Fig. 2
TRAF6-mediated AKT K8/14 ubiquitination leads to AKT phosphorylation, which is required for receptor desensitization. (A-C) Cells were treated with 100 nM Quin for 5 min according to desensitization protocol. Cell lysates were immunoprecipitated with anti-FLAG beads. Co-IP/IP was immunoblotted with corresponding antibodies. A HEK293 cells expressing D3R (1.9 ~ 2.1 pmol/mg protein) were transfected with HA-Ub and FLAG-WT-AKT or FLAG-T308A/S473A-AKT. Cell lysates were immunoprecipitated with anti-FLAG beads. ***p < 0.001, **p < 0.01 compared to the corresponding “WT/−” group (n = 3). B HEK293 cells expressing D3R (1.9 ~ 2.1 pmol/mg protein) were transfected with TRAF6 and FLAG-WT-AKT or FLAG-K8/14R-AKT. ***p < 0.001 compared to “WT/−” group, ##p < 0.01 compared to “WT/w+” group (n = 3). C HEK293 cells expressing D3R (1.9 ~ 2.1 pmol/mg protein) were transfected with HA-Ub and FLAG-WT-AKT or FLAG-K8/14R-AKT. ****p < 0.0001 compared to corresponding veh group (n = 3). (D-H) After the cells were pretreated, they were treated with 100 nM Quin for 5 min, washed three times with warm serum-free medium, and then treated with increasing concentrations of Quin. The cellular levels of cAMP were indirectly evaluated by using the CRE-luciferase reporter gene assay. ***p < 0.001, **p < 0.01, *p < 0.05 (n = 3) (D) HEK293 cells expressing D3R were pretreated with either vehicle or 200 nM triciribine for 6 h. Veh/Quin group was significantly different from Veh/veh group at treatment concentrations of quinpirole between 3 × 10−10 and 10−8 M. E CTRL-shRNA and TRAF6-KD cells were transfected with D3R. CTRL-shRNA/Quin group was significantly different from CTRL-shRNA /Veh group at concentrations ranging from 3 × 10−10 to 10−8 M. The cell lysates were immunoblotted with antibodies against TRAF6 and β-actin. The knockdown efficiency of TRAF6 was approximately 87%. F TRAF6-KD cells were transfected with D3R and WT-TRAF6 or C70A-TRAF6. The WT-TRAF6/Quin group was significantly different from WT-TRAF6/Veh group at concentrations of quinpirole between 10−9 and 10−8 M. G HEK293 cells expressing D3R were transfected with WT-AKT or K8/14R-AKT. The WT-AKT/Quin group was significantly different from other groups at concentrations of quinpirole between 10−9 and 10−8 M. H CTRL-shRNA and TRAF6-KD cells were transfected with K149C-D2R. The CTRL-shRNA/Quin group was significantly different from CTRL-shRNA/veh group at concentrations of quinpirole between 10−9 and 10−8 M