Schematic representation of human APC and APCL and rat Axin.

<p>Functional modules are shown, including the two dimerisation domains of APC (DIM), the armadillo repeat domain (Arm), the 15 (15RA to D) and 20 (20R1 to 7) amino acid repeats that function as β-catenin binding sites, the β-catenin inhibitory domain (CiD) involved in β-catenin degradation and the SAMP repeats that represent Axin or Axin2 binding sites. The mutation cluster region (MCR) contains most of the APC truncating mutations that have been observed in colon cancer. The RGS (SAMP-binding region), DIX (oligomerisation), β-catenin and GSK3β domains in Axin are indicated. The numbers indicate the amino acid positions. The numbers with arrows correspond to the size of the different constructs analysed in this study, which were fused at the N-terminus to YFP (APC and APCL) or myc (Axin).</p

Summary of the co-oligomerisation experiments.

<p>Summary of the co-oligomerisation experiments.</p

Stimulation of co-oligomerisation occurs only with the Axin/APC pair.

<p>DLD1 cells were transiently transfected on day 1 with full-length APC or APCL constructs or N-terminal flag-tagged Axin or Axin2, either individually or in combination. The cells were fixed on day 3 and were stained with an anti-flag antibody. Where applicable, the percentages indicate the proportion of different localisation patterns in the transfected cells. The imaging parameters were identical for the flag tag; the yAPCL signal was more intense than the yAPC signal. Bar, 10 μM.</p

Working model.

<p>The interactions between the 20R2 of APC and amino acids 508-713 of Axin and between the SAMP repeat of APC and the RGS domain of Axin are shown with double arrows. We attribute putative functions to these interactions. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094413#pone-0094413-g001" target="_blank">figure 1</a> for details.</p

The 20R2 of APCL is required for Axin colocalisation in the absence of the SAMP repeats.

<p>DLD1 cells were transiently transfected on day 1 with the indicated APCL constructs or the indicated N-terminal flag-tagged Axin (<b>A</b>) or Axin2 (<b>B</b>) constructs, either individually or in combination. The cells were stained on day 3 with an anti-flag antibody and Hoechst dye. When expressed alone, truncated yAPCL displayed a dotty localisation pattern, flag-Axin exhibited a diffuse or dotty expression pattern and fAxin2 demonstrated diffuse localisation. <b>A</b>, APCL constructs lacking the 20R2 (i.e., 1147 and shorter) did not colocalise with fAxin (<b>c</b>, <b>d</b>), whereas colocalisation occurred in the presence of the 20R2 (yAPCL1179 and yAPCL1728) (<b>a</b>, <b>b</b>). <b>B</b>, Axin2 interacted with the N-terminus of APC. The percentages indicate the proportion of different localisation patterns in the transfected cells. The imaging parameters were identical for each type of tag. Bar, 10 μM.</p

The first SAMP repeat of truncated APC is required for Axin oligomerisation, whereas the 20R2 is not necessary.

<p>DLD1 cells were transiently transfected on day 1 with the indicated wild-type or mutant APC constructs and N-terminal myc-tagged Axin, either individually or in combination. yAPC1641-2μ contains the mutations presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094413#pone-0094413-g004" target="_blank">figure 4A</a>. The cells were fixed on day 3 and were stained with an anti-myc or an anti-flag antibody. Where applicable, the percentages indicate the proportion of different localisation patterns in the transfected cells. The imaging parameters were identical for each type of tag. Bar, 10 μM.</p

The RGS domain of Axin is not necessary for colocalisation with APC or APCL.

<p>DLD1 cells were transiently transfected on day 1 with the indicated APC (<b>A</b>) or APCL (<b>B</b>) constructs or with the indicated N-terminal myc-tagged Axin constructs, either individually or in combination. The cells were stained on day 3 with an anti-myc antibody and Hoechst dye. Cells transfected with yAPCL alone displayed dotty and fibre-like expression patterns, with yAPC alone exhibited a fibre-like expression pattern, with myc-Axin alone displayed diffuse and dotty expression patterns and with myc-Axin(298–832) alone exhibited only a dotty expression pattern. Coexpression of myc-Axin and APC or APCL resulted in colocalisation, which was also observed with when the RGS domain (298–832) was deleted from Axin. The percentages indicate the proportion of different localisation patterns in the transfected cells. The imaging parameters were identical for the myc constructs; the yAPCL signal was more intense than the yAPC signal. Bar, 10 μM.</p

Amino acids 508–713 of Axin are required for colocalisation with truncated APCL.

