scholarly journals NF-κB Activation by Double-Stranded-RNA-Activated Protein Kinase (PKR) Is Mediated through NF-κB-Inducing Kinase and IκB Kinase

2000 ◽  
Vol 20 (4) ◽  
pp. 1278-1290 ◽  
Author(s):  
Maryam Zamanian-Daryoush ◽  
Trine H. Mogensen ◽  
Joseph A. DiDonato ◽  
Bryan R. G. Williams
Keyword(s):  
Protein Kinase ◽  
Kinetic Studies ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Null Cell ◽  
Necrosis Factor Alpha ◽  
Double Stranded Rna ◽  
Iκb Kinase ◽  
Tnf Α ◽  
Factor Alpha

ABSTRACT The interferon (IFN)-inducible double-stranded-RNA (dsRNA)-activated serine-threonine protein kinase (PKR) is a major mediator of the antiviral and antiproliferative activities of IFNs. PKR has been implicated in different stress-induced signaling pathways including dsRNA signaling to nuclear factor kappa B (NF-κB). The mechanism by which PKR mediates activation of NF-κB is unknown. Here we show that in response to poly(rI) · poly(rC) (pIC), PKR activates IκB kinase (IKK), leading to the degradation of the inhibitors IκBα and IκBβ and the concomitant release of NF-κB. The results of kinetic studies revealed that pIC induced a slow and prolonged activation of IKK, which was preceded by PKR activation. In PKR null cell lines, pIC failed to stimulate IKK activity compared to cells from an isogenic background wild type for PKR in accord with the inability of PKR null cells to induce NF-κB in response to pIC. Moreover, PKR was required to establish a sustained response to tumor necrosis factor alpha (TNF-α) and to potentiate activation of NF-κB by cotreatment with TNF-α and IFN-γ. By coimmunoprecipitation, PKR was shown to be physically associated with the IKK complex. Transient expression of a dominant negative mutant of IKKβ or the NF-κB-inducing kinase (NIK) inhibited pIC-induced gene expression from an NF-κB-dependent reporter construct. Taken together, these results demonstrate that PKR-dependent dsRNA induction of NF-κB is mediated by NIK and IKK activation.

scholarly journals Activation of IκB Kinase β by Protein Kinase C Isoforms

1999 ◽  
Vol 19 (3) ◽  
pp. 2180-2188 ◽  
Author(s):  
Maria-José Lallena ◽  
María T. Diaz-Meco ◽  
Gary Bren ◽  
Carlos V. Payá ◽  
Jorge Moscat
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Cell Functions ◽  
Kinase C ◽  
Iκb Kinase ◽  
Tnf Α

ABSTRACT The atypical protein kinase C (PKC) isotypes (λ/ιPKC and ζPKC) have been shown to be critically involved in important cell functions such as proliferation and survival. Previous studies have demonstrated that the atypical PKCs are stimulated by tumor necrosis factor alpha (TNF-α) and are required for the activation of NF-κB by this cytokine through a mechanism that most probably involves the phosphorylation of IκB. The inability of these PKC isotypes to directly phosphorylate IκB led to the hypothesis that ζPKC may use a putative IκB kinase to functionally inactivate IκB. Recently several groups have molecularly characterized and cloned two IκB kinases (IKKα and IKKβ) which phosphorylate the residues in the IκB molecule that serve to target it for ubiquitination and degradation. In this study we have addressed the possibility that different PKCs may control NF-κB through the activation of the IKKs. We report here that αPKC as well as the atypical PKCs bind to the IKKs in vitro and in vivo. In addition, overexpression of ζPKC positively modulates IKKβ activity but not that of IKKα, whereas the transfection of a ζPKC dominant negative mutant severely impairs the activation of IKKβ but not IKKα in TNF-α-stimulated cells. We also show that cell stimulation with phorbol 12-myristate 13-acetate activates IKKβ, which is entirely dependent on the activity of αPKC but not that of the atypical isoforms. In contrast, the inhibition of αPKC does not affect the activation of IKKβ by TNF-α. Interestingly, recombinant active ζPKC and αPKC are able to stimulate in vitro the activity of IKKβ but not that of IKKα. In addition, evidence is presented here that recombinant ζPKC directly phosphorylates IKKβ in vitro, involving Ser177 and Ser181. Collectively, these results demonstrate a critical role for the PKC isoforms in the NF-κB pathway at the level of IKKβ activation and IκB degradation.

