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Although prominent FRAT/GBP exhibits a limited degree of homology to Axin, the binding sites on GSK3 for FRAT/GBP and Axin may overlap to prevent the effect of FRAT/GBP in stabilizing β-catenin in the Wnt pathway. Using a yeast two-hybrid screen, we identified a novel protein, GSK3β interaction protein (GSKIP), which binds to GSK3β. We have defined a 25-amino acid region in the C-terminus of GSKIP that is highly similar to the GSK3β interaction domain (GID) of Axin. Using an in vitro kinase assay, our results indicate that GSKIP is a good GSK3β substrate, and both the full-length protein and a C-terminal fragment of GSKIP can block phosphorylation of primed and nonprimed substrates in different fashions. Similar to Axin GID381–405 and FRATtide, synthesized GSKIPtide is also shown to compete with and/or block the phosphorylation of Axin and β-catenin by GSK3β. Furthermore, our data indicate that overexpression of GSKIP induces β-catenin accumulation in the cytoplasm and nucleus as visualized by immunofluorescence. A functional assay also demonstrates that GSKIP-transfected cells have a significant effect on the transactivity of Tcf-4. Collectively, we define GSKIP as a naturally occurring protein that is homologous with the GSK3β interaction domain of Axin and is able to negatively regulate GSK3β of the Wnt signaling pathway. [ABSTRACT FROM AUTHOR]