Article

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

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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.

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... 4(a) and S5(A) for SFM control]. HMECs have been shown to express approximately 1 Â 10 5 EGFR per cell in HMECs; 19 therefore, for our cell density of 1000 cells/device, the expected number of EGFR copies per device is approximately 10 8 receptors. At a flow rate of (c) Linear dosing confirmed by fluorescently tagged dextran (left), heatmap of single cell ERK activity for cells (middle), and average ERK activity (right) for linear ramps of SFM!SFM and SFM!2 ng/ml EGF over 24 h. ...
... As a test of this potential mechanism, we repeated our experimental paradigm using HaCaT keratinocytes, which express approximately 8.6 Â 10 5 EGFR per cell 34 compared to 1 Â 10 5 EGFR per cell in HMECs. 19 At this much higher 4. (a) ERK activity patterns in response to a slow ramp of EGF from 0 to 2 ng/ml over 24 h followed by a step increase to 3 ng/ml. Shown are dosing confirmed by fluorescently tagged dextran (left), heatmap of single-cell ERK activity for cells (middle), and average ERK activity (right). ...
... Human mammary epithelial cells (HMECs 184a1, a gift from H. Steven Wiley, Pacific Northwest National Laboratory) were selected for use in this study. A key feature of this cell line is that while they do produce some EGF-family ligands, they do not produce autocrine EGF 19 and express an order of magnitude more EGFR compared to HER2 and HER3. 20 These features result in a simpler cellular system to study EGF signaling than many cell lines. ...
Article
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Utilizing microfluidics to mimic the dynamic temporal changes of growth factor and cytokine concentrations in vivo has greatly increased our understanding of how signal transduction pathways are structured to encode extracellular stimuli. To date, these devices have focused on delivering pulses of varying frequency, and there are limited cell culture models for delivering slowly increasing concentrations of stimuli that cells may experience in vivo. To examine this setting, we developed and validated a microfluidic device that can deliver increasing concentrations of growth factor over periods ranging from 6 to 24 h. Using this device and a fluorescent biosensor of extracellular-regulated kinase (ERK) activity, we delivered a slowly increasing concentration of epidermal growth factor (EGF) to human mammary epithelial cells and surprisingly observed minimal ERK activation, even at concentrations that stimulate robust activity in bolus delivery. The cells remained unresponsive to subsequent challenges with EGF, and immunocytochemistry suggested that the loss of an epidermal growth factor receptor was responsible. Cells were then challenged with faster rates of change of EGF, revealing an increased ERK activity as a function of rate of change. Specifically, both the fraction of cells that responded and the length of ERK activation time increased with the rate of change. This microfluidic device fills a gap in the current repertoire of in vitro microfluidic devices and demonstrates that slower, more physiological changes in growth factor presentation can reveal new regulatory mechanisms for how signal transduction pathways encode changes in the extracellular growth factor milieu.
... The challenge in this context is to acquire a large number of system-specific biochemical parameters. This task has become more amenable as the field progresses and quantitative approaches have been delivering more and more estimates: protein abundances, biophysical parameters, enzymatic rates [96,[109][110][111]. ...
... In order to observe a situation described by the deterministic equations in Eq. (110), the mutant population needs to grow to a sizeable fraction of the population. However, every mutant appears first in only one individual and undergoes a subsequent random walk of reproduction and death, which means that the most likely fate is for it go immediately extinct. ...
... whose deterministic part reproduces Eq. (110) in the absence of mutations. ...
Article
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The adaptive immune system is a dynamical, self-organized multiscale system that protects vertebrates from both pathogens and internal irregularities, such as tumors. For these reasons it fascinates physicists, yet the multitude of different cells, molecules and sub-systems is often also petrifying. Despite this complexity, as experiments on different scales of the adaptive immune system become more quantitative, many physicists have made both theoretical and experimental contributions that help predict the behavior of ensembles of cells and molecules that participate in an immune response. Here we review some recent contributions with an emphasis on quantitative questions and methodologies. We also provide a more general methods section that presents some of the wide array of theoretical tools used in the field.
... On the basis of these results, we hypothesized that cell types naturally expressing different levels of these adaptor proteins would display different phosphorylation patterns. Protein copy numbers have been assayed using both global and targeted mass spectrometrybased proteomics in various cell lines (Kulak et al., 2014;Shi et al., 2016). These estimates include the protein copy numbers (per cell) for EGFR, Grb2, and Shc1 in the nontumorigenic mammary epithelial HMEC and MCF10A cells, as well as in HeLa cervical cancer cells (see Supplemental Table S1). ...
... However, these rate constants are potentially cell specific. Phosphatase abundances are known to vary across cell type (Shi et al., 2016), giving rise to the possibility that effective dephosphorylation rates may be cell specific. Furthermore, membrane properties are known to vary across cell type (Lajoie et al., 2007), with potential impacts on EGFR localization and dimerization, which could affect autophosphorylation. ...
... Our results provide novel insight into the regulation of EGFR phosphorylation, demonstrating critical roles for adaptor protein abundance and receptor dimerization lifetimes. The abundances of adaptor proteins vary across cell types (Shi et al., 2016) and it has been proposed that these variations explain cellspecific functions of EGFR and other receptor tyrosine kinases (RTKs). Here, we have seen that there is an interplay between singlemolecule patterns of phosphorylation and adaptor protein abundances. ...
Article
Differential EGFR phosphorylation is thought to couple receptor activation to distinct signaling pathways. However, the molecular mechanisms responsible for biased signaling are unresolved due to a lack of insight into the phosphorylation patterns of full-length EGFR. We extended a single molecule pull-down (SiMPull) technique previously used to study protein-protein interactions to allow for robust measurement of receptor phosphorylation. We found that EGFR is predominantly phosphorylated at multiple sites, yet phosphorylation at specific tyrosines is variable and only a subset of receptors share phosphorylation at the same site, even with saturating ligand concentrations. We found distinct populations of receptors as soon as one min after ligand stimulation, indicating early diversification of function. To understand this heterogeneity, we developed a mathematical model. The model predicted that variations in phosphorylation are dependent on the abundances of signaling partners, while phosphorylation levels are dependent on dimer lifetimes. The predictions were confirmed in studies of cell lines with different expression levels of signaling partners, and in experiments comparing low- and high-affinity ligands and oncogenic EGFR mutants. These results reveal how ligand-regulated receptor dimerization dynamics and adaptor protein concentrations play critical roles in EGFR signaling.
... To establish the concentrations and modification states of these proteins in response to MAPK activity, we performed quantitative targeted mass spectrometry on A375 cells treated for 24 hr with four doses of vemurafenib (from 0.01 to 1 µM), yielding absolute abundances for 21 proteins across a range of MAPK activities ( Figure 3B). Phosphorylation stoichiometry at key regulatory sites was also measured by mass spectrometry and mRNA transcript levels by RNA-seq ( Figure 3C-E); see also (Shi et al., 2016). ...
... Targeted quantification of protein abundances and phosphorylation was performed as previously described (Shi et al., 2016). Briefly, cell pellets from A375 cell lines treated with different doses of vemurafenib were lysed in 100 μL of lysis buffer containing 8 M urea in 100 mM NH4HCO3 (pH 7.8). ...
... For protein abundance quantification (Shi et al., 2016) (Yi et al., 2018), crude heavy-isotope labeled synthetic peptides were purchased from Thermo Scientific and the two best response peptides were selected to configure final selected reaction monitoring (SRM) assays for each target protein. All samples were measured by regular LC-SRM using the scheduled SRM algorithm (Shi et al., 2017) for simultaneous quantification of the selected target proteins. ...
Preprint
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Anti-cancer drugs commonly target signal transduction proteins activated by mutation. In patients with BRAFV600E melanoma, small molecule RAF and MEK kinase inhibitors cause dramatic but often transient tumor regression. Emerging evidence suggests that cancer cells adapting by non-genetic mechanisms constitute a reservoir for the development of drug-resistant tumors. Here, we show that few hours after exposure to RAF/MEK inhibitors, BRAFV600E melanomas undergo adaptive changes involving disruption of negative feedback and sporadic pulsatile reactivation of the MAPK pathway, so that MAPK activity is transiently high enough in some cells to drive proliferation. Quantitative proteomics and computational modeling show that pulsatile MAPK reactivation is possible due to the co-existence in cells of two MAPK cascades: one driven by BRAFV600E that is drug-sensitive and a second driven by receptors that is drug-resistant. Paradoxically, this may account both for the frequent emergence of drug resistance and for the tolerability of RAF/MEK therapy in patients.
... To monitor the intensity of multiple fragment ions in parallel, PRM uses MS 2-D ata with high-resolution MS 2 full scans, whereas SRM utilizes only fragment ion monitoring, for quantification PRM also uses MS 3 scan which can be highly accurate but is highly selective and may have reduced sensitivity. For qualitative proteome analysis, targeted LC-MS using MS 3 is employed where a neutral loss ion is fragmented to identify a phosphopeptide or to discern two highly similar peptides (Shi et al. 2016). The relative quantification of proteins in multiple biological samples uses the 'label-free' approach where the analytes are not labeled using isotopes and LC-MS of the sample is performed separately with the same parameters. ...
... The recombinant expression of a quantification concatemer (QconCAT) is another novel method used to prepare peptides and intact purified protein standards which are used in protein standard absolute quantification (PSAQ) workflows (Manes and Nita-Lazar 2018). Signaling pathways have been studied using quantitative LC-MS like the EGFR-MAPK pathway between a variety of normal and cancerous human cell types; a combination of targeted proteomics, phosphoproteomics along with transcriptomic data were used (Shi et al. 2016). Investigation of docetaxel inhibition of prostate cancer cell division and involvement of a network of proteins associated with this have been done showing proteome-wide capability of targeted LC-MS technology (Lescarbeau and Kaplan 2014). ...