scholarly journals Coordinate Regulation of IκB Kinases by Mitogen-Activated Protein Kinase Kinase Kinase 1 and NF-κB-Inducing Kinase

1998 ◽  
Vol 18 (12) ◽  
pp. 7336-7343 ◽  
Author(s):  
Shino Nemoto ◽  
Joseph A. DiDonato ◽  
Anning Lin
Keyword(s):  
Protein Kinase ◽  
Interleukin 1 ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Mutant ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Ikk Complex ◽  
Mitogen Activated Protein ◽  
Protein Kinase Kinase

ABSTRACT IκB kinases (IKKα and IKKβ) are key components of the IKK complex that mediates activation of the transcription factor NF-κB in response to extracellular stimuli such as inflammatory cytokines, viral and bacterial infection, and UV irradiation. Although NF-κB-inducing kinase (NIK) interacts with and activates the IKKs, the upstream kinases for the IKKs still remain obscure. We identified mitogen-activated protein kinase kinase kinase 1 (MEKK1) as an immediate upstream kinase of the IKK complex. MEKK1 is activated by tumor necrosis factor alpha (TNF-α) and interleukin-1 and can potentiate the stimulatory effect of TNF-α on IKK and NF-κB activation. The dominant negative mutant of MEKK1, on the other hand, partially blocks activation of IKK by TNF-α. MEKK1 interacts with and stimulates the activities of both IKKα and IKKβ in transfected HeLa and COS-1 cells and directly phosphorylates the IKKs in vitro. Furthermore, MEKK1 appears to act in parallel to NIK, leading to synergistic activation of the IKK complex. The formation of the MEKK1-IKK complex versus the NIK-IKK complex may provide a molecular basis for regulation of the IKK complex by various extracellular signals.

scholarly journals A Novel Inhibitor of the NF-κB Signaling Pathway Encoded by the Parapoxvirus Orf Virus

2010 ◽  
Vol 84 (8) ◽  
pp. 3962-3973 ◽  
Author(s):  
D. G. Diel ◽  
G. Delhon ◽  
S. Luo ◽  
E. F. Flores ◽  
D. L. Rock
Keyword(s):  
Immune Responses ◽  
Signaling Pathway ◽  
Viral Infections ◽  
Nuclear Translocation ◽  
Orf Virus ◽  
Necrosis Factor Alpha ◽  
Iκb Kinase ◽  
Genes Expression ◽  
Tnf Α ◽  
Factor Alpha

ABSTRACT The parapoxvirus orf virus (ORFV) is a pathogen of sheep and goats that has been used as a preventive and therapeutic immunomodulatory agent in several animal species. However, the functions (genes, proteins, and mechanisms of action) evolved by ORFV to modulate and manipulate immune responses are poorly understood. Here, the novel ORFV protein ORFV024 was shown to inhibit activation of the NF-κB signaling pathway, an important modulator of early immune responses against viral infections. Infection of primary ovine cells with an ORFV024 deletion mutant virus resulted in a marked increase in expression of NF-κB-regulated chemokines and other proinflammatory host genes. Expression of ORFV024 in cell cultures significantly decreased lipopolysaccharide (LPS)- and tumor necrosis factor alpha (TNF-α)-induced NF-κB-responsive reporter gene expression. Further, ORFV024 expression decreased TNF-α-induced phosphorylation and nuclear translocation of NF-κB-p65, phosphorylation, and degradation of IκBα, and phosphorylation of IκB kinase (IKK) subunits IKKα and IKKβ, indicating that ORFV024 functions by inhibiting activation of IKKs, the bottleneck for most NF-κB activating stimuli. Although ORFV024 interferes with activation of the NF-κB signaling pathway, its deletion from the OV-IA82 genome had no significant effect on disease severity, progression, and time to resolution in sheep, indicating that ORFV024 is not essential for virus virulence in the natural host. This represents the first description of a NF-κB inhibitor encoded by a parapoxvirus.

