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beta-Catenin is efficiently phosphorylated by glycogen synthase kinase-3beta in the Axin complex in the cytoplasm, resulting in the down-regulation. In response to Wnt, beta-catenin is stabilized and translocated into the nucleus where it stimulates gene expression through Tcf/Lef. Here we report a novel protein, designated Duplin (for axis duplication inhibitor), which negatively regulates the function of beta-catenin in the nucleus. Duplin was located in the nucleus. Duplin bound directly to the Armadillo repeats of beta-catenin, thereby inhibiting the binding of Tcf to beta-catenin. It did not affect the stability of beta-catenin but inhibited Wnt- or beta-catenin-dependent Tcf activation. Furthermore, expression of Duplin in Xenopus embryos inhibited the axis formation and beta-catenin-dependent axis duplication, and prevented the beta-catenin''s ability to rescue ventralizing phenotypes induced by ultraviolet light irradiation. Thus, Duplin is a nuclear protein that inhibits beta-catenin signaling.
FIG. 4. Effect of Duplin on axis formation of Xenopus embryos. A, ventralizing activity of Duplin. Embryos were injected dorsally with
Duplin (1 ng) (a), ventrally with Duplin (1 ng) (b), dorsally with Duplin-(1–482) (1 ng) (c), or dorsally with Duplin-(482–749) (1 ng) (d). B, inhibition
of siamois expression. Expression of siamois in injected embryos was detected with RT-PCR analysis. Dorsal injection with Xglobin (lane 1, 1 ng)
and Duplin (lane 2, 100 pg; lane 3, 500 pg; lane 4, 1 ng). Ventral injection with Duplin (lane 5, 1 ng). Dorsal injection with Duplin-(1–482) and
Duplin-(482–749) (lanes 6 and 7, 1 ng). The amounts of cDNA were standardized with EF-1a. Sia, siamois; RT 2, experiments without RT-PCR.
C, inhibition by Duplin of Wnt signal-dependent axis formation. Embryos were injected dorsally (a, c, e, g) or ventrally (b, d, f, h) with Xwnt-8 (100
pg) and Xglobin (1 ng) (a and b), Xwnt-8 (100 pg) and Duplin (1 ng) (c and d), Xb-catenin (500 pg) and Xglobin (1 ng) (e and f), or Xb-catenin (500 pg) and Duplin (1 ng) (g and h). D, left panel indicates the results of average DAI of A. DAI 0, completely ventralized; DAI 5, normal. The average
DAI used is not an accurate concept but is used for illustrative purposes. Right panel shows the frequency of the secondary axis in C. Black and
white columns indicate the complete and incomplete axes, respectively. E, inhibition by Duplin of b-catenin-dependent rescue of the axis formation.
Injections were done into UV-treated embryos with Xglobin (2 ng) (a), Xglobin (1 ng) and Duplin (1 ng) (b), Xb-catenin (500 pg) and Xglobin (1 ng)
(c), or Xb-catenin (500 pg) and Duplin (1 ng) (d). F, inhibition by Duplin of b-catenin-induced expression of siamois. Embryos without UV light
irradiation (lane 1). UV light-irradiated embryos injected with Xglobin (2 ng) (lane 2), Xglobin (1 ng) and Duplin (1 ng) (lane 3), Xb-catenin (500
pg) and Xglobin (1 ng) (lane 4), or Xb-catenin (500 pg) and Duplin (1 ng) (lane 5). G, the results in E were expressed with DAI. H, expression of
Duplin in Xenopus development. Aliquots of the extracts from the indicated stages of embryos were probed with the anti-Duplin antibody.