Andrea Ciliberto

Steady States and Oscillations in the p53/Mdm2 Network

p53 is activated in response to events compromising the genetic integrity of a cell. Recent data show that p53 activity does not increase steadily with genetic damage but rather fluctuates in an oscillatory fashion (Lahav et al., Nature Genetics, 36, 147-150, 2004). Theoretical studies suggest that oscillations can arise from a combination of positive and negative feedbacks or from a long negative feedback loop alone. Both negative and positive feedbacks are present in the p53/Mdm2 network, but it is not known what roles they play in the oscillatory response to DNA damage. We developed a mathematical model of p53 oscillations based on positive and negative feedbacks in the p53/Mdm2 network. According to the model, the system reacts to DNA damage by moving from a stable steady state into a region of stable limit cycles. Oscillations in the model are born with large amplitude, which guarantees an all-or-none response to damage. As p53 oscillates, damage is repaired and the system moves back to a stable steady state with low p53 activity. The model reproduces experimental data in quantitative detail. We suggest new experiments for dissecting the contributions of negative and positive feedbacks to the generation of oscillations.

Regulation of cell shape by Cdc42 is mediated by the synergic actin-bundling activity of the Eps8-IRSp53 complex.

Actin-crosslinking proteins organize actin into highly dynamic and architecturally diverse subcellular scaffolds that orchestrate a variety of mechanical processes, including lamellipodial and filopodial protrusions in motile cells. How signalling pathways control and coordinate the activity of these crosslinkers is poorly defined. IRSp53, a multi-domain protein that can associate with the Rho-GTPases Rac and Cdc42, participates in these processes mainly through its amino-terminal IMD (IRSp53 and MIM domain). The isolated IMD has actin-bundling activity in vitro and is sufficient to induce filopodia in vivo. However, the manner of regulation of this activity in the full-length protein remains largely unknown. Eps8 is involved in actin dynamics through its actin barbed-ends capping activity and its ability to modulate Rac activity. Moreover, Eps8 binds to IRSp53. Here, we describe a novel actin crosslinking activity of Eps8. Additionally, Eps8 activates and synergizes with IRSp53 in mediating actin bundling in vitro, enhancing IRSp53-dependent membrane extensions in vivo. Cdc42 binds to and controls the cellular distribution of the IRSp53–Eps8 complex, supporting the existence of a Cdc42–IRSp53–Eps8 signalling pathway. Consistently, Cdc42-induced filopodia are inhibited following individual removal of either IRSp53 or Eps8. Collectively, these results support a model whereby the synergic bundling activity of the IRSp53–Eps8 complex, regulated by Cdc42, contributes to the generation of actin bundles, thus promoting filopodial protrusions.

Evidence that Aurora B is implicated in spindle checkpoint signalling independently of error correction

Fidelity of chromosome segregation is ensured by a tension‐dependent error correction system that prevents stabilization of incorrect chromosome–microtubule attachments. Unattached or incorrectly attached chromosomes also activate the spindle assembly checkpoint, thus delaying mitotic exit until all chromosomes are bioriented. The Aurora B kinase is widely recognized as a component of error correction. Conversely, its role in the checkpoint is controversial. Here, we report an analysis of the role of Aurora B in the spindle checkpoint under conditions believed to uncouple the effects of Aurora B inhibition on the checkpoint from those on error correction. Partial inhibition of several checkpoint and kinetochore components, including Mps1 and Ndc80, strongly synergizes with inhibition of Aurora B activity and dramatically affects the ability of cells to arrest in mitosis in the presence of spindle poisons. Thus, Aurora B might contribute to spindle checkpoint signalling independently of error correction. Our results support a model in which Aurora B is at the apex of a signalling pyramid whose sensory apparatus promotes the concomitant activation of error correction and checkpoint signalling pathways.

Modeling Networks of Coupled Enzymatic Reactions Using the Total Quasi-Steady State Approximation

In metabolic networks, metabolites are usually present in great excess over the enzymes that catalyze their interconversion, and describing the rates of these reactions by using the Michaelis–Menten rate law is perfectly valid. This rate law assumes that the concentration of enzyme–substrate complex (C) is much less than the free substrate concentration (S 0). However, in protein interaction networks, the enzymes and substrates are all proteins in comparable concentrations, and neglecting C with respect to S 0 is not valid. Borghans, DeBoer, and Segel developed an alternative description of enzyme kinetics that is valid when C is comparable to S 0. We extend this description, which Borghans et al. call the total quasi-steady state approximation, to networks of coupled enzymatic reactions. First, we analyze an isolated Goldbeter–Koshland switch when enzymes and substrates are present in comparable concentrations. Then, on the basis of a real example of the molecular network governing cell cycle progression, we couple two and three Goldbeter–Koshland switches together to study the effects of feedback in networks of protein kinases and phosphatases. Our analysis shows that the total quasi-steady state approximation provides an excellent kinetic formalism for protein interaction networks, because (1) it unveils the modular structure of the enzymatic reactions, (2) it suggests a simple algorithm to formulate correct kinetic equations, and (3) contrary to classical Michaelis–Menten kinetics, it succeeds in faithfully reproducing the dynamics of the network both qualitatively and quantitatively.

In Vitro FRAP Identifies the Minimal Requirements for Mad2 Kinetochore Dynamics

BACKGROUND Mad1 and Mad2 are constituents of the spindle-assembly checkpoint, a device coupling the loss of sister-chromatid cohesion at anaphase to the completion of microtubule attachment of the sister chromatids at metaphase. Fluorescence recovery after photobleaching (FRAP) revealed that the interaction of cytosolic Mad2 with kinetochores is highly dynamic, suggesting a mechanism of catalytic activation of Mad2 at kinetochores followed by its release in a complex with Cdc20. The recruitment of cytosolic Mad2 to kinetochores has been attributed to a stable receptor composed of a distinct pool of Mad2 tightly bound to Mad1. Whether specifically this interaction accounts for the kinetochore dynamics of Mad2 is currently unknown. RESULTS To gain a precise molecular understanding of the interaction of Mad2 with kinetochores, we reconstituted the putative Mad2 kinetochore receptor and developed a kinetochore recruitment assay with purified components. When analyzed by FRAP in vitro, this system faithfully reproduced the previously described in vivo dynamics of Mad2, providing an unequivocal molecular account of the interaction of Mad2 with kinetochores. Using the same approach, we dissected the mechanism of action of p31(comet), a spindle-assembly checkpoint inhibitor. CONCLUSIONS In vitro FRAP is a widely applicable approach to dissecting the molecular bases of the interaction of a macromolecule with an insoluble cellular scaffold. The combination of in vitro fluorescence recovery after photobleaching with additional fluorescence-based assays in vitro can be used to unveil mechanism, stoichiometry, and kinetic parameters of a macromolecular interaction, all of which are important for modeling protein interaction networks.

Bub3 reads phosphorylated MELT repeats to promote spindle assembly checkpoint signaling

Regulation of macromolecular interactions by phosphorylation is crucial in signaling networks. In the spindle assembly checkpoint (SAC), which enables errorless chromosome segregation, phosphorylation promotes recruitment of SAC proteins to tensionless kinetochores. The SAC kinase Mps1 phosphorylates multiple Met-Glu-Leu-Thr (MELT) motifs on the kinetochore subunit Spc105/Knl1. The phosphorylated MELT motifs (MELTP) then promote