Activation of noncanonical NF-kappaB signaling by the oncoprotein Tio

Abstract

NF-kappaB transcription factors are key regulators of cellular proliferation and frequently contribute to oncogenesis. The herpesviral oncoprotein Tio, which promotes growth transformation of human T cells in a recombinant herpesvirus saimiri background, potently induces canonical NF-kappaB signaling through membrane recruitment of the ubiquitin ligase tumor necrosis factor receptor-associated factor 6 (TRAF6). Here, we show that, in addition to Tio-TRAF6 interaction, the Tio-induced canonical NF-kappaB signal requires the presence of the regulatory subunit of the inhibitor of kappaB kinase (IKK) complex, NF-kappaB essential modulator (NEMO), and the activity of its key kinase, IKKbeta, to up-regulate expression of endogenous cellular inhibitor of apoptosis 2 (cIAP2) and interleukin 8 (IL-8) proteins. Dependent on TRAF6 and NEMO, Tio enhances the expression of the noncanonical NF-kappaB proteins, p100 and RelB. Independent of TRAF6 and NEMO, Tio mediates stabilization of the noncanonical kinase, NF-kappaB-inducing kinase (NIK). Concomitantly, Tio induces efficient processing of the p100 precursor molecule to its active form, p52, as well as DNA binding of nuclear p52 and RelB. In human T cells transformed by infection with a Tio-recombinant virus, sustained expression of p100, RelB, and cIAP2 depends on IKKbeta activity, yet processing to p52 remains largely unaffected by IKKbeta inhibition. However, long term inhibition of IKKbeta disrupts the continuous growth of the transformed cells and induces cell death. Hence, the Tio oncoprotein triggers noncanonical NF-kappaB signaling through NEMO-dependent up-regulation of p100 precursor and RelB, as well as through NEMO-independent generation of p52 effector.

FIGURE 1.

NF-κB-driven luciferase reporter activity and induction of endogenous protein expression by pEF1-Tio. Jurkat T cells were electroporated with expression plasmids coding for FLAG-tagged Tio or Tio mutants (mT6b, Y136F, mSH3b, mT6b-mSH3b) and were harvested 48 h after transfection. Transfection of pEF1 vector DNA (vec) served as a negative control. An average of at least three independent experiments is shown. A, co-transfection of an NF-κB luciferase reporter. The relative NF-κB activity is depicted in percent, and the average of Tio-transfected samples was set to 100%. B, immunoblot analysis of whole cell lysates for endogenous cIAP2 protein expression. Protein expression of Tio was verified with a FLAG antibody. An unspecific band was used as loading control (lc). C, endogenous IL-8 in supernatants of transfected cells detected by ELISA.

FIGURE 2.

Induction of an NF-κB-responsive CD14 reporter by pEF1-Tio in the presence or absence of NEMO. A, Jurkat T cells (NEMO⁺ and NEMO⁻), stably transduced with an NF-κB-driven CD14 reporter, were transfected with Tio and NEMO expression constructs, alone or in combination. Vector-transfected cells served as a negative control. CD14 surface expression was detected 48 h after transfection with a phycoerythrin-Cy5-labeled CD14 antibody by flow cytometry. B, NEMO⁻ Jurkat T cells were transfected with Tio or Tio mutants (mT6b, Y136F, mSH3b, mT6b-mSH3b) alone or in combination with a NEMO expression plasmid. Whole cell lysates were prepared 48 h after transfection and analyzed by immunoblotting for expression of endogenous cIAP2. Expression of FLAG-Tio and NEMO was verified. Hsp90α/β expression served as a loading control. vec, vector.

FIGURE 3.

Processing of p100 to p52 and expression of endogenous RelB in presence and absence of NEMO. A, NEMO⁻ Jurkat T cells were transfected with Tio or mutant expression plasmids. NEMO was reconstituted by co-transfection of a NEMO expression construct. Immunoblot analysis was performed 48 h after transfection for endogenous p100/p52 and RelB. Tio expression was confirmed with a FLAG antibody, and NEMO was detected by a specific antiserum. Hsp90α/β expression served as loading control. vec, vector. B, experiment analogous to A was performed in the absence of NEMO. A NEMO-transfected sample served as positive expression control.

