AP-1 Is a Component of the Transcriptional Network Regulated by GSK-3 in Quiescent Cells

Abstract

BackgroundThe 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 FindingsUsing 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/SignificanceThese 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.

A p38 Mitogen-Activated Protein Kinase-Regulated Myocyte Enhancer Factor 2–β-Catenin Interaction Enhances Canonical Wnt Signaling

“…We further analyzed c-Jun protein levels, since c-Jun expression has been shown to be regulated by canonical Wnt signaling through a TCF/LEF binding motif on its endogenous promoter, which is truncated in the pJC6 reporter (12). Based on reports in the literature, induction of quiescence results in a reduction of c-Jun (46,47), and this was observed even with coexpression of both MEF2C and ␤-catenin (act) (Fig. 4C, left panel).…”

Identification of Two Forms of TNF Tolerance in Human Monocytes: Differential Inhibition of NF-κB/AP-1– and PP1-Associated Signaling

“…It has also been shown that GSK3 inhibits the activation of JNK and p38 (54) and is also involved in the inhibition of c-jun-mediated transcription (28). For example, it has been suggested that AP-1, CREB, and NF-kB form an integrated transcriptional network largely responsible for maintaining repression of genes downstream of GSK3 signaling (55). It should also be mentioned that GSK3 phosphorylates c-jun targeting this protein for degradation (56).…”

A GSK-3-mediated transcriptional network maintains repression of immediate early genes in quiescent cells

“…Consistent with this, ChIP assays indicated that c-Jun and JunD but not JunB were bound to predicted AP-1 sites upstream of eight of the GSK-3-repressed genes. 37 The binding of c-Jun increased in response to both PDGF stimulation and direct GSK-3 inhibition, whereas the binding of JunD was unaffected, suggesting a direct effect of GSK-3 on c-Jun. In addition, targeted siRNA knockdown confirmed that c-Jun is required for the induction of three genes following GSK-3 inhibition.…”

“…in quiescent T98G cells, 37 as their expression requires growth factor signaling. [38][39][40][41] In contrast, c-Jun, JunD and JunB were all expressed in quiescent cells, and, therefore, were considered as candidate targets for regulation by GSK-3.…”