Guo J , Cagatay T , Zhou G , Chan CC , Blythe S , Suyama K , Zheng L , Pan K , Qian C , Hamelin R , Thibodeau SN , Klein PS , Wharton KA , Liu W .

R01-CA60117 NCI NIH HHS , R01-GM65404 NIGMS NIH HHS , Howard Hughes Medical Institute , R01 GM076621-03 NIGMS NIH HHS , R01 GM076621 NIGMS NIH HHS , R01 GM065404 NIGMS NIH HHS

	Figure 1. Nkd mutations in fly and human.(A) Wild-type, axin, apc, and nkd Drosophila cuticles. Wild type has alternating denticle bands (arrow) and naked cuticle (arrowhead), with each mutant lacking denticle bands. (B) NKD1 locus has 10 exons. Nkd1 schematic (orange) includes N-terminal myristoylation, EFX, 30aa (blue), and carboxy-terminal His-rich motifs. Exon 10 sequences around poly-(C) tracts (red) above native (black) and mutant (blue) residues are shown. (C) NKD1 electropherograms showing wild-type (WT) poly-(C)7, cell line TC7 with C-deletion (C6), cell line RKO with C-insertion (C8), and cell line HCT15 with G>A mutation (arrow) 3′ of poly-(C)7. (D, E) α-Nkd1 blots of whole cell extracts (D) and Triton X-100 soluble and insoluble fractions (E) from cell lines with indicated NKD1 mutation. Arrows designate Nkd1 proteins. β-actin is loading control in D. CCD841 has full length Nkd1, with a minor degradation product at ∼35 kDa also seen with transfected NKD1 (e.g. Fig. 1E, 5C), whereas SW480 with more abundant but wild-type Nkd1 has several degradation products.
	Figure 2. Wild-type Nkd1 but not mutant Nkd1 inhibits Wnt/β-catenin signaling.(A) % Xenopus embryos with indicated axis phenotype after injection of XWnt8+/−wild-type or mutant NKD1. (n) = # embryos injected. Panels at right show representative lateral (L) and dorsal (D) views of embryos scored as single axis (white), short A/P axis (light gray), partial axis duplication (dark gray), and full axis duplication (black) according to the Materials and Methods. In embryos with single axis in the left panels, note trunk (white arrow) and cement gland (black arrow). Arrowheads designate each axis in embryos with axis duplications (right panels). Embryos with partial axis duplication have duplicated trunk tissue but a single cement gland, while embryos with full axis duplication have duplicated trunk tissues and cement glands. (B) Normalized TOPflash luciferase activity (RLU) in HEK-293 cells transfected with reporter +/−Dvl2 +/− indicated Nkd1 construct.
	Figure 3. Effect of NKD1-mutations on β-catenin and cell proliferation.(A) Western blot of cytosolic and nuclear extracts of cells with wild-type (CCD841) or mutant (Co115) NKD1. GAPDH and HDAC2 were probed as loading controls. (B–B″, C–C″) β-catenin (red) and DNA (blue) distribution in CCD841 cells (B–B″) and Co115 (C–C″) cells. Arrows designate nuclei. Merged images in B″ and C″. (D) Western blot of cytosolic extracts of HEK-293 cells transfected with lacZ control (-), wild type Nkd1 (WT), or indicated mutant Nkd1 construct, and probed for β-catenin, Nkd1, and loading control GAPDH. Note that each mutant Nkd1 but not wild type Nkd1 increases β-catenin levels. (E) Relative cell number as a function of days post retroviral infection of CCD841 cells with empty vector control, wild type Nkd1, or indicated Nkd1 mutant (p = 0.016, 0.012, and 0.0091 for C6, C8, and R288H mutants as compared to control) (F) Relative cell number as a function of days post retroviral infection of Co115 cells with control or wild-type Nkd1 (p = 0.022). α-Nkd1 western blots of cell extracts, with GAPDH or β-actin loading control, are shown below each plot in E and F.
