(A) Proteasome and p97/VCP inhibitors inhibited the degradation of endogenous Vangl proteins. HEK293T cells were treated with CHX (100 μg/ml) alone or with proteasome inhibitor MG132 (10 μM), lysosome inhibitor CQ (25 μM) or p97/VCP inhibitor DBeQ (10 μM), NMS-873 (2 μM), or CB-5083 (5 μM) for the indicated time. Endogenous Vangl1 and Vangl2 were detected, and the relative Vangl protein levels were quantified below. (B) Knockdown of VCP by small interfering RNA (siRNA) increased Vangl protein levels in HEK293T cells. (C) Single guide RNA (sgRNA)–mediated knockdown of ERAD component HRD1 or MARCH6, but not SEL1L or DERL1, increased Vangl protein levels in HEK293T cells. The Vangl protein levels were quantified in the right panel. (D) Vangl2 was poly-ubiquitinated. Treatment of proteasome inhibitor MG132 (10 μM) for 4 hours significantly increased Vangl2 ubiquitination. (E) Vangl2 underwent K48-linked poly-ubiquitination. HEK293T cells were treated with MG132 (10 μM) or CQ (25 μM) for 4 hours, and ubiquitination was examined by total ubiquitin FK2, K48 linkage–, or K63 linkage–specific antibodies. The bands at 55 and 100 kDa were immunoglobulin G and nonspecific bands, respectively. Ub, ubiquitin. (F) K300 and K306 contribute to Vangl2 ubiquitination. The corresponding Vangl2 lysines (K) were mutated to arginines (R), which reduced the level of Vangl2 ubiquitination. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

(A) Endogenous interaction between Vangl and KBTBD7 in HEK293T cells. Vangl2 binds to KBTBD7 and Vangl1 but not KBTBD6 or CUL3 (left). KBTBD7 binds to Vangl1, Vangl2, CUL3, and KBTBD6 (right). IgG, immunoglobulin G. (B) KBTBD7 directly binds to Vangl2. Glutathione S-transferase (GST)–fused KBTBD7 and His-fused C-terminal Vangl2 (amino acids 254 to 521) recombinant proteins were coincubated and subjected to GST pull-down. CBB, Coomassie brilliant blue staining. (C) Schematic domain structures of various KBTBD7. M99A, methionine to alanine mutation within the BTB domain. (D) Interaction between various Flag-tagged KBTBD7 (FG-K7) and HA-tagged CUL3 or Vangl2 in HEK293T cells. KBTBD7-M99A mutation abolished its binding to CUL3 (left). Kelch repeats of KBTBD7 were required for binding to Vangl2 (right). Note that overexpressed HA-Vangl2 was largely degraded by full-length KBTBD7 (red arrow in input). (E) KBTBD7 alone or with KBTBD6 and CUL3, but not KBTBD6 or CUL3 alone, promoted Vangl2 ubiquitination in HEK293T cells. (F) KBTBD7 abolished the plasma membrane localization of Vangl2. KBTBD6, CUL3, or mutant KBTBD7 was incapable of degrading Vangl2. Endogenous Vangl2 (green) in Madin-Darby canine kidney (MDCK) cells was examined by immunofluorescent staining (IF). The cells expressing KBTBD6, KBTBD7, or CUL3 were marked by FLAG IF (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 10 μm. (G) KBTBD7-M99A mutant was unable to degrade Vangl proteins but showed a dominant negative effect on wild-type (WT) KBTBD7 (K7). (H) Loss of KBTBD7 in HEK293T cells increased endogenous Vangl1 and Vangl2 protein levels. KBTBD7 was knocked out in HEK293T cells by CRISPR-Cas9–mediated genome editing, and two single KBTBD7 null clones were analyzed. The PX459 empty vector served as a control. (I) Loss of KBTBD7 decreased Vangl2 ubiquitination.

(A) Inhibition of proteasome or p97/VCP attenuated the KBTBD7-induced Vangl degradation. HEK293T cells were transfected with KBTBD7 in an increasing dose (0, 250, 500, and 1000 ng) and treated with dimethyl sulfoxide (DMSO), MG132 (10 ?M), CQ (25 ?M), clathrin inhibitor CPZ (30 ?M), caveolin inhibitor Nystatin (25 ?g/ml) or p97/VCP inhibitor DBeQ (10 ?M), NMS-873 (2 ?M), or CB-5083 (5 ?M) for 6 hours (4 hours for DBeQ). The relative Vangl protein levels were quantified below. (B) VCP is required for KBTBD7-induced Vangl degradation. Upon siRNA knockdown of VCP in HEK293T cells, KBTBD7 (K7) failed to induce Vangl degradation, which was restored by reconstitution of VCP. (C) HRD1 and MARCH6 are required for KBTBD7-induced Vangl degradation. sgRNA-mediated silencing of HRD1 or MARCH6, but not SEL1L or DERL1, protected Vangl from KBTBD7-induced degradation. (D) HRD1 and MARCH6 are required for KBTBD7-induced Vangl2 ubiquitination. sgRNA-mediated silencing of HRD1 or MARCH6, but not SEL1L or DERL1, blocked KBTBD7-induced Vangl2 ubiquitination in HEK293T cells. (E) VCP is required for KBTBD7-induced Vangl2 ubiquitination. KBTBD7 was unable to induce Vangl2 ubiquitination upon VCP knockdown by siRNA. (F) KBTBD7-induced Vangl2 ubiquitination mainly occurred in the ER but not in the cytosol. Total lysates, ER, and cytosolic fractions of HEK293T cells were isolated and subjected to ubiquitination assay. Activating transcription factor 6 (ATF6) served as an ER marker. T, total cell lysate; ER, ER fraction; C, cytosolic fraction. (G) The endogenous Vangl2-KBTBD7 interaction depends on VCP. Upon siRNA knockdown of VCP in HEK293T cells, the binding between Vangl2 and KBTBD7 was significantly reduced.

