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Extracellular signal-regulated kinases phosphorylate 5-lipoxygenase and stimulate 5-lipoxygenase product formation in leukocytes¹

OLIVER WERZ², EVA BÜRKERT, LUTZ FISCHER, DAGMAR SZELLAS, DAVID DISHART^*, BENGT SAMUELSSON^*, OLOF RÅDMARK^* and DIETER STEINHILBER

Institute of Pharmaceutical Chemistry, University of Frankfurt, D-60439 Frankfurt, Germany; and
^* Department of Medical Biochemistry and Biophysics, Division of Physiological Chemistry II Karolinska Institutet, S-171 77 Stockholm, Sweden

²Correspondence: Institute of Pharmaceutical Chemistry, University of Frankfurt, Marie-Curie Strasse 9, D-60439 Frankfurt, Germany. E-mail: o.werz{at}pharmchem.uni-frankfurt.de

SPECIFIC AIMS

>5-Lipoxygenase (LO) is the key enzyme in the biosynthesis of the proinflammatory leukotrienes (LTs). The aim of this study was to determine whether extracellular signal-regulated kinases (ERKs) are able to phosphorylate 5-LO and if ERK-mediated phosphorylation could stimulate cellular 5-LO product synthesis.

PRINCIPAL FINDINGS

1. 5-LO is phosphorylated by ERK2, which is promoted by unsaturated fatty acids
Purified 5-LO was dose-dependently phosphorylated by active ERK2 in vitro (Fig. 1 A). Compared with the excellent ERK substrate myelin basic protein (MBP), 5-LO was 20- to 30-fold less efficiently phosphorylated by ERK2. However, AA or the unsaturated fatty acids (UFAs) oleic acid and linoleic acid (10–50 µM) increased 5-LO phosphorylation by ERK2 in a dose-dependent fashion up to 25-fold (Fig. 1B ), whereas the saturated fatty acids arachidic acid and palmitic acid failed to enhance 5-LO phosphorylation rates (Fig. 1C ). Opposite effects of AA were observed when MBP was used as substrate. The putative phosphorylation site Ser-663 within the ERK motif in 5-LO (YLSP at residues 661–664) was mutated to alanine (S663A-5-LO) and subjected to phosphorylation by ERK2. In contrast to wt 5-LO, AA led to an only marginal increase in the phosphorylation rate for the S663A mutant (Fig. 1D ), indicating that enhanced phosphate incorporation requires the Ser-663 residue in 5-LO.

View larger version (51K): [in this window] [in a new window]   Figure 1. ERK2 phosphorylates 5-LO in vitro: effects of UFAs. A) Phosphorylation of 5-LO (left panel) and MBP (right panel). Phosphorylation of the proteins (40 pmol each) was determined by in vitro kinase assay. Arrows indicate the positions of the corresponding proteins. B) AA promotes 5-LO phosphorylation by ERK2. C) Effects of fatty acids on 5-LO phosphorylation by ERK2. D) Ser-663 is required for AA-induced increase in 5-LO phosphorylation by ERK2. The proteins (40 pmol each) were incubated with 10 mU active ERK2 in the absence or presence of the fatty acids and phosphorylation was determined by in vitro kinase assays. Equal amount of proteins was determined by Coomassie staining. Results are representative of three separate experiments, respectively.

2. ERK activation correlates to 5-LO activation in MM6 cells
Stimulation of MM6 cells with ionophore alone failed to activate ERK1/2, but priming with PMA resulted in a clear kinase activation. Inhibition of protein kinase C (PKC) by GF109203x (IC₅₀0.1 µM) or MEK1/2 by U0126 (IC₅₀0.3 µM) or PD98059 (IC₅₀3 µM) blocked ERK1/2 activation by PMA. In immunocomplex kinase assays, purified 5-LO was efficiently phosphorylated by endogenous ERKs, immunoprecipitated from PMA-primed (but not from unprimed) MM6 cells stimulated with ionophore, and pretreatment of cells with U0126 (3 µM) prevented the PMA effect.

Priming with PMA enhanced the ionophore-induced [³H]AA-release up to sixfold, and inhibition of PKC by GF109203x (0.3 µM) or ERK1/2 activation by U0126 (1 µM) or PD98059 (10 µM) suppressed the liberation of AA. Priming with PMA also increased ionophore-stimulated 5-LO product formation up to sevenfold; in the presence of exogenous AA, a twofold increase was observed. These up-regulative effects of PMA were efficiently attenuated by GF109203x (IC₅₀0.2 µM) as well as by U0126 (IC₅₀0.3 µM) or PD98059 (IC₅₀5 µM), whereas ERK-independent 5-LO product formation (induced by ionophore plus AA) was not inhibited.

