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Address correspondence to Jackie Papkoff, diaDexus, Inc., 343 Oyster Point Blvd., South San Francisco, CA 94080. Tel.: (650) 246-6502. Fax: (650) 246-6597. E-mail: jpapkoff{at}diadexus.com
We have developed a novel Saccharomyces cerevisiae model system to dissect the molecular events of ß-catenin (ß-cat) signaling. Coexpression of mammalian ß-cat with TCF4 or LEF1 results in nuclear accumulation of these proteins and a functional complex that activates reporter gene transcription from constructs containing leukocyte enhancer factor (LEF)/T cell factor (TCF) response elements. Reporter transcription is constitutive, requires expression of both ß-cat and TCF4 or LEF1, and is not supported by mutated LEF/TCF binding elements or by TCF4 or LEF1 mutants. A cytoplasmic domain of E-cadherin or a functional fragment of adenomatous polyposis coli (APC) protein (APC-25) complexes with ß-cat, reduces ß-cat binding to TCF4, and leads to increased cytoplasmic localization of ß-cat and a reduction in reporter activation. Systematic mutation of putative nuclear export signal sequences in APC-25 decreases APC-25 binding to ß-cat and restores reporter gene transcription. Additional ß-cat signaling components, Axin and glycogen synthase kinase 3ß, form a multisubunit complex similar to that found in mammalian cells. Coexpression of the F-box protein ß-transducin repeat-containing protein reduces the stability of ß-cat and decreases reporter activation. Thus, we have reconstituted a functional ß-cat signal transduction pathway in yeast and show that ß-cat signaling can be regulated at multiple levels, including protein subcellular localization, protein complex formation, and protein stability.
Key Words: Saccharomyces cerevisiae; active transport; cell nucleus; cytoskeletal proteins; adenomatous polyposis coli
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