Lan K. Nguyen

When ubiquitination meets phosphorylation: a systems biology perspective of EGFR/MAPK signalling

Ubiquitination, the covalent attachment of ubiquitin to target proteins, has emerged as a ubiquitous post-translational modification (PTM) whose function extends far beyond its original role as a tag for protein degradation identified three decades ago. Although sharing parallel properties with phosphorylation, ubiquitination distinguishes itself in important ways. Nevertheless, the interplay and crosstalk between ubiquitination and phosphorylation events have become a recurrent theme in cell signalling regulation. Understanding how these two major PTMs intersect to regulate signal transduction is an important research question. In this review, we first discuss the involvement of ubiquitination in the regulation of the EGF-mediated ERK signalling pathway via the EGF receptor, highlighting the interplay between ubiquitination and phosphorylation in this cancer-implicated system and addressing open questions. The roles of ubiquitination in pathways crosstalking to EGFR/MAPK signalling will then be discussed. In the final part of the review, we demonstrate the rich and versatile dynamics of crosstalk between ubiquitination and phosphorylation by using quantitative modelling and analysis of network motifs commonly observed in cellular processes. We argue that given the overwhelming complexity arising from inter-connected PTMs, a quantitative framework based on systems biology and mathematical modelling is needed to efficiently understand their roles in cell signalling.

A dynamic model of the hypoxia-inducible factor 1α (HIF-1α) network

Summary Activation of the hypoxia-inducible factor (HIF) pathway is a critical step in the transcriptional response to hypoxia. Although many of the key proteins involved have been characterised, the dynamics of their interactions in generating this response remain unclear. In the present study, we have generated a comprehensive mathematical model of the HIF-1&agr; pathway based on core validated components and dynamic experimental data, and confirm the previously described connections within the predicted network topology. Our model confirms previous work demonstrating that the steps leading to optimal HIF-1&agr; transcriptional activity require sequential inhibition of both prolyl- and asparaginyl-hydroxylases. We predict from our model (and confirm experimentally) that there is residual activity of the asparaginyl-hydroxylase FIH (factor inhibiting HIF) at low oxygen tension. Furthermore, silencing FIH under conditions where prolyl-hydroxylases are inhibited results in increased HIF-1&agr; transcriptional activity, but paradoxically decreases HIF-1&agr; stability. Using a core module of the HIF network and mathematical proof supported by experimental data, we propose that asparaginyl hydroxylation confers a degree of resistance upon HIF-1&agr; to proteosomal degradation. Thus, through in vitro experimental data and in silico predictions, we provide a comprehensive model of the dynamic regulation of HIF-1&agr; transcriptional activity by hydroxylases and use its predictive and adaptive properties to explain counter-intuitive biological observations.

Bistability in the Rac1, PAK, and RhoA signaling network drives actin cytoskeleton dynamics and cell motility switches

Summary Dynamic interactions between RhoA and Rac1, members of the Rho small GTPase family, play a vital role in the control of cell migration. Using predictive mathematical modeling, mass spectrometry-based quantitation of network components, and experimental validation in MDA-MB-231 mesenchymal breast cancer cells, we show that a network containing Rac1, RhoA, and PAK family kinases can produce bistable, switch-like responses to a graded PAK inhibition. Using a small chemical inhibitor of PAK, we demonstrate that cellular RhoA and Rac1 activation levels respond in a history-dependent, bistable manner to PAK inhibition. Consequently, we show that downstream signaling, actin dynamics, and cell migration also behave in a bistable fashion, displaying switches and hysteresis in response to PAK inhibition. Our results demonstrate that PAK is a critical component in the Rac1-RhoA inhibitory crosstalk that governs bistable GTPase activity, cell morphology, and cell migration switches.

Signalling by protein phosphatases and drug development: a systems‐centred view

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.

The ErbB4 CYT2 variant protects EGFR from ligand-induced degradation to enhance cancer cell motility

Dimerization of EGFR with an ErbB4 receptor variant increases growth factor–induced migration of breast cancer cells. Drug Resistance Through Dimerization The epidermal growth factor receptor (EGFR) is often targeted in various cancers, including breast cancer. The EGFR can dimerize with related receptors in the ErbB family, and formation of these heterodimers is associated with the development of resistance to EGFR inhibitors. Kiuchi et al. found that binding of EGFR to a naturally occurring variant of the receptor ErbB4 prevented a ubiquitin E3 ligase from associating with EGFR and triggering its breakdown. The migration of breast cancer cells to EGFR ligands was increased when EGFR was overexpressed with the ErbB4 variant, but not with a mutant that could not dimerize with EGFR. Furthermore, the transcript for this ErbB4 variant was increased in a subset of breast cancer patients. The epidermal growth factor receptor (EGFR) is a member of the ErbB family that can promote the migration and proliferation of breast cancer cells. Therapies that target EGFR can promote the dimerization of EGFR with other ErbB receptors, which is associated with the development of drug resistance. Understanding how interactions among ErbB receptors alter EGFR biology could provide avenues for improving cancer therapy. We found that EGFR interacted directly with the CYT1 and CYT2 variants of ErbB4 and the membrane-anchored intracellular domain (mICD). The CYT2 variant, but not the CYT1 variant, protected EGFR from ligand-induced degradation by competing with EGFR for binding to a complex containing the E3 ubiquitin ligase c-Cbl and the adaptor Grb2. Cultured breast cancer cells overexpressing both EGFR and ErbB4 CYT2 mICD exhibited increased migration. With molecular modeling, we identified residues involved in stabilizing the EGFR dimer. Mutation of these residues in the dimer interface destabilized the complex in cells and abrogated growth factor–stimulated cell migration. An exon array analysis of 155 breast tumors revealed that the relative mRNA abundance of the ErbB4 CYT2 variant was increased in ER+ HER2– breast cancer patients, suggesting that our findings could be clinically relevant. We propose a mechanism whereby competition for binding to c-Cbl in an ErbB signaling heterodimer promotes migration in response to a growth factor gradient.

