Bak is ubiquitinated and degraded by proteasomes in resistant B-NHL cell lines. (A) Dose-dependent inhibition of proteasome activity in resistant cells correlated with dose-dependent induction of Bak expression. Resistant cells (RL 4RH shown here) were incubated with 0-25nM bortezomib for 24 hours, then assayed for chymotrypsin-like activity of the 26S proteasome. Bak expression within the same cell population was determined by Western blot. Proteasome inhibition occurred at similar doses in other resistant and sensitive cell lines (data not shown). Bak induction correlated with proteasome inhibition in other resistant but not sensitive cell lines (data not shown). Data shown are from a representative experiment repeated 3 times. Points on the proteasome activity graph represent the average of triplicate wells ± SD (B) Increased Bak expression was observed in resistant cells treated with proteasome inhibitors and not other stress-inducing agents. Western blot analysis of whole cell lysates demonstrated increased Bak expression in resistant cells (Raji 2R shown here) treated for 48 hours with the proteasome inhibitors bortezomib (Bort.; 25nM) or MG132 (5μM) but not the standard chemotherapeutic agents cisplatinum (CDDP; 100μM) or adriamycin (ADR; 50μM), or the endoplasmic reticulum (ER) stress-inducers brefeldin A (BfA; 1 μg/mL), tunicamycin (Tunic.; 1 μg/mL), or thapsigargin (Thaps.; 1μM). (C) Incubation of resistant cells with bortezomib did not induce mRNA coding for Bak. Quantitative real-time PCR analysis of RNA extracted from resistant cells (Raji 4RH cells shown here) treated with bortezomib over a 24-hour time course showed no increased expression of BAK1, while Bak protein level within the same cell population increased approximately 2.5-fold. Data shown are from a representative experiment repeated twice. Real-time PCR data were analyzed using the ΔΔCt method and is expressed as fold change compared with time = 0 hours ± SD. (D) Bortezomib treatment of resistant cell lines stabilized expression of Bak protein. Bak stability was determined by standard ³⁵S-methionine pulse-chase experiments. Bak was rapidly degraded in vehicle-treated (DMSO) resistant cells (open triangles) but not sensitive cells (closed circles). Incubation of resistant cells with bortezomib (50nM) stabilized the expression of Bak to a level comparable with untreated sensitive cells. Data shown are from a representative experiment repeated twice. (E) Exogenously expressed Bak appears ubiquitinated in resistant but not sensitive cells. FLAG-tagged Bak was immunoprecipitated using FLAG beads and subjected to Western blot for Bak expression. Higher molecular weight bands were detected in FLAG-IP/Bak Western blots from resistant (Raji 2R shown here) but not sensitive cells (Raji shown here). (F) Bortezomib treatment increases the ubiquitination of Bak in resistant cells. After 24 hours of bortezomib treatment (25nM), endogenous Bak or ubiquitin were immunoprecipitated from lysates of sensitive (RL shown here) or resistant (RL 4RH shown here) cells. Ubiquitin detected in the Bak IP from resistant cells increased to a greater degree following bortezomib treatment than did the ubiquitin detected in the Bak IP from sensitive cells while total ubiquitin levels increased similarly in sensitive and resistant cells treated with bortezomib.