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. 2004 Oct 25;167(2):339–349. doi: 10.1083/jcb.200402153

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Copyright © 2004, The Rockefeller University Press

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Figure 8. — Cadherin phosphorylation reverses β-catenin binding selectivity during Wnt signaling. (A) Phosphorylation of cad-GST increases β-catenin binding to cadherin compared with TCF. A cytosolic fraction from L cells transfected with Wnt3a were incubated with equimolar amounts of cad-GST, TCF-GST, and CK2-P-cad-GST-glutathione–coupled beads for 1 h at 4°C (see Fig. S1 for characterization of GST fusion proteins). The resulting anti–β-catenin and anti-GST immunoblots are shown. (B) Fraction of β-catenin that binds cadherin is a subset of fraction of β-catenin that binds TCF. Cytosolic fraction of Wnt cells was sequentially affinity precipitated with cad-GST (lanes 1–3) or TCF-GST (lanes 6–8) proteins. After cad-GST depletion (lanes 1–3), half of the cad-GST non-binding fraction (NB/2) was precipitated with TCF-GST (lane 4); the other half was precipitated with TCA to show amount remaining (lane 5, far right). After TCF-GST depletion (lanes 6–8), half of the TCF-GST non-binding fraction (NB/2, lane 9) was precipitated with cad-GST, whereas the other half was precipitated with TCA to show amount remaining (lane 10, far right). Lanes 5 and 10 reveal a fraction of β-catenin that binds neither TCF nor cadherin. This fraction is likely due to β-catenin already complexed with partners such as ICAT (Gottardi and Gumbiner, 2004). (C) Phosphorylated cadherin-GST and TCF-GST bind the same pool of β-catenin in Wnt-activated cells. Cytosolic fraction was precipitated with cad-GST (top blot), TCF-GST (bottom left) or P-cadherin-GST (bottom right) fusion proteins. After cad-GST depletion (lanes 2–4 and 7–9), there is a fraction of β-catenin that binds TCF-GST (lane 5) and P-cadherin-GST (lane 10). Note that after TCF-GST depletion (lanes 13–15), there is no β-catenin remaining to bind P-cadherin-GST (lane 16). After P-cadherin-GST depletion (lanes 18–20), there is no β-catenin remaining to bind TCF-GST (lane 21). Reciprocal depletions suggest that P-cadherin-GST and TCF-GST bind the same form of β-catenin.