Conservation of protein abundance patterns reveals the regulatory architecture of the EGFR-MAPK pathway

Abstract

Various genetic mutations associated with cancer are known to alter cell signaling, but it is not clear whether they dysregulate signaling pathways by altering the abundance of pathway proteins. Using a combination of RNA sequencing and ultrasensitive targeted proteomics, we defined the primary components—16 core proteins and 10 feedback regulators—of the epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) pathway in normal human mammary epithelial cells and then quantified their absolute abundance across a panel of normal and breast cancer cell lines as well as fibroblasts. We found that core pathway proteins were present at very similar concentrations across all cell types, with a variance similar to that of proteins previously shown to display conserved abundances across species. In contrast, EGFR and transcriptionally controlled feedback regulators were present at highly variable concentrations. The absolute abundance of most core proteins was between 50,000 and 70,000 copies per cell, but the adaptors SOS1, SOS2, and GAB1 were found at far lower amounts (2000 to 5000 copies per cell). MAPK signaling showed saturation in all cells between 3000 and 10,000 occupied EGFRs, consistent with the idea that adaptors limit signaling. Our results suggest that the relative stoichiometry of core MAPK pathway proteins is very similar across different cell types, with cell-specific differences mostly restricted to variable amounts of feedback regulators and receptors. The low abundance of adaptors relative to EGFR could be responsible for previous observations that only a fraction of total cell surface EGFR is capable of rapid endocytosis, high-affinity binding, and mitogenic signaling.

EGFR Ligands Differentially Stabilize Receptor Dimers to Specify Signaling Kinetics

“…The receptor density range below which epiregulin or epigen induce less FRET than EGF (∼500 receptors per μm 2 ) in Figures 5B and C corresponds to a local EGFR concentration of ∼80 μM, or ∼200,000 receptors/cell. Most responsive EGFR-expressing cells have substantially lower numbers of receptors than this (Shi et al, 2016), suggesting that the dimerization affinity differences we observe are physiologically relevant.…”

“…Remarkably, however, we found exactly the opposite (Figure 6). We performed these studies in MCF-7 breast adenocarcinoma cells, which express only a few thousand EGFR molecules per cell (Shi et al, 2016), so that signaling differences arising from reduced receptor dimerization strength would be most evident. As shown in Figure 6A, tyrosine autophosphorylation of EGFR was substantially more sustained following activation with epiregulin or epigen than with EGF.…”

Facile carrier-assisted targeted mass spectrometric approach for proteomic analysis of low numbers of mammalian cells

“…We chose to target EGFR pathway proteins due to the availability of heavy peptide internal standards with established SRM assays (Supplementary Data 4) and established protein copies per cell from our recent study (Supplementary Data 6) 37 . Another reason we chose this pathway is because EGFR pathway is one of the most important signaling pathways in cancer 38,39 .…”

Analysis of K-Ras Interactions by Biotin Ligase Tagging

“…Interestingly, a significant number of cell juncture/adhesion proteins were biotinylated, including mucin 13; integrins β1, β5, and α6, CD44, catenin δ, desmoglein, the coxsackie virus and adenovirus receptor, and the 4F2 cell-surface antigen heavy chain ( Table I). All of these have been identified as potential cancer biomarkers (79)(80)(81)(82)(83)92), but their specific interactions with K-Ras and contributions to the progression of pancreatic cancers have yet to be thoroughly elucidated. We anticipate that analysis of these and other K-Ras interactions in pancreatic cancer cells, and in other cancers will be worthy of further scrutiny.…”