HTLV-1 Tax Stimulates Ubiquitin E3 Ligase, Ring Finger Protein 8, to Assemble Lysine 63-Linked Polyubiquitin Chains for TAK1 and IKK Activation

Abstract

Human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, impacts a multitude of cellular processes, including I-κB kinase (IKK)/NF-κB signaling, DNA damage repair, and mitosis. These activities of Tax have been implicated in the development of adult T-cell leukemia (ATL) in HTLV-1-infected individuals, but the underlying mechanisms remain obscure. IKK and its upstream kinase, TGFβ-activated kinase 1 (TAK1), contain ubiquitin-binding subunits, NEMO and TAB2/3 respectively, which interact with K63-linked polyubiquitin (K63-pUb) chains. Recruitment to K63-pUb allows cross auto-phosphorylation and activation of TAK1 to occur, followed by TAK1-catalyzed IKK phosphorylation and activation. Using cytosolic extracts of HeLa and Jurkat T cells supplemented with purified proteins we have identified ubiquitin E3 ligase, ring finger protein 8 (RNF8), and E2 conjugating enzymes, Ubc13:Uev1A and Ubc13:Uev2, to be the cellular factors utilized by Tax for TAK1 and IKK activation. In vitro, the combination of Tax and RNF8 greatly stimulated TAK1, IKK, IκBα and JNK phosphorylation. In vivo, RNF8 over-expression augmented while RNF8 ablation drastically reduced canonical NF-κB activation by Tax. Activation of the non-canonical NF-κB pathway by Tax, however, is unaffected by the loss of RNF8. Using purified components, we further demonstrated biochemically that Tax greatly stimulated RNF8 and Ubc13:Uev1A/Uev2 to assemble long K63-pUb chains. Finally, co-transfection of Tax with increasing amounts of RNF8 greatly induced K63-pUb assembly in a dose-dependent manner. Thus, Tax targets RNF8 and Ubc13:Uev1A/Uev2 to promote the assembly of K63-pUb chains, which signal the activation of TAK1 and multiple downstream kinases including IKK and JNK. Because of the roles RNF8 and K63-pUb chains play in DNA damage repair and cytokinesis, this mechanism may also explain the genomic instability of HTLV-1-transformed T cells and ATL cells.

Fig 1. Ubc13-containing E2 enzymes are crucial for IKK activation by Tax in vitro and in vivo.

(A) Ubc13-containing E2 conjugating enzyme complexes enhanced IKK activation by Tax in cell-free HeLa-G S100 extracts. The HeLa-G cytosolic S100 extract [17,20] was incubated with recombinant TaxH₆ alone (lanes 2), Ubc13, Ubc13:Uev2, or Ubc13:Uev1AH₆ with (lanes 4–5) or without TaxH₆ (lanes 6–8) as indicated at 30°C for 1 hour. IKK activation was detected by immunoblot with anti-p-IκBα. Uev1A was detected using anti-poly-Histidine antibody. (B & C) IKK activation by Tax is reduced by the depletion of Ubc13, Uev1A, or Uev2, but restored by exogenously added Ubc13, Uev2 or Uev1A. (B) HeLa-G, Ubc13_KD, Uev1A_KD and Uev2_KD S100 extracts were generated and incubated with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) recombinant TaxH₆, or (C) in the absence of exogenous factors (lanes 1, 4, and 7), in the presence of Tax without (lanes 2, 5, and 8) or with Ubc13, Uev2, or Ubc13:Uev1A H₆ (lanes 3, 6, and 9) as indicated at 30°C for 1 hour. Uev1A protein was not detectable in immunoblot, and Uev1A_KD is determined via mRNA levels shown in S1 Fig. (D) Tax-induced IKK activation is attenuated in HeLa-G cells knocked down for Ubc13, Uev2, or Uev1A expression. The Ubc13_KD, Uev1A_KD and Uev2_KD cell clones were transiently co-transfected with E-Selectin-Luc with or without Tax (Materials and Methods). Firefly luciferase activity was measured at 48 hours post-transfection. Fold of activation was calculated as the ratio of luciferase activities in cells with Tax over those in cells without Tax.

Fig 2. The E3 ubiquitin ligase, RNF8, supports IKK activation by Tax in vitro.

(A) RNF8 co-purifies with Tax_S-tag expressed in transiently transfected 293T cells. 293T cells that were transiently transfected with an S-tagged Tax expression plasmid were lysed and incubated with RNase S-agarose beads. The supernatant (S) and pull-down (P) fractions are immunoblotted with the indicated antibodies. (B) Commassie brilliant blue-stained full-length RNF8 and truncated RNF8_345–485 purified from Sf9 insect cells and E. coli respectively. (C) Domain organization of RNF8 and a truncation mutant, RNF8_345–485 . Amino acid residues that encompass the FHA domain, the coiled coil (CC) dimerization domain, and the RING finger domain are as indicated. (D) Depletion of RNF8 prevents IKK activation by Tax in vitro. HeLa-G (lanes 1 & 2) and RNF8_KD (lanes 3 & 4) S100 extracts were generated and incubated with or without recombinant TaxH₆ as indicated at 30°C for 1 hour. IKK activation was detected by immunoblot with anti-p-IκBα. (E) Addition of RNF8 greatly enhances Tax-mediated IKK activation in S100 extracts from wild-type and RNF8_KD cells respectively. HeLa-G (lanes 1–4) and RNF8_KD (lanes 5–10) S100 extracts were incubated with recombinant TaxH₆, RNF8 and/or RNF8_345-485 as indicated at 30°C for 1 hour. Significant IKK activation was detected by immunoblot with anti-p-IκBα and anti-p-IKKα/β.

