Shao Cong Sun

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.

Regulation of the NF-κB-inducing Kinase by Tumor Necrosis Factor Receptor-associated Factor 3-induced Degradation

The NF-κB family of transcription factors plays a pivotal role in regulation of diverse biological processes, including immune responses, cell growth, and apoptosis. Activation of NF-κB is mediated by both canonical and noncanonical signaling pathways. Although the canonical pathway has been extensively studied, the mechanism mediating the noncanonical pathway is still poorly understood. Recent studies have identified the NF-κB-inducing kinase (NIK) as a key component of the noncanonical pathway of NF-κB activation; however, how the signaling function of NIK is regulated remains unknown. We report here that one important mechanism of NIK regulation is through its dynamic interaction with the tumor necrosis factor receptor-associated factor 3 (TRAF3). TRAF3 physically associates with NIK via a specific sequence motif located in the N-terminal region of NIK; this molecular interaction appears to target NIK for degradation by the proteasome. Interestingly, induction of noncanonical NF-κB signaling by extracellular signals involves degradation of TRAF3 and the concomitant enhancement of NIK expression. These results suggest that induction of noncanonical NF-κB signaling may involve the rescue of NIK from TRAF3-mediated negative regulation.

IKKγ Serves as a Docking Subunit of the IκB Kinase (IKK) and Mediates Interaction of IKK with the Human T-cell Leukemia Virus Tax Protein

The tax gene product of human T-cell leukemia virus type I induces activation of transcription factor NF-κB, which contributes to deregulated expression of various cellular genes. Tax expression triggers persistent phosphorylation and degradation of the NF-κB inhibitory proteins IκBα and IκBβ, resulting in constitutive nuclear expression of NF-κB. Recent studies demonstrate that Tax activates the IκB kinase (IKK), although the underlying mechanism remains unclear. In this report, we show that Tax physically interacts with a regulatory component of the IKK complex, the NF-κB essential modulator or IKKγ (NEMO/IKKγ). This molecular interaction appears to be important for recruiting Tax to the IKK catalytic subunits, IKKα and IKKβ. Expression of NEMO/IKKγ greatly promotes binding of Tax to IKKα and IKKβ and stimulates Tax-mediated IKK activation. Interestingly, a mutant form of Tax defective in IKK activation exhibited a markedly diminished level of NEMO/IKKγ association. These findings suggest that the physical interaction of Tax with NEMO/IKKγ may play an important role in Tax-mediated IKK activation.

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.

Bcl-2 Prevents CD95 (Fas/APO-1)-induced Degradation of Lamin B and Poly(ADP-ribose) Polymerase and Restores the NF-κB Signaling Pathway

In the study presented here, we investigated the possible interactions between CD95 (Fas/APO-1) and Bcl-2 by studying the effects of Bcl-2 on the modulation of cellular pathways activated by CD95 using HeLa cells as a model system. We report that stable expression of Bcl-2 in HeLa cells is associated with multiple phenotypic changes. Treatment of HeLa cells with anti-CD95 monoclonal antibody (mAb) resulted in preferential degradation of lamin B compared with lamins A and C. Significant lamin B degradation was detected as early as 1 h after anti-CD95 mAb treatment. In contrast, lamins A and C as well as actin remained unchanged until 4 h after treatment with anti-CD95 mAb, a time point that correlated with the period of DNA fragmentation. These results indicate that selective degradation of lamin B is an early cellular event in response to activation of the CD95 pathway and that it precedes DNA fragmentation. Overexpression of Bcl-2 resulted in prevention of lamin B degradation and DNA fragmentation into oligonucleosome fragments in response to the apoptotic signal by anti-CD95 mAb. In addition, in Bcl-2-overexpressing cells that were protected against apoptosis, anti-CD95 mAb-induced cleavage of poly(ADP-ribose) polymerase was completely blocked. Overexpression of Bcl-2 also resulted in restoration of the CD95-mediated signaling pathway involving activation of the transcription factor NF-κB (p50/RelA). These findings suggest that Bcl-2 prevents apoptosis in part by preventing the degradation of major nuclear polypeptides such as lamin B and poly(ADP-ribose) polymerase. In addition, our results demonstrate that CD95-mediated signaling involves activation of NF-κB (p50/RelA).

