An RNA interference screen identifies the Deubiquitinase STAMBPL1 as a critical regulator of human T-cell leukemia virus type 1 tax nuclear export and NF-κB activation

Abstract

The human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein actively shuttles between the nucleus, where it interacts with transcriptional and splicing regulatory proteins, and the cytoplasm, where it activates NF-κB. Posttranslational modifications of Tax such as ubiquitination regulate its subcellular localization and hence its function; however, the regulation of Tax trafficking and NF-κB activation by host factors is poorly understood. By screening a deubiquitinating (DUB) enzyme small interfering RNA (siRNA) library, we identified the metalloprotease STAM-binding protein-like 1 (STAMBPL1) as a positive regulator of Tax-mediated NF-κB activation. Overexpression of wild-type STAMBPL1, but not a catalytically inactive mutant, enhanced Tax-mediated NF-κB activation, whereas silencing of STAMBPL1 with siRNA impaired Tax activation of both the canonical and noncanonical NF-κB signaling pathways. STAMBPL1 regulated Tax-induced NF-κB signaling indirectly by controlling Tax nuclear/cytoplasmic transport and was required for DNA damage-induced Tax nuclear export. Together, these results reveal that the deubiquitinase STAMBPL1 is a key regulator of Tax trafficking and function.

Fig 1

STAMBPL1 enhances Tax-mediated activation of canonical and noncanonical NF-κB pathways. (A) STAMBPL1 regulates Tax-mediated NF-κB activation. 293T cells were transfected with pCMV4-Tax and either control or STAMBPL1 siRNA, together with a κB-driven firefly luciferase reporter (κB-Luc) and thymidine kinase-driven Renilla luciferase reporter (pRL-TK). The luciferase activity is presented as fold induction compared to vector-transfected cells. (B) STAMBPL1 potentiates the induction of IκBα mRNA by Tax. 293T cells were transfected with the indicated expression vectors. As a positive control, cells were also treated with TNF-α (20 ng/ml) for 4 h. IκBα mRNA was detected by qRT-PCR and normalized according to control 18S rRNA. (C) Positive regulation of Tax-mediated NF-κB activation by STAMBPL1. Jurkat cells were transfected with the indicated expression vectors, together with κB-Luc and pRL-TK reporters. Cells were also treated with TNF-α (20 ng/ml) as a positive control. The luciferase activity is presented as fold induction compared to vector-transfected cells. (D and E) 293T cells were transfected with either control (CTR) or STAMBPL1 siRNA together with κB-Luc and pRL-TK plasmids. After 24 h, cells were treated with either TNF-α or IL-1β (both at 20 ng/ml) for the indicated times. The luciferase activity is presented as fold induction compared to vector-transfected cells. Error bars represent SDs of triplicate samples. (F) Overexpression of wild-type STAMBPL1, but not a catalytically inactive mutant (E292A), in HTLV-1-transformed MT-2 cells enhances the activation of NF-κB. MT-2 cells were transfected with the indicated expression vectors, together with κB-Luc and pRL-TK reporters. The luciferase activity is presented as fold induction compared to vector-transfected cells. The lysates were subjected to immunoblotting with anti-Tax (α-Tax), anti-STAMBPL1 (α-STAMBPL1), and anti-β-actin (α-β-actin). (G) STAMBPL1 is essential for Tax to activate the noncanonical NF-κB pathway. 293T cells were transfected with the indicated expression vectors. The lysates were subjected to immunoblotting with anti-p100 (α-p100), anti-Tax, anti-STAMBPL1, and anti-β-actin. (H) Knockdown of STAMBPL1 in MT-2 cells impairs IκBα phosphorylation and degradation. MT-2 cells were transfected with scrambled control or STAMBPL1 siRNA. After 48 h, the cells were lysed and subjected to SDS-PAGE and immunoblotting using anti-IκBα (α-IκBα), anti-phospho-IκBα (α-P-IκBα), anti-Tax, anti-STAMBPL1, and anti-β-actin. *, P < 0.05; **, P < 0.01.

Fig 2

STAMBPL1 stabilizes Tax protein and inhibits its K48-linked polyubiquitination. (A) Tax is degraded in the absence of STAMBPL1. 293T cells were transfected with pCMV4-Tax and either control (siCTR) or STAMBPL1 (siSTAMBPL1) siRNA. After 24 h cells were treated with CHX and/or MG-132 (10 μM) for the indicated times. Cells were lysed and subjected to SDS-PAGE and immunoblotting using anti-Tax (α-Tax), anti-STAMBPL1 (α-STAMBPL1), and anti-β-actin (α-β-actin). (B) STAMBPL1 stabilizes Tax in HTLV-1-transformed cells. MT-2 cells were transfected with control, STAMBPL1, or ELKS siRNA. After 48 h the cells were treated with CHX and/or MG-132 (10 μM) for the indicated times, lysed, and subjected to SDS-PAGE and immunoblotting using anti-Tax, anti-STAMBPL1, and anti-β-actin. (C) STAMBPL1 inhibits K48-linked polyubiquitination of Tax. 293T cells were transfected with the indicated expression vectors and either control (siCTR) or STAMBPL1 (siSTAMBPL1) siRNA. After 24 h, cells were treated with MG-132 (10 μM) for 3 h. Cells were lysed and subjected to immunoprecipitation with anti-Flag and immunoblotting using anti-HA (α-HA). The lysates were subjected to immunoblotting with anti-Tax, anti-STAMBPL1, anti-HA, and anti-β-actin.

