Protein tyrosine kinase 7 has a conserved role in Wnt/β-catenin canonical signalling

Abstract

The receptor protein tyrosine kinase 7 (PTK7) was recently shown to participate in noncanonical Wnt/planar cell polarity signalling during mouse and frog embryonic development. In this study, we report that PTK7 interacts with β-catenin in a yeast two-hybrid assay and mammalian cells. PTK7-deficient cells exhibit weakened β-catenin/T-cell factor transcriptional activity on Wnt3a stimulation. Furthermore, Xenopus PTK7 is required for the formation of Spemann's organizer and for Siamois promoter activation, events that require β-catenin transcriptional activity. Using epistatic assays, we demonstrate that PTK7 functions upstream from glycogen synthase kinase 3. Taken together, our data reveal a new and conserved role for PTK7 in the Wnt canonical signalling pathway.

Figure 1

Protein tyrosine kinase 7 interacts with β-catenin. (A) Schematic representation of PTK7 and results of two-hybrid analysis in yeast. Interactions were positive (+) when β-galactosidase activity and auxotrophy for histidine were detected in the presence of 10 mM 3-aminotriazole. (B) Myc–β-catenin was coexpressed with Flag-tagged PTK7–Flag or Flag–PTK7^1–788 in COS7 cells. Proteins were immunoprecipitated using PTK7 antibody and revealed with PTK7 (upper panel) and β-catenin (middle panel) antibodies. Equal amounts of β-catenin or PTK7 (data not shown) were present in the lysates (bottom panel). (C) GFP or indicated PTK7 regions fused to GFP were coexpressed with Myc–β-catenin in COS7 cells. After immunoprecipitation with GFP antibody (upper panel), bound partners were detected by β-catenin antibody (middle panel). Comparable amounts of β-catenin and GFP proteins (data not shown) were detected in the total lysates (bottom panel). (D) Indicated Myc-tagged β-catenin constructs and Flag–PTK7 were coexpressed in COS7 cells. Proteins were immunoprecipitated with PTK7 antibody (upper panel). Bound partners were then detected using Myc antibody (middle panel), asterisk indicates a nonspecific band. Total lysates were blotted using Myc and PTK7 (bottom panel and not shown) antibodies. (E) Proteins extracted from Caco2 cells were immunoprecipitated with PTK7 or Scrib antibodies (or control antibodies of same species) and bound proteins were revealed by western blot with the mentioned antibodies (PTK7, Scrib, β-catenin or βPIX). (F) Subcellular localization of endogenous proteins was revealed by using immunofluorescence and confocal analysis in polarized MDCK cells. PTK7 is shown in green, E-cadherin is shown in red and β-catenin is shown in purple. Nuclei were stained with Hoechst blue dye. (G) After transfection of Wnt3a–Myc-, Myc–β-catenin- and Flag–PTK7-expressing plasmids in MDCK cells, total proteins were extracted and immunoprecipitated with PTK7 antibody; bound proteins were revealed by western blotting, as indicated. Total lysates were blotted using PTK7, β-catenin, Myc and α-tubulin (right panel) antibodies. Ab, antibody; β-PIX, β-PAK-interacting exchange factor; EC, extracellular; GFP, green fluorescent protein; IP, immunoprecipitation; MDCK, Madin–Darby canine kidney; PTK7, protein tyrosine kinase 7; TK, tyrosine kinase domains; TM, transmembrane.

