Predicted amino acid sequence of T6BP. The amino acid sequence deduced from the nucleotide sequence of the full-length T6BP cDNA is shown. The single underlines show the coiled-coil regions. The 34-amino acid peptide used for generating anti-T6BP antibody is indicated by a double underline. The two amino acids that are different from TXBP151 are indicated by asterisks. The full-length T6BP nucleotide sequence has been deposited in GenBank (accession no. AF268075).

The N-terminal ring finger and zinc finger regions of TRAF6 are required for its interaction with T6BP. (A) 293 cells were transiently transfected with the Myc-epitope tagged T6BP (amino acids 185–747) and the GST-tagged TRAF6 deletion mutants. Cell extracts were prepared and immunoprecipitated (IP) with glutathione beads. Coprecipitating Myc-T6BP was detected by immunoblotting (IB) analysis with the anti-Myc polyclonal antibody (Top). The amounts of GST-TRAF6 deletion mutants immunoprecipitated and the expression levels of Myc-T6BP in total cell extracts were determined by immunoblotting with anti-GST antibody (Middle) and anti-Myc antibody (Bottom). (B) Myc-epitope tagged T6BP (amino acids 185–747) and the Flag-tagged TRAF6 deletion mutants were transiently expressed in 293 cells. Cell extracts were prepared and immunoprecipitated with anti-Flag monoclonal antibody (F) or control mouse IgG (C). Coprecipitating Myc-T6BP was detected by immunoblotting analysis with the anti-Myc polyclonal antibody (Top). The amounts of TRAF6 deletion mutants immunoprecipitated were determined by immunoblotting with anti-Flag polyclonal antibody (Middle). The expression of Flag-TRAF6 (amino acids 1–354) is weak but can be detected with anti-TRAF6 monoclonal antibody (Bottom). KD, kilodalton.

Self-association of T6BP. Myc-epitope tagged T6BP deletion mutants and the Flag-tagged full-length T6BP were coexpressed in 293 cells. Cell extracts were prepared and immunoprecipitated (IP) with anti-Flag monoclonal antibody (F) or control mouse IgG (C). Coprecipitating Myc-T6BP deletion mutants were detected by immunoblotting (IB) analysis with the anti-Myc polyclonal antibody (Top). The amounts of Flag-T6BP immunoprecipitated and the expression level of Myc-T6BP deletion mutants in total cell extracts were determined by immunoblotting with anti-Flag polyclonal antibody (Middle) and anti-Myc antibody (Lower).

IL-1-inducible interactions among TRAF6, T6BP, and IRAK. 293.IL-1RI cells were stimulated with 10 ng/ml IL-1 for various times. Cell lysates were immunoprecipitated (IP) with an anti-T6BP rabbit polyclonal antibody (A), anti-TRAF6 polyclonal antibody (B), or anti-IRAK polyclonal antibody (C). Coprecipitating endogenous proteins were detected by immunoblotting (IB) with anti-TRAF6 monoclonal antibody (Top), anti-T6BP polyclonal antibody (Middle), or anti-IRAK polyclonal antibody (Bottom). The positions of TRAF6, T6BP, and IRAK are indicated.

IRAK is required for the IL-1-inducible TRAF6–T6BP complex formation. (A) 293.IL-1RI cells or I1A cells were stimulated with 10 ng/ml IL-1 for various times. Cell lysates were immunoprecipitated (IP) with anti-T6BP antibody and immunoblotted (IB) with anti-TRAF6 monoclonal antibody (Upper). The amounts of T6BP in the immunoprecipitates were determined by immunoblotting with anti-T6BP antibody (Lower). (B) I1A cells were transiently transfected with HA-tagged IRAK in pRK expression vector and stimulated with 10 ng/ml IL-1 for various times. Cell lysates were immunoprecipitated with anti-T6BP antibody and immunoblotted with anti-TRAF6 monoclonal antibody (Upper). The amounts of T6BP in the immunoprecipitates were determined by immunoblotting with anti-T6BP antibody (Lower).

The TRAF6–T6BP interaction is IL-1-specific. (A) HeLa cells were stimulated with 10 ng/ml IL-1 or 100 ng/ml TNF for various times. Cell lysates were immunoprecipitated (IP) with anti-T6BP antibody and immunoblotted (IB) with anti-TRAF6 monoclonal antibody (Upper). The amounts of T6BP in the immunoprecipitates were determined by immunoblotting with anti-T6BP antibody (Lower). (B) 293.CD40 cells were stimulated with a membrane preparation of CD40 ligand for various times. Cell lysates were immunoprecipitated with anti-T6BP antibody and immunoblotted with anti-TRAF6 monoclonal antibody (Upper Left). The amounts of T6BP in the immunoprecipitates were determined by immunoblotting with anti-T6BP antibody (Lower Left). The same cell lysates were also immunoprecipitated with anti-Flag antibodies that recognize the Flag-tagged CD40 receptor and were immunoblotted with anti-TRAF6 monoclonal antibody (Upper Right). The amounts of CD40 receptor in the immunoprecipitates were determined by immunoblotting with anti-CD40 antibody (Lower Right).

T6BP is not an activator of NF-κB or JNK pathways. (A) 293 cells were transiently cotransfected with an E-selectin-luciferase reporter gene plasmid and various amounts (given in micrograms per 35-mm plate) of TRAF6 or T6BP expression plasmids. At 24 h after transfection, luciferase activities were determined and normalized on the basis of β-galactosidase expression. (B) 293 cells were transiently cotransfected with HA-JNK plasmid and various amounts (given in micrograms per 100-mm plate) of TRAF6, T6BP, or MEKK1 expression plasmids. Cell lysates were immunoprecipitated (IP) with anti-HA antibody and incubated with [γ-³²P]ATP in the presence of bacterially expressed GST-Jun (amino acids 1–79). The proteins were resolved by SDS/PAGE and analyzed by autoradiography (Upper). The HA-JNK protein expression levels were determined by immunoblotting (IB) with anti-HA antibody (Lower). The positions of GST-Jun (1–79) and HA-JNK are indicated.

T6BP specifically interacts with TRAF6. 293 cells were transiently transfected with expression vectors encoding Myc-epitope-tagged T6BP and the Flag-tagged TRAFs 1–6. Cell extracts were prepared and immunoprecipitated (IP) with anti-Flag monoclonal antibody (F) or control mouse IgG (C). Coprecipitating Myc-T6BP was detected by immunoblotting (IB) analysis with the anti-Myc polyclonal antibody (Top). The amount of TRAFs immunoprecipitated and the expression level of Myc-T6BP in total cell extracts were determined by immunoblotting with anti-Flag polyclonal antibody (Middle) and anti-Myc polyclonal antibody (Bottom). The positions of T6BP and TRAFs are indicated. KD, kilodalton.

The TRAF6-interacting domains in T6BP. 293 cells were transiently transfected with expression vectors encoding Myc-epitope tagged T6BP deletion mutants and Flag-tagged TRAF6. Cell extracts were prepared and immunoprecipitated (IP) with anti-Flag monoclonal antibody (F) or control mouse IgG (C). Coprecipitating Myc-T6BP deletion mutants were detected by immunoblotting (IB) analysis with the anti-Myc polyclonal antibody (Top). The amounts of TRAF6 immunoprecipitated and the expression level of Myc-T6BP deletion mutants in total cell extracts were determined by immunoblotting with anti-Flag polyclonal antibody (Middle) and anti-Myc antibody (Lower). KD, kilodalton.