scholarly journals c-Jun N-Terminal Protein Kinase 1 (JNK1), but Not JNK2, Is Essential for Tumor Necrosis Factor Alpha-Induced c-Jun Kinase Activation and Apoptosis

2004 ◽  
Vol 24 (24) ◽  
pp. 10844-10856 ◽  
Author(s):  
Jing Liu ◽  
Yuzuru Minemoto ◽  
Anning Lin
Keyword(s):  
Protein Kinase ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Terminal Protein ◽  
Necrosis Factor Alpha ◽  
Kinase Activation ◽  
Jun Kinase ◽  
Tnf Α ◽  
Factor Alpha ◽  
Necrosis Factor

ABSTRACT Two ubiquitously expressed isoforms of c-Jun N-terminal protein kinase (JNK), JNK1 and JNK2, have shared functions and different functions. However, the molecular mechanism is unknown. Here we report that JNK1, but not JNK2, is essential for tumor necrosis factor alpha (TNF-α)-induced c-Jun kinase activation, c-Jun expression, and apoptosis. Using mouse fibroblasts deficient in either Jnk1 or Jnk2, we found that JNK1 was activated by TNF-α, whereas JNK2 activation was negligible. In addition, JNK2 interfered with JNK1 activation via its “futile” phosphorylation by upstream kinases. Consequently, expression and activation of c-Jun, which depends on JNK activity, were impaired in Jnk1 null cells but enhanced in Jnk2 null cells. TNF-α-induced apoptosis was also suppressed in Jnk1 null fibroblasts but increased in Jnk2 null cells. Thus, our results provide a molecular mechanism underlying the different biological functions of JNK isoforms.

scholarly journals Production of Tumor Necrosis Factor Alpha in Human T Lymphocytes by Staphylococcal Enterotoxin B Correlates with Toxin-Induced Proliferation and Is Regulated through Protein Kinase C

1999 ◽  
Vol 67 (12) ◽  
pp. 6611-6618 ◽  
Author(s):  
Zhengyin Yan ◽  
David C. H. Yang ◽  
Roger Neill ◽  
Marti Jett
Keyword(s):  
T Cells ◽  
Protein Kinase ◽  
Mhc Class Ii ◽  
Staphylococcal Enterotoxin B ◽  
Necrosis Factor Alpha ◽  
Kinase C ◽  
Tnf Α ◽  
Factor Alpha ◽  
Enterotoxin B ◽  
Necrosis Factor

ABSTRACT The superantigen staphylococcal enterotoxin B (SEB) simultaneously binds both the major histocompatibility complex (MHC) class II receptor on monocytes and the T-cell receptor (TCR) on T lymphocytes, resulting in a range of cell responses including induction of tumor necrosis factor alpha (TNF-α). In this study, we have used mixed cultures of human peripheral blood monocytes and lymphocytes to investigate biochemical events controlling SEB induction of TNF-α. TNF-α production induced by SEB in mixed cultures is more closely associated with T cells than with monocytes: (i) a TCR-binding-site mutant of SEB (N23F) is less active in TNF-α induction than an MHC class II receptor-binding-site mutant (F44R), and (ii) flow cytometric analysis indicated that SEB induced TNF-α production in T cells but not in monocytes. Pretreatment of cells with inhibitors of signal transduction pathways was employed to further define events in SEB-induced TNF-α production. Neither protein kinase A inhibitors nor two protein tyrosine kinase inhibitors altered SEB-induced TNF-α production. In contrast, SEB induced protein kinase C (PKC) translocation, and pretreatment of cultures with inhibitors of PKC blocked TNF-α induction. Alteration of levels of diacylglycerol (DAG), an activator of PKC, by treatment with inhibitors of phospholipase C or DAG kinase also altered SEB-induced TNF-α production. These data suggest that PKC activation plays a critical role in SEB-induced TNF-α production in human T cells.

scholarly journals βTrCP-Mediated Proteolysis of NF-κB1 p105 Requires Phosphorylation of p105 Serines 927 and 932

2003 ◽  
Vol 23 (1) ◽  
pp. 402-413 ◽  
Author(s):  
Valerie Lang ◽  
Julia Janzen ◽  
Gregory Zvi Fischer ◽  
Yasmina Soneji ◽  
Sören Beinke ◽  
...  
Keyword(s):  
Target Sequence ◽  
Double Knockout ◽  
Necrosis Factor Alpha ◽  
Ubiquitin E3 Ligase ◽  
Iκb Kinase ◽  
Tnf Α ◽  
Ikk Complex ◽  
Factor Alpha ◽  
Necrosis Factor

