FIG. 7.

GSK-3β acts downstream of Bim mRNA induction and translation to stabilize Bim leading to increased proapoptotic protein expression. (A) Cerebellar granule neurons were deprived of trophic support for 8 h in the presence or absence (−) of LiCl (10 mM) to inhibit GSK-3, SB216763 (3 μM) to inhibit GSK-3, or SP600125 (3 μM) to inhibit JNK. bim-EL and actin mRNA levels were assessed by reverse transcription-PCR. Representative gel images are shown. Neither inhibition of GSK-3 or JNK prevents the stress-induced increase in bim-EL expression. (B) Quantitated data (means plus standard errors of the means [SEM] [error bars] for 3 to 12 replicates) are shown. Values that are significantly from the control values are indicated (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (C) To examine the effect of GSK-3 or JNK inhibition on stress-induced Bim protein increase, cerebellar granule neurons (7 DIV) were treated as described above for panel A, except that cells were lysed in Laemmli sample buffer and immunoblotted with a Bim antibody. A representative gel (inset) and quantitative data from 5 to 10 experiments are shown. GSK-3, but not JNK, activity is critical for Bim protein induction in response to stress. (D) To measure the influence of GSK-3 on Bim stability cerebellar granule neurons (7 DIV) were deprived of trophic support (trophic deprivation [TD]) for 8 h after which trophic deprivation was continued for 4 h in the presence (+) or absence (−) of cycloheximide (50 μΜ) to inhibit translation and SB216763 (3 μM) to inhibit GSK-3. Blocking of translation allowed us to monitor the influence of GSK-3 on Bim protein turnover. GSK-3 inhibition significantly reduced Bim levels in the presence of cycloheximide, suggesting that GSK-3 activity blocks Bim degradation following trophic deprivation. Values that were significantly different (P < 0.05) from the control values are indicated by an asterisk.