Fig. 4.

Cdh1 suppresses Src kinase activity by locking Src in a closed conformation. a Immuno-purified HA-Src from 293T cells were incubated with bacterially purified and cleaved recombinant Cdh1 as indicated. The reaction was performed at 30 °C for 60 min followed by SDS-PAGE and Immunoblot (IB) analyses. b Recombinant Cdh1 inhibited the kinase activity of Src to promote the C-terminal domain of p85 cortactin (CTTN-C) phosphorylation in vitro. c A schematic illustration of the open and closed conformations of the Src kinase. d IB analysis of whole-cell lysates (WCL) and immunoprecipitates (IP) derived from 293T cells transfected with HA-Cdh1 and the indicated Src constructs. e IB analysis of WCL and IP derived from 293T cells transfected with HA-Cdh1 and the indicated Src constructs. f A schematic illustration of how Cdh1 secures the closed, inhibitory conformation of Src by reinforcing the binding between N- and C- terminus of Src. g Immuno-purified HA-Src proteins from 293T cells were incubated with bacterially purified GST-Cdh1. The reaction was performed at 30 °C for 60 min and followed by SDS-PAGE and IB analyses. h IB analysis of WCL derived from MDA-MB-231 cells transfected with EV, WT, D-box1-mutated, and D-box2-mutated HA-Src as indicated. Cells were harvested 48 h after transfection. i IB analysis of WCL and IP derived from 293T cells transfected with HA-Cdh1 and the indicated Src constructs. j Immuno-purified WT and Y530F-HA-Src from 293T cells were incubated with bacterially purified Cdh1 and CTTN-C. The reaction was performed at 30 °C for 60 min and followed by SDS-PAGE and IB analyses. k IB analysis of WCL and IP derived from 293T cells transfected with HA-Cdh1 and the indicated Src constructs