The serine/threonine kinase glycogen synthase kinase-3 (GSK-3) is a master regulator of a variety of cellular processes. First characterized as the kinase responsible for phosphorylating and inactivating glycogen synthase, GSK-3 now has recognized roles in controlling cell proliferation, survival and differentiation. Abnormal GSK-3 regulation has been associated with many human diseases including diabetes, heart disease, cancer, Alzheimer's disease and schizophrenia 
GSK-3 has two widely expressed mammalian isoforms, GSK-3α and GSK-3β, both of which are subject to regulation by the PI 3-kinase/Akt pathway 
. GSK-3 has been shown to regulate cell survival and proliferation downstream of PI 3-kinase signaling through phosphorylation of cyclin D1 
, Mcl-1 
, and eukaryotic translation initiation factor 2B (eIF2B) 
, as well as a variety of transcription factors 
. GSK-3 is also regulated through the Wnt pathway. Wnt signaling results in a decrease in the phosphorylation of β-catenin by GSK-3, causing a corresponding increase in the transcriptional activation of β-catenin/TCF target genes 
Unlike most protein kinases, GSK-3 is constitutively active in quiescent cells, and undergoes an inhibitory phosphorylation by Akt (on serine 9 for GSK-3β, and on serine 21 for GSK-3α) in the presence of growth factors 
. The activity of GSK-3 in quiescent cells suggests that it may actively maintain repression of growth factor-regulated genes in the absence of PI 3-kinase signaling. We have investigated the role of GSK-3 in quiescence by combining global expression profiling and computational analyses to examine gene expression downstream of PI 3-kinase/Akt/GSK-3 signaling 
. These studies identified a set of twelve immediate early genes whose induction following growth factor stimulation of quiescent T98G human glioblastoma cells was dependent upon PI 3-kinase and which could also be induced by direct inhibition of GSK-3 without growth factor stimulation 
. These genes mainly encoded growth factors and transcription factors involved in cell proliferation, so their repression by GSK-3 presumably contributed to maintenance of the quiescent state of the cell.
The identification of a set of genes that required GSK-3 to maintain their repression during quiescence allowed us to investigate the transcriptional network downstream of GSK-3 signaling. Since the expression of co-regulated genes may be mediated by common transcription factors, we examined the upstream sequences of the twelve GSK-3 repressed genes to identify statistically over-represented and evolutionarily conserved transcription factor binding sites. This computational analysis predicted AP-1, as well as CREB and NFκB transcription factors, as potential regulators of these genes downstream of GSK-3 
In the present study, we have investigated the role of AP-1 family members in GSK-3 mediated transcriptional regulation. Two AP-1 family members, c-Jun and JunD, bound to predicted upstream regulatory sequences in 8 of the 12 GSK-3-regulated genes. Consistent with previous studies demonstrating inhibition of c-Jun by GSK-3 
, c-Jun was phosphorylated by GSK-3 in quiescent cells. The association of c-Jun with its target sequences was increased by growth factor stimulation as well as by GSK-3 inhibition, and a physiological role for c-Jun was demonstrated by siRNA inhibition of gene induction. These results indicate that inhibition of c-Jun by GSK-3 contributes to the repression of growth factor-regulated genes during quiescence. Moreover, together with previous studies, these findings delineate an integrated transcriptional network in which AP-1, CREB and NFκB play major roles in GSK-3-mediated repression of target genes in quiescent cells.