PKC phosphorylation of TRAF2 mediates IKKalpha/beta recruitment and K63-linked polyubiquitination

Abstract

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is a key mediator in TNF signaling. Previous studies suggested that TRAF2 functions as an adaptor in the NF-kappaB and AP-1 pathways. However, the precise molecular mechanisms by which TRAF2 relays signals are unknown. We previously reported that TRAF2 is phosphorylated following TNF stimulation and now identify the PKC kinases responsible for phosphorylation. Phosphorylated TRAF2 facilitates recruitment of IKKalpha and IKKbeta to the TNF receptor. Phosphorylation also determines K63-linked polyubiquitination of TRAF2 at lysine 31. TRAF2 K63-linked ubiquitination contributes to associations with TAB2/3 and activation of the downstream IKK and JNK kinases. The combined data reveal that phosphorylation of TRAF2 plays a critical role in TNF signaling by directing the IKK complex to the membrane, promoting TRAF2 K63-linked ubiquitination, and positioning the IKKalpha and IKKbeta chains with the TAK1/TAB kinase.

Figure 1. TRAF2 phosphorylation is induced by TNF and required for IKK activation

(A) TRAF2 knockout (KO) MEFs were infected with retrovirus expressing FLAG-tagged human TRAF2 or a point mutant TRAF2(T117A). After puromycin selection the two stable cell lines, TRAF2^−/−(hTRAF2) and TRAF2^−/−(hT117A) were stimulated with 20 ng/ml human TNFα (hTNFα) for the designated times. Immunoblots (IB) were probed with antibodies specific for phospho-peptide or TRAF2 protein. (B) TNFα-induced IKKβ kinase activity in wild type and traf2-/- stable cell lines. Cells were treated with hTNFα for the indicated times then cell extracts were immunoprecipitated with anti-IKKβ antibody for analysis of kinase activity using GST-IκBα as substrate. (C) TNFα-induced IKKβ kinase activity in wild type and traf2-/- stable cell lines transfected with TRAF5 siRNA oligo. (D) Relative mRNA levels of A20 and IL-6 in wild type and traf2-/- stable cell lines. After TNF stimulation for the designated times, cells were collected for mRNA extraction and reverse transcription. cDNAs were then subjected to real-time PCR and the relative mRNA levels were calculated. (E) IκBα phosphorylation and degradation in wild type and traf2-/- stable cell lines with or without TRAF5 RNAi.

Figure 2. PKC controls TRAF2 Thr¹¹⁷ phosphorylation and promotes NF-κB activation

(A) FLAG-TRAF2 was cotransfected with various PKC constructs into 293T cells; after 48 h cell lysates were collected for Western Blot and probed with the indicated antibodies. Double mutation PKCε(K437W/T566A) is a dominant negative of PKCε (DN-PKCε). (B) In vitro PKC phosphorylation of the indicated GST tagged Thr¹¹⁷ peptide. Purified GST tagged T117 or A117 peptides were incubated with recombinant PKCα, PKCδ, or PKCε in PKC kinase assay buffer for 30 min at 30°C. The reaction buffer was then boiled for Western Blot and probed with anti-phospho-TRAF2 (Thr¹¹⁷) antibody. Coomassie staining was shown as the peptide loading control. The upper band detected by anti-phospho-TRAF2 antibody was due to Thr¹¹⁷ phosphorylation plus phosphorylation of a site in GST. (C) Purified FLAG tagged TRAF2 was incubated in vitro with recombinant PKCδ, in PKC kinase assay buffer for 30 min at 30 °C. (D) TRAF2 phosphorylation and IKKβ kinase activity in wild type, PKCε deficient cells and PKCε deficient cells transfected with PKCδ siRNA oligo. Forty-eight hours after transfection cells were stimulated with 20 ng/ml mouse TNFα for the designated times. Cell lysates were immunoprecipitated with anti-TRAF2 or IKKβ antibody to examine TRAF2 phosphorylation and IKK kinase activity. (E) TNF-induced mRNA expression of A20 and IL-6 in wild type, PKCε deficient cells and PKCε deficient cells with PKCδ RNAi.

Figure 3. Thr¹¹⁷ phosphorylation controls TRAF2 interactions with IKKα and IKKβ

(A) After TNF treatment, 293T cell lysates were immunoprecipitated with anti-TRAF2 antibody. (B) Myc-tagged IKKα or IKKβ was cotransfected with FLAG-tagged TRAF2 or TRAF2(T117A) into 293T cells. Cells were treated with media or 20 ng/ml hTNFα for 10 min before harvest and immunoprecipitation with anti-FLAG antibody. (C) Stable traf2-/-(hTRAF2) and traf2-/-(hT117A) cells were treated with TNFα (20 ng/ml) for the indicated times before harvest and cell lysates were immunoprecipitated with anti-FLAG antibody. (D) Thr¹¹⁷ phosphorylation is required for fluorescence resonance energy transfer (FRET) between TRAF2 and IKKβ. The insert includes a red line (ab) to indicate the line of photobleaching. Average CFP fluorescence intensities were measured before and after photobleaching. The arrow highlights the dramatic increase in intensity of CFP tagged IKKβ after bleaching YFP tagged TRAF2. (E) Purified FLAG-TRAF2 or TRAF2(T117A) was phosphorylated in vitro with recombinant PKCδ, and then precipitated with GST-IKKβ by GST pull-down or anti-FLAG antibody. (F) Wild type, PKCε deficient cells, and PKCε deficient cells with PKCδ RNAi were treated with TNFα for the indicated times. Cell extracts were then immunoprecipitated with anti-TRAF2 antibody.

