Figure 5.

TRIP6 enhances TRAF6-NF-κB signaling by inhibiting the binding of A20 to TRAF6, but promotes LPA-induced TRAF6-JNK-AP-1 activation in an A20-independent manner. (a) The K63-linked polyubiquitination of TRAF6 is enhanced by wild-type TRIP6, but not E52A-TRIP6 mutant defective in blocking the recruitment of A20 to TRAF6. SKOV-3 cells stably expressing TRIP6 shRNA (SKOV-3-shTRIP6) were transiently transfected with the expression vectors of HA-K63-Ubiquitin, FLAG-TRAF6 and either EGFP, EGFP-TRIP6 or EGFP-E52A-TRIP6. After starvation for 24 h, cells were treated with LPA for 1 h. The levels of K63-linked polyubiquitinated FLAG-TRAF6 were determined as described in Figure 3a. The expression of HA-K63-ubiquitin, endogenous TRIP6, EGFP or EGFP-TRIP6 in the whole-cell lysates was detected by immunoblotting using antibody specific to HA, TRIP6 or GFP. (b and c) Mutation of E52 to Ala greatly eliminates the function of TRIP6 in promoting IκBα phosphorylation, but barely or only mildly attenuates its ability to promote LPA-induced JNK activation. SKOV-3 cells stably expressing TRIP6 shRNA (b) or Cas9/TRIP6 sgRNA (sgTRIP6-1) (c) were transiently transfected with EGFP- (b) or FLAG-tagged (c) wild-type or E52A TRIP6. Cells were starved for 24 h, followed by addition of LPA for 30 min. Immunoblotting was performed to detect the levels of indicated proteins in the whole-cell lysates. Data shown in (a–c) are representative of two or three independent experiments. (d) The effect of TRIP6 on promoting LPA-stimulated NF-κB activity is attenuated by the E52A mutation. SKOV-3-shTRIP6 cells were transiently transfected with pNF-κB-Luc, the β-galactosidase expression vector and either pEGFP, pEGFP-TRIP6 or pEGFP-E52A-TRIP6. Cells were starved for 24 h, followed by treatment with LPA for 3 h. The NF-κB-driven luciferase activity was determined and normalized to the β-galactosidase activity. Data shown are the mean±s.e.m. of four independent experiments done in duplicates (*P<0.001 versus untreated EGFP cells; **P<0.01, ***P<0.05 versus treated EGFP cells; ^#P<0.05 versus untreated EGFP-TRIP6 cells; ^##P<0.05 versus treated EGFP-TRIP6 cells; Student’s t-test). (e) The effect of TRIP6 on promoting LPA-induced AP-1 activity is not significantly affected by the E52A mutation. The expression vectors of AP-1-Luc and β-galactosidase were co-transfected with either pEGFP, pEGFP-TRIP6 or pEGFP-E52A-TRIP6 into SKOV-3-shTRIP6 cells. After starvation for 24 h, cells were treated with LPA for 3 h. The AP-1-driven luciferase activity was determined and normalized to the β-galactosidase activity. Data shown are the mean±s.e.m. of three independent experiments done in triplicates (*P<0.01 versus untreated EGFP cells; **P<0.001 versus treated EGFP cells; ^#P<0.05 versus untreated cells; Student’s t-test). (f) A20 specifically inhibits LPA-stimulated IκBα phosphorylation, but not JNK activation; however, this effect is partially reversed by TRIP6 overexpression. SKOV-3 cells transiently overexpressing EGFP-A20 and/or FLAG-TRIP6 were starved in 0.1% fatty acid-free BSA-containing medium for 6 h, followed by stimulation with LPA for 30 min. Immunoblotting was performed to determine the levels of indicated proteins in the whole-cell lysates. Data shown are representative of two independent experiments. (g) The effect of A20 on the restriction of NF-κB activity is partially reversed by TRIP6 overexpression. NF-κB-Luc and β-galactosidase were transiently co-expressed with FLAG-A20 and/or FLAG-TRIP6 in SKOV-3 cells. Cells were starved overnight, followed by stimulation with LPA for 2 h. The NF-κB-driven luciferase activity was determined and normalized to the β-galactosidase activity. Data shown are the mean±s.e.m. of four independent experiments (*P<0.001, **P<0.05 versus treated mock cells; ***P<0.05 versus untreated mock cells; ^#P<0.001, ^##P<0.05 versus untreated cells; Student’s t-test). The bottom three panels are representative immunoblots showing the expression of FLAG-TRIP6, FLAG-A20 or GAPDH in the whole-cell lysates.