Fig 3. Tax and RNF8 potently activate IKK and the canonical NF-κB pathway.

(A) HeLa-G cells with ablation of RNF8 were generated via CRISPR/Cas9 system and confirmed by immunoblotting. (B) Tax-mediated NF-κB activation is significantly diminished in RNF8_KO cells. HeLa-G or RNF8_KO cells (5x10⁴) were transiently co-transfected with E-Selectin-Luc (250 ng/ml) and increasing concentrations of Tax (0, 25, 50 and 100 ng/ml). Firefly luciferase activity and fold of activation for (B) and (C) below were measured and calculated as in Fig 1D. (C) RNF8 augmented and restored respectively Tax-mediated NF-κB activation in wild-type and RNF8_KO cells. As in (B), HeLa-G or RNF8_KO cells were co-transfected with E-Selectin-Luc (250 ng/ml), Tax (50 ng/ml), and increasing amounts of RNF8 (0, 50, 100, and 200 ng/ml). (D) RNF8 ablation does not affect non-canonical NF-κB activation by Tax. HeLa-G and RNF8_KO cells were mock transduced (lanes 1 & 4), or transduced with Ad-Tax (lanes 2 & 5) or Ad-tTa (for tet trans-activator) vector (lanes 3 & 6) for 48 hours. Both mock- and Ad-tTa-transduced cells were used as negative controls. Cells were harvested and immunoblotted for the indicated proteins. (E) Tax and RNF8 promote TAK1 and JNK activation in vitro. HeLa-G (lanes 1–4) or TAK1_KD (lanes 5–8) S100 extract was incubated with Tax and/or RNF8 as indicated at 30°C for 1 hour and immunoblotted for the indicated proteins. (NS: non-specific).

Fig 4. Tax redistributes RNF8 from the nucleus to the cytoplasm.

(A) RNF8 is localized to the cytoplasm when Tax is present. HeLa-G cells were transduced with Ad-Tax at an MOI of 0.5 for 48 hours. HeLa-G cells contain a Tax-inducible GFP cassette and turn brightly green upon Tax expression as indicated by white arrows. The cells were then fixed, permeabilized and stained with an antibody for RNF8 (Red) and DAPI (Blue). (B) HeLa cells were transduced with Ad-Tax or Ad-tTa for 48 hours and fractionated following manufacturer’s protocol. The cytosolic and nuclear fractions were immunoblotted for the indicated proteins. (C) Much RNF8 is localized in the cytoplasm of Jurkat and HTLV-1-transformed MT4 T cell lines. Cells were fixed, permeabilized and stained with an antibody for RNF8 (Red) and DAPI (Blue). (D) Jurkat and MT4 cells were harvested and fractionated following manufacturer’s protocol. The cytosolic and nuclear fractions were immunoblotted for the indicated proteins. HDAC1 and GAPDH are used as nuclear and cytosolic protein control respectively for both (B) and (D).

Fig 5. Tax stimulates RNF8 and Ubc13:Uev1A/2 to assemble long K63-pUb.

(A) In vitro assembly of unanchored K63-pUb by RNF8 and RNF8_345–485 in the presence or absence of Tax. Each ubiquitination reaction contained E1, ubiquitin, ATP, TaxH₆, RNF8 or RNF8_345–485, and Ubc13:Uev1A or Ubc13:Uev2 as indicated and incubated at 37^°C for 4 hours. Reactions products were resolved in 15% (upper panel), 4–20% (middle panel), and 4–20% (bottom panel) polyacrylamide gels, respectively. They are probed with indicated antibodies (ubiquitin, upper and middle panels and K63-pUb, bottom panel). Reactions in lanes 2–6 and 7–11 contained Ubc13:Uev1A and Ubc13:Uev2 E2 enzyme complexes respectively. RNF8 and RNF8_345–485 were used in reactions in lanes 3, 4, 8, 9 and 5, 6, 10, 11 respectively. (B) Polyubiquitin chain assembly by RNF8 and Ubc13:Uev1A in the presence of Tax requires ubiquitin molecules that contain lysine at amino acid residue 63. In vitro reactions were carried out as described in (A) with RNF8, Ubc13:Uev1A, ATP and E1, in the presence (lanes 2–7) or absence (lane 1) of TaxH₆. Different variants of ubiquitin were used: wild-type ubiquitin (WT); K63R and K43R, mutants with lysine to arginine substitution at amino acid residue 63 and 48 respectively; K63 and K48, mutants with all lysine residues substituted by arginine except residue 63 and 48; and K0, a mutant with all lysine residues substituted by arginine. The protein concentration of the K63R ubiquitin mutant was based on the data sheet provided by the vender and was approximately ~1/4 of the other ubiquitin mutants, making it harder to detect by immunoblotting. (C) HeLa-G cells (10⁵) were co-transfected with Ub-HA (50 ng/ml), Tax (50 ng/ml), and/or increasing amounts of RNF8 (0, 50, 100, 200 ng/ml) as indicated for 48 hours. Cells were lysed with SDS sample buffer and immunoblotted for the indicated proteins.

Fig 6. Tax hijacks RNF8 and Ubc13:Uev1A/Uev2 to activate IKK and the canonical NF-κB pathway.

Tax interacts with and stimulates RNF8 and Ubc13:Uev1A/2 to assemble free K63-pUb chains, which serve as platforms for TAK1 and IKK to convene and activate the canonical NF-κB and other signaling pathways.