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.

Author Summary

Activation of the NF-κB family of transcription factors by the HTLV-1 oncoprotein, Tax, is causally linked to adult T cell leukemia (ATL) development in HTLV-1-infected individuals, but the underlying mechanisms are not fully understood. NF-κB activation requires the phosphorylation of its inhibitor, IκBα, by IκB kinase (IKK), which marks IκBα for degradation. In this study, we demonstrate that Tax inappropriately activates a ubiquitin E3 ligase, RNF8, and ubiquitin E2 conjugating enzymes, Ubc13:Uev1A/Uev2, to assemble long lysine 63-linked polyubiquitin (K63-pUb) chains, which function as signaling platforms for polyubiquitin-binding TGFβ-activated kinase 1 (TAK1) and IKK to congregate and become activated. Because TAK1 mediates the activation of multiple downstream signaling pathways, the mechanism described here can explain the complex effect of Tax on cell signaling. The major functions of RNF8 are to signal cellular DNA damage repair (DDR) and cell division by assembling K63-pUb chains at the site of DNA damage and cell cleavage. As such, the inappropriate activation of RNF8 and the over-abundance of K63-pUb chains in Tax-expressing cells may explain how Tax causes DNA damage and cell division defect.