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• W2088996645 abstract "The enzyme 5-lipoxygenase initiates the synthesis of leukotrienes from arachidonic acid. Protein kinase A phosphorylates 5-lipoxygenase on Ser523, and this reduces its activity. We report here that phosphorylation of Ser523 also shifts the subcellular distribution of 5-lipoxygenase from the nucleus to the cytoplasm. Phosphorylation and redistribution of 5-lipoxygenase could be produced by overexpression of the protein kinase A catalytic subunit α, by pharmacological activators of protein kinase A, and by prostaglandin E2. Mimicking phosphorylation by replacing Ser523 with glutamic acid caused cytoplasmic localization; replacement of Ser523 with alanine prevented phosphorylation and redistribution in response to protein kinase A activation. Because Ser523 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 Ser523 with glutamic acid within the peptide impaired nuclear accumulation; overexpression of the protein kinase A catalytic subunit α and pharmacological activation of protein kinase caused phosphorylation of the fusion protein at Ser523, and the phosphorylated protein was found chiefly in the cytoplasm. Taken together, these results indicate that phosphorylation of Ser523 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 Ser523 serves to inhibit leukotriene production by both impairing catalytic activity and by placing the enzyme in a site that is unfavorable for action. The enzyme 5-lipoxygenase initiates the synthesis of leukotrienes from arachidonic acid. Protein kinase A phosphorylates 5-lipoxygenase on Ser523, and this reduces its activity. We report here that phosphorylation of Ser523 also shifts the subcellular distribution of 5-lipoxygenase from the nucleus to the cytoplasm. Phosphorylation and redistribution of 5-lipoxygenase could be produced by overexpression of the protein kinase A catalytic subunit α, by pharmacological activators of protein kinase A, and by prostaglandin E2. Mimicking phosphorylation by replacing Ser523 with glutamic acid caused cytoplasmic localization; replacement of Ser523 with alanine prevented phosphorylation and redistribution in response to protein kinase A activation. Because Ser523 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 Ser523 with glutamic acid within the peptide impaired nuclear accumulation; overexpression of the protein kinase A catalytic subunit α and pharmacological activation of protein kinase caused phosphorylation of the fusion protein at Ser523, and the phosphorylated protein was found chiefly in the cytoplasm. Taken together, these results indicate that phosphorylation of Ser523 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 Ser523 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 2The abbreviations used are: LTleukotriene5-LO5-lipoxygenasePKAprotein kinase ANLSnuclear localization sequenceWTwild typeGFPgreen fluorescent proteinp5-LOphosphoSer523 5-LOCαprotein kinase A catalytic subunit α6-bnzN6-benzoyladenosine8-br8-bromoDAPI4′,6-diamidino-2-phenylindoleMAPKmitogen-activated protein kinasePBSphosphate-buffered salinePGprostaglandin.(s)) are intercellular mediators that have important roles in pathologic as well as homeostatic inflammation (reviewed in Ref. 1Funk C.D. Science. 2001; 294: 1871-1875Crossref PubMed Scopus (3070) Google Scholar). For instance, the overproduction of LTs contributes to a variety of diseases, including asthma (2Drazen J.M. Lilly C.M. Sperling R. Rubin P. Israel E. Adv. Prostaglandin Thromboxane Leukotriene Res. 1994; 22: 251-262PubMed Google Scholar), atherosclerosis (3Dwyer J.H. Allayee H. Dwyer K.M. Fan J. Wu H. Mar R. Lusis A.J. Mehrabian M. N. Engl. J. Med. 2004; 350: 29-37Crossref PubMed Scopus (558) Google Scholar), and fibrosis (4Peters-Golden M. Bailie M. Marshall T. Wilke C. Phan S.H. Toews G.B. Moore B.B. Am. J. Respir. Crit. Care Med. 2002; 165: 229-235Crossref PubMed Scopus (175) Google Scholar, 5Wilborn J. Bailie M. Coffey M. Burdick M. Strieter R. Peters-Golden M. J. Clin. Investig. 1996; 97: 1827-1836Crossref PubMed Scopus (175) Google Scholar). By contrast, the underproduction of LTs, as occurs in human immunodeficiency virus infection (6Coffey M. Phare S.M. Kazanjian P.H. Peters-Golden M. J. Immunol. 1996; 157: 393-399PubMed Google Scholar, 7Thorsen S. Busch-Sorensen M. Sondergaard J. AIDS. 1989; 3: 651-653Crossref PubMed Google Scholar) or malnutrition (8Skerrett S.J. Henderson W.R. Martin T.R. J. Immunol. 1990; 144: 1052-1061PubMed Google Scholar), 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. leukotriene 5-lipoxygenase protein kinase A nuclear localization sequence wild type green fluorescent protein phosphoSer523 5-LO protein kinase A catalytic subunit α N6-benzoyladenosine 8-bromo 4′,6-diamidino-2-phenylindole mitogen-activated protein kinase phosphate-buffered saline prostaglandin. 