DIX domains of Dvl and axin are necessary for protein interactions and their ability to regulate beta-catenin stability

Abstract

The N-terminal region of Dvl-1 (a mammalian Dishevelled homolog) shares 37% identity with the C-terminal region of Axin, and this related region is named the DIX domain. The functions of the DIX domains of Dvl-1 and Axin were investigated. By yeast two-hybrid screening, the DIX domain of Dvl-1 was found to interact with Dvl-3, a second mammalian Dishevelled relative. The DIX domains of Dvl-1 and Dvl-3 directly bound one another. Furthermore, Dvl-1 formed a homo-oligomer. Axin also formed a homo-oligomer, and its DIX domain was necessary. The N-terminal region of Dvl-1, including its DIX domain, bound to Axin directly. Dvl-1 inhibited Axin-promoted glycogen synthase kinase 3beta-dependent phosphorylation of beta-catenin, and the DIX domain of Dvl-1 was required for this inhibitory activity. Expression of Dvl-1 in L cells induced the nuclear accumulation of beta-catenin, and deletion of the DIX domain abolished this activity. Although expression of Axin in SW480 cells caused the degradation of beta-catenin and reduced the cell growth rate, expression of an Axin mutant that lacks the DIX domain did not affect the level of beta-catenin or the growth rate. These results indicate that the DIX domains of Dvl-1 and Axin are important for protein-protein interactions and that they are necessary for the ability of Dvl-1 and Axin to regulate the stability of beta-catenin.

FIG. 1

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

FIG. 2

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.

FIG. 3

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.

FIG. 4

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.

FIG. 5

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.

FIG. 6

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.

FIG. 7

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.

FIG. 8

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.