D W Ballard

Vanderbilt University, Нашвилл, Michigan, United States

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Publications (96)689.5 Total impact

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    ABSTRACT: NEMO and CYLD are intracellular proteins that regulate the NF-kappa B signaling pathway. Although mice with either CYLD deficiency or an alteration in the zinc finger domain of NEMO (K392R) are born healthy, we found that the combination of these two gene defects in double mutant (DM) mice is early embryonic lethal but can be rescued by the absence of TNF receptor 1 (TNFR1). Notably, NEMO was not recruited into the TNFR1 complex of double mutant (DM) cells, and consequently NF-kappa B induction by TNF was severely impaired and DM cells were sensitized to TNF-induced cell death. Interestingly, the TNF signaling defects can be fully rescued by reconstitution of DM cells with CYLD lacking ubiquitin hydrolase activity but not with CYLD mutated in TRAF2 or NEMO binding sites. Therefore, our data demonstrate an unexpected non-catalytic function for CYLD as an adapter protein between TRAF2 and the NEMO zinc finger that is important for TNF-induced NF-kappa B signaling during embryogenesis. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Full-text · Article · Jul 2015 · Journal of Biological Chemistry
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    ABSTRACT: PMMA particles released from bone implants are considered major contributor to osteolysis and subsequent implant failure. Although the ensuing inflammatory response has been described, the mechanisms underlying PMMA particulate-induced osteolysis remain enigmatic. In previous studies, we have established that activation of Nuclear factor kappa-B (NF-κB) and MAP kinase pathways plays a central role in the pathogenesis of inflammatory osteolysis. Specifically, we have shown that impeding IKK complex assembly, and thus subsequent NF-κB activation, dampens particle-induced osteolysis. The IKK complex consists of IKKα, IKKβ, and IKKγ, also known as NEMO. NEMO has no catalytic activity and serves as a scaffold protein facilitating assembly and distal activation of NF-κB signaling. In fact, blocking binding of NEMO with IKKα/β abolishes NF-κB activity. In the current study, we identify Lysine 392 residue in NEMO as crucial mediator of PMMA particle-induced inflammatory osteoclastogenesis and osteolysis. Using mice in which NEMO-K392R mutation has been introduced, we provide evidence that PMMA-induced osteoclasts and osteolytic responses are impaired. Furthermore, we show that this impairment is likely due to poor activation of NF-κB and Erk, but not other MAP kinases. Our findings suggest that NEMO Lysine392, a well-established K63-linked polyubiquitination site, is an important mediator of PMMA-induced osteolysis. Therefore, this NEMO motif should be considered as a target to combat PMMA particle-induced osteolysis.
    Full-text · Article · Apr 2012 · Journal of Orthopaedic Research
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    ABSTRACT: Transcription factor NF-kappaB controls the expression of multiple genes involved in immunity and inflammation. The initial activation and duration of NF-kappaB signaling is regulated by posttranslational modifications to IkappaB kinase, which earmarks inhibitors of NF-kappaB for degradation. Prior studies suggest that K63-linked ubiquitination of NEMO (NF-kappaB essential modulator), an IkappaB kinase regulatory subunit, is critical for NF-kappaB and MAPK signaling following engagement of Ag receptors. We now demonstrate that NF-kappaB and MAPK pathways are largely unaffected in primary cells from mice harboring a ubiquitination-defective form of NEMO, NEMO-KR. TLR- but not Ag receptor-induced cellular responses are impaired in NEMO-KR mice, which are more resistant to LPS-induced endotoxic shock than wild-type animals. Thus, one function of NEMO ubiquitination is to fine tune innate immune responses under TLR control.
    Full-text · Article · Jul 2008 · The Journal of Immunology
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    ABSTRACT: Intracellular detection of RNA virus infection is mediated by the RNA helicase RIG-I, which is recruited to mitochondria by the adaptor protein MAVS and triggers activation of the transcription factors NF-kappaB, IRF3 and IRF7. Here we demonstrate that virus-induced activation of IRF3 and IRF7 depended on the NF-kappaB modulator NEMO, which acted 'upstream' of the kinases TBK1 and IKKepsilon. IRF3 phosphorylation, formation of IRF3 dimers and DNA binding, as well as IRF3-dependent gene expression, were abrogated in NEMO-deficient cells. IRF3 phosphorylation and interferon production were restored by ectopic expression of NEMO. Thus, NEMO, like MAVS, acts as an adaptor protein that allows RIG-I to activate both the NF-kappaB and IRF signaling pathways.
