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Signaling by the Wnt family of extracellular proteins is critical in a variety of developmental processes in which cell and tissue polarity are established [1-5]. Wnt signal transduction has been studied mostly by the genetic approach in Drosophila and Caenorhabditis elegans [1,2,5], but the biochemical mechanisms involved remain to be elucidated. The Wnt pathway also operates during axis determination in vertebrates [3,5]. Frizzled receptors transduce a signal to Dishevelled, leading to inactivation of glycogen synthase kinase 3 (GSK3) and regulation of gene expression by the complex of beta-catenin with LEF/TCF (lymphocyte enhancer factor/T-cell factor) transcription factors [3,5]. Axin is a negative regulator of Wnt signaling and dorsal axial development in vertebrates [6]. Here, we demonstrate that axin is associated with GSK3 in the Xenopus embryo and we localize the GSK3-binding domain to a short region of axin. Binding of GSK3 correlates with the ability of axin to inhibit axial development and with the axis-inducing activity of its dominant-negative form (delta RGS). We also find that wild-type axin, but not delta RGS, forms a complex with beta-catenin. Thus, axin may act as a docking station mediating negative regulation of beta-catenin by GSK3 during dorsoventral axis determination in vertebrate embryos.
Figure 1.
(a) Axin constructs used in this study. (b) Defining the GSK3-binding domain in axin. Myc-tagged axin constructs were overexpressed in Xenopus embryos by microinjection of the corresponding mRNAs into all blastomeres at the four-cell stage. Embryo lysates were immunoprecipitated (IP) at the early gastrula stage (stage 10+) with 9E10 antibodies followed by western analysis with anti-GSK3β (α-GSK3β) monoclonal antibodies. The arrow indicates a position corresponding to GSK3. (c) Expression of axin derivatives detected with 9E10 antibodies. The same membrane was used in (b) and (c). The asterisk in (b,c) denotes the immunoglobulin heavy chain recognized by the secondary antibodies; the double asterisk in (c) marks a nonspecific protein band.
Figure 2.
Association of GSK3 with axin correlates with the effects of axin and its dominant-negative form on axis determination in Xenopus embryos. Overexpression of (a) C2 mRNA, but not (b) C3 mRNA, in both dorsovegetal blastomeres of four-cell embryos leads to inhibition of dorsal axial development. Overexpression of (c)δRGS-C2 mRNA, but not (d)δRGS-C3 mRNA, in a ventralblastomere induces a complete secondary axis. Embryos shown in (d) are indistinguishable from uninjected control siblings. (e) Inhibition of dorsal development by different axin constructs. DAI, dorso-anterior index. (f) Axis-inducing activity of different δRGS constructs. Filled bars represent embryos with complete axes, including eyes and cement glands; open bars represent embryos with partial neural tubes and hindbrain, but lacking fore/midbrain structures.
Figure 3.
(a) Axin-associated kinase activity is down-regulated by Xwnt8. Embryos were injected with different RNAs as indicated. Embryonic lysates were prepared at stage 10+ for precipitation with specific antibodies. An immune complex kinase assay was carried out with myelin basic protein (MBP) as a substrate. Expression levels of axin, ΔRGS, C3 and GSK3 (arrowhead) were analysed with specific antibodies. (b) Comparative analysis of GSK3 activity by immune complex kinase assay. Relative kinase activity was calculated as a ratio of MBP phosphorylation to the amount of GSK3β. Data were normalized by the median between the highest value and background and are shown as the mean ± standard error from three experiments. C, uninjected control embryos, 9E10 antibodies (left panel) or anti-GSK3β antibodies (right panel) were used for immunoprecipitations. (c) Association of β-catenin with axin. Embryos were injected with RNAs encoding different forms of axin as indicated. Axin complexes were precipitated from cell lysates with 9E10 antibodies and the presence of β-catenin (arrowhead) was evaluated on western blots with specific antiserum (α-βcat, bottom panel). The lower band corresponds to an unknown cross-reacting protein. Expression of axin constructs was assessed with 9E10 antibodies (top panel).
