Figure 2.

HEXIM1, which is phosphorylated on the serine at position 158, neither binds to RNA nor inhibits P-TEFb. (A) Only the WT HEXIM1 protein is phosphorylated by PKC in vitro. Purified His epitope-tagged WT HEXIM1 and mutant HEXIM1 (S158A) proteins (Hex and HexS158A) were incubated with native PKC catalytic subunit purified from rat brain in the presence of [γ-³²P] ATP for indicated times at 30°C. The phosphorylated HEXIM1 protein ((P)Hex) was detected by autoradiography. (B) Only the unmodified HEXIM1 protein binds to RNA. Indicated amounts of WT and in vitro PKC-phosphorylated HEXIM1 proteins were incubated with dsRNA for 10 min. at room temperature. RNA:protein mixtures were then separated by non-denaturing gels, and visualized by silver staining. The positions for Hex:dsRNA complex and free Hex protein are indicated on the left of the gel. (C) The phosphorylated HEXIM1 protein no longer inhibits the kinase activity of CDK9. IVKAs were performed by incubating purified P-TEFb with DSIF as a substrate in the presence of WT HEXIM1 (lane 2, Hex) or the phosphorylated HEXIM1 protein alone (lane 3, (P)Hex) or in the presence of dsRNA or 7SK snRNA (lanes 4 and 5, dsRNA and 7SK) or with combinations of increasing amounts of HEXIM1 and 7SK snRNA (lanes 6–8, 7SK + Hex), phosphorylated HEXIM1 proteins (lanes 9–11, dsRNA + (P)Hex; or, lanes 12–14, 7SK + (P)Hex). Phosphorylated DSIF was visualized by autoradiography.