    Full-text · Article · Jul 2007 · Nature Immunology
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    ABSTRACT: Reoviruses induce apoptosis both in cultured cells and in vivo. Apoptosis plays a major role in the pathogenesis of reovirus encephalitis and myocarditis in infected mice. Reovirus-induced apoptosis is dependent on the activation of transcription factor NF-kappaB and downstream cellular genes. To better understand the mechanism of NF-kappaB activation by reovirus, NF-kappaB signaling intermediates under reovirus control were investigated at the level of Rel, IkappaB, and IkappaB kinase (IKK) proteins. We found that reovirus infection leads initially to nuclear translocation of p50 and RelA, followed by delayed mobilization of c-Rel and p52. This biphasic pattern of Rel protein activation is associated with the degradation of the NF-kappaB inhibitor IkappaBalpha but not the structurally related inhibitors IkappaBbeta or IkappaBepsilon. Using IKK subunit-specific small interfering RNAs and cells deficient in individual IKK subunits, we demonstrate that IKKalpha but not IKKbeta is required for reovirus-induced NF-kappaB activation and apoptosis. Despite the preferential usage of IKKalpha, both NF-kappaB activation and apoptosis were attenuated in cells lacking IKKgamma/Nemo, an essential regulatory subunit of IKKbeta. Moreover, deletion of the gene encoding NF-kappaB-inducing kinase, which is known to modulate IKKalpha function, had no inhibitory effect on either response in reovirus-infected cells. Collectively, these findings indicate a novel pathway of NF-kappaB/Rel activation involving IKKalpha and IKKgamma/Nemo, which together mediate the expression of downstream proapoptotic genes in reovirus-infected cells.
    Full-text · Article · Mar 2007 · Journal of Virology
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    ABSTRACT: Covalent modification of proteins by reactive intermediates is a hallmark of xenobiotic toxicity, but has been poorly understood from a mechanistic perspective. The use of affinity tagged electrophile probes together with liquid chromatography-tandem mass spectrometry (LC-MS-MS)-based proteomics methods have enabled global identification of protein targets of electrophiles. Site specific mapping of hundreds of adducts by LC-MS-MS has revealed significant differences in specificity between different electrophile chemistries, as a prototypical SN2 alkylator (the iodoacetamido probe IAB) and a prototypical Michael acceptor (the N-alkylmaleimide probe BMCC) display only about 20% overlap in target sites. Although both probes alkylate comparable numbers of proteins in intact cells, kinetic analyses reveal that BMCC adducts rapidly accumulate and then decline, whereas IAB adducts form more slowly, but persist. This suggests either possible repair pathways or enhanced degradation for some types of adducts. IAB adduction induces apoptosis in HEK293 cells, whereas BMCC does not. IAB induced c-jun-N-terminal kinase (JNK) activation and inhibited nuclear factor kappa B (NF-B) activation in HEK293 cells whereas BMCC did neither. JNK activates pro-apoptotic signaling whereas NF-B signals pro-survival. To determine the protein targets of the electrophiles in these pathways, adducted (biotinylated) proteins from IAB- and BMCC-treated cells were enriched by streptavidin capture and analyzed by Western blot analysis. JNK, its regulators and upstream kinases were adducted by IAB, but to a much lesser extent by BMCC. Similar results were observed for inhibitor of kappaB kinase beta (IKK-b) and inhibitor of NF-kB (IkB), two key NF-kB pathway proteins. Immunoprecipitation and Western blot studies were carried out to determine the effect of the electrophiles on apoptosis signaling kinase 1 (ASK1) activation, an upstream trigger for JNK activation. IAB adducted both ASK1 and thioredoxin (Trx1), dissociated ASK1 from Trx1, and induced ASK1 activation. In contrast, BMCC did not induce these events. These data suggest that adduction of the JNK activator, ASK1, and its negative regulator, thioredoxin 1, contribute to JNK activation and apoptosis. Components of the JNK and NF-B pathways are important sensors of stress responses and are differentially regulated to promote cell death in response to reactive electrophiles. (Supported by NIH grants ES010056 and ES000267).