Figure 2

Protein tyrosine kinase 7 is involved in Wnt/β-catenin canonical signalling. (A) HCT116 cells were stably transfected with shRNAs directed against PTK7 (shPTK7.1 or shPTK7.2) or luciferase (shLuc). PTK7 expression was assessed by FACS analysis and western blotting. (B) pSuper8xTOP or FOPFlash firefly luciferase reporter plasmids and pBeta–Renilla vector were transfected into HCT116 shPTK7 or shLuc cells along with Wnt3a–Myc-expressing plasmid or a relative empty vector (pCAGGS–Myc). Relative luciferase units normalized to pCAGGS–Myc TOP values (RLU fold) are represented. Error bars represent s.e. values and statistical analyses were performed by using a Student's t-test in at least three independent experiments. (C) Indicated proteins were revealed by western blotting in protein extracts of MEFs deficient in (−/−) or expressing (+/+) PTK7. (D) MEFs were nucleofected with pSuper8xTOP/FOPFlash (pSuper8xFOPFlash not shown) reporter plasmids, pBeta–Renilla vector and the indicated expression vector combinations. Relative luciferase units normalized to TOP-empty vectors values (RLU fold) are represented. Expression of Wnt3a–Myc and PTK7 was revealed by western blotting in protein extracts and cell culture supernatants (Wnt3a). Error bars represent s.e. values and statistical analyses were performed by using a Student's t-test in at least three independent experiments. (E) After nucleofection of Wnt3a–Myc-expressing plasmid and proteasome inhibition treatment, by MG132 and NEM, proteins extracted from MEF cells were immunoprecipitated with β-catenin mouse antibody and bound proteins were revealed by western blot with the mentioned antibodies (ubiquitin, PTK7 and β-catenin). Expression of Wnt3a–Myc was revealed by western blotting in cell culture supernatants (Myc). Blots are representative of at least three independent experiments. FACS, fluorescence-activated cell sorting; MEF, mouse embryonic fibroblast; MG132, N-(benzyloxycarbonyl)leucinylleucinylleucinal; NEM, N-ethylmaleimide; PTK7, protein tyrosine kinase 7; RLU, relative luciferase unit; shRNA, short hairpin RNA.

Figure 3

Protein tyrosine kinase 7 is required for organizer gene expression. (A) Four-cell embryos were injected with 10 ng per cell control MO in the two dorsal cells, or with a mixture of 5 ng each per cell of PTK7 MOb and MOc and stained for sox2 expression at the tailbud stage. (B) Embryos injected as in (A) or with 2.5 ng per cell β-catenin MO were processed for WISH analysis at gastrula stage 10. For the rescue assay, four-cell PTK7 MO-injected embryos received a second injection of 10 ng PTK7–Venus mRNA in the two dorsal cells. A similar rescue resulted from injection of 1 ng PTK7 mRNA. (C) Embryos injected as in (A) and (B) were collected at stage 10 and processed for RT–qPCR. (D) Embryos were injected with 1 pg per cell pRL-TK, 40 pg per cell 833pSia-Luc plasmids and MOs, as indicated, collected at stage 10 and processed for luciferase assays. (E) Four-cell embryos were injected dorsally with 625 pg per cell PTK7-ECD mRNA and processed for WISH analysis at stage 10 (chd, gsc) and at tailbud stage (sox2, front view). For all qPCR graphs, error bars represent s.e.m. values of three independent experiments with two technical duplicates. For all luciferase reporter assays, error bars represent s.e.m. values of three independent experiments with three technical replicates. MO, morpholino; PTK7, protein tyrosine kinase 7; RT–qPCR, reverse transcriptase–quantitative PCR; WISH, whole-mount in situ hybridization.

Figure 4

Protein tyrosine kinase 7 is required for Wnt canonical signalling, upstream from glycogen synthase kinase 3. (A) Four-cell embryos were first injected with MOs in the two ventral cells, followed by a second injection of 10 pg per cell wnt8 mRNA in the same cells. (B) Embryos were injected as in (A), but in all four cells. (C) Embryos were injected as in (A), together with pRL-TK and -833pSia-Luc plasmids. (D–F) Four-cell embryos were injected with MOs first and then with 500 pg per cell dsh mRNA in the two ventral cells. (G–I) Four-cell embryos were injected with MOs first and then with 500 pg per cell dnAxin mRNA in the two ventral cells. (J,K) Four-cell embryos were injected in all four cells with MOs first and then with 500 pg per cell dnGSK3 mRNA. (L) Four-cell embryos were injected with MOs and dnGSK3 mRNA in the two ventral cells. (M,N) Four-cell embryos were injected in all cells with MOs and incubated in 0.3 M LiCl in 5% Ficoll for 10 min at 64-cell stage, washed in 0.1 × Modified Barth's solution (MBS) and further cultured to stage 10. (O) Four-cell embryos were first injected in the two ventral cells with MOs and then with 500 pg per cell ΔN-βcat mRNA. WISH analysis: (A,D,G,J,M); RT–qPCR: (B,E,H,K,N); luciferase assay: (C,F,I,L,O). β-cat, β-catenin; Cont, control; dnAxin, dominant-negative Axin; dnGSK3, dominant-negative glycogen synthase kinase 3; MOs, morpholinos; mRNA, messenger RNA; RT–qPCR, reverse transcriptase–quantitative PCR; Uninj, uninjected; WISH, whole-mount in situ hybridization.