We have discovered a previously uncharacterized glucocorticoid-regulated cell signaling cascade and identified steroid-regulated target genes that control cell-cell interactions and polarization of cultured monolayers of tumorigenic and nontumorigenic mammary epithelial cells. The mammary epithelial cell system we have developed is unique because hormone signals can be used to acutely control, in a reversible manner, apical junction organization and tight junction dynamics in a physiologically appropriate manner. The overall goal of this amended proposal is to test the hypothesis that the functional coordination of ubiquitin-proteasome mediated degradation of GSK3 (Glycogen Synthase Kinase-3), a multifunctional serine/threonine kinase, and of CAS (Cellular Apoptosis Susceptibility gene), a component of the nuclear transport machinery that binds to importin-alpha, directly controls beta-catenin dynamics and promotes steroid dependent adherens junction organization and tight junction sealing. We have demonstrated that phosphorylation of GSK-3 by the serum- and glucocorticoid-inducible protein kinase (Sgk), or by Akt, at Serine-9 of GSK3-beta is required for GSK3 protein degradation. As a direct result of GSK3 degradation, a stabilized hypophosphorylated form of beta-catenin is produced that is accessible to be localized to its site of function in the adherens junction. We have previously established that Sgk is a primary glucocorticoid induced gene and an importin-alpha cargo protein. We propose that the glucocorticoid stimulation of Sgk expression, in combination with activated Akt, phosphorylates both the GSK3-beta and GSK3-alpha isoforms to trigger their ubiquitination by a specific E3 ubiquitin ligase complex and subsequent 26S proteasome-mediated degradation. We further propose that the glucocorticoid induced degradation of CAS results in the cytoplasmic localization of importin-alpha and its Sgk cargo protein, thereby allowing Sgk to be accessible to GSK3 in glucocorticoid treated cells. One goal of this proposal is to use mutagenic and protein biochemical approaches to define the precise structural domains in GSK-3 and CAS that control their glucocorticoid regulated degradation and to functionally characterize components of this regulated degradative pathway in vitro and in transfected cells. A complementary goal is to use mutagenesis along with in vitro and intact cell nuclear/cytoplasmic trafficking assays to examine how the CAS/importin-alpha interaction regulates Sgk localization and how beta-catenin compartmentalization is regulated by glucocorticoids. A final goal is to functionally characterize the roles of GSK3 and CAS and their degradative systems in the steroid dependent control of beta-catenin dynamics, apical junction organization and tight junction formation.