title: The Intracellular Localisation and Phosphorylation Profile of the Human 5-Lipoxygenase Δ13 Isoform Differs from That of Its Full Length Counterpart.
abstract: 5-Lipoxygenase (5-LO) catalyzes leukotriene (LT) biosynthesis by a mechanism that involves interactions with 5-lipoxygenase activating protein (FLAP) and coactosin-like protein (CLP). 5-LO splice variants were recently identified in human myeloid and lymphoid cells, including the catalytically inactive ∆13 isoform (5-LO∆13) whose transcript lacks exon 13. 5-LO∆13 inhibits 5-LO product biosynthesis when co-expressed with active full length 5-LO (5-LO1). The objective of this study was to investigate potential mechanisms by which 5-LO∆13 interferes with 5-LO product biosynthesis in transfected HEK293 cells. When co-expressed with 5-LO1, 5-LO∆13 inhibited LT but not 5-hydroxyeicosatetraenoic acid (5-HETE) biosynthesis. This inhibition was independent of 5-LO∆13-FLAP interactions since it occurred in cells expressing FLAP or not. In cell-free assays CLP enhances 5-LO activity through interactions with tryptophan-102 of 5-LO. In the current study, the requirement for W102 was extended to whole cells, as cells expressing the 5-LO1-W102A mutant produced little 5-LO products. W102A mutants of 5-LO∆13 inhibited 5-LO product biosynthesis as effectively as 5-LO∆13 suggesting that inhibition is independent of interactions with CLP. Confocal microscopy showed that 5-LO1 was primarily in the nucleoplasm whereas W102A mutants showed a diffuse cellular expression. Despite the retention of known nuclear localisation sequences, 5-LO∆13 was cytosolic and concentrated in ER-rich perinuclear regions where its effect on LT biosynthesis may occur. W102A mutants of 5-LO∆13 showed the same pattern. Consistent with subcellular distribution patterns, 5-LO∆13 was hyper-phosphorylated on S523 and S273 compared to 5-LO1. Together, these results reveal a role for W102 in nuclear targeting of 5-LO1 suggesting that interactions with CLP are required for nuclear localization of 5-LO1, and are an initial characterisation of the 5-LO∆13 isoform whose inhibition of LT biosynthesis appears independent of interactions with CLP and FLAP. Better knowledge of the regulation and properties of alternative 5-LO isoforms will contribute to understanding the complex regulation of LT biosynthesis.
authors.affiliation: Département de Chimie et Biochimie, Université de Moncton, Moncton, Canada.
authors.forename: Eric P
authors.initials: EP
authors.lastname: Allain
authors.affiliation: Département de Chimie et Biochimie, Université de Moncton, Moncton, Canada.
authors.forename: Luc H
authors.initials: LH
authors.lastname: Boudreau
authors.affiliation: Centre de recherche de l'Institut universitaire de cardiologie et de pneumologie de Québec, Département de médecine et Faculté de médecine, Université Laval, Québec, Canada.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Flamand
authors.affiliation: Département de Chimie et Biochimie, Université de Moncton, Moncton, Canada.
authors.forename: Marc E
authors.initials: ME
authors.lastname: Surette
mesh_terms: D058927:5-Lipoxygenase-Activating Proteins / Q000378:metabolism; D017398:Alternative Splicing; D019943:Amino Acid Substitution; D001094:Arachidonate 5-Lipoxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000096:biosynthesis; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015289:Leukotrienes / Q000096:biosynthesis; D008840:Microfilament Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D058927:5-Lipoxygenase-Activating Proteins; C548592:ALOX5AP protein, human; C118115:COTL1 protein, human; D006893:Hydroxyeicosatetraenoic Acids; D007527:Isoenzymes; D015289:Leukotrienes; D008840:Microfilament Proteins; D011994:Recombinant Proteins; C022022:5-hydroxy-6,8,11,14-eicosatetraenoic acid; D001094:Arachidonate 5-Lipoxygenase