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 beta-catenin in the Wnt pathway. Using a yeast two-hybrid screen, we identified a novel protein, GSK3beta interaction protein (GSKIP), which binds to GSK3beta. We have defined a 25-amino acid region in the C-terminus of GSKIP that is highly similar to the GSK3beta interaction domain (GID) of Axin. Using an in vitro kinase assay, our results indicate that GSKIP is a good GSK3beta 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 GID(381-405) and FRATtide, synthesized GSKIPtide is also shown to compete with and/or block the phosphorylation of Axin and beta-catenin by GSK3beta. Furthermore, our data indicate that overexpression of GSKIP induces beta-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 GSK3beta interaction domain of Axin and is able to negatively regulate GSK3beta of the Wnt signaling pathway.

Emerging evidence shows that glycogen synthase kinase 3␤ (GSK3␤) is involved in mitotic division and that inhibiting of GSK3␤ kinase activity causes defects in spindle microtubule length and chromosome alignment. However, the purpose of GSK3␤ involvement in spindle microtubule assembly and accurate chromosome segregation remains obscure. Here, we report that GSK3␤ interacts with the spindle-associated protein Astrin both in vitro and in vivo. Additionally, Astrin acts as a substrate for GSK3␤ and is phosphorylated at Thr-111, Thr-937 ((S/T)P motif) and Ser-974/Thr-978 ((S/T)XXX(S/T)-p motif; p is a phosphorylatable residue). Inhibition of GSK3␤ impairs spindle and kinetochore accumulation of Astrin and spindle formation at mitosis, suggesting that Astrin association with the spindle microtubule and kinetochore may be dependent on phosphorylation by GSK3␤. Conversely, depletion of Astrin by small interfering RNA has no detectable influence on the localization of GSK3␤. Interestingly, in vitro assays demonstrated that Astrin enhances GSK3␤-mediated phosphorylation of other substrates. Moreover, we showed that coexpression of Astrin and GSK3␤ differentially increases GSK3␤-mediated Tau phosphorylation on an unprimed site. Collectively, these data indicate that GSK3␤ interacts with and phosphorylates the spindle-associated protein Astrin, resulting in targeting Astrin to the spindle microtubules and kinetochores. In turn, the GSK3␤-Astrin complex may also facilitate further physiological and pathological phosphorylation.Glycogen synthase kinase 3 (GSK3), 2 a serine/threonine kinase active in several signaling pathways, is involved in the regulation of cell fate, including Wnt and Hedgehog signal transduction, protein synthesis, glycogen metabolism, mitosis and apoptosis (1-4). GSK3 has two structurally similar isoforms in mammals, GSK3␣ and GSK3␤, that are ubiquitously expressed and differ in their N-and C-terminal regions (2, 5). Earlier reports indicated that not only are the developmental profiles of GSK3␣ and GSK3␤ expression different but also the regulation and functions of these two proteins are not always identical (6 -9). Factors known to influence the functions of GSK3␤ include the phosphorylation of GSK3␤ itself, the subcellular localization of GSK3␤, the protein-protein interaction of GSK3␤, and the phosphorylation state of GSK3␤ substrates (1, 3, 4, 10). Insulin-mediated inhibition of GSK3 was mediated through a phosphorylation-dependent mechanism, with the phosphorylation at position Ser-21 and Ser-9 in GSK3␣ and GSK3␤, respectively (11).GSK3␤ also has a preference for pre-phosphorylated substrates, recognizing the consensus sequence (S/T)XXX(S/T)-p. In this sequence the first S/T residue is the target for GSK3␤ phosphorylation, X is any amino acid, P denotes the phosphorylatable residue, and the S/T located at the C terminus is the phosphorylation priming site (12). On the other hand, a number of proteins, including Axin and Tau, are phosphorylated by GSK3␤ without pre-phosphorylation. Recombinant Tau...