FIGURE 5.

Loss of JNK2 impairs EGFR internalization. A, cells were treated with 100 ng/ml FITC-EGF at 37 °C for 15 min. Media were aspirated, and membrane-bound ligand was acid-stripped with 0.01% acetic acid solution. FITC-EGF uptake was captured live using ImagePro 6.1 software and color overlay (EGF = green, ×100). Data are representative of two independent experiments. B, conditions were similar to A, except PyVMTjnk2^−/−gipz and PyVMTjnk2^−/−sheps8 cells were treated with Texas Red-EGF. Acid stripping removed unbound EGF (EGF = red, ×100). C, subconfluent cell lines were lysed and later probed with EGFR antibody. GAPDH was used as the internal loading control. D, subconfluent PyVMTjnk2^−/−gipz and PyVMTjnk2^−/−sheps8 cells were serum-starved overnight and then stimulated with 1% FBS for 0, 0.5, 1, 2, and 4 h. Cell lysates were probed using primary antibodies, as indicated. GAPDH was used to evaluate equal loading among samples. E, model diagram. In the leading edge of a migrating cell, activated EGFR is internalized into endosomes via active Rab5 GTPase. EPS8, when complexed with Abi-1 and Sos-1, transmits signals from EGFR to activate Rac and JNK, which facilitate actin remodeling and migration. JNK2 inhibits eps8 expression such that low levels of EPS8 bind to Abi-1 and Sos-1. With JNK2 deficiency, eps8 transcription is up-regulated, and the Abi-1/Sos-1 pool becomes rate-limiting. Excess EPS8 binds to RN-Tre. EPS8 binding activates RN-Tre, a GTPase-activating enzyme for Rab5, resulting in Rab5 inactivation and reduced EGFR internalization.