3. Involvement of ERKs in 5-LO product formation in polymorphonuclear leukocytes (PMNL)
AA activated ERK1/2 and induced 5-LO product formation in PMNL. U0126 (0.3–3 µM) partially reduced (63±5.9%) AA-induced 5-LO product formation. Similarly, 5-LO product synthesis induced by 1 µM fMLP (which activates ERKs) was suppressed by U0126, particularly in the absence of exogenous AA. In contrast, 5-LO product formation induced by AA plus ionophore or AA plus NaCl (which activate 5-LO independent of ERK1/2, respectively) was hardly affected by U0126.

4. Role of putative phosphorylation sites in 5-LO for product formation
HeLa cells transformed with plasmids encoding wt-5-LO or S663A-5-LO gave similar prominent product formation on stimulation with ionophore plus AA (10–60 µM), whereas 5-LO product formation for the S663A mutant was significantly lower compared with wt-5-LO, when cells were treated with AA alone.

5. ERKs act in conjunction with MK2 in 5-LO activation
AA-induced 5-LO product formation in PMNL was also partially suppressed (by 40±11.3%) by the p38 MAPK inhibitor SB203580 (10 µM). Intriguingly, a combination of U0126 (3 µM) with SB203580 (10 µM) caused 5-LO suppression by 83 ± 3.8%, suggesting that ERKs and p38 MAPK pathways are both involved in AA-induced 5-LO activation (Fig. 2 A). To confirm that phosphorylation by MK2 (at Ser-271) and by ERKs (at Ser-663) are both important, 5-LO product formation was determined in HeLa cells expressing wt-5-LO, S663A-5-LO, and S271A-S663A-5-LO. For wt-5-LO, the ratio of AA to AA plus ionophore-induced 5-LO product synthesis at 10 to 60 µM AA was 24 to 43%, for S663A-5-LO 11 to 22%, and for the S271A-S663A-5-LO 5 to 14% (Fig. 2B ). Thus, phosphorylation at both Ser-663 and Ser-271 may be required for 5-LO product formation at low intracellular Ca²⁺ levels.

View larger version (26K): [in this window] [in a new window]   Figure 2. 5-LO activation involving phosphorylation pathways A) Effects of U0126 and SB203580 on 5-LO product formation in PMNL. Human PMNL (freshly isolated from leukocyte concentrates obtained from healthy donors) were preincubated with U0126 (3 µM) and SB203580 (10 µM) for 30 min and stimulated with 60 µM AA or 2.5 µM ionophore plus 10 µM AA for another 10 min. 5-LO product formation was determined by HPLC. The 100% values in the absence of inhibitors were 57.1 ± 7.7 and 100.7 ± 2.2 ng/106 cells in the absence or presence of ionophore, respectively. B) Ratio of 5-LO product formation in transformed HeLa cells stimulated with AA and with AA plus ionophore. HeLa cells transiently transformed with plasmid DNA encoding wt-5-LO, S663A-5-LO, or S271A-S663A-5-LO were stimulated for 10 min with the indicated concentrations of AA in the absence or presence of 10 µM ionophore, and 5-LO product formation was determined. Results (mean+SE, n=3) are presented as the quotient of AA-induced to AA plus ionophore-induced 5-LO product formation. The expression of 5-LO proteins was analyzed by Western blot (insert).

CONCLUSIONS

Based on several inhibitor studies, the ERK1/2 pathway has been implicated in the activation of 5-LO, but the precise molecular mechanisms involved have not been elucidated. Although 5-LO phosphorylation by ERKs could not be detected by others, we found that recombinant active ERK2 and ERK1/2-IPs from stimulated MM6 cells phosphorylate 5-LO in vitro. These opposite findings could be related to different assay conditions, e.g., purity, integrity, amount of 5-LO, etc. Thus, compared with MBP, 5-LO is a rather poor substrate for ERK2. However, the presence of UFAs such as AA enhanced 5-LO phosphorylation rates of ERKs up to 25-fold. In another study we found that 5-LO phosphorylation by MK2 was also up-regulated by UFAs, whereas the activity of the 5-LO kinases PKA or CaMKII was not increased. Thus, the ability of UFAs to promote protein phosphorylation seems to be specific for 5-LO as substrate and is restricted to particular kinases. Presumably UFAs lead to exposure of the serine residues or favor substrate recognition and access by the kinases.

