NF-kappaB activation by double-stranded-RNA-activated protein kinase (PKR) is mediated through NF-kappaB-inducing kinase and IkappaB kinase

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-kappaB). The mechanism by which PKR mediates activation of NF-kappaB is unknown. Here we show that in response to poly(rI). poly(rC) (pIC), PKR activates IkappaB kinase (IKK), leading to the degradation of the inhibitors IkappaBalpha and IkappaBbeta and the concomitant release of NF-kappaB. 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-kappaB in response to pIC. Moreover, PKR was required to establish a sustained response to tumor necrosis factor alpha (TNF-alpha) and to potentiate activation of NF-kappaB by cotreatment with TNF-alpha and IFN-gamma. By coimmunoprecipitation, PKR was shown to be physically associated with the IKK complex. Transient expression of a dominant negative mutant of IKKbeta or the NF-kappaB-inducing kinase (NIK) inhibited pIC-induced gene expression from an NF-kappaB-dependent reporter construct. Taken together, these results demonstrate that PKR-dependent dsRNA induction of NF-kappaB is mediated by NIK and IKK activation.     

Constitutive nuclear expression of the I kappa B kinase complex and its activation in human neutrophils

A singular feature of human neutrophils is that they constitutively express substantial amounts of NF-kappaB/Rel proteins and IkappaB-alpha in the nucleus. In this study, we show that in these cells, IkappaB kinase alpha (IKKalpha), IKKbeta, and IKKgamma also partially localize to the nucleus, whereas IKK-related kinases (IKKepsilon, TANK-binding kinase-1) are strictly cytoplasmic, and the NF-kappaB-inducing kinase is strictly nuclear. Following neutrophil activation, IKKbeta and IKKgamma become transiently phosphorylated in both the cytoplasm and nucleus, whereas IKKalpha transiently vanishes from both compartments in what appears to be an IKKbeta-dependent process. These responses are paralleled by the degradation of IkappaB-alpha, and by the phosphorylation of RelA on serine 536, in both compartments. Although both proteins can be IKK substrates, inhibition of IKK prevented IkappaB-alpha phosphorylation, while that of RelA was mostly unaffected. Finally, we provide evidence that the nuclear IKK isoforms (alpha, beta, gamma) associate with chromatin following neutrophil activation, which suggests a potential role in gene regulation. This is the first study to document IKK activation and the phosphorylation of NF-kappaB/Rel proteins in primary neutrophils. More importantly, our findings unveil a hitherto unsuspected mode of activation for the IKK/IkappaB signaling cascade within the cell nucleus.     

CARD9 is a novel caspase recruitment domain-containing protein that interacts with BCL10/CLAP and activates NF-kappa B

BCL10/CLAP is an activator of apoptosis and NF-kappaB signaling pathways and has been implicated in B cell lymphomas of mucosa-associated lymphoid tissue. Although its role in apoptosis remains to be determined, BCL10 likely activates NF-kappaB through the IKK complex in response to upstream stimuli. The N-terminal caspase recruitment domain (CARD) of BCL10 has been proposed to function as an activation domain that mediates homophilic interactions with an upstream CARD-containing NF-kappaB activator. To identify upstream signaling partners of BCL10, we performed a mammalian two-hybrid analysis and identified CARD9 as a novel CARD-containing protein that interacts selectively with the CARD activation domain of BCL10. When expressed in cells, CARD9 binds to BCL10 and activates NF-kappaB. Furthermore, endogenous CARD9 is found associated with BCL10 suggesting that both proteins form a pre-existing signaling complex within cells. CARD9 also self-associates and contains extensive coiled-coil motifs that may function as oligomerization domains. We propose here that CARD9 is an upstream activator of BCL10 and NF-kappaB signaling.     

Nonstructural 5A protein of hepatitis C virus modulates tumor necrosis factor alpha-stimulated nuclear factor kappa B activation

The hepatitis C virus nonstructural protein 5A (NS5A) is a multifunctional phosphoprotein that leads to pleiotropic responses, in part by regulating cell growth and cellular signaling pathways. Here we show that overexpression of NS5A inhibits tumor necrosis factor (TNF)-alpha-induced nuclear factor kappaB (NF-kappaB) activation in HEK293 cells, as determined by luciferase reporter gene expression and by electrophoretic mobility shift assay. When overexpressed, NS5A cannot inhibit the recruitment of TNF receptor-associated factor 2 (TRAF2) and IkappaB kinase (IKK)beta into the TNF receptor 1-TNF receptor-associated death domain complex. In contrast, NS5A is a part of the TNF receptor 1 signaling complex. NF-kappaB activation by TNF receptor-associated death domain and TRAF2 was inhibited by NS5A, whereas MEKK1 and IKKbeta-dependent NF-kappaB activation was not affected, suggesting that NS5A may inhibit NF-kappaB activation signaled by TRAF2. Coimmunoprecipitation and colocalization of NS5A and TRAF2 expressed in vivo provide compelling evidence that NS5A directly interacts with TRAF2. This interaction was mapped to the middle one-third (amino acids 148-301) of NS5A and the TRAF domain of TRAF2. Our findings suggest a possible molecular mechanism that could explain the ability of NS5A to negatively regulate TNF-alpha-induced NF-kappaB activation.     

