Schematic representations of Dvl-1, Dvl-3, and rAxin constructs used in this study.

Interaction of Dvl-1 with Dvl-3. (A) Intact cells. Lysates (20 μg of protein) of COS cells expressing Myc–Dvl-3 (lane 2), HA–Dvl-1 (lane 3), HA–Dvl-1-(140-670) (lane 4), Myc–Dvl-3 and HA–Dvl-1 (lane 5), or Myc–Dvl-3 and HA–Dvl-1-(140-670) (lane 6) were simultaneously probed with anti-Myc and anti-HA antibodies. The same lysates (200 μg of protein) were immunoprecipitated with the anti-Myc antibody, and the immunoprecipitates were probed with the anti-Myc and anti-HA antibodies (lanes 7 to 11). The lysates of COS cells transfected with empty vectors were used as a control (lane 1). IP, immunoprecipitation; Ab, antibody; Ig, immunoglobulin. The arrows and arrowhead indicate the positions of HA–Dvl-1 or HA–Dvl-1-(140-670) and Myc–Dvl-3, respectively. (B) Direct binding. After GST–Dvl-1-(1-82) (lanes 1 to 3) or GST–Dvl-1-(140-670) (lanes 4 to 6) (50 pmol of each) was incubated with MBP–Dvl-3 (full length) (lanes 1 and 4), MBP–Dvl-3-(1-80) (lanes 2 and 5), and MBP (lanes 3 and 6) (30 pmol of each) immobilized on amylose resin, MBP fusion proteins were precipitated by centrifugation. The precipitates were probed with the anti-GST antibody. The positions of GST–Dvl-1-(1-82) and GST–Dvl-1-(140-670) are shown in lanes 7 and 8, respectively. The results shown are representative of three independent experiments.

Self-association of Dvl-1. (A) Intact cells. Lysates (20 μg of protein) of COS cells expressing HA–Dvl-1 (lane 2), Myc–Dvl-1 (lane 3), or HA–Dvl-1 and Myc-Dvl-1 (lane 4) were sequentially probed with anti-HA and anti-Myc antibodies. The same lysates (200 μg of protein) were immunoprecipitated with the anti-Myc antibody, and the immunoprecipitates were probed with the anti-HA and anti-Myc antibodies (lanes 5 to 7). The lysates of COS cells transfected with empty vectors were used as a control (lane 1). IP, immunoprecipitation; Ab, antibody; Ig, immunoglobulin. The arrow and arrowhead indicate the positions of HA–Dvl-1 and Myc–Dvl-1, respectively. (B) Direct binding. After GST–Dvl-1-(1-82) was incubated with MBP–Dvl-1-(1-82) (lane 1), MBP–Dvl-3-(1-80) (lane 2), or MBP (lane 3) immobilized on amylose resin, MBP fusion proteins were precipitated by centrifugation. The precipitates were probed with the anti-GST antibody. The results shown are representative of three independent experiments.

Self-association of Axin. (A) Intact cells. Lysates (20 μg of protein) of COS cells expressing HA-rAxin (lane 2), Myc-rAxin (lane 3), or HA-rAxin and Myc-rAxin (lane 4) were probed with anti-HA and anti-Myc antibodies. The same lysates (200 μg of protein) were immunoprecipitated with the anti-Myc antibody, and the immunoprecipitates were probed with the anti-HA and anti-Myc antibodies (lanes 5 to 7). The lysates of COS cells transfected with empty vectors were used as a control (lane 1). IP, immunoprecipitation; Ab, antibody. The arrow and arrowhead indicate the positions of HA-rAxin and Myc-rAxin, respectively. (B) In vitro. The lysates of COS cells expressing Myc-rAxin (full length) (lanes 1 and 6), Myc–rAxin-(1-713) (lanes 2 and 7), Myc–rAxin-(1-229) (lanes 3 and 8), Myc–rAxin-(298-506) (lanes 4 and 9), or Myc–rAxin-(713-832) (lanes 5 and 10) were directly probed with the anti-Myc antibody (lanes 1 to 5) or incubated with MBP-rAxin (full length) (10 pmol) immobilized on amylose resin, and then MBP-rAxin was precipitated by centrifugation (lanes 6 to 10). The lysates of COS cells expressing Myc-rAxin (full length) were incubated with MBP-rAxin (full length) (lane 11), MBP–rAxin-(298-832) (lane 12), or MBP–rAxin-(508-832) (lane 13) (10 pmol of each) immobilized on amylose resin. MBP-rAxin and its deletion mutants were precipitated by centrifugation, and the precipitates were probed with the anti-Myc antibody. The arrow indicates the position of Myc-rAxin (full length). (C) Direct binding. GST–rAxin-(298-832) (lanes 1 and 2) or GST (lane 3) (50 pmol of each) was incubated with MBP (lane 1) or MBP-rAxin (full length) (lanes 2 and 3) (10 pmol of each) immobilized on amylose resin, and then MBP-rAxin and MBP were precipitated by centrifugation. The precipitates were probed with the anti-GST antibody. The arrows indicate the positions of GST–rAxin-(298-832) and GST. The results shown are representative of three independent experiments.

