FIGURE 5.

CK1 phosphorylates GST-OREBP-(146-167) in vitro and regulates OREBP nuclear export in vivo. A, sequence alignment of relevant OREBP regions from human, mouse, and zebrafish (containing residues equivalent to Ser-155 and Ser-158 of human OREBP). Conserved residues have been boxed. B, CKI-7 inhibits in vitro phosphorylation of Ser-158. Nuclear extracts were obtained from HeLa cells treated with hypotonic growth medium for 30 min. GST-OREBP-(146-167) and its mutants were incubated with the nuclear extracts in the presence of [γ-³²P]ATP and CKI-7 or ethanol (solvent) for 20 min at 30 °C. The proteins were separated by electrophoresis, stained with Coomassie Blue (lower panel), and submitted to autoradiography (upper panel). C, recombinant CK1α phosphorylates the GST-OREBP-(146-167) mutant containing a phosphomimetic mutation at Ser-155 (S155D). CK1α1 (40 ng) was incubated with GST-OREBP-(146-167) or its mutants in the presence of [γ-³²P]ATP for 20 min at 30 °C. The proteins were separated by electrophoresis, stained with Coomassie Blue (lower panel), and submitted to autoradiography (upper panel). D, left, CKI-7 inhibits nuclear export of OREBP in vivo. HeLa cells transfected with FLAG-OREBP-(1-581)-Δ1-131 were pretreated with CKI-7 (100 or 200 μm) or ethanol (-) for 4 h in growth medium (Isotonic). The cells were moved to hypotonic medium supplemented with the same amount of the inhibitor or solvent for another 90 min (Hypotonic). FLAG-tagged recombinant protein was visualized after fixation with a FLAG antibody and a FITC-labeled secondary antibody; right, quantification of the subcellular localization of OREBP in the presence of CKI-7. For each condition >100 cells were counted. The data shown are the average of three independent experiments. E, real time quantitative PCR of different CK1 isoforms after siRNA knockdown. HeLa cells were transfected with the indicated siRNAs, respectively. After 72 h, mRNA was harvested from the cells and reverse-transcribed and quantified by primers specific to the isoform. Data were expressed as percentage relative to cells that were not transfected(Untreated control). Cells transfected with scramble siRNA (Nontargeting control) were also included to demonstrate the specificity of siRNA knockdown. F, left, effect of CK1 knockdown on nuclear export of OREBP. HeLa cells transfected with FLAG-OREBP-(1-581)-Δ1-131 and the indicated siRNA were treated with hypotonic medium for 90 min. FLAG-tagged recombinant protein was visualized after fixation with a FLAG antibody and a FITC-labeled secondary antibody and counterstained with DAPI; right, quantification of the subcellular localization of OREBP. For each condition >100 cells were counted. The data represented are the average of three independent experiments.