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PMID:17135271

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Citation

Lamothe, B, Besse, A, Campos, AD, Webster, WK, Wu, H and Darnay, BG (2007) Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of I kappa B kinase activation. J. Biol. Chem. 282:4102-12

Abstract

Tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) is a key mediator in proximal signaling of the interleukin-1/Toll-like receptor and the TNF receptor superfamily. Analysis of TRAF6-deficient mice revealed a fundamental role of TRAF6 in osteoclastogenesis; however, the molecular mechanism underlying TRAF6 signaling in this biological process is not understood. Recent biochemical evidence has indicated that TRAF6 possesses ubiquitin ligase activity that controls the activation of IKK and NF-kappaB. Because these studies are primarily based on cell-free systems, the role of the ubiquitin ligase activity of TRAF6 and its auto-ubiquitination to initiate the NF-kappaB pathway in vivo remain elusive. Here we show that an intact RING domain of TRAF6 in conjunction with the E2 enzyme Ubc13/Uev1A is necessary for Lys-63-linked auto-ubiquitination of TRAF6 and for its ability to activate IKK and NF-kappaB. Furthermore, a RING mutant of TRAF6 abolishes its ability to induce receptor activator of NF-kappaB-independent osteoclast differentiation and nuclear accumulation of the transcription factor NFATc1. Notably, we map the auto-ubiquitination site of TRAF6 to a single Lys residue, which if mutated renders TRAF6 unable to activate transforming growth factor-beta-activated kinase 1 and IKK and to cause spontaneous osteoclast differentiation. Additionally, we provide biochemical and in vivo evidence that TRAF6 serves as an E3 to directly ubiquitinate NEMO. Reconstituting TRAF6-deficent cells with various TRAF6 mutants, we clearly demonstrate the requirement for the TRAF6 RING domain and site-specific auto-ubiquitination of TRAF6 to activate IKK in response to interleukin-1. These data establish a signaling cascade in which regulated site-specific Lys-63-linked TRAF6 auto-ubiquitination is the critical upstream mediator of IKK.

Links

PubMed PMC3221607 Online version:10.1074/jbc.M609503200

Keywords

Animals; Catalysis; Cell Line; Enzyme Activation; Humans; I-kappa B Kinase/metabolism; Lysine/metabolism; Mice; Mice, Knockout; NF-kappa B/metabolism; Protein Structure, Tertiary/genetics; Signal Transduction/physiology; TNF Receptor-Associated Factor 6/chemistry; TNF Receptor-Associated Factor 6/deficiency; TNF Receptor-Associated Factor 6/metabolism; TNF Receptor-Associated Factor 6/physiology; Transcription Factors/metabolism; Transcription Factors/physiology; Ubiquitin/metabolism; Ubiquitin-Conjugating Enzymes/metabolism; Ubiquitin-Conjugating Enzymes/physiology

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

HUMAN:TRAF6

enables

GO:0061630: ubiquitin protein ligase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:TRAF6

involved_in

GO:0070555: response to interleukin-1

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:TRAF6

involved_in

GO:0070534: protein K63-linked ubiquitination

ECO:0000316: genetic interaction evidence used in manual assertion

UniProtKB:P0CG47

P

Seeded From UniProt

complete

HUMAN:TRAF6

involved_in

GO:0051865: protein autoubiquitination

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:TRAF6

involved_in

GO:0045672: positive regulation of osteoclast differentiation

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:TRAF6

involved_in

GO:0043507: positive regulation of JUN kinase activity

ECO:0000303: author statement without traceable support used in manual assertion

P

Seeded From UniProt

complete

HUMAN:TRAF6

involved_in

GO:0043123: positive regulation of I-kappaB kinase/NF-kappaB signaling

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:TRAF6

involved_in

GO:0032147: activation of protein kinase activity

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

HUMAN:TRAF6

enables

GO:0004842: ubiquitin-protein transferase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

HUMAN:TRAF6

enables

GO:0004842: ubiquitin-protein transferase activity

ECO:0000269: experimental evidence used in manual assertion

F

Seeded From UniProt

complete


See also

References

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