Site-specific Lys-63-linked tumor necrosis factor receptor-associated factor 6 auto-ubiquitination is a critical determinant of I kappa B kinase activation

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.

FIGURE 1. Lys-63-linked auto-ubiquitination of TRAF6 requires an intact RING domain and Ubc13/Uev1A

A, The RING domain of TRAF6 is required for auto-ubiquitination in vivo. HEK293 cells were transfected with the indicated plasmids encoding FLAG-TRAF6 or TRAF6-C70A in the absence (−) or presence (+) of HA-Ub (1 μg). Cells were lysed in Buffer A and immunoprecipitated with anti-FLAG or mouse IgG in Buffer C. Bound proteins were subjected to SDS-PAGE and immunoblotted first with anti-HA, and then the membrane was stripped and reprobed with the indicated antibody (left panels). Cell lysates were immunoblotted with the indicated antibodies (right panels). B, In vitro Lys-63-linked auto-ubiquitination of TRAF6 requires Ubc13/Uev1A. HEK293 cells were transfected with the indicated plasmids encoding FLAG-TRAF6 or TRAF6-C70A and cells were lysed (Buffer A) and immunoprecipitated with anti-FLAG in Buffer C followed by an in vitro ubiquitination assay with Ub (left panel) or Ub-Lys-63 (right panel) in the absence (−) or presence (+) of Ubc13/Uev1A (E2). The extent of TRAF6 ubiquitination was determined by immunoblotting with anti-Ub (top). The membrane was stripped and reprobed with anti-FLAG (middle). Cell lysates were immunoblotted with anti-FLAG (bottom). C, Bacterial expressed TRAF6 is auto-ubiquitinated through Lys-63-linked poly-Ub. The indicated GST-fusion proteins were subjected to an in vitro ubiquitination assay in the absence (−) or presence (+) of Ubc13/Uev1A with Ub-WT (left panel) or the indicated Ub mutants (right panel). After the ubiquitination assay, the GST-fusion proteins bound to glutathione-agarose were washed with Buffer C and then subjected to SDS-PAGE and immunoblotted with anti-Ub. The membrane was stained with Ponceau S (bottom).

FIGURE 2. The TRAF6 RING structure is required for signaling

A, The RING domain structure of TRAF6 is required for NF-κB activation. HEK293 cells were co-transfected with empty vector or with increasing amounts of FLAG-TRAF6 or FLAG-TRAF6-C70A together with an NF-κB-luciferase reporter. Thirty-six hours after transfection, cells were harvested for luciferase activity and processed according to the manufacturers protocol (Promega). Expression of the transfected TRAF6 and TRAF6-C70A constructs was determined by immunoblotting with anti-FLAG. B, Ubc13-C87A block TRAF6-dependent NF-κB activation. HEK293 cells were co-transfected with empty vector or with increasing amounts of FLAG-Ubc13-C87A in the absence or presence of TRAF6 together with an NF-κB-luciferase reporter and processed essentially as described in (A). Expression of the transfected TRAF6 and Ubc13-C87A constructs was determined by immunoblotting with anti-FLAG. C, TRAF6, but not TRAF6-C70A, induces spontaneous osteoclast differentiation. RAW cells (left panels) or mouse BMM (right panels) were infected with the indicated retrovirus as described in Experimental Procedures. Pictures were then taken with a 10x-objective lens for phase (Bright Field) and fluorescence (GFP), after which some of the samples were fixed and stained for TRAP. D, Osteoclasts derived from TRAF6 infection resorb bone and form actin rings. For pit formation, infected RAW cells were seeded in 24-well plates containing dentine slices (ALPCO) and after one week, the dentine slices were processed for pit formation. Cells from TRAF6 infection were plated on glass cover slips, fixed, and stained with rhodamine-phalloidin and Hoechst. E, TRAF6, but not TRAF6-C70A, induces nuclear expression of NFATc1. Cells from (C) were harvested and cytoplasmic and nuclear fractions were prepared. Protein from the nuclear extracts (NE) was immunoblotted with anti-NFATc1, and then the membrane was stripped and reprobed with anti-tubulin. Cytoplasmic extracts (CE) were immunoblotted with anti-FLAG, and then the membrane was stripped and reprobed with anti-actin.

FIGURE 3. Identification of the auto-ubiquitination site(s) in TRAF6

A, Schematic diagram of the TRAF6 RING domain. Shown is an alignment of the RING domain and the region before the first zinc finger of TRAF6 from 6 different species (h, human; m, mouse; c, chicken; z, zebra fish; r, rat; d, dog). Conserved Lys (K) residues are colored in blue and the Cys (C) and His (H) residues that constitute the RING domain are colored in red. Residue numbers are for mouse TRAF6. B, A molecular model of TRAF6 from the RING domain to the first zinc finger domain (residues D61-L159), modeled onto the Rag1 dimerization domain. Magenta: RING domain; blue: residues between the RING and the first zinc finger; cyan: the first zinc finger. Side chains of the seven conserved Lys residues are shown and labeled. C, K124 is the predominant site of TRAF6 auto-ubiquitination. HEK293 cells were transfected with vector (vector), FLAG-TRAF6 (WT) or the indicated TRAF6 mutants in the presence of HA-Ub (0.2 μg). Cells were lysed (Buffer A) and immunoprecipitated with anti-FLAG in Buffer A followed by boiling in 1% SDS and a second immunoprecipitation with anti-FLAG. The bound proteins were subjected to SDS-PAGE and immunoblotted with anti-HA. The membrane was stripped and reprobed with anti-FLAG. Cell lysates were immunoblotted with the indicated antibody (bottom). D, Lysine mutants of TRAF6 retain ubiquitin ligase activity. The indicated GST-fusion proteins were subjected to in vitro ubiquitination assay as described in Figure 1C, except the supernatant of the reaction was subjected to SDS-PAGE and immunoblotted with anti-Ub (top). The membrane was stained with Ponceau S (bottom).

