14 research outputs found
The role of CD44v6 in RTK internalization and trafficking
Internalization of receptor tyrosine kinases (RTKs) is crucial for RTK signaling and cellular responses such as cell migration. This work shows that CD44v6 mediates the internalization of the RTK Met and traffics together with Met through endosomal compartments suggesting that CD44v6 is required for Met-signaling from endosomes. The RTK VEGFR-2 also interacts with CD44v6. However Met and VEGFR-2 are activated with strikingly different kinetics suggesting a different mechanism of internalization
Correction: Internalization of Met Requires the Co-Receptor CD44v6 and Its Link to ERM Proteins
Correction: Internalization of Met Requires the Co-Receptor CD44v6 and Its Link to ERM Proteins.
Internalization of Met Requires the Co-Receptor CD44v6 and Its Link to ERM Proteins
<div><p>Receptor Tyrosine Kinases (RTKs) are involved in many cellular processes and play a major role in the control of cell fate. For these reasons, RTK activation is maintained under tight control. Met is an essential RTK that induces proliferation, differentiation, migration, survival and branching morphogenesis. Deregulation of Met by overexpression, amplification or lack of effective degradation leads to cancer and metastasis. We have shown that Met relies on CD44v6 for its activation and for signaling in several cancer cell lines and also in primary cells. In this paper, we show that internalization of Met is dependent on CD44v6 and the binding of Ezrin to the CD44v6 cytoplasmic domain. Both CD44v6 and Met are co-internalized upon Hepatocyte Growth Factor induction suggesting that Met-induced signaling from the endosomes relies on its collaboration with CD44v6 and the link to the cytoskeleton provided by ERM proteins.</p></div
Correction: Internalization of Met Requires the Co-Receptor CD44v6 and Its Link to ERM Proteins
Colocalization of Met and CD44v6 in Rab5 positive endosomes upon HGF stimulation.
<p>(<b>A</b>) HeLa cells were transfected with mRFP-Rab5 wild type and human CD44v6. After 24 hours the cells were serum starved and treated with 50 µM cycloheximide for 2 hours. Cells were stimulated with 25 ng/ml HGF for 1hour on ice (cold start) and subsequently shifted to 37°C for the indicated time. Cells were fixed, permeabilized, and stained for Met (green) and CD44v6 (cyan) with specific antibodies (Materials and Methods). Nuclei were stained with Dapi (blue) and images were taken with a confocal microscope (Leica SPE) using a 63× objective. The last image is an enlargement of the square indicated in the middle image. Intensity profiles of the respective emission wavelengths of the mRFP-Rab5, Met, and CD44v6 signals were measured along lines drawn across the plasma membrane (a) or across vesicles (b,c). (<b>B</b>) HeLa cells were transfected with a constitutive active Rab5-GFP construct (Rab5 Q79L-GFP) and a human CD44v6 construct (see Materials and Methods) and treated as in A. Rab5 Q79L-GFP (green), Met (red), CD44v6 (cyan), Dapi (blue). Intensity profiles of the respective emission wavelengths of Rab5 Q79L-GFP, Met and CD44v6 are shown on the right side for the indicated endosomes at 15 minutes. Intensity profiles corresponding to the plasma membrane (a) and to an endosome (b) are included for the 0 minute time point (below).</p
The link between CD44v6 and the cytoskeleton through Ezrin is required for Met internalization.
<p>(<b>A</b>) HeLa cells were transfected with a rat CD44v4-7 construct deleted from the cytoplasmic domain (CD44v4-7Δcyt) or a control vector. The kinetic of Met internalization upon HGF induction was measured in a MESNA experiment (Material and Methods). In the first sample cells were kept at 4°C. –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 rat specific CD44v6 antibody 1.1ASML (Material and Methods). (<b>B</b>) HeLa cells were transfected either with human CD44v6 wild type or with human CD44v6 tailless constructs as indicated. After 24 hours cells were serum starved, treated with 50 µM cycloheximide for 2 hours prior to HGF-induction, stimulated with 25 ng/ml HGF for 1 hour on ice (cold start) and incubated at 37°C for the indicated time periods. Cells were then fixed, stained, and imaged using a confocal microscope (Leica SPE) with a 63× objective. Met (red), CD44v6 wild type (green), CD44v6 tailless (green), Dapi (blue). Scale bar = 15 µm. The quantification of three independent experiments (n = 20) is shown. The percentage of transfected cells with Met exclusively located on endosomes or exclusively at the plasma membrane or on both was calculated for each time point. Values are the means of three independent experiments. Student´s t test: *p<0,05 and **p<0,01. (<b>C</b>) HeLa cells were transfected with an Ezrin construct deleted from the actin-binding domain (EzrinΔABD) or a control vector. The cells were serum starved for 24 hours followed by biotinylation (0,5 mg/ml), induced with HGF (50 ng/ml) and treated with MESNA. Upon cell lysis proteins were pulled down by NeutrAvidin beads. The precipitates were subjected to Western Blot analysis for Met and CD44v6. For the first sample the cell were kept at 4°C. –M: cells were not treated with MESNA. The experiment was repeated at least three times.</p
Co-localization of Met and CD44v6 in internal compartments after HGF stimulation.