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.

Regulation of the interleukin-2 CD28-responsive element by NF-ATp and various NF-kappaB/Rel transcription factors.

The CD28 costimulatory signal enhances antigen-mediated induction of interleukin-2 (IL-2) gene transcription through activation of an enhancer termed the CD28-responsive element (CD28RE). Although various nuclear proteins have been shown to bind to CD28RE, their in vivo functions in the regulation of this enhancer remain elusive. In this report, we show that CD28RE binds distinct transcription factors in cells treated with different mitogenic stimuli. Stimulation of the T-cell receptor (TCR) complex in the absence of a CD28 costimulatory signal induces a member of the nuclear factor of the activated T cells, NF-ATp; however, this treatment fails to activate the CD28RE enhancer activity. Significant activation of CD28RE was detected when the cells were treated with both the TCR stimulators and an anti-CD28 monoclonal antibody (anti-CD28), which induces the NF-kappaB/Rel enhancer binding proteins in addition to NF-ATp. The costimulatory activity of anti-CD28 can be further enhanced by a phorbol ester. Kinetic analyses demonstrate that activation of endogenous IL-2 gene transcription is correlated with the binding of CD28RE by NF-ATp and different NF-kappaB/Rel species. Transient-transfection studies reveal that expression of either NF-ATp or the p50-RelA NF-kappaB heterodimer leads to the potent transactivation of both the CD28RE enhancer and the intact IL-2 promoter in mitogen-stimulated cells. Remarkably, coexpression of these two families of enhancer-binding proteins in Jurkat T cells results in the transactivation of the CD28RE enhancer even in the absence of any cellular stimuli. Together, these results suggest that activation of IL-2 gene transcription by the TCR- and CD28-mediated signals involves the interaction of CD28RE with NF-ATp and various NF-kappaB/Rel transcription factors.

Gene expression profiles in HTLV-I-immortalized T cells: deregulated expression of genes involved in apoptosis regulation

Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia, an acute and often fatal T-cell malignancy. A key step in HTLV-I-induced leukemigenesis is induction of abnormal T-cell growth and survival. Unlike antigen-stimulated T cells, which cease proliferation after a finite number of cell division, HTLV-I-infected T cells proliferate indefinitely (immortalized), thus facilitating occurrence of secondary genetic changes leading to malignant transformation. To explore the molecular basis of HTLV-I-induced abnormal T-cell survival, we compared the gene expression profiles of normal and HTLV-I-immortalized T cells using `gene array. These studies revealed a strikingly altered expression pattern of a large number of genes along with HTLV-I-mediated T-cell immortalization. Interestingly, many of these deregulated genes are involved in the control of programmed cell death or apoptosis. These findings indicate that disruption of the cellular apoptosis-regulatory network may play a role in the HTLV-I-mediated oncogenesis.

IκB kinases serve as a target of CD28 signaling

Optimal T cell activation and interleukin-2 production requires a second signal in addition to antigen-mediated T cell receptor (TCR) signaling. The CD28 molecule has been demonstrated to act as an effective costimulatory molecule upon binding by B7.1 or B7.2 present on antigen-presenting cells. The CD28 signal acts in concert with the TCR signal to significantly augment activation of the NF-κB family of transcription factors. The interleukin-2 gene is regulated by NF-κB among other transcription factors, in part, via a CD28 responsive element (CD28RE) present in the IL-2 promoter. Enhanced activation of NF-κB by CD28 is mediated by rapid phosphorylation and proteasome-mediated degradation of the NF-κB inhibitory proteins IκBα and IκBβ, which allows for accelerated nuclear expression of the liberated NF-κB. Herein, we provide evidence that the catalytic activities of two recently identified IκB kinases, IKKα and IKKβ, are significantly elevated when T cells are stimulated through CD28 in addition to mitogen treatment. Catalytically inactive forms of IKKs are able to block the in vivo phosphorylation of IκBα induced by mitogen and CD28. Furthermore, CD28-mediated reporter gene transactivation of the CD28RE/AP-1 composite element is consistently attenuated by the IKK mutants. These findings suggest that cellular signaling pathways initiated at the TCR and CD28 converge at or upstream of IKK, resulting in more robust kinase activity and enhanced and prolonged NF-κB activation.