Figure 4.

Alpha subunit of stimulatory heterotrimeric GTP‐binding protein (Gαs) upregulates B‐cell leukemia/lymphoma‐2 gene homologous antagonist killer protein (Bak) expression by delaying degradation of Bak protein and by increasing transcription of the Bak gene. (a) Gαs reduced the degradation rate of the Bak protein. Subconfluent A549 cells were treated with a protein synthesis inhibitor, cycloheximide (30 µg/mL), and the remaining Bak protein was monitored at the indicated times by western blotting analysis. (b) Gαs increased Bak mRNA levels. Subconfluent A549 cells were treated with cisplatin for 24 h and Bak mRNA level was measured by reverse transcription–polymerase chain reaction. The expression level of Bak mRNA was normalized to that of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). (c) Gαs increased the activity of Bak promoter. For luciferase assays, A549 cells were transfected with 2.5 µg of (–3500 bp) Bak‐pLuc and 2.5 µg of a β‐galactosidase construct. After 24 h, cells were pretreated with H89 (15 mM) for 30 min or transfected with cyclic AMP response element (CRE) decoy (200 nM), and then with 40 µM cisplatin for 24 h. Bak luciferase activities were measured and normalized against β‐galactosidase activity and are presented as ratio against vector‐transfected controls. Three independent experiments were carried out in duplicate, and asterisks indicate significant difference from vector‐transfected controls (P < 0.05, Mann–Whitney U‐test). G, GαsQL; V, vector.