Gutian Xiao

NF-κB-Inducing Kinase Regulates the Processing of NF-κB2 p100

Processing of the nf(kappa)b2 gene product p100 to generate p52 is an important step in NF-kappaB regulation. We show that this step is negatively regulated by a processing-inhibitory domain (PID) within p100 and positively regulated by the NF-kappaB-inducing kinase (NIK). While the PID suppresses the constitutive processing of p100, NIK induces p100 processing by stimulating site-specific phosphorylation and ubiquitination of this precursor protein. Further, a natural mutation of the gene encoding NIK in alymphoplasia (aly) mice cripples the function of NIK in p100 processing, causing a severe defect in p52 production. These data suggest that NIK is a specific kinase regulating p100 processing and explain why the aly and nf(kappa)b2 knockout mice exhibit similar immune deficiencies.

Retroviral oncoprotein Tax induces processing of NF‐κB2/p100 in T cells: evidence for the involvement of IKKα

IκB kinase (IKK) is a key mediator of NF‐κB activation induced by various immunological signals. In T cells and most other cell types, the primary target of IKK is a labile inhibitor of NF‐κB, IκBα, which is responsible for the canonical NF‐κB activation. Here, we show that in T cells infected with the human T‐cell leukemia virus (HTLV), IKKα is targeted to a novel signaling pathway that mediates processing of the nfκb2 precursor protein p100, resulting in active production of the NF‐κB subunit, p52. This pathogenic action is mediated by the HTLV‐encoded oncoprotein Tax, which appears to act by physically recruiting IKKα to p100, triggering phosphorylation‐dependent ubiquitylation and processing of p100. These findings suggest a novel mechanism by which Tax modulates the NF‐κB signaling pathway.

NF-κB-inducing Kinase and IκB Kinase Participate in Human T-cell Leukemia Virus I Tax-mediated NF-κB Activation

The tax gene product of human T-cell leukemia virus I induces aberrant expression of various cellular genes, which contributes to transformation of host cells. Induction of many Tax target genes is mediated through transcription factor NF-κB. Here we show that Tax triggers activation of cellular protein kinases, IκB kinase α (IKKα) and IKKβ, which phosphorylate the NF-κB inhibitory protein IκBα, resulting in its degradation and NF-κB activation. Constitutive IKK activation occurs in both Tax-transfected and human T-cell leukemia virus I-infected T cells. We further demonstrate that Tax-mediated NF-κB signaling also requires the NF-κB-inducing kinase (NIK). Consistently, inactive forms of either IKKs or NIK attenuate Tax-mediated NF-κB activation. Therefore, Tax activates NF-κB by targeting cellular signaling molecules, including both IKKs and NIK.

Somatic mutagenesis studies of NF-κB signaling in human T cells : evidence for an essential role of IKKγ in NF-κB activation by T-cell costimulatory signals and HTLV-I Tax protein

NF-κB plays a pivotal role in normal T-cell activation and may also mediate human T-cell leukemia virus (HTLV)-induced T-cell transformation. Activation of NF-κB by both T-cell costimulatory signals and the HTLV Tax protein involves stimulation of IκB kinase (IKK). As a genetic approach to dissect the intermediate steps involved in NF-κB activation in human T cells, we performed somatic cell mutagenesis to isolate signaling-defective mutant Jurkat T-cell lines. One of the mutant cell lines was shown to have a specific blockade in the IKK signaling pathway but remained competent in the c-Jun N-terminal kinase and MAP kinase pathways. Interestingly, this mutant cell line lacks expression of IKKγ, a non-catalytic component of the IKK complex. Expression of exogenous IKKγ in the mutant cells restored NF-κB activation by both the T-cell costimulation agents and Tax. These findings provide genetic evidence for the requirement of IKKγ in NF-κB signaling triggered by both T-cell costimulatory signals and HTLV-I Tax protein.

Domain-specific Interaction with the IκB Kinase (IKK) Regulatory Subunit IKKγ Is an Essential Step in Tax-mediated Activation of IKK

The human T-cell leukemia virus type 1 Tax oncoprotein deregulates the NF-κB signaling pathway by persistently stimulating a key signal transducer, the IκB kinase (IKK). Tax physically associates with the IKK regulatory subunit, IKKγ, although the underlying biochemical mechanism and functional significance remain unclear. We show that the Tax-IKKγ interaction requires two homologous leucine zipper domains located within IKKγ. These leucine zipper domains are unique for the presence of a conserved upstream region that is essential for Tax binding. Site-directed mutagenesis analysis revealed that a leucine-repeat region of Tax is important for IKKγ binding. Interestingly, all the Tax mutants defective in IKKγ binding failed to engage the IKK complex or stimulate IKK activity, and these functional defects can be rescued by fusing the Tax mutants to IKKγ. These results provide mechanistic insights into how Tax specifically targets and functionally activates the cellular kinase IKK.