Article
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Cancer is a disease with second largest fatalities in humans and the sheer complexity of the disease is an enigma to the scientific community. It continues to be a highly significant research area for the development of diagnostics and therapeutics. While a great deal of focus has been on genomics study of cancer, the fact that cancer tissues have altered protein expression and unique protein–protein interactions demands more attention. The role of proteins and peptides which are unique and aberrant in cancer tissues has generated a great deal of interest in cancer proteome and serve as important diagnostic and therapeutic targets in the form of biomarkers. Oncoproteomics along with computational and genomic advances is utilized for the discovery of biomarkers and modern therapeutics indispensable in cancer management and care; the focus areas in cancer research involving proteomic studies, technologies used and their advances, identification and quantification of proteins with greater sensitivity and resolution, and detection of post-translational modifications. In this review, we have discussed an overview of various technological advances in oncoproteomics and the therapeutic milestones achieved using the oncoproteome data leading to antibody engineering, peptidomimetics, bispecific antibodies, and other techniques.
... T cells have near singlemolecule antigen-sensing capabilities (54)(55)(56)(57) and can activate with only few triggered TCRs. In contrast, EGFR stimulation often involves thousands or even millions of activated receptors per cell (58)(59)(60). Among molecules in the EGFR-MAPK pathway, SOS is expressed at very low levels and is likely a limiting component (60). ...
... In contrast, EGFR stimulation often involves thousands or even millions of activated receptors per cell (58)(59)(60). Among molecules in the EGFR-MAPK pathway, SOS is expressed at very low levels and is likely a limiting component (60). Thus, unlike the LAT:Grb2:SOS condensate, an EGFR:Grb2:SOS condensate would seem implausible if SOS was a key crosslinking element that needed to be present in stoichiometric ratios with EGFR. ...
Article
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Significance Two-dimensional condensates of proteins on the membrane surface, driven by tyrosine phosphorylation, are beginning to emerge as important players in signal transduction. This work describes discovery of a protein condensation phase transition of EGFR and Grb2 on membrane surfaces, which is poised to have a significant impact on how we understand EGFR signaling and misregulation in disease. EGFR condensation is mediated through a Grb2-Grb2 crosslinking element, which itself is regulatable through a specific phosphotyrosine site on Grb2. Furthermore, the EGFR condensate exerts significant control over the ability of SOS to activate Ras, thus implicating the EGFR condensate as a regulator of signal propagation from EGFR to Ras and the MAPK pathway.
... T cells have near single molecule antigen sensing capabilities (50)(51)(52)(53), and can activate with only few triggered TCRs. In contrast, EGFR stimulation often involves thousands or even millions of activated receptors per cell (54)(55)(56). Among molecules in the EGFR-MAPK pathway, SOS is expressed at very low levels and is likely a limiting component (56). ...
... In contrast, EGFR stimulation often involves thousands or even millions of activated receptors per cell (54)(55)(56). Among molecules in the EGFR-MAPK pathway, SOS is expressed at very low levels and is likely a limiting component (56). Thus unlike the LAT:Grb2:SOS condensate, an EGFR:Grb2:SOS condensate would seem implausible if SOS was a key crosslinking element that needed to be present in stoichiometric ratios with EGFR. ...
Preprint
We reconstitute a phosphotyrosine-mediated protein condensation phase transition of the ~200 residue cytoplasmic tail of the epidermal growth factor receptor (EGFR) and the adaptor protein, Grb2, on a membrane surface. The phase transition depends on phosphorylation of the EGFR tail, which recruits Grb2, and the dimerization of Grb2, which provides the crosslinking element for condensation with EGFR. The Grb2 Y160 residue plays a structurally critical role in dimer formation, and phosphorylation or mutation of Y160 prevents EGFR:Grb2 condensation. By extending the reconstitution experiment to include the guanine nucleotide exchange factor, SOS, and its substrate Ras, we further find that EGFR condensation controls the ability of SOS to activate Ras. These results identify an EGFR:Grb2 protein condensation phase transition as a regulator of signal propagation from EGFR to the MAPK pathway.
... Quantitative understanding of this effect is critical for relating bulk biochemical data (an average over essentially infinite copy number) and single-molecule measurements to functional kinetics in cellular systems. Moreover, the relatively low protein copy number per cell of SOS in the MAPK pathway (26) implicates SOS as a kinetic bottleneck and suggests that timing and delays at this step may propagate downstream in the signaling cascade. The processivity of SOS and the resulting saturation behavior in the activation of Ras likely reflect common phenomena in cellular signal transduction. ...
... Slightly counterintuitive for a signaling mechanism that relies on rare events, this sensitivity is relatively robust to protein expression variation since activation only depends on early activators and not the entire average. In contrast, activation based on the population average is relatively prone to crippling by variations in protein expression [the CV 2 of SOS copy number in cells is about 50% (26)]. ...
Article
Significance Timing in cellular signal transduction is generally observed as a functional property of an ensemble, but it is fundamentally governed by the reaction kinetics of individual signaling molecules. Here, we combined stochastic modeling and reconstitution experiments to show that the functional timescale of Ras activation by multiple Son of Sevenless (SOS) molecules is much shorter than the average activation time of individual SOS molecules at the membrane. These results illustrate how common mechanistic features of cellular signaling reactions can establish a complex relationship between individual molecular kinetics and the apparent functional kinetics of the system. They also underscore the importance of physiological protein copy numbers in establishing the functional output from a signaling module.
... Finally, the enzymatic digestion for shotgun proteomics also presents an issue in terms of sequence coverage since mutation containing peptides based on enzymatic cleavage patterns are sometimes simply too short or too long to be identifiable. In contrast, MS-based targeted proteomics has the potential for precise broad targeted discovery of functionally important protein mutations because it selects the predefined targeted peptides without bias selection, and it has higher detection sensitivity, quantitation accuracy (accurate or absolute quantification), and reproducibility (≤10% CV) (Shi, Song, et al., 2016;Shi, Su, et al., 2012;Shi, Niepel, et al., 2016) with the same level of detection specificity (easy to distinguish SAAVs) as global proteomics (Lesur et al., 2015;Lichti et al., 2015;Tan et al., 2020;Wang et al., 2011) ( Figure 1). Furthermore, the shortcoming of targeted proteomics, lower multiplexing for target analytes, has recently been addressed effectively with enabling simultaneous quantification of~1000 analytes in a single analysis (Stopfer et al., 2021). ...
... PRISM-SRM allows for accurate and reproducible quantification of plasma proteins at the 50-100 pg/ml range in human blood with IgY14 immunoaffinity depletion (Shi, Fillmore, et al., 2012) and the sub ng/ml to low ng/ ml range without immunoaffinity depletion . When applied to human cell lines it can reliably quantify proteins at 10-100 copies per cell (Shi, Niepel, et al., 2016). Such sensitivity is comparable to or even better than most analytically validated ELISAs (Shi, Fillmore, et al., 2012;Shi et al., 2014). ...
Article
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Cancers are caused by accumulated DNA mutations. This recognition of the central role of mutations in cancer and recent advances in next‐generation sequencing, has initiated the massive screening of clinical samples and the identification of 1000s of cancer‐associated gene mutations. However, proteomic analysis of the expressed mutation products lags far behind genomic (transcriptomic) analysis. With comprehensive global proteomics analysis, only a small percentage of single nucleotide variants detected by DNA and RNA sequencing have been observed as single amino acid variants due to current technical limitations. Proteomic analysis of mutations is important with the potential to advance cancer biomarker development and the discovery of new therapeutic targets for more effective disease treatment. Targeted proteomics using selected reaction monitoring (also known as multiple reaction monitoring) and parallel reaction monitoring, has emerged as a powerful tool with significant advantages over global proteomics for analysis of protein mutations in terms of detection sensitivity, quantitation accuracy and overall practicality (e.g., reliable identification and the scale of quantification). Herein we review recent advances in the targeted proteomics technology for enhancing detection sensitivity and multiplexing capability and highlight its broad biomedical applications for analysis of protein mutations in human bodily fluids, tissues, and cell lines. Furthermore, we review recent applications of top‐down proteomics for analysis of protein mutations. Unlike the commonly used bottom‐up proteomics which requires digestion of proteins into peptides, top‐down proteomics directly analyzes intact proteins for more precise characterization of mutation isoforms. Finally, general perspectives on the potential of achieving both high sensitivity and high sample throughput for large‐scale targeted detection and quantification of important protein mutations are discussed.
... The aim of the study was to quantify the different protein species involved in the Erk cascade. The analysis revealed that most of these proteins have a similar level of expression between different cell lines (Shi 2016). Through approaches like this, proteomics reinforces the reliability of models applied to different cell lines. ...
... MAPK signaling showed saturation in all cells between 3000 and 10,000 occupied EGFRs, consistent with the idea that adaptors limit signalling. Shi (2016) shows how 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. ...
Thesis
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Systems biology allows analytical investigation of intracellular dynamics, analyzing complex processes and taking into account the interactions among the various subsystems. In this study, biochemical models describing the behavior of regulatory molecular networks were created and interfaced with a simulation system able to reproduce motility and proliferation of eukaryotic cell cultures. The primary focus was on MAPK cascades, particularly Erk1/2 activation by growth factors and mitogens such as EGF through tyrosine kinase receptors (RTKs) as Egfr, which represent a fundamental signal transduction and regulatory network affecting many cellular processes, including proliferation, motility, differentiation and survival. Erk1/2 predicted levels were related to reactions representing the progression of the cell cycle and used to modulate cell growth in a cell simulator. The biochemical model was built starting from literature data and a database of estimated protein concentrations representative of different cell types and experimental conditions and may be run for prolonged time frames and in various experimental conditions, including a vast array of cell lines. A software tool developed on purpose is able to run the model and interface with the cell simulator.
