HTLV-1 Tax Induces Formation of the Active Macromolecular IKK Complex by Generating Lys63- and Met1-Linked Hybrid Polyubiquitin Chains

PLoS Pathog. 2017 Jan 19;13(1):e1006162. doi: 10.1371/journal.ppat.1006162. eCollection 2017 Jan.

Abstract

The Tax protein of human T-cell leukemia virus type 1 (HTLV-1) is crucial for the development of adult T-cell leukemia (ATL), a highly malignant CD4+ T cell neoplasm. Among the multiple aberrant Tax-induced effects on cellular processes, persistent activation of transcription factor NF-κB, which is activated only transiently upon physiological stimulation, is essential for leukemogenesis. We and others have shown that Tax induces activation of the IκB kinase (IKK) complex, which is a critical step in NF-κB activation, by generating Lys63-linked polyubiquitin chains. However, the molecular mechanism underlying Tax-induced IKK activation is controversial and not fully understood. Here, we demonstrate that Tax recruits linear (Met1-linked) ubiquitin chain assembly complex (LUBAC) to the IKK complex and that Tax fails to induce IKK activation in cells that lack LUBAC activity. Mass spectrometric analyses revealed that both Lys63-linked and Met1-linked polyubiquitin chains are associated with the IKK complex. Furthermore, treatment of the IKK-associated polyubiquitin chains with Met1-linked-chain-specific deubiquitinase (OTULIN) resulted in the reduction of high molecular weight polyubiquitin chains and the generation of short Lys63-linked ubiquitin chains, indicating that Tax can induce the generation of Lys63- and Met1-linked hybrid polyubiquitin chains. We also demonstrate that Tax induces formation of the active macromolecular IKK complex and that the blocking of Tax-induced polyubiquitin chain synthesis inhibited formation of the macromolecular complex. Taken together, these results lead us to propose a novel model in which the hybrid-chain-dependent oligomerization of the IKK complex triggered by Tax leads to trans-autophosphorylation-mediated IKK activation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Activation / physiology*
  • Gene Products, tax / metabolism*
  • HEK293 Cells
  • HTLV-I Infections / metabolism*
  • Human T-lymphotropic virus 1 / pathogenicity
  • Humans
  • I-kappa B Kinase / metabolism*
  • Immunoblotting
  • Immunoprecipitation
  • Jurkat Cells
  • NF-kappa B / metabolism*
  • Real-Time Polymerase Chain Reaction
  • Signal Transduction / physiology
  • Transfection

Substances

  • Gene Products, tax
  • NF-kappa B
  • I-kappa B Kinase

Grant support

This work was supported in part by grants-in-aid from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (MEXT; http://www.mext.go.jp/, grant number; 22117002, 15H01176 to JI) and from the Japanese Society for the Promotion of Science (JSPS; https://www.jsps.go.jp, grant number; 26290036 to JI and 26840062 to YSh). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.