    No preview · Conference Paper · Jan 2007
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    ABSTRACT: Reovirus infection activates NF-kappaB, which leads to programmed cell death in cultured cells and in the murine central nervous system. However, little is known about how NF-kappaB elicits this cellular response. To identify host genes activated by NF-kappaB following reovirus infection, we used HeLa cells engineered to express a degradation-resistant mutant of IkappaBalpha (mIkappaBalpha) under the control of an inducible promoter. Induction of mIkappaBalpha inhibited the activation of NF-kappaB and blocked the expression of NF-kappaB-responsive genes. RNA extracted from infected and uninfected cells was used in high-density oligonucleotide microarrays to examine the expression of constitutively activated genes and reovirus-stimulated genes in the presence and absence of an intact NF-kappaB signaling axis. Comparison of the microarray profiles revealed that the expression of 176 genes was significantly altered in the presence of mIkappaBalpha. Of these genes, 64 were constitutive and not regulated by reovirus, and 112 were induced in response to reovirus infection. NF-kappaB-regulated genes could be grouped into four distinct gene clusters that were temporally regulated. Gene ontology analysis identified biological processes that were significantly overrepresented in the reovirus-induced genes under NF-kappaB control. These processes include the antiviral innate immune response, cell proliferation, response to DNA damage, and taxis. Comparison with previously identified NF-kappaB-dependent gene networks induced by other stimuli, including respiratory syncytial virus, Epstein-Barr virus, tumor necrosis factor alpha, and heart disease, revealed a number of common components, including CCL5/RANTES, CXCL1/GRO-alpha, TNFAIP3/A20, and interleukin-6. Together, these results suggest a genetic program for reovirus-induced apoptosis involving NF-kappaB-directed expression of cellular genes that activate death signaling pathways in infected cells.
    Full-text · Article · Mar 2006 · Journal of Virology
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    ABSTRACT: Transcription factor NF-kappaB governs the expression of multiple genes involved in cell growth, immunity, and inflammation. Nuclear translocation of NF-kappaB is regulated from the cytoplasm by IkappaB kinase-beta (IKKbeta), which earmarks inhibitors of NF-kappaB for polyubiquination and proteasome-mediated degradation. Activation of IKKbeta is contingent upon signal-induced phosphorylation of its T loop at Ser-177/Ser-181. T loop phosphorylation also renders IKKbeta a substrate for monoubiquitination in cells exposed to chronic activating cues, such as the Tax oncoprotein or sustained signaling through proinflammatory cytokine receptors. Here we provide evidence that the T loop-proximal residue Lys-163 in IKKbeta serves as a major site for signal-induced monoubiquitination with significant regulatory potential. Conservative replacement of Lys-163 with Arg yielded a monoubiquitination-defective mutant of IKKbeta that retains kinase activity in Tax-expressing cells but is impaired for activation mediated by chronic signaling from the type 1 receptor for tumor necrosis factor-alpha. Phosphopeptide mapping experiments revealed that the Lys-163 --> Arg mutation also interferes with proper in vivo but not in vitro phosphorylation of cytokine-responsive serine residues located in the distal C-terminal region of IKKbeta. Taken together, these data indicate that chronic phosphorylation of IKKbeta at Ser-177/Ser-181 leads to monoubiquitin attachment at nearby Lys-163, which in turn modulates the phosphorylation status of IKKbeta at select C-terminal serines. This mechanism for post-translational cross-talk may play an important role in the control of IKKbeta signaling during chronic inflammation.
