HRas signaling is required for HGF-induced Met internalization.

(A) HGF-dependent ubiquitination of Met in HeLa cells transfected with a tailless mutant of CD44. HeLa cells were transfected as indicated with either the rat CD44v4-7Δcyt construct or a control vector as described in Material and Methods. 24 hours after transfection cells were starved and incubated with HGF (25 ng/ml) at 37°C for the indicated time periods. The Met immunoprecipitates were loaded for SDS-PAGE and subjected to Western Blot analysis using antibodies against ubiquitin and Met (Materials and Methods). For quantification, the intensities of the bands corresponding to three independent experiments were used. (B) Constitutively inactive Ras (HRasS17N) blocks HGF-induced Erk-activation. HeLa cells were co-transfected with HA-Erk and either the constitutively inactive Ras (HRasS17N-EGFP) or the control vector. After 24 hours cells were serum starved and induced with HGF (25 ng/ml) for 10 minutes or left untreated. HA-Erk was immunoprecipitated with a HA-antibody and blotted for phospho-Erk and Erk. (C) Confocal images of endogenous Met after HGF-induction in HeLa cells transfected with a Ras dominant negative mutant (HRasS17N) or a control vector. HeLa cells were transfected as indicated either with the HRasS17N-EGFP construct or with a control vector (Material and Methods). After 24 hours cells were serum starved, treated with 50 µM cycloheximide for 2 hours, then incubated with HGF for 1 hour on ice (cold start) and subsequently shifted to 37°C for the indicated time period. Cells were then fixed, stained, and imaged using a confocal microscope (Leica SPE) with a 63× objective. Met (red), HRasS17N-EGFP (green), EGFP (green), Dapi (blue). Scale bar = 15 µm. The quantification of three independent experiments (n = 20) is shown. For quantification the percentage of transfected cells with Met exclusively located on endosomes was calculated. Student´s t test: **p<0,01. (D) HeLa cells were transfected with the HRasS17N construct or a control vector. The kinetic of Met internalization upon HGF induction was measured in a MESNA internalization assay. - M refers to a sample obtained from cells that were not treated with MESNA. The Western Blot analysis was performed with a Met specific antibody and the TfR antibody. (E) Membrane and endosomal fractions were blotted against Met. Na+/K+ ATPase is used as a control for the membrane fraction (middle). EEA1 is used as a control for the endosomal fraction (right). Left: Phosphorylation of Erk is blocked in the presence of the Ras inhibitor (farnesyl thiosalicylic acid).