Phosphorylated EGFR Dimers Are Not Sufficient to Activate Ras

Liang, Samantha I.; Lengerich, Bettina van; Eichel, Kelsie; Cha, Minkwon; Patterson, David M.; Yoon, Tae–Young; Zastrow, Mark von; Jura, Natalia; Gartner, Zev J.

doi:10.1016/j.celrep.2018.02.031

Cited by 66 publications

(54 citation statements)

References 38 publications

(39 reference statements)

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“…Mutations at the EGFR asymmetric kinase dimer interface alter the ensemble of ligand-independent EGFR oligomers but do not significantly alter their stability, implying that ligand-independent EGFR oligomers are distinct from the active state dimer. We also show that although levels of phosphorylated EGFR increase with increasing EGFR concentrations, this phosphorylation does not lead to increased phosphorylation of either Erk1/2 or STAT1, indicating that receptor phosphorylation is not sufficient to trigger pathway activation as recently observed for artificially forced EGFR dimers (53,54).…”

Section: Discussionsupporting

confidence: 58%

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Ligand-independent EGFR oligomers do not rely on the active state asymmetric kinase dimer

Byrne

Hristova²,

Leahy

2020

Preprint

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The human Epidermal Growth Factor Receptor (EGFR/ERBB1) is a Receptor Tyrosine Kinase (RTK) that forms active oligomers in response to ligand. Much evidence indicates that EGFR/ERBB1 forms oligomers in the absence of ligand, but the structure and physiological role of these ligandindependent dimers remain unclear. We use fluorescence microscopy to measure the oligomer stability and FRET efficiency for homo-and hetero-oligomers of fluorescent-protein labeled forms of EGFR and its paralog, Human Epidermal Growth Factor Receptor 2 (HER2/ERBB2) in vesicles derived from native cell membranes. Both receptors form ligand-independent oligomers at physiological plasma membrane concentrations. Mutations introduced in the EGFR kinase region at a key interface within the active state dimer alter the FRET efficiency within ligand-independent EGFR oligomers but do not affect their stability. These results indicate that ligand-independent EGFR oligomers do not require this interface and that the inactive state ensemble is distinct from the EGFR active state ensemble.

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Section: Discussionsupporting

confidence: 58%

“…The presence of a substantial fraction of phosphorylated oligomeric EGFR in the absence of ligand (24,53,58) (Fig. 4) raises the question of whether EGFR is sampling the active dimer state, which involves a specific asymmetric dimer interaction between intracellular kinase domains (37).…”

Section: Discussionmentioning

confidence: 99%

Ligand-independent EGFR oligomers do not rely on the active state asymmetric kinase dimer

Byrne

Hristova²,

Leahy

2020

Preprint

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“…Further, Her2-EGFR dimerization, driven by overexpression of one or both proteins, has been shown to lead to a more aggressive breast cancer phenotype 20 . Notably, new evidence supports the hypothesis that not only the formation of dimers but also of higher-order receptor assemblies at cell membrane regulates Her2 function [21][22][23][24][25] . Although there are well established correlations between the levels of Her2 homo-and heterodimers and cancer aggressiveness, the roles of the composition of Her2 protein nanoenvironments for downstream signalling are poorly understood 19 .…”