Phosphorylation of a very small amount of 5-LO in vivo was demonstrated by others. We failed to convincingly assess in vivo phosphorylation after 5-LO immunoprecipitation from various cell types labeled with ³²P_i. This might be due to inefficient ³²P-labeling of 5-LO (due to experimental settings), but it is also possible that only a small fraction of 5-LO is phosphorylated in the cell. This small activated pool of 5-LO may be sufficient to activate the bulk of enzyme via 5-LO-derived hydroperoxides that convert the active site iron from the ferrous to the ferric state, which is important for initializing the 5-LO catalytic redox cycle. Others have implied that upon cell stimulation by AA, only a small amount of 5-LO is initially activated by Ca²⁺-independent mechanisms before activation of the bulk of 5-LO. The stimulus-dependent difference in 5-LO product synthesis of the S663A 5-LO mutant compared with wt-5-LO suggests that 5-LO phosphorylation by ERKs indeed plays a role for 5-LO activation in intact cells.

It was proposed that 5-LO in intact PMNL can be activated by at least two different pathways: either by elevation of intracellular Ca²⁺ (using ionophore as stimulus) or by a cell stress-induced, p38 MAPK-regulated pathway that is Ca²⁺ independent. For cPLA₂, similar mechanisms of enzyme activation have been suggested. Thus, stimuli that lead to cPLA₂ phosphorylation caused AA release at basal Ca²⁺ levels whereas ionophore induced AA release when phosphorylation of cPLA₂ was blocked. Our studies confirm the hypothesis of Ca²⁺- and/or phosphorylation-mediated 5-LO activation. Thus, 5-LO product formation in HeLa cells expressing wt- or S663A-5-LO was similar when cells had been stimulated by ionophore, where Ca²⁺ is the predominant 5-LO activator and phosphorylation might be of minor importance. However, the S663A-5-LO mutant produced significantly lower amounts of 5-LO products when cells had been stimulated with only AA (causing pronounced ERK activation but only moderate Ca²⁺ fluxes).

Our inhibitor studies using PMNL or MM6 cells clearly indicate an involvement of ERKs in the 5-LO activation. Stimuli that activate ERKs induced (AA for PMNL) or up-regulated (PMA for MM6 cells) 5-LO product synthesis, which was highly sensitive to specific ERK activation inhibitors at quite similar concentrations required to prevent ERK activation. When 5-LO was activated by ionophore or by ERK-independent phosphorylation pathways (via p38 MAPK), the inhibitors failed to suppress 5-LO product synthesis, again indicating that phosphorylation is crucial for 5-LO activation at low intracellular Ca²⁺.

Finally, our data suggest that enzyme phosphorylation by ERK2 and MK2 at multiple sites can act in conjunction to activate 5-LO. In fact, for HeLa cells the ratio of AA to AA plus ionophore-induced 5-LO product formation of S271A-S663A-5-LO, lacking phosphorylation sites for MK2 and ERKs, was significantly lower than 5-LO mutants lacking only one phosphorylation site (S271A-5-LO or S663A-5-LO). Moreover, U0126 or SB203580 by themselves each suppressed AA-induced 5-LO product formation in PMNL only partially, whereas the combination of both inhibitors caused almost complete 5-LO inhibition.

Taken together, ERKs, particularly in the presence of UFAs, can be considered as potential 5-LO kinases, which in conjunction with p38 MAPK-regulated MKs can stimulate cellular 5-LO for product formation. These findings might provide the molecular basis for 5-LO activation in leukocytes in response to particular agonists and priming agents and may provide new concepts for the pharmacological intervention with leukotriene biosynthesis during inflammatory diseases.

View larger version (29K): [in this window] [in a new window]   Figure 3. Schematic illustration of cellular 5-LO activation and AA metabolism. Extracellular stimuli lead to elevation of intracellular Ca2+ and activate MAPK pathways (1). These events stimulate translocation and activation of cPLA2, which in turn releases AA from phospholipids at the nuclear membrane (2). Free AA may stimulate 5-LO phosphorylation by ERKs and MK2 (3). Upon activation, 5-LO translocates to the nuclear membrane and colocalizes with 5-LO-activating protein (FLAP) (4). Free AA is transferred via FLAP to 5-LO for conversion to LTs (5). Depending on the stimulus and the cell type, phosphorylation and/or Ca2+ may be required for activation of 5-LO (see text).

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0909fje; to cite this article, use FASEB J. (July 1, 2002) 10.1096/fj.01-0909fje

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