Fig 3

STAMBPL1 is required for the nuclear export of Tax. (A) STAMBPL1 regulates the nuclear export of Tax. 293T cells were seeded on poly-l-lysine-coated coverslips and transfected with Flag-Tax and either control (siCTR) or STAMBPL1 (siSTAMBPL1) siRNA. Cells were irradiated with UV light (30 J/m2) for 30 min and immunostained with anti-Tax. The coverslips were subjected to confocal microscopy to visualize Tax (red) and nuclei using DAPI (blue). (B) Quantitation of Tax immunostaining experiments. Cytoplasmic Tax (either completely cytoplasmic or cytoplasmic/nuclear) was quantitated by enumerating 100 cells for each of the indicated experimental conditions, and is presented as the percentage of cytoplasmic Tax. (C) STAMBPL1 inhibits Tax-induced HTLV-1–LTR transcriptional activity. 293T cells were transfected with pCMV4-Tax and either control or STAMBPL1 siRNA together with an HTLV-1–LTR-driven firefly luciferase reporter (pU3R-Luc) and pRL-TK. Cells were also treated with UV light (30 J/m2) as indicated. The luciferase activity is presented as fold induction compared to vector-transfected cells. (D) STAMBPL1 is predominantly localized in the nucleus. 293T cells were seeded on poly-l-lysine-coated coverslips and transfected with Flag-Tax and Flag-HA-STAMBPL1. Cells were immunostained with anti-HA (α-HA). The coverslips were subjected to confocal microscopy to visualize STAMBPL1 (red) and nuclei (blue) using DAPI. Cell lysates were also subjected to immunoblotting using anti-HA, anti-Tax (α-Tax), and anti-β-actin (α-β-actin). NT, not transfected. *, P < 0.05; **, P < 0.01.

Fig 4

Tax localization in the cis-Golgi is dependent on STAMBPL1. 293T cells were seeded on poly-l-lysine-coated coverslips and transfected with GFP-Tax and either control or STAMBPL1 siRNA. Cells were irradiated with UV light (30 J/m²) for 30 min and subjected to immunostaining using anti-GM-130. Coverslips were analyzed by confocal microscopy to visualize GFP-Tax (green), GM-130 (red), and nuclei (blue) using DAPI. Cell lysates were subjected to immunoblotting using anti-STAMBPL1 (α-STAMBPL1) and anti-Tax (α-Tax). NT, not transfected; NS, nonspecific band.

Fig 5

A proteasome inhibitor rescues Tax degradation in the nucleus elicited by STAMBPL1 knockdown. (A) A proteasome inhibitor rescues degradation of nuclear Tax. 293T cells were seeded on poly-l-lysine-coated coverslips and transfected with pCMV4-Tax and either control (CTR) or STAMBPL1 siRNA. Cells were then irradiated with UV light (30 J/m²) and/or treated with MG-132 (10 μM) and then processed for immunostaining using anti-Tax (α-Tax). Coverslips were analyzed by confocal microscopy to visualize Tax (red) and nuclei (blue) using DAPI. Cell lysates were subjected to immunoblotting using anti-STAMBPL1, anti-Tax and anti-β-actin (α-β-actin). (B) Knockdown of STAMBPL1 with siRNA inhibits Tax nuclear/cytoplasmic transport. 293T cells were transfected with the indicated expression vectors and either control or STAMBPL1 siRNA, and after 24 h, cells were irradiated with UV light (30 J/m²) and/or treated with MG-132 (10 μM). Cells were lysed and subjected to nuclear (N) and cytoplasmic (C) subcellular fractionation and immunoblotting using anti-Tax, anti-STAMBPL1 (α-STAMBPL1), anti-TATA-binding protein (α-TATA BP), and anti-β-actin. NT, not transfected.

Fig 6

STAMBPL1 indirectly stabilizes Tax by promoting its nuclear export. LMB blocks the shuttling of Tax to the cytoplasm mediated by wild-type STAMBPL1, but not by the mutant STAMBPL1 E292A. 293T cells were seeded on poly-l-lysine-coated coverslips and transfected with GFP-Tax, STAMBPL1, or STAMBPL1 E292A. Cells were then irradiated with UV light (30 J/m²) and/or treated with MG-132 (10 μM) and LMB (40 ng/ml) and then processed for immunostaining. Coverslips were analyzed by confocal microscopy to visualize Tax (green) and nuclei (blue) using DAPI. Cell lysates were subjected to immunoblotting using anti-STAMBPL1 (α-STAMBPL1), anti-Tax (α-Tax), and anti-β-actin (α-β-actin).

Fig 7

STAMBPL1 does not directly deubiquitinate Tax. (A) STAMBPL1 is not a DUB for Tax in vitro. 293T cells were transfected with pCMV4-Tax and HA-ubiquitin (Ub), K63-only HA-Ub, or K48-only HA-Ub. The lysates were subjected to immunoprecipitation using anti-Flag (α-Flag). The immunoprecipitated proteins were eluted with 3× Flag peptide and incubated with immunoaffinity-purified human STAMBPL1 (1 μg) for 18 h at 37°C in DUB buffer. Proteins were resolved by SDS-PAGE and subjected to immunoblotting using anti-HA (α-HA) and anti-Tax (α-Tax). (B) STAMBPL1 is a DUB for K63-linked polyubiquitin chains. Purified STAMBPL1 (1 μg) was incubated with K63-linked penta-ubiquitin chains (Ub⁵) at 37°C for 18 h. Proteins were resolved by SDS-PAGE and subjected to immunoblotting using anti-K63-specific Ub (α-Ub).