Identification of Two Novel Nuclear Import Sequences on the 5-Lipoxygenase Protein

Jones, Scott; Luo, Ming; Peters‐Golden, Marc; Brock, Thomas G.

doi:10.1074/jbc.m211021200

Cited by 33 publications

(22 citation statements)

References 42 publications

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“…Activation of PKA, as following PGE 2 treatment, typically resulted in strong cytoplasmic localization of 5-LO in some cells, with normal import in others. These results are strongly reminiscent of those obtained with mutation of NLS 518 , which left two functional but regulated NLSs intact (16,19). This suggests that 5-LO that is phosphorylated in the cytoplasm may conceivably be imported through the action of other NLSs.…”

Section: Discussionsupporting

confidence: 50%

“…Plasmid containing the NLS 518 peptide of 5-LO (encoding Val 514 -Leu 535 ) in fusion with GFP in pEGFP has also been described previously (16); substitution of Ser 523 with Glu in 5-LO or the NLS 518 peptide was performed using the QuikChange sitedirected mutagenesis kit (Stratagene) following the manufacturer's directions. The double mutant, mutNLS 112 ϩS271A, was produced by site-directed mutagenesis of Ser 271 in the previously described and characterized mutNLS 112 of GFP/5-LO (19), where mutNLS 112 is R115Q/ K117Q/R120Q. Mutations and protein frame reading were verified by DNA sequence analysis (DNA Sequencing Core, University of Michigan).…”

Section: Methodsmentioning

confidence: 99%

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Phosphorylation by Protein Kinase A Inhibits Nuclear Import of 5-Lipoxygenase

Luo

Jones

Flamand

et al. 2005

Journal of Biological Chemistry

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523 is positioned within the nuclear localization sequence-518 of 5-lipoxygenase, the ability of protein kinase A to phosphorylate and alter the localization of green fluorescent protein fused to the nuclear localization sequence-518 peptide was also tested. Site-directed replacement of Ser 523 with glutamic acid within the peptide impaired nuclear accumulation; overexpression of the protein kinase A catalytic subunit ␣ and pharmacological activation of protein kinase caused phosphorylation of the fusion protein at Ser 523 , and the phosphorylated protein was found chiefly in the cytoplasm. Taken together, these results indicate that phosphorylation of Ser 523 inhibits the nuclear import function of a nuclear localization sequence, resulting in the accumulation of 5-lipoxygenase enzyme in the cytoplasm. As cytoplasmic localization can be associated with reduced leukotriene synthetic capacity, phosphorylation of Ser 523 serves to inhibit leukotriene production by both impairing catalytic activity and by placing the enzyme in a site that is unfavorable for action.Leukotrienes (LT 2 (s)) are intercellular mediators that have important roles in pathologic as well as homeostatic inflammation (reviewed in Ref. 1). For instance, the overproduction of LTs contributes to a variety of diseases, including asthma (2), atherosclerosis (3), and fibrosis (4, 5). By contrast, the underproduction of LTs, as occurs in human immunodeficiency virus infection (6, 7) or malnutrition (8), results in compromised antimicrobial defense. The enzyme 5-lipoxygenase (5-LO) catalyzes the first two steps of LT synthesis from arachidonic acid. Therefore, the regulation of 5-LO action has been a focus of interest.Previous studies have shown that the subcellular localization of soluble 5-LO before cell stimulation can affect the amount of LT secreted following cell stimulation. For example, import of 5-LO into the nucleus in neutrophils upon adherence increases LTB 4 secretion upon subsequent stimulation (9). On the other hand, nuclear import of 5-LO following adherence rapidly inhibits LTC 4 synthetic capacity in eosinophils (10). Whereas adherence can change 5-LO localization and LT production rapidly, cytokines alter these parameters more slowly. For example, interleukin-3 has been shown to increase the nuclear localization of 5-LO and increase LTC 4 synthetic capacity, in eosinophils treated for 6 h (11). Also, differentiation of human cord blood-derived mast cells with interleukin-3 or interleukin-5 for 5 days increased both nuclear localization of 5-LO and LTC 4 production upon cell stimulation (12). These results demonstrate that different factors can alter 5-LO localization and that 5-LO redistribution affects LT generation upon subsequent cell activation. However, little is known about the intracellular signaling pathways that alter 5-LO localization and consequent LT production.We recently demonstrated that 5-LO can be phosphorylated by protein kinase A (PKA) on Ser 523 (13). Numerous studies have demonstrated that factors that elevate ...

