��-catenin is a multi functional protein that is involved in cell-cell adhesion
and cell signaling. In non-stimulated cells, ��-catenin is tightly down-regulated by
GSK-3��-dependent phosphorylation at Ser and Thr residues, followed by rapid
ubiquitination and proteasomal degradation. It is well established that mutations
within the regulatory GSK-3�� region lead to stabilized ��-catenin and constitutive
��-catenin/TCF-dependent gene activation. Furthermore, it has been shown that
amino acids adjacent to codon 33, namely 32 and 34 of ��-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 ��-catenin
interfere with phosphorylation and ubiquitination of ��-catenin.
Site-directed mutagenesis was used to create defined ��-catenin mutants,
namely D32G, D32N, and D32Y. The signaling potential of various ��-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 ��-catenin mutants
compared to WT ��-catenin after 48 hours of transfection. Protein analysis by
Western blotting showed massive accumulation of mutant ��-catenin. Antibody
specific for phosphorylated ��-catenin showed that the accumulated D32G and
D32N ��-catenin proteins were strongly phosphorylated both in vivo and in vitro,
whereas D32Y ��-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 ��-catenin, which was transcriptionally
inactive. This suppression of ��-catenin transcriptional capacity was independent of
ALLN-associated apoptosis in the transfected cells. Furthermore, exogenous ��-catenin mediated modest cell survival and rendered cells sensitive to apoptotic stimuli.
Thus, although codon 32 of ��-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 ��-catenin,
which is a direct target of GSK-3��. In addition, these results showed for the first
time that the phosphorylation step of ��-catenin is not enough to regulate
transcriptional activity, and that ��-catenin still needs to be ubiquitinated for
successful down-regulation. / Graduation date: 2003
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/31639 |
| Date | 12 February 2003 |
| Creators | Al-Fageeh, Mohamed B. |
| Contributors | Dashwood, Roderick H. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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