HTLV-1 Tax Stimulates Ubiquitin E3 Ligase, Ring Finger Protein 8, to Assemble Lysine 63-Linked Polyubiquitin Chains for TAK1 and IKK Activation.
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.
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America.
Yik-Khuan
YK
Ho
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America.
Huijun
H
Zhi
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America.
Tara
T
Bowlin
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America.
Batsukh
B
Dorjbal
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America.
Subha
S
Philip
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America.
Muhammad Atif
MA
Zahoor
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan.
Hsiu-Ming
HM
Shih
Department of Microbiology and Molecular Cell Biology, The Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School, Norfolk, Virginia, United States of America.
Oliver John
OJ
Semmes
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America.
Brian
B
Schaefer
Department of Biochemistry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada.
J N Mark
JN
Glover
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland, United States of America.
Chou-Zen
CZ
Giam
D002460:Cell Line; D002471:Cell Transformation, Neoplastic / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D055785:Gene Knockdown Techniques; D016356:Gene Products, tax / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D015151:Immunoblotting; D020930:MAP Kinase Kinase Kinases / Q000378:metabolism; D016133:Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D000099151:MAP Kinase Kinase Kinase 7
D004268:DNA-Binding Proteins; D016356:Gene Products, tax; D051550:I-kappa B Kinase; D020930:MAP Kinase Kinase Kinases; D044767:Ubiquitin-Protein Ligases; D000099151:MAP Kinase Kinase Kinase 7; C486521:RNF8 protein, human; C514970:tax protein, Human T-lymphotrophic virus 1