... Targeted quantification of protein abundances and phosphorylation was performed as previously described (Shi et al., 2016). Briefly, cell pellets from A375 cell lines treated with different doses of vemurafenib were lysed in 100 mL of lysis buffer containing 8 M urea in 100 mM NH4HCO3 (pH 7.8). ...
... For protein abundance quantification (Shi et al., 2016)(Yi et al., 2018, crude heavy-isotope labeled synthetic peptides were purchased from Thermo Scientific and the two best response peptides were selected to configure final selected reaction monitoring (SRM) assays for each target protein. All samples were measured by regular LC-SRM using the scheduled SRM algorithm (Shi et al., 2017) for simultaneous quantification of the selected target proteins. ...
Article
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Targeted inhibition of oncogenic pathways can be highly effective in halting the rapid growth of tumors but often leads to the emergence of slowly dividing persister cells, which constitute a reservoir for the selection of drug-resistant clones. In BRAFV600E melanomas, RAF and MEK inhibitors efficiently block oncogenic signaling, but persister cells emerge. Here, we show that persister cells escape drug-induced cell-cycle arrest via brief, sporadic ERK pulses generated by transmembrane receptors and growth factors operating in an autocrine/paracrine manner. Quantitative proteomics and computational modeling show that ERK pulsing is enabled by rewiring of mitogen-activated protein kinase (MAPK) signaling: from an oncogenic BRAFV600E monomer-driven configuration that is drug sensitive to a receptor-driven configuration that involves Ras-GTP and RAF dimers and is highly resistant to RAF and MEK inhibitors. Altogether, this work shows that pulsatile MAPK activation by factors in the microenvironment generates a persistent population of melanoma cells that rewires MAPK signaling to sustain non-genetic drug resistance.
... For example, the top 50 and bottom 50 genes together identify the first-degree neighbors of targets for 33.5% of CTRP drugs (18.6% and 14.9% in the top and the bottom 50 genes, respectively). Take an EGFR inhibitor lapatinib for example, the top 10 genes from the gene essentiality signature not only identified the primary targets, namely ERBB1(EGFR) and ERBB2 (HER2), but also revealed several important downstream genes in the EGFR signaling pathway, including ERBB3 27 , GAB1 28,29 , and SOS1 29 ( Figure 4D). This showed the gene essentiality signature's ability to reveal both binding targets and downstream changes upon initial target gene inhibition. ...
Preprint
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Background Cancer drugs often kill cells independent of their putative targets, suggesting the limitation of existing drug target information. The lack of understanding of a drug’s mechanism of action may prevent biomarker identification and ultimately lead to attrition in clinical trials. Current experimental strategies, such as binding affinity assays provide limited coverage at the proteome scale. In this study, we explored whether the integration of loss-of-function genetic and drug sensitivity screening data could define a novel signature to better understand the mechanisms of action of drugs. Methods Loss-of-function genetic screening data was collected from the DepMap database, while drug sensitivity data were collected from three extensive screening studies, namely CTRP (n = 545), GDSC (n = 198), and PRISM (n = 1448). An L1 penalized regression model using the gene essentiality features was constructed for each drug to predict its sensitivity on multiple cell lines. The optimized model coefficients were then considered as the gene essentiality signature of the drug. We compared the gene essentiality signature with structure-based fingerprints and the gene expression signature of cancer drugs in predictions of their known targets. Finally, we applied the gene essentiality signature to predict the novel targets for a panel of noncancer drugs with potential anticancer efficacy. Results We showed that the gene essentiality signature can predict drug targets and their downstream signaling pathways. Both supervised and unsupervised prediction accuracies were higher than those using chemical fingerprints and gene expression signatures. Pathway analyses of these gene essentiality signatures confirmed key mechanisms previously reported, including the EGFR signaling network for lapatinib, and DNA mismatch repair drugs. Finally, we showed that the gene essentiality signature of noncancer drugs can discover novel targets. Conclusions Integrating drug sensitivity data and loss-of-function genetic data enables the construction of gene essentiality signatures that help discover drug targets and their downstream signaling pathways. We found novel targets for noncancer drugs that explain their anticancer efficacy, paving the way for the rational design of drug repurposing.
... Note that many of the proteins involved in membrane translocation, and their membrane-bound targets, are in fact scarce. For example, in several mammalian cell lines, Sos is ~5 nM, Ras is ~130 nM, and Raf-1 is ~20 nM (33). ...
Preprint
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Translocation of cytoplasmic molecules to the plasma membrane is commonplace in cell signaling. Membrane localization has been hypothesized to increase intermolecular association rates; however, it has also been argued that association should be faster in the cytosol because membrane diffusion is slow. Here we directly compare an identical association reaction in solution and on supported membranes. The measured rate constants show that for 10 μm-radius spherical cell, association is 15-25-fold faster at the membrane than in the cytoplasm. The advantage is cell size-dependent, and for typical ~1 μm prokaryotic cells it should be essentially negligible. Rate enhancement is attributable to a combination of closer proximity of the signaling molecule to its targets after translocation and the higher efficiency of a two-dimensional search.
... highly expressed in many cell lines (Shi et al., 2016). This ensures that the Btk signal remains 577 responsive to activation of the B cell receptor. ...
Preprint
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The Tec-family kinase Btk contains a lipid-binding Pleckstrin homology and Tec homology (PH22 TH) module connected by a proline-rich linker to a “Src module”, an SH2-SH3-kinase unit also found in Src-family kinases and Abl. We showed previously that Btk is activated by PH-TH dimerization, which is triggered on membranes by the phosphatidyl inositol phosphate PIP3, or in solution by hexakisinositol phosphate (IP6) (Wang et al. 2015, https://doi.org/10.7554/eLife.06074). We now report that the ubiquitous adaptor protein growth factor-receptor-bound protein 2 (Grb2) binds to and substantially increases the activity of PIP3- bound Btk on membranes. Using reconstitution on supported-lipid bilayers, we find that Grb2 can be recruited to membrane-bound Btk through interaction with the proline-rich linker in Btk. This interaction requires intact Grb2, containing both SH3 domains and the SH2 domain, but does not require that the SH2 domain be able to bind phosphorylated tyrosine residues – thus Grb2 bound to Btk is free to interact with scaffold proteins via the SH2 domain. We show that the Grb2-Btk interaction recruits Btk to scaffold-mediated signaling clusters in reconstituted membranes. Our findings indicate that PIP3-mediated dimerization of Btk does not fully activate Btk, and that Btk adopts an autoinhibited state at the membrane that is released by Grb2.
... pTyr signalling is often dysregulated in cancers and other diseases. For example, EGF receptors can be mutationally activated or present in high copy numbers vis-à-vis their cognate adaptors, and oncogenic mutations frequently map to SH2 domains (Li et al., 2012a;Li et al., 2021;Shi et al., 2016;Sigismund et al., 2018). It therefore remains important to understand the molecular principles of how pTyr-dependent signalling networks function. ...
Article
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Phosphotyrosine (pTyr) motifs in unstructured polypeptides orchestrate important cellular processes by engaging SH2-containing adaptors to assemble complex signalling networks. The concept of phase separation has recently changed our appreciation of multivalent networks, however, the role of pTyr motif positioning in their function remains to be explored. We have now investigated this parameter in the operation of the signalling cascade driving actin-based motility and spread of Vaccinia virus. This network involves two pTyr motifs in the viral protein A36 that recruit the adaptors Nck and Grb2 upstream of N-WASP and Arp2/3 complex-mediated actin polymerization. Manipulating the position of pTyr motifs in A36 and the unrelated p14 from Orthoreovirus, we find that only specific spatial arrangements of Nck and Grb2 binding sites result in robust N-WASP recruitment, Arp2/3 complex driven actin polymerization and viral spread. This suggests that the relative position of pTyr adaptor binding sites is optimised for signal output. This finding may explain why the relative positions of pTyr motifs are frequently conserved in proteins from widely different species. It also has important implications for regulation of physiological networks, including those undergoing phase transitions.
... This limits their usefulness for predicting the impact of drugs or genetic changes, such as copy number variations. Several recent studies, however, have quantified pathway protein abundance as explicit model parameters and have shown that this improves the ability of models to predict the impact of both drugs and altered protein abundance [81]. As the technologies needed to quantify the oftenlow levels of signaling proteins improve, inclusion of their abundance values in models should improve their predictive power. ...
Article
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Purpose of Review Signaling pathways serve to communicate information about extracellular conditions into the cell, to both the nucleus and cytoplasmic processes to control cell responses. Genetic mutations in signaling network components are frequently associated with cancer and can result in cells acquiring an ability to divide and grow uncontrollably. Because signaling pathways play such a significant role in cancer initiation and advancement, their constituent proteins are attractive therapeutic targets. In this review, we discuss how signaling pathway modeling can assist with identifying effective drugs for treating diseases, such as cancer. An achievement that would facilitate the use of such models is their ability to identify controlling biochemical parameters in signaling pathways, such as molecular abundances and chemical reaction rates, because this would help determine effective points of attack by therapeutics. Recent Findings We summarize the current state of understanding the sensitivity of phosphorylation cycles with and without sequestration. We also describe some basic properties of regulatory motifs including feedback and feedforward regulation. Summary Although much recent work has focused on understanding the dynamics and particularly the sensitivity of signaling networks in eukaryotic systems, there is still an urgent need to build more scalable models of signaling networks that can appropriately represent their complexity across different cell types and tumors.
... The AML (MOLM-14, K562 and CMK), MCF-7, A549 and MCF10A breast cell lines were obtained from the American Type Culture Collection and were prepared as previously described 60 . Cell pellets were washed with ice-cold phosphate-buffered saline (PBS), lysed in a lysis buffer containing 50 mM TEABC, pH 8.0, 8 M urea, and a 1% protease and phosphatase inhibitor. ...
