scholarly journals p38-MAPK Signals Survival by Phosphorylation of Caspase-8 and Caspase-3 in Human Neutrophils

2004 ◽  
Vol 199 (4) ◽  
pp. 449-458 ◽  
Author(s):  
Maria Alvarado-Kristensson ◽  
Fredrik Melander ◽  
Karin Leandersson ◽  
Lars Rönnstrand ◽  
Christer Wernstedt ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Caspase 3 ◽  
Mitogen Activated Protein Kinase ◽  
Human Neutrophils ◽  
Caspase 8 ◽  
Neutrophil Apoptosis ◽  
Mapk Activity ◽  
Mitogen Activated Protein ◽  
Phosphopeptide Mapping

Neutrophil apoptosis occurs both in the bloodstream and in the tissue and is considered essential for the resolution of an inflammatory process. Here, we show that p38–mitogen-activated protein kinase (MAPK) associates to caspase-8 and caspase-3 during neutrophil apoptosis and that p38-MAPK activity, previously shown to be a survival signal in these primary cells, correlates with the levels of caspase-8 and caspase-3 phosphorylation. In in vitro experiments, immunoprecipitated active p38-MAPK phosphorylated and inhibited the activity of the active p20 subunits of caspase-8 and caspase-3. Phosphopeptide mapping revealed that these phosphorylations occurred on serine-364 and serine-150, respectively. Introduction of mutated (S150A), but not wild-type, TAT-tagged caspase-3 into primary neutrophils made the Fas-induced apoptotic response insensitive to p38-MAPK inhibition. Consequently, p38-MAPK can directly phosphorylate and inhibit the activities of caspase-8 and caspase-3 and thereby hinder neutrophil apoptosis, and, in so doing, regulate the inflammatory response.

scholarly journals p38 Mitogen-Activated Protein Kinase (MAPK) Is a Key Mediator in Glucocorticoid-Induced Apoptosis of Lymphoid Cells: Correlation between p38 MAPK Activation and Site-Specific Phosphorylation of the Human Glucocorticoid Receptor at Serine 211

2005 ◽  
Vol 19 (6) ◽  
pp. 1569-1583 ◽  
Author(s):  
Aaron L. Miller ◽  
M. Scott Webb ◽  
Alicja J. Copik ◽  
Yongxin Wang ◽  
Betty H. Johnson ◽  
...  
Keyword(s):  
P38 Mapk ◽  
Mapk Signaling ◽  
Lymphoid Cells ◽  
Mitogen Activated Protein Kinase ◽  
Enzymatic Assays ◽  
Mapk Activity ◽  
Mitogen Activated Protein ◽  
Elevated Activity ◽  
Induced Apoptosis

Abstract Glucocorticoids (GCs) induce apoptosis in lymphoid cells through activation of the GC receptor (GR). We have evaluated the role of p38, a MAPK, in lymphoid cell apoptosis upon treatment with the synthetic GCs dexamethasone (Dex) or deacylcortivazol (DAC). The highly conserved phosphoprotein p38 MAPK is activated by specific phosphorylation of its threonine180 and tyrosine182 residues. We show that Dex and DAC stimulate p38 MAPK phosphorylation and increase the mRNA of MAPK kinase 3, a specific immediate upstream activator of p38 MAPK. Enzymatic assays confirmed elevated activity of p38 MAPK. Pharmacological inhibition of p38 MAPK activity was protective against GC-driven apoptosis in human and mouse lymphoid cells. In contrast, inhibition of the MAPKs, ERK and cJun N-terminal kinase, enhanced apoptosis. Activated p38 MAPK phosphorylates specific downstream targets. Because phosphorylation of the GR is affected by MAPKs, we examined its phosphorylation state in our system. We found serine 211 of the human GR to be a substrate for p38 MAPK both in vitro and intracellularly. Mutation of this site to alanine greatly diminished GR-driven gene transcription and apoptosis. Our results clearly demonstrate a role for p38 MAPK signaling in the pathway of GC-induced apoptosis of lymphoid cells.

scholarly journals Proteomic Identification of 14-3-3ζ as a Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 Substrate: Role in Dimer Formation and Ligand Binding