<p>(<b>A</b>) Confocal images of endogenous Met and CD44v6 at different time points after HGF-induction. HeLa cells were serum starved, treated for 2 hours with 50 µM cycloheximide, incubated at 4°C for 1 hour with HGF (25 ng/ml) (cold start) and then shifted to 37°C for the indicated times. After fixation cells were stained for Met (red, AF276) and CD44v6 (green, VFF18) and nuclei (blue, Draq5) and imaged with the confocal microscope (LSM510) with a 63× objective (Material and Methods). For each time point, the mean Pearson Coefficient of 4–6 images was calculated using the Imaris software. Scale bar = 15 µm (<b>B</b>) Intensity profiles of the emission wavelengths of the Met and CD44v6 signals along lines drawn across the plasma membrane or vesicles containing both Met and CD44v6. The intensity of the respective wavelengths is plotted against the distance in µm. (<b>C</b>) Co-immunoprecipitation of CD44v6 and Met upon HGF induction. Serum-starved HeLa cells were induced with HGF (50 ng/ml) for the indicated time periods and Met was immunoprecipitated. The precipitate was subjected to Western Blot analysis and the membrane was blotted for Met, phospho-Met and CD44v6. The experiment was repeated at least three times.</p
CD44v6 controls Met internalization.
<p>(<b>A</b>) Left side: HeLa cells were transfected either with a pool of control siRNAs or a mixture of two different CD44v6 specific siRNAs and starved for 24 hours (Material and Methods). The cells were biotinylated (0,5 mg/ml), induced with HGF (50 ng/ml) and treated with MESNA. After cell lysis, proteins were pulled down with a NeutrAvidin resin and subjected to Western Blot analysis with Met or the Transferrin receptor (TfR) antibodies. For the first sample the cells were kept at 4°C. –M: the cells were not treated with MESNA. Right side: Western Blot analysis of cell lysates of ctrl siRNAs and CD44v6 siRNAs transfected HeLa cells using the CD44v6 and the TfR antibodies. (<b>B</b>) Starved HeLa cells respectively HT29 cells were incubated with the v6 peptide or a control peptide for 10 minutes at 37°C and then induced with 25 ng/ml of HGF for the indicated time points. Cells were then either lysed and the lysates were subjected to Western Blot analysis for phospho-Met and Met (below) or cells were fixed, permeabilized and stained for Met with specific antibodies (red) (above). Nuclei were stained with Dapi and images were taken with a confocal microscope (Leica SPE) using a 63× objective. The quantification of three independent experiments (n = 40) is shown. The percentage of cells with Met exclusively located at the plasma membrane was calculated for each time point. Student´s t test: ***p<0,001.</p
CD44v6 is internalized upon HGF induction.
<p>(<b>A</b>) Left side: Lysates of HT29, HeLa and HepG2 cells were immunoprecipitated with the CD44v6 antibody (VFF18) and subjected to SDS-PAGE. The blots were probed with the same antibody. Apparent MW is indicated. Right side: starved HeLa cells were induced for 5 min with HGF (25 ng/ml) and Met and Erk phosphorylation was determined as described in Material and Methods. Where indicated cells were pre-treated with CD44v6 peptides (v6pep1: 5mer, v6pep2: 14mer) or a control peptide (see Material and Methods). Met and Erk hybridization was used as loading controls. The experiment was repeated at least three times. (<b>B</b>) Top: The optimal biotin concentration for efficient biotinylation was determined (50–1000 µg/ml). Serum starved cells were biotinylated for 15 minutes on ice and biotinylated proteins were pulled down using a NeutrAvidin resin. Both fractions, the pull down (PD) and the supernatant (sup) were subjected to Western Blot analysis using Met (25H2) and CD44v6 antibodies as indicated. The MESNA treatment (last lane) was tested with the highest concentration of biotin (1 mg/ml). Below: Serum starved cells were biotinylated (0,5 mg/ml), induced with HGF (50 ng/ml) and treated with MESNA (Material and Methods). After lysis, internalized (still biotinylated) proteins were pulled down with a NeutrAvidin resin. The fractions were subjected to Western Blot analysis and Met, CD44v6 and the Transferrin receptor (TfR) were detected with the respective antibodies (Material and Methods). The first sample (lane 1) was kept at 4°C. –M: the cells were not treated with MESNA. (<b>C</b>) HGF-induced internalization of surface proteins detected by the trypsin assay. Serum starved cells were induced by HGF (25 ng/ml) for the indicated times and then cooled down on ice followed by a treatment with trypsin for 30 minutes (Materials and Methods). FCS was added to stop the trypsin treatment and the cells were then lysed. CD44v6 was immunoprecipitated and detected by Western Blot analysis. First lane: immunoprecipitation with IgG.</p