(A) Endogenous interaction between Vangl2 and VCP in HEK293T cells. rWnt5a, recombinant Wnt5a (200 ng/ml, 2 hours). (B) Sequence alignment identified a well-defined VCP-interacting motif (VIM) RX₅AAX₂R that is highly conserved across multiple species in both Vangl1 and Vangl2. (C) Mutations in Vangl2 VIM (R334A or A330/331L) nearly abrogated its interaction with p97/VCP. (D) VCP directly binds to Vangl2 via VIM. GST-fused VCP and His-fused C-terminal (amino acids 254 to 521) WT or VIM mutant (R334A) Vangl2 were purified, coincubated, and then subjected to GST pull-down. (E) KBTBD7 failed to induce the ubiquitination of VIM mutant (R334A) Vangl2. (F) VIM mutant Vangl2 (R334A or A330/331L) is resistant to the KBTBD7-induced degradation. HEK293T cells were transfected with an increasing dose (0, 250, 500, and 1000 ng) of KBTBD7. (G) KBTBD7 (red) failed to abolish the membrane localization of VIM mutant Vangl2 (R334A or A330/331L, green) in MDCK cells. Nuclei were stained with DAPI (blue). Scale bar, 10 μm. (H) KBTBD7 was enriched in the ER by WT Vangl2. KBTBD7 (green) was expressed with or without Vangl2 (WT or VIM mutant R334A, purple) in MDCK cells. The expression of WT but not VIM mutant Vangl2 led to an enrichment of KBTBD7 in the ER (arrows in the enlarged panel). ER was stained by ER dye (red). Scale bar, 10 μm in the top panels and 2 μm in the enlarged panel. (I) p97/VCP adaptor UBA-UBX proteins (SAKS1, UBXD7, or FAF1) bind to KBTBD7. (J) Knockdown of SAKS1 or UBXD7 by sgRNA significantly increased endogenous Vangl protein levels. (K) sgRNA-mediated knockdown of p97/VCP adaptors (SAKS1, UBXD7, and FAF1) compromised the KBTBD7-induced Vangl degradation.

(A) Wnt5a blocked the degradation of endogenous Vangl proteins. HEK293T cells were treated with CHX (100 μg/ml) alone or with rWnt5a (200 ng/ml) for the indicated time. The relative Vangl protein levels are quantified on the bottom panel. (B) Wnt5a inhibited KBTBD7-induced Vangl2 ubiquitination through CK1-mediated Vangl2 phosphorylation. CHO cells were treated with or without CK1 inhibitor D4476 (100 μM) for 6 hours. (C) Vangl2 phosphorylation inhibited its ubiquitination. HEK293T cells expressing WT, phospho-mutant (A), or phospho-mimetic (E) Vangl2 were treated with MG132 (10 μM) for 4 hours. Because phospho-mutant Vangl2 was known to be unstable, more plasmids were transfected to increase the input level. (D) CK1 protected Vangl2 from the KBTBD7-induced degradation. (E) Wnt5a and CK1 induced Vangl2 basal phosphorylation in the ER. CHO cells were treated with rWnt5a (200 ng/ml) for 2 hours or transfected with CK1δ. Vangl2 was examined in total cell lysates (T) and ER fractions (ER). ATF6 served as an ER marker. The lower black, middle blue, and upper red arrows point to the unphosphorylated, basal phosphorylated, and hyperphosphorylated Vangl2, respectively. The percentage of basal phosphorylated Vangl2 (blue) and unphosphorylated Vangl2 (black) in the ER fractions is quantified in the bottom panel. (F) Phosphorylation facilitated ER export of Vangl2. The transient expression of GFP-HA-tagged WT, phospho-mutant (A), phospho-mimetic (E), VIM mutant (R334A), or Lp mutant (S464N) Vangl2 (green) was induced by doxycycline (1 μg/ml) in MDCK stable cell lines for 1 or 2 hours (upper two rows). Doxycycline was removed after 2 hours of treatment, and the fate of Vangl2 (green) was traced for another 2 or 4 hours (lower two rows). ER, ER dye (red); nuclei, DAPI (blue). Scale bar, 10 μm.

