Fig 2: LPA and Thr-induced rapid synthesis of free K63-linked polyubiquitin chains is required for T-loop phosphorylation of TAK1 and its downstream effectors. A HaCaT cells were serum-starved for 24 h and then exposed to IL-1β (10 ng/mL) or LPA (10 μM) for the indicated times. WCE were prepared and subjected to GST-ZnF UBP pull-down before immunoblotting analysis using both anti-GST and anti-K63 ubiquitin antibodies simultaneously. Data are representative of two independent experiments. B-C HaCaT stable cell lines expressing an empty vector or 4 isoT-derived ZnF domains in tandem were serum-starved for 24 h and then exposed to LPA (10 μM) (B) or Thr (1 U/mL) (C) for the indicated times. WCE were prepared, equally divided, and run simultaneously on different acrylamide gels and subjected to immunoblotting analysis using the indicated antibodies. Data are representative of at least three independent experiments. D-E The quantification of the phosphorylating events shown in panels B and C is depicted as histogram bars, representing the ratio of the phosphorylated protein to the non-phosphorylated form. Data (n = 3) are represented as mean ± SEM. * P < 0.05; ** p < 0.01; *** p < 0.001, 2-way ANOVA with Tukey’s multiple-comparison test

Fig 3: TRAF6 is rapidly activated and produces free K63-linked polyubiquitin chains, which interact with TAK1-TAB2 complexes in response to GPCR stimulation. A VSMC were serum-starved for 24 h and then exposed to Ang II (100 nM) and Il-1β (10 ng/mL) for the indicated times. Cytosolic and membrane fractions were prepared, and cell extracts were immunoblotted with the indicated antibodies. Data are representative of 2 independent experiments. The quantification of TRAF6 is shown below the TRAF6 immunoblot and represents the ratio of TRAF6 to β-actin. B VSMC were serum-starved for 24 h and then exposed to Ang II (100 nM) for the indicated times. WCE were prepared and subjected to GST or GST-ZnF UBP pull-down. GST-ZnF UBP-trapped proteins were treated with or without isoT (200 nM) prior to immunoblotting analysis using both anti-GST and anti-K63 ubiquitin antibodies simultaneously. Arrows indicate the generation of constant HMW free K63-linked polyubiquitin chain species. Data are representative of a minimum of 3 independent experiments. C and D Wild-type and TRAF6 knockout MEFs were serum-starved for 5 h and then exposed to Thr (1 U/mL) and LPA (10 μM) for 10 min. WCE were prepared and subjected to immunoprecipitation using anti-TAB2 antibody before immunoblotting analysis with the indicated antibodies. The data are representative of two independent experiments. E VSMC were serum-starved for 24 h and then exposed to Thr (1 U/mL) and LPA (10 μM) for 5 min. WCE were prepared and subjected to immunoprecipitation using anti-TAB2 antibody. Immunoprecipitated proteins were treated with or without isoT (200 nM) before immunoblotting analysis using the indicated antibodies. Data are representative of two independent experiments. F HASMC were serum-starved for 24 h and then exposed to Ang II (1 μM) for the indicated times. WCE were prepared and subjected to immunoprecipitation using anti-TAK1 antibody. Immunoprecipitated proteins were treated with or without isoT (200 nM) before immunoblotting analysis using the indicated antibodies. Data are representative of a minimum of 3 independent experiments