Glucocorticoid hormones stimulate adherens junction and tight junction formation in Con8 mammary epithelial tumor cells through a multistep pathway involving numerous cell signaling components. The multifunctional adherens junction protein β-catenin is regulated in several ways by glucocorticoids. β-catenin mRNA transcripts are upregulated in response to treatment with the synthetic glucocorticoid dexamethasone, possibly through promoter regulation by the Id-1 transcriptional repressor protein. Dexamethasone treatment also induces the production of unphosphorylated, stabilized β-catenin protein that is localized exclusively to the cell periphery, whereas in the absence of dexamethasone, phosphorylated β-catenin is either degraded or localized to the nucleus. The localization of β-catenin at the cell periphery in steroid-treated cells correlates with the glucocorticoid-induced changes in localization of the nuclear export factor CAS, the nuclear import factor, importin-α, the protein kinases Sgk (Serum- and glucocorticoid-induced kinase), and ERK (Extracellular signal-regulated kinase). GSK3 (Glycogen Synthase Kinase-3) phosphorylation of β-catenin is known to trigger the degradation of β-catenin suggesting that steroid activated cascades may be targeting this protein kinase. Dexamethasone induces the ubiquitin-26S proteasome mediated degradation of GSK3 protein with no change in GSK3 transcript levels. This ubiquitin conjugation is not mediated through the β-TrCP E3 ligase. In transfected cells, deletion of the N-terminal nine amino acids or mutation of the serine-9 phosphorylation site on GSK3-β prevented its glucocorticoid-induced ubiquitination and degradation. Expression of stabilized GSK3 proteins ablated the glucocorticoid-induced tight junction sealing and resulted in production of a nonphosphorylated β-catenin that localizes to both the nucleus and the cell periphery in steroid treated cells. Serine-9 on GSK3 can be phosphorylated by Sgk and by Akt. Expression of dominant negative forms of either Sgk or Akt inhibited ubiquitination and degradation of GSK3 by glucocorticoids, and disrupted the dexamethasone-induced effects on β-catenin dynamics. Furthermore, the steroid-induced tight junction sealing is attenuated in cells expressing dominant negative forms of either Sgk or Akt, although the effect of blunting Sgk signaling was significantly greater. I have uncovered a new cellular cascade in which Sgk and Akt trigger the glucocorticoid-regulated phosphorylation, ubiquitination and degradation of GSK3, which alters β-catenin dynamics, leading to the formation of the apical junctional complex.