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 LTB4 secretion upon subsequent stimulation (9Brock T.G. McNish R.W. Bailie M.B. Peters-Golden M. J. Biol. Chem. 1997; 272: 8276-8280Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). On the other hand, nuclear import of 5-LO following adherence rapidly inhibits LTC4 synthetic capacity in eosinophils (10Brock T.G. Anderson J.A. Fries F.P. Peters-Golden M. Sporn P.H.S. J. Immunol. 1999; 162: 1669-1676PubMed Google Scholar). 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 LTC4 synthetic capacity, in eosinophils treated for 6 h (11Cowburn A.S. Holgate S.T. Sampson A.P. J. Immunol. 1999; 163: 456-465PubMed Google Scholar). 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 LTC4 production upon cell stimulation (12Hsieh F.H. Lam B.K. Penrose J.F. Austen K.F. Boyce J.A. J. Exp. Med. 2001; 193: 123-133Crossref PubMed Scopus (181) Google Scholar). 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 Ser523 (13Luo M. Jones S.M. Coffey M.J. Peters-Golden M. Brock T.G. J. Biol. Chem. 2004; 279: 41512-41520Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Numerous studies have demonstrated that factors that elevate cellular cAMP levels rapidly inhibit LT synthesis (e.g. Refs. 14Kooman W.J. Orange R.P. Austen K.F. J. Immunol. 1970; 105: 1096-1102PubMed Google Scholar and 15Flamand N. Surette M.E. Picard S. Bourgoin S. Borgeat P. Mol. Pharmacol. 2002; 62: 250-256Crossref PubMed Scopus (110) Google Scholar). We found that phosphorylation of 5-LO, in cells or in vitro, as well as mimicking phosphorylation by substituting Ser523 with Glu, reduced the enzymatic activity of 5-LO. Thus, the direct phosphorylation of 5-LO by PKA may contribute to the reduction in LT synthesis that occurs following elevation of cellular cAMP. Interestingly, Ser523 is embedded in one of the three nuclear localization sequences (NLS) of 5-LO, NLS518 (16Jones S.M. Luo M. Healy A.M. Peters-Golden M. Brock T.G. J. Biol. Chem. 2002; 277: 38550-38556Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Because the function of a classical NLS is to bind with a karyopherin (importin) protein (17Pemberton L.F. Paschal B.M. Traffic. 2005; 6: 187-198Crossref PubMed Scopus (574) Google Scholar) to initiate import, we asked whether phosphorylation at Ser523 would inhibit the nuclear import of 5-LO. We report here, for the first time to our knowledge, that phosphorylation of 5-LO on Ser523 results in an accumulation of 5-LO in the cytoplasm. Plasmids, Mutagenesis, and DNA Construction—Plasmids containing the wild type (WT) 5-LO or the S523A mutant of 5-LO, alone in pcDNA or fused to green fluorescent protein (GFP) in pEGFP, have been described previously (13Luo M. Jones S.M. Coffey M.J. Peters-Golden M. Brock T.G. J. Biol. Chem. 2004; 279: 41512-41520Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 18Healy A.M. Peters-Golden M. Yao J.P. Brock T.G. J. Biol. Chem. 1999; 274: 29812-29818Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Plasmid containing the NLS518 peptide of 5-LO (encoding Val514-Leu535) in fusion with GFP in pEGFP has also been described previously (16Jones S.M. Luo M. Healy A.M. Peters-Golden M. Brock T.G. J. Biol. Chem. 2002; 277: 38550-38556Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar); substitution of Ser523 with Glu in 5-LO or the NLS518 peptide was performed using the QuikChange site-directed mutagenesis kit (Stratagene) following the manufacturer's directions. The double mutant, mutNLS112+S271A, was produced by site-directed mutagenesis of Ser271 in the previously described and characterized mutNLS112 of GFP/5-LO (19Jones S.M. Luo M. Peters-Golden M. Brock T.G. J. Biol. Chem. 2003; 278: 10257-10263Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), where mutNLS112 is R115Q/K117Q/R120Q. Mutations and protein frame reading were verified by DNA sequence analysis (DNA Sequencing Core, University of Michigan). All oligonucleotides were synthesized by Integrated DNA Technologies Inc. (Coralville, IA). Plasmid and oligonucleotide sequences are available upon request. The plasmid encoding the mouse PKA catalytic subunit α (Cα) was a gift of Dr. Michael D. Uhler (Department of Biological Chemistry, University of Michigan). Cell Culture, Transfection, and Immunoblotting—NIH 3T3 cells obtained from ATCC (Manassas, VA) were grown under 5% CO2 in Dulbecco's modified Eagle's medium (Invitrogen) with 10% calf serum and 100 units/ml each of penicillin and streptomycin (complete medium). Cells were plated at 70% confluency and transfected with plasmids using Polyfect transfection reagent (Qiagen) following the manufacturer's instructions. 