FIGURE 4.
The dimerization mutant, A52-M65E, can still interact with TRAF6 but cannot cause TRAF6-TAK1 association or TRAF6 self-association. A, schematic diagram of residue interactions across the A52 dimer interface generated by PDBsum (48). Parallel lines represent non-bonded contacts, and solid lines represent hydrogen bonds. For non-bonded contacts, the width of the parallel line is proportional to the number of atomic contacts. B, dimer interface of A52 homodimer generated using ICM Molsoft browser. The Phe-154 and Met-65 residues are represented as red and blue stick models, respectively. The dimer interface comprises of the N terminus (N), α1, and α6 of the Bcl-2 fold domain. C, HEK293T cells were transfected with 4 μg each of Myc-A52 and HA-A52 (lane 1), Myc-A52 F154A, and HA-A52 F154A (lane 2), or Myc-A52 M65E, and HA-A52 M65E (lane 3). After 48 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting (IB) with the indicated antibodies. Immunoblots were subjected to densitometric analysis with levels of co-immunoprecipitated HA-A52, HA-A52 F154A, and HA-A52 M65E normalized to total levels of immunoprecipitated Myc-A52, Myc-A52 F154A, and Myc-A52 M65E, respectively. D, HEK293T cells were transfected with 4 μg each of HA-A52 M65E, Myc-TRAF6, and Myc-IRAK2. After 48 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. E, HEK293T cells were seeded into 10-cm dishes (1.5 × 106 cells) 24 h before transfection with 4 μg each of empty vector, Myc-A52, or Myc-A52-M65E. After 48 h, lysates were analyzed by either native gel electrophoresis or SDS-PAGE. Immunoblots were subjected to densitometric analysis with levels of TRAF6 oligomer normalized to total levels of TRAF6 monomer. F, HEK293T cells were transfected with the indicated amounts of Myc-TRAF6, FLAG-TAK1, HA-A52, and HA-A52 M65E. After 48 h, lysates were subject to immunoprecipitation, SDS-PAGE, and immunoblotting with the indicated antibodies. G–I, HEK293-R1 cells were transfected for 24 h with 150 ng of Myc-A52, Myc-A52 F154A, Myc-A52 M65E, or pCMV-Myc empty vector (EV), along with either the NFκB luciferase reporter gene (G), the pFR luciferase reporter gene and CHOP-Gal4 (H) or the IL-10 promoter luciferase reporter gene (I). MEK3 is the positive control for the PathdetectTM CHOP assay (H). Cells were stimulated with 50 ng/ml IL-1α for 6 h (G), and luciferase reporter gene activity was measured. The data are mean ± S.D. of triplicate samples and are representative of at least three separate experiments. *, p < 0.05; **, p < 0.005; or ***, p < 0.0005 compared with empty vector.