Siva-1 promotes K-48 polyubiquitination of TRAF2 and inhibits TCR-mediated activation of NF-kappaB

Abstract

The proapoptotic protein Siva-1 plays an important role in some of the extrinsic and intrinsic apoptosis signaling pathways in cancer cells. Previously, we showed that Siva-1 inhibited the activity of the prosurvival transcription factor NF-kappaB. In the present study, upon TCR cross-linking of Jurkat T leukemia cells, we demonstrated that the inhibitory target of Siva-1 is upstream of the IKK complex in the NF-kappaB signaling pathway. Additionally, Siva-1 also suppressed the activity of another crucial transcription factor AP-1, and a common mediator of both these pathways is the adaptor protein TRAF2. Further, we observed that Siva-1 indeed interacted with TRAF2 and negatively regulated its activity by promoting K48-hnked polyubiquitination. Siva-1 specifically interacted with the ring finger domain of TRAF2, which is essential for its E3 hgase activity and its ability to subsequently activate NF-kappaB. TCR cross-linking of Jurkat T cells that lacked Siva-1 revealed significantly lowered K48- but elevated K63-ubiquitinated TRAF2 levels upon TCR cross-linking, suggesting that the differential pattern of ubiquitination in these cells essentially contributed to a robust and sustained activation of NF-kappaB. The above results demonstrated an important role for endogenous Siva-1 in negatively regulating NF-kappaB activation by targeting TRAF2.

FIGURE 1

Siva-1 acts upstream of the IKK complex in the TCR-mediated NF-κB signaling pathway. Jurkat cells were co-transfected with either Siva-1-myc and/or IKKβ vectors along with the NF-κB-luciferase reporter and β-galactosidase vectors. The inset shows immunoblots of whole-cell lysates with anti-myc antibody and anti-IKKβ antibodies. Results shown are drawn from three independent experiments. Abrogation of endogenous Siva-1 resulted in a significant increase in TCR-induced AP-1 reporter activity (p < 0.01), and, conversely, expression of Siva-1 led to a dramatic inhibition of both basal and TCR-induced AP-1 reporter activity (p < 0.001).

FIGURE 2

Siva-1 inhibits TCR-induced AP1 activity. Jurkat cells were co-transfected with either control siRNA or siSiva (A) or human Siva-1-expressing or control vector (B) along with the AP-1 luciferase reporter vector, and the Siva-1 expression was confirmed (inset). Luciferase assay was performed after 6 hours, and the normalized fold activity was plotted in comparison with unstimulated control cells. Results shown are representative of three independent experiments and are statistically significant (p < 0.01). Lysates of lentivirus-infected Jurkat cells expressing either control siRNA or siSiva-1 siRNA were immunoblotted with polyclonal anti-Siva-1 antibody to demonstrate the knockdown of endogenous Siva-1 expression in them. Actin levels serve as loading controls (C).

FIGURE 3

Siva-1 inhibits TRAF2- and NIK- but not TRAF6-induced NF-κB activity. Jurkat cells were co-transfected with control and Siva-1-myc vectors with either Flag-TRAF2 or Flag-TRAF6 (A) or myc-NIK constructs (B), along with the NF-κB-luciferase reporter vector. Cells were left unstimulated or stimulated by TCR cross-linking, and the normalized fold activity was plotted in comparison with unstimulated cells transfected with control vector. Expression levels were confirmed by immunoblotting (inset). Results shown were compiled from a minimum of three independent experiments. Co-expression of Siva-1 with TRAF2 or NIK resulted in a significant inhibition of TCR-induced activation of NF-κB (p < 0.01) and are statistically significant (p < 0.01), but not when TRAF6 was co-expressed.

FIGURE 4

Siva-1 interacts with TRAF2. 293T cells were co-transfected with either control or Siva-1-myc/His vectors along with either Flag-TRAF2 or Flag-TRAF6 constructs. Anti-flag and anti-myc antibodies were used for immunoblotting (A). 293T cells were transfected with control or myc-tagged Siva-1 vectors. Siva-1 was immunoprecipitated using anti-myc antibody, and immunoblotting was performed using anti-TRAF2 and anti-myc antibodies (B). Endogenous complexes of Siva-1 and TRAF2 were immunoprecipitated from Jurkat cells using anti-Siva-1 and anti-TRAF2 antibodies (C). Ring finger motif of TRAF2 is required for association with Siva-1. 293T cells were co-transfected with either GST or GST-Siva-1 and Flag-TRAF2 (left panel) or GST or GST-Siva-1 and Flag-TRAF2ΔN (right panel) expressing plasmids. Glutathione beads were used to precipitate GST-tagged proteins, and the complexes were separated and immunoblotted with anti-FLAG and anti-GST antibodies (D).

FIGURE 5

Exogenous expression of Siva-1 dramatically inhibits the K63- but not K48-ubiquitylated TRAF2-mediated NF-κB activation. Jurkat cells were co-transfected with either the control or Siva-1 vectors along with the TRAF2 construct and either K63-Ub- (A) or K48-Ub-expressing plasmids (B) and the NF-κB luciferase reporter vector. Luciferase assay was performed after 6 hours, and the fold activity was plotted in comparison with unstimulated control siRNA cells. Results were compiled from three independent experiments.

FIGURE 6

Siva-1-expression results in increased K48 but not K63 polyubiquitination of TRAF2. 293T cells were co-transfected with control or Siva-1 and TRAF2 constructs along with either HA-K63-Ub- (A) or HA-K48-Ub-expressing plasmids (B). Immunoprecipitation of the lysates was performed using anti-TRAF2 antibody. The complexes were separated and immunoblotted with the HA antibody to visualize the ubiquitination of TRAF2. Siva-1- and TRAF2-expression levels in the lysates were determined using anti-myc and anti-Flag antibodies.

FIGURE 7

Siva-1 knockdown T cells demonstrate decreased K48 and increased K63 ubiquitination of TRAF2. Control siRNA or siSiva Jurkat T cells were co-transfected with Flag-TRAF2 and K63-Ub (A) or K48-Ub constructs (B). After 36 hours, T cells were either left unstimulated or subjected to TCR cross-linking for 30 minutes. TRAF2 complexes from detergent cell lysates containing 0.01% SDS were collected by immunoprecipitation with anti-Flag antibody. Complexes were separated on gradient gels, and immunoblotting was performed with anti-HA and anti-TRAF2 antibodies to check ubiquitina-tion and TRAF2 levels. Data from K48-Ub-transfected cells pretreated with MG-132 for 1 hour and then subjected to TCR cross-linking are shown in the right panel of B.