FIG. 5.

Activated MEK1-ERK signaling up-regulates nuclear CDK9 and enhances its association with cyclin T1 to form P-TEFb. (A) Cyclin T1, free or incorporated into P-TEFb, was immunoprecipitated from nuclear extracts of GH4C1 cells prepared at various time points of TRH stimulation (0 [basal condition], 12, and 48 min) in the presence or absence of U0126 (10 μM) using an anti-cyclin T1 antibody. Cyclin T1 and CDK9 were analyzed by Western blotting with an anti-cyclin T1 and an anti-CDK9 antibody. Shown is a typical experiment repeated three times. (B) Nuclear and cytosolic extracts prepared at various time points after TRH stimulation (0, 12, and 48 min) in the presence or absence of U0126 were subjected to Western blotting with an anti-CDK9, an anti-cyclin T1, an anti-TFIIB (nuclear extracts), and an anti-actin (cytosolic extracts) antibody. Nuclear extracts were separately analyzed for phospho-ERKs and ERKs by Western blotting with an anti-phospho-ERK and an anti-ERK antibody. Shown are typical experiments repeated three times. (C) Nuclear extract prepared with or without TRH stimulation or ERK activation inhibitor peptide I treatment (2 μM) were subjected to immunoprecipitation using an anti-cyclin T1 antibody. The nuclear extract and the immunoprecipitated proteins were subjected to Western blotting with an anti-CDK9, an anti-cyclin T1, an anti-TFIIB, an anti-phospho-ERK, and an anti-ERK antibody. Shown are typical experiments repeated two times. (D and E) The reduction of CDK9/cyclin T1 dimers (i.e., P-TEFb) (D) or that of CDK9 (E) in the nucleus was calculated by quantification of the bands derived from experiments shown in panels A to C and is shown as a graph. The relative ratio is shown as the mean ± standard error of the mean (n = 3; U0126) or the mean of two experiments with the range (ERK inhibitor).