ABSTRACT NF-κB1 p105 functions both as a precursor of NF-κB1 p50 and as a cytoplasmic inhibitor of NF-κB. Following the stimulation of cells with tumor necrosis factor alpha (TNF-α), the IκB kinase (IKK) complex rapidly phosphorylates NF-κB1 p105 on serine 927 in the PEST region. This phosphorylation is essential for TNF-α to trigger p105 degradation, which releases the associated Rel/NF-κB subunits to translocate into the nucleus and regulate target gene transcription. Serine 927 resides in a conserved motif (Asp-Ser927-Gly-Val-Glu-Thr-Ser932) homologous to the IKK target sequence in IκBα. In this study, TNF-α-induced p105 proteolysis was revealed to additionally require the phosphorylation of serine 932. Experiments with IKK1−/− and IKK2−/− double knockout embryonic fibroblasts demonstrate that the IKK complex is essential for TNF-α to stimulate phosphorylation on p105 serines 927 and 932. Furthermore, purified IKK1 and IKK2 can each phosphorylate a glutathione S-transferase-p105758-967 fusion protein on both regulatory serines in vitro. IKK-mediated p105 phosphorylation generates a binding site for βTrCP, the receptor subunit of an SCF-type ubiquitin E3 ligase, and depletion of βTrCP by RNA interference blocks TNF-α-induced p105 ubiquitination and proteolysis. Phosphopeptide competition experiments indicate that βTrCP binds p105 more effectively when both serines 927 and 932 are phosphorylated. Interestingly, however, βTrCP affinity for the IKK-phosphorylated sequence on p105 is substantially lower than that on IκBα. Thus, it appears that reduced p105 recruitment of βTrCP and subsequent ubiquitination may contribute to delayed p105 proteolysis after TNF-α stimulation relative to that for IκBα.

scholarly journals Mitogen-Activated Protein Kinase p38 Controls the Expression and Posttranslational Modification of Tristetraprolin, a Regulator of Tumor Necrosis Factor Alpha mRNA Stability

2001 ◽  
Vol 21 (19) ◽  
pp. 6461-6469 ◽  
Author(s):  
Kamal R. Mahtani ◽  
Matthew Brook ◽  
Jonathan L. E. Dean ◽  
Gareth Sully ◽  
Jeremy Saklatvala ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Rna Binding ◽  
Mitogen Activated Protein Kinase ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Mitogen Activated Protein ◽  
Factor Alpha ◽  
The Stability ◽  
Necrosis Factor ◽  
P38 Pathway

ABSTRACT Signal transduction pathways regulate gene expression in part by modulating the stability of specific mRNAs. For example, the mitogen-activated protein kinase (MAPK) p38 pathway mediates stabilization of tumor necrosis factor alpha (TNF-α) mRNA in myeloid cells stimulated with bacterial lipopolysaccharide (LPS). The zinc finger protein tristetraprolin (TTP) is expressed in response to LPS and regulates the stability of TNF-α mRNA. We show that stimulation of RAW264.7 mouse macrophages with LPS induces the binding of TTP to the TNF-α 3′ untranslated region. The p38 pathway is required for the induction of TNF-α RNA-binding activity and for the expression of TTP protein and mRNA. Following stimulation with LPS, TTP is expressed in multiple, differentially phosphorylated forms. We present evidence that phosphorylation of TTP is mediated by the p38-regulated kinase MAPKAPK2 (MAPK-activated protein kinase 2). Our findings demonstrate a direct link between a specific signal transduction pathway and a specific RNA-binding protein, both of which are known to regulate TNF-α gene expression at a posttranscriptional level.

scholarly journals Mixed-Lineage Kinase 3 Delivers CD3/CD28-Derived Signals into the IκB Kinase Complex

