Figure 6.

Activation of the JNK but not ERK pathway in vivo increases GR phosphorylation. HeLa cells (1 × 10⁶) were transiently transfected in 100-mm dishes with pCMV-GR S224A S232A (2.3 μg) and (A) pRK5-M2 JNK (4.5 μg) with pCMV5-racQ61L (12 μg) or an empty pCMV5 vector (12 μg) or (B) pRK5-HA ERK (4.5 μg) with pRK5-HA f-sos (12 μg) or an empty pRK5 vector (12 μg) by using Lipofectamine. After 18 h, GR was metabolically labeled for 2 h with 1 mCi/ml of [³²P]orthophosphate in the presence of 100 nM Dex. In vivo labeled receptor was immunoprecipitated from cell lysates with the rat GR-specific monoclonal antibody, BuGR2. Immunoprecipitates were separated on 9% SDS/PAGE gels, transferred to Immobilon membrane, and exposed to film to visualize the phosphorylated GR (Upper). To normalize to GR protein expression, Western blotting of the same membrane with anti-GR rabbit polyclonal antibody was performed (Lower). The densitometric value obtained in the absence of cotransfected racQ61L (A) or f-sos (B) is arbitrarily set as 1.