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

Nicolas Flamand,Ming Luo,Marc Peters-Golden,Thomas G Brock

doi:10.1074/jbc.m805593200

Nicolas Flamand, Ming Luo + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m805593200

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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 kinase-deficient p38 MAPK. Nuclear export of 5-LO can also be induced by KN-93, an inhibitor of Ca2+/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.

Highlights

In vitro experiments have shown that recombinant 5-LO can be phosphorylated on Ser-271, by MAPK-activated protein kinase-2 (MK-2) and possibly by MK-3 [43], by Ca2ϩ/calmodulin-dependent kinase II (CaMKII) and by protein kinase A [44]
Ser-271 Is Phosphorylated on 5-LO in Untreated NIH 3T3 Cells—The finding that 5-LO could be phosphorylated on Ser271 by MAPK-activated protein kinase-2 in vitro [43] was the first unequivocal report of direct phosphorylation of the 5-LO protein
In the same study we found that mutation phosphorylated on Ser-271 when combined in vitro with spe- of Ser-271 to Ala, preventing phosphorylation at that site, cific kinases, including MAPK-activated protein kinase-2, led to both reduced nuclear 5-LO and a 60% reduction in LTB4 CaMKII, and protein kinase A

Summary

Introduction

Cellular proteins from NIH 3T3 cells overexpressing WT 5-LO or GFP/5-LO, when assayed for 5-LO with phosphorylated Ser271 (p271 5-LO) by immunoblot, produced bands at ϳ78 kDa for WT 5-LO and ϳ105 kDa for GFP/5-LO, and these corresponded to bands detected with an antibody for total 5-LO (Fig. 1). These results indicate that at least a portion of the total cellular 5-LO in transfected, but otherwise untreated, NIH 3T3 cells is phosphorylated on Ser-271.

Results

Conclusion