scholarly journals Cross-talk between IRAK-4 and the NADPH oxidase1

2007 ◽  
Vol 403 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Sandrine Pacquelet ◽  
Jennifer L. Johnson ◽  
Beverly A. Ellis ◽  
Agnieszka A. Brzezinska ◽  
William S. Lane ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
P38 Mapk ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Free System ◽  
Mitogen Activated Protein ◽  
A Cell ◽  
Tlr4 Activation

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

Phosphorylation of Argonaute 2 at serine-387 facilitates its localization to processing bodies

2008 ◽  
Vol 413 (3) ◽  
pp. 429-436 ◽  
Author(s):  
Yan Zeng ◽  
Heidi Sankala ◽  
Xiaoxiao Zhang ◽  
Paul R. Graves
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Kinase Inhibitor ◽  
Mapk Pathway ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Processing Bodies ◽  
Mitogen Activated Protein

Ago (Argonaute) proteins are essential effectors of RNA-mediated gene silencing. To explore potential regulatory mechanisms for Ago proteins, we examined the phosphorylation of human Ago2. We identified serine-387 as the major Ago2 phosphorylation site in vivo. Phosphorylation of Ago2 at serine-387 was significantly induced by treatment with sodium arsenite or anisomycin, and arsenite-induced phosphorylation was inhibited by a p38 MAPK (mitogen-activated protein kinase) inhibitor, but not by inhibitors of JNK (c-Jun N-terminal kinase) or MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase]. MAPKAPK2 (MAPK-activated protein kinase-2) phosphorylated bacterially expressed full-length human Ago2 at serine-387 in vitro, but not the S387A mutant. Finally, mutation of serine-387 to an alanine residue or treatment of cells with a p38 MAPK inhibitor reduced the localization of Ago2 to processing bodies. These results suggest a potential regulatory mechanism for RNA silencing acting through Ago2 serine-387 phosphorylation mediated by the p38 MAPK pathway.

scholarly journals Identification of RAS-Mitogen-Activated Protein Kinase Signaling Pathway Modulators in an ERF1 Redistribution® Screen

2006 ◽  
Vol 11 (4) ◽  
pp. 423-434 ◽  
Author(s):  
Charlotta Grånäs ◽  
Betina Kerstin Lundholt ◽  
Frosty Loechel ◽  
Hans-Christian Pedersen ◽  
Sara Petersen Bjørn ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Signaling Pathway ◽  
Kinase Inhibitors ◽  
Mapk Pathway ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
A Cell ◽  
Protein Kinase Signaling

The RAS-mitogen-activated protein kinase (MAPK) signaling pathway has a central role in regulating the proliferation and survival of both normal and tumor cells. This pathway has been 1 focus area for the development of anticancer drugs, resulting in several compounds, primarily kinase inhibitors, in clinical testing. The authors have undertaken a cell-based, high-throughput screen using a novel ERF1 Redistribution® assay to identify compounds that modulate the signaling pathway. The hit compounds were subsequently tested for activity in a functional cell proliferation assay designed to selectively detect compounds inhibiting the proliferation of MAPK pathway-dependent cancer cells. The authors report the identification of 2 cell membrane-permeable compounds that exhibit activity in the ERF1 Redistribution® assay and selectively inhibit proliferation of MAPK pathway-dependent malignant melanoma cells at similar potencies (IC50 =< 5 μM). These compounds have drug-like structures and are negative in RAF, MEK, and ERK in vitro kinase assays. Drugs belonging to these compound classes may prove useful for treating cancers caused by excessive MAPK pathway signaling. The results also show that cell-based, high-content Redistribution® screens can detect compounds with different modes of action and reveal novel targets in a pathway known to be disease relevant.

GLUT4 translocation precedes the stimulation of glucose uptake by insulin in muscle cells: potential activation of GLUT4 via p38 mitogen-activated protein kinase

2001 ◽  
Vol 359 (3) ◽  
pp. 639-649 ◽  
Author(s):  
Romel SOMWAR ◽  
David Y. KIM ◽  
Gary SWEENEY ◽  
Carol HUANG ◽  
Wenyan NIU ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Glucose Uptake ◽  
P38 Mapk ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Assay ◽  
Glut4 Translocation ◽  
L6 Myotubes ◽  
Mitogen Activated Protein ◽  
Stimulation Of

