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. 2009 Dec 28;285(8):5347–5360. doi: 10.1074/jbc.M109.076976

FIGURE 5.

FIGURE 5.

TRAF2 and TRAF6 mediate Lys63-linked TAK1 polyubiquitination at the Lys158 residue in vivo and in vitro. A and B, overexpression of TRAF2 (A) and TRAF6 (B) induces Lys63-linked TAK1 polyubiquitination. Expression vectors encoding TAK1-V5-His and TRAF2 (A) or TRAF6 (B) were co-transfected into HEK-293T cells with control vector and expression vectors encoding HA-ubiquitin wild type, Lys63-only and Lys48-only, respectively. TAK1-V5-His proteins in the transfected cells were immunoprecipitated (IP) with anti-V5 antibodies and immunoblotted (IB) with anti-HA antibodies to detect the presence of ubiquitinated TAK1-V5-His. C and D, TRAF2 (C) and TRAF6 (D) mediate Lys63-linked TAK1 polyubiquitination in vitro. Recombinant GST-TAK1-V5-(1–292), GST-TAB1, E1, E2, (Ubc13/Uev1a), and GST-TRAF2 (C) or GST-TRAF6 (D) were co-incubated in the ubiquitination buffer with recombinant ubiquitin wild type, Lys48-only, and Lys63-only, respectively. After 2 h, GST-TAK1-V5-(1–292) proteins were immunoprecipitated with anti-V5 antibodies and immunoblotted with anti-ubiquitin antibodies to detect the presence of ubiquitinated TAK1-V5-(1–292). E and F, TRAF2 (E) and TRAF6 (F) mediate Lys63-linked TAK1 polyubiquitination at Lys158 in vitro. Recombinant ubiquitin wild type, GST-TAB1, E1, E2 (Ubc13/Uev1a), and GST-TRAF2 (E) or GST-TRAF6 (F) were co-incubated in the ubiquitination buffer with GST-TAK1-V5-(1–292) wild type, K158R, or K150R, respectively. After 2 h, GST-TAK1-V5-(1–292) wild type and mutant proteins were immunoprecipitated with anti-V5 antibodies and immunoblotted with anti-ubiquitin antibodies to detect the presence of ubiquitinated TAK1-V5-(1–292). G and H, TAK1 lysine 158 to arginine mutant inhibits TRAF2-induced (G) and TRAF6-induced (H) NF-κB activation. TRAF2 or TRAF6 expression vectors, NF-κB luciferase reporter, and control Renilla luciferase reporter vectors were co-transfected into TAK1-deficient MEF cells with empty vector or expression vectors encoding TAK1 wild type and lysine 158 to arginine (K158R) mutant. The relative luciferase activity was measured 48 h later and normalized with the Renilla activity. Error bars, ±S.D. in triplicate experiments.