Cell fate decisions are specified by the dynamic ERK interactome

Signalling and Proteomics Laboratory, The Beatson Institute for Cancer Research, Glasgow G61 1BD, UK.
Nature Cell Biology (Impact Factor: 19.68). 12/2009; 11(12):1458-64. DOI: 10.1038/ncb1994
Source: PubMed

ABSTRACT Extracellular signal-regulated kinase (ERK) controls fundamental cellular functions, including cell fate decisions. In PC12, cells shifting ERK activation from transient to sustained induces neuronal differentiation. As ERK associates with both regulators and effectors, we hypothesized that the mechanisms underlying the switch could be revealed by assessing the dynamic changes in ERK-interacting proteins that specifically occur under differentiation conditions. Using quantitative proteomics, we identified 284 ERK-interacting proteins. Upon induction of differentiation, 60 proteins changed their binding to ERK, including many proteins that were not known to participate in differentiation. We functionally characterized a subset, showing that they regulate the pathway at several levels and by different mechanisms, including signal duration, ERK localization, feedback, crosstalk with the Akt pathway and differential interaction and phosphorylation of transcription factors. Integrating these data with a mathematical model confirmed that ERK dynamics and differentiation are regulated by distributed control mechanisms rather than by a single master switch.

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Available from: Alexander Von Kriegsheim, Sep 27, 2015
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    • "With this compromise, proteomic measurements of time-dependent changes in signaling pathways can be obtained using targeted, multiplexed and quantitative approaches, such as MRM-MS coupled with stable isotope dilution (SID) [53-55], in vitro kinase assays [56], quantitative immunoblotting and enzyme-linked immunosorbent assays (ELISAs) [57] or protein arrays [58-60]. By monitoring dynamic changes in these PPIs, temporal data have been used to reconstruct signaling pathways involved in cell differentiation and apoptosis [61-63]. In addition, immuno-enrichment of phosphotyrosine residues and quantitative MS methods have previously explored time-dependent changes in signaling downstream of epidermal growth factor receptor (EGFR) [64,65]; the combination of MS, phosphorylation motif–directed antibodies, and phosphorylated serine-threonine–binding modules (e.g., 14-3-3 proteins or the Polo-box domain of Polo-like kinases) identified signaling networks involved in cell migration, metabolism, mitosis, and DNA damage [66-68]; and the use of analog-specific protein kinase mutants and MS identified comprehensive lists of substrates, e.g., those previously unknown to cyclin-dependent kinase 1-cyclin B, with the potential of expanding our understanding of kinase-substrate connections in signaling networks [69,70]. "
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    ABSTRACT: During the past several decades, the understanding of cancer at the molecular level has been primarily focused on mechanisms on how signaling molecules transform homeostatically balanced cells into malignant ones within an individual pathway. However, it is becoming more apparent that pathways are dynamic and crosstalk at different control points of the signaling cascades, making the traditional linear signaling models inadequate to interpret complex biological systems. Recent technological advances in high throughput, deep sequencing for the human genomes and proteomic technologies to comprehensively characterize the human proteomes in conjunction with multiplexed targeted proteomic assays to measure panels of proteins involved in biologically relevant pathways have made significant progress in understanding cancer at the molecular level. It is undeniable that proteomic profiling of differentially expressed proteins under many perturbation conditions, or between normal and "diseased" states is important to capture a first glance at the overall proteomic landscape, which has been a main focus of proteomics research during the past 15-20 years. However, the research community is gradually shifting its heavy focus from that initial discovery step to protein target verification using multiplexed quantitative proteomic assays, capable of measuring changes in proteins and their interacting partners, isoforms, and post-translational modifications (PTMs) in response to stimuli in the context of signaling pathways and protein networks. With a critical link to genotypes (i.e., high throughput genomics and transcriptomics data), new and complementary information can be gleaned from multi-dimensional omics data to (1) assess the effect of genomic and transcriptomic aberrations on such complex molecular machinery in the context of cell signaling architectures associated with pathological diseases such as cancer (i.e., from genotype to proteotype to phenotype); and (2) target pathway- and network-driven changes and map the fluctuations of these functional units (proteins) responsible for cellular activities in response to perturbation in a spatiotemporal fashion to better understand cancer biology as a whole system.
