Abstract
Epidermal growth factor (EGF) receptor (EGFR) signal transduction is organized by scaffold and adaptor proteins, which have specific subcellular distribution. On a way from the plasma membrane to the lysosome EGFRs are still in their active state and can signal from distinct subcellular locations. To identify organelle-specific targets of EGF receptor signaling on endosomes a combination of subcellular fractionation, two-dimensional DIGE, fluorescence labeling of phosphoproteins, and MALDI-TOF/TOF mass spectrometry was applied. All together 23 EGF-regulated (phospho)proteins were identified as being differentially associated with endosomal fractions by functional organelle proteomics; among them were proteins known to be involved in endosomal trafficking and cytoskeleton rearrangement (Alix, myosin-9, myosin regulatory light chain, Trap1, moesin, cytokeratin 8, septins 2 and 11, and CapZbeta). Interestingly R-Ras, a small GTPase of the Ras family that regulates cell survival and integrin activity, was associated with endosomes in a ligand-dependent manner. EGF-dependent association of R-Ras with late endosomes was confirmed by confocal laser scanning immunofluorescence microscopy and Western blotting of endosomal fractions. EGFR tyrosine kinase inhibitor gefitinib was used to confirm EGF-dependent regulation of all identified proteins. EGF-dependent association of signaling molecules, such as R-Ras, with late endosomes suggests signaling specification through intracellular organelles.
Highlights
Epidermal growth factor (EGF) receptor (EGFR) signal transduction is organized by scaffold and adaptor proteins, which have specific subcellular distribution
Serum-starved cells were treated with EGF (100 ng/ml) for the indicated time intervals (Fig. 1), and total cell lysates were analyzed by Western blotting
New EGF-regulated Proteins on Endosomes Revealed by DIGE— we looked at proteins that were present either in higher or lower amounts on purified endosomal membranes after 40 min of EGF treatment
Summary
Cell Culture—EpH4 mouse mammary epithelial cells [15] were cultured in high glucose Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum, 100 units/ml penicillin, 100 units/ml streptomycin, 10 mM HEPES, pH 7.3. After scanning selected gels were fixed and poststained with Pro-Q Diamond phosphospecific fluorescent dye (Molecular Probes). The images of the same gel scanned before and after poststaining were compared using DeCyder BVA software (Amersham Biosciences). Cells were incubated at room temperature with primary anti-mouse CD107a (LAMP1) rat monoclonal antibody (Pharmingen) diluted 1:300 for 1 h following PBS washing and for 30 min with secondary antibody (goat anti-rat IgG AlexaFluor 594, Molecular Probes, 1:1000) following PBS washing. Total cell lysates were analyzed by Western blotting using specific antibodies against phosphorylated ERK1/2 (p-ERK1/2; A, top panel), stripped, and reprobed with anti-ERK1/2 antibodies to verify equal protein loading (A, bottom panel). Confocal laser scanning fluorescence microscopy (LSM 510 META, Carl Zeiss GmbH) using Zeiss LSM Software, version 3.2
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
