1. The TRAF6 Ubiquitin Ligase and TAK1 Kinase Mediate IKK Activation by BCL10 and MALT1 in T Lymphocytes 
Lijun Sun, Li Deng, Chee-Kwee Ea, Zong-Ping Xia, Zhijian J. Chen 
Molecular Cell 
Volume 14, Issue 3, Pages (May 2004)
DOI: /S (04)

2. Figure 1 BCL10-Dependent Activation of IKK
(A) BCL10 activates IKK in Jurkat cell extracts. Cytosolic extracts were prepared from Jurkat (WT, lanes 1 and 2) or a NEMO-deficient Jurkat cell line (lanes 3–6), and then incubated with recombinant BCL10 protein in the presence of ATP. Phosphorylation of IκBα by IKK in the extracts was determined by immunoblotting with an IκBα antibody. In lanes 5 and 6, recombinant NEMO was added back to the cell extracts to confirm the requirement of NEMO for IKK activation by BCL10.
(B) An intact CARD domain is required for BCL10 to activate IKK in vitro. Wild-type BCL10 protein (150 nM, lane 2) or mutants harboring point mutations in the CARD domain (L41Q or G78R, lanes 3–8) were added to HeLa cytosolic extracts (S100) at increasing concentrations (30 nM, lanes 3 and 6; 150 nM, lanes 4 and 7; 750 nM, lanes 5 and 8). IKK activity was determined by immunoblotting of IκBα as described in (A).
(C) An intact CARD domain is required for NF-κB activation. The CARD domain mutants or wild-type BCL10 was transfected into HEK293 cells together with an NF-κB-dependent luciferase reporter gene to assess their ability to activate NF-κB. The results shown were from duplicate experiments and normalized for transfection efficiency.
Molecular Cell  , DOI: ( /S (04) )

3. Figure 2 IKK Activation by BCL10 Requires Ubc13 and TRAF6
(A) Dominant-negative Ubc13 mutant inhibits IKK activation by TCR stimulation. 107 Jurkat cells were transfected with 10 μg of pEF-HA-Ubc13 (C87A, lanes 4–6), pcDNA3-FLAG-Ubc5 (C85A, lanes 7–9), or pEF vector (lanes 1–3) by electroporation. Twenty-four hours after transfection, cells were stimulated with antibodies against CD3 and CD28 (2 μg/ml each) for the indicated times. The IKK complex was immunoprecipitated with a NEMO antibody and then assayed for its kinase activity using GST-IκBαNT (N-terminal fragment) and γ-32P-ATP as the substrates (top panel). An aliquot of the immunoprecipitated IKK complex was immunoblotted with an IKKβ antibody (middle panel). To measure ERK activation, the cell extracts were immunoblotted with an antibody specific for phosphorylated ERK.
(B) Ubc13/Uev1A and an associated factor are required for IKK activation by BCL10 in vitro. HeLa cytosolic extracts (S100) were loaded onto an immobilized Ubc13-Sepharose column to deplete the endogenous Uev1A as well as other Ubc13-associated proteins. The depleted extracts (S100[−]Uev1A) were incubated with recombinant BCL10 protein, 35S-labeled IκBα, and ATP to determine IKK activity (lanes 3 and 4). Recombinant Ubc13/Uev1A protein and the Ubc13 eluate (0.5 M salt eluate) were then added individually or in combination (lanes 5–9) to the depleted extracts to attempt to restore IKK activation.
(C) Purification of TRAF6 as an IKK activator downstream of BCL10 (IKAB). The Ubc13-associated IKK activator was purified through five steps of chromatography as shown in the diagram. The fractions from the MonoQ column were subjected to SDS-PAGE, followed by silver staining (top panel). The same fractions were also assayed for IKK activity in the presence of BCL10 (bottom panel). The arrow indicates the protein band that was identified as TRAF6 by mass spectrometry and confirmed by immunoblotting (data not shown).
Molecular Cell  , DOI: ( /S (04) )