    Clinical Proteomics 06/2014; 11(1):22. DOI:10.1186/1559-0275-11-22
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    • "Neurite outgrowth is an important aspect of neuronal plasticity and regeneration in neuropathological conditions and neural injury [27]. Consistent with previous reports [28,29], in this study, more than 20% of NGF-stimulated PC12 cells had neurites that were twice the cell body length after exposure to RA4. Although the effects of IH on neuronal cell differentiation remain poorly understood, a previous study reported that 10 cycles of IH (1% O2 for 24 h, following a 24-h recovery period under RA) suppressed retinoic acid-induced differentiation of neuroblastoma cells [9]. "
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    ABSTRACT: Background Intermittent hypoxia (IH) plays a critical role in sleep breathing disorder-associated hippocampus impairments, including neurocognitive deficits, irreversible memory and learning impairments. IH-induced neuronal injury in the hippocampus may result from reduced precursor cell proliferation and the relative numbers of postmitotic differentiated neurons. However, the mechanisms underlying IH-induced reactive oxygen species (ROS) generation effects on cell proliferation and neuronal differentiation remain largely unknown. Results ROS generation significantly increased after 1–4 days of IH without increased pheochromocytoma-12 (PC12) cell death, which resulted in increased protein phosphatase 2A (PP2A) mRNA and protein levels. After 3–4 days of IH, extracellular signal-regulated kinases 1/2 (ERK1/2) protein phosphorylation decreased, which could be reversed by superoxide dismutase (SOD), 1,10-phenanthroline (Phe), the PP2A phosphorylation inhibitors, okadaic acid (OKA) and cantharidin, and the ERK phosphorylation activator nicotine (p < 0.05). In particular, the significantly reduced cell proliferation and increased proportions of cells in the G0/G1 phase after 1–4 days of IH (p < 0.05), which resulted in decreased numbers of PC12 cells, could be reversed by treatment with SOD, Phe, PP2A inhibitors and an ERK activator. In addition, the numbers of nerve growth factor (NGF)-induced PC12 cells with neurite outgrowths after 3–4 days of IH were less than those after 4 days of RA, which was also reversed by SOD, Phe, PP2A inhibitors and an ERK activator. Conclusions Our results suggest that IH-induced ROS generation increases PP2A activation and subsequently downregulates ERK1/2 activation, which results in inhibition of PC12 cell proliferation through G0/G1 phase arrest and NGF-induced neuronal differentiation.
    Journal of Biomedical Science 05/2014; 21(1):46. DOI:10.1186/1423-0127-21-46 · 2.76 Impact Factor
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    • "Thus, growth factor‐stimulated activation of the ERK pathway is typically transient. The kinetics of ERK activation and adaptation have been quantitatively characterized (Cirit et al, 2010; Sturm et al, 2010; Fritsche‐Guenther et al, 2011) and have proven to be important for proliferation and cell‐fate decisions (Marshall, 1995; von Kriegsheim et al, 2009; Chung et al, 2010; Albeck et al, 2013). "
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    ABSTRACT: The extracellular signal-regulated kinase (ERK) signaling pathway controls cell proliferation and differentiation in metazoans. Two hallmarks of its dynamics are adaptation of ERK phosphorylation, which has been linked to negative feedback, and nucleocytoplasmic shuttling, which allows active ERK to phosphorylate protein substrates in the nucleus and cytosol. To integrate these complex features, we acquired quantitative biochemical and live-cell microscopy data to reconcile phosphorylation, localization, and activity states of ERK. While maximal growth factor stimulation elicits transient ERK phosphorylation and nuclear translocation responses, ERK activities available to phosphorylate substrates in the cytosol and nuclei show relatively little or no adaptation. Free ERK activity in the nucleus temporally lags the peak in nuclear translocation, indicating a slow process. Additional experiments, guided by kinetic modeling, show that this process is consistent with ERK's modification of and release from nuclear substrate anchors. Thus, adaptation of whole-cell ERK phosphorylation is a by-product of transient protection from phosphatases. Consistent with this interpretation, predictions concerning the dose-dependence of the pathway response and its interruption by inhibition of MEK were experimentally confirmed.
    Molecular Systems Biology 01/2014; 10(1):718. DOI:10.1002/msb.134708 · 10.87 Impact Factor
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