Preprint
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Effective phosphoproteome of nanoscale sample analysis remains a daunting task, primarily due to significant sample loss associated with non-specific surface adsorption during enrichment of low stoichiometric phosphopeptide. We developed a novel tandem tip phosphoproteomics sample preparation method that is capable of sample cleanup and enrichment without additional sample transfer, and its integration with our recently developed SOP (Surfactant-assisted One-Pot sample preparation) and iBASIL (improved Boosting to Amplify Signal with Isobaric Labeling) approaches provides a streamlined workflow enabling sensitive, high-throughput nanoscale phosphoproteome measurements. This approach significantly reduces both sample loss and processing time, allowing the identification of >3,000 (>9,500) phosphopeptides from 1 (10) μg of cell lysate using the label-free method without a spectral library. It also enabled precise quantification of ~600 phosphopeptides from 100 cells sorted by FACS (single-cell level input for the enriched phosphopeptides) and ~700 phosphopeptides from human spleen tissue voxels with a spatial resolution of 200 μm (equivalent to ~100 cells) in a high-throughput manner. The new workflow opens avenues for phosphoproteome profiling of mass-limited samples at the low nanogram level.
... To visualize active Ras in EGF-stimulated cells, we tagged RAF1 with mCh by gene editing in cells expressing endogenous mNG-NRAS ( Fig. 9 A). Because RAF1 is a low-abundance protein (Shi et al., 2016), detection of endogenous RAF1-mCh required lengthy image acquisition times and high laser power. This resulted in the detection of substantial vesicular autofluorescence through the 561-nm channel that was highly colocalized with vesicles imaged through the nonspecific 640-nm channel, regardless of EGF stimulation ( Fig. 9 B). ...
Article
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The subcellular localization of RAS GTPases defines the operational compartment of the EGFR-ERK1/2 signaling pathway within cells. Hence, we used live-cell imaging to demonstrate that endogenous KRAS and NRAS tagged with mNeonGreen are predominantly localized to the plasma membrane. NRAS was also present in the Golgi apparatus and a tubular, plasma-membrane derived endorecycling compartment, enriched in recycling endosome markers (TERC). In EGF-stimulated cells, there was essentially no colocalization of either mNeonGreen-KRAS or mNeonGreen-NRAS with endosomal EGFR, which, by contrast, remained associated with endogenous Grb2-mNeonGreen, a receptor adaptor upstream of RAS. ERK1/2 activity was diminished by blocking cell surface EGFR with cetuximab, even after most ligand-bound, Grb2-associated EGFRs were internalized. Endogenous mCherry-tagged RAF1, an effector of RAS, was recruited to the plasma membrane, with subsequent accumulation in mNG-NRAS–containing TERCs. We propose that a small pool of surface EGFRs sustain signaling within the RAS-ERK1/2 pathway and that RAS activation persists in TERCs, whereas endosomal EGFR does not significantly contribute to ERK1/2 activity.
... The MCF10A breast cell line was obtained from the American Type Culture Collection (Manassas, VA) and was grown in culture media, as previously described. 22 Briefly, MCF10A cells were cultured and maintained in 15 cm dishes in ATCCformulated Eagle's minimum essential medium (Thermo Fisher Scientific) supplemented with 0.01 mg/mL human recombinant insulin and a final concentration of 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) with 1% penicillin/streptomycin (Thermo Fisher Scientific). Cells were grown at 37°C in 95% O 2 and 5% CO 2 . ...
Article
Recent advances in sample preparation enable label-free mass spectrometry (MS)-based proteome profiling of small numbers of mammalian cells. However, specific devices are often required to downscale sample processing volume from the standard 50-200 μL to sub-μL for effective nanoproteomics, which greatly impedes the implementation of current nanoproteomics methods by the proteomics research community. Herein, we report a facile one-pot nanoproteomics method termed SOPs-MS (surfactant-assisted one-pot sample processing at the standard volume coupled with MS) for convenient robust proteome profiling of 50-1000 mammalian cells. Building upon our recent development of SOPs-MS for label-free single-cell proteomics at a low μL volume, we have systematically evaluated its processing volume at 10-200 μL using 100 human cells. The processing volume of 50 μL that is in the range of volume for standard proteomics sample preparation has been selected for easy sample handling with a benchtop micropipette. SOPs-MS allows for reliable label-free quantification of ∼1200-2700 protein groups from 50 to 1000 MCF10A cells. When applied to small subpopulations of mouse colon crypt cells, SOPs-MS has revealed protein signatures between distinct subpopulation cells with identification of ∼1500-2500 protein groups for each subpopulation. SOPs-MS may pave the way for routine deep proteome profiling of small numbers of cells and low-input samples.
... A substrate other than IRS may be responsible for the effect; however, IRS is central to other model predictions, and the literature supports its role. IRS is an adaptor protein that is considered one of the bottlenecks of signaling activation [50,51], and recent structural analysis of the two RTKs suggests a differential binding of IRS proteins [34]. Relieving the negative feedback from S6K on IRS was previously shown to sensitize colorectal cancer cells to EGFR inhibition [28]. ...
Article
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Aberrant signaling through insulin (Ins) and insulin-like growth factor I (IGF1) receptors contribute to the risk and advancement of many cancer types by activating cell survival cascades. Similarities between these pathways have thus far prevented the development of pharmacological interventions that specifically target either Ins or IGF1 signaling. To identify differences in early Ins and IGF1 signaling mechanisms, we developed a dual receptor (IGF1R & InsR) computational response model. The model suggested that ribosomal protein S6 kinase (RPS6K) plays a critical role in regulating MAPK and Akt activation levels in response to Ins and IGF1 stimulation. As predicted, perturbing RPS6K kinase activity led to an increased Akt activation with Ins stimulation compared to IGF1 stimulation. Being able to discern differential downstream signaling, we can explore improved anti-IGF1R cancer therapies by eliminating the emergence of compensation mechanisms without disrupting InsR signaling.
... In any case, it is apparent that a definitive quantitation of the steadystate, real intracellular concentration of SOS1 and SOS2 in different biological contexts can be achieved only by accurate mass-spectrometric determination and quantitation of the amounts of specific peptides unique for either SOS1 or SOS2 in each sample analyzed. In this regard, a recent proteomic study performed across different cell types has revealed that the absolute abundance of SOS1 and SOS2 proteins is quite similar [49]. ...
Article
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The SOS family of Ras-GEFs encompasses two highly homologous and widely expressed members, SOS1 and SOS2. Despite their similar structures and expression patterns, early studies of constitutive KO mice showing that SOS1-KO mutants were embryonic lethal while SOS2-KO mice were viable led to initially viewing SOS1 as the main Ras-GEF linking external stimuli to downstream RAS signaling, while obviating the functional significance of SOS2. Subsequently, different genetic and/or pharmacological ablation tools defined more precisely the functional specificity/redundancy of the SOS1/2 GEFs. Interestingly, the defective phenotypes observed in concomitantly ablated SOS1/2-DKO contexts are frequently much stronger than in single SOS1-KO scenarios and undetectable in single SOS2-KO cells, demonstrating functional redundancy between them and suggesting an ancillary role of SOS2 in the absence of SOS1. Preferential SOS1 role was also demonstrated in different RASopathies and tumors. Conversely, specific SOS2 functions, including a critical role in regulation of the RAS–PI3K/AKT signaling axis in keratinocytes and KRAS-driven tumor lines or in control of epidermal stem cell homeostasis, were also reported. Specific SOS2 mutations were also identified in some RASopathies and cancer forms. The relevance/specificity of the newly uncovered functional roles suggests that SOS2 should join SOS1 for consideration as a relevant biomarker/therapy target
... This limits their usefulness for predicting the impact of drugs or genetic changes, such as copy number variations. Several recent studies, however, have quantified pathway protein abundance as explicit model parameters and have shown that this improves the ability of models to predict the impact of both drugs and altered protein abundance [67]. As the technologies needed to quantify the often-low levels of signaling proteins improve, inclusion of their abundance values in models should improve their predictive power. ...
Preprint
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Signaling pathways serve to communicate information about extracellular conditions into the cell, to both the nucleus and cytoplasmic processes to control cell responses. Genetic mutations in signaling network components are frequently associated with cancer and can result in cells acquiring an ability to divide and grow uncontrollably. Because signaling pathways play such a significant role in cancer initiation and advancement, their constituent proteins are attractive therapeutic targets. In this review, we discuss how signaling pathway modeling can assist with identifying effective drugs for treating diseases, such as cancer. An achievement that would facilitate the use of such models is their ability to identify controlling biochemical parameters in signaling pathways, such as molecular abundances and chemical reaction rates, because this would help determine effective points of attack by therapeutics.
... The genes involved in each pathway were obtained from the Kyoto encyclopedia of genes and genomes (KEGG) database (Kanehisa and Goto 2000) and further manually curated to remove overlap between pathways. The genes involved in the MAPK pathway were obtained from Shi et al. (2016). The gene sets are provided in supplementary table S2, Supplementary Material online. ...
Article
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Mechanical properties such as substrate stiffness are a ubiquitous feature of a cell's environment. Many types of animal cells exhibit canonical phenotypic plasticity when grown on substrates of differing stiffness, in vitro and in vivo. Whether such plasticity is a multivariate optimum due to hundreds of millions of years of animal evolution, or instead is a compromise between conflicting selective demands, is unknown. We addressed these questions by means of experimental evolution of populations of mouse fibroblasts propagated for ∼90 cell generations on soft or stiff substrates. The ancestral cells grow twice as fast on stiff substrate as on soft substrate and exhibit the canonical phenotypic plasticity. Soft-selected lines derived from a genetically diverse ancestral population increased growth rate on soft substrate to the ancestral level on stiff substrate and evolved the same multivariate phenotype. The pattern of plasticity in the soft-selected lines was opposite of the ancestral pattern, suggesting that reverse plasticity underlies the observed rapid evolution. Conversely, growth rate and phenotypes did not change in selected lines derived from clonal cells. Overall, our results suggest that the changes were the result of genetic evolution and not phenotypic plasticity per se. Whole-transcriptome analysis revealed consistent differentiation between ancestral and soft-selected populations, and that both emergent phenotypes and gene expression tended to revert in the soft-selected lines. However, the selected populations appear to have achieved the same phenotypic outcome by means of at least two distinct transcriptional architectures related to mechano-transduction and proliferation.
