Figure 1.
TRAF6 interacts with STAT3 and mediates the ubiquitination of STAT3.
(A) TRAF6 interacts with STAT3 in mammalian cells. Myc-TRAF6 and Flag-STAT3 were co-transfected into HEK293 cells. After 24 h, the cell lysates were immunoprecipitated with anti-Flag antibody and subjected to Western blot with the anti-Flag or anti-Myc antibodies. (B) The cell lysates of HEK293 cells were immunoprecipitated with anti-TRAF6 antibody and subjected to Western blot with the anti-TRAF6 or anti-STAT3 antibodies. (C) Myc-TRAF6 was transfected into HEK293 cells in a dose-dependent manner. After 24 h, the cell lysates were probed with anti-Myc, anti-STAT3 and anti-GAPDH antibodies. (D, E and F) TRAF6 mediates the ubiquitination of STAT3. HA-Ub(WT) (D), HA-Ub(K63R) (E) or HA-Ub(K48R) (F) and Flag-STAT3 were co-expressed in HEK293 cells with Myc-TRAF6 or empty vectors. The cell lysates and immunoprecipitates were resolved by SDS-PAGE and immunoblotted with anti-HA or anti-Flag antibodies. The data are representative of at least three independent experiments.
Figure 2.
TRAF6 inhibits the transcriptional activity of STAT3.
(A, B) TRAF6 represses the activity of STAT3 with IFNα stimulation. HEK293 cells were transiently transfected with the indicated combinations of m67 luciferase reporter or 4×IRF luciferase reporter, pRL-TK control, STAT3 and TRAF6. Then the cells were treated with IFNα (50 ng/ml) for 6 h before lysis. After 24 h, the cell lysates were collected for luciferase activity measurements. Data are presented as the means ± S.D. (n = 3). (C, D) TRAF6 inhibits the activity of STAT3 with the forced expression of JAK2. HEK293 cells were transiently transfected with GAS luciferase reporter or m67 luciferase reporter, TRAF6, STAT3 and JAK2. After 24 h, the cell lysates were collected for luciferase activity measurements. Data are presented as the means ± S.D. (n = 3).
Figure 3.
The RING finger domain and the TRAF-type zinc finger domain play key roles in the regulation of the transcriptional activity of STAT3.
(A) A schematic illustration of TRAF6 domains. The asterisk indicates the expression of TRAF6 mutants that was detected by Western blot with anti-Flag antibody. (B and C) Reporter gene assays of STAT3. HEK293 cells were transiently transfected with various TRAF6 truncated mutants, m67 luciferase plasmids (B) or 4×IRF luciferase plasmids (C), STAT3 and pRL-TK plasmids. After 24 h, the cell lysates were collected for luciferase activity measurements. Data are presented as the means ± S.D. (n = 3).
Figure 4.
TRAF6 negatively regulates the expression of the STAT3 target genes.
(A) TRAF6 inhibited the expression of the STAT3 target gene, CRP. The HEK293 cells were transfected with the indicated amounts of TRAF6. After 24 h, the HEK293 cells were stimulated with IFNα for 15 min and the mRNA levels of CRP were examined by real-time PCR. The expression values were normalized to the expression of GAPDH. Data are presented as the means ± S.D. (n = 3). (B) TRAF6 inhibited the expression of ACT. HEK293 cells were transiently transfected with the indicated combinations of pACT luciferase reporter, pRL-TK control TRAF6, STAT3 and JAK2. After 24 h, the cell lysates were collected for luciferase activity measurements. Data are presented as the means ± S.D. (n = 3). These results are representative of three independent experiments. (**P<0.01 compared with control samples which are not transfected with TRAF6, Student's t test).