We previously reported that SB203580, an inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), attenuates insulin-stimulated glucose uptake without altering GLUT4 translocation. These results suggested that insulin might activate GLUT4 via a p38 MAPK-dependent pathway. Here we explore this hypothesis by temporal and kinetic analyses of the stimulation of GLUT4 translocation, glucose uptake and activation of p38 MAPK isoforms by insulin. In L6 myotubes stably expressing GLUT4 with an exofacial Myc epitope, we found that GLUT4 translocation (t1/2 = 2.5min) preceded the stimulation of 2-deoxyglucose uptake (t1/2 = 6min). This segregation of glucose uptake from GLUT4 translocation became more apparent when the two parameters were measured at 22°C. Preincubation with the p38 MAPK inhibitors SB202190 and SB203580 reduced insulin-stimulated transport of either 2-deoxyglucose or 3-O-methylglucose by 40–60%. Pretreatment with SB203580 lowered the apparent transport Vmax of insulin-mediated 2-deoxyglucose and 3-O-methylglucose without any significant change in the apparent Km for either hexose. The IC50 values for the partial inhibition of 2-deoxyglucose uptake by SB202190 and SB203580 were 1 and 2μM respectively, and correlated with the IC50 for full inhibition of p38 MAPK by the two inhibitors in myotubes (2 and 1.4μM, respectively). Insulin caused a dose- (EC50 = 15nM) and time- (t1/2 = 3min) dependent increase in p38 MAPK phosphorylation, which peaked at 10min (2.3±0.3-fold). In vitro kinase assay of immunoprecipitates from insulin-stimulated myotubes showed activation of p38α (2.6±0.3-fold) and p38β (2.3±0.2-fold) MAPK. These results suggest that activation of GLUT4 follows GLUT4 translocation and that both mechanisms contribute to the full stimulation of glucose uptake by insulin. Furthermore, activation of GLUT4 may occur via an SB203580-sensitive pathway, possibly involving p38 MAPK.

scholarly journals MSK1 activity is controlled by multiple phosphorylation sites

2005 ◽  
Vol 387 (2) ◽  
pp. 507-517 ◽  
Author(s):  
Claire E. McCOY ◽  
David G. CAMPBELL ◽  
Maria DEAK ◽  
Graham B. BLOOMBERG ◽  
J. Simon C. ARTHUR
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Kinase Domain ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Cascades ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Ribosomal S6 Kinase ◽  
In Cells

MSK1 (mitogen- and stress-activated protein kinase) is a kinase activated in cells downstream of both the ERK1/2 (extracellular-signal-regulated kinase) and p38 MAPK (mitogen-activated protein kinase) cascades. In the present study, we show that, in addition to being phosphorylated on Thr-581 and Ser-360 by ERK1/2 or p38, MSK1 can autophosphorylate on at least six sites: Ser-212, Ser-376, Ser-381, Ser-750, Ser-752 and Ser-758. Of these sites, the N-terminal T-loop residue Ser-212 and the ‘hydrophobic motif’ Ser-376 are phosphorylated by the C-terminal kinase domain of MSK1, and their phosphorylation is essential for the catalytic activity of the N-terminal kinase domain of MSK1 and therefore for the phosphorylation of MSK1 substrates in vitro. Ser-381 is also phosphorylated by the C-terminal kinase domain, and mutation of Ser-381 decreases MSK1 activity, probably through the inhibition of Ser-376 phosphorylation. Ser-750, Ser-752 and Ser-758 are phosphorylated by the N-terminal kinase domain; however, their function is not known. The activation of MSK1 in cells therefore requires the activation of the ERK1/2 or p38 MAPK cascades and does not appear to require additional signalling inputs. This is in contrast with the closely related RSK (p90 ribosomal S6 kinase) proteins, whose activity requires phosphorylation by PDK1 (3-phosphoinositide-dependent protein kinase 1) in addition to phosphorylation by ERK1/2.

scholarly journals Differential activation of stress-activated protein kinase kinases SKK4/MKK7 and SKK1/MKK4 by the mixed-lineage kinase-2 and mitogen-activated protein kinase kinase (MKK) kinase-1

1998 ◽  
Vol 333 (1) ◽  
pp. 11-15 ◽  
Author(s):  
Ana CUENDA ◽  
Donna S. DOROW
Keyword(s):  
Protein Kinase ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Kinase ◽  
Mixed Lineage Kinase ◽  
Mitogen Activated Protein ◽  
Necrosis Factor ◽  
Pro Inflammatory Cytokines ◽  
Protein Kinase Kinase

Overexpression of the protein kinases mixed-lineage kinase-2 (MLK2) or mitogen-activated protein kinase (MAPK) kinase kinase-1 (MEKK1) is known to trigger the activation of stress-activated protein kinase (SAPK1)/c-Jun N-terminal kinase (JNK). Here we demonstrate that MLK2 activates SAPK kinase-1 (SKK1)/MAPK kinase 4 (MKK4) and SKK4/MKK7, the two known direct activators of SAPK1/JNK (both in transfection studies and in vitro). In contrast, MEKK1 activates SKK1/MKK4 more efficiently than MLK2, but barely activates SKK4/MKK7. Since SKK4/MKK7 (but not SKK1/MKK4) is activated by interleukin-1 and tumour necrosis factor in several cells and tissues, we suggest that MEKK1 does not mediate the activation of SKK4/MKK7 and SAPK1/JNK induced by these pro-inflammatory cytokines. MLK2 and MEKK1 also activated SKK2/MKK3 and SKK3/MKK6, the direct upstream activators of SAPK2a/p38.

scholarly journals Selective Regulation of c-jun Gene Expression by Mitogen-Activated Protein Kinases via the 12-O-Tetradecanoylphorbol-13-Acetate- Responsive Element and Myocyte Enhancer Factor 2 Binding Sites