FIGURE 4.

DNA binding of Tio-induced nuclear p52 and RelB. Oligonucleotide pulldown was performed from nuclear extracts of Tio-transfected NEMO⁺ and NEMO⁻ Jurkat T cells. Double-stranded biotinylated oligonucleotides, derived from the HIV LTR and the human IFNβ promoter, were incubated with nuclear extracts in the presence or absence of unbiotinylated competitor oligonucleotide (comp). Proteins bound to the biotinylated probes were captured from the lysate with streptavidin-coupled Sepharose beads and subjected to immunoblot analysis with p52 and RelB antibodies. Input, untreated nuclear lysate; nc, streptavidin-Sepharose beads incubated with lysate in the absence oligonucleotides served as negative control.

FIGURE 5.

Stabilization of NIK in Jurkat T cells and virus-transformed human T cells. A, Jurkat T cells (NEMO⁺ and NEMO⁻) were transfected with Tio or mutant expression plasmids. Cells were lysed after 48 h, and endogenous NIK was enriched by immunoprecipitation (IP). Immunoblot analysis was performed to detect NIK. Depleted lysates (post IP) and a polyclonal rabbit antiserum were used to confirm Tio expression. Hsp90α/β expression served as loading control. vec, vector. B, experiment analogous to A was performed using virus-transformed PBLs (1763 YYYY, 1765 YYYY, 1766 YYYY). Jurkat T cells served as negative controls.

FIGURE 6.

Effects of IKKβ inhibition on Tio-induced NF-κB reporter activity and IL-8 expression in Jurkat T cells. Jurkat T cells were transfected with Tio expression plasmid and subsequently incubated with the indicated concentrations of IKKβ inhibitor, ACHP, starting 4 h after transfection. Cells were harvested 48 h after transfection. Three independent experiments were performed. A, co-transfection of an NF-κB reporter. NF-κB activity is depicted as relative response ratio with reference to the average of untreated Tio-transfected cells. vec, vector. B, determination of IL-8 levels in the supernatants by ELISA. C, protein expression of Tio verified using a FLAG-antibody. Detection of Hsp90α/β expression served as loading control.

FIGURE 7.

Survival of human T cells transformed with Tio-recombinant herpesvirus saimiri in the presence of IKKβ inhibitor. Human PBLs transformed with Tio-recombinant herpesvirus saimiri (1763 YYYY, 1765 YYYY, and 1766 YYYY) were treated with the indicated concentrations of IKKβ inhibitor, ACHP. Jurkat T cells served as negative control. Every 24 h cells were harvested, stained with propidium iodide (PI, 10 μg/ml) and subjected to flow cytometry. Inhibitor was added freshly every 24 h. Propidium iodide-negative cells are depicted in percent of the total population. A, time response curves for 4 days treatment. B, end point analysis at day 4.

FIGURE 8.

Expression of p100/p52, RelB, and cIAP2 in human T cells transformed with Tio-recombinant herpesvirus saimiri in the presence of IKKβ inhibitor. Virus-transformed human PBLs (1763 YYYY, 1765 YYYY, and 1766 YYYY) were treated with 2.5 μm IKKβ inhibitor ACHP for 48 h. A, immunoblot analysis was performed to detect endogenous p100, RelB, and cIAP2 expression and p100 processing. Tio expression was verified using a polyclonal rabbit anti-Tio serum. Hsp90α/β expression served as a loading control; pc, whole cell lysate from Tio-transfected Jurkat T cells served as positive control. B, densitometric quantification of p100 and p52 protein levels was performed. The ratio of p100 to p52 expression was calculated as ((p100 sample)/(p52 sample))/((p100 untreated)/(p52 untreated)) for each cell line.