    No preview · Article · Dec 2005 · Journal of Biological Chemistry
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    ABSTRACT: Transcription factor NF-kappaB plays a key regulatory role in the cellular response to pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF). In the absence of TNF, NF-kappaB is sequestered in the cytoplasm by inhibitory IkappaB proteins. Phosphorylation of IkappaBby the beta-catalytic subunit of IKK, a multicomponent IkappaB kinase, targets the inhibitor for proteolytic destruction and facilitates nuclear translocation of NF-kappaB. This pathway is initiated by TNF-dependent phosphorylation of T loop serines in IKKbeta, which greatly stimulates IkappaB kinase activity. Prior in vitro mixing experiments indicate that protein serine/threonine phosphatase 2A (PP2A) can dephosphorylate these T loop serines and inactivate IKK, suggesting a negative regulatory role for PP2A in IKK signaling. Here we provided several in vivo lines of evidence indicating that PP2A plays a positive rather than a negative role in the regulation of IKK. First, TNF-induced degradation of IkappaB is attenuated in cells treated with okadaic acid or fostriecin, two potent inhibitors of PP2A. Second, PP2A forms stable complexes with IKK in untransfected mammalian cells. This interaction is critically dependent on amino acid residues 121-179 of the IKKgamma regulatory subunit. Third, deletion of the PP2A-binding site in IKKgamma attenuates T loop phosphorylation and catalytic activation of IKKbeta in cells treated with TNF. Taken together, these data provide strong evidence that the formation of IKK.PP2A complexes is required for the proper induction of IkappaB kinase activity in vivo.
    Preview · Article · Nov 2005 · Journal of Biological Chemistry
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    ABSTRACT: Staphylococcal enterotoxin B and related toxins that target T cells have the capacity to elicit systemic inflammation, tissue injury, and death. Genes that encode mediators of inflammation can be globally inhibited by blocking the nuclear import of stress-responsive transcription factors. Here we show that cell-permeant peptides targeting Rch1/importin alpha/karyopherin alpha 2, a nuclear import adaptor protein, are delivered to T cells where they inhibit the staphylococcal enterotoxin B-induced production of inflammatory cytokines ex vivo in cultured primary spleen cells and in vivo. The systemic production of tumor necrosis factor alpha, interferon gamma, and interleukin-6 was attenuated in mice either by a cell-permeant cyclized form of SN50 peptide or by a transgene whose product suppresses the nuclear import of transcription factor nuclear factor kappa B in T cells. The extent of liver apoptosis and hemorrhagic necrosis was also reduced, which correlated with significantly decreased mortality rates. These findings highlight nuclear import inhibitors as a potentially useful countermeasure for staphylococcal enterotoxin B and other toxins that trigger harmful systemic inflammatory responses.
    Full-text · Article · May 2004 · Journal of Biological Chemistry
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    ABSTRACT: Initiation of the genetic programs for inflammation and immunity involves nuclear mobilization of transcription factor NF-κB. This signal-dependent process is controlled in part by the β-catalytic subunit of IκB kinase (IKKβ), which marks IκBα and other cytoplasmic inhibitors of NF-κB for proteolytic destruction. The catalytic activity of IKKβ is stimulated by pathologic and physiologic inducers of NF-κB, such as the Tax oncoprotein and proinflammatory cytokines. We now report evidence that these NF-κB inducers target IKKβ for conjugation to ubiquitin (Ub) in mammalian cells. The apparent molecular size of modified IKKβ is compatible with monoubiquitination rather than attachment of a multimeric Ub chain. The modification is contingent upon signal-induced phosphorylation of the activation T loop in IKKβ at Ser-177/Ser-181. The formation of IKKβ-Ub conjugates is disrupted in cells expressing YopJ, a Ub-like protein protease that interferes with the NF-κB signaling pathway. These findings indicate an important mechanistic link between phosphorylation, ubiquitination, and the biologic action of IKKβ.