... Are some of these behaviors observed in biological systems? One example where the signaling response becomes maximal when only a small fraction of receptors are bound (ΘY * < ΘX * ) is the EGFR-MAPK pathway, which elicits a full MAPK response at less than 5% receptor occupancy (57). Our analysis explains this by an activation mechanism or a concerted mechanism, in which the activation strength dominates the repression strength. ...
Article
Significance Molecular switches are key components of intracellular signal-transduction pathways. Experimental work has established that the activity of molecular switches is regulated by three distinct mechanisms: 1) activation, 2) derepression, and 3) concerted (activation and derepression). However, it remains unclear how the choice of signaling mechanism influences the performance properties of the switch. Here, we characterize each switch design in terms of dose–response relationship, response time, and abilities to process upstream fluctuations. Our results highlight unique features of each mechanism and provide insight into the operating principles that underlie many different signaling pathways.
... Cell culture. The MCF10A (MCF7) breast cancer cell line was obtained from the American Type Culture Collection (Manassas, VA) and was grown in culture media 63 . Briefly, MCF10A (MCF7) cells were cultured and maintained in 15 cm dishes in ATCC-formulated Eagle's minimum essential medium (Thermo Fisher Scientific) supplemented with 0.01 mg/mL human recombinant insulin and a final concentration of 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) with 1% penicillin/streptomycin (Thermo Fisher Scientific). ...
Article
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Large numbers of cells are generally required for quantitative global proteome profiling due to surface adsorption losses associated with sample processing. Such bulk measurement obscures important cell-to-cell variability (cell heterogeneity) and makes proteomic profiling impossible for rare cell populations (e.g., circulating tumor cells (CTCs)). Here we report a surfactant-assisted one-pot sample preparation coupled with mass spectrometry (MS) method termed SOP-MS for label-free global single-cell proteomics. SOP-MS capitalizes on the combination of a MS-compatible nonionic surfactant, n-Dodecyl-β-D-maltoside, and hydrophobic surface-based low-bind tubes or multi-well plates for ‘all-in-one’ one-pot sample preparation. This ‘all-in-one’ method including elimination of all sample transfer steps maximally reduces surface adsorption losses for effective processing of single cells, thus improving detection sensitivity for single-cell proteomics. This method allows convenient label-free quantification of hundreds of proteins from single human cells and ~1200 proteins from small tissue sections (close to ~20 cells). When applied to a patient CTC-derived xenograft (PCDX) model at the single-cell resolution, SOP-MS can reveal distinct protein signatures between primary tumor cells and early metastatic lung cells, which are related to the selection pressure of anti-tumor immunity during breast cancer metastasis. The approach paves the way for routine, precise, quantitative single-cell proteomics.
... Furthermore, knowing and accounting for the cellular abundances of receptors and their effectors may help to identify rate-limiting steps of signaling pathways and elucidate mechanisms by which endocytosis controls these pathway bottlenecks. Copy numbers of the entire HeLa cell proteome have been quantitated using mass spectrometry (187), and a targeted analysis was performed to quantitate copy numbers of major signaling proteins in multiple human mammary cell lines (188). Studying the signaling and endocytosis of GPCRs and RTKs in mammalian organisms in vivo is the next inevitable, yet feasible, step in this research area. ...
Article
Intricate relationships between endocytosis and cellular signaling, first recognized nearly 40 years ago through the study of tyrosine kinase growth factor receptors, are now known to exist for multiple receptor classes and to affect myriad physiological and developmental processes. This review summarizes our present understanding of how endocytosis orchestrates cellular signaling networks, with an emphasis on mechanistic underpinnings and focusing on two receptor classes—tyrosine kinase and G protein–coupled receptors—that have been investigated in particular detail. Together, we believe that these examples provide a useful survey of the current consensus, uncertainties, and controversies in this rapidly advancing area of cell biology. Expected final online publication date for the Annual Review of Biochemistry, Volume 90 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... In this study, we focus on EGFR signaling transductions. After activation by EGF, Grb2 and SOS are recruited to EGFR, which further catalyzes the Ras-GDP exchange to Ras-GTP, and activates MAPK signaling cascade (37). The GTP-loaded Ras can be detected by a Ras-binding domain (RBD) truncated from Raf (36). ...
Article
Significance Multichannel single-molecule tracking is a powerful tool to probe molecular interactions with high spatial-temporal resolution. Current methods have at least two major limitations: the lack of high-performance intracellular single-molecule fluorescence probes and the difficulty of multispecies single-molecule labelling. Here, we employ a nanopore-electroporation technique to deliver organic dye-labelled proteins into living cells to achieve multi-color single molecule tracking. These organic dyes have better optical and chemical properties than the probes currently used. Therefore, we significantly expand the selection of intracellular single-molecule probes to widely available dyes. We demonstrate the application of this technique by revealing detailed spatial resolution and dynamics of membrane EGFR signaling transductions, which will shed light on the understanding of its fundamental mechanism.
... While our analysis indicates that the expression level of pathway components is insignificant in determining ERK activity in the cell lines assayed, this rules out neither activity-modifying mutations that do not alter expression, nor the existence of an activity threshold above which cells are eliminated by selection. Furthermore, while our limited immunoblot-based analysis was unable to identify differences in expression that explain the divergence of ERK signaling from the expected, a more precise and comprehensive proteomic analysis could reveal overlooked correlations (Shi et al, 2016). Naturally, the same caveats apply to the vast majority of cell-based experiments on RAS signaling (including transient expression experiments that typically exceed at least one cell cycle). ...
Article
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Activating mutations in RAS are present in ~ 30% of human tumors, and the resulting aberrations in ERK/MAPK signaling play a central role in oncogenesis. However, the form of these signaling changes is uncertain, with activating RAS mutants linked to both increased and decreased ERK activation in vivo. Rationally targeting the kinase activity of this pathway requires clarification of the quantitative effects of RAS mutations. Here, we use live-cell imaging in cells expressing only one RAS isoform to quantify ERK activity with a new level of accuracy. We find that despite large differences in their biochemical activity, mutant KRAS isoforms within cells have similar ranges of ERK output. We identify roles for pathway-level effects, including variation in feedback strength and feedforward modulation of phosphatase activity, that act to rescale pathway sensitivity, ultimately resisting changes in the dynamic range of ERK activity while preserving responsiveness to growth factor stimuli. Our results reconcile seemingly inconsistent reports within the literature and imply that the signaling changes induced by RAS mutations early in oncogenesis are subtle.
... Fig. 2). These results illustrate the importance of receptor expression levels and their ratios to capture the distinct signaling features observed for each cell line (44). ...
Preprint
Targeted therapies have shown significant patient benefit in about 5-10% of solid tumors that are addicted to a single oncogene. Here, we explore the idea of ligand addiction as a driver of tumor growth. High ligand levels in tumors have been shown to be associated with impaired patient survival, but targeted therapies have not yet shown great benefit in unselected patient populations. Using a novel approach of applying Bagged Decision Trees (BDT) to high-dimensional signaling features derived from a computational model, we can predict ligand dependent proliferation across a set of 58 cell lines. This mechanistic, multi-pathway model that features receptor heterodimerization, was trained on seven cancer cell lines and can predict signaling across two independent cell lines by adjusting only the receptor expression levels for each cell line. Interestingly, for patient samples the predicted tumor growth response correlates with high growth factor expression in the tumor microenvironment, which argues for a co-evolution of both factors in vivo . Summary Prediction of ligand-induced growth of cancer cell lines, which correlates with ligand-blocking antibody efficacy, could be significantly improved by learning from features of a mechanistic signaling model, and was applied to reveal a correlation between growth factor expression and predicted response in patient samples.
... ENSG00000100485-SOS2/cell). Interestingly, an elegant quantitative proteomic analysis of the core components of the EGFR-MAPK pathway across different cell types has recently revealed that the absolute abundance of SOS1 and SOS2 proteins (2000-5000 copies per cell) is far lower than that of most other core proteins (50000-70000 copies per cell) in the EGFR-MAPK pathway, suggesting that the low-abundance of SOS GEFs may serve as a regulatory bottleneck in this pathway [58]. ...
Article
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SOS1 and SOS2 are the most universal and widely expressed family of guanine exchange factors (GEFs) capable or activating RAS or RAC1 proteins in metazoan cells. SOS proteins contain a sequence of modular domains that are responsible for different intramolecular and intermolecular interactions modulating mechanisms of self-inhibition, allosteric activation and intracellular homeostasis. Despite their homology, analyses of SOS1/2-KO mice demonstrate functional prevalence of SOS1 over SOS2 in cellular processes including proliferation, migration, inflammation or maintenance of intracellular redox homeostasis, although some functional redundancy cannot be excluded, particularly at the organismal level. Specific SOS1 gain-of-function mutations have been identified in inherited RASopathies and various sporadic human cancers. SOS1 depletion reduces tumorigenesis mediated by RAS or RAC1 in mouse models and is associated with increased intracellular oxidative stress and mitochondrial dysfunction. Since WT RAS is essential for development of RAS-mutant tumors, the SOS GEFs may be considered as relevant biomarkers or therapy targets in RAS-dependent cancers. Inhibitors blocking SOS expression, intrinsic GEF activity, or productive SOS protein-protein interactions with cellular regulators and/or RAS/RAC targets have been recently developed and shown preclinical and clinical effectiveness blocking aberrant RAS signaling in RAS-driven and RTK-driven tumors.