2000 ◽  
Vol 20 (7) ◽  
pp. 2556-2568 ◽  
Author(s):  
Steffen P. Hehner ◽  
Thomas G. Hofmann ◽  
Alexej Ushmorov ◽  
Oliver Dienz ◽  
Irene Wing-Lan Leung ◽  
...  
Keyword(s):  
T Cell ◽  
Tumor Necrosis Factor ◽  
Tumor Necrosis Factor Alpha ◽  
Tumor Necrosis ◽  
Interleukin 1 ◽  
Dominant Negative ◽  
Necrosis Factor Alpha ◽  
Iκb Kinase ◽  
Factor Alpha ◽  
Necrosis Factor

ABSTRACT The phosphorylation of IκB by the multiprotein IκB kinase complex (IKC) precedes the activation of transcription factor NF-κB, a key regulator of the inflammatory response. Here we identified the mixed-lineage group kinase 3 (MLK3) as an activator of NF-κB. Expression of the wild-type form of this mitogen-activated protein kinase kinase kinase (MAPKKK) induced nuclear immigration, DNA binding, and transcriptional activity of NF-κB. MLK3 directly phosphorylated and thus activated IκB kinase alpha (IKKα) and IKKβ, revealing its function as an IκB kinase kinase (IKKK). MLK3 cooperated with the other two IKKKs, MEKK1 and NF-κB-inducing kinase, in the induction of IKK activity. MLK3 bound to components of the IKC in vivo. This protein-protein interaction was dependent on the central leucine zipper region of MLK3. A kinase-deficient version of MLK3 strongly impaired NF-κB-dependent transcription induced by T-cell costimulation but not in response to tumor necrosis factor alpha or interleukin-1. Accordingly, endogenous MLK3 was phosphorylated and activated by T-cell costimulation but not by treatment of cells with tumor necrosis factor alpha or interleukin-1. A dominant negative version of MLK3 inhibited NF-κB- and CD28RE/AP-dependent transcription elicited by the Rho family GTPases Rac and Cdc42, thereby providing a novel link between these GTPases and the IKC.

scholarly journals p38 Mitogen-Activated Protein Kinase Controls NF-κB Transcriptional Activation and Tumor Necrosis Factor Alpha Production through RelA Phosphorylation Mediated by Mitogen- and Stress-Activated Protein Kinase 1 in Response to Borrelia burgdorferi Antigens

2006 ◽  
Vol 75 (1) ◽  
pp. 270-277 ◽  
Author(s):  
Chris M. Olson ◽  
Michael N. Hedrick ◽  
Hooman Izadi ◽  
Tonya C. Bates ◽  
Elias R. Olivera ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Map Kinase ◽  
Transcriptional Activation ◽  
P38 Map Kinase ◽  
Phagocytic Cells ◽  
Necrosis Factor Alpha ◽  
Tnf Α ◽  
Mitogen Activated Protein ◽  
Factor Alpha ◽  
Necrosis Factor

ABSTRACT The interaction of Borrelia burgdorferi, the causative agent of Lyme borreliosis, with phagocytic cells induces the activation of NF-κB and the expression of proinflammatory cytokines including tumor necrosis factor alpha (TNF-α). B. burgdorferi-induced TNF-α production is also dependent on the activation of p38 mitogen-activated protein (MAP) kinase. The specific contribution of these signaling pathways to the response of phagocytic cells to the spirochete and the molecular mechanisms underlying this response remain unresolved. We now show that p38 MAP kinase activity regulates the transcriptional activation of NF-κB in response to spirochetal lysate stimulation of phagocytic cells. The regulation occurs at the nuclear level and is independent of the translocation of the transcription factor to the nucleus or its capacity to bind to specific DNA target sequences. In RAW264.7 cells, p38α MAP kinase regulates the phosphorylation of NF-κB RelA. p38 MAP kinase phosphorylates the nuclear kinase mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 in turn phosphorylates the transcriptionally active subunit of NF-κB, RelA. The repression of MSK1 expression with small interfering RNA results in reduced RelA phosphorylation and a significant decrease in the production of TNF-α in response to B. burgdorferi lysates. Overall, these results clarify the contribution of the signaling pathways that are activated in response to the interaction of spirochetes with phagocytic cells to TNF-α production. Our results situate p38 MAP kinase activity as a central regulator of the phagocytic proinflammatory response through MSK1-mediated transcriptional activation of the transcription factor NF-κB.

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