    Preview · Article · Jan 2004 · Journal of Biological Chemistry
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    ABSTRACT: Initiation of the genetic programs for inflammation and immunity involves nuclear mobilization of transcription factor NF-κB. This signal-dependent process is controlled in part by the β-catalytic subunit of IκB kinase (IKKβ), which marks IκBα and other cytoplasmic inhibitors of NF-κB for proteolytic destruction. The catalytic activity of IKKβ is stimulated by pathologic and physiologic inducers of NF-κB, such as the Tax oncoprotein and proinflammatory cytokines. We now report evidence that these NF-κB inducers target IKKβ for conjugation to ubiquitin (Ub) in mammalian cells. The apparent molecular size of modified IKKβ is compatible with monoubiquitination rather than attachment of a multimeric Ub chain. The modification is contingent upon signal-induced phosphorylation of the activation T loop in IKKβ at Ser-177/Ser-181. The formation of IKKβ-Ub conjugates is disrupted in cells expressing YopJ, a Ub-like protein protease that interferes with the NF-κB signaling pathway. These findings indicate an important mechanistic link between phosphorylation, ubiquitination, and the biologic action of IKKβ.
    No preview · Article · Dec 2003 · Journal of Biological Chemistry
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    AL Mora · RA Corn · AK Stanic · S Goenka · M Aronica · S Stanley · D W Ballard · S Joyce · M Boothby
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    ABSTRACT: Inducible protection from apoptosis in vivo controls the size of cell populations. An important question in this respect is how differentiation affects mechanisms of apoptosis regulation. Among mature T lymphocytes, the NF-kappaB/Rel transcription factors are coupled to receptors that control cell population sizes by concurrently regulating survival and multiplication. In the present study, we used a transgenic inhibitor of NF-kappaB/Rel signaling to investigate the role of this pathway in proliferation and death of mature T cells in vivo. The results indicate that NF-kappaB integrates two critical yet distinct molecular pathways preventing apoptosis affected by the death receptor Fas, coordinately regulating levels of FLIP and Bcl-x(L) in primary T cells. Surprisingly, NF-kappaB blockade preferentially impacted naive as compared to memory T cells. The Fas/FasL pathway was linked to these findings by evidence that the abnormalities imposed by NF-kappaB inhibition were ameliorated by Fas deficiency, particularly for the CD4(+) lineage. Moreover, levels of an inhibitor of Fas-mediated apoptosis, c-FLIP, were diminished in cells expressing the transgenic inhibitor. NF-kappaB was also linked to T cell survival in vivo by mediating induction of Bcl-x(L): restoration of Bcl-x(L) levels reversed the preferential deficit of naive T cells, differentially impacting the CD4 and CD8 subsets. These results show that promoting survival and effective multiplication are central roles for NF-kappaB in T lymphoid homeostasis in vivo, but this effect and its underlying mechanisms are influenced by the developmental state of the lymphocyte.
    Full-text · Article · Oct 2003 · Cell Death and Differentiation
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    ABSTRACT: Transcription factor NF-kappaB plays a pivotal regulatory role in the genetic programs for cell cycle progression and inflammation. Nuclear translocation of NF-kappaB is controlled by an inducible protein kinase called IKK, which earmarks cytoplasmic inhibitors of NF-kappaB for proteolytic destruction. IKK contains two structurally related catalytic subunits termed IKKalpha and IKKbeta as well as a noncatalytic subunit called IKKgamma/NEMO. Mutations in the X-linked gene encoding IKKgamma can interfere with NF-kappaB signaling and lead to immunodeficiency disease. Although its precise mechanism of action remains unknown, IKKgamma is phosphorylated in concert with the induction of NF-kappaB by the viral oncoprotein Tax and the proinflammatory cytokine tumor necrosis factor alpha (TNF). We now demonstrate that TNF-induced phosphorylation of IKKgamma is blocked in cells deficient for IKKbeta but not IKKalpha. Phosphopeptide-mapping experiments with metabolically radiolabeled cells indicate that IKKbeta phosphorylates human IKKgamma at Ser-31, Ser-43, and Ser-376 following the enforced expression of either the Tax oncoprotein or the type 1 TNF receptor. Inducible phosphorylation of IKKgamma is attenuated following the deletion of its COOH-terminal zinc finger domain (amino acids 397-419), a frequent target for mutations that occur in IKKgamma-associated immunodeficiencies. As such, IKKbeta-mediated phosphorylation of IKKgamma at these specific serine targets may facilitate proper regulation of NF-kappaB signaling in the immune system.
