Fig 1: Abl kinases Stabilize Ptpn12 to Regulate p130Cas Phosphorylation.(A) Deletion of Abl/Arg in MEF cells led to an increased phosphorylation of p130Cas. (B) Elevated expression of phosphorylated p130Cas was observed in Abl/Arg mutant lenses (arrowheads). (C) Inhibition of Abl kinase activity in NIH3T3 cells by Imatinib led to increased phosphorylation of p130Cas and enhanced Crk-p130Cas interaction, as demonstrated by co-immunoprecipitation. (D) Analysis from the DepMap database indicates positive co-dependency between Ptpn12 and Abl and negative co-dependency with Crk and p130Cas. (E) Expression of constitutively active AblPP in HEK293 cells led to tyrosine phosphorylation of Ptpn12 and an increase in its expression level. (F) Mutating of tyrosine residues in Ptpn12 significantly reduced its expression, which was mitigated by proteosome inhibitor MG132. (G) Increased p-p130Cas levels were observed following siRNA-mediated knockdown of Ptpn12, but not Ptpn14. (H) Deletion of p130Cas in Abl/Arg mutants rescued the lens vesicle separation phenotype. (I) Quantification of the lens stalk phenotype.
Fig 2: FGF and Abl signaling regulates lens vesicle separation via the p130Cas-Crk-Rac1-RhoA axis.(A) Cdh1 deletion did not exacerbate the lens vesicle separation defect in Abl/Arg mutants. (B) Combined ablation of Cdh2 and 3 did not disrupt lens development. (C) Phosphorylated MLC (pMLC) was downregulated in Abl/Arg mutant lens vesicles (arrowheads). (D) The corneal-lens attachment phenotype was similar in Abl/Arg and RhoALSL-G17V single mutants, but was significantly more severe in the double mutants (arrowheads). (E) Quantification of the corneal-lens contact length. One-way ANOVA n=3 for Abl/Arg and RhoALSL-G17V single mutants, n=5 for double mutants. (F) Rac1 deletion rescued lens vesicle separation in RhoALSL-G17V, Abl/Arg, KrasG12D and Tg-Fgf3 mutants. (G) Quantification of the lens stalk phenotype. (H) The model FGF and Abl kinase signaling in lens vesicle development. FGF and Abl kinase signaling regulates the p130Cas via Src phosphorylation and Ptpn12 dephosphorylation, respectively. This modulation affects the recruitment of Crk and CrkL to p130Cas and the activation of Erk and Rac1, the latter antagonizes RhoA activity. RhoA regulates actomyosin contractility, crucial for separating the lens vesicle from the surface ectoderm.
Fig 3: FGF and ABL signaling regulates lens vesicle separation via the p130CAS-CRK-RAC1-RHOA axis.A Cdh1 deletion did not exacerbate the lens vesicle separation defect in Abl1/2 mutants. Scale bar: 100 µm. B Combined ablation of Cdh2 and 3 did not disrupt lens development. Scale bar: 100 µm. C Phospho-MLC (pMLC) was downregulated in Abl1/2 mutant lens vesicles (arrowheads). Scale bar: 50 µm. D The corneal-lens attachment phenotype in Abl1/2 and RhoALSL-G17V double mutants was significantly more severe compared to either single mutant (arrowheads). Scale bar: 50 µm. E Quantification of the corneal-lens contact length. Data are presented as mean ± SD and analyzed using one-way ANOVA with Tukey’s multiple comparison test; n = 3 for Abl1/2 and RhoALSL-G17V single mutants, n = 5 for double mutants. P = 0.021 and 0.0234. Source data are provided as a Source Data file. F Imatinib-mediated ABL inhibition in αTN4 lens epithelial cells eliminated pCRK and elevated RAC1–GTP, as shown by the PAK-RBD pulldown assay. G Rac1 deletion rescued lens vesicle separation in RhoALSL-G17V, Abl1/2, KrasG12D and Tg-Fgf3 mutants. Scale bar: 100 µm. H Quantification of the lens stalk phenotype. H Pregnant mice carrying Abl1/2 mutants were injected with the RAC1 inhibitor at E9.5 and E10.5 and embryos were harvested at E14.5. I The RAC1 inhibitor significantly reduced the lens stalk size in Abl1/2 mutant embryos as shown by both whole mount GFP expression and Cadherin staining. Scale bar: 100 µm. J Quantification of the lens stalk size. Data are presented as mean ± SD and analyzed using two-sided Student’s t-test; n = 14 for control and mutant. P = 0.046. K The model FGF and ABL kinase signaling in lens vesicle development. FGF and ABL kinase signaling regulate the p130CAS via SRC phosphorylation and PTPN12 dephosphorylation, respectively. This modulation affects the recruitment of CRK and CRKL to p130CAS and the activation of ERK and RAC1, the latter antagonizes RHOA activity. RHOA regulates actomyosin contractility, crucial for separating the lens vesicle from the surface ectoderm. Source data are provided as a Source Data file.
Fig 4: ABL kinases stabilize PTPN12 to regulate p130CAS Phosphorylation.A Deletion of Abl1/2 in MEF cells led to an increased phosphorylation of p130CAS. B Elevated expression of phosphorylated p130CAS was observed in Abl1/2 mutant lenses (arrowheads). Scale bar: 100 µm. C Inhibition of ABL kinase activity in NIH3T3 cells by Imatinib led to increased phosphorylation of p130CAS and enhanced CRK-p130CAS interaction, as demonstrated by co-immunoprecipitation. D Analysis from the DepMap database indicates positive co-dependency between Ptpn12 and Abl and negative co-dependency with Crk and p130Cas. E Expression of constitutively active Abl1PP in HEK293 cells led to tyrosine phosphorylation of PTPN12 and an increase in its expression level. F Mutating of tyrosine residues in PTPN12 significantly reduced its expression, which was mitigated by proteosome inhibitor MG132. G Increased p-p130CAS levels were observed following siRNA-mediated knockdown of PTPN12, but not PTPN14. H Deletion of p130CAS in Abl1/2 mutants rescued the lens vesicle separation phenotype. Scale bars: 50 µm. I Quantification of the lens stalk phenotype. J siRNA-mediated knockdown of PTPN12, but not GAPDH, results in elevated pERK levels. K Deletion of p130CAS reduced pERK staining in Abl1/2 mutant lens vesicle. Scale bars: 100 µm. L Relative pERK vs ERK level in the primary lens fiber cells at E12.5. Data are presented as mean ± SD and analyzed using two-sided Student’s t-test; n = 3 for control and mutant. P = 0.0002. Source data are provided as a Source Data file.
Supplier Page from Sino Biological, Inc. for Human PTPN12 Gene ORF cDNA clone expression plasmid, N-HA tag