Retarded proteasome-mediated turnover and inefficient ubiquitination of Myc mutants.
(A) Immunoblot analysis of v- and c-Myc (upper panel) and β-actin (lower panel) in U-937-myc-6 cells after 2 hours treatment with the proteasome inhibitors MG115, MG132, and LLnL, the protease inhibitors chloroquine (CQ), E64, and pepstatin or DMSO vehicle in the presence of cycloheximide (CHX) as indicated. (B) Pulse chase analysis of c-Myc turnover in the presence of the proteasome inhibitor MG115. Daudi (wt c-Myc) and CA46 (mutated c-Myc) Burkitt's lymphoma cells were treated with the proteasome inhibitor for 1.5 hours, pulsed with35S methionine followed by chase in the presence of MG115 for the indicated time points or were pulse labeled in the absence of the inhibitor. Cell lysates were then immunoprecipitated with pan-Myc antibodies and analyzed as in Figure 3. (C) Reduced ubiquitination of the T58A c-Myc mutant. U2OS cells were transfected with wt c-Myc, the T58A c-Myc mutant, His6-Ub or HA-Ub vectors alone or in combination as indicated in the figure. The transfected cells were treated with MG115 during the last 2 hours before harvest. His6-Ub-conjugated proteins were purified as described in “Materials and Methods” and subjected to Western blot analysis using Myc antibodies (upper panel) or dot blot analysis using ubiquitin antibodies (lower panel). The smears of c-Myc-His6-Ub-conjugates are indicated. The middle panel shows c-Myc immunoblot analysis of 1/10 of the input material (note that the exposure time for this blot is shorter than for the upper panel).
