Fig. 4
AKT ubiquitination occurs in the nucleus under receptor desensitization conditions. A HEK293 cells expressing D3R were transfected with HA-Ub and FLAG-AKT. The cells were treated with 100 nM Quin for 5 min, washed, and re-challenged. Cell lysates were fractionated according to the protocol described in the “Materials and methods” section. AKT ubiquitination was assessed using nuclear and cytosolic fractions. NF, nuclear fraction; CF, cytosolic fraction. ****p < 0.0001 compared to “Veh/NF” group (n = 3). B HEK293 cells expressing D3R were transfected with GFP-AKT and FLAG-TRAF6. Cell lysates were fractionated according to the protocol described in the “Materials and methods” section. The interaction of TRAF6 with AKT was assessed in nuclear and cytosolic fractions. NF, nuclear fraction; CF, cytosolic fraction. ****p < 0.0001 compared to corresponding “Veh/NF” group (n = 3). C The bold letters represent the critical residues in the putative NLS of AKT (upper line). These residues were mutated to alanine residues in NLSX-AKT (lower line). D HEK293 cells were transfected with GFP-WT-AKT or GFP-NLSX-AKT. The cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature. After being washed 3 times with PBS, the cells were visualized via a laser-scanning confocal microscope (TCS SP5/AOBS/Tandem; Germany). The scale bar represents 10 μm. E HEK293 cells expressing D3R were transfected with HA-Ub and FLAG-WT-AKT or FLAG-NLSX-AKT. The cells were treated with 100 nM Quin for 5 min, washed, and re-challenged. Cell lysates were immunoprecipitated with anti-FLAG beads. Co-IP/IPs was immunoblotted with corresponding antibodies. ***p < 0.001, ****p < 0.0001 compared to the corresponding Veh group (n = 3). F HEK293 cells expressing D3R were transfected with WT-AKT or NLSX-AKT in AKT-KD cells. The cellular levels of cAMP were indirectly evaluated by using the CRE-luciferase reporter gene assay. WT/Quin group was significantly different from WT/Veh group at concentrations of quinpirole between 10−10 and 10−8 M.****p < 0.0001, ***p < 0.001, **p < 0.01 (n = 3)