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AP-1 is a component of the transcriptional network regulated by GSK-3 in quiescent cells.

Tullai JW, Tacheva S, Owens LJ, Graham JR, Cooper GM - PLoS ONE (2011)

Bottom Line: Inhibition of GSK-3 attenuated this phosphorylation, resulting in the stabilization of c-Jun.These results indicate that inhibition of c-Jun by GSK-3 contributes to the repression of growth factor-inducible genes in quiescent cells.Together, AP-1, CREB and NFκB form an integrated transcriptional network that is largely responsible for maintaining repression of target genes downstream of GSK-3 signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Boston University, Boston, Massachusetts, United States of America.

ABSTRACT

Background: The protein kinase GSK-3 is constitutively active in quiescent cells in the absence of growth factor signaling. Previously, we identified a set of genes that required GSK-3 to maintain their repression during quiescence. Computational analysis of the upstream sequences of these genes predicted transcription factor binding sites for CREB, NFκB and AP-1. In our previous work, contributions of CREB and NFκB were examined. In the current study, the AP-1 component of the signaling network in quiescent cells was explored.

Methodology/principal findings: Using chromatin immunoprecipitation analysis, two AP-1 family members, c-Jun and JunD, bound to predicted upstream regulatory sequences in 8 of the 12 GSK-3-regulated genes. c-Jun was phosphorylated on threonine 239 by GSK-3 in quiescent cells, consistent with previous studies demonstrating inhibition of c-Jun by GSK-3. Inhibition of GSK-3 attenuated this phosphorylation, resulting in the stabilization of c-Jun. The association of c-Jun with its target sequences was increased by growth factor stimulation as well as by direct GSK-3 inhibition. The physiological role for c-Jun was also confirmed by siRNA inhibition of gene induction.

Conclusions/significance: These results indicate that inhibition of c-Jun by GSK-3 contributes to the repression of growth factor-inducible genes in quiescent cells. Together, AP-1, CREB and NFκB form an integrated transcriptional network that is largely responsible for maintaining repression of target genes downstream of GSK-3 signaling.

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Effect of GSK-3 inhibition on c-Jun phosphorylation and stability.T98G cells were rendered quiescent by serum starvation for 72 hours. Cells were then left untreated (NT), or treated with SB-216763 or vehicle control (DMSO) for the times indicated. Cell extracts were immunoblotted in parallel with anti-phospho threonine 239-c-Jun, pan-c-Jun and β-actin antibodies. Data shown are representative of three separate experiments. The left and right panels of the immunoblot in (B) were taken from the same autoradiography film, and therefore are identical exposure times.
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pone-0020150-g004: Effect of GSK-3 inhibition on c-Jun phosphorylation and stability.T98G cells were rendered quiescent by serum starvation for 72 hours. Cells were then left untreated (NT), or treated with SB-216763 or vehicle control (DMSO) for the times indicated. Cell extracts were immunoblotted in parallel with anti-phospho threonine 239-c-Jun, pan-c-Jun and β-actin antibodies. Data shown are representative of three separate experiments. The left and right panels of the immunoblot in (B) were taken from the same autoradiography film, and therefore are identical exposure times.

Mentions: Since the recruitment of c-Jun to its target sites was stimulated by inhibition of GSK-3, we investigated the effect of GSK-3 inhibition on c-Jun phosphorylation. It has been previously shown that GSK-3 phosphorylates c-Jun on threonine 239 [21]. This phosphorylation event has been shown to block c-Jun DNA binding activity [13] and transactivation [22], and target it for ubiquitination and proteasomal degradation [14]. We therefore assessed the phosphorylation status of c-Jun with the use of phospho-specific antibodies (Figure 4A). Quiescent T98G cells were treated for a time course up to 60 minutes with the GSK-3 inhibitor SB-216763, or the corresponding vehicle control, without growth factor stimulation. This was the treatment time corresponding to the gene inductions described previously [10]. As expected, phosphorylation of c-Jun threonine 239 was readily detectable in quiescent cells, consistent with the increased kinase activity of GSK-3 [10], [12]. As compared to the untreated sample (NT), phosphorylation at threonine 239 decreased as rapidly as 15 minutes following addition of SB-216763, and declined for the duration of the time course. The 60-minute vehicle control was unchanged.


