5-lipoxygenase is phosphorylated by p38 kinase-dependent MAPKAP kinases - PubMed (original) (raw)

5-lipoxygenase is phosphorylated by p38 kinase-dependent MAPKAP kinases

O Werz et al. Proc Natl Acad Sci U S A. 2000.

Abstract

5-lipoxygenase (5-LO) catalyzes the initial steps in the formation of leukotrienes, a group of inflammatory mediators derived from arachidonic acid (AA). Here we describe that activation of p38 mitogen-activated protein kinase in human polymorphonuclear leukocytes and in Mono Mac 6 cells leads to activation of downstream kinases, which can subsequently phosphorylate 5-LO in vitro. Different agents activated the 5-LO kinase activities, including stimuli for cellular leukotriene biosynthesis (A23187, thapsigargin, N-formyl-leucyl-phenylalanine), compounds that up-regulate the capacity for leukotriene biosynthesis (phorbol 12-myristate 13-acetate, tumor necrosis factor alpha, granulocyte/macrophage colony-stimulating factor), and well known p38 stimuli as sodium arsenite and sorbitol. For all stimuli, 5-LO kinase activation was counteracted by SB203580 (3 microM or less), an inhibitor of p38 kinase. At least two p38-dependent 5-LO kinase activities were found. Based on migration properties in in-gel kinase assays and immunoreactivity, one of these was identified as mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP kinase 2). The other appeared to be MAPKAP kinase 3; however, it could not be excluded that also other p38-dependent kinases contributed. When polymorphonuclear leukocytes were incubated with sodium arsenite (strong activator of 5-LO kinases), platelet-activating factor and exogenous AA, there was a 4-fold increase in 5-LO activity as compared with incubations with only platelet-activating factor and AA. This indicates that 5-LO phosphorylation can be one factor determining cellular 5-LO activity.

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Figures

Figure 1

Figure 1

Phosphorylation of 5-LO, in-gel kinase assays. Total cell extracts of differentiated MM6 cells (A) and human PMNL (B) were examined by in-gel kinase assays. MM6 cells (2.5 × 107) and PMNL (5 × 107) in 1 ml PBS containing 1 mg/ml glucose and 1 mM CaCl2 were incubated for 3 min at 37°C in absence or presence of calcium ionophore A23187 (5 μM and 2.5 μM, respectively). Incubations were terminated by addition of the same volume of SDS-b, heated at 95°C for 6 min, and aliquots (corresponding to 0.25 × 106 MM6 cells; 0.5 × 106 PMNL) were electrophoresed on a 10% SDS/polyacrylamide gel, which had been polymerized with or without 0.15 mg/ml purified recombinant 5-LO. In-gel kinase assay was performed as described in Materials and Methods. Positions of standard proteins are indicated. Similar results were obtained in two additional independent experiments.

Figure 2

Figure 2

Effects of kinase inhibitors on phosphorylation of 5-LO. (A) Differentiated MM6 cells (2.5 × 107 in 1 ml) were preincubated for 30 min at 37°C with the kinase inhibitors indicated in the figure. Cells were then stimulated with ionophore (5 μM) for 3 min at 37°C. Incubations were terminated by addition of the same volume of SDS-b, heated at 95°C for 6 min. Aliquots of total cell lysates (corresponding to 0.25 × 106 cells) were electrophoresed and analyzed for 5-LO phosphorylation by in-gel kinase assay. (B) Total cell lysates of 0.5 × 106 human PMNL (lanes 1 and 3) and 0.25 × 106 differentiated MM6 cells (lanes 2 and 4) were examined for p38 kinase and MAPKAPK-2 protein expression by Western blot analysis. Results are representative of at least two separate experiments.

Figure 3

Figure 3

MAPKAPK-2 phosphorylates 5-LO, in-gel kinase assays. Differentiated MM6 cells (2.5 × 107 in 1 ml) were incubated with ionophore (5 μM) for 3 min at 37°C. To prepare total cell lysates, incubations were stopped by addition of the same volume of SDS-b, heated at 95°C for 6 min. Alternatively, incubations were stopped by addition of 2 volumes of ice-cold stop-buffer and supernatants and immunoprecipitates were prepared. (Left) Aliquots of total cell lysates, whole supernatants, supernatants remaining after MK2 immunoprecipitation (corresponding to 0.25 × 106 cells, respectively), and MK2-IPs were electrophoresed and assayed for 5-LO phosphorylation by in-gel kinase assay. (Right) The indicated amounts of active MK2 (rabbit skeletal muscle) were electrophoresed and assayed for 5-LO phosphorylation by in-gel kinase assay.

Figure 4

Figure 4

MAPKAPK-2 phosphorylates 5-LO, in vitro kinase assays. Phosphorylation of 5-LO by MK2 was determined by in vitro kinase assays. Arrows indicate the positions of 5-LO in the gels. (Left) Differentiated MM6 cells (2.5 × 107 in 1 ml) were incubated with or without A23187 (5 μM) for 3 min at 37°C as indicated. Incubations were stopped by addition of 2 volumes of ice-cold stop-buffer, MK2 was immunoprecipitated, and aliquots of the MK2-IPs corresponding to the same cell numbers were incubated with 5-LO (3 μg). (Right) Different amounts of active MK2 (rabbit skeletal muscle) were incubated with 5-LO (3 μg). Results are representative of at least three separate experiments.

Figure 5

Figure 5

Stimulation of 5-LO kinase activities in PMNL. Activation of 5-LO kinases by different stimuli, and inhibition by SB203580. PMNL (5 × 107 in 1 ml) were preincubated pairwise (with or without 3 μM SB203580) for 30 min at 37°C and subsequently were stimulated by addition of 2.5 μM ionophore A23187, 1 μM thapsigargin, 1 μM fMLP, 100 nM phorbol 12-myristate 13-acetate, 10 ng/ml TNF, or 100 μM sodium arsenite. Incubations were terminated after 3 min at 37°C, and aliquots of total cell lysates (corresponding to 0.5 × 106 cells) were analyzed by in-gel kinase assay.

Figure 6

Figure 6

Effects of sodium arsenite (SA) on PAF-induced leukotriene synthesis and 5-LO kinase activity. (Left) Up-regulation of 5-LO kinase activity by SA. PMNL (5 × 107 in 1 ml) were stimulated by addition of PAF (1 μM), AA (10 μM), SA (100 μM), or ionophore A23187 as indicated in the figure. Incubations were terminated after 3 min at 37°C, and aliquots of total cell lysates (corresponding to 0.5 × 106 cells) were analyzed by in-gel kinase assay. (Right) Up-regulation of leukotriene synthesis by SA. PMNL (5 × 106 in 1 ml PBS containing 1 mg/ml glucose and 1 mM CaCl2) were stimulated by addition of PAF (1 μM), AA (10 μM), and SA (100 μM) as indicated in the figure. After 10 min at 37°C, 5-LO activity was determined. Results represent the mean ± SE (n = 3).

Figure 7

Figure 7

MAPKAP kinase 2 phosphorylation motif in 5-lipoxygenases, heat shock protein 27 (Hsp27), and lymphocyte-specific protein 1 (LSP1).

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