The N-terminus of PKR is responsible for the activation of the NF-kappaB signaling pathway by interacting with the IKK complex

The interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) has been shown to activate NF-kappaB independently of its kinase function after interaction with the IKK complex. In order to investigate the mechanism of NF-kappaB activation by PKR, we identified the domain of PKR responsible for stimulating the NF-kappaB pathway in PKR-deficient fibroblasts using an NF-kappaB dependent reporter assay. The N-terminal 1-265 AA of PKR activates NF-kappaB, whereas the 1-180 AA N-terminus restricted to the two dsRNA Binding Domains (DRBD), the third basic domain alone (AA 181-265), or the C-terminus of PKR (AA 266-550) were unable to stimulate the expression of the NF-kappaB dependent reporter gene. Using confocal microscopy, we confirmed that PKR full length as well as PKR N-terminus colocalized with IKKbeta. By GST-pulldown analysis, using different PKR domains, we then revealed the specific ability of the PKR N-terminus 1-265 to bind to and activate IKK and showed that this activation requires the integrity of the IKK complex. This activation is not only due to DRBDs since the DRBD fragment 1-180 failed to inhibit PKR 1-265 induced NF-kappaB activation. Our results therefore demonstrate that the ability of PKR to mediate NF-kappaB activation resides in its full N-terminus, and requires both DRBDs and the third basic domain.     

Acetyl-11-keto-beta-boswellic acid potentiates apoptosis, inhibits invasion, and abolishes osteoclastogenesis by suppressing NF-kappa B and NF-kappa B-regulated gene expression

Acetyl-11-keto-beta-boswellic acid (AKBA), a component of an Ayurvedic therapeutic plant Boswellia serrata, is a pentacyclic terpenoid active against a large number of inflammatory diseases, including cancer, arthritis, chronic colitis, ulcerative colitis, Crohn's disease, and bronchial asthma, but the mechanism is poorly understood. We found that AKBA potentiated the apoptosis induced by TNF and chemotherapeutic agents, suppressed TNF-induced invasion, and inhibited receptor activator of NF-kappaB ligand-induced osteoclastogenesis, all of which are known to require NF-kappaB activation. These observations corresponded with the down-regulation of the expression of NF-kappaB-regulated antiapoptotic, proliferative, and angiogenic gene products. As examined by DNA binding, AKBA suppressed both inducible and constitutive NF-kappaB activation in tumor cells. It also abrogated NF-kappaB activation induced by TNF, IL-1beta, okadaic acid, doxorubicin, LPS, H2O2, PMA, and cigarette smoke. AKBA did not directly affect the binding of NF-kappaB to the DNA but inhibited sequentially the TNF-induced activation of IkappaBalpha kinase (IKK), IkappaBalpha phosphorylation, IkappaBalpha ubiquitination, IkappaBalpha degradation, p65 phosphorylation, and p65 nuclear translocation. AKBA also did not directly modulate IKK activity but suppressed the activation of IKK through inhibition of Akt. Furthermore, AKBA inhibited the NF-kappaB-dependent reporter gene expression activated by TNFR type 1, TNFR-associated death domain protein, TNFR-associated factor 2, NF-kappaB-inducing kinase, and IKK, but not that activated by the p65 subunit of NF-kappaB. Overall, our results indicated that AKBA enhances apoptosis induced by cytokines and chemotherapeutic agents, inhibits invasion, and suppresses osteoclastogenesis through inhibition of NF-kappaB-regulated gene expression.     

Theileria-induced constitutive IKK activation is independent of functional Hsp90

The intracellular parasite Theileria induces uncontrolled proliferation and host cell transformation. Parasite-induced transformation is accompanied by constitutive activation of IkappaB kinase (IKK), resulting in permanently high levels of activated nuclear factor (NF)-kappaB. IKK activation pathways normally require heat shock protein 90 (Hsp90), a chaperone that regulates the stability and activity of signalling molecules and can be blocked by the benzoquinone ansamycin compound geldanamycin (GA). In Theileria-transformed cells, IkappaBalpha and p65 phosphorylation, NF-kappaB nuclear translocation and DNA binding activity are largely resistant to GA and also NF-kappaB-dependent reporter gene expression is only partly affected. Our findings indicate that parasite-induced IKK activity does not require functional Hsp90.     