Hypoxia-inducible factor (HIF) network: insights from mathematical models

Oxygen is a crucial molecule for cellular function. When oxygen demand exceeds supply, the oxygen sensing pathway centred on the hypoxia inducible factor (HIF) is switched on and promotes adaptation to hypoxia by up-regulating genes involved in angiogenesis, erythropoiesis and glycolysis. The regulation of HIF is tightly modulated through intricate regulatory mechanisms. Notably, its protein stability is controlled by the oxygen sensing prolyl hydroxylase domain (PHD) enzymes and its transcriptional activity is controlled by the asparaginyl hydroxylase FIH (factor inhibiting HIF-1).To probe the complexity of hypoxia-induced HIF signalling, efforts in mathematical modelling of the pathway have been underway for around a decade. In this paper, we review the existing mathematical models developed to describe and explain specific behaviours of the HIF pathway and how they have contributed new insights into our understanding of the network. Topics for modelling included the switch-like response to decreased oxygen gradient, the role of micro environmental factors, the regulation by FIH and the temporal dynamics of the HIF response. We will also discuss the technical aspects, extent and limitations of these models. Recently, HIF pathway has been implicated in other disease contexts such as hypoxic inflammation and cancer through crosstalking with pathways like NFκ B and mTOR. We will examine how future mathematical modelling and simulation of interlinked networks can aid in understanding HIF behaviour in complex pathophysiological situations. Ultimately this would allow the identification of new pharmacological targets in different disease settings.

Switches, excitable responses and oscillations in the Ring1B/Bmi1 ubiquitination system.

In an active, self-ubiquitinated state, the Ring1B ligase monoubiquitinates histone H2A playing a critical role in Polycomb-mediated gene silencing. Following ubiquitination by external ligases, Ring1B is targeted for proteosomal degradation. Using biochemical data and computational modeling, we show that the Ring1B ligase can exhibit abrupt switches, overshoot transitions and self-perpetuating oscillations between its distinct ubiquitination and activity states. These different Ring1B states display canonical or multiply branched, atypical polyubiquitin chains and involve association with the Polycomb-group protein Bmi1. Bistable switches and oscillations may lead to all-or-none histone H2A monoubiquitination rates and result in discrete periods of gene (in)activity. Switches, overshoots and oscillations in Ring1B catalytic activity and proteosomal degradation are controlled by the abundances of Bmi1 and Ring1B, and the activities and abundances of external ligases and deubiquitinases, such as E6-AP and USP7.

A Review of Systems Biology Perspective on Genetic Regulatory Networks with Examples

We present an extensive review of genetic regulatory networks (GRNs) from a system biology perspective, and discuss pertinent research issues related to GRNs such as roles of feedback loops, and internal and external noise. A suc- cinct review of mathematical modelling is also given as mathematical modelling is central to understand the complex mo- lecular living systems. We discuss tryptophan production system in Escherichia coli bacteria, as an example, to illustrate how feedback loops originate, and finally we present a development of a mathematical model of circadian rhythms of Drosophila (fruit fly) as a comprehensive illustration of building a system model from components. We attempt to inte- grate experimentally acquired molecular biology knowledge in the model developments and discussions to emphasise the importance of being true to the data and insights in the experimental molecular biology. The experimental knowledge and insights should drive the efforts in development of mathematical and computational models of living systems, not the other way around, and this is a challenging task.

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.

Regulation of oscillation dynamics in biochemical systems with dual negative feedback loops

Feedback controls are central to cellular regulation. Negative-feedback mechanisms are well known to underline oscillatory dynamics. However, the presence of multiple negative-feedback mechanisms is common in oscillatory cellular systems, raising intriguing questions of how they cooperate to regulate oscillations. In this work, we studied the dynamical properties of a set of general biochemical motifs with dual, nested negative-feedback structures. We showed analytically and then confirmed numerically that, in these motifs, each negative-feedback loop exhibits distinctly different oscillation-controlling functions. The longer, outer feedback loop was found to promote oscillations, whereas the short, inner loop suppresses and can even eliminate oscillations. We found that the position of the inner loop within the coupled motifs affects its repression strength towards oscillatory dynamics. Bifurcation analysis indicated that emergence of oscillations may be a strict parametric requirement and thus evolutionarily tricky. Investigation of the quantitative features of oscillations (i.e. frequency, amplitude and mean value) revealed that coupling negative feedback provides robust tuning of the oscillation dynamics. Finally, we demonstrated that the mitogen-activated protein kinase (MAPK) cascades also display properties seen in the general nested feedback motifs. The findings and implications in this study provide novel understanding of biochemical negative-feedback regulation in a mixed wiring context.