16-20 h posttransfection, cells were treated with complete medium containing various compounds including 100 μm forskolin (Calbiochem) with and without 200 μm 3-isobutyl-1-methylxanthine (Calbiochem), 1 mm each of N6-benzoyladenosine-(6-bnz-) cAMP, 8-n-hexylaminoadenosine-cAMP, 8-bromo (8-br-)-cAMP, dibutyryl-cAMP (all from Biolog Life Science Institute, Germany), with and without 1-30 μm H89 or 1-30 μm SB203580 (Calbiochem) for 3 h or various time points from 0 to 8 h. Following treatment, cells were washed with PBS, harvested by a rubber policeman, and lysed with 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid (Sigma) with complete protease inhibitor and phosphatase inhibitor cocktails (Sigma). In some cases, cell pellets were directly lysed in 1× SDS loading buffer (50 mm Tris-HCl, pH 6.8, 100 mm dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol) and heated twice in boiling water for 3 min. Protein samples were separated by 10% SDS-PAGE and transferred to nitrocellulose. Membranes were probed with the previously described rabbit polyclonal antibody raised against phosphorylated 5-LO Ser523 (titer: 1:3000) and antibodies against purified human leukocyte 5-LO (a generous gift of Dr. J. Evans, Merck Research Laboratories; titer 1:3000) or GFP (Santa Cruz Biotechnology, Inc.; titer 1:500) followed by peroxidase-conjugated secondary antibody and enhanced chemiluminescence detection (Amersham Biosciences). Densitometry was performed using NIH Image software. Indirect Immunofluorescence and Confocal Microscopy—NIH 3T3 cells grown on two-well chamber slides (BD Falcon™) were transfected with 0.5 μg of plasmid DNA overnight, then washed, and treated as described. For immunostaining, cells were washed with PBS, fixed in 4% paraformaldehyde in PBS for 25 min at room temperature, and permeabilized with 0.3% Triton X-100 in PBS containing 0.1% bovine serum albumin. The cells were then blocked with 1% bovine serum albumin in PBS, 0.3% Triton X-100 and incubated with rabbit phospho-5-LO antibody (p5-LO, titer 1: 200) for 1 h at room temperature. Cells were washed in permeabilization buffer and incubated with rhodamine-conjugated goat anti-rabbit secondary antibody (titer 1:250) in blocking buffer for 1 h. Mounting was done in mounting medium containing 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Inc., Burlingame, CA). Cells were visualized and imaged using a Nikon E600 microscope equipped for epifluorescence and digital image capture using a SPOT RT camera. Confocal microscopy was performed with a Bio-Rad MRC-600 laser confocal microscope. Quantitation of Subcellular Distribution following PKA Activation— As described previously (16Jones S.M. Luo M. Healy A.M. Peters-Golden M. Brock T.G. J. Biol. Chem. 2002; 277: 38550-38556Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), 3T3 cells at 16 h posttransfection were treated with 1 mm 8-br-cAMP for various time points from 0 to 6 h. After fixation with 4% paraformaldehyde, 100 positive cells were scored as to whether nuclear fluorescence was greater than, equal to, or less than cytosolic fluorescence. Care was taken to avoid damaged, dead, or autofluorescent cells. Results from at least three independent transfections were used for statistical analysis. As a second approach, 100 individual cells from each time point after 8-br-cAMP treatment were scored for cytosolic and nuclear fluorescence intensity. Using Adobe Photoshop 6.0, grayscale digital images were adjusted to include the full black-to-white range, and representative gray values, from 0 (white) to 100 (black), were obtained for the cytoplasm and nucleoplasm. Cytoplasmic and nuclear values for each cell were summed to give total cellular fluorescence, and the percent fluorescence values for the nuclear compartment were calculated. Measurement of Intracellular cAMP Production—After (20Aronoff D.M. Canetti C. Peters-Golden M. J. Immunol. 2004; 173: 559-565Crossref PubMed Scopus (285) Google Scholar), 3T3 cells were plated until confluent in 6-well tissue culture dishes in complete medium. The medium was then replaced with serum-free medium, and the cells were exposed to prostaglandin (PG) E2, the EP2-selective agonist butaprost, the EP4-selective agonist ONO-AE1-329 (final concentration of each 1 μm), or vehicle for the times indicated. Culture supernatants were aspirated, and the cells were lysed by incubation for 20 min with 0.1 m HCl (22 °C), followed by disruption using a cell scraper. Intracellular cAMP levels were determined by enzyme-linked immunosorbent assay kit according to the manufacturer (Cayman Chemical, Ann Arbor, MI). PGE2 and butaprost (supplied as the free acid) were from Cayman Chemical; ONO-AE1-329 was a generous gift from ONO Pharmaceutical. Compounds were dissolved in Me2SO4, and stock solutions were stored at -80 °C until used in assays. Required dilutions of all compounds were prepared immediately before use, and equivalent quantities of vehicle were added to the appropriate controls. Statistical Analysis—Statistical significance was evaluated by