Two carboxyl-terminal activation regions of Epstein-Barr virus latent membrane protein 1 activate NF-kappaB through distinct signaling pathways in fibroblast cell lines

Latent membrane protein 1 (LMP1), an Epstein-Barr virus transforming protein, is able to activate NF-kappaB through its carboxyl-terminal activation region 1 (CTAR1) and 2 (CTAR2), but the exact role of each domain is not fully understood. Here we show that LMP1 activates NF-kappaB in different NF-kappaB essential modulator (NEMO)-defective cell lines, but not in cells lacking both IkappaB kinase 1 (IKK1) and 2 (IKK2). Mutational studies reveal that CTAR1, but not CTAR2, mediates NEMO-independent NF-kappaB activation and that this process largely depends on IKK1. Retroviral expression of LMP1 mutants in cells lacking either functional NF-kappaB inducing kinase (NIK), NEMO, IKK1, or IKK2 further illustrates distinct signals from the two activation regions of LMP1 for persistent NF-kappaB activation. One originates in CTAR2, operates through the canonical NEMO-dependent pathway, and induces NFKB2 p100 production; the second signal originates in CTAR1, utilizes NIK and IKK1, and induces the processing of p100. Our results thus help clarify how two functional domains of LMP1 persistently activate NF-kappaB through distinct signaling pathways.     

Regulation of I(kappa)B kinase complex by phosphorylation of (gamma)-binding domain of I(kappa)B kinase (beta) by Polo-like kinase 1

IkappaB kinase (IKK) complex is a key regulator of NF-kappaB pathways. Signal-induced interaction of the IKKgamma (NEMO) subunit with the C-terminal IKKgamma/NEMO-binding domain (gammaBD) of IKKbeta is an essential interaction for IKK regulation. Underlying regulatory mechanism(s) of this interaction are not known. Phosphorylation of gammaBD has been suggested to play a regulatory role for IKK activation. However, a kinase that phosphorylates gammaBD has not been identified. In this study, we used a C-terminal fragment of IKKbeta as substrate and purified Polo-like kinase 1 (Plk1) from HeLa cell extracts by standard chromatography as a gammaBD kinase. Plk1 phosphorylates serines 733, 740, and 750 in the gammaBD of IKKbeta in vitro. Phosphorylating gammaBD with Plk1 decreased its affinity for IKKgamma in pulldown assay. We generated phosphoantibodies against serine 740 and showed that gammaBD is phosphorylated in vivo. Expressing a constitutively active Plk1 in mammalian cells reduced tumor necrosis factor (TNF)-induced IKK activation, resulting in decreased phosphorylation of endogenous IkappaBalpha and reduced NF-kappaB activation. To activate endogenous Plk1, cells were treated with nocodazole, which reduced TNF-induced IKK activation, and increased the phosphorylation of gammaBD. Knocking down Plk1 in mammalian cells restored TNF-induced IKK activation in nocodazole-treated cells. Activation of Plk1 inhibited TNF-induced expression of cyclin D1. In cells in which Plk1 was knocked down, TNFalpha increased expression of cyclin D1 and the proportion of cells in the S phase of the cell cycle. Taken together, this study shows that phosphorylation regulates the interaction of gammaBD of IKKbeta with IKKgamma and therefore plays a critical role for IKK activation. Moreover, we identify Plk1 as a gammaBD kinase, which negatively regulates TNF-induced IKK activation and cyclin D1 expression, thereby affecting cell cycle regulation. Untimely activation of cyclin D1 by TNFalpha can provide a potential mechanism for an involvement of TNFalpha in inflammation-induced cancer.     

Induction of in vitro reprogramming by Toll-like receptor (TLR)2 and TLR4 agonists in murine macrophages: effects of TLR "homotolerance" versus "heterotolerance" on NF-kappa B signaling pathway components

In this study, tolerance induction by preexposure of murine macrophages to Toll-like receptor (TLR)2 and TLR4 agonists was revisited, focusing on the major signaling components associated with NF-kappaB activation. Pretreatment of macrophages with a pure TLR4 agonist (protein-free Escherichia coli (Ec) LPS) or with TLR2 agonists (Porphyromonas gingivalis LPS or synthetic lipoprotein Pam3Cys) led to suppression of TNF-alpha secretion, IL-1R-associated kinase-1, and IkappaB kinase (IKK) kinase activities, c-jun N-terminal kinase, and extracellular signal-regulated kinase phosphorylation, and to suppression of NF-kappaB DNA binding and transactivation upon challenge with the same agonist (TLR4 or TLR2 "homotolerance," respectively). Despite inhibited NF-kappaB DNA binding, increased levels of nuclear NF-kappaB were detected in agonist-pretreated macrophages. For all the intermediate signaling elements, heterotolerance was weaker than TLR4 or TLR2 homotolerance with the exception of IKK kinase activity. IKK kinase activity was unperturbed in heterotolerance. TNF-alpha secretion was also suppressed in P. gingivalis LPS-pretreated, Ec LPS-challenged cells, but not vice versa, while Pam3Cys and Ec LPS did not induce a state of cross-tolerance at the level of TNF-alpha. Experiments designed to elucidate novel mechanisms of NF-kappaB inhibition in tolerized cells revealed the potential contribution of IkappaBepsilon and IkappaBxi inhibitory proteins and the necessity of TLR4 engagement for induction of tolerance to Toll receptor-IL-1R domain-containing adapter protein/MyD88-adapter-like-dependent gene expression. Collectively, these data demonstrate that induction of homotolerance affects a broader spectrum of signaling components than in heterotolerance, with selective modulation of specific elements within the NF-kappaB signaling pathway.