<p>DLD1 cells were transiently transfected on day 1 with wild-type or mutant N-terminal myc-tagged Axin, either individually or in combination with the yAPCL1179 expression vector. The cells were fixed on day 3 and were stained with an anti-myc antibody and Hoechst dye. The presence of amino acids 508–713 in the myc-Axin deletion mutants resulted in colocalisation with yAPCL1179. Where applicable, the percentages indicate the proportion of different localisation patterns in the transfected cells. The imaging parameters were identical for each type of tag. Bar, 10 μM.</p

The 20R2 of APCL is required to inhibit β-catenin transcriptional activity (A) and to down-regulate β-catenin (B) but is not necessary to recruit Axin (C) in the presence of SAMP repeats.

<p>A, SW480 cells were transiently transfected on day 1 with reporter plasmids and 100(flag) or the indicated APCL constructs. yAPCL-2μ contains the mutations shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094413#pone-0094413-g004" target="_blank">figure 4A</a>. TOP/FOP reporter assays were performed on day 3 to measure β-catenin transcriptional activity. The data are presented as the mean ± standard deviation of three independent values from a representative experiment. * p<0.004 compared with flag, Student's t-test. ns, not significant. In a parallel experiment, cells were transiently transfected with 1 μg of the indicated plasmids on day 1. Cell extracts were prepared on day 3 and were subjected to western blotting using the indicated antibodies. The molecular weights are presented in kDa. B, SW480 cells were transiently transfected with control vector (flag) or the indicated APCL constructs, fixed on day 3 and stained with an anti-β-catenin antibody. Bar, 10 μM. C, DLD1 cells were transiently transfected on day 1 with the indicated APCL constructs or N-terminal flag-tagged Axin, either individually or in combination. The cells were fixed on day 3 and were stained with an anti-flag antibody. Where applicable, the percentages indicate the proportion of different localisation patterns in the transfected cells. The imaging parameters were identical for each type of tag. Bar, 10 μM.</p

The 20R2s of APC and APCL mediate Axin colocalisation, β-catenin down-regulation and the inhibition of β-catenin transcriptional activity in the absence of the SAMP repeats.

<p>A, 20R2 sequences of wild-type and mutant human APC (residues 1370–1404) and APCL (residues 1145–1179). Red indicates amino acids common to APC and APCL from various species (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0094413#pone.0094413.s002" target="_blank">fig. S2</a>), blue represents the residues common to human APC and APCL, and the small bold black letters signify the residues that have been mutated. The underlined sequence comes from the human APC 20R2. B, Mutation of the 20R2 abolished the colocalisation of APCL with Axin, whereas the 20R2 of APC restored colocalisation with Axin. DLD1 cells were transiently transfected on day 1 with the indicated APCL constructs or N-terminal flag-tagged Axin, either individually or in combination. The cells were fixed on day 3 and were stained with an anti-flag antibody and Hoechst dye. Bar, 10 μM. C, SW480 cells were transiently transfected with a control vector (flag) or the indicated APCL constructs, fixed on day 3 and stained with an anti-β-catenin antibody. Bar, 10 μM. D, SW480 cells were transiently transfected on day 1 with reporter plasmids and 100 ng of an empty vector (flag) or the indicated APCL constructs. TOP/FOP reporter assays were performed on day 3 to measure β-catenin transcriptional activity. The data are presented as the mean ± standard deviation of three independent values from a representative experiment. * p<0.05, *** p<0.001, ns, not significant using Student's t-test. In a parallel experiment, cells were transiently transfected with 1 μg of the indicated plasmids on day 1. Cell extracts were prepared on day 3 and subjected to western blotting using the indicated antibodies. The molecular weights are shown in kDa. The imaging parameters were identical for each type of tag.</p