Nuclear localization of 5-lipoxygenase as a determinant of leukotriene B₄synthetic capacity

Abstract

The enzyme 5-lipoxygenase (5-LO) initiates the synthesis of leukotrienes from arachidonic acid. In resting cells, 5-LO can accumulate in either the cytoplasm or the nucleoplasm and, upon cell stimulation, translocates to membranes to initiate leukotriene synthesis. Here, we used mutants of 5-LO with altered subcellular localization to assess the role that nuclear positioning plays in determining leukotriene B4 (LTB4) synthesis. Mutation of either a nuclear localization sequence or a phosphorylation site reduced LTB4 synthesis by 60%, in parallel with reduced nuclear localization of 5-LO. Mutation of both sites together or mutation of all three nuclear localization sequences on 5-LO inhibited LTB4 synthesis by 90% and abolished nuclear localization. Reduced LTB4 generation in mutants could not be attributed to differences in 5-LO amount, enzymatic activity, or membrane association. Instead, 5-LO within the nucleus acts at a different site, the nuclear envelope, than does cytosolic 5-LO, which acts at cytoplasmic and perinuclear membranes. The significance of this difference was suggested by evidence that exogenously derived arachidonic acid colocalized with activated nuclear 5-LO. These results unequivocally demonstrate that the positioning of 5-LO within the nucleus of resting cells is a powerful determinant of the capacity to generate LTB4 upon subsequent activation. L eukotriene B 4 (LTB 4 ) is a lipid mediator that is important for host defense, as it stimulates leukocyte chemotaxis, degranulation, phagocytosis, and superoxide generation (reviewed in ref. 1). The underproduction of leukotrienes (LTs) results in susceptibility to infection (2, 3), whereas the overproduction of LTB 4 contributes to inflammatory disease (4, 5). The enzyme 5-lipoxygenase (5-LO) initiates the synthesis of LTB 4 from arachidonic acid (AA). Thus, the action of 5-LO is a key point of regulation in LTB 4 synthesis.In resting leukocytes, 5-LO is a soluble enzyme. Elevated intracellular calcium, as occurs upon cell stimulation, causes translocation of 5-LO to nuclear and perinuclear membranes (6, 7). Membrane association places the enzyme close to its substrate and initiates LT synthesis. LT synthesis can also be stimulated by activation of p38 mitogen-activated protein kinase (MAPK) (8), which is thought to be due to phosphorylation of 5-LO on Ser-271 by MAPK-activated protein (MAPKAP) kinase 2 (9) and phosphorylation-dependent nuclear association of 5-LO (10).Entirely distinct from its ability to move to membranes upon cell stimulation, 5-LO can redistribute in unstimulated cells; 5-LO can be imported into the nucleus (11, 12), and it can be exported from the nucleus (13). The import of soluble 5-LO protein, through nuclear pores on the nuclear envelope, is mediated by three distinct nuclear localization signals (NLSs) on 5-LO (14, 15). The import process can be triggered by adherence (11,12) or by cytokines (16,17). Movement of 5-LO from the cytoplasm into the interior of the nucleus also occurs during leukocyte recruitment into site...

Resolvin D1 limits 5-lipoxygenase nuclear localization and leukotriene B 4 synthesis by inhibiting a calcium-activated kinase pathway

“…Phosphorylation of 5-LOX at Ser271 by the P38-activated kinase mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2) (MK2) leads to nuclear localization of 5-LOX, which allows 5-LOX to align with other enzymes required for LTB 4 synthesis (17,(23)(24)(25). We found that RvD1 blocked AA-stimulated phosphorylation of both kinases, which is a measure of their activation state ( Fig.…”

Phosphorylation of Serine 271 on 5-Lipoxygenase and Its Role in Nuclear Export

“…Plasmids, Mutagenesis, and DNA Construction-Plasmids containing the wild type (WT) 5-LO and the S523A and the S271A mutants of 5-LO alone in pcDNA or fused to green fluorescent protein (GFP) in pEGFP have been described previously (34,36,46). Substitutions of Leu-270 and Leu-272 with Ala in 5-LO were performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) following the manufacturer's instructions.…”

“…4B). Importantly, NIH 3T3 cells overexpressing S271A 5-LO produce significantly less than half as much LTB 4 when stimulated with calcium ionophore (10 M) with added arachidonic acid (10 M) versus cells with WT 5-LO (1912 ϩ 232 versus 4570 ϩ 807 pg/ml; p Ͻ 0.01) (34). Treatment of NIH 3T3 cells expressing S271A 5-LO with the nuclear export inhibitor lmb (5 ng/ml, 2 h) resulted in the redistribution of 5-LO to the nucleus (Fig.…”

“…These sequences determine the degree of nuclear localization of 5-LO, as they act additively rather than redundantly (32). This is important because the amount of LTB 4 produced upon 5-LO activation increases with the amount of 5-LO present within the nucleus (33,34). Furthermore, phosphorylation of Ser-523 on 5-LO via the cAMP/ protein kinase A pathway serves to inhibit nuclear import (35) and inhibit LTB 4 biosynthesis (36).…”

Inhibition of 5-Lipoxygenase Product Synthesis by Natural Compounds of Plant Origin

“…It is clear that not only the supply of AA but also the access towards 5-LO is a prerequisite for LT biosynthesis [44], [45], and one important class of synthetic inhibitors of LT biosynthesis are so-called FLAP inhibitors (e. g., MK-886, Bay-X-1005). Among plant-derived compounds, the phenylanthraquinone knipholone 54 ( Table 3), from the roots of Kniphofia foliosa, was suggested to suppress cellular 5-LO product synthesis by inhibition of FLAP [46].…”