2003 ◽  
Vol 23 (15) ◽  
pp. 5376-5387 ◽  
Author(s):  
David W. Powell ◽  
Madhavi J. Rane ◽  
Brian A. Joughin ◽  
Ralitsa Kalmukova ◽  
Jeong-Ho Hong ◽  
...  
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Adaptor Protein ◽  
Mitogen Activated Protein Kinase ◽  
Hek293 Cells ◽  
Human Neutrophils ◽  
Ionization Mass ◽  
Protein Database ◽  
Mitogen Activated Protein

ABSTRACT Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MAPKAPK2) mediates multiple p38 MAPK-dependent inflammatory responses. To define the signal transduction pathways activated by MAPKAPK2, we identified potential MAPKAPK2 substrates by using a functional proteomic approach consisting of in vitro phosphorylation of neutrophil lysate by active recombinant MAPKAPK2, protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and phosphoprotein identification by peptide mass fingerprinting with matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and protein database analysis. One of the eight candidate MAPKAPK2 substrates identified was the adaptor protein, 14-3-3ζ. We confirmed that MAPKAPK2 interacted with and phosphorylated 14-3-3ζ in vitro and in HEK293 cells. The chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) stimulated p38-MAPK-dependent phosphorylation of 14-3-3 proteins in human neutrophils. Mutation analysis showed that MAPKAPK2 phosphorylated 14-3-3ζ at Ser-58. Computational modeling and calculation of theoretical binding energies predicted that both phosphorylation at Ser-58 and mutation of Ser-58 to Asp (S58D) compromised the ability of 14-3-3ζ to dimerize. Experimentally, S58D mutation significantly impaired both 14-3-3ζ dimerization and binding to Raf-1. These data suggest that MAPKAPK2-mediated phosphorylation regulates 14-3-3ζ functions, and this MAPKAPK2 activity may represent a novel pathway mediating p38 MAPK-dependent inflammation.

scholarly journals Anaplasma phagocytophilum Delay of Neutrophil Apoptosis through the p38 Mitogen-Activated Protein Kinase Signal Pathway

2005 ◽  
Vol 73 (12) ◽  
pp. 8209-8218 ◽  
Author(s):  
Kyoung-Seong Choi ◽  
Joon Tae Park ◽  
J. Stephen Dumler
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Anaplasma Phagocytophilum ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Human Neutrophils ◽  
Neutrophil Apoptosis ◽  
Intracellular Infection ◽  
Granulocytic Anaplasmosis ◽  
Mitogen Activated Protein

ABSTRACT Human granulocytic anaplasmosis is caused by the obligate intracellular bacterium Anaplasma phagocytophilum. The bacterium avoids host innate defenses in part by infecting, surviving in, and propagating in neutrophils, as well as by inhibiting neutrophil apoptosis. However, the mechanisms of A. phagocytophilum survival in neutrophils and the inhibition of spontaneous apoptosis are not well understood. In this study, we demonstrated that antiapoptotic Mcl-1 protein (Bcl-2 family) expression is maintained and that inhibition of procaspase-3 processing occurs in A. phagocytophilum-infected human neutrophils. An evaluation of p38 mitogen-activated protein kinase (MAPK) showed evidence of increased phosphorylation with infection. Moreover, antagonism of p38 MAPK by the inhibitor SB203580 reversed apoptosis inhibition in live or heat-killed A. phagocytophilum-infected neutrophils. A role for the autocrine or paracrine production of antiapoptotic interleukin 8 (IL-8) expressed with A. phagocytophilum infection was excluded by the use of IL-8-, IL-8R1 (CXCR1)-, and IL-8R2 (CXCR2)-blocking antibodies. As previously demonstrated, the antiapoptotic effect was initially mediated by exposure to A. phagocytophilum components in heat-killed bacteria. However, an important role for active infection is demonstrated by the additional delay in apoptosis with intracellular growth and the refractory abrogation of this response by the p38 MAPK inhibitor 3 to 6 h after neutrophil infection. These results suggest that the initial activation of the p38 MAPK pathway leading to A. phagocytophilum-delayed neutrophil apoptosis is bypassed with active intracellular infection. Moreover, active intracellular infection contributes more to the overall delay in apoptosis than do components of heat-killed A. phagocytophilum alone.

