Nuclear localization of Duplin, a beta-catenin-binding protein, is essential for its inhibitory activity on the Wnt signaling pathway.
Duplin binds to beta-catenin and inhibits the Wnt signaling pathway, thereby leading to repression of the beta-catenin-mediated transactivation and Xenopus axis formation. To find an additional function of Duplin, yeast two-hybrid screening was carried out. Importin alpha was isolated as a binding protein of Duplin. Importin alpha bound directly to basic amino acid clusters of Duplin. Although Duplin was present in the nucleus, deletion of the basic amino acid clusters (Duplin(Delta 500-584)) retained Duplin in the cytoplasm. Duplin(Delta 500-584) bound to beta-catenin as efficiently as wild-type Duplin, but it neither repressed Wnt-dependent Tcf transcriptional activation in mammalian cells nor showed ventralization in Xenopus embryos. The Duplin mutant without a beta-catenin-binding region lost the ability to inhibit the Wnt-dependent Tcf activation, but retained its ventralizing activity. Furthermore, Duplin not only suppressed beta-catenin-dependent axis duplication and expression of siamois, a Wnt-regulated gene, but also inhibited siamois-dependent axis duplication. These results indicate that Duplin is translocated to the nucleus by interacting with importin alpha, and that nuclear localization is essential for the function of Duplin. Moreover, Duplin has an additional activity of inhibiting the Wnt signaling pathway by affecting the downstream beta-catenin target genes.
Kobayashi
Masashi
M
Department of Biochemistry, Faculty of Medicine, Hiroshima University 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
Kishida
Shosei
S
Fukui
Akimasa
A
Michiue
Tatsuo
T
Miyamoto
Yoichi
Y
Okamoto
Tetsuji
T
Yoneda
Yoshihiro
Y
Asashima
Makoto
M
Kikuchi
Akira
A
D021581:Active Transport, Cell Nucleus; D020816:Amino Acid Motifs; D000818:Animals; D001921:Brain / Q000502:physiology; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000502:physiology; D015723:Gene Library; D028884:Karyopherins / Q000378:metabolism; D007739:L Cells; D051379:Mice; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D011518:Proto-Oncogene Proteins / Q000037:antagonists & inhibitors*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015534:Trans-Activators; D015533:Transcriptional Activation; D051153:Wnt Proteins; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000196:embryology; D029961:Zebrafish Proteins; D051178:alpha Catenin; D051176:beta Catenin
C495266:CTNNB1 protein, Xenopus; C495267:CTNNB1 protein, mouse; D002352:Carrier Proteins; C415830:Chd8 protein, rat; C495272:Ctnna1 protein, mouse; D003598:Cytoskeletal Proteins; D028884:Karyopherins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D051153:Wnt Proteins; D029867:Xenopus Proteins; D029961:Zebrafish Proteins; D051178:alpha Catenin; D051176:beta Catenin; D011505:Protein-Tyrosine Kinases