one-way analysis of variance, using p < 0.05 as indicative of statistical significance. Pairs of group means were analyzed using the Tukey-Kramer posttest. Persistent Phosphorylation on Ser523 of 5-LO—As phosphorylation of proteins can be transient, the effects of phosphorylation may also be transitory. To clearly evaluate the effects of phosphorylation of Ser523 on the localization of 5-LO, we co-transfected NIH 3T3 cells with plasmids encoding GFP/5-LO with or without Cα. In cells expressing only GFP/5-LO, the majority of the fluorescence was in the nucleus, co-localizing with DAPI-stained DNA (Fig. 1, A-C). As expected, these cells were negative for p5-LO. In cells expressing GFP/5-LO with Cα, the fluorescence was outside of the nucleus, in the cytoplasmic compartment (Fig. 1D). Staining for p5-LO matched the fluorescence pattern of GFP/5-LO almost exactly (Fig. 1E). These results demonstrated that the overexpression of the active catalytic subunit of PKA with GFP/5-LO resulted in the accumulation of 5-LO in the cytoplasm, rather than in the nucleus. The phosphorylation of specific residues on proteins can be mimicked by substitution of the residue with an acidic amino acid, such as glutamic acid. The replacement of Ser523 with Glu on GFP/5-LO resulted in a striking redistribution from the nucleus (WT, Fig. 2, A and B) to the cytoplasm (S523E, Fig. 2, C and D). Thus, a single amino acid change, like co-transfection with Cα, produced cytoplasmic localization of GFP/5-LO. Activation of PKA Leads to Phosphorylation and Redistribution of 5-LO—To study the effects of PKA activation on 5-LO localization, we first tested different agonists for their ability to phosphorylate 5-LO. The adenylyl cyclase activator forskolin, with or without the phosphodiesterase IV inhibitor 3-isobutyl-1-methylxanthine, as well as PKA-specific agonist 6-bnz-cAMP and the cAMP analogues 8-br-cAMP and dibutyryl-cAMP, were all able to elicit phosphorylation of 5-LO on Ser523, as indicated by positive immunostaining using the p5-LO antibody (Fig. 3A). However, the B site activator of PKA, 8-n-hexylaminoadenosine-cAMP, appeared to be a poor agonist. The treatment of cells overexpressing 5-LO with Ser523 replaced with Ala did not show phosphorylation in response to the PKA-specific agonist 6-bnz-cAMP (Fig. 3B). Based on these results, additional experiments focused on the effects of 6-bnz-cAMP and 8-br-cAMP on 5-LO phosphorylation and localization. The kinetics of phosphorylation of 5-LO by PKA was evaluated using 6-bnz-cAMP, a potent, selective activator of PKA (21Christensen A.E. Selheim F. de Rooij J. Dremier S. Schwede F. Dao K.K. Martinez A. Maenhaut C. Bos J.L. Genieser H.G. Doskeland S.O. J. Biol. Chem. 2003; 278: 35394-35402Abstract Full Text Full Text PDF PubMed Scopus (362) Google Scholar). Although significant phosphorylation of 5-LO was evident within 1 h after the addition of 6-bnz-cAMP, phosphorylation continued to increase over 6 h (Fig. 4). Modest phosphorylation of Ser523, as indicated by positive staining with the p5-LO antibody, was evident in untreated cultures. Enhanced phosphorylation could be seen as early as 15 min after treatment (data not shown). Also, treatment with the phosphatase inhibitors okadaic acid or calyculin A significantly increased phosphorylation at earlier time points (data not shown). Taken together, these results indicate that PKA activation results in a relatively slow but continuous phosphorylation of 5-LO, countered in part by dephosphorylation by endogenous phosphatase activity, with phosphorylated 5-LO accumulating over time. Because persistent phosphorylation altered 5-LO localization, it was of interest to determine whether a time-dependent redistribution of 5-LO paralleled the time-dependent phosphorylation. Live cell imaging of individual cells indicated that treatment with either 6-bnz-cAMP or 8-br-cAMP resulted in a significant change in the subcellular localization of 5-LO within 1 h in some cells, whereas other cells did not respond at all (data not shown). To quantitate the change in localization within a population of cells, cultures were treated with 1 mm 8-br-cAMP for various times, then fixed, photographed, and scored for subcellular distribution of GFP/5-LO. In untreated cells overexpressing GFP/5-LO, essentially all cells had fluorescent nuclei (Fig. 5A). Following PKA activation, the difference in the fluorescence between the nucleus and cytoplasm diminished, with some cells having nuclei that were darker than the cytoplasm (Fig. 5, B and C). Quantitative analysis of nuclear fluorescence of untreated cells indicated that most cells exhibited moderate (Fig. 5D, N1) or strong (Fig. 5D, N2) nuclear accumulation of WT 5-LO. PKA activation resulted in a decrease in the percentage of cells with strong nuclear fluorescence and an increase in cells with predominantly