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.

scholarly journals The major phosphorylation site of the NADPH oxidase component p67phox is Thr233

1999 ◽  
Vol 338 (1) ◽  
pp. 99-105 ◽  
Author(s):  
Louisa V. FORBES ◽  
Oanh TRUONG ◽  
Frans B. WIENTJES ◽  
Stephen J. MOSS ◽  
Anthony W. SEGAL
Keyword(s):  
Protein Kinase ◽  
Cyanogen Bromide ◽  
Tryptic Peptide ◽  
Phosphorylation Site ◽  
Mitogen Activated Protein Kinase ◽  
Rich Domain ◽  
Mitogen Activated Protein ◽  
Phosphopeptide Mapping ◽  
Major Phosphorylation Site

Phosphorylation of p67phox was shown to increase two- to three-fold upon stimulation by PMA, N-formylmethionyl-leucylphenylalanine or serum-opsonized zymosan. Phosphopeptide mapping showed one major tryptic peptide for p67phox immunoprecipitated from resting or stimulated cells. In vitro phosphorylation of p67phox by isolated cytosol or mitogen-activated protein kinase also generated the same phosphopeptide. Results of cyanogen bromide digestion and HPLC–MS suggested that Thr233 was the phosphorylated residue. Mutagenesis of Thr233 to alanine resulted in loss of phosphorylation in vitro. In the present work, Thr233 has been identified as the major phosphorylation site of p67phox, which is situated in a proline-rich domain.

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.

Mitigation of Direct Neurotoxic Effects of Lidocaine and Amitriptyline by Inhibition of p38 Mitogen-activated Protein Kinase In Vitro  and In Vivo 

2006 ◽  
Vol 104 (6) ◽  
pp. 1266-1273 ◽  
Author(s):  
Philipp Lirk ◽  
Ingrid Haller ◽  
Robert R. Myers ◽  
Lars Klimaschewski ◽  
Yi-Chuan Kau ◽  
...  
Keyword(s):  
Sciatic Nerve ◽  
P38 Mapk ◽  
Local Anesthetic ◽  
Apoptotic Cell ◽  
Mitogen Activated Protein Kinase ◽  
Mitogen Activated Protein ◽  
P38 Mapk Inhibitor ◽  
Mapk Inhibitor

Background Local anesthetic-induced direct neurotoxicity (paresthesia, failure to regain normal sensory and motor function) is a potentially devastating complication of regional anesthesia. Local anesthetics activate the p38 mitogen-activated protein kinase (MAPK) system, which is involved in apoptotic cell death. The authors therefore investigated in vitro (cultured primary sensory neurons) and in vivo (sciatic nerve block model) the potential neuroprotective effect of the p38 MAPK inhibitor SB203580 administered together with a clinical (lidocaine) or investigational (amitriptyline) local anesthetic. Methods Cell survival and mitochondrial depolarization as marker of apoptotic cell death was assessed in rat dorsal root ganglia incubated with lidocaine or amitriptyline either with or without the addition of SB203580. Similarly, in a sciatic nerve block model, the authors assessed wallerian degeneration by light microscopy to detect a potential mitigating effect of MAPK inhibition. Results Lidocaine at 40 mm/approximately 1% and amitriptyline at 100 microm reduce neuron count, but coincubation with the p38 MAPK inhibitor SB203580 at 10 mum significantly reduces cytotoxicity and the number of neurons exhibiting mitochondrial depolarization. Also, wallerian degeneration and demyelination induced by lidocaine (600 mm/approximately 15%) and amitriptyline (10 mm/approximately 0.3%) seem to be mitigated by SB203580. Conclusions The cytotoxic effect of lidocaine and amitriptyline in cultured dorsal root ganglia cells and the nerve degeneration in the rat sciatic nerve model seem, at least in part, to be mediated by apoptosis but seem efficiently blocked by an inhibitor of p38 MAPK, making it conceivable that coinjection might be useful in preventing local anesthetic-induced neurotoxicity.

