Fig. 6.

Src phosphorylates Cdh1 at Y148 to inhibit APC^Cdh1 E3 ligase activity. a, b In vitro kinase assays showing that bacterially purified GST-Cdh1 N-terminus could be phosphorylated by immuno-purified wild type (WT) Src, but not the enzymatic deficient K298R-HA-Src mutant, by autoradiography (a) or immunoblot (IB) analysis using the phospho-tyrosine (p-Y) antibody (b). c IB analysis of whole-cell lysates (WCL) and GST-pull down precipitates (GST PD) derived from 293T cells transfected with pCMV-GST-Cdh1 and the indicated HA-Src constructs. d In vitro kinase assays showing that immuno-purified Src kinase phosphorylated both bacterially purified GST-Cdh1 and its N terminus. e IB analysis of WCL and GST PD derived from 293T cells transfected with pCMV-GST-Cdh1 and the indicated HA-tyrosine kinases (TKs) constructs. f A schematic illustration of the previously identified phospho-serine/threonine sites as well as the candidate phospho-tyrosine sites of Cdh1 that could be phosphorylated by the Src kinase. g IB analysis of WCL and GST PD derived from 293T cells transfected with HA-Src and the indicated pCMV-GST-Cdh1 constructs. h In vitro kinase assays showing that immuno-purified WT-HA-Src failed to promote the phosphorylation of bacterially purified Y148F mutated-GST-Cdh1 N-terminus. i IB analysis of WCL derived from MDA-MB-231 cells that were treated with 4 ng ml⁻¹ PDGF for 30 min as indicated. Cells were serum-starved overnight before treatment. j IB analysis of WCL and anti-Cdh1 IP derived from control or sgSrc-infected MDA-MB-231 cells. Cells were pretreated with 4 ng ml⁻¹ PDGF for 30 min before harvest. k IB analysis of WCL and anti-Cdh1 IP derived from DMSO or dasatinib-treated MDA-MB-231 cells. Cells were pretreated with 4 ng ml⁻¹ PDGF for 30 min before harvest. l IB analysis of WCL derived from WT and Src^−/− MEFs. m A schematic illustration of phosphorylation of Cdh1 at Y148 by Src disrupts the binding between Cdh1 N terminus and the APC core complex