... that peaked at around ~30-min of exposure ( Fig. 4.1 A), indicating activation of these three protein kinases families. Since MAPKs are commonly activated downstream of EGFR in epithelial cells (Shi et al., 2016), we also tested EGFR activation by the biofilm extract. This also showed activation-dependent Y1068 phosphorylation after a 30-min biofilm extract exposure ( Fig. 4.1 A). ...
Thesis
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Globally, about half of the adult population suffers from periodontal diseases. In periodontal diseases, multispecies bacterial biofilms accumulate between the epithelium of gingiva and teeth resulting in inflammation, periodontal pocket formation and alveolar bone loss. Integrin αvβ6 maintains anti-inflammatory transforming growth factor-β1 (TGF-β1) signaling in healthy junctional epithelium. However, it is significantly reduced in the pocket epithelium in periodontal disease. In this study, we show that β6 integrin mRNA and protein expression is suppressed by bacterial biofilms in cultured gingival epithelial cells (GECs). We found that biofilm-induced suppression of β6 integrin expression is driven by autocrine epidermal growth factor receptor (EGFR) signaling and attenuation of TGF-β1 signaling which leads to enhanced pro-inflammatory response. The biofilm-initiated β6 integrin downregulation in GEC can be prevented by blocking EGFR signaling. In addition, selective EGFR inhibitors significantly reduce periodontal inflammation and bone loss in an experimental periodontitis model in vivo. Therefore, blocking EGFR signaling could serve as a novel approach to reduce inflammation and bone loss in periodontal disease.
... Cell Culture and Bulk-scale Protein Digestion-The MCF-7 and MCF10A breast cell line were obtained from the American Type Culture Collection and was prepared as previously described (19). ...
Article
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Mass spectrometry (MS)-based proteomics has great potential for overcoming the limitations of antibody-based immunoassays for antibody-independent, comprehensive, and quantitative proteomic analysis of single cells. Indeed, recent advances in nanoscale sample preparation have enabled effective processing of single cells. In particular, the concept of using boosting/carrier channels in isobaric labeling to increase the sensitivity in MS detection has also been increasingly used for quantitative proteomic analysis of small-sized samples including single cells. However, the full potential of such boosting/carrier approaches has not been significantly explored, nor has the resulting quantitation quality been carefully evaluated. Herein, we have further evaluated and optimized our recent boosting to amplify signal with isobaric labeling (BASIL) approach, originally developed for quantifying phosphorylation in small number of cells, for highly effective analysis of proteins in single cells. This improved BASIL (iBASIL) approach enables reliable quantitative single-cell proteomics analysis with greater proteome coverage by carefully controlling the boosting-to-sample ratio (e.g., in general <100x) and optimizing MS automatic gain control (AGC) and ion injection time settings in MS/MS analysis (e.g., 5E5 and 300 ms, respectively, which is significantly higher than that used in typical bulk analysis). By coupling with a nanodroplet-based single cell preparation (nanoPOTS) platform, iBASIL enabled identification of ~2,500 proteins and precise quantification of ~1,500 proteins in the analysis of 104 FACS-isolated single cells, with the resulting protein profiles robustly clustering the cells from three different acute myeloid leukemia cell lines. This study highlights the importance of carefully evaluating and optimizing the boosting ratios and MS data acquisition conditions for achieving robust, comprehensive proteomic analysis of single cells.
... Although there are usually~10 5 -10 6 EGFR receptors on mammalian cell surface , the downstream network response, for example Akt phosphorylation, often saturates when only a relatively small fraction (5-10%) of the receptors are bound to their cognate ligands (Chen et al., 2009;Shi et al., 2016). Our study suggests that one potential advantage of such a system architecture is that, beyond simple signal activation, it may endow cells with a large dynamic range of receptor abundances to memorize stimulation levels of multiple extracellular ligands (Hart et al., 2013;Nandagopal et al., 2018). ...
Article
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Detecting relative rather than absolute changes in extracellular signals enables cells to make decisions in fluctuating environments. However, how mammalian signaling networks store the memories of past stimuli and subsequently use them to compute relative signals, i.e. perform fold change detection, is not well understood. Using the growth factor-activated PI3K-Akt signaling pathway, we develop computational and analytical models, and experimentally validate a novel mechanism of relative sensing in mammalian cells. This mechanism relies on a new form of cellular memory, where cells effectively encode past stimulation levels in the abundance of cognate receptors on the cell surface. We show the robustness and specificity of the relative sensing for two physiologically important ligands, epidermal growth factor (EGF) and hepatocyte growth factor (HGF), and across wide ranges of background stimuli. Our results suggest that similar mechanisms of memory and fold change detection are likely to be important across diverse signaling cascades and biological contexts.
... MAPK8) that peaked at ∼30 min of exposure (Fig. 1A), indicating activation of these three protein kinases families. Since MAPKs are commonly activated downstream of EGFR in epithelial cells (Shi et al., 2016), we also tested EGFR activation by the biofilm extract. This also showed activation-dependent Y1068 phosphorylation after a 30-min biofilm extract exposure (Fig. 1A). ...
Article
In periodontal disease (PD), bacterial biofilms cause gingival inflammation, leading to bone loss. In healthy individuals, αvβ6 integrin in junctional epithelium maintains anti-inflammatory transforming growth factor-β1 (TGF-β1) signaling, whereas its expression is lost in individuals with PD. Bacterial biofilms suppress β6 integrin expression in cultured gingival epithelial cells (GECs) by attenuating TGF-β1 signaling, leading to an enhanced pro-inflammatory response. In the present study, we show that GEC exposure to biofilms induced activation of mitogen-activated protein kinases and epidermal growth factor receptor (EGFR). Inhibition of EGFR and ERK stunted both the biofilm-induced ITGB6 suppression and IL1B stimulation. Furthermore, biofilm induced the expression of endogenous EGFR ligands that suppressed ITGB6 and stimulated IL1B expression, indicating that the effects of the biofilm were mediated by autocrine EGFR signaling. Biofilm and EGFR ligands induced inhibitory phosphorylation of the TGF-β1 signaling mediator Smad3 at S208. Overexpression of a phosphorylation-defective mutant of Smad3 (S208A) reduced the β6 integrin suppression. Furthermore, inhibition of EGFR signaling significantly reduced bone loss and inflammation in an experimental PD model. Thus, EGFR inhibition may provide a target for clinical therapies to prevent inflammation and bone loss in PD. This article has an associated First Person interview with the first author of the paper.
... Note that, for prokaryotic cells this is the case for the majority of the proteins 33 . Although in eukaryotic cells proteins are in general more abundant there is still a significant portion of the cases for which the copy numbers appear to be low (see for example, Schwanhausser et al. 34 , Shi et al. 35 , Nguyen et al. 36 ). We would like to remark that extrinsic noise is not taken into account in this study since we are quantifying the effect of intrinsic noise in hysteresis. ...
Article
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Cell fate determination, the process through which cells commit to differentiated states is commonly mediated by gene regulatory motifs with mutually exclusive expression states. The classical deterministic picture for cell fate determination includes bistability and hysteresis, which enables the persistence of the acquired cellular state after withdrawal of the stimulus, ensuring a robust cellular response. However, stochasticity inherent to gene expression dynamics is not compatible with hysteresis, since the stationary solution of the governing Chemical Master Equation does not depend on the initial conditions. We provide a quantitative description of a transient hysteresis phenomenon reconciling experimental evidence of hysteretic behaviour in gene regulatory networks with inherent stochasticity: under sufficiently slow dynamics hysteresis is transient. We quantify this with an estimate of the convergence rate to the equilibrium and introduce a natural landscape capturing system’s evolution that, unlike traditional cell fate potential landscapes, is compatible with coexistence at the microscopic level.
Article
Cellular signaling dynamics are sensitive to differences in ligand identity, levels, and temporal patterns. These signaling patterns are also impacted by the larger context that the cell experiences (i.e., stimuli such as media formulation or substrate stiffness that are constant in an experiment exploring a particular variable but may differ between independent experiments which explore that variable) although the reason for different dynamics is not always obvious. Here, we compared extracellular-regulated kinase (ERK) signaling in response to epidermal growth factor treatment of human mammary epithelial cells cultures in either well culture or a microfluidic device. Using a single-cell ERK kinase translocation reporter, we observed extended ERK activation in well culture and only transient activity in microfluidic culture. The activity in microfluidic culture resembled that of the control condition, suggesting that shear stress led to the early activity and a loss of autocrine factors dampened extended signaling. Through experimental analysis we identified growth differentiation factor-15 as a candidate factor that led to extended ERK activation through a protein kinase C-α/β dependent pathway. Our results demonstrate that context impacts ERK dynamics and that comparison of distinct contexts can be used to elucidate new aspects of the cell signaling network.
Article
The ERK signaling pathway, consisting of core protein kinases Raf, MEK and effector kinases ERK1/2, regulates various biological outcomes such as cell proliferation, differentiation, apoptosis, or cell migration. Signal transduction through the ERK signaling pathway is tightly controlled at all levels of the pathway. However, it is not well understood whether ERK pathway signaling can be modulated by the abundance of ERK pathway core kinases. In this study, we investigated the effects of low-level overexpression of the ERK2 isoform on the phenotype and scattering of cuboidal MDCK epithelial cells growing in discrete multicellular clusters. We show that ERK2 overexpression reduced the vertical size of lateral membranes that contain cell-cell adhesion complexes. Consequently, ERK2 overexpressing cells were unable to develop cuboidal shape, remained flat with increased spread area and intercellular adhesive contacts were present only on the basal side. Interestingly, ERK2 overexpression was not sufficient to increase phosphorylation of multiple downstream targets including transcription factors and induce global changes in gene expression, namely to increase the expression of pro-migratory transcription factor Fra1. However, ERK2 overexpression enhanced HGF/SF-induced cell scattering as these cells scattered more rapidly and to a greater extent than parental cells. Our results suggest that an increase in ERK2 expression primarily reduces cell-cell cohesion and that weakened intercellular adhesion synergizes with upstream signaling in the conversion of the multicellular epithelium into single migrating cells. This mechanism may be clinically relevant as the analysis of clinical data revealed that in one type of cancer, pancreatic adenocarcinoma, ERK2 overexpression correlates with a worse prognosis.
