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Biochemical coupling of transcription factor NF-κB to antigen and co-stimulatory receptors is required for the temporal control of T-cell proliferation. In contrast to its transitory activation during normal growth-signal transduction, NF-κB is constitutively deployed in T-cells transformed by the type 1 human T-cell leukemia virus (HTLV-1). This viral/host interaction is mediated by the HTLV-1-encoded Tax protein, which has potent oncogenic properties. As reviewed here, Tax activates NF-κB primarily via a pathway leading to the chronic phosphorylation and degradation of IκBα, a cytoplasmic inhibitor of NF-κB. To access this pathway, Tax associates stably with a cytokine-inducible IκB kinase (IKK), which contains both catalytic (IKKα and IKKβ) and noncatalytic (IKKγ) subunits. Unlike their transiently induced counterparts in cytokine-treated cells, Tax-associated forms of IKKα and IKKβ are persistently activated in HTLV-1-infected T cells. Acquisition of the deregulated IKK phenotype is contingent on the presence of IKKγ, which functions as a molecular adaptor in the assembly of pathologic Tax/IκB kinase complexes. These findings highlight a key mechanistic role for IKK in the Tax/NF-κB signaling axis and define new intracellular targets for the therapeutic control of HTLV-1-associated disease.

The inhibitor protein IκBα controls the nuclear import of the transcription factor NF-κB. The inhibitory activity of IκBα is regulated from the cytoplasmic compartment by signal-induced proteolysis. Previous studies have shown that signal-dependent phosphorylation of serine residues 32 and 36 targets IκBα to the ubiquitin-proteasome pathway. Here we provide evidence that lysine residues 21 and 22 serve as the primary sites for signal-induced ubiquitination of IκBα. Conservative Lys$\\longrightarrow$Arg substitutions at both Lys-21 and Lys-22 produce dominant-negative mutants of IκBα in vivo. These constitutive inhibitors are appropriately phosphorylated but fail to release NF-κB in response to multiple inducers, including viral proteins, cytokines, and agents that mimic antigenic stimulation through the T-cell receptor. Moreover, these Lys$\\longrightarrow$Arg mutations prevent signal-dependent degradation of IκBα in vivo and ubiquitin conjugation in vitro. We conclude that site-specific ubiquitination of phosphorylated IκBα at Lys-21 and/or Lys-22 is an obligatory step in the activation of NF-κB.

The eukaryotic transcription factor nuclear factor-kappa B (NF-κB) participates in many parts of the genetic program mediating T lymphocyte activation and growth. Nuclear expression of NF-κB occurs after its induced dissociation from its cytoplasmic inhibitor IκBα. Phorbol ester and tumor necrosis factor-alpha induction of nuclear NF-κB is associated with both the degradation of preformed IκBα and the activation of IκBα gene expression. Transfection studies indicate that the IκBα gene is specifically induced by the 65-kilodalton transactivating subunit of NF-κB. Association of the newly synthesized IκBα with p65 restores intracellular inhibition of NF-κB DNA binding activity and prolongs the survival of this labile inhibitor. Together, these results show that NF-κB controls the expression of IκBα by means of an inducible autoregulatory pathway.

RelA (p65) functions as the critical transactivating component of the heterodimeric p50-p65 NF-κB complex and contains a high-affinity binding site for its cytoplasmic inhibitor, IκBα. After cellular activation, IκBα is rapidly degraded in concert with the induced nuclear translocation of NF-κB. The present study demonstrates that tumor necrosis factor α-induced degradation of IκBα in human T cells is preceded by its rapid phosphorylation in vivo. However, these effects on IκBα result in nuclear mobilization of only a fraction of the entire cytoplasmic pool of RelA. Subsequent studies have revealed that (i) cytoplasmic RelA is stably associated not only with IκBα but also with other ankyrin motifrich proteins including the products of the NF-κB2 (p100) and NF-κB1 (p105) genes; (ii) in contrast to RelA-IκBα, RelA-p100 cytoplasmic complexes are not dissociated following tumor necrosis factor α activation; (iii) p100 functions as a potent inhibitor of RelA-mediated transcription in vivo; (iv) the interaction of RelA and p100 involves the conserved Rel homology domain of both proteins but not the nuclear localization signal of RelA, which is required for IκBα binding; (v) p100 inhibition of RelA function requires the C-terminal ankyrin motif domain, which mediates cytoplasmic retention of RelA; and (vi) as observed with IκBα, nuclear RelA stimulates p100 mRNA and protein expression. These findings thus reveal the presence of a second inducible autoregulated inhibitory pathway that helps ensure the rapid but transient action of nuclear NF-κB.

Molecular cloning of the polypeptide component of the Rel-related human p75 nucleoprotein complex has revealed its identity with the 65-kDa (p65) subunit of NF-κB. Functional analyses of chimeric proteins composed of NF-κB p65 C-terminal sequences linked to the DNA-binding domain of the yeast GAL4 polypeptide have indicated that the final 101 amino acids of NF-κB p65 comprise a potent transcriptional activation domain. Transient transfection of human T cells with an expression vector encoding NF-κB p65, but not NF-κB p50, produced marked transcriptional activation of a basal promoter containing duplicated κB enhancer motifs from the long terminal repeat of type 1 human immunodeficiency virus. These stimulatory effects of NF-κB p65 were synergistically enhanced by coexpression of NF-κB p50 but were completely inhibited by coexpression of the v-rel oncogene product. Together, these functional studies demonstrate that NF-κB p65 is a transactivating subunit of the heterodimeric NF-κB complex and serves as one cellular target for v-Rel-mediated transcriptional repression.