4. Figure 3 TRAF6 and TAK1 Mediate IKK and NF-κB Activation
(A) Dominant-negative mutants of TRAF6 and TAK1 inhibit NF-κB activation by BCL10 and PKCθ. Expression constructs encoding a FLAG-tagged mouse TRAF6 mutant lacking the N-terminal RING and zinc finger domains (TRAF6DN, residues 294–530), or the catalytically inactive mutant of TAK1 (TAK1DN, K63W, also FLAG-tagged), were transfected into HEK293 cells together with the expression constructs encoding BCL10 or a constitutively active form of PKCθ (PKCθ-A/E). An NF-κB luciferase reporter construct was cotransfected to measure NF-κB activation.
(B) Dominant-negative mutants of TRAF6 and TAK1 inhibit IKK activation by TCR stimulation. Expression constructs encoding FLAG-tagged TRAF6DN (lanes 4–6), TAK1DN (lanes 7–9), or vector DNA were transfected into Jurkat cells. Twenty-four hours after transfection, cells were stimulated with antibodies against CD3 and CD28, and then IKK and ERK activation assays were carried out as described in the Experimental Procedures. The expression of TRAF6 and TAK1 mutants was assessed by immunoblotting with a FLAG antibody (right panel).
Molecular Cell  , DOI: ( /S (04) )

5. Figure 4
TRAF6, TRAF2, TAK1, and MALT1 Are Essential for IKK Activation by T Cell Receptor Stimulation
(A) TRAF6 is required for IKK activation by BCL10 in vitro. Double-stranded siRNA oligos corresponding to the sequence of GFP (control, lanes 1 and 2) or human TRAF6 (lanes 3–6) were transfected into HEK293 cells. Cytosolic extracts prepared from the RNAi cells were incubated with recombinant BCL10 (lanes 2, 4, and 6) or purified native TRAF6 proteins (lanes 5 and 6) in the presence of ATP and 35S-labeled IκBα as a substrate. The efficiency of RNAi was assessed by immunoblotting with a TRAF6 antibody (right panel).
(B) Jurkat cells were transfected with siRNA oligos corresponding to the sequences of GFP (control, lanes 1–3), TRAF6 (lanes 4–6), TRAF2 (lanes 7–9), or both TRAF6 and TRAF2 (lanes 10–12). Three days after transfection, cells were stimulated with antibodies against CD3 and CD28 for the indicated times. Cell lysates were prepared for immunoblotting with an antibody specific for phosphorylated ERK (bottom panel) or for IKK assays after immunoprecipitation of the IKK complex with a NEMO antibody (top panel). The amount of immunoprecipitated IKK in each sample was detected by immunoblotting with an IKKβ antibody (middle panel). The efficiency of RNAi was determined by immunoblotting with an antibody against TRAF2 or TRAF6 (right panel).
(C and D) RNAi and kinase assays were performed as described in (B), except that siRNA oligos corresponding to TAK1 (C) or MALT1 (D) were used.
(E) RNAi experiments were carried out as in (B)–(D), except that Jurkat cells were stimulated for 8 hr, and then the culture media were harvested for ELISA assays to determine the production of interleukin-2 (IL-2).
Molecular Cell  , DOI: ( /S (04) )

6. Figure 5 Reconstitution of the BCL10 → IKK Pathway In Vitro
(A) IKK activation by BCL10 requires MALT1, TAK1/TAB2, and TRAF6-mediated polyubiquitination. Native IKK complex and TRAF6 proteins were purified from HeLa cytosolic extracts and then incubated with recombinant proteins of BCL10, MALT1, TAK1/TAB2 complex (labeled as TAK1), and Ubc13/Uev1A as indicated. The reaction mixture also contained E1, Ub, ATP, and 35S-labeled IκBα as a substrate. TAK1-K63W indicates the “kinase-dead” mutant of TAK1.
(B) MALT1 functions downstream of BCL10 to activate IKK. A MALT1 mutant containing the C-terminal half (MALT1C, residues 334–824) was expressed and purified from Sf9 cells, and then assayed for its ability to activate IKK in a reconstituted system as described in (A).
Molecular Cell  , DOI: ( /S (04) )