    Preview · Article · Jun 2003 · Journal of Biological Chemistry
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    ABSTRACT: The Tax transforming protein encoded by human T-cell leukemia virus type 1 (HTLV1) persistently activates transcription factor NF-κB and deregulates the expression of downstream genes that mediate cell cycle entry. We recently found that Tax binds to and chronically stimulates the catalytic function of IκB kinase (IKK), a cellular enzyme complex that phosphorylates and inactivates the IκB inhibitory subunit of NF-κB. We now demonstrate that the IKKβ catalytic subunit and IKKγ regulatory subunit of IKK are chronically phosphorylated in HTLV1-infected and Tax-transfected cells. Alanine substitutions at Ser-177 and Ser-181 in the T loop of IKKβ protect both of these IKK subunits from Tax-directed phosphorylation and prevent the induction of IκB kinase activity. Each of these inhibitory effects is recapitulated in Tax transfectants expressing the bacterial protein YopJ, a potent in vivo agonist of T loop phosphorylation. Moreover, ectopically expressed forms of IKKβ that contain glutamic acid substitutions at Ser-177 and Ser-181 have the capacity to phosphorylate a recombinant IKKγ substrate in vitro. We conclude that Tax-induced phosphorylation of IKKβ is required for IKKβ activation, phosphoryl group transfer to IKKγ, and acquisition of the deregulated IKK phenotype.
    Preview · Article · Aug 2001 · Journal of Biological Chemistry
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    ABSTRACT: The Tax transforming protein encoded by human T-cell leukemia virus type 1 (HTLV1) persistently activates transcription factor NF-κB and deregulates the expression of downstream genes that mediate cell cycle entry. We recently found that Tax binds to and chronically stimulates the catalytic function of IκB kinase (IKK), a cellular enzyme complex that phosphorylates and inactivates the IκB inhibitory subunit of NF-κB. We now demonstrate that the IKKβ catalytic subunit and IKKγ regulatory subunit of IKK are chronically phosphorylated in HTLV1-infected and Tax-transfected cells. Alanine substitutions at Ser-177 and Ser-181 in the T loop of IKKβ protect both of these IKK subunits from Tax-directed phosphorylation and prevent the induction of IκB kinase activity. Each of these inhibitory effects is recapitulated in Tax transfectants expressing the bacterial protein YopJ, a potent in vivo agonist of T loop phosphorylation. Moreover, ectopically expressed forms of IKKβ that contain glutamic acid substitutions at Ser-177 and Ser-181 have the capacity to phosphorylate a recombinant IKKγ substrate in vitro. We conclude that Tax-induced phosphorylation of IKKβ is required for IKKβ activation, phosphoryl group transfer to IKKγ, and acquisition of the deregulated IKK phenotype.
    No preview · Article · Jul 2001 · Journal of Biological Chemistry
  • Dean W Ballard
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    ABSTRACT: Transcription factor NF-kappaB is biochemically coupled to the T cell antigen receptor (TCR) and activated transiently during an adaptive immune response. The author's laboratory is investigating the signal-dependent regulation of NF-kappaB, its downstream gene targets, and its function in lymphocyte biology. Our studies have revealed novel enzymatic checkpoints in the NF-kappaB signaling pathway and constitutive repressors of NF-kappaB that might be clinically applicable for therapeutic control of the immune system. We have also found that the Tax transforming protein encoded by human T cell leukemia virus type 1 (HTLV1) binds to and persistently activates an inducible protein kinase in the TCR/NF-kappaB axis. This viral/host interaction appears to trigger the inappropriate expression of NF-kappaB and the development of HTLV1-associated disease.
