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

Abstract

The enzyme 5-lipoxygenase (5-LO) initiates the biosynthesis of leukotrienes, inflammatory mediators involved in immune diseases and defense. The subcellular localization of 5-LO is regulated, with nuclear import commonly leading to increased leukotriene production. We report here that 5-LO is constitutively phosphorylated on Ser-271 in transfected NIH 3T3 cells. This residue is nested in a classical nuclear export sequence, and phosphorylated Ser-271 5-LO was exclusively found in the nucleus by immunofluorescence and by fractionation techniques. Mutation of Ser-271 to Ala allowed nuclear export of 5-LO that was blocked by the specific nuclear export inhibitor leptomycin b, suggesting that phosphorylation of Ser-271 serves to interfere with exportin-1-mediated nuclear export. Consistent with previous reports that purified 5-LO can be phosphorylated on Ser-271 in vitro by MAPK-activated protein kinase 2, the nuclear export of 5-LO was increased by either treatment with the p38 inhibitor SB 203,580 or co-expression of a kinasedeficient p38 MAPK. Nuclear export of 5-LO can also be induced by KN-93, an inhibitor of Ca 2؉ /calmodulin-dependent kinase II, and the effects of SB 203,580 plus KN-93 are additive. Finally, HeLa cells, which lack nuclear 5-LO, also lack constitutive phosphorylation of Ser-271. Taken together, these results indicate that the phosphorylation of Ser-271 serves to inhibit the nuclear export of 5-LO. This action works in concert with nuclear import, which is regulated by phosphorylation on Ser-523, to determine the subcellular distribution of 5-LO, which in turn regulates leukotriene biosynthesis. Leukotrienes (LTs)2 are intercellular messengers that play critical roles in inflammatory and allergic diseases (for review, see Ref. 1). In particular, LTB 4 promotes the recruitment and activation of leukocytes, stimulates phagocytosis and killing of pathogens, and promotes the synthesis of proteins involved in the inflammatory process. Through these effects and others, the overproduction of LTB 4 helps drive the chronic inflammation that characterizes asthma (2-4), bronchitis (5-7), and rhinitis (8). On the other hand, the underproduction of LTB 4 represents a form of immune suppression, resulting in impaired host defense against infectious pathogens. Thus, LTB 4 promotes immune defense against pathogenic bacteria (9 -11), fungi (12), viruses (13), and parasites (14 -16). Significantly, reduced LT biosynthesis correlates with increased susceptibility to infectious disease in numerous conditions, including cigarette smoking (17, 18), malnutrition (19 -22), vitamin D deficiency (23), human immunodeficiency virus infection (24, 25), and type II diabetes (26).The enzyme 5-lipoxygenase (5-LO) initiates the biosynthesis of LTs from the polyunsaturated fatty acid arachidonic acid and represents a key point of regulation. In its resting state 5-LO is a soluble enzyme that can reside in either the cytoplasm or the nucleus (27-29). The 5-LO protein has three nuclear import sequences that can be regulated and f...

Analysis of 5‐lipoxygenase phosphorylation on molecular level by MALDI‐MS

“…Detection limits of the corresponding phosphopeptides have been determined and tagged 5-LO isolated from MM6 cells was shown to be phosphorylated on Ser271, regardless of the cell treatment. This is in accordance with findings from another group showing that 5-LO is constitutively phosphorylated at Ser271 in transfected NIH 3T3 cells [31].…”

Anti-proliferative and pro-apoptotic effects of lichen-derived compound protolichesterinic acid are not mediated by its lipoxygenase-inhibitory activity

“…In this study we confirmed previously obtained results by us and others that indicate changes in expression and sub-cellular location of 5-and 12-LOX enzymes in relation to the progression of the cell cycle [36]. The pure compound PA, isolated from the traditional medicinal herb Iceland moss, inhibits 5-and 12-LOX [23,27] and has anti-proliferative effects on various cancer cell lines [26].…”

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

“…One mechanism of control of 5-LOX location is phosphorylation. For instance, phosphorylation of Ser-271 interferes with the export of 5-LOX from the nucleus (44). Thus, cell stimulation results in the Ca 2ϩ -dependent targeting of 5-LOX to the inner and/or outer nuclear membrane(s), depending upon the compartment(s) in which the unstimulated enzyme resides.…”