Fig 2: Mutation of the palmitoylation sites and the Lys-1403 ubiquitination site accelerate LRP6 targeting to ERAD.(A) Metabolic 35SCys/Met pulse chase experiment (20 min pulse) on transiently expressed myc-LRP6 wild type (WT, curve corresponding to the one in Figure 1D), palmitoylation deficient (CC-SS, n = 3) or K1403R (KR, n = 6) mutants in HeLa cells. (B) Metabolic 35SCys/Met pulse chase experiment (20 min pulse) on transiently expressed myc-LRP6 wild type (WT, n = 7), or K1403R (K1403R, n = 7) mutant in HeLa cells silenced or not for mesd gene (siRNA mesd, n = 3). Errors represent standard deviation,*LRP6 WT and K1403R. (C) Metabolic 35SCys/Met pulse chase experiment (20 min pulse) on transiently expressed myc-LRP6K1403R in HeLa cells supplemented or not (n = 6, curve corresponding to the one in Figure 2A) with MG132 (K1403 + MG123, n = 3) or Bafilomycin A (K1403 + BafA, n = 3). (D) Immunoprecipitation of myc-tagged LRP6 Wild Type (WT) and K1403 mutant (KR) revealed with anti-Ubiquitin antibody, with or without MG132 treatment. (E) Metabolic 35SCys/Met pulse chase experiment (2 hr pulse) on transiently expressed myc-LRP6 wild type (WT, n = 4, curve corresponding to the one in Figure 1D) or palmitoylation deficient (CC-SS, n = 3) in HeLa cells. (F) Metabolic 35SCys/Met pulse chase experiment (16 hr pulse) on transiently expressed myc-LRP6 wild type (WT, curve corresponding to the one in Figure 1D), palmitoylation deficient (CC-SS, n = 3), K1403R (K1403R, n = 3) or K1403R in the palmitoyl deficient background (CC-SS + KR, n = 3) mutants in HeLa cells.DOI: http://dx.doi.org/10.7554/eLife.19083.00710.7554/eLife.19083.008Figure 2—source data 1.Numeric data for graphs of Figure 2A,B,C and E,F.DOI: http://dx.doi.org/10.7554/eLife.19083.008

Fig 3: CD44 is in complex with LRP6, AXIN, and DVL after Wnt induction.a Mean fluorescence of chromobodies (CB) against active ß-catenin in the nucleus of DMSO, CHIR, control CM, or Wnt3a CM-treated HeLa_BC1-TagGFP2 or CD44-/-_HeLa_BC1-TagGFP2 cells 24 h after induction. Control CM vs. Wnt3a CM: Mann–Whitney U-test. DMSO vs. CHIR: Student’s t test. Error bars, ±SE. b, c CD44/LRP6, CD44/DVL, and CD44/AXIN d, e in situ PLA complexes (dots) in HeLa cells treated with Wnt3a CM for 15 and 45 min, compared to control CM-treated cells. Cells were incubated with primary antibodies against CD44 and LRP6, DVL, or AXIN. Error bars, ±SE. Scale bar: b 20 µm, d 10 µm. c, e Fold induction of average interactions per cell, compared to control CM treatment. Error bars, ±SE. Student’s t test. f, g Co-immunoprecipitation of AXIN/CD44 (f) and CD44/DVL (g) on lysates of HeLa cells induced with Wnt3a CM for the indicated time points. Student’s t test/Mann–Whitney U-test: *p < 0.05, **p < 0.01, ns not significant. n = number of analyzed cells.

Fig 4: Constitutively active LRP6 promoted both cell migration and invasion.Cell migration and invasion assays were performed using LRP6-stably expressing BEL-7402 cells. (A) Overexpression of myc-CA LRP6 enhanced cell migration in BEL-7402 cells. The numbers of migrated cells in CA LRP6 Clones #3 and #8 were significantly higher than the vector control (P<0.001 for both). (B) Overexpression of myc-CA LRP6 promoted cell invasion in BEL-7402 cells. The numbers of invaded cells in CA LRP6 Clones #3 and #8 were significantly higher than the vector control cells (P<0.001 and = 0.009, respectively).

Fig 5: De-ubiquitination of LRP6 by USP19 on Lys-1403 promotes LRP6 biogenesis.(A) Immunoprecipitation of endogenous LRP6 and (B) cellular level in RPE1 cells upon usp19 silencing. C/D/E/F: Metabolic 35SCys/Met pulse chase experiment (20 min pulse) on transiently expressed myc-LRP6 wild type (WT), palmitoylation deficient mutant (CC–SS), K1403R mutant (K1403R) or K1403R mutation in the palmitoyl deficient background (CC-SS + KR) mutants in HeLa cells upon over expression of GFP-tagged USP19 (O.E. USP19) or GFP-tagged USP19 catalytically inactive (O.E. USP19 C-S) or upon usp19 gene silencing (siRNA USP19). (C) WT n = 6, same as in Figure 1D, other conditions n = 3, (D) (CC-SS curve corresponding to the one in Figure 2A) and (F) all conditions n = 3, (E) K1403R n = 6, other conditions n = 3.DOI: http://dx.doi.org/10.7554/eLife.19083.00910.7554/eLife.19083.010Figure 3—source data 1.Numeric data for graphs of Figure 3C, D and E, F.DOI: http://dx.doi.org/10.7554/eLife.19083.010