FIGURE 4. TRAF6-K124R is required for its signaling capacity

A, Activation of an NF-κB luciferase reporter by TRAF6 and its mutants. HEK293 cells were transfected with vector, FLAG-TRAF6 (WT) or the indicated TRAF6 mutants together with an NF-κB-luciferase reporter. Thirty-six hours after transfection, cells were harvested and then luciferase activity was measured. Expression of TRAF6 and its mutants was determined by immunoblotting with anti-FLAG (inset). B, K124 of TRAF6 is required for IKK activity. Lysates from the indicated HEK293 stable cell lines were immunoprecipitated with anti-NEMO in Buffer A followed by an in vitro kinase assay with GST-IκBα. The membrane was probed with anti-NEMO. Cell lysates were immunoblotted with the indicated antibodies. C, K124 of TRAF6 is required for TAB2-TAK1 kinase activity. Lysates from the indicated HEK293 stable cell lines were immunoprecipitated with anti-TAB2 in Buffer A followed by an in vitro kinase assay with catalytically inactive MKK6. The membrane was probed with anti-TAB2 and then stripped and reprobed with anti-TAK1. Expression of the variants of TRAF6 was determined by immunoblotting with anti-FLAG.

FIGURE 5. Effect of TRAF6 mutants on spontaneous osteoclast differentiation

A–C, K124 of TRAF6 is required for osteoclast differentiation. RAW cells were infected with the indicated retrovirus and processed as described in Figure 2C. Prior to seeding in 24-well plates, the total number of GFP positive cells was counted to ensure equivalent infection (A). The total number of TRAP⁺, multi-nucleated osteoclasts was counted from wells plated in quadruplicate and shown as the average with standard deviation (B). Shown are representative pictures taken with a 10x objective lens (C). Similar results were obtained in at least three independent experiments. D, TRAF6-K124 is required for efficient induction of nuclear NFATc1 expression. Cells from (B) were harvested and cytoplasmic and nuclear fractions were prepared, and processed as described in Figure 2E.

FIGURE 6. K124 of TRAF6 is required for its auto-ubiquitination and signaling in TRAF6-deficient cells

A, TRAF6-K124R is not ubiquitinated in TRAF6-deficient MEFs. TRAF6^−/− MEFs which express vector (pMX) or TRAF6 and the indicated TRAF6 mutants were serum starved overnight. Cell lysates prepared from the indicated cell lines were immunoprecipitated with anti-FLAG in Buffer C and then immunoblotted with anti-Ub. The membrane was stripped and reprobed with anti-FLAG. Cell lysates were immunoblotted with indicated antibodies. B, K124 of TRAF6 is required to activate the IKK complex. Cell lysates from the indicated cell lines were immunoblotted with anti-p-IκBα, and then the membranes were stripped and reprobed with anti-actin and anti-FLAG. Lysates were also immunoprecipitated with anti-NEMO antibody followed by an in vitro kinase assay with GST-IκBα essentially as described in Figure 4B. Cell lysates were immunoblotted with the indicated antibodies. C, NEMO ubiquitination is impaired in TRAF6-K124R expressing cells. Cell lysates from the indicated cell lines were immunoprecipitated with anti-NEMO in Buffer A, followed by boiling in 1% SDS and immunoprecipitated again with anti-NEMO. The eluted proteins were subjected to SDS-PAGE and immunoblotted with anti-Ub. The membrane was stripped and reprobed with anti-NEMO. Cell lysates were immunoblotted with the indicated antibodies. D, TRAF6 directly ubiquitinates NEMO. Bacterial expressed and purified GST, GST-TRAF6, and GST-NEMO bound to glutathione-agarose beads were processed for in vitro ubiquitination assays as described (left) and NEMO immunoprecipitates were immunoblotted with anti-Ub. The membrane was stripped and reprobed with anti-NEMO. E, Effect of TRAF6 mutants on spontaneous osteoclast differentiation in TRAF6-deficient monocytes. Spleen-derived monocytes from a TRAF6-deficient mouse were infected with the indicated retrovirus and processed as described in Figure 2C.

FIGURE 7. K124 of TRAF6 is required for IL-1-dependent ubiquitination and IKK activation

A, IL-1-dependent ubiquitination of TRAF6 at K124. Stable TRAF6^−/− MEFs which express vector (pMX) or TRAF6 and the indicated TRAF6 mutants were serum starved overnight and then treated with IL-1 (0.1 ng/ml) for the indicated times. Cell lysates were prepared in Buffer A and immunoprecipitated with anti-FLAG followed by boiling in 1% SDS and immunoprecipitated again with anti-FLAG. The eluted proteins were subjected to SDS-PAGE and immunoblotted with anti-Ub. The membrane was stripped and reprobed with anti-FLAG. Cell lysates were immunoblotted with the indicated antibodies. B, IL-1-dependent IKK activation is impaired in TRAF6-K124R expressing cells. Stable TRAF6^−/− MEFs which express vector (pMX) or TRAF6 and the indicated TRAF6 mutants were serum starved overnight and then treated with IL-1 (0.1 ng/ml) for the indicated times. Lysates from the indicated stable cell lines were immunoprecipitated with anti-NEMO in Buffer A followed by an in vitro kinase assay with GST-IκBα. The membrane was probed with anti-NEMO. Cell lysates were immunoblotted with the indicated antibodies.