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Section: Discussionsupporting

confidence: 50%

Section: Methodsmentioning

confidence: 99%

Phosphorylation by Protein Kinase A Inhibits Nuclear Import of 5-Lipoxygenase

Luo

Jones

Flamand

et al. 2005

Journal of Biological Chemistry

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“…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).…”

Section: Regulation Of Lt Synthesismentioning

confidence: 99%

Leukotriene pathway genetics and pharmacogenetics in allergy

Duroudier

Tulah

Sayers

2009

Allergy

102

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Leukotrienes (LT) are a family of potent eicosanoid lipid mediators with central importance in disease processes such as inflammation and proliferation most notably observed in allergic conditions like asthma (1). There are two general classes of LT according to the presence of a cysteine residue in their amino acid chain: the cysteinyl leukotrienes (CysLTs) including LTC 4 , LTD 4 and LTE 4 and the dihydroxyleukotriene LTB 4 (2). The 5-lipoxygenase pathwayLeukotrienes are produced in a multi-step enzyme pathway called the 5-lipoxygenase (5-LO) pathway, which is active in leucocytes such as neutrophils, eosinophils, mast cells and monocytes (Fig. 1). Arachidonic acid is the precursor for LT synthesis and is hydrolysed from the plasma membrane by cytosolic phospholipase A 2 (and other isoforms) (3, 4) in a calcium-dependent process (5). Upon activation, the rate-limiting enzyme 5-LO oxygenates first free arachidonic acid into the unstable intermediate 5-hydroperoxyeicosatetraenoic acid (5-HPETE) which is then either hydrolysed to 5-hydroxyeicosatetraenoic acid (5-HETE) or transformed into the unstable epoxide leukotriene A 4 (LTA 4 ) by forming a conjugated triene system through dehydration (6). 5-LO translocates from either the nucleus (in macrophages) or cyotosol (in neutrophils) to the nuclear envelope in response to cell activation (7,8). This movement occurs as 5-LO is dependent on a 5-LO activating protein (FLAP) for its function. FLAP remains associated with the nuclear membrane (9) and acts as a Ôtransfer proteinÕ presenting arachidonic acid to 5-LO, a system which allows the favourable conversion of 5-HPETE to LTA 4 compared to 5-HETE (10). Both 5-LO and FLAP are expressed in cells of myeloid lineage (11) restricting the pathway to these cell types. LTA 4 can be further metabolised to the cysteinyl leukotrienes (CysLTs) or LTB 4 . The specific glutathione S-transferase leukotriene C 4 synthase (LTC 4 S) conjugates LTA 4 to form LTC 4 which can then be rapidly converted to LTD 4 by a gammaglutamyl transpeptidase and to LTE 4 by a dipeptidase once exported out the cell by membrane transport proteins such as multi-drug related protein 1 (MRP1) (12-15). A specific zinc metallohydrolase, LTA 4 hydrolase (LTA 4 H) is responsible for the conversion of LTA 4 to LTB 4 (16). CysLTs and LTB 4 act on different G-protein coupled receptors (GPCRs). Each bind to at least two different receptors: CysLTs to CYSLTR1 and CYSLTR2 and LTB 4 to LTB 4 R1 and LTB 4 R2 (or BLT1 and BLT2) (17).Leukotrienes (LT) are biologically active lipid mediators known to be involved in allergic inflammation. Leukotrienes have been shown to mediate diverse features of allergic conditions including inflammatory cell chemotaxis/activation and smooth muscle contraction. Cysteinyl leukotrienes (LTC 4 , LTD 4 and, LTE 4 ) and the dihydroxy leukotriene LTB 4 are generated by a series of enzymes/ proteins constituting the LT synthetic pathway or 5-lipoxygenase (5-LO) pathway. Their function is mediated by interacting with multiple receptors. Leukotr...

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“…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).…”

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

Flamand

Luo

Peters‐Golden

et al. 2009

Journal of Biological Chemistry

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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...

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Identification of Two Novel Nuclear Import Sequences on the 5-Lipoxygenase Protein

Cited by 33 publications

References 42 publications

Phosphorylation by Protein Kinase A Inhibits Nuclear Import of 5-Lipoxygenase

Phosphorylation by Protein Kinase A Inhibits Nuclear Import of 5-Lipoxygenase

Leukotriene pathway genetics and pharmacogenetics in allergy

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

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