Activation of IKKα and IKKβ through their fusion with HTLV-I Tax protein

Human T-cell leukemia virus type I (HTLV-I) Tax protein persistently stimulates the activity of IκB kinase (IKK), resulting in constitutive activation of the transcription factor NF-κB. Tax activation of IKK requires physical interaction of this viral protein with the IKK regulatory subunit, IKKγ. The Tax/IKKγ interaction allows Tax to engage the IKK catalytic subunits, IKKα and IKKβ, although it remains unclear whether this linker function of IKKγ is sufficient for supporting the Tax-specific IKK activation. To address this question, we have examined the sequences of IKKγ required for modulating the Tax/IKK signaling. We demonstrate that when fused to Tax, a small N-terminal fragment of IKKγ, containing its minimal IKKα/β-binding domain, is sufficient for bringing Tax to and activating the IKK catalytic subunits. Disruption of the IKKα/β-binding activity of this domain abolishes its function in modulating the Tax/IKK signaling. We further demonstrate that direct fusion of Tax to IKKα and IKKβ leads to activation of these kinases. These findings suggest that the IKKγ-directed Tax/IKK association serves as a molecular trigger for IKK activation.

The NF-κB Signaling Pathway Is Not Required for Fas Ligand Gene Induction but Mediates Protection from Activation-induced Cell Death

Stimulation of T cells by antigens or mitogens triggers multiple signaling pathways leading to activation of genes encoding interleukin-2 and other growth-regulatory cytokines. The same stimuli also activate the gene encoding an apoptosis-inducing molecule, Fas ligand (FasL), which contributes to activation-induced cell death. It has been proposed that the signaling pathways involved in cytokine gene induction also contribute to activation-induced FasL expression; however, genetic evidence for this proposal is lacking. In the present study, the role of the NF-κB signaling pathway in FasL gene expression was examined using a mutant T cell line deficient in an essential NF-κB signaling component, IκB kinase γ. These mutant cells have a blockade in signal-induced activation of NF-κB but remained normal in the activation of NF-AT and AP-1 transcription factors. Interestingly, the NF-κB signaling defect has no effect on mitogen-stimulated FasL gene expression, although it completely blocks the interleukin-2 gene induction. We further demonstrate that NF-κB activation is required for protecting T cells from apoptosis induction by mitogens and an agonistic anti-Fas antibody. These genetic results suggest that the NF-κB signaling pathway is not required for activation-induced FasL expression but rather mediates cell growth and protection from activation-induced cell death.

Activation of I-κB Kinase by the HTLV Type 1 Tax Protein: Mechanistic Insights into the Adaptor Function of IKKγ

The Tax protein encoded by human T cell leukemia virus type 1 (HTLV-1) induces constitutive nuclear expression of the transcription factor NF-κB, causing aberrant expression of a large array of cellular genes. Tax activates NF-κB by stimulating the activity of the I-κB kinase (IKK), which in turn leads to phosphorylation and degradation of the NF-κB inhibitor I-κBα. In normal T cells, IKK activation occurs transiently on cellular stimulation through the T cell receptor (TCR) and the CD28 costimulatory molecule. However, this inducible kinase is constitutively activated in Tax-expressing and HTLV-1-infected T cells, which contributes to the deregulated nuclear expression of NF-κB. As a genetic approach to dissect the pathways mediating IKK activation by Tax and T cell activation signals, somatic cell mutagenesis was performed to isolate signaling-defective mutant Jurkat T cell lines. One of the mutant cell lines was shown to have a defect in NF-κB activation by both T cell mitogens and Tax. Interestingly, ...

Study of T-cell signaling by somatic cell mutagenesis and complementation cloning.

Somatic cell mutagenesis is a powerful genetic approach used in dissection of signal transduction pathways in mammalian cells. Here we describe a method that has been successfully used to identify and analyze components of the NF-kappaB signaling pathway in Jurkat T cells using the combination of somatic cell mutagenesis and a complementation cloning strategy. By treating Jurkat T cells with the chemical mutagen ICR191, mutant T-cell lines can be selected that have a deficiency in a given biological response or in the expression of a particular selectable marker. Mutant phenotypes can be rescued by retroviral-mediated delivery of cDNA libraries and subsequent selection of rescued cell clones by flow cytometric cell sorting. Cell lines reverting back to the wild-type phenotype due to the ectopic expression of the exogenous gene can then be evaluated by functional assays. The gene rendering the reversion of the mutant phenotype may be isolated by PCR using library vector-specific primers. Clearly, creation of a somatic cell genetic system can yield exciting new advances in deciphering signal transduction events by discovery of novel pathway participants.