cytoplasmic fluorescence. Cells overexpressing GFP/5-LO with the S523A mutation showed moderate to strong nuclear fluorescence that was not affected by PKA activation (data not shown). A statistically significant decrease in the percentage of cells with predominantly nuclear fluorescence, paralleled by a significant increase in cytoplasmic predominant cells, was documented at 6 h after 8-br-cAMP treatment (Fig. 6A). Immunofluorescent staining of these cells with the p5-LO antibody revealed strong positive staining within the cytoplasm (Fig. 6B). Thus, PKA activation produced a time-dependent redistribution of 5-LO with accumulation of phosphorylated 5-LO in the cytoplasm.FIGURE 6Quantitative and visual characterization of GFP/5-LO subcellular localization after treatment with 8-br-cAMP for 6 h. A, after treating GFP/5-LO-overexpressing cells with 8-br-cAMP (1 mm), subcellular distribution of fluorescence was scored as nuclear (N>C), balanced between nucleus and cytoplasm (N=C), or cytoplasmic (N<C) in 100 cells. *, p < 0.05 versus untreated. B, fluorescent visualization of total GFP/5-LO (GFP), phosphorylated GFP/5-LO (p5-LO), or DNA (DAPI). Results are from one experiment and are representative of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) From these results, it was not clear whether PKA phosphorylation of 5-LO occurred in the cytoplasm, in the nucleus, or both. Further examination of cells treated with 8-br-cAMP for 6 h and stained for p5-LO revealed that several cells with more nuclear than cytoplasmic GFP/5-LO also stained positive for p5-LO. Confocal analysis of these cells indicated that the majority of the p5-LO was in the cytoplasm (Fig. 7, top and bottom), although some was evident within the nucleus, particularly at the periphery (Fig. 7, middle). To further test whether cytoplasmic 5-LO is phosphorylated more than nuclear 5-LO, we compared the ability of PKA to target WT GFP/5-LO versus a mutant developed to alter 5-LO localization but not activity. This mutant, with substitutions to inactivate NLS112 and block phosphorylation of Ser271, has significantly stronger cytoplasmic localization than the WT protein (Fig. 8A). Activation of PKA with 8-br-cAMP produced greater phosphorylation of the mutant than WT GFP/5-LO, as determined by immunoblot analysis (Fig. 8B). These results indicate that although PKA may be able to phosphorylate 5-LO within the nucleus there appears to be greater targeting of 5-LO within the cytoplasm.FIGURE 8Greater phosphorylation of a mutant of 5-LO with more cytoplasmic protein. A, comparison of the subcellular distribution of WT GFP/5-LO versus GFP/5-LO with inactivation of the NLS112 import sequence plus mutation of the Ser271 site (mutNLS112+S271A). Results are from five independent experiments. B, comparison of the phosphorylation of 5-LO at various time points after activation of PKA using 8-br-cAMP. The ratio of band densities (×100) from samples probed for phosphorylated 5-LO and total 5-LO is presented graphically. Results are from one experiment, representative of two independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Effect of p38 Mitogen-activated Protein Kinase (MAPK) Inhibition on PKA Modulation of 5-LO—5-LO can be phosphorylated on Ser271 in response to stresses that activate p38 MAPK (22Werz O. Klemm J. Samuelsson B. Radmark O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5261-5266Crossref PubMed Scopus (185) Google Scholar). We asked whether phosphorylation of Ser523 was independent of p38 MAPK activity in vivo by treating cells with the selective p38 MAPK inhibitor SB203580. The PKA/PKG inhibitor H-89 demonstrated a dose-dependent inhibition of phosphorylation of 5-LO by 8-br-cAMP, whereas SB203580 was without effect up to 30 μm (Fig. 9A). It has been reported that the IC50 of SB203580 on p38 MAPK is 0.5 μm (23Davies S.P. Reddy H. Caivano M. Cohen P. Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (3956) Google Scholar). In addition, 3T3 cells overexpressing GFP/5-LO retained nuclear localization of 5-LO and did not stain with the p5-LO antibody when treated with 8-br-cAMP (1 mm) plus H-89 (30 μm) (Fig. 9B). The inhibition of p38 MAPK with SB203580 (30 μm) did not block cytoplasmic localization and phosphorylation of Ser523 on 5-LO in response to 8-br-cAMP (Fig. 9B). PKA Can Phosphorylate and Alter the Localization of the NLS518 Peptide—We previously reported that the NLS518 peptide was sufficient to drive nuclear accumulation of GFP (16Jones S.M. Luo M. Healy A.M. Peters-Golden M. Brock T.G. J. Biol. Chem. 2002; 277: 38550-38556Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). We asked whether phosphorylation of GFP-tagged NLS518 on Ser523 would alter nuclear import. As expected, GFP alone was distributed diffusely throughout the cell (Fig. 10A), whereas GFP/NLS518 was strongly accumulated within the nucleus (Fig. 10C). Substitution of Ser523 with Glu, to produce a phosphorylation mimic within the GFP/NLS518 construct, resulted in a relatively diffuse distribution, consistent with little or no active nuclear import (Fig. 10E). Co-transfection of 3T3 cells with GFP/NLS518 and Cα resulted in more cytoplasmic than nuclear GFP fluorescence, with strong positive staining with the p5-LO antibody, particularly in the cytoplasm (Fig. 11, A-C). Finally, activation of PKA with 8-br-cAMP also resulted in strong positive staining with p5-LO, again in the cytoplasm (Fig. 11, D-F). In this treatment, the effect of phosphorylation on localization becomes most apparent, as abundant fluorescent protein is able to be accumulated in the nucleus when NLS518 is not phosphorylated and the majority of phosphorylated NLS518 is found in the cytoplasm. Taken together, these results suggest that the NLS518 peptide itself can be targeted by PKA and that when phosphorylated its ability to drive nuclear import is impaired.FIGURE 11Phosphorylation of the NLS518 peptide by the catalytic subunit of PKA and by PKA activation. A-C, 3T3 cells were co-transfected with plasmids encoding both GFP/NLS518 and Cα and, after 16 h, were fixed and stained for p5-LO. D-F, cells overexpressing GFP/NLS518 were stimulated with 8-br-cAMP for 6 h then were fixed and stained for p5-LO. Images show GFP/NLS518 (GFP), p5-LO and DNA (DAPI). Results are from one experiment, representative of four independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) PGE2 Increases cAMP, Causes Phosphorylation of 5-LO, and Increases Cytoplasmic GFP/5-LO Localization—PGE2 can inhibit leukotriene synthesis via a cAMP-dependent process (24Ham E.A. Soderman D.D. Zanetti M.E. Dougherty H.W. McCauley E. Kuehl Jr., F.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4349-4353Crossref PubMed Scopus (255) Google Scholar). PGE2 acts through four distinct G protein-coupled receptor subtypes (EP1-4), with distinct signaling pathways. Because the Gs-coupled EP2 and EP4 receptors activate adenylate cyclase activity (25Breyer M.D. Breyer R.M. Annu. Rev. Physiol. 2001; 63: 579-605Crossref PubMed Scopus (196) Google Scholar), we measured changes in intracellular cAMP in response to PGE2 (Fig. 12A). Treatment of 3T3 cells with PGE2 (1 μm) provoked an immediate elevation of cAMP, resulting in a 50% increase in cAMP 5 min after PGE2 exposure. Levels remained elevated for ∼60 min. To determine the contribution of either EP2 or EP4 receptors to this increase, cells were treated with equimolar amounts of the EP2-selective agonist butaprost or the EP4-selective agonist ONO-AE1-329 for various times. Both butaprost and ONO-AE1-329 evoked an ∼20% increase in intracellular cAMP within 15 min that returned to baseline levels by 60 min (data not shown). Treatment with the same concentration of PGE2 also resulted in phosphorylation of 5-LO within 15 min, as indicated by immunoblot analysis (Fig. 12B). Phosphorylation of 5-LO in response to PGE2 continued to increase over 6 h, although the level of phosphorylation obtained with PGE2 was less than that induced by 6-bnz-cAMP. The vehicle, Me2SO, did not induce phosphorylation. After 6 h of treatment with PGE2, 5-LO was clearly cytosolic in some cells, as indicated by redistribution of GFP/5-LO (Fig. 12C). Some cells showed little redistribution following PGE2 treatment, with 5-LO remaining largely within the nucleus; other cells had a balanced distribution between nucleus and cytoplasm. Following fixation and staining for p5-LO, cells with cytoplasmic 5-LO based on GFP fluorescence (Fig. 12D) also had positive staining for p5-LO in the cytoplasm (Fig. 12E). Cells that had predominantly nuclear 5-LO following PGE2 treatment did not typically stain positive for p5-LO (not shown). As 5-LO serves the key function of initiating LT synthesis from arachidonic acid, it represents a primary point for regulating the generation of these potent proinflammatory mediators. Several studies have demonstrated that changes in the subcellular localization of 5-LO in resting leukocytes significantly affects the amount of LT produced when those leukocytes are activated. For example, we have recently used molecular modification of the import sequences of 5-LO to show that LTB4 production decreased, as less 5-LO could be imported into the nucleus (26Luo M. Jones S.M. Peters-Golden M. Brock T.G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 12165-12170Crossref PubMed Scopus (117) Google Scholar). In that study and others, the positioning of 5-LO in the cytoplasm of resting leukocytes typically correlated with reduced production of LTs upon cell stimulation. In this study, we have examined the effects of phosphorylation of Ser523 on the localization of 5-LO. This residue is nested in NLS518, identified as 518RGRKSSGFPKSVK530 (16Jones S.M. Luo M. Healy