Fig 8. Wingless stimulation results in increased membrane-associated ADP-ribosylated Axin.

(A, left panel) Subcellular fractionation of lysates from S2R+ cells treated with either control medium (CTR) or Wingless conditioned medium (Wg CM) for 1 hour. The total cell lysates (T), cytoplasmic (C), and membrane (M) fractions were analyzed by SDS-PAGE. Immunoblotting with phosphorylated LRP6 antibody shows activation of the Wnt pathway following treatment with Wg CM. Immunoblotting with Axin antibody shows no significant shift from the cytoplasmic fraction to membrane fraction. Tubulin, a cytoplasmic marker, and Nervana, a membrane marker, were used to assay the efficiency of fractionation. Asterisk indicates a non-specific band with p-LRP6 antibody. (A, right panel) Following cell fractionation, GST-WWE pulldown was performed with cytoplasmic (C) and membrane (M) fractions. Immunoblotting with Axin antibody reveals that the majority of ADP-ribosylated Axin is in the membrane fraction, with little ADP-ribosylated Axin detected in the cytoplasm. Phospho-LRP6 is pulled down by GST-WWE in the membrane fraction in response to Wingless. (B) Model for dual roles of membrane-associated Axin. In the absence of Wnt stimulation, a pool of Axin is localized in membrane-proximal puncta, which might represent the sites of the destruction complex. Membrane-associated Axin is targeted for degradation by Tnks in the unstimulated state, which contributes to maintaining Axin at the limiting concentrations important for regulation of Wnt signaling. Wnt stimulation induces a rapid increase in the level of Axin, and in particular the membrane-associated ADP-ribosylated Axin pool. ADP-ribosylation enhances the interaction of Axin with phospho-LRP6/Arrow, and thus promotes the activation of signaling. R: ADP-ribosylation.