UBC3 is a target of SCF^βTrCP. A, UBC3 protein levels in CSN knockdown cells are rescued by CUL1 suppression. shRNA-transduced 293T cells were transfected with a control oligonucleotide or with two different siRNAs targeting CUL1. UBC3 content in total lysates was analyzed by Western blotting (upper panel). Cullin mRNA levels were analyzed by quantitative reverse transcription-PCR (lower panel). B, endogenous UBC3 and βTrCP interact. UBC3 (middle panel) and βTrCP (right panel) were immunoprecipitated (IP) from HEK293T cell lysates with specific antibodies. The control used was the irrelevant mouse (left panel) or rabbit (right panel) IgG antibody. WB, Western blot. C, the F-box is not required for βTrCP interaction with UBC3. 293T cells were transfected with the indicated vectors (full-length βTrCP (βTrCP-FL) and the F-box deletion mutant βTrCPΔFbox), and βTrCP was immunoprecipitated with anti-FLAG antibodies. UBC3 wt, wild-type UBC3. D, βTrCP overexpression promotes UBC3 ubiquitination. 293T cells were transfected as indicated. HA-UBC3 was immunoprecipitated with anti-HA antibody, and ubiquitinated forms of UBC3 were detected by anti-Myc Western blotting. E, down-regulation of βTrCP increases UBC3 protein levels. 293T cells were transfected with two different siRNA oligonucleotides that target both βTrCP1 and βTrCP2. F, down-regulation of SKP2 has no effect on UBC3 protein levels. 293T cells were transfected with two siRNA oligonucleotides targeting SKP2.

The acidic UBC3 C-terminal extension is a destabilizing element. A, shown is a schematic representation of the UBCH5 constructs. UBC3 WT, wild-type UBC3. B, the addition of the UBC3 C-terminal extension reduces UBCH5A half-life. 293T cells were transfected with HA-wild-type UBCH5A or HA-UBCH5A+3tail and treated with CHX for the indicated times. Lysates were analyzed by Western blotting. UBCH5A/actin ratios are represented relative to time 0. C, the UBC3 C-terminal extension destabilizes UBCH5B. 293T cells were transfected with the indicated UBCH5 constructs and a GFP vector. Cells were treated and analyzed as described in the legend to Fig. 3D. D, the UBC3 C-terminal extension promotes interaction with βTrCP. 293T cells were transfected with the indicated constructs. βTrCP was immunoprecipitated (IP) with anti-FLAG antibodies. Total lysates (lower panel) and immunoprecipitated samples (upper panels) were analyzed by Western blotting. Lower molecular weight bands in the fourth lane represent proteolytic products of UBCH5B+3tail. E, the UBC3 C-terminal extension directs βTrCP-dependent ubiquitination of UBCH5B. 293T cells were transfected with the indicated constructs. UBCH5B was immunoprecipitated with anti-HA antibody.

UBC3 is destabilized upon CSN down-regulation. A, UBC3 levels are decreased in CSN-deficient cells. 293T cells were transduced with lentiviral vectors expressing either a control shRNA or shRNA targeting CSN4 or CSN5. Left panel, shown are the results of Western blot analysis. Right panel, CSN4, CSN5, and UBC3 mRNA levels were analyzed by real-time reverse transcription-PCR and are represented relative to the control sample. Error bars represent S.D. of three independent experiments. B, UBC3 protein levels were analyzed after CSN down-regulation in HeLa cells, human primary fibroblasts, and T cells. C, treatment with the proteasome inhibitor MG132 recovered UBC3 protein levels in CSN4-deficient 293T cells (upper panel). Total RNA from the same cultures was analyzed by quantitative reverse transcription-PCR for UBC3 expression (lower panel). cont, control. D, CSN down-regulation affects UBC3 half-life. Upper panel, 293T cells were transduced with control or CSN4 shRNA vectors and treated with CHX for the indicated times. Lysates were analyzed by Western blotting. Lower panel, densitometric quantification of the blots. UBC3/actin ratios are plotted relative to time 0.

UBC3 C-terminal extension is required for interaction with βTrCP and UBC3 ubiquitination. A, schematic representation of UBC3 constructs. The UBC3-(1–229) deletion construct lacks the βTrCP consensus motif. UBC3-(1–200) lacks the acidic domain. UBC3 WT, wild-type UBC3. B, the C-terminal extension of UBC3 is required for interaction with βTrCP. 293T cells were transfected with the indicated vectors, and UBC3 constructs were immunoprecipitated (IP) with anti-HA antibody. Endogenous βTrCP was visualized by Western blotting. C, βTrCP-dependent ubiquitination of UBC3 requires the acidic C-terminal domain. 293T cells were transfected with the indicated expression constructs, and UBC3 was immunoprecipitated with anti-HA antibody. Total lysates (lower panel) and immunoprecipitated proteins (upper panels) were analyzed by Western blotting. D, the acidic domain affects UBC3 stability. 293T cells were transfected with the indicated HA-UBC3 constructs and with a GFP construct as a control of transfection efficiency. Cultures were divided in two after 24 h and treated with CHX alone (60 μg/ml) or in combination with MG132 (20 μm) for 6 h. UBC3 levels in lysates were quantified by phosphorimaging and normalized to GFP levels. The graph represents -fold increases in protein levels of the indicated constructs in the presence of MG132 relative to the values in the absence of MG132.

Down-regulation of βTrCP, but not of USP15, recovers UBC3 levels in CSN-deficient cells. A, shRNA-transduced cells were transfected with control siRNA oligonucleotides or oligonucleotides targeting βTrCP. Cell lysates were analyzed by Western blotting. B, βTrCP is maintained in CSN-deficient cells. Lysates of shRNA-transduced cells were analyzed by Western blotting. C, HeLa cells were transfected in duplicates with control RNA oligonucleotides or two different RNA oligonucleotides targeting USP15. Total lysates were analyzed by Western blotting with the indicated antibodies.