A.M. Peters-Golden M. Brock T.G. J. Biol. Chem. 2002; 277: 38550-38556Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Previous work demonstrated that phosphorylation of Ser523 significantly reduced the intrinsic enzymatic activity of 5-LO (13Luo M. Jones S.M. Coffey M.J. Peters-Golden M. Brock T.G. J. Biol. Chem. 2004; 279: 41512-41520Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). We report here, for the first time to our knowledge, that phosphorylation of Ser523 also blocks nuclear import, resulting in the accumulation of 5-LO in the cytoplasm. Thus, phosphorylation of 5-LO achieves two effects that both serve to reduce cellular LT generation: a direct molecular effect on the intrinsic catalytic activity of 5-LO and a slower, cellular effect of placing 5-LO in a subcellular compartment that is less favorable for arachidonic acid metabolism. A very surprising result is the apparently slow rate of phosphorylation of 5-LO following PKA activation. One contributing factor is the activity of endogenous phosphatases. Elevated cAMP levels increase activity of phosphatases (27Revan S. Montesinos M.C. Naime D. Landau S. Cronstein B.N. J. Biol. Chem. 1996; 271: 17114-17118Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 28Feschenko M.S. Stevenson E. Nairn A.C. Sweadner K.J. J. Pharmacol. Exp. Ther. 2002; 302: 111-118Crossref PubMed Scopus (87) Google Scholar), suggesting that our results might underestimate the true rate of phosphorylation. Consistent with this interpretation, we found that pretreatment with the phosphatase inhibitors okadaic acid or calyculin A resulted in stronger phosphorylation of 5-LO at earlier time points (data not shown). Thus, phosphorylation of 5-LO by PKA may occur in minutes, as observed in response to PGE2 treatment. Dephosphorylation by phosphatases, then, would serve to limit the time of phosphorylation and its impact on LT synthesis. The positioning of Ser523 within NLS518 suggests that this NLS is functional in promoting import when Ser523 is not phosphorylated, with phosphorylation inhibiting import perhaps by interfering with docking of karyopherins with the NLS. Activation of PKA, as following PGE2 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 NLS518, which left two functional but regulated NLSs intact (16Jones S.M. Luo M. Healy A.M. Peters-Golden M. Brock T.G. J. Biol. Chem. 2002; 277: 38550-38556Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 19Jones S.M. Luo M. Peters-Golden M. Brock T.G. J. Biol. Chem. 2003; 278: 10257-10263Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). This suggests that 5-LO that is phosphorylated in the cytoplasm may conceivably be imported through the action of other NLSs. That is, the presence of three NLSs allows several grades of import of 5-LO (29Luo M. Pang C.W.M. Gerken A.E. Brock T.G. Traffic. 2004; 5: 847-854Crossref PubMed Scopus (42) Google Scholar). Silencing of NLS518 leaves two functional sequences, NLS112 and NLS158, which can mediate import of 5-LO. As nuclear export appears also to be regulated (30Hanaka H. Shimizu T. Izumi T. Biochem. J. 2002; 361: 505-514Crossref PubMed Scopus (38) Google Scholar, 31Covin R.B. Brock T.G. Bailie M.B. Peters-Golden M. Am. J. Physiol. 1998; 275: L303-L310Crossref PubMed Google Scholar), other factors besides phosphorylation of NLS518 must be able to influence the final positioning of 5-LO. Activated PKA can phosphorylate nuclear targets, like the cAMP response element binding protein, and so it may phosphorylate 5-LO within the nucleus. This raises the possibility that, in addition to reducing 5-LO activity, phosphorylation may promote the export of 5-LO. We cannot rule out this possibility. However, our results using the GFP/NLS518 fusion peptide, which showed that unphosphorylated protein was found in the nucleus and that phosphorylated protein remained cytoplasmic (Fig. 11), strongly indicated that phosphorylation inhibits the import function of this NLS. PGE2 generally down-regulates the function of leukocytes, and this is mediated by elevation of cAMP through activation of the EP2 and EP4 receptors. For example, PGE2 inhibits macrophage phagocytosis (20Aronoff D.M. Canetti C. Peters-Golden M. J. Immunol. 2004; 173: 559-565Crossref PubMed Scopus (285) Google Scholar) and neutrophil phospholipase D (32Burelout C. Thibault N. Levasseur S. Simard S. Naccache P.H. Bourgoin S.G. Mol. Pharmacol. 2004; 66: 293-301Crossref PubMed Scopus (28) Google Scholar) through EP2, monocyte IL-12 production through EP4 (33Iwasaki K. Noguchi K. Endo H. Kondo H. Ishikawa I. Oral Microbiol. Immunol. 2003; 18: 150-155Crossref PubMed Scopus (31) Google Scholar), and major histocompatibility complex class II expression in dendritic cells (34Harizi H. Grosset C. Gualde N. J. Leukoc. Biol. 2003; 73: 756-763Crossref PubMed Scopus (145) Google Scholar) and the release of tumor necrosis factor-α, endothelin-1, and interleukin-1α by macrophages via both EP2 and EP4 (35Treffkorn L. Scheibe R. Maruyama T. Dieter P. Prostaglandins Other Lipid Mediat. 