	Figure 4. Nkd1 truncation alters subcellular localization and Dvl colocalization.(A, B) HEK-293 cells expressing HA/Flag-tagged Nkd1 (A) or Nkd1C8 (B) stained with α-HA (green). Nkd1 accumulates in puncta (arrows), while Nkd1C8 is diffusely distributed in the cytoplasm. Nkd1C6 distributed in a pattern similar to Nkd1C8 (not shown). (C, D) Cells co-expressing Dvl3 and wild-type (C) or C8 (D) Nkd1GFP constructs. Nkd1-GFP (green, C) shows near complete colocalization (arrows) with Dvl3 (red, C′), while Nkd1C8-GFP (D) accumulates in cytoplasmic aggregates surrounded by Dvl3 (arrows). Merged images are in C″, D″, DNA is blue in A–D. Similar results were observed in cells coexpressing Nkd1C8-GFP and Dvl1 or Dvl2, and in cells expressing Nkd1C6-GFP and Dvl1, Dvl2, or Dvl3 (not shown). (E) Salivary gland from wild-type third instar Drosophila larva stained with α-Dsh (red) showing diffuse and punctate staining. (F–F″) A8-Gal4/UAS-NkdGFP salivary gland stained with α-Dsh and imaged for GFP (F) and Dsh (F′; merged in F″). Fly NkdGFP relocalizes Dsh to perinuclear aggregates (arrows) similar to those observed with Nkd1GFP/Dvl3 in C.
	Figure 5. Mutant Nkd1 proteins exhibit reduced Dsh/Dvl associations.(A) Y2H between Nkd1-baits and Dvl-prey assayed for growth on triple dropout (3D+3AT) or double dropout (2D) media. Bar graphs on right show ONPG units from quantitative Y2H assay. Western blot indicates that Nkd1 mutant proteins are of comparable abundance (not shown). (B) GST-pulldown assay of in vitro translated, 35[S]-labelled Dvl1-3 with each GST-Nkd1 protein. Bar graph is quantitative band intensity. Coomassie-stained gel shows each GST-fusion protein, with arrowheads indicating full-length proteins. (C) Western blots of Flag-IPs from HEK-293 cell extracts with equal amounts of each HA/Flag-tagged Nkd1, and then probed with α-HA (upper) and α-Dvl3 (middle). β-actin is loading control (lower). Note that less Dvl3 is IP'd by Nkd1C6 or Nkd1C8 as compared to full-length Nkd1. No co-IP was observed between wild-type or mutant Nkd1s and Dvl1 or Dvl2, reminiscent of the lack of stable co-IP between Drosophila Nkd and Dsh [13].
	Figure 6. Mutant Nkd1s do not limit the abundance of Dsh/Dvl as well as wild type Nkd1.(A–A″) 71B-Gal4/UAS-NkdGFP third-instar Drosophila salivary gland stained with α-Dsh and imaged for GFP (A) and Dsh (A′) distributions (merged image in A″). Quantitation of Dsh pixel intensity (white box in A′) reveals reduced staining in cell expressing more (right, green asterisk) NkdGFP than in adjacent cell expressing less NkdGFP. (B) Western blots of HEK-293 cells transfected with indicated Flag/HA-tagged Nkd1 and Dvl1, Dvl2, or Dvl3 constructs probed with α-HA, α-Dvl1-3, and α-βtubulin as a loading control. Each of the Dvl1-3 blots was loaded with equal amounts of extract, as confirmed by probing each blot with α-βtubulin. (C) HEK-293 cells co-expressing Nkd1GFP and Dvl1, stained with α-Dvl1, and imaged for GFP (green), Dvl1 (red), and DNA (blue) showing fine punctate Dvl1 distribution in cells expressing low to absent Nkd1GFP (white arrow). In adjacent cells expressing Nkd1GFP, Dvl1 is relocalized to Nkd1GFP/Dvl1 aggregates (yellow arrow), with loss of fine punctate Dvl1 staining (arrowheads). (D, E) Models of Nkd function in Drosophila (D) and NKD1-mutant CRC (E). Double lines: upper = plasma membrane; lower = nuclear membrane. (D) Fly Wg(Wnt) binds Fz/Arrow(Lrp5/6) receptors, which inhibits the Apc/Axin/CK1/GSK3β complex that promotes degradation of Arm(β-catenin). Arm complexes with Pan(TCF) to activate target genes including nkd. Nkd promotes Dsh turnover to partially inhibit signaling, and employs the nuclear import factor Imp-α3 to enter the nucleus and further inhibit signaling through unknown mechanisms [31]. (E) In NKD1-mutant CRC, the mutant Nkd1 protein no longer binds and promotes Dvl turnover, stabilizing β-catenin and activating TCF-dependent transcription of target genes.

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