Interaction of Dvl-1 with Axin. (A) Intact cells. Lysates (20 μg of protein) of COS cells expressing Myc-rAxin (lane 2), HA–Dvl-1 (lane 3), or Myc-rAxin and HA–Dvl-1 (lane 4) were probed with anti-Myc and anti-HA antibodies. The lysates of COS cells transfected with empty vectors served as the control (lane 1). The same lysates (250 μg of protein) of COS cells prepared in lanes 2 to 4 were immunoprecipitated with the anti-HA antibody (lanes 5 to 7). The immunoprecipitates were probed with the anti-Myc and anti-HA antibodies. IP, immunoprecipitation; Ab, antibody; Ig, immunoglobulin. The arrow and arrowhead indicate the positions of Myc-rAxin and HA–Dvl-1, respectively. (B) Binding region. The lysates (250 to 500 μg of protein) of COS cells coexpressing HA–Dvl-1 and Myc-rAxin (full length) (lanes 1 and 7), Myc–rAxin-(1-713) (lanes 2 and 8), Myc–rAxin-(1-437) (lanes 3 and 9), Myc–rAxin-(1-229) (lanes 4 and 10), Myc–rAxin-(298-506) (lanes 5 and 11), or Myc–rAxin-(713-832) (lanes 6 and 12) were immunoprecipitated with the anti-Myc antibody, and the immunoprecipitates were probed with the anti-Myc (lanes 1 to 6) and anti-HA (lanes 7 to 12) antibodies, respectively. IB, immunoblotting. The arrow indicates the positions of HA–Dvl-1. (C) Direct binding. GST–Dvl-1-(1-282) (lane 1), GST–Dvl-1-(1-140) (lane 2), GST–Dvl-1-(1-82) (lane 3), GST–Dvl-1-(83-282) (lane 4), GST–Dvl-1-(281-670) (lane 5), or GST (lane 6) (25 pmol of each) was incubated with MBP-rAxin (10 pmol) immobilized on amylose resin, and then MBP-rAxin was precipitated by centrifugation. The precipitates were probed with the anti-GST antibody. After GST–Dvl-1-(1-282) (25 pmol) was incubated with MBP-rAxin (full length) (lane 7), MBP–rAxin-(1-529) (lane 8), MBP–rAxin-(508-832) (lane 9), MBP–rAxin-(713-832) (lane 10), or MBP alone (lane 11) (10 pmol each) immobilized on amylose resin, MBP fusion proteins were precipitated by centrifugation. The precipitates were probed with the anti-GST antibody. The arrows and arrowhead indicate the positions of GST–Dvl-1-(1-282) and GST–Dvl-1-(281-670), respectively. (D) Effects of Dvl-1 on the binding of GSK-3β, β-catenin, and APC to rAxin. MBP-rAxin (full length) (10 pmol) immobilized on amylose resin was incubated with 1 μM GST–GSK-3β, 1.4 μM GST–β-catenin, or 250 nM GST–APC-(1211-2075) in the presence of the indicated concentrations of GST–Dvl-1 (full length). MBP-rAxin was precipitated by centrifugation, and then the precipitates were probed with the anti-GSK-3β, anti-β-catenin, or anti-GST [for GST–APC-(1211-2075)] antibody. The positions of GST–APC-(1211-2075), GST–β-catenin, and GST–GSK-3β are indicated by the arrows. The results shown are representative of three independent experiments.

Inhibition of GSK-3β-dependent phosphorylation of β-catenin in the presence of Axin by Dvl. (A) Time course. Ninety nanomolar MBP-rAxin and 90 nM GST–GSK-3β were incubated with 1.4 μM His₆–β-catenin in the presence of 1 μM MBP or MBP–Dvl-1 for the indicated periods. (B) Effects of the DIX and PDZ domains of Dvl on its activity. GST–GSK-3β was incubated with His₆–β-catenin and MBP-rAxin in the presence of the indicated concentrations of MBP–Dvl-1 (●), MBP–Dvl-1-(140-670) (▴), MBP–Dvl-1^ΔPDZ (■), or MBP (○) for 15 min. (C) Inhibition of GSK-3β-dependent phosphorylation of APC in the presence of Axin by Dvl. GST–GSK-3β was incubated with 250 nM GST–APC-(1211-2075) and the indicated concentrations of MBP–Dvl-1 in the presence (●) or absence (○) of MBP-rAxin for 15 min. (D) Phosphorylation of synthetic peptide. GST–GSK-3β was incubated with 10 μM GSK peptide 1 and the indicated concentrations of MBP–Dvl-1 in the presence (●) or absence (○) of MBP-rAxin for 15 min. The results shown are representative of five independent experiments.

Roles of the DIX domains of Dvl-1 and rAxin on the nuclear accumulation of β-catenin. L cells grown on coverslips were microinjected with pCGN/Dvl-1 (full length) (A and B), pCGN/Dvl-1-(140-670) (C and D), pCGN/Dvl-1^ΔPDZ (E and F), pBJ-Myc/rAxin (full length) (G and H), pEF-BOS-Myc/rAxin-(1-713) (I and J), or pBJ-Myc/rAxin-(713-832) (K and L). Cells were fixed, permeabilized, and stained with anti-HA (A, C, and E), anti-Myc (G, I, and K), and anti-β-catenin (B, D, F, H, J, and L) antibodies. The arrows indicate injected cells. The results shown are representative of three independent experiments.

Effects of the DIX domain of rAxin on its function. (A) Effects of rAxin-(1-713) and rAxin-(298-832) on the degradation of β-catenin. Lysates (20 μg of each protein) of wild-type SW480 cells (lane 1) or SW480 stably expressing Myc–rAxin-(298-832) (lane 2) or Myc–rAxin-(1-713) (lane 3) were probed with anti-Myc and anti-β-catenin antibodies. The arrows and arrowhead indicate the positions of Myc–rAxin-(298-832) or Myc–rAxin-(1-713) and endogenous β-catenin, respectively. (B) Growth rates. The numbers (35-mm-diameter dish) of wild-type SW480 cells (○) and SW480 cells stably expressing Myc–rAxin-(1-713) (●) or Myc–rAxin-(298-832) (▵) were determined. The results shown are representative of three independent experiments.