scholarly journals p38 Mitogen-Activated Protein Kinase Is Critical for Synergistic Induction of the FSHβ Gene by Gonadotropin-Releasing Hormone and Activin through Augmentation of c-Fos Induction and Smad Phosphorylation

2007 ◽  
Vol 21 (12) ◽  
pp. 3071-3086 ◽  
Author(s):  
Djurdjica Coss ◽  
Cameron M. Hand ◽  
Karen K. J. Yaphockun ◽  
Heather A. Ely ◽  
Pamela L. Mellon
Keyword(s):  
P38 Mapk ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Limiting Factor ◽  
Activator Protein 1 ◽  
Mapk Activity ◽  
C Terminus ◽  
Smad Phosphorylation ◽  
Mitogen Activated Protein ◽  
Smad 3

Abstract GnRH and activin independently and synergistically activate transcription of the FSH β-subunit gene, the subunit that provides specificity and is the limiting factor in the synthesis of the mature hormone. This synergistic interaction, as determined by two-way ANOVA, is specific for FSHβ and may, therefore, contribute to differential expression of the two gonadotropin hormones, which is critical for the reproductive cycle. We find that the cross-talk between the GnRH and activin signaling pathways occurs at the level of p38 MAPK, because the synergy is dependent on p38 MAPK activity, which is activated by GnRH, and activin cotreatment augments p38 activation by GnRH. Both the Smad and activator protein-1 binding sites on the FSHβ promoter are necessary and sufficient for synergy. After cotreatment, Smad 3 proteins are more highly phosphorylated on the activin-receptor signaling-dependent residues on the C terminus than with activin treatment alone, and c-Fos is more highly expressed than with GnRH treatment alone. Inhibition of p38 by either of two different inhibitors or a dominant-negative p38 kinase abrogates synergy on FSHβ expression, reduces c-Fos induction by GnRH, and prevents the further increase in c-Fos levels that occurs with cotreatment. Additionally, p38 is necessary for maximal Smad 3 C-terminal phosphorylation by activin treatment alone and for the further increase caused by cotreatment. Thus, p38 is the pivotal signaling molecule that integrates GnRH and activin interaction on the FSHβ promoter through higher induction of c-Fos and elevated Smad phosphorylation.

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 Molecular mechanisms of neutrophil apoptosis (review)

2018 ◽  
pp. 5-18
Author(s):  
И.А. Щепеткин ◽  
О.П. Буданова ◽  
И.Ю. Малышев ◽  
Д.Н. Аточин
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Mitogen Activated Protein Kinase ◽  
Neutrophil Apoptosis ◽  
Nuclear Factor ◽  
Phosphatidylinositol 3 Kinase ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Nuclear Factor Kb

В обзоре представлены современные данные о механизмах инициации, регуляции и выполнении процесса апоптоза нейтрофилов с участием «рецепторов смерти», митохондрий, белков семейства Bcl-2, PI3-K (phosphatidylinositol 3-kinase), протеинкиназных каскадов p38 MAPK (mitogen-activated protein kinase), ERK (extracellular signal regulated kinase) и JNK (c-Jun N-terminal kinase), протеинкиназ А, В и С, сAMP, белков теплового шока, NF-kB (nuclear factor-kB), кальпаинов, каспаз и их ингибиторов, активных форм кислорода и других факторов. Предложена гипотетическая модель вовлечения апоптотических процессов в регуляцию дифференцировки и реактивности нейтрофилов. This review presented recent data on initiation, regulation, and execution of neutrophil apoptosis with participation of «death receptors», mitochondria, Bcl-2 family proteins, PI3-K (phosphatidylinositol 3-kinase), p38 MAPK (mitogen-activated protein kinase), ERK (extracellular signal regulated kinase) and JNK (c-Jun N-terminal kinase) cascades, protein kinases A, B and C, сAMP, heat shock proteins, NF-kB (nuclear factor-kB), calpains, caspases and theirs inhibitors, reactive oxygen species, and other factors. A speculative model of the apoptotic processes involvement in the regulation of neutrophil differentiation and reactivity was proposed.

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