AP-1 is a component of the transcriptional network regulated by GSK-3 in quiescent cells.

Tullai JW, Tacheva S, Owens LJ, Graham JR, Cooper GM - PLoS ONE (2011)

Effect of GSK-3 inhibition on c-Jun phosphorylation and stability.T98G cells were rendered quiescent by serum starvation for 72 hours. Cells were then left untreated (NT), or treated with SB-216763 or vehicle control (DMSO) for the times indicated. Cell extracts were immunoblotted in parallel with anti-phospho threonine 239-c-Jun, pan-c-Jun and β-actin antibodies. Data shown are representative of three separate experiments. The left and right panels of the immunoblot in (B) were taken from the same autoradiography film, and therefore are identical exposure times.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102068&req=5

pone-0020150-g004: Effect of GSK-3 inhibition on c-Jun phosphorylation and stability.T98G cells were rendered quiescent by serum starvation for 72 hours. Cells were then left untreated (NT), or treated with SB-216763 or vehicle control (DMSO) for the times indicated. Cell extracts were immunoblotted in parallel with anti-phospho threonine 239-c-Jun, pan-c-Jun and β-actin antibodies. Data shown are representative of three separate experiments. The left and right panels of the immunoblot in (B) were taken from the same autoradiography film, and therefore are identical exposure times.
Mentions: Since the recruitment of c-Jun to its target sites was stimulated by inhibition of GSK-3, we investigated the effect of GSK-3 inhibition on c-Jun phosphorylation. It has been previously shown that GSK-3 phosphorylates c-Jun on threonine 239 [21]. This phosphorylation event has been shown to block c-Jun DNA binding activity [13] and transactivation [22], and target it for ubiquitination and proteasomal degradation [14]. We therefore assessed the phosphorylation status of c-Jun with the use of phospho-specific antibodies (Figure 4A). Quiescent T98G cells were treated for a time course up to 60 minutes with the GSK-3 inhibitor SB-216763, or the corresponding vehicle control, without growth factor stimulation. This was the treatment time corresponding to the gene inductions described previously [10]. As expected, phosphorylation of c-Jun threonine 239 was readily detectable in quiescent cells, consistent with the increased kinase activity of GSK-3 [10], [12]. As compared to the untreated sample (NT), phosphorylation at threonine 239 decreased as rapidly as 15 minutes following addition of SB-216763, and declined for the duration of the time course. The 60-minute vehicle control was unchanged.

Bottom Line: Inhibition of GSK-3 attenuated this phosphorylation, resulting in the stabilization of c-Jun.These results indicate that inhibition of c-Jun by GSK-3 contributes to the repression of growth factor-inducible genes in quiescent cells.Together, AP-1, CREB and NFκB form an integrated transcriptional network that is largely responsible for maintaining repression of target genes downstream of GSK-3 signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Boston University, Boston, Massachusetts, United States of America.

ABSTRACT

Background: The protein kinase GSK-3 is constitutively active in quiescent cells in the absence of growth factor signaling. Previously, we identified a set of genes that required GSK-3 to maintain their repression during quiescence. Computational analysis of the upstream sequences of these genes predicted transcription factor binding sites for CREB, NFκB and AP-1. In our previous work, contributions of CREB and NFκB were examined. In the current study, the AP-1 component of the signaling network in quiescent cells was explored.

Methodology/principal findings: Using chromatin immunoprecipitation analysis, two AP-1 family members, c-Jun and JunD, bound to predicted upstream regulatory sequences in 8 of the 12 GSK-3-regulated genes. c-Jun was phosphorylated on threonine 239 by GSK-3 in quiescent cells, consistent with previous studies demonstrating inhibition of c-Jun by GSK-3. Inhibition of GSK-3 attenuated this phosphorylation, resulting in the stabilization of c-Jun. The association of c-Jun with its target sequences was increased by growth factor stimulation as well as by direct GSK-3 inhibition. The physiological role for c-Jun was also confirmed by siRNA inhibition of gene induction.

Conclusions/significance: These results indicate that inhibition of c-Jun by GSK-3 contributes to the repression of growth factor-inducible genes in quiescent cells. Together, AP-1, CREB and NFκB form an integrated transcriptional network that is largely responsible for maintaining repression of target genes downstream of GSK-3 signaling.

Show MeSH
Related in: MedlinePlus