Journal: bioRxiv

Article Title: Chk1 Phosphorylates Cdh1 to Promote SCF βTRCP -Dependent Degradation of Cdh1 During S-Phase

doi: 10.1101/535799

Figure Lengend Snippet: (A) Chk1 phosphorylates Cdh1 in-vitro . GST-Cdh1 was phosphorylated with Chk1 through In-vitro kinase assays. Immunoblot represents immunoprecipitated GST-Cdh1 on glutathione beads following in-vitro kinase assays. PIMAGO western blot kit was used to detect phospho-GST-Cdh1. Phosphorylation was further confirmed by including Chk1 inhibitor where indicated during the kinase assays or by treating phosphorylated GST-Cdh1 with phosphatase. (B) Schematic diagram of Cdh1. Chk1-mediated phosphorylation sites identified from mass-spectrometry analysis are indicated in blue. Cdh1 phosphorylation sites mediated by Cyclin A-Cdk2 and Plk1 are in red and magenta, respectively. The SCF-βTRCP1 phosphodegron is indicated. (C). Chk1 mediated phosphorylation of Cdh1 creates a binding site for βTRCP1. 293T cells were transfected with the indicated HA-Cdh1 constructs together with Flag-βTRCP1. Cells were treated with proteasome inhibitor MG132 (10 µM) for 5 hr and the interaction between HA-Cdh1 and Flag-βTRCP1 was analyzed. Immunoprecipiated HA - Cdh1 WT and the mutant band intensities were first normalized to their relative amounts in lysates. The relative amount of the indicated Cdh1 proteins bound to Flag-βTRCP1 was normalized to wild type HA-Cdh1. See also Figure EV2A . (D) Cdh1 interacts with Chk1 in vivo. Both HA-Cdh1 and constitutively active Myc-Chk1 L449R were co-expressed in 293T cells. 30 hr post-transfection, cells were treated with proteasome inhibitor MG132 (10 µM) for 5 hr. The interaction between HA-Cdh1 and Myc-Chk1 L449R proteins was monitored by immunoprecipitation. (E) Constitutively active Chk1 promotes Cdh1 and SCF βTRCP1 interaction in-vivo . Immunoblot analysis of immunoprecipitates and whole-cell lysates derived from 293T cells transfected with Flag-βTRCP1, HA-Cdh1 and Myc-Chk1 L449R (where indicated). 30 hr post-transfection, cells were treated with proteasome inhibitor MG132 (10 µM) for 5 hr. Immunoprecipiated HA - Cdh1 band intensities (EV2B) were normalized to their respected Flag-βTRCP1 IP bands and then further normalized to control. (F) n-vivo ubiquitination assays shows that SCF βTRCP1 promotes Cdh1 ubiquitination in a Chk1 dependent manner. (G) Mutations of Chk1 mediated phosphorylation sites in Cdh1 increases the stability of Cdh1. 293T cells were transfected with the indicated HA-Cdh1 constructs together with Flag-βTRCP1 and Myc-Chk1 L449R (where indicated). Cells were treated with 50 µg/ml cycloheximide (CHX). At the indicated time points, whole-cell lysates were prepared for immunoblot analysis. The intensities of Cdh1 bands were normalized to actin, then normalized to the t=0 time point. The plots represent the relative fraction of indicated Cdh1 protein at final time point (t=320 min) after adding CHX. Data are represented as mean +/- SD, n=3 biological replicates. See also (EV2C) .

Article Snippet: Phosphorylation of GST-Cdh1 was detected by pIMAGO western-blot kit (Tymora Chemicals).

Techniques: In Vitro, Western Blot, Immunoprecipitation, Mass Spectrometry, Binding Assay, Transfection, Construct, Mutagenesis, In Vivo, Derivative Assay