Article
Epithelial morphogenesis and oncogenic transformation can cause loss of cell adhesion, and detached cells are eliminated by anoikis. Here, we reveal that transforming growth factor beta receptor 3 (TGFBR3) acts as an anoikis mediator through the coordination of activating transcription factor 4 (ATF4). In breast cancer, TGFBR3 is progressively lost, but elevated TGFBR3 is associated with a histologic subtype characterized by cellular adhesion defects. Dissecting the impact of extracellular matrix (ECM) deprivation, we demonstrate that ECM loss promotes TGFBR3 expression, which in turn differentiates cell aggregates to a prosurvival phenotype and drives the intrinsic apoptotic pathway. We demonstrate that inhibition of TGFBR3 impairs epithelial anoikis by activating ATF4 signaling. These preclinical findings provide a rationale for therapeutic inhibition of ATF4 in the subgroup of breast cancer patients with low TGFBR3 expression.
Article
Cells sense a variety of extracellular growth factors and signaling molecules through numerous distinct receptor tyrosine kinases (RTKs) on the cell surface. In many cases, the same intracellular signaling molecules interact with more than one type of RTK. How signals from different RTKs retain the identity of the triggering receptor and how (or if) different receptors may synergize or compete remains largely unknown. Here we utilize an experimental strategy, combining microscale patterning and single molecule imaging, to measure the competition between ephrin-A1:EphA2 and EGF:EGFR ligand-receptor complexes for the shared downstream signaling molecules, Grb2 and SOS. The results reveal a distinct hierarchy, in which newly formed EGF:EGFR complexes outcompete ephrin-A1:EphA2 for Grb2 and SOS, revealing a type of negative crosstalk interaction fundamentally controlled by chemical mass action and protein copy number limitations.
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Phosphotyrosine (pTyr) motifs in unstructured polypeptides orchestrate important cellular processes by engaging SH2-containing adaptors to nucleate complex signalling networks. The concept of phase separation has recently changed our appreciation of such multivalent networks, however, the role of pTyr motif positioning in their function remains to be explored. We have now explored this parameter in the assembly and operation of the signalling cascade driving actin-based motility and spread of Vaccinia virus. This network involves two pTyr motifs in the viral protein A36 that recruit the adaptors Nck and Grb2 upstream of N-WASP and Arp2/3-mediated actin polymerization. We generated synthetic networks on Vaccinia by manipulating pTyr motifs in A36 and the unrelated p14 from Orthoreovirus. In contrast to predictions, we find that only specific spatial arrangements of Grb2 and Nck binding sites result in robust N-WASP recruitment, Arp2/3 driven actin polymerization and viral spread. Our results suggest that the relative position of pTyr adaptor binding sites is optimised for signal output. This finding may explain why the relative positions of pTyr motifs are usually conserved in proteins from widely different species. It also has important implications for regulation of physiological networks, including those that undergo phase transitions.
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KRAS forms transient dimers and higher-order multimers (nanoclusters) on the plasma membrane, which drive MAPK signaling and cell proliferation. KRAS is a frequently mutated oncogene, and while it is well known that the most prevalent mutation, G12D, impairs GTP hydrolysis, thereby increasing KRAS activation, G12D has also been shown to enhance nanoclustering. Elucidating structures of dynamic KRAS assemblies on a membrane has been challenging, thus we have refined our NMR approach that uses nanodiscs to study KRAS associated with membranes. We incorporated paramagnetic relaxation enhancement (PRE) titrations and interface mutagenesis, which revealed that, in addition to the symmetric 'α-α' dimerization interface shared with wild-type KRAS, the G12D mutant also self-associates through an asymmetric 'α-β' interface. The 'α-β' association is dependent on the presence of phosphatidylserine lipids, consistent with previous reports that this lipid promotes KRAS self-assembly on the plasma membrane in cells. Experiments using engineered mutants to spoil each interface, together with PRE probes attached to the membrane or free in solvent, suggest that dimerization through the primary 'α-α' interface releases β interfaces from the membrane promoting formation of the secondary 'α-β' interaction, potentially initiating nanoclustering. In addition, the small molecule BI-2852 binds at a β-β interface, stabilizing a new dimer configuration that outcompetes native dimerization and blocks the effector-binding site. Our data indicate that KRAS self-association involves a delicately balanced conformational equilibrium between transient states, which is sensitive to disease-associated mutation and small molecule inhibitors. The methods developed here are applicable to biologically important transient interactions involving other membrane-associated proteins.
Article
Drug resistance poses a major challenge for targeted cancer therapy. To be able to functionally screen large randomly mutated target gene libraries for drug resistance mutations, we developed a biochemically defined high-throughput assay termed PhosphoFlowSeq. Instead of selecting for proliferation or resistance to apoptosis, PhosphoFlowSeq directly analyzes the enzymatic activities of randomly mutated kinases, thereby reducing the dependency on the signaling network in the host cell. Moreover, simultaneous analysis of expression levels enables compensation for expression-based biases on a single cell level. Using EGFR and its kinase inhibitor erlotinib as a model system, we demonstrate that the clinically most relevant resistance mutation T790M is reproducibly detected at high frequencies after four independent PhosphoFlowSeq selection experiments. Moreover, upon decreasing the selection pressure, also mutations which only confer weak resistance were identified, including T854A and L792H. We expect that PhosphoFlowSeq will be a valuable tool for the prediction and functional screening of drug resistance mutations in kinases.
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Using 11 proteomics datasets, mostly available through the PRIDE database, we assembled a reference expression map for 191 cancer cell lines and 246 clinical tumour samples, across 13 lineages. We found unique peptides identified only in tumour samples despite a much higher coverage in cell lines. These were mainly mapped to proteins related to regulation of signalling receptor activity. Correlations between baseline expression in cell lines and tumours were calculated. We found these to be highly similar across all samples with most similarity found within a given sample type. Integration of proteomics and transcriptomics data showed median correlation across cell lines to be 0.58 (range between 0.43 and 0.66). Additionally, in agreement with previous studies, variation in mRNA levels was often a poor predictor of changes in protein abundance. To our knowledge, this work constitutes the first meta-analysis focusing on cancer-related public proteomics datasets. We therefore also highlight shortcomings and limitations of such studies. All data is available through PRIDE dataset identifier PXD013455 and in Expression Atlas.
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Significance Signaling pathways form complex networks of biochemical reactions, but inferring the topology of such networks and measuring how they are remodeled in disease is still challenging. Using MS, our study defined the circuitry and plasticity of a kinase signaling network de novo, with unprecedented depth and without prior assumptions of its topology. In addition, we observed a degree of stochasticity in how the network was remodeled upon chronic inhibition of phosphoinositide 3-kinase (PI3K) or mammalian target of rapamycin complexes 1/2 (mTORC1/2), suggesting that the initial condition of the system was not the only determinant of how cells become resistant to targeted therapies. These observations may have implications for our ability to predict the evolution of signaling networks during therapy to prevent the acquisition of resistance.
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Targeted disruption of both alleles of mouse sos1, which encodes a Ras-specific exchange factor, conferred mid-gestational embryonic lethality that was secondary to impaired placental development and was associated with very low placental ERK activity. The trophoblastic layers of sos1-/- embryos were poorly developed, correlating with high sos1 expression in wild-type trophoblasts. A sos1-/- cell line, which expressed readily detectable levels of the closely related Sos2 protein, formed complexes between Sos2, epidermal growth factor receptor (EGFR) and Shc efficiently, gave normal RasGTP and ERK responses when treated with EGF for 10 min and was transformed readily by activated Ras. However, the sos1-/- cells were resistant to transformation by v-Src or by overexpressed EGFR and continuous EGF treatment, unlike sos1+/- or wild-type cells. This correlated with Sos2 binding less efficiently than Sos1 to EGFR and Shc in cells treated with EGF for 90 min or to v-Src and Shc in v-Src-expressing cells, and with less ERK activity. We conclude that Sos1 participates in both short- and long-term signaling, while Sos2-dependent signals are predominantly short-term.
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While the number and identity of proteins expressed in a single human cell type is currently unknown, this fundamental question can be addressed by advanced mass spectrometry (MS)-based proteomics. Online liquid chromatography coupled to high-resolution MS and MS/MS yielded 166 420 peptides with unique amino-acid sequence from HeLa cells. These peptides identified 10 255 different human proteins encoded by 9207 human genes, providing a lower limit on the proteome in this cancer cell line. Deep transcriptome sequencing revealed transcripts for nearly all detected proteins. We calculate copy numbers for the expressed proteins and show that the abundances of >90% of them are within a factor 60 of the median protein expression level. Comparisons of the proteome and the transcriptome, and analysis of protein complex databases and GO categories, suggest that we achieved deep coverage of the functional transcriptome and the proteome of a single cell type.
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Phosphatases are important regulators of intracellular signaling events, and their functions have been implicated in many biological processes. Dual-specificity phosphatases (DUSPs), whose family currently contains 25 members, are phosphatases that can dephosphorylate both tyrosine and serine/threonine residues of their substrates. The archetypical DUSP, DUSP1/MKP1, was initially discovered to regulate the activities of MAP kinases by dephosphorylating the TXY motif in the kinase domain. However, although DUSPs were discovered more than a decade ago, only in the past few years have their various functions begun to be described. DUSPs can be categorized based on the presence or absence of a MAP kinase-interacting domain into typical DUSPs and atypical DUSPs, respectively. In this review, we discuss the current understanding of how the activities of typical DUSPs are regulated and how typical DUSPs can regulate the functions of their targets. We also summarize recent findings from several in vivo DUSP-deficient mouse models that studied the involvement of DUSPs during the development and functioning of T cells. Finally, we discuss briefly the potential roles of DUSPs in the regulation of non-MAP kinase targets, as well as in the modulation of tumorigenesis.