Figure 4. TNF signaling through TNFR1 mediates endogenous TRAF2 phosphorylation and recruitment of IKK complex

(A) Immortalized wild-type and TNFR1 deficient astrocytes were stimulated with 20 ng/ml mTNFα for the indicated times. Cell extracts were then immunoprecipitated with anti-TRAF2 antibody. (B) Immortalized TNFR1 and TNFR2 double deficient MEFs were transfected with hTNFR1 or hTNFR2 and stimulated with 20 ng/ml mTNFα for the indicated times. Cell extracts were then immunoprecipitated with anti-TRAF2 antibody. (C-D) Indicated MEFs or reconstituted cells were stimulated with 20 ng/ml mTNFα for the designated times. Cells were harvested and immunoprecipitated with anti-mTNFR1 antibody.

Figure 5. Phosphorylation controls K63-linked ubiquitination of TRAF2 at lysine 31

(A) Stably reconstituted traf2^−/−(hTRAF2) and traf2^−/−(hT117A) cells were stimulated with 20 ng/ml TNFα for the designated times. Immunoblots were probed with antibodies specific for TRAF2 phospho-peptide or human TRAF2. (B) FLAG-TRAF2 or TRAF2(T117A) constructs were transfected into 293T cells with HA-Ub or its mutants containing only one lysine at either K48 or K63. Ub-K48 expressing cells were also treated with the proteasome inhibitor MG132 to prevent the degradation of K48-linked TRAF2. The cell lysates were immunoprecipitated with anti-FLAG antibody. (C) FLAG-TRAF2 or TRAF2(T117A) constructs were transfected into 293T cells with or without UbcH13. (D) TRAF2 knockout MEFs were infected with retrovirus expressing FLAG-tagged human TRAF2 or lysine point mutants TRAF2(K31R), TRAF2(K115R) and TRAF2(K119R). After puromycin selection these stable cell lines were stimulated with 20 ng/ml TNFα for the designated times. (E) FLAG-TRAF2, TRAF2(K31R) or TRAF2(K115R) constructs were transfected into 293T cells with UbcH13. (F) FLAG-TRAF2 or TRAF2(K31R) constructs were transfected into 293T cells with HA-Ub K63 only. After TNF stimulation, cells were harvested for IP with anti-FLAG antibody.

Figure 6. K63-linked ubiquitination of TRAF2 mediates the association with TAB2/3

(A-B) HA-tagged TAB2 was cotransfected with FLAG-tagged TRAF2, TRAF2(T117A) or TRAF2(K31R) into 293T cells. Indicated groups were treated with 20 ng/ml hTNFα for 10 min before harvest and immunoprecipitated with anti-FLAG antibody. (C) After TNF stimulation reconstituted traf2^−/−(hTRAF2) vs. traf2^−/−(hK31R) MEFs were immunoprecipitated with anti-TAB2 antibody. (D) Epitope-tagged IKKβ and TRAF2 or TRAF2(K31R) were cotransfected into 293T cells. After TNF treatment TRAF2 was immunoprecipitated and blots were probed with the indicated antibodies. (E) Indicated reconstituted traf2-/- cells were stimulated with 20 ng/ml mTNFα for the designated times. Cells were harvested and immunoprecipitated with anti-mTNFR1 antibody.

Figure 7. Ubiquitination of TRAF2 regulates IKK and JNK activity

(A) TNF-induced mRNA expression of A20 and IL-6 in stably reconstituted traf2^−/−(hTRAF2) vs. traf2^−/−(hK31R) MEFs. (B) TNFα-induced IKKβ kinase activity in stably reconstituted traf2^−/−(hTRAF2) vs. traf2^−/−(hK31R) MEFs or in the same cell lines with TRAF5 RNAi. (C) TNFα-induced JNK kinase activity in stably reconstituted traf2^−/−(hTRAF2) vs. traf2^−/−(hK31R) MEFs. (D) TNF-induced p38, ERK and JNK activation in wild type, traf2^-/-, and MEFs stably reconstituted with the indicated mutants. Cells were treated with 20 ng/ml hTNFα as designated. Immunoblots were probed as indicated. (E) MTT assay with wild type MEFs and various traf2-/- stable cell lines treated for 24h with cycloheximide (0.25 μg/ml; CHX), or TNFα (10 ng/ml), or both. The percent live cells ±SD is indicated. (F) A model of TNF-induced IKK activation.