Fig 2: A schematic illustration of the proposed model of how Cdh1 exerts its tumor suppressor role in part by the negative regulation of WWP2 E3 ligase activity to stabilize PTEN and to suppress the oncogenic Akt signaling. In most cancer cells, where Cdh1 is largely APC-free due to relatively high Cdk activity, Cdh1 mainly directly binds and inhibits WWP2, which leads to the accumulation of various WWP2 substrates including PTEN, TRIF and Rpb1. But further phosphorylation of Cdh1 by Cdk and Plk promotes Cdh1 degradation and the loss of Cdh1 eventually leads to PTEN destabilization and subsequent Akt activation to facilitate tumorigenesis. APC, anaphase-promoting complex/cyclosome.

Fig 3: Cdh1 promotes the intramolecular interaction of WWP2. (a) Immunoblot (IB) analysis of whole-cell lysates (WCL) and immunoprecipitates (IP) derived from 293 T cells transfected with the indicated Cdh1 and WWP2 constructs. Thirty-six hours post transfection, the cells were pretreated with 10 μM MG132 before collecting. (b) Autoradiography of 35S-labeled Cdh1 bound to the indicated GST-fusion proteins. (c) Gel filtration experiments to illustrate that depletion of endogenous Cdh1 promotes WWP2 oligomer formation in cells. IB analysis of the indicated fractionations derived from the gel filtration experiment with shGFP or shCdh1 infected HCT116 cells. Before running cell lysates, the molecular weight resolution of the column was first estimated by running native molecular weight markers (thyroglobulin ~669 KDa, ferritin ~440 KDa, aldolase ~158 KDa, conalbumin ~75 KDa and ovalbumin ~44 KDa) and determining their retention times on Coomassie-stained SDS–PAGE protein gels. (d) A proposed model of how the WWP2 E3 ligase activity is suppressed by Cdh1 via enhancing WWP2 intramolecular interaction-mediated auto-inhibition. PAGE, polyacrylamide gel electrophoresis.

Fig 4: APC/CCdh1 promotes FAH polyubiquitination and decreases FAH protein half-life. (A) Flag-Cdh1, sgRNA-1-Cdh1 and HA-ubiquitin were co-transfected into HEK293 cells with Myc-FAH and treated with MG132 (5 µM) for 8 h. The cells were immunoprecipitated with Myc-antibody followed by immunoblotting with HA and Myc antibodies and the polyubiquitination of FAH was examined. (B) Determination of FAH protein half-life. HEK293 cells were transfected with Myc-FAH and control vector and treated with cycloheximide (CHX) (150 µg/mL). Cells were harvested at specific time intervals and analyzed by Western blotting with the Myc antibody indicated. (C) FAH protein half-life is extended upon the transfection of sgRNA targeting Cdh1. HEK293 cells were transfected with the control vector and sgRNA-Cdh1, treated with CHX and analyzed by Western blotting. (D) FAH protein half-life is reduced upon transfection of Flag-Cdh1. HEK293 cells were transfected with control vector and Flag-Cdh1 and treated with CHX and expression levels were analyzed by Western blotting. GAPDH was used as a loading control. The FAH band intensities for (B–D) were estimated by ImageJ software and normalized with GAPDH for graphical representation. Data were presented as the mean and standard deviation of three independent experiments. Two way ANOVA followed by Tukey’s post hoc test was used for comparison between two groups and the p-values are indicated.

Fig 5: E3 ligase APC/CCdh1 destabilizes FAH protein. (A) Screening E3 ligases for FAH. Several ligases were transfected transiently along with Myc-FAH in HEK293 cells and analyzed through Western blotting. (B) HEK293 cells were co-transfected with a constant amount of Myc-FAH along with Flag-Cdh1 at increasing concentrations (0, 0.25, 0.5, 0.75, 1.0 and 1.25 µg). The Myc-FAH band intensity was estimated by ImageJ software and normalized with GAPDH for graphical representation. Data were presented as the mean and standard deviation of three independent experiments. One-way ANOVA followed by Tukey’s post hoc test was used and p-values are indicated. (C) HHSteCs were co-transfected with Flag-Cdh1 at increasing concentrations (0, 0.25, 0.5, 0.75, 1.0 and 1.25 µg) and bands were analyzed by endogenous FAH antibody through Western blotting. The FAH band intensity was estimated by ImageJ software and normalized with GAPDH for graphical representation. Data were presented as the mean and standard deviation of three independent experiments. One-way ANOVA followed by Tukey’s post hoc test was used and p-values are indicated. (D) Myc-FAH was transfected with HA-ubiquitin along with Flag-Cdh1 in HEK293 cells to see the effect of Cdh1 on the ubiquitination and proteolysis of FAH. Cell lysates were immunoprecipitated with Myc antibody followed by immunoblotting with HA or Myc antibody. GAPDH was used as a loading control.