J Biochem. 2009 Dec;146(6):821-32. doi: 10.1093/jb/mvp127. Epub 2009 Aug 12.

Ro52-mediated monoubiquitination of IKK{beta} down-regulates NF-{kappa}B signalling.

Source

Department of Cardiology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA.

Abstract

Upon activation, NF-kappaB translocates into the nucleus and initiates biological events. This NF-kappaB signalling is mainly regulated by the protein kinase IKKbeta. Early in this signalling pathway, IKKbeta is phosphorylated for activation by several factors, such as pro-inflammatory cytokines and the Tax oncoprotein of HTLV-1. In cells infected by HTLV-1, IKKbeta is persistently phosphorylated and conjugated with monoubiquitin due to Tax expression. Although this Tax-induced monoubiquitination appears to be an important regulation system for IKKbeta, how the monoubiquitination occurs is unknown and its role in NF-kappaB signalling is still unclear. Here, we show that an E3-ubiquitin ligase Ro52 interacts weakly with wild-type IKKbeta but strongly with a phosphomimetic mutant IKKbeta to conjugate monoubiquitin in cooperation with an E2-ubiquitin-conjugating enzyme UbcH5B. These results suggest that the Tax-induced phosphorylation of IKKbeta causes an interaction with Ro52 for the subsequent monoubiquitination. NF-kappaB reporter assays have shown that the IKKbeta activity is suppressed by wild-type Ro52, but not by its inactive mutant. In addition, monoubiquitin fusion of IKKbeta reduced its activity for NF-kappaB signalling. We also found that Ro52 dramatically reduces the level of Tax. These results suggest that Ro52 down-regulates Tax-induced NF-kappaB signalling by monoubiquitinating IKKbeta and by reducing the level of Tax.

PMID:: 19675099; [PubMed - indexed for MEDLINE]
PMCID:: PMC2917061

Free PMC Article

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Fig. 6.

In vitro monoubiquitination of IKKβ. (A) UbcH5B-dependent monoubiquitination of IKKβ. GST-fused IKKβ.SE was immobilized on beads and incubated in the reaction mixture containing RH-tagged ubiquitin, recombinant E1 enzyme and various poly-His-tagged recombinant E2 enzymes (UbcH2, UbcH5B, UbcH7, UbcH10 and hCDC34). After this reaction, GST-IKKβ.SE on the beads was washed to remove the reaction mixture and solubilized in SDS-treating solution. GST-IKKβ.SE was then analysed by western blotting using anti-RH antibody to detect ubiquitinated GST-IKKβ.SE (upper panel) and anti-GST antibody to detect both unubiquitinated and ubiquitinated GST-IKKβ.SE (lower panel). Molecular size markers are shown on the left in kilodaltons. (B) Minimum requirements for the in vitro monoubiquitination of IKKβ. In the in vitro ubiquitination assay shown above, the complete reaction mixture contained RH-ubiquitin, E1 enzyme, UbcH5B as an E2 enzyme and Ro52 as an E3 enzyme. To determine the minimum requirements for the in vitro monoubiquitination of IKKβ, GST-IKKβ.SE immobilized on beads was incubated in the incomplete reaction mixture lacking one of these components (lanes 1–4). As a positive control, GST-IKKβ.SE immobilized on beads was incubated in the complete reaction mixture (lane 5). As a negative control, glutathione-sepharose beads alone without immobilization of GST-IKKβ.SE were incubated in the complete reaction mixture (lane 6). After the reaction, the beads were washed and treated in SDS-containing solution to solubilize GST-IKKβ.SE. Then, GST-IKKβ.SE was analysed by western blotting using anti-RH antibody to detect ubiquitinated GST-IKKβ.SE (upper panel) and anti-GST antibody to detect both unubiquitinated and ubiquitinated GST-IKKβ.SE (lower panel). The incomplete reaction mixture shown in lanes 1, 2, 3 and 4 lacked ubiquitin, E1 enzyme, UbcH5B and Ro52, respectively.

Ro52-mediated Monoubiquitination of IKK? Down-regulates NF-?B Signalling

J Biochem. 2009 December;146(6):821-832.

Fig. 8.

NF-κB reporter assays. (A) Role of Ro52 in Tax-induced NF-κB signalling. Tax (FLAG-tagged) was co-expressed with wild-type Ro52 or its C16A mutant (RH-tagged) in HeLa cells transfected with the NF-κB reporter plasmid. Fifteen hours after transfection, the cell extract was prepared and assayed for luciferase activity. Each bar represents the mean ± standard error (*P < 0.01, ^**P > 0.05) (upper panel). Exogenously expressed proteins for the reporter assay were analysed by western blotting using anti-Tax antibody to detect Tax and anti-RH antibody to detect Ro52-RH (lower panel). (B) Enhanced activity of IKKβ mutant (SE) to promote NF-κB signalling. Empty vector or RH-tagged IKKβ (wild-type or SE mutant) was expressed in HEK293 cells transfected with the NF-κB reporter plasmid. Fifteen hours after transfection, the cell extract was prepared and assayed for luciferase activity. Each bar represents the mean ± standard error (upper panel). Exogenously expressed RH-IKKβ (wild-type and SE mutant) for the reporter assay were analysed by western blotting using anti-RH antibody (lower panel). (C) Role of Ro52 in IKKβ-mediated activation of NF-κB signalling. RH-tagged IKKβ (wild-type or SE mutant) was co-expressed with empty vector or FLAG-tagged Ro52 (wild-type or C16A mutant) in HEK293 cells transfected with the NF-κB reporter plasmid. Fifteen hours after transfection, cell extracts were prepared and assayed for luciferase activity. Each bar represents the mean ± standard error (*P < 0.01) (upper panel). Exogenously expressed proteins for the reporter assay were analysed by western blotting using anti-RH antibody to detect RH-IKKβ and anti-FLAG antibody to detect FLAG-Ro52 (lower panel). (D) Effect of monoubiquitin fusion to IKKβ on IKKβ-mediated activation of NF-κB signalling. To determine the effect of monoubiquitin fusion, wild-type IKKβ (control), IKKβ.SE or monoubiquitin-fused IKKβ.SE was expressed with empty vector, Ro52 (wild type) or Ro52 (C16A) in HEK293 cells transfected with the NF-κB reporter plasmid. Fifteen hours after transfection, cell extracts were prepared and assayed for luciferase activity. Each bar represents the mean ± standard error (*P < 0.01) (upper panel). Exogenously expressed RH-tagged IKKβ (wild-type, SE mutant and monoubiquitin-fused SE mutant) for the reporter assay were analysed by western blotting (lower panel).

Ro52-mediated Monoubiquitination of IKK? Down-regulates NF-?B Signalling

J Biochem. 2009 December;146(6):821-832.