2005 ◽  
Vol 25 (9) ◽  
pp. 3784-3792 ◽  
Author(s):  
Midori Kayahara ◽  
Xin Wang ◽  
Cathy Tournier
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Cell Fate ◽  
P38 Mapk ◽  
Physiological Role ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Cascades ◽  
Mitogen Activated Protein

ABSTRACT To further understand how the mitogen-activated protein kinase (MAPK) signaling pathways regulate AP-1 activity, we have elucidated the physiological role of these cascades in the regulation of c-jun gene expression. c-Jun is a crucial component of AP-1 complexes and has been shown in vitro to be a point of integration of numerous signals that can differentially affect its expression as well as its transcriptional activity. Our strategy was based on the use of (i) genetically modified fibroblasts deficient in components of the MAPK cascades and (ii) pharmacological reagents. The results demonstrate that c-Jun NH2-terminal protein kinase (JNK) is essential for a basal level of c-Jun expression and for c-Jun phosphorylation in response to stress. In addition to JNK, p38 MAPK or ERK1/2 and ERK5 are required for mediating UV radiation- or epidermal growth factor (EGF)-induced c-Jun expression, respectively. Further studies indicate that p38 MAPK inhibits the activation of JNK in response to EGF, causing a down-regulation of c-Jun. Overall, these data provide important insights into the mechanisms that ultimately determine the function of c-Jun as a regulator of cell fate.

An Inhibitor of p38 Mitogen-Activated Protein Kinase Abrogates Procoagulant and Proinflammatory Effects of Antiphospholipid Antibodies In Vivo.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 136-136
Author(s):  
Silvia S. Pierangeli ◽  
Mariano E. Vega-Ostertag ◽  
Xiaowei Liu
Keyword(s):  
Protein Kinase ◽  
Carotid Artery ◽  
P38 Mapk ◽  
Specific Inhibitor ◽  
Mitogen Activated Protein Kinase ◽  
Peritoneal Cells ◽  
Mitogen Activated Protein ◽  
Pre Treatment

Abstract Background: Activation of p38 mitogen-activated protein kinase (p38 MAPK) has been shown to play a fundamental role in antiphospholipid-induced up regulation of tissue factor (TF) expression and function in monocytes and in endothelial cells (ECs) and increased expression of intercellular adhesion molecule -1 (ICAM-1) in vitro. Those effects correlate with the thrombogenic and pro-inflammatory effects of aPL in vivo. However, It is not clear whether aPL-induceTF in vivo. Methods: To examine this question, we treated CD1 male mice, in groups of 4, with IgG from 3 patients with Antiphospholipid Syndrome (IgG-APS) or with control IgG from healthy controls (IgG-NHS), twice. Seventy-two hours after the first injection, the adhesion of leukocytes per capillary venule (#WBC) to EC in cremaster muscle (as an indication of EC activation in vivo), as well the size of an induced thrombus in the femoral vein of the mice were examined. Some mice were infused i.p. with 25 mg/kg of SB203580 (a p38 MAPK-specific inhibitor) 30 minutes prior to the each IgG-APS injection. TF activity was determined using a chromogenic assay that measures the conversion of factor X into Factor Xa, in homogenates of carotid artery, and in peritoneal cells of mice treated with IgG-APS or with IgG-NHS. Expression of TF and ICAM-1 was determined by cyto-ELISA on cultured HUVECs after treatment of the cells with IgG-APS or with IgG-NHS. Results: At the time of the surgical procedures, the mean aCL titer in the sera of the mice injected with IgG-APS was 73 ± 34 GPL. In vivo, IgG-APS increased significantly the #WBC adhering to EC, when compared to control mice (5.25 ± 0.96 vs 1.85 ± 0.72) and these effects were significantly reduced (2.1 ± 0.74), when mice were pre-treated with SB203580. IgG-APS increased significantly the thrombus size when compared to IgG-NHS-treated mice (3189 ± 558 μm2 vs 1468 ± 401 μm2) and SB203580 inhibited this effect by 65%. Treatment of the mice with IgG-APS also induced significantly increased TF function in peritoneal cells and in homogenates of carotid artery when compared to IgG-NHS-treated mice (17.5 ±11.1 pM vs. 0.8 ±0.2 pM and 8.31 ± 1.59 vs 0.69 ± 0.03, respectively). Pre-treatment of the mice with SB203580 abrogated completely those effects (0.61 ± 0.06 pM in peritoneal cells and 0.75 ± 0.28 pM in carotid artery preparations of mice treated with IgG-APS). Significant expression of TF and ICAM-1 was observed in vitro when HUVECs were treated with any of the three IgG-APS. TF upregulation and ICAM-1 expression were significantly reduced by pre-treatment of the cells with SB203580 (49–97% for TF and 25–69% for ICAM-1). Conclusions: The data show that IgG-APS up regulates TF function in vivo, and this correlates with an in vivo pro-inflammatory and pro-thrombotic effect. Importantly, those effects were abrogated in vivo by a p38 MAPK specific inhibitor. These findings may be important in designing new modalities of targeted therapies to treat thrombosis in patients with APS.

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