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Selected reaction monitoring (SRM) - also known as multiple reaction monitoring (MRM) - has emerged as a promising high-throughput targeted protein quantification technology for candidate biomarker verification and systems biology applications. A major bottleneck for current SRM technology, however, is insufficient sensitivity for, e.g. detecting low-abundance biomarkers likely present at the low ng/mL to pg/mL range in human blood plasma or serum, or extremely low-abundance signaling proteins in cells or tissues. Herein, we review recent advances in methods and technologies, including front-end immunoaffinity depletion, fractionation, selective enrichment of target proteins/peptides including posttranslational modifications, as well as advances in MS instrumentation which have significantly enhanced the overall sensitivity of SRM assays and enabled the detection of low-abundance proteins at low- to sub-ng/mL level in human blood plasma or serum. General perspectives on the potential of achieving sufficient sensitivity for detection of pg/mL level proteins in plasma are also discussed.
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Understanding the information-processing capabilities of signal transduction networks, how those networks are disrupted in disease, and rationally designing therapies to manipulate diseased states require systematic and accurate reconstruction of network topology. Data on networks central to human physiology, such as the inflammatory signalling networks analyzed here, are found in a multiplicity of on-line resources of pathway and interactome databases (Cancer CellMap, GeneGo, KEGG, NCI-Pathway Interactome Database (NCI-PID), PANTHER, Reactome, I2D, and STRING). We sought to determine whether these databases contain overlapping information and whether they can be used to construct high reliability prior knowledge networks for subsequent modeling of experimental data. We have assembled an ensemble network from multiple on-line sources representing a significant portion of all machine-readable and reconcilable human knowledge on proteins and protein interactions involved in inflammation. This ensemble network has many features expected of complex signalling networks assembled from high-throughput data: a power law distribution of both node degree and edge annotations, and topological features of a "bow tie" architecture in which diverse pathways converge on a highly conserved set of enzymatic cascades focused around PI3K/AKT, MAPK/ERK, JAK/STAT, NFκB, and apoptotic signaling. Individual pathways exhibit "fuzzy" modularity that is statistically significant but still involving a majority of "cross-talk" interactions. However, we find that the most widely used pathway databases are highly inconsistent with respect to the actual constituents and interactions in this network. Using a set of growth factor signalling networks as examples (epidermal growth factor, transforming growth factor-beta, tumor necrosis factor, and wingless), we find a multiplicity of network topologies in which receptors couple to downstream components through myriad alternate paths. Many of these paths are inconsistent with well-established mechanistic features of signalling networks, such as a requirement for a transmembrane receptor in sensing extracellular ligands. Wide inconsistencies among interaction databases, pathway annotations, and the numbers and identities of nodes associated with a given pathway pose a major challenge for deriving causal and mechanistic insight from network graphs. We speculate that these inconsistencies are at least partially attributable to cell, and context-specificity of cellular signal transduction, which is largely unaccounted for in available databases, but the absence of standardized vocabularies is an additional confounding factor. As a result of discrepant annotations, it is very difficult to identify biologically meaningful pathways from interactome networks a priori. However, by incorporating prior knowledge, it is possible to successively build out network complexity with high confidence from a simple linear signal transduction scaffold. Such reduced complexity networks appear suitable for use in mechanistic models while being richer and better justified than the simple linear pathways usually depicted in diagrams of signal transduction.
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To understand how integration of multiple data types can help decipher cellular responses at the systems level, we analyzed the mitogenic response of human mammary epithelial cells to epidermal growth factor (EGF) using whole genome microarrays, mass spectrometry-based proteomics and large-scale western blots with over 1000 antibodies. A time course analysis revealed significant differences in the expression of 3172 genes and 596 proteins, including protein phosphorylation changes measured by western blot. Integration of these disparate data types showed that each contributed qualitatively different components to the observed cell response to EGF and that varying degrees of concordance in gene expression and protein abundance measurements could be linked to specific biological processes. Networks inferred from individual data types were relatively limited, whereas networks derived from the integrated data recapitulated the known major cellular responses to EGF and exhibited more highly connected signaling nodes than networks derived from any individual dataset. While cell cycle regulatory pathways were altered as anticipated, we found the most robust response to mitogenic concentrations of EGF was induction of matrix metalloprotease cascades, highlighting the importance of the EGFR system as a regulator of the extracellular environment. These results demonstrate the value of integrating multiple levels of biological information to more accurately reconstruct networks of cellular response.
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Deep proteomic analysis of mammalian cell lines would yield an inventory of the building blocks of the most commonly used systems in biological research. Mass spectrometry-based proteomics can identify and quantify proteins in a global and unbiased manner and can highlight the cellular processes that are altered between such systems. We analyzed 11 human cell lines using an LTQ-Orbitrap family mass spectrometer with a "high field" Orbitrap mass analyzer with improved resolution and sequencing speed. We identified a total of 11,731 proteins, and on average 10,361 ± 120 proteins in each cell line. This very high proteome coverage enabled analysis of a broad range of processes and functions. Despite the distinct origins of the cell lines, our quantitative results showed surprisingly high similarity in terms of expressed proteins. Nevertheless, this global similarity of the proteomes did not imply equal expression levels of individual proteins across the 11 cell lines, as we found significant differences in expression levels for an estimated two-third of them. The variability in cellular expression levels was similar for low and high abundance proteins, and even many of the most highly expressed proteins with household roles showed significant differences between cells. Metabolic pathways, which have high redundancy, exhibited variable expression, whereas basic cellular functions such as the basal transcription machinery varied much less. We harness knowledge of these cell line proteomes for the construction of a broad coverage "super-SILAC" quantification standard. Together with the accompanying paper (Schaab, C. MCP 2012, PMID: 22301388) (17) these data can be used to obtain reference expression profiles for proteins of interest both within and across cell line proteomes.
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Recent advances in next-generation DNA sequencing and proteomics provide an unprecedented ability to survey mRNA and protein abundances. Such proteome-wide surveys are illuminating the extent to which different aspects of gene expression help to regulate cellular protein abundances. Current data demonstrate a substantial role for regulatory processes occurring after mRNA is made - that is, post-transcriptional, translational and protein degradation regulation - in controlling steady-state protein abundances. Intriguing observations are also emerging in relation to cells following perturbation, single-cell studies and the apparent evolutionary conservation of protein and mRNA abundances. Here, we summarize current understanding of the major factors regulating protein expression.
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Measurement precision determines the power of any analysis to reliably identify significant signals, such as in screens for differential expression, independent of whether the experimental design incorporates replicates or not. With the compilation of large-scale RNA-Seq datasets with technical replicate samples, however, we can now, for the first time, perform a systematic analysis of the precision of expression level estimates from massively parallel sequencing technology. This then allows considerations for its improvement by computational or experimental means. We report on a comprehensive study of target identification and measurement precision, including their dependence on transcript expression levels, read depth and other parameters. In particular, an impressive recall of 84% of the estimated true transcript population could be achieved with 331 million 50 bp reads, with diminishing returns from longer read lengths and even less gains from increased sequencing depths. Most of the measurement power (75%) is spent on only 7% of the known transcriptome, however, making less strongly expressed transcripts harder to measure. Consequently, <30% of all transcripts could be quantified reliably with a relative error<20%. Based on established tools, we then introduce a new approach for mapping and analysing sequencing reads that yields substantially improved performance in gene expression profiling, increasing the number of transcripts that can reliably be quantified to over 40%. Extrapolations to higher sequencing depths highlight the need for efficient complementary steps. In discussion we outline possible experimental and computational strategies for further improvements in quantification precision. rnaseq10@boku.ac.at
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LC-MS-based quantitative proteomics has become increasingly applied to a wide range of biological applications due to growing capabilities for broad proteome coverage and good accuracy and precision in quantification. Herein, we review the current LC-MS-based quantification methods with respect to their advantages and limitations and highlight their potential applications.
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Gene expression is a multistep process that involves the transcription, translation and turnover of messenger RNAs and proteins. Although it is one of the most fundamental processes of life, the entire cascade has never been quantified on a genome-wide scale. Here we simultaneously measured absolute mRNA and protein abundance and turnover by parallel metabolic pulse labelling for more than 5,000 genes in mammalian cells. Whereas mRNA and protein levels correlated better than previously thought, corresponding half-lives showed no correlation. Using a quantitative model we have obtained the first genome-scale prediction of synthesis rates of mRNAs and proteins. We find that the cellular abundance of proteins is predominantly controlled at the level of translation. Genes with similar combinations of mRNA and protein stability shared functional properties, indicating that half-lives evolved under energetic and dynamic constraints. Quantitative information about all stages of gene expression provides a rich resource and helps to provide a greater understanding of the underlying design principles.
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Signaling mediated by the Epidermal Growth Factor Receptor (EGFR) is crucial in normal development, and aberrant EGFR signaling has been implicated in a wide variety of cancers. Here we find that the high- and low-affinity interactions between EGFR and its ligands activate different signaling pathways. While high-affinity ligand binding is sufficient for activation of most canonical signaling pathways, low-affinity binding is required for the activation of the Signal transducers and activators of transcription (Stats) and Phospholipase C-gamma 1 (PLCγ1). As the Stat proteins are involved in many cellular responses including proliferation, migration and apoptosis, these results assign a function to low-affinity interactions that has been omitted from computational models of EGFR signaling. The existence of receptors with distinct signaling properties provides a way for EGFR to respond to different concentrations of the same ligand in qualitatively different ways.
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Endocytosis of the epidermal growth factor receptor (EGFR) is important for the regulation of EGFR signaling. However, EGFR endocytosis mechanisms are poorly understood, which precludes development of approaches to specifically inhibit EGFR endocytosis and analyze its impact on signaling. Using a