supporting

confidence: 56%

A DNA nanoassembly-based approach to map membrane protein nanoenvironments

Ambrosetti

Bernardinelli

Hoffecker

et al. 2019

Preprint

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Super-resolution imaging has revealed that most proteins at the plasma membrane are not uniformly distributed but localize to dynamic domains of nanoscale dimensions. To investigate their functional relevance, there is a need for methods that enable comprehensive mapping of the compositions and spatial organizations of membrane protein nanodomains in cell populations. However, current superresolution methods are limited to analysing small, preselected subsets of proteins, at very low sampling fractions. Here we describe the development of a non-microscopy based super-resolution method for unbiased ensemble analysis of membrane protein nanodomains. The method, termed NANOscale DEciphEring of membrane Protein nanodomains (NanoDeep), is based on the use of DNA nanoassemblies to translate membrane protein organization information into a DNA sequencing readout. Using NanoDeep, we characterized the nanoenvironments of Her2, a membrane receptor of critical relevance in cancer. We found that the occupancies of Her2, Her3 and EGFR in the nanoenvironments surrounding Her2 were similar in two cell lines with vastly different expression levels of Her2. Further, we found that adding Heregulin-β1 to cancer cells led to increased occupancy of Her2 and Her3, and to a lesser extent EGFR, in Her2 nanoenvironments. NanoDeep has the potential to provide new insights into the roles of the composition and spatial organization of protein nanoenvironments in the regulation of membrane protein function. MainCells sense extracellular signals, such as protein ligands, through specialized proteins present on the cell surface called membrane receptors. The protein nanoenvironment, i.e. the composition and spatial organization of proteins surrounding membrane receptors, is dynamic and often modulated by ligand binding, suggesting that it has functional relevance 1, 2 . Super-resolution microscopy has enabled the characterization of the nanoscale spatial distributions of proteins at the cell membrane but is limited to simultaneously analysing only a few proteins. Further, using super-resolution microscopy, it is only feasible to image a small fraction of all protein nanodomains present in a cell population 3-5 . DNA detection as a proxy for protein detection through the use of oligo-conjugated affinity binders has been used extensively for signal amplification 6 and analysis of proximity between pairs of proteins 7, 8 . Further, DNA sequencing is used as a readout in DNA microscopy, a new method to visualize the spatial organization of RNA and DNA molecules inside cells 9-11 . Here we present NanoDeep, a method that uses DNA sequencing to decipher the nanoscale spatial distribution of membrane proteins. This method allows for the detection en masse of the inventory of proteins that forms the nanoenvironment of any reference membrane protein in cell populations. We demonstrate the application of NanoDeep to the analysis of protein nanoenviroments surrounding Human Epidermal Growth Factor Receptor 2 (Her2). Her2 cooperates with members of the Ep...

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“…Upon EGF (epidermal growth factor) treatment, PKM2 formed ac omplex with HDAC3t or egulate histone H3 acetylation; without EGF,the HDAC3-PKM2 association was completely abolished (Figure 7b). [26a,b] Since the cells used here were not EGF-stimulated, we hypothesized that PKM2 might bind to other HDACsorbind to HDAC3without the need for EGFstimulation in HeLa cells.T oinvestigate this,wefirst verified the status of EGFR signalling pathway.Serum-starved HeLa cells,either non-stimulated or EGF-stimulated, [27] were lysed, and then three protein phosphorylation markers in the EGFR pathway:E GFR/Y1068, Akt/S473, and ERK/T202/Y204, [28] were analysed with the respective assays (Abcam;E GFR: ab12638;A kt:a b253299;E RK:a b176660), which measured and compared the quantities of total protein and the phosphorylated protein. As shown in Figure S10, non-stimulated HeLa cells exhibited very low phosphorylation level for all three phosphorylation markers,w hile the total protein expression remained largely unaffected upon stimulation.…”

Section: Methodsmentioning

confidence: 99%

Identification of Histone deacetylase (HDAC)‐Associated Proteins with DNA‐Programmed Affinity Labeling

et al. 2020

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Histone deacetylase (HDAC) is a major class of deacetylation enzymes. Many HDACs exist in large protein complexes in cells and their functions strongly depend on the complex composition. The identification of HDAC‐associated proteins is highly important in understanding their molecular mechanisms. Although affinity probes have been developed to study HDACs, they were mostly targeting the direct binder HDAC, while other proteins in the complex remain underexplored. We report a DNA‐based affinity labeling method capable of presenting different probe configurations without the need for preparing multiple probes. Using one binding probe, 9 probe configurations were created to profile HDAC complexes. Notably, this method identified indirect HDAC binders that may be inaccessible to traditional affinity probes, and it also revealed new biological implications for HDAC‐associated proteins. This study provided a simple and broadly applicable method for characterizing protein‐protein interactions.

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Phosphorylated EGFR Dimers Are Not Sufficient to Activate Ras

Cited by 66 publications

References 38 publications

Ligand-independent EGFR oligomers do not rely on the active state asymmetric kinase dimer

Ligand-independent EGFR oligomers do not rely on the active state asymmetric kinase dimer

A DNA nanoassembly-based approach to map membrane protein nanoenvironments

Identification of Histone deacetylase (HDAC)‐Associated Proteins with DNA‐Programmed Affinity Labeling

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