Alex von Kriegsheim

Cell fate decisions are specified by the dynamic ERK interactome.

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.

Inferring signaling pathway topologies from multiple perturbation measurements of specific biochemical species.

Bayesian inference–based modeling identifies the most likely paths through a signaling network. Picking the Right Path Signaling networks have become increasingly complex as large-scale analysis and experiments in multiple systems add new potential connections and players. Xu et al. present a mathematical approach to rank the possible paths through a signaling pathway and develop hypotheses that can be rationally tested. They call their approach BIBm for Bayesian inference–based modeling and apply BIBm to explore the signaling pathways by which epidermal growth factor (EGF) stimulates extracellular signal–regulated kinase (ERK). Using a limited set of biochemical experiments, the authors tested four models and found that the one that relied on two Raf family members ranked the highest. This model was then experimentally validated in two cell lines to show that both Raf-1 and B-Raf contribute to ERK activation in response to EGF. The specification of biological decisions by signaling pathways is encoded by the interplay between activation dynamics and network topologies. Although we can describe complex networks, we cannot easily determine which topology the cell actually uses to transduce a specific signal. Experimental testing of all plausible topologies is infeasible because of the combinatorially large number of experiments required to explore the complete hypothesis space. Here, we demonstrate that Bayesian inference–based modeling provides an approach to explore and constrain this hypothesis space, permitting the rational ranking of pathway models. Our approach can use measurements of a limited number of biochemical species when combined with multiple perturbations. As proof of concept, we examined the activation of the extracellular signal–regulated kinase (ERK) pathway by epidermal growth factor. The predicted and experimentally validated model shows that both Raf-1 and, unexpectedly, B-Raf are needed to fully activate ERK in two different cell lines. Thus, our formal methodology rationally infers evidentially supported pathway topologies even when a limited number of biochemical and kinetic measurements are available.

Regulation of the Raf-MEK-ERK pathway by protein phosphatase 5

The Raf–MEK–ERK pathway couples growth factor, mitogenic and extracellular matrix signals to cell fate decisions such as growth, proliferation, migration, differentiation and survival. Raf-1 is a direct effector of the Ras GTPase and is the initiating kinase in this signalling cascade. Although Raf-1 activation is well studied, little is known about how Raf-1 is inactivated. Here, we used a proteomic approach to identify molecules that may inactivate Raf-1 signalling. Protein phosphatase 5 (PP5) was identified as an inactivator that associates with Raf-1 on growth factor stimulation and selectively dephosphorylates an essential activating site, Ser 338. The PP5-mediated dephosphorylation of Ser 338 inhibited Raf-1 activity and downstream signalling to MEK, an effect that was prevented by phosphomimetic substitution of Ser 338, or by ablation of PP5 catalytic function. Furthermore, depletion of endogenous PP5 increased cellular phospho-Ser 338 levels. Our results suggest that PP5 is a physiological regulator of Raf-1 signalling pathways.

Spatial regulation of RhoA activity during pancreatic cancer cell invasion driven by mutant p53

The ability to observe changes in molecular behavior during cancer cell invasion in vivo remains a major challenge to our understanding of the metastatic process. Here, we demonstrate for the first time, an analysis of RhoA activity at a subcellular level using FLIM-FRET (fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer) imaging in a live animal model of pancreatic cancer. In invasive mouse pancreatic ductal adenocarcinoma (PDAC) cells driven by mutant p53 (p53(R172H)), we observed a discrete fraction of high RhoA activity at both the leading edge and rear of cells in vivo which was absent in two-dimensional in vitro cultures. Notably, this pool of active RhoA was absent in noninvasive p53(fl) knockout PDAC cells, correlating with their poor invasive potential in vivo. We used dasatanib, a clinically approved anti-invasive agent that is active in this model, to illustrate the functional importance of spatially regulated RhoA. Dasatanib inhibited the activity of RhoA at the poles of p53(R172H) cells in vivo and this effect was independent of basal RhoA activity within the cell body. Taken together, quantitative in vivo fluorescence lifetime imaging illustrated that RhoA is not only necessary for invasion, but also that subcellular spatial regulation of RhoA activity, as opposed to its global activity, is likely to govern invasion efficiency in vivo. Our findings reveal the utility of FLIM-FRET in analyzing dynamic biomarkers during drug treatment in living animals, and they also show how discrete intracellular molecular pools might be differentially manipulated by future anti-invasive therapies.

Phosphatase and feedback regulation of Raf-1 signaling.

The Raf-1 kinase is an effector of Ras GTPases that lies at the apex of the three-tier Raf/MEK/ERK pathway. Raf-1 activation is a complex process that entails two major events – relief of autoinhibition imposed by the regulatory domain and kinase domain activation. Recent studies indicate that the transition of Raf-1 from an active to an inactive state bears similar complexity to the activation process. Both these events require dynamic changes in Raf-1 phosphorylation. Here, we discuss the critical role of phosphatases and feedback phosphorylation during activation and inactivation of Raf-1 signalling.