A sensitive DNA-protein crosslinking approach has been used to characterize four inducible T-cell proteins (50 kDa, 55 kDa, 75 kDa, and 85 kDa) that specifically bind to κB enhancer elements. Partial proteolytic mapping revealed a distinct cleavage pattern for three of these proteins. These polypeptides are sequestered as inactive precursors in the cytosol of unstimulated T cells but can be converted into active forms in vivo by phorbol ester stimulation or in vitro by detergent treatment. The induction of these proteins by phorbol ester results in a strikingly biphasic pattern of nuclear expression with the 55-kDa and 75-kDa species appearing within minutes, whereas the 50-kDa and 85-kDa species appear only several hours after cellular stimulation. These data suggest that NF-κB-binding activity may not correspond to a single polypeptide but rather a family of at least four inducible and differentially regulated DNA-binding proteins that are expressed with distinct kinetics in human T lymphocytes.

Oncogenic mutations in ras lead to constitutive activation of downstream signaling pathways that modulate the activities of transcription factors. In turn, these factors control the expression of a subset of genes responsible for neoplastic cell transformation. Recent studies suggest that transcription factor NF-kappa B contributes to cell transformation by inhibiting the cell death signal activated by oncogenic Ras. In this study, inhibition of NF-kappa B activity by forced expression of a super-repressor form of I kappa B alpha, the major inhibitor of NF-kappa B, markedly decreased the growth rate, saturation density and tumorigenicity of oncogenic H-Ras transformed rat embryo fibroblasts. Such clonally isolated cells overexpressing I kappa B alpha super-repressor not only were viable but also exhibited no sign of spontaneous apoptosis. Inhibition of NF-kappa B in these cells was functionally demonstrated by both the loss of cytokine induced DNA binding activity and a profoundly increased sensitivity to cell death in response to TNF-alpha treatment. In contrast, inhibition of NF-kappa B activity in non-transformed fibroblasts had minimal effect on growth, but rendered the cells resistant to a subsequent transformation by H-ras oncogene. Similar results were also obtained with rat intestinal epithelial cells harboring an inducible ras oncogene. Taken together, these findings suggest that NF-kappa B activity is essential for abnormal cell proliferation and tumorigenicity activated by the ras oncogene and highlight an alternative functional role for NF-kappa B in oncogenic Ras-mediated cell transformation that is distinct from its anti-apoptotic activity. Oncogene (2000) 19, 841 - 849.

We have investigated the biochemical basis for the activation of interleukin 2 receptor α -subunit (IL-2Rα ) gene expression in primary human T lymphocytes by a cytokine (tumor necrosis factor α ), a T-cell mitogen (phorbol 12-myristate 13-acetate), and the transactivator protein (Tax) from the type I human T-cell leukemia virus. Using in vivo transfection techniques specifically designed for these primary T cells in conjunction with in vitro gel retardation and DNA footprinting assays, we found that activation of the IL-2Rα promoter by each of these agents involves the induction of nuclear proteins that specifically interact with a κ B-like enhancer element (i.e., an element resembling the immunoglobulin κ -chain enhancer sequence recognized by transcription factor NF-κ B). DNA-protein crosslinking studies revealed that primary T cells express at least three different inducible DNA-binding proteins (50-55, 70-75, and 80-90 kDa) that specifically interact with this IL-2Rα κ B element.

Transcriptional activation of the human interleukin-2 (IL-2) gene, like induction of the IL-2 receptor $\\alpha $ (IL-2R$\\alpha $) gene and the type 1 human immunodeficiency virus (HIV-1), is shown to be modulated by a $\\kappa $B-like enhancer element. Mutation of a $\\kappa $B core sequence identified in the IL-2 promoter (-206 to -195) partially inhibits both mitogen- and HTLV-I Tax-mediated activation of this transcription unit and blocks the specific binding of two inducible cellular factors. These $\\kappa $B-specific proteins (80 to 90 and 50 to 55 kilodaltons) similarly interact with the functional $\\kappa $B enhancer present in the IL-2R$\\alpha $ promoter. These data suggest that these $\\kappa $B-specific proteins have a role in the coordinate regulation of this growth factor-growth factor receptor gene system that controls T cell proliferation.

Members of the NF-κ B/Rel and inhibitor of apoptosis (IAP) protein families have been implicated in signal transduction programs that prevent cell death elicited by the cytokine tumor necrosis factor α (TNF). Although NF-κ B appears to stimulate the expression of specific protective genes, neither the identities of these genes nor the precise role of IAP proteins in this anti-apoptotic process are known. We demonstrate here that NF-κ B is required for TNF-mediated induction of the gene encoding human c-IAP2. When overexpressed in mammalian cells, c-IAP2 activates NF-κ B and suppresses TNF cytotoxicity. Both of these c-IAP2 activities are blocked in vivo by coexpressing a dominant form of Iκ B that is resistant to TNF-induced degradation. In contrast to wild-type c-IAP2, a mutant lacking the C-terminal RING domain inhibits NF-κ B induction by TNF and enhances TNF killing. These findings suggest that c-IAP2 is critically involved in TNF signaling and exerts positive feedback control on NF-κ B via an Iκ B targeting mechanism. Functional coupling of NF-κ B and c-IAP2 during the TNF response may provide a signal amplification loop that promotes cell survival rather than death.