FIG. 1.

The axin GID inhibits GSK-3β activity. (A) Schematic of Myc-tagged constructs used in coimmunoprecipitation and activity assays. N-terminal (N-term; aa 63 to 288), GID-1 (aa 277 to 545), C-terminal (C-term; aa 429 to 713), and FL Xaxin (aa 1 to 842) constructs were expressed in oocytes. Shading differentiates the RGS domain (grey), GID (black), and DIX domain (striped). Results from panels B and C for each construct are summarized at the right “Binding” refers to coimmunoprecipitation of GSK-3β with Myc-tagged axin; “Activity” refers to GSK-3β-dependent tau phosphorylation; “act” and “inh” stand for active and inhibited GSK-3β, respectively. (B) Tau phosphorylation in oocytes expressing GSK-3β (lanes 2 to 6), C-terminal (lane 3), GID (lane 4), and N-terminal (lane 5) fragments, and FL Xaxin (lane 6). Top, immunoblot of oocyte lysates, using an antibody specific for phosphorylated tau (tau-P; lanes 1 to 6), middle, immunoblot with antibodies that recognize both phosphorylated and unphosphorylated tau (tau; lanes 1 to 6); bottom, immunoblot with an antibody to GSK-3β (lanes 1 to 5). (C) The axin GID binds GSK-3β in oocytes. Oocytes were injected with GSK-3β mRNA alone (lane 1) or with RNA encoding Myc-tagged C-terminal (lane 2), GID (lane 3), and N-terminal (lane 4) fragments of axin and FL Xaxin (lane 5). After 16 h, Myc-tagged axin fragments were immunoprecipitated with anti-Myc antibodies and immunoblotted with the anti-GSK-3β antibody. GSK-3β coimmunoprecipitates only with the GID (lane 3) and FL Xaxin (lane 5). Approximately equal amounts of axin or axin fragments were present in the immunoprecipitates (data not shown). IgG, immunoglobulin G.