Structural and Functional Criteria Reveal a New Nuclear Import Sequence on the 5-Lipoxygenase Protein

Abstract

Leukotrienes are lipid mediators with important roles in immunity. The enzyme 5-lipoxygenase initiates leukotriene synthesis; nuclear import of 5-lipoxygenase modulates leukotriene synthetic capacity. In this study, we used structural and functional criteria to identify potential nuclear import sequences. Specifically, we sought basic residues that 1) were common to different 5-lipoxygenases but not shared with other lipoxygenases, 2) were found on random coil/loop structures, and 3) could be replaced without eliminating catalytic activity. Application of these criteria to the putative bipartite nuclear import sequence of 5-lipoxygenase revealed that this region formed an ␣-helix rather than a random coil, that the critical residue arginine 651 serves a structural role, and that mutation of this residue eliminated catalytic activity. A previously unrecognized region corresponding to residues 518 -530 on human 5-lipoxygenase was found to be unique to 5-lipoxygenase and on a random coil. This region alone was sufficient to drive import of green fluorescent protein to the same degree as complete 5-lipoxygenase. Replacement of basic residues in this region of the complete protein was capable of eliminating nuclear import without abolishing catalytic activity. Surprisingly, two subpopulations of cells expressing 5-lipoxygenase with this mutated region could be discerned: those with strongly impaired import and those with normal import. Taken together, these results show that the previously identified region with a bipartite motif is not a functional import sequence, whereas the newly identified basic region constitutes a true nuclear import sequence. Moreover, we suggest that another sequence that can mediate nuclear import of 5-lipoxygenase remains to be identified.Leukotrienes are lipid messengers that play central roles in immune response and tissue homeostasis (reviewed in Ref. 1). However, abnormal production of leukotrienes contributes to a variety of diseases. The enzyme 5-lipoxygenase (5-LO) 1 catalyzes the initial steps in the synthesis of leukotrienes from arachidonic acid. For this reason, leukotriene synthesis is critically dependent on processes that modulate 5-LO activity.One such process is nuclear import of 5-LO. In leukocytes circulating in peripheral blood, 5-LO is found exclusively in the cytoplasm (2-4). However, import of 5-LO into the nucleus can be induced by recruitment of cells into sites of inflammation (2, 5), by adherence to different surfaces (2, 5, 6), or by cytokine treatment (3). Import of 5-LO is linked to activity since nuclear import of 5-LO can strongly enhance (2, 3) or suppress (5) leukotriene synthesis upon subsequent cell stimulation.The regulation of nuclear import of 5-LO is poorly understood. In particular, our understanding of the regions on 5-LO that act as nuclear import sequences (NISs) is incomplete. Previously, Funk and colleagues (7) identified three candidate regions based on the abundance of the basic amino acids Arg or Lys. Partial mutagenesis of these basic region...

Leukotriene pathway genetics and pharmacogenetics in allergy

“…5-LO is concentrated in the cytosol of resting neutrophils, but is located in the nucleus of alveolar macrophages and mast cells (23). This localisation is possible because of three nuclear localisation signals (24,25). The mechanisms involved here are still not fully understood, but there is evidence for cells with nuclear 5-LO to produce more LTB 4 than the same cells with cytoplasmic 5-LO (26).…”

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

“…Quantitation of Subcellular Distribution-As described previously (30), 100 positively transfected NIH 3T3 cells were scored by blinded evaluators as to whether nuclear fluorescence was greater than, equal to, or less than cytosolic fluorescence.…”

“…In its resting state 5-LO is a soluble enzyme that can reside in either the cytoplasm or the nucleus (27)(28)(29). The 5-LO protein has three nuclear import sequences that can be regulated and function independently from one another (30,31). These sequences determine the degree of nuclear localization of 5-LO, as they act additively rather than redundantly (32).…”

Location, Location, Location: Compartmentalization of Early Events in Leukotriene Biosynthesis

“…Compartmentalization of enzyme activities has been proposed to play a significant role in the regulation of LT production. Prior to activation, 5-LOX can reside in the cytoplasm or the nucleus (due to the presence of multiple nuclear location sequences (42,43)); furthermore, its cellular location is dynamic and can vary with cell type. One mechanism of control of 5-LOX location is phosphorylation.…”