Proteomics and phosphoproteomics for the mapping of cellular signalling networks.

Proteomics is transitioning from inventory mapping to the mapping of functional cellular contexts. This has been enabled by progress in technologies as well as conceptual strategies. Here, we review recent advances in this area with focus on cellular signalling pathways. We discuss genetics‐based methods such as yeast two hybrid methods as well as biochemistry‐based methods such as two‐dimensional gel electrophoresis, quantitative proteomics, interaction proteomics, and phosphoproteomics. A central tenet is that by its ability to capture dynamic changes in protein expression, localisation and modification modern proteomics has become a powerful tool to map signal transduction pathways and deliver the functional information that will promote insights in cell biology and systems biology.

Dnmt3a and Dnmt3b Associate with Enhancers to Regulate Human Epidermal Stem Cell Homeostasis

The genome-wide localization and function of endogenous Dnmt3a and Dnmt3b in adult stem cells are unknown. Here, we show that in human epidermal stem cells, the two proteins bind in a histone H3K36me3-dependent manner to the most active enhancers and are required to produce their associated enhancer RNAs. Both proteins prefer super-enhancers associated to genes that either define the ectodermal lineage or establish the stem cell and differentiated states. However, Dnmt3a and Dnmt3b differ in their mechanisms of enhancer regulation: Dnmt3a associates with p63 to maintain high levels of DNA hydroxymethylation at the center of enhancers in a Tet2-dependent manner, whereas Dnmt3b promotes DNA methylation along the body of the enhancer. Depletion of either protein inactivates their target enhancers and profoundly affects epidermal stem cell function. Altogether, we reveal novel functions for Dnmt3a and Dnmt3b at enhancers that could contribute to their roles in disease and tumorigenesis.

HGF Induces Epithelial-to-Mesenchymal Transition by Modulating the Mammalian Hippo/MST2 and ISG15 Pathways

Epithelial to mesenchymal transition (EMT) is a fundamental cell differentiation/dedifferentiation process which is associated with dramatic morphological changes. Formerly polarized and immobile epithelial cells which form cell junctions and cobblestone-like cell sheets undergo a transition into highly motile, elongated, mesenchymal cells lacking cell-to-cell adhesions. To explore how the proteome is affected during EMT we profiled protein expression and tracked cell biological markers in Madin-Darby kidney epithelial cells undergoing hepatocyte growth factor (HGF) induced EMT. We were able to identify and quantify over 4000 proteins by mass spectrometry. Enrichment analysis of this revealed that expression of proteins associated with the ubiquitination machinery was induced, whereas expression of proteins regulating apoptotic pathways was suppressed. We show that both the mammalian Hippo/MST2 and the ISG15 pathways are regulated at the protein level by ubiquitin ligases. Inhibition of the Hippo pathway by overexpression of either ITCH or A-Raf promotes HGF-induced EMT. Conversely, ISG15 overexpression is sufficient to induce cell scattering and an elongated morphology without external stimuli. Thus, we demonstrate for the first time that the Hippo/MST2 and ISG15 pathways are regulated during growth-factor induced EMT.

FIH Regulates Cellular Metabolism through Hydroxylation of the Deubiquitinase OTUB1

The asparagine hydroxylase, factor inhibiting HIF (FIH), confers oxygen-dependence upon the hypoxia-inducible factor (HIF), a master regulator of the cellular adaptive response to hypoxia. Studies investigating whether asparagine hydroxylation is a general regulatory oxygen-dependent modification have identified multiple non-HIF targets for FIH. However, the functional consequences of this outside of the HIF pathway remain unclear. Here, we demonstrate that the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1) is a substrate for hydroxylation by FIH on N22. Mutation of N22 leads to a profound change in the interaction of OTUB1 with proteins important in cellular metabolism. Furthermore, in cultured cells, overexpression of N22A mutant OTUB1 impairs cellular metabolic processes when compared to wild type. Based on these data, we hypothesize that OTUB1 is a target for functional hydroxylation by FIH. Additionally, we propose that our results provide new insight into the regulation of cellular energy metabolism during hypoxic stress and the potential for targeting hydroxylases for therapeutic benefit.

Signalling by protein phosphatases and drug development

Protein modification cycles catalysed by opposing enzymes, such as kinases and phosphatases, form the backbone of signalling networks. Although, historically, kinases have been at the research forefront, a systems‐centred approach reveals predominant roles for phosphatases in controlling the network response times and spatio‐temporal profiles of signalling activities. Emerging evidence suggests that phosphatase kinetics are critical for network function and cell‐fate decisions. Protein phosphatases operate as both immediate and delayed regulators of signal transduction, capable of attenuating or amplifying signalling. This versatility of phosphatase action emphasizes the need for systems biology approaches to understand cellular signalling networks and predict the cellular outcomes of combinatorial drug interventions.