ARE-mRNA stabilization by PKB. (A) The Tet-β-globin-IL3UTR reporter gene was transfected alone (lanes 1–3) or in combination with constitutively activated m/pPKB (lanes 4–6). After 24 h, transcription was stopped by addition of doxycycline. Cytoplasmic RNA was isolated at the indicated time points and processed for northern blotting. (B) The graph shows the quantifications of five independent decay assays normalized to the actin signal. Standard errors are shown unless too small to be represented.

PKB phosphorylates BRF1 on S92. (A) Alignment of BRF1 sequences from human, mouse, rat and X. leavis. S90 and S92 are indicated. (B) In vitro phosphorylation reactions were performed for the times indicated using 40 ng of activated recombinant PKB and 20 ng of either full length rBRF1-wt, a N-terminal fragment (aa 3–110), the Zn-finger domain (aa 111–179) or a C-terminal fragment (aa 180–338). The asterisk denotes the full-length BRF1 protein, lower bands are degradation products. (C) In vitro phosphorylation reactions were performed for the times indicated, using 40 ng of activated recombinant PKB and 20 ng of either rBRF1-wt, rBRF1-S90A or rBRF1-S92A.

BRF1 is phosphorylated in vivo at S92. (A) Indicated amounts of rBRF1-wt were incubated with buffer alone or activated PKB for 30′ at 30°C and processed for western blotting. Phosphorylated (upper panel) or unphosphorylated BRF1 (lower panel) were detected by corresponding antibodies directed against a p-S92 containing or a C-terminal peptide (see Materials and methods). (B) rBRF1 phosphorylated as in (A) was processed for western blotting. Antibodies recognizing phosphorylated BRF1 (upper panel), BRF1 (middle panel) or PKB (lower panel) were pre-incubated on ice for 30′ with or without 50 μg of the PP used for immunization before adding to the blot. (C) HIRc-B cells were serum starved overnight, treated with WM (200 nM) for 30′ where indicated before stimulation with insulin (20 μg/ml) for 15′ (lanes 1–3). Whole-cell extracts were analyzed by western blot, detecting p-BRF1, phospho-PKB and α-tubulin as a loading control. (D) NIH3T3 B₂A₂-23 cells were transfected with m/pPKB or kinase-dead PKB (PKBkd) and BRF1-wt or BRF1S90A/S92A (BRF1AA). Whole-cell extracts were analyzed by western blot, detecting p-BRF1, phospho-PKB and α-tubulin as a loading control.

BRF1 S92 phosphorylation regulates ARE-dependent RNA decay in vitro. (A) Radioactively labeled ARE_IL3 and ΔARE transcripts were incubated together in S100 extract from slowC cells. Decay reactions were performed at 37°C in the presence of increasing amounts of rBRF1-wt, stopped at the indicated time points and resolved on a 10% urea-polyacrylamide gel. ΔARE transcripts served as specificity and loading control. (B) Decay reactions were carried out in the absence (lanes 1–4) or in the presence of 20 ng of rBRF1-wt (lanes 5–12) or rBRF1-S92A (lanes 13–20) after pretreatment with buffer (lanes 5–8/13–16) or activated PKB (lanes 9–12/17–20). The graph below shows quantification of at least four independent experiments after normalizing to the ΔARE signal. Standard error bars were too small to be represented.

Effect of insulin on reporter mRNA decay. (A) At 24 h after co-transfection of the Tet-β-globin-IL3UTR reporter gene and the Tet-responsive transcriptional activator tTA (pTET-on), HIRc-B cells were split into fresh medium for 16 h. Where indicated cells were pretreated with WM (200 nM) for 30′ before addition of insulin (20 μg/ml) for another 15′. Then doxycycline was added (2 μg/ml) to stop transcription, cytoplasmic RNA was isolated at the indicated time points and processed for northern blotting. (B) Quantification of three independent experiments is shown normalized to the 18S rRNA signal. (C) MEF-PKBα−/− and +/+ cells were pretreated with WM for 30′ prior to insulin (Ins) stimulation for 15′ as indicated. p-BRF1, phospho-PKB, PKB and tubulin protein levels were analyzed by western blot. (D) MEF-PKBα−/− and +/+ cells were treated as described for HIRc-B in (A). Reporter RNA was analyzed by northern blot. One representative experiment is shown.

S92 phosphorylation of BRF1 induces complex formation with 14-3-3. (A) Electrophoretic mobility shift assay. Indicated amounts of rBRF1 were pre-incubated with or without activated PKB, mixed with radio-labeled ARE_IL3 RNA and resolved on 4% nondenaturing PAGE. (B) His-tagged rBRF1 pretreated with buffer (lane 1) or activated PKB (lanes 2–11) was mixed with S100 extract from slowC cells. Increasing concentrations of the phosphorylated peptide surrounding S92 (aa 86–98) of BRF1 (PP) or the corresponding S92A mutant peptide (AP) were added. rBRF1 was purified using Ni-NTA beads, and 14-3-3 and BRF1 were detected with the corresponding antibodies. The lower band represents 14-3-3, the upper His-tagged rBRF1. (C) In vitro decay reactions were carried out as described in Figure 3 in the presence of 0.4 mM of PP (lanes 9–12) or AP (lanes 13–16). The graph on the right shows quantification of four independent experiments after normalizing to the ΔARE signal. Standard error bars were too small to be represented. (D) COS7 cells were transfected with vector, bsdHisBRF1 wt, bsdHisBRF1 aa, either alone (lanes 1–3) or in combination with m/pPKB (lanes 5–8), as indicated. In the experiment shown on the right (lanes 4–8), FCS was withdrawn for 40 h. Expression of His-tagged BRF1, phospho-PKB and 14-3-3 in the cell extracts was determined by western blotting (load). After purification of BRF1 by Ni-NTA beads, co-precipitation of 14-3-3 was tested by western blotting (Co-P).