7. Figure 6
Oligomerization of BCL10 and MALT1 Induces TRAF6 Oligomerization and IKK Activation
(A) BCL10 forms oligomers that activate IKK. Recombinant BCL10 protein was fractionated by glycerol gradient ultracentrifugation (10%–50%; the density of the glycerol gradient increases from the top to the bottom). Fractions were collected and immunoblotted with an antibody against BCL10 (top panel). The same fractions were assayed for their ability to activate IKK, which phosphorylates 35S-labeled IκBα in Jurkat extracts (bottom panel). The 20S and 26S proteasomes were used as the size marker for the glycerol gradient ultracentrifugation.
(B) The oligomerized forms of MALT1C activate IKK. Recombinant MALT1C protein was fractionated by glycerol gradient ultracentrifugation and assayed for IKK activity as described in (A).
(C) MALT1C induces TRAF6 oligomerization. Native TRAF6 protein was partially purified from HeLa cells and then incubated alone (top panel), with recombinant BCL10 (middle panel), or MALT1C (bottom two panels) at 4°C for 60 min. The mixtures were then fractionated by glycerol gradient ultracentrifugation as described in (A). Fractions were analyzed by immunoblotting with a TRAF6 antibody.
(D) MALT1 associates with TRAF6 in cells. FLAG-TRAF6 expression construct was transfected into a HEK293 cell line stably expressing MALT1C-HA. The binding between TRAF6 and MALT1C was analyzed by immunoprecipitation with a MALT1 antibody or a control IgG. The precipitated proteins were detected by antibodies against FLAG (TRAF6) or HA (MALT1C).
(E) Diagram of the C terminus of MALT1 containing the TRAF6 binding sites.
(F) MALT1 binds to TRAF6 in vitro. Recombinant protein of the wild-type MALT1C (WT) or mutant E653A/E806A (2EA) was incubated with TRAF6 protein and then immunoprecipitated with a MALT1 antibody. The precipitated proteins were detected by immunoblotting with an antibody against TRAF6 or MALT1.
(G) Mutations of the TRAF6 binding sites of MALT1 abolished its ability to induce TRAF6 oligomerization. Recombinant MALT1C or 2EA protein was incubated with TRAF6 and then subjected to glycerol gradient ultracentrifugation as described in (C). Fractions were analyzed by immunoblotting with a TRAF6 antibody.
(H) Mutations of the TRAF6 binding sites of MALT1 abolish its ability to activate IKK. Wild-type MALT1 or 2EA mutant was added to ATP-supplemented Jurkat cell extracts at the indicated amounts, and 35S-labeled IκBα was used as a substrate for IKK.
(I) MALT1C binds to TRAF2 in vitro. Sepharose beads coated with MALT1C were incubated with HeLa S100, and then bound proteins were analyzed by immunoblotting with antibodies against TRAF6, TRAF2, or TRAF5.
Molecular Cell  , DOI: ( /S (04) )

8. Figure 7
MALT1 Stimulates the Ubiquitin Ligase Activity of TRAF6 and Polyubiquitination of NEMO
(A) Oligomerized MALT1 promotes TRAF6 ubiquitin ligase activity. MALT1C was fractionated by glycerol gradient ultracentrifugation as described in Figure 6B, and fractions containing MALT1C of different sizes were incubated with purified native TRAF6 protein in the presence of ubiquitination components including E1, Ubc13/Uev1A, ubiquitin, and ATP. After incubation at 37°C for 1 hr, polyubiquitin chain synthesis was analyzed by immunoblotting with a Ub antibody.
(B) Mutations of the TRAF6 binding sites of MALT1 abolish its ability to promote polyubiquitin chain synthesis by TRAF6. Recombinant MALT1C (lanes 2 and 4) or 2EA mutant protein (lanes 6 and 8) was added to in vitro ubiquitination reactions as described in (A), and the synthesis of polyUb chains was analyzed by immunoblotting with a Ub antibody.
(C) MALT1C facilitates the polyubiquitination of TRAF6. Ubiquitination of TRAF6 was carried out in the presence of MALT1C (lane 2 and 3) or 2EA mutant protein (lane 4), and the reaction products were analyzed by immunoblotting with a TRAF6 antibody.
(D) Synergistic effects of MALT1 and TRAF6 on the polyubiquitination of NEMO. Ubiquitination reactions were carried out as described in (A), except that in vitro-translated, 35S-labeled NEMO (lanes 1–7) or a mutant lacking the C-terminal zinc finger domain of NEMO (ΔC, lanes 8–14) was also added to the reactions. The reaction products were resolved by SDS-PAGE and then detected by phosphoimaging. In lanes 6 and 13, a lysine-less Ub mutant (KO) was used instead of wild-type Ub.
(E) Mechanism of IKK activation by BCL10: an oligomerization/ubiquitination cascade activates IKK in T cells. Stimulation of TCR leads to the activation of PKCθ, which then promotes the membrane recruitment and subsequent aggregation/oligomerization of CARMA1 and BCL10. Oligomerized BCL10 binds to MALT1 and induces the oligomerization of MALT1. Oligomerized MALT1 in turn binds to TRAF6 and enhances oligomerization and subsequent ubiquitination of TRAF6 and NEMO, thereby resulting in the activation of TAK1 and IKK.
Molecular Cell  , DOI: ( /S (04) )