(A to C) Kbtbd7 degraded Vangl2 and caused convergent extension (CE) defects in zebrafish. (A) Three hundred picograms of zebrafish Kbtbd7 WT or M99A mutant mRNA was synthesized and microinjected into one-cell stage of embryos. Endogenous Vangl2 was examined. (B) Forty-eight hours after injection, zebrafish embryos were analyzed and classified into four groups (normal, mild, moderate, and severe) according to their CE phenotype. (C) Low level of Vangl2 (20 pg) partially rescued the CE defects caused by Kbtbd7 expression. The results are summarized from three independent experiments (t = 3). The total number of injected embryos (N =) for each group is labeled on the top. The error bars are the SD values. Scale bar, 1 mm. (D) The effects of Vangl2 MOs and HA-Vangl2 mRNA in zebrafish embryos were examined by Vangl2 and HA immunoblotting, respectively. (E) Knockdown of Vangl2 showed synergistic effects with Kbtbd7 expression in causing CE defects. Vangl2 MO (0.25, 0.5, or 2 ng) was injected alone or with Kbtbd7 mRNA (300 pg). (F) Two or 4 ng of Kbtbd7 MO was injected into one-cell stage of embryos. Kbtbd7 knockdown increased endogenous Vangl2 protein levels in a dose-dependent manner. (G) Typical morphological defects of Kbtbd7 MO–injected embryos, exhibiting ventral body curvature. Scale bar, 1 mm. (H) Vangl2 expression exhibited synergistic effects with Kbtbd7 MO in causing CE defects. Twenty or 60 pg of Vangl2 mRNA was injected alone or with 4 ng of Kbtbd7 MO. The results are summarized from three independent experiments (t = 3). The total number of injected embryos (N =) for each experimental setting is labeled on the top. The error bars are the SD values.

(A) TCGA data analysis of breast cancer. KBTBD7 was down-regulated in breast carcinoma compared to normal breast tissues, Student’s t test, P < 0.001. (B) Kaplan-Meier survival analysis of 4295 patients for overall survival and 2637 patients for metastatic relapse–free (MR-free) survival (univariate Cox analysis, P = 0.0004 and 0.0002). Patients with lower KBTBD7 expression level have decreased overall survival and MR-free survival. HR, hazard ratio. CI, confidence intervals. (C) Establishment of a HCC1806 breast cancer cell line stably expressing FLAG-tagged KBTBD7 (HCC1806-KBTBD7, left). Vangl2 was further stably expressed in HCC1806-KBTBD7 cells (right). (D) XTT assays of HCC1806 stable cancer cell lines. Expression of KBTBD7 slightly inhibited but further expression of Vangl2 promoted the proliferation of HCC1806 cells. (n = 3 repetitions; Student’s t test, *P < 0.05, ***P < 0.001). (E and F) Transwell migration (E) and wound healing (F) assays of HCC1806 stable cancer cell lines. KBTBD7 inhibited but Vangl2 promoted the migration of HCC1806 cancer cells (n = 3 repetitions; Student’s t test, **P < 0.01, ***P < 0.001). (G and H) KBTBD7 expression slightly inhibited but Vangl2 expression strongly promoted tumor growth in mouse xenograft models. (G) Ex vivo luciferase–based bioluminescence imaging of NOD SCID female mice 25 days after injection of HCC1806 stable cancer cells into the fat pads (n = 5 for each group). (H) Primary mammary tumors isolated from NOD SCID female mice 25 days after injection (left). Their body weights were measured (right) (n = 5 for each group; Student’s t test, *P = 0.0113, ****P < 0.0001). (I) Luciferase-based bioluminescence imaging (left) and quantification (right) of the lungs of NOD SCID female mice 8 weeks after injection of HCC1806 stable cancer cells into mammary fat pads (n = 4 for each group, Student’s t test, **P < 0.01, ***P < 0.001). KBTBD7 expression suppressed but Vangl2 expression strongly promoted lung metastasis of HCC1806 breast cancer cells.

(I) Newly synthesized Vangl proteins are basally phosphorylated by CK1 in the ER; (II) the basal phosphorylated Vangl are transported to the cell surface where they undergo further phosphorylation and become stabilized; (III) some of unmodified Vangl escape ERAD and reach the cell surface; (IV) the unphosphorylated Vangl are not stable and are internalized and degraded via the lysosomal pathway; (V) most of the unmodified or unfolded Vangl proteins are degraded through the ERAD pathway. ERAD components HRD1 and MARCH6 are required for the degradation of Vangl and may initiate Vangl ubiquitination. (VI) VCP directly binds to Vangl at a highly conserved VIM; (VII) VCP recruits cytosolic E3 ligase KBTBD7 via its UBA-UBX adaptors (SAKS1, UBXD7, and FAF1), resulting in enhanced poly-ubiquitination of Vangl; and (VIII) extraction of highly ubiquitinated Vangl molecules for proteasomal degradation.