Abstract
Despite its central role in cell survival and proliferation, the transcriptional program controlled by GSK-3 is poorly understood. We have employed a systems level approach to characterize gene regulation downstream of PI 3-kinase/Akt/GSK-3 signaling in response to growth factor stimulation of quiescent cells. Of 31 immediate-early genes whose induction was dependent on PI 3-kinase signaling, 12 were induced directly by inhibition of GSK-3. Most of the GSK-3-regulated genes encoded transcription factors, growth factors, and signaling molecules. Binding sites for CREB were highly over-represented in the upstream regions of these genes, with 9 genes containing CREB sites that were conserved in mouse orthologs. Binding sites predicted in 6 genes were confirmed by CREB chromatin immunoprecipitation and forskolin induction of CBP binding. Moreover, CREB siRNA substantially blocked induction of 5 genes by forskolin and of 3 genes following inhibition of GSK-3. These results indicate that GSK-3 actively represses gene expression in quiescent cells, with inhibition of CREB playing a key role in this transcriptional response.
Highlights
As a target of PI 3-kinase/Akt signaling, GSK-3 plays a key role in regulating cell metabolism, proliferation, and survival
Its targets include a variety of transcription factors, the transcriptional program controlled by GSK-3 downstream of
26), which binds to CREB phosphorylated at serine 133
Summary
Cell Culture and Treatments—T98G human glioblastoma cells were grown in Minimal Essential Medium (Invitrogen) containing 10% fetal calf serum. Using a set of sequences bound by human TATAbinding protein (hTBP) in vitro (18), the threshold was based on the collection of highest scoring positions within each sequence These 68 sequences, derived from a restriction endonuclease protection selection and amplification (REPSA) assay, were grouped into four categories based on their consensus sequence as TATAAA, TAAATA, TATATA, or Other. Chromatin Immunoprecipitation—Chromatin immunoprecipitations were performed as described (11), except that cells were resuspended in lysis buffer (5 mM PIPES, pH 8.0, 85 mM KCl, 0.5% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml pepstatin A, and aprotinin) for 10 min after formaldehyde fixation. Transfection mixtures were prepared in 400 l of total volume by adding 24 l HiPerfect and either CREB1 siRNA (Ambion siRNA ID 109994, 5Ј-GGUGGAAAAUGGACUGGCUtt-3Ј) or nonspecific siRNA (Ambion negative control siRNA 1, catalog 4611) to serum-free medium, mixed, and incubated at room temperature for 10 min. Total RNA-extracted, and analyzed by real time RT-PCR
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