    No preview · Article · Feb 2001 · Immunologic Research
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    ABSTRACT: The immunoglobulin superfamily member CD83 is expressed on the surface of mature dendritic cells that present processed antigens to T lymphocytes. In addition, T cells acquire CD83 expression following mitogenic stimulation in vitro. Here we report two lines of evidence demonstrating that this inducible lymphocyte response is genetically programmed by transcription factor NF-κB and contingent upon proteolytic breakdown of its cytoplasmic inhibitor IκBα. First, signal-dependent induction of CD83 mRNA expression is blocked in both transformed and primary T cells harboring a degradation-resistant mutant of IκBα that constitutively represses NF-κB. Second, as revealed in gel retardation assays, the IκBα constitutive repressor prevents the inducible interaction of NF-κB with consensus recognition sites identified in the CD83 promoter. Given that IκBα is functionally coupled to the T-cell antigen receptor, these findings suggest that the downstream transcription unit for CD83 is triggered by NF-κB during an adaptive immune response.
    No preview · Article · Sep 2000 · Molecular Immunology
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    ABSTRACT: Mutations in the gene encoding Bruton's tyrosine kinase (btk) cause the B cell deficiency diseases X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (xid) in mice. In vivo and in vitro studies indicate that the BTK protein is essential for B cell survival, cell cycle progression, and proliferation in response to B cell antigen receptor (BCR) stimulation. BCR stimulation leads to the activation of transcription factor nuclear factor (NF)-kappaB, which in turn regulates genes controlling B cell growth. We now demonstrate that a null mutation in btk known to cause the xid phenotype prevents BCR-induced activation of NF-kappaB. This defect can be rescued by reconstitution with wild-type BTK. This mutation also interferes with BCR-directed activation of IkappaB kinase (IKK), which normally targets the NF-kappaB inhibitor IkappaBalpha for degradation. Taken together, these findings indicate that BTK couples IKK and NF-kappaB to the BCR. Interference with this coupling mechanism may contribute to the B cell deficiencies observed in XLA and xid.
    Full-text · Article · Jun 2000 · Journal of Experimental Medicine
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    ABSTRACT: Reovirus infection induces apoptosis in cultured cells and in vivo. To identify host cell factors that mediate this response, we investigated whether reovirus infection alters the activation state of the transcription factor nuclear factor kappa B (NF-kappaB). As determined in electrophoretic mobility shift assays, reovirus infection of HeLa cells leads to nuclear translocation of NF-kappaB complexes containing Rel family members p50 and p65. Reovirus-induced activation of NF-kappaB DNA-binding activity correlated with the onset of NF-kappaB-directed transcription in reporter gene assays. Three independent lines of evidence indicate that this functional form of NF-kappaB is required for reovirus-induced apoptosis. First, treatment of reovirus-infected HeLa cells with a proteasome inhibitor prevents NF-kappaB activation following infection and substantially diminishes reovirus-induced apoptosis. Second, transient expression of a dominant-negative form of IkappaB that constitutively represses NF-kappaB activation significantly reduces levels of apoptosis triggered by reovirus infection. Third, mutant cell lines deficient for either the p50 or p65 subunits of NF-kappaB are resistant to reovirus-induced apoptosis compared with cells expressing an intact NF-kappaB signaling pathway. These findings indicate that NF-kappaB plays a significant role in the mechanism by which reovirus induces apoptosis in susceptible host cells.
    Full-text · Article · May 2000 · Journal of Virology

Publication Stats

11k Citations
689.50 Total Impact Points

Institutions

  • 1993-2015
    • Vanderbilt University
      • • Department of Pathology, Microbiology and Immunology
      • • Department of Medicine
      Нашвилл, Michigan, United States
  • 2006
    • Texas A&M University - Galveston
      Galveston, Texas, United States
  • 1987-2000
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
    • Yale University
      New Haven, Connecticut, United States
  • 1999
    • Nagasaki University
      • Institute of Tropical Medicine
      Nagasaki, Nagasaki, Japan
  • 1995
    • Harvard University
      • Department of Molecular and Cell Biology
      Cambridge, Massachusetts, United States
  • 1989
    • IST Austria
      Klosterneuberg, Lower Austria, Austria
    • Mount Sinai Medical Center
      New York, New York, United States
  • 1982-1989
    • University of Illinois, Urbana-Champaign
      • Department of Microbiology
      Urbana, Illinois, United States