2004; 74: 113-123Crossref PubMed Scopus (59) Google Scholar). Through these suppressive effects on leukocytes, PGE2 can contribute to the resolution of inflammation. PGE2 has been shown to inhibit LT synthesis through the elevation of cAMP (24Ham E.A. Soderman D.D. Zanetti M.E. Dougherty H.W. McCauley E. Kuehl Jr., F.A. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 4349-4353Crossref PubMed Scopus (255) Google Scholar, 36Wightman P.D. Dallob A. J. Biol. Chem. 1990; 265: 9176-9180Abstract Full Text PDF PubMed Google Scholar). Our finding that PGE2 promotes phosphorylation of 5-LO on Ser523 and causes redistribution of 5-LO to the cytoplasm, both effects that can reduce the synthesis of proinflammatory LTs, provides insight into the mechanism by which this effect is mediated. Previously, we reported that PKA modulators did not alter the subcellular distribution of 5-LO in the first 30 min after treatment (13Luo M. Jones S.M. Coffey M.J. Peters-Golden M. Brock T.G. J. Biol. Chem. 2004; 279: 41512-41520Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). As shown in Fig. 5, the changes in 5-LO localization are slow, with only modest changes observable at 2-h posttreatment. We also found that the effects of PKA modulators were heterogeneous, with phosphorylation as well as redistribution of 5-LO occurring faster in some cells, slower in others, and not at all in some cells (data not shown). This may be because of the fact that a non-synchronized cell culture is heterogeneous. The lag between phosphorylation and redistribution of 5-LO suggests that phosphorylation may be the initial factor affecting 5-LO activity, with redistribution serving to increase or prolong the effects of phosphorylation. It is important to note that elevation of intracellular cAMP also inhibits the release of arachidonic acid (37Flamand N. Boudreault S. Picard S. Austin M. Surette M.E. Plante H. Krump E. Vallee M.J. Gilbert C. Naccache P. Laviolette M. Borgeat P. Am. J. Respir. Crit. Care Med. 2000; 161: 88-94Crossref PubMed Scopus (75) Google Scholar, 38Godfrey R. Manzi R. Gennaro D. Hoffstein S. J. Cell. Physiol. 1987; 131: 384-392Crossref PubMed Scopus (21) Google Scholar, 39Fonteh A.N. Winkler J.D. Torphy T.J. Heravi J. Undem B.J. Chilton F.H. J. Immunol. 1993; 151: 339-350PubMed Google Scholar). This effect is rapid, whereas the effects of PKA activation on 5-LO phosphorylation and subcellular distribution appear to be slower. Whether arachidonic acid release remains impaired after prolonged exposure to cAMP-elevating agents is, to our knowledge, not known. It is possible that at early time points inhibition of LT synthesis by elevated cAMP results primarily from decreased arachidonic acid release. At later time points, 5-LO redistribution to the cytosolic compartment and its sustained phosphorylation on Ser523 will likely lead to a dramatic decrease in the capacity to synthesize LTs even by trans-cellular metabolism (i.e. where arachidonic acid originates from other cell types unaffected by the cAMP-elevating agent). Additional kinetic experiments with leukocytes are necessary to assess this possible sequential cascade of molecular events involved in the down-regulation of LT synthesis by elevated cAMP concentrations. The experiments presented in this study take advantage of the ease-of-transfection of 3T3 cells to address molecular aspects of the regulation of 5-LO. However, an important question is the relevance of these findings to primary leukocytes. Cell transformation can be achieved by reduced phosphatase activity, (40Schuchner S. Wintersberger E. J. Virol. 1999; 73: 9266-9273Crossref PubMed Google Scholar), and it is likely that the transformed 3T3 cells used here have reduced phosphatase activity when compared with primary, differentiated leukocytes. Consistent with this possibility, we found that inhibition of endogenous phosphatases is required to demonstrate phosphorylation of Ser523 on 5-LO in leukocytes (data not shown). Additional studies on the regulation of the phosphorylation of Ser523 on 5-LO in leukocytes, in vitro and in vivo, are in progress. In summary, we report here that elevation of cAMP results in redistribution of 5-LO from the nucleus to the cytoplasm through phosphorylation of Ser523 and inhibition of nuclear import through NLS518. As both phosphorylation on Ser523 and positioning 5-LO in the cytoplasm serve to reduce LT synthesis, these events may be important in the resolution of inflammation." @default.
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• W2088996645 title "Phosphorylation by Protein Kinase A Inhibits Nuclear Import of 5-Lipoxygenase" @default.
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