The intracellular localisation and phosphorylation profile of the human 5-lipoxygenase A13 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 Δ13 isoform (5-LOΔ13) whose transcript lacks exon 13.5-LOΔ13 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-LOΔ13 interferes with 5-LO product biosynthesis in transfected HEK293 cells. When co-expressed with 5-LO1, 5-LOΔ13 inhibited LT but not 5-hydroxyeicosatetraenoic acid (5-HETE) biosynthesis. This inhibition was independent of 5-LOΔ13 - 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-LOΔ13 inhibited 5-LO product biosynthesis as effectively as 5-LOΔ13 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-LOΔ13 was cytosolic and concentrated in ER-rich perinuclear regions where its effect on LT biosynthesis may occur. W102A mutants of 5-LOΔ13 showed the same pattern. Consistent with subcellular distribution patterns, 5-LOΔ13 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-LOΔ13 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.