Our long-term goal is to understand the molecular mechanisms regulating disabled-2 (Dab2) protein expression levels. Dab2 is a cytoplasmic adaptor molecule that is associated with clathrin mediated endocytosis. It was originally identified as a protein down-regulated in ovarian carcinoma and it has since been established that its expression levels have significant impact on cellular proliferation, differentiation, and cancer development. It has been designated as a putative tumor suppressor due to its decreased expression levels in ovarian, breast, prostate and pancreatic carcinoma cells. We have obtained preliminary data suggesting that, in addition to transcriptional regulation, cellular Dab2 expression levels are also maintained through post-transcriptional mechanisms involving transcript-specific translational control and ubiquitin-mediated proteasomal degradation. In two cellular differentiation systems, a TGFbeta-mediated epithelial to mesenchymal transdifferentiation model in mouse mammary epithelial (NMuMG) cells and a retinoic acid (RA)-induced visceral endoderm differentiation model in F9 teratocarcinoma cells, we present data suggesting that transcriptional induction of Dab2 mRNA alone can not account for induced Dab2 protein expression levels. We demonstrate that under basal .conditions, Dab2 expression levels are translationally silenced through a mechanism driven by sequence elements contained in the Dab2 3'-untranslated region (3'-UTR). Cytosolic extracts from TGFbeta-treated NMuMG or RA-treated F9 cells can overcome this 3'- UTR-mediated silencing, resulting in increased Dab2 protein expression. We further show that once synthesized, Dab2 levels are regulated through a GSK-3beta-dependent phosphorylation and ubiquitin-mediated degradation pathway. These results demonstrate that Dab2 expression is highly regulated and support the hypothesis that its expression levels underlie its proliferate and differentiative functions. In this proposal, we wish to further explore the molecular mechanisms through which Dab2 expression levels are regulated and determine whether dysregulation of these mechanisms might underlie its aberrant expression in cancer.
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