sites on CLP appear to be distinct. However, they may be overlapping, or simultaneous binding of 5LO (78 kDa) and actin (42 kDa) to the small CLP molecule (16 kDa) may suffer from steric hindrance. Our attempts to identify a ternary complex of 5LO, CLP, and filamentous actin were unsuccessful. Rather, data suggest that 5LO prevents CLP from the interaction with actin. This is relevant since preliminary experiments suggest that CLP might antagonize the activity of gelsolin as a capper of the plus ends of actin-filaments (data not shown), a function reported for coactosin, which interfered with the actin-capping activity of fragment S1 of severin, a gelsolin-related protein in Dictyostelium (38Röhrig U. Gerisch G. Morozova L. Schleicher M. Wegner A. FEBS Lett. 1995; 374: 284-286Crossref PubMed Scopus (33) Google Scholar). In the presence of CLP and gelsolin, 5LO may restore the capping activity by sequestering CLP. Thus, one could speculate that 5LO may inhibit the polymerization of actin in two ways: by maintaining the activity of capping proteins in the presence of CLP and by acting itself as an inhibitor of actin polymerization. Considering the role of 5LO in vivo, the caveat has to be taken into account that modification of the proteins involved, such as phosphorylation (3Werz O. Klemm J. Samuelsson B. Rådmark O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5261-5266Crossref PubMed Scopus (182) Google Scholar, 39Lepley R.A. Muskardin D.T. Fitzpatrick F.A. J. Biol. Chem. 1996; 271: 6179-6184Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar), or the presence of cofactors may turn the inhibitory effect of 5LO into a supporting one. In conclusion, our results demonstrate that 5LO directly interacts with CLP. We also show that 5LO inhibits actin polymerization and interferes with the binding of CLP to F-actin. It may be hypothesized that 5LO, in addition to its key role in leukotriene synthesis, modulates the actin dynamics in inflammatory cells, thus representing a novel regulator of actin function. We thank Timo Pikkarainen, Alexander Stock, Gerard Marriott, and Patrik Andersson for fruitful discussions and Agneta Nordberg for excellent technical assistance. We are grateful to Philip James for providing the yeast strain PJ69–4A and Jesper Z. Haeggström for providing purified LTA4 hydrolase." @default.
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W2008238416 title "5-Lipoxygenase Interacts with Coactosin-like Protein" @default.
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