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Dev Biol
2019 Sep 01;4531:48-55. doi: 10.1016/j.ydbio.2019.05.007.
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PTK7 proteolytic fragment proteins function during early Xenopus development.
Lichtig H
,
Cohen Y
,
Bin-Nun N
,
Golubkov V
,
Frank D
.
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Protein Tyrosine Kinase 7 (PTK7) is as a critical regulator of canonical and non-canonical Wnt-signaling during embryonic development and cancer cell formation. Disrupting PTK7 activity perturbs vertebrate nervous system development, and also promotes human cancer formation. Observations in different model systems suggest a complex cross-talk between PTK7 protein and Wnt signaling. During Xenopus laevis nervous system development, we previously showed that PTK7 protein positively regulates canonical Wnt signaling by maintaining optimal LRP6 protein levels, but PTK7 also acts in concert with LRP6 protein to repress non-canonical Wnt activity. PTK7 is a transmembrane protein, but studies in cancer cells showed that PTK7 undergoes "shedding" by metalloproteases to different proteolytic fragments. Some PTK7 proteolytic fragments are oncogenic, being localized to alternative cytoplasmic and nuclear cell compartments. In this study we examined the biological activity of two proteolytic carboxyl-terminal PTK7 proteolytic fragments, cPTK7 622-1070 and cPTK7 726-1070 during early Xenopus nervous system development. We found that these smaller PTK7 proteolytic fragments have similar activity to full-length PTK7 protein to promote canonical Wnt-signaling via regulation of LRP6 protein levels. In addition to cancer systems, this study shows in vivo proof that these smaller PTK7 proteolytic fragments can recapitulate full-length PTK7 protein activity in diverse systems, such as vertebrate nervous system development.
Fig. 1. The cPTK7 622â1070 protein undergoes co-IP with LRP6 protein in HEK cells
A. A schematic description of the PTK7 constructs used in this study: Full-length 1â1070 PTK7-Flag-tagged, full-length 1â1070 PTK7-Myc tagged, cPTK7 622â1070 V5/His-tagged and cPTK 726â1070-V5/His-tagged. S, signal peptide, Ig, immunoglobulin-like domain, TM, transmembrane domain, JM, juxtamembrane region, KINASE, the catalytically dead kinase domain. The amino acid residue numbering is shown below the construct structure. MT1-MMP, ADAM and γ-secretase cleavage sites are indicated by arrows. The full-length 1â1070 PTK7-Myc-tagged protein was used in the co-IP assays in HEK293â¯cell culture. The full-length 1â1070 PTK7-Flag-tagged protein was a more biologically active protein than the Myc-tagged protein in Xenopus embryos.
B. HEK293 cells (lane 1) were co-transfected with plasmids encoding the full length VSVG-LRP6 protein (lanes 2â4) and the full-length PTK7-Myc protein (lane 2) and the short PTK7 proteins, PTK7 622â1070-V5 (lane 3) and PTK7 726â1070-V5 (lane 4). Whole-cell extracts were immune-precipitated (left panel) by VSVG antibody (targeting LRP6 protein) on protein G Agarose beads. The co-IP proteins were blotted with VSVG, myc and V5 antibodies. Note that two proteins underwent co-IP with VSVG-LRP6 protein, full length PTK7 and PTK7 622â1070; the PTK7 726â1070 protein did not undergo co-IP with the VSVG-LRP6 proteins. The left panel shows the IP (lanes 1â4) and the right panel shows the total protein expression input (lanes 5â8) before co-IP. α-tubulin protein (right panel) serves as a positive control for protein loading in the protein input samples before co-IP.
Fig. 2. The cPTK7 622â1070 and cPTK 726â1070 proteins rescue NTDs and posterior neural cell fates in PTK7 knockdown/morphant embryos
A. Embryos were examined for morphological rescue at neurula (stage 17â18, top two rows of panels) and tailbud (stage 24â26, lower two panels). Embryos were injected with 12â¯ng PTK7-MO and rescued by co-injection of 100â400â¯pg of cPTK7 622â1070 protein encoding RNA. Four representative embryos are shown per injected group. The control embryos (CE) are the two panels of four on the left side (upper four: neurula stage and lower four: tailbud stage). The middle two panels of four are the PTK7-MO injected embryos. The two panels of four on the right are the PTK7-MO/cPTK7 622â1070 co-injected rescued embryos. At neurula stages, stronger neural folding is observed in the PTK7-MO/cPTK7 622â1070 co-injected embryos versus the PTK7-MO injected embryos (upper-right four panels, versus upper-center four panels). At tailbud stages the embryos are less squat and more elongated the in the PTK7-MO/cPTK7 622â1070 co-injected embryos versus the PTK7-MO injected embryos (lower right four panels, versus lower center four panels). Rescue experiment statistics and data bar graphs are shown in Fig. 2C.
B. Embryos were examined for morphological rescue at neurula (stage 17â18, top two rows of panels) and tailbud (stage 24â26, lower two panels). Embryos were injected with 12â¯ng PTK7-MO and rescued by co-injection of 100â400â¯pg of cPTK7 726â1070 protein encoding RNA. Four representative embryos are shown per injected group. The control embryos (CE) are the two panels of four on the left side (upper four: neurula stage and lower four: tailbud stage). The middle two panels of four are the PTK7-MO injected embryos. The two panels of four on the right are the PTK7-MO/cPTK7 726â1070 co-injected rescued embryos. At neurula stages, stronger neural folding is observed in the PTK7-MO/cPTK7 726â1070 co-injected embryos versus the PTK7-MO injected embryos (upper-right four panels, versus upper-center four panels). At tailbud stages the embryos are less squat and more elongated the in the PTK7-MO/cPTK7 726â1070 co-injected embryos versus the PTK7-MO injected embryos (lower right four panels, versus lower center four panels). Rescue experiment statistics and data bar graphs are shown in Fig. 2C.
C. Statistical analysis of embryos shown in Fig. 2AâB. Embryos were scored at tailbud stage for rescue as described in the text. Control embryos (CE) nâ¯=â¯434, 95% normal neural plate folding; PTK7-MO injected embryos (MO) nâ¯=â¯390, 6% normal neural plate folding; PTK7-MO/PTK7 full-length protein co-injected embryos (MO â+ âPTK7) n â= â418, 61% normal neural plate folding; PTK7-MO/cPTK7 622â1070 protein co-injected embryos (MO+622) n â= â296, 50% normal neural plate folding; cPTK7 726â1070 protein co-injected embryos (MO+726) n â= â250, 42% normal neural plate folding;
D. Control embryos (lane 2) were injected at the one-cell stage with PTK7-MO (12â¯ng, lane 3) or the PTK7-MO and RNA encoding cPTK7 622â1070 protein (400â¯pg, lane 4) Total RNA was isolated from pools of eight neurula (st.17) embryos from each group. Gene expression was analysed by Semi-quantitative (sq)RT-PCR analysis to: krox20, hoxb3 (hindbrain), n-tub (primary neuron), sox2 and ptk7 (panneural). âRT was performed (lane 1) to total RNA from the control uninjected embryos. EF1α is as positive control for RNA levels. Markers are rescued by the cPTK7 622â1070 protein (lanes 2â4)
E. Control embryos (lane 2) were injected at the one-cell stage with PTK7-MO (12â¯ng, lane 3) or the PTK7-MO and RNA encoding the full-length human PTK7 protein (50â¯pg, lane 4), which serves as a positive control for rescue (ref. 11). In parallel, the PTK7-MO was co-injected with increasing concentrations of RNA encoding the cPTK7 726â1070 protein (100â¯pg, 200â¯pg, 400â¯pg, lanes 5â7). Total RNA was isolated from pools of eight neurula (st.17) embryos from each injected group and sqRT-PCR was performed to analyse expression of the krox20, hoxb3 (hindbrain), n-tub (primary neuron), xanf1 (forebrain), sox2 and ptk7 (panneural) genes. âRT was performed (lane 1) to total RNA from the control uninjected embryos. ODC serves as positive control for RNA levels. Markers are rescued by either the full-length PTK7 or cPTK7 726â1070 proteins (compare lane 3â4 to 5â7).
Fig. 3. PTK7 cPTK7 622â1070 and 726â1070 proteins support LRP6 activation of Wnt direct-target genes
A. Embryos were injected at the one-cell stage with PTK7-MO (10â¯ng, lanes 5â7). In parallel, these embryos co-injected animally with mRNA encoding the ligand-independent form of the LRP6 protein, LRP6ÎN (175â¯pg) that activates the Wnt pathway (lane 4), and mRNA encoding the full-length PTK7 protein (200â¯pg, lane 7). Animal caps (ACs) removed at the blastula stage were grown until gastrula st.10.5, when total RNA was isolated from five control embryos (lane 2) and 18â¯ACs from each group (lanes 3â7). Wnt direct-target gene expression was examined by sq (semiquantiative) RT-PCR: xnr3, siamois and hoxd1. Ef1α serves as positive control for RNA levels.
B. Embryos were injected at the one-cell stage with PTK7-MO (10â¯ng, lanes 5â8). In parallel, these embryos co-injected animally with mRNA encoding the ligand-independent form of the LRP6 protein, LRP6ÎN (175â¯pg) that activates the Wnt pathway (lane 4), and mRNA encoding the short PTK7 proteins cPTK7 622â1070 and cPTK7 726â1070 (200â¯pg, lanes 7â8). ACs removed at the blastula stage were grown until gastrula st.10.5, when total RNA was isolated from five control embryos (lane 2) and 18â¯ACs from each group (lanes 3â9). Wnt direct-target gene expression was examined by sqRT-PCR: xnr3, siamois and hoxd1. Ef1α serves as positive control for RNA levels
C. Embryos were injected at the one-cell stage with PTK7-MO (12â¯ng, lanes 4â6). In parallel, these embryos co-injected animally with mRNA encoding LRP6 protein (600â¯pg, lane 3), and mRNA encoding the short PTK7 proteins cPTK7 622â1070 and cPTK7 726â1070 (200â¯pg, lanes 5â6). Embryos were grown until gastrula st.10.5, when total RNA was isolated from five embryos in each group. Wnt direct-target gene expression was examined by sqRT-PCR: hoxd1 and gbx2. Siamois expression is unchanged as described in text. Ef1α serves as positive control for RNA levels.
Fig. 4. LRP6 protein levels are maintained by small PTK7 proteolytic fragments
A. Control embryos (lane 1) were injected at the one-cell stage with PTK7-MO (12â¯ng, lane 2) and/or RNA encoding the activated LRP6ÎN-VSVG protein (200â¯pg, lane 3â4), and RNA encoding the human PTK7 726â1070 protein (lane 5). Protein was isolated for Western blot analysis at gastrula stages. Note the increased levels of LRP6ÎN-VSVG protein in lane 5 versus lane 4. α-tubulin protein serves as a positive control for protein loading.
B. Control embryos (lane 1) Embryos were animally injected at the one-cell stage with mRNA encoding the full-length LRP6 (600â¯pg, lanes 2â6). The PTK7-MO (12â¯ng, lanes 3â6) is co-injected parallel, along with mRNAs encoding the full-length PTK7 protein (200â¯pg, lane 4), mRNA encoding the cPTK7 622â1070 protein (200â¯pg, lane 5) or mRNA encoding the cPTK7 726â1070 protein (200â¯pg, lane 6). Embryos were grown to gastrula (stage 10.5). Total protein was isolated from ten embryos per group for Western analysis. The VSVG antibody detects the full-length LRP6 protein. α-tubulin protein serves as a positive control for protein loading.
