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. 2016 Mar 1;2:15048. doi: 10.1038/celldisc.2015.48

Figure 3.

Figure 3

TRIP6 regulates LPA-induced K63-linked polyubiquitination of TRAF6, and antagonizes the recruitment of A20 and CYLD to TRAF6. (a) Knockdown of TRIP6 attenuates K63-linked polyubiquitination of transfected TRAF6. SKOV-3 cells stably expressing scrambled shRNA or TRIP6 shRNA were transfected with the expression vectors of HA-K63-ubiquitin and FLAG-TRAF6. Cells were starved for 6 h, followed by LPA stimulation for 1 h. Heat-denatured FLAG-TRAF6 was immunoprecipitated with anti-FLAG M2 monoclonal antibody-conjugated agarose beads, followed by immunoblotting using anti-HA antibody to detect K63-linked polyubiquitinated FLAG-TRAF6. After stripping, the immunoblot was reprobed with anti-FLAG antibody to detect immunoprecipitated FLAG-TRAF6. The bottom two panels show the expression of total HA-K63-ubiquitin and TRIP6 in the whole-cell lysates. (b and c) Depletion of TRIP6 eliminates LPA-stimulated K63-linked polyubiquitination of endogenous TRAF6. HEK293T cells stably harboring scrambled shRNA or TRIP6 shRNA (b), or SKOV-3 cells stably harboring Cas9 alone (sgC) or Cas9/TRIP6 sgRNA (sgTRIP6-1, sgTRIP6-2) (c) were transfected with the HA-K63-ubiquitin expression vector. Cells were starved overnight, followed by stimulation with LPA for 1 h. Heat-denatured endogenous TRAF6 was immunoprecipitated with anti-TRAF6 mouse monoclonal antibody, followed by immunoblotting with anti-HA antibody to detect K63-linked polyubiquitinated TRAF6. The immunoblot was reprobed with anti-TRAF6 rabbit polyclonal antibody to detect immunoprecipitated TRAF6. (d) TRIP6 barely enhances the in vitro autoubiquitination of TRAF6. The in vitro ubiquitination assay was performed by incubating E1, Ubc13/Uev1A (E2), ubiquitin and ATP without or with purified recombinant TRAF6 and TRIP6 at 30 °C for 1 h. After heat denaturation, TRAF6 was immunoprecipitated with anti-TRAF6 mouse monoclonal antibody, followed by immunoblotting with anti-ubiquitin rabbit antibody to detect autoubiquitinated TRAF6. The blot was reprobed with anti-TRAF6 rabbit antibody. The right panel shows coomassie blue staining of purified TRAF6 and TRIP6 used in this experiment. (e) LPA stimulation decreases the association of TRAF6 with CYLD, but promotes its binding to A20, whereas TRIP6 binds to TRAF6 constitutively and associates with NF-κB p65 following LPA stimulation. HEK293T cells expressing FLAG-TRAF6 were starved for 6 h, followed by LPA stimulation for the indicated times. FLAG-TRAF6 was immunoprecipitated with anti-FLAG M2 mouse monoclonal antibody-conjugated agarose beads, and co-immunoprecipitated endogenous CYLD, A20 or TRIP6 was detected by immunoblotting using rabbit antibody specific to each protein. Endogenous TRIP6 was immunoprecipitated with anti-TRIP6 mouse monoclonal antibody or control mouse IgG, followed by immunoblotting to detect co-immunoprecipitated endogenous NF-κB p65. The bottom four panels show the expression of endogenous CYLD, A20, TRIP6 or NF-κB p65 in the whole-cell lysates. (f) Overexpression of TRIP6 eliminates LPA-promoted interaction between TRAF6 and A20, and mildly attenuates the association of TRAF6 with CYLD in the absence of LPA. HEK293T cells expressing FLAG-TRAF6 alone or with HA-TRIP6 were starved for 6 h, followed by stimulation with LPA for 1.5 or 3 h. FLAG-TRAF6 was immunoprecipitated with anti-FLAG M2 mouse monoclonal antibody-conjugated agarose beads. Co-immunoprecipitated A20, CYLD or HA-TRIP6 was detected by immunoblotting using rabbit antibody specific to A20, CYLD or HA epitope. (g) TRIP6 physically interferes with the binding of TRAF6 to A20. HEK293T cells harboring an empty vector or FLAG-A20 were treated with LPA for 1.5 h, and then harvested in RIPA buffer. FLAG-A20 was immunoprecipitated with anti-FLAG M2 mouse monoclonal antibody-conjugated agarose beads. The beads were then aliquoted and incubated with 0.1 μg purified recombinant TRAF6 in the absence or presence of 0.1 μg purified recombinant TRIP6 at 4 °C for 3 h. After washing four times, the beads were subjected to immunoblotting with anti-TRAF6 rabbit antibody to detect co-immunoprecipitated recombinant TRAF6. The blot was stripped and reprobed with anti-FLAG antibody to detect immunoprecipitated FLAG-A20. The right panel shows coomassie blue staining of purified recombinant TRAF6 and TRIP6. (h and i) Depletion of TRIP6 greatly enhances the binding of TRAF6 to A20 and CYLD in SKOV-3 cells. SKOV-3 cell lines stably expressing shRNA (shScr, shTRIP6) (h) or Cas9/sgRNA (sgControl, sgTRIP6-1) (i) were starved overnight, followed by LPA treatment for various times as indicated. Endogenous TRAF6 was immunoprecipitated with anti-TRAF6 mouse monoclonal antibody or control mouse IgG, followed by immunoblotting with rabbit antibody specific to A20, CYLD, TRIP6 or TRAF6. The bottom four panels in each figure show the expression of endogenous A20, CYLD, TRIP6 or GAPDH in the whole-cell lysates. Data shown in each figure are representative of two to four independent experiments.