Graduate Thesis Or Dissertation

 

Functional analysis of novel β-catenin mutants Public Deposited

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  • β-catenin is a multi functional protein that is involved in cell-cell adhesion and cell signaling. In non-stimulated cells, β-catenin is tightly down-regulated by GSK-3β-dependent phosphorylation at Ser and Thr residues, followed by rapid ubiquitination and proteasomal degradation. It is well established that mutations within the regulatory GSK-3β region lead to stabilized β-catenin and constitutive β-catenin/TCF-dependent gene activation. Furthermore, it has been shown that amino acids adjacent to codon 33, namely 32 and 34 of β-catenin, are hotspots for substitution mutations in carcinogen-induced animal tumors. Thus, a major hypothesis of this thesis was that substitution mutations at codon 32 of β-catenin interfere with phosphorylation and ubiquitination of β-catenin. Site-directed mutagenesis was used to create defined β-catenin mutants, namely D32G, D32N, and D32Y. The signaling potential of various β-catenin was analyzed in a gene reporter assay by co-transfection with a hTcf cDNA with a reporter plasmid containing a Tcf-dependent promoter (TOPFlash). There was a significant enhancement of the reporter gene activity with all β-catenin mutants compared to WT β-catenin after 48 hours of transfection. Protein analysis by Western blotting showed massive accumulation of mutant β-catenin. Antibody specific for phosphorylated β-catenin showed that the accumulated D32G and D32N β-catenin proteins were strongly phosphorylated both in vivo and in vitro, whereas D32Y β-catenin exhibited significantly attenuated phosphorylation in vivo. Further studies showed, however, that none of the mutants was sufficiently ubiquitinated. In addition, inhibition of the proteasome activity by ALLN was associated with accumulation of cytosolic β-catenin, which was transcriptionally inactive. This suppression of β-catenin transcriptional capacity was independent of ALLN-associated apoptosis in the transfected cells. Furthermore, exogenous β-catenin mediated modest cell survival and rendered cells sensitive to apoptotic stimuli. Thus, although codon 32 of β-catenin is not a direct target for phosphorylation, results from this thesis suggested that it affects the phosphorylation and ubiquitination of the adjacent Ser-33 residue of β-catenin, which is a direct target of GSK-3β. In addition, these results showed for the first time that the phosphorylation step of β-catenin is not enough to regulate transcriptional activity, and that β-catenin still needs to be ubiquitinated for successful down-regulation.
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