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. 2024 Apr 15;15:3220. doi: 10.1038/s41467-024-47427-w

Fig. 4. PIN1 catalyzes trans to cis isomerization of the pS163-P motif in CDH1 to prevent CDH1 dephosphorylation.

Fig. 4

a 293T cells were transfected with indicated constructs for 36 hrs. Input is 5% of the total lysates used in IP. b In vitro kinase assay showing that CDK4 phosphorylates CDH1 at Ser163. c FACS analysis of APC/C-degron reporter levels in MCF-7 cells expressing either WT CDH1, S163A or S163E mutants CDH1. d IB analysis of indicated proteins derived from MCF-7 cells expressing either WT CDH1, S163A or S163E mutants CDH1 and treated with 1 μM palbociclib for 3 days. e IB analysis of GST pull-down precipitates derived from 293T cells transfected with GST-PIN1 and HA-CDH1 mutants for 36 h. f NMR analysis of phosphorylated peptide bound to PIN1. Average chemical shift perturbation in PIN1 backbone amide resonances on the binding of the CDH1 phosphopeptide. g Overlay of two-dimensional (2D) 1H-15N Heteronuclear single quantum coherence (HSQC) spectrum from the backbone of R17, S18, W34, and E35, and the W34 sidechain of 15N-labeled PIN1 (blue) and its complex with the CDH1 phosphopeptide (orange). h HADDOCK model demonstrating putative interaction between the CDH1 phosphopeptide shown as red sticks and PIN1 WW (magenta) and PPIase domain (cyan; PDB: 1PIN). i Overlay of 13C-HSQC spectra acquired on free peptide (red) and its complex with PIN1 (green). The peak volumes were used to derive isomer population estimates. j DIA-MS analysis of the relative abundance of the peptide containing phosphorylation site of CDH1-S163 derived from WT or PIN1 KO MCF-7 cells stably expressing HA-CDH1. k IB analysis of indicated immunoprecipitates derived from WT or PIN1 KO MCF-7 cells expressing either WT CDH1, S163A or S163E mutants CDH1 and pulled down by anti-HA antibody. Input is 5% of the total lysates used in IP. l Schematic diagram illustrating PIN1-catalyzed trans to cis prolyl-isomerization of the CDH1-pS163-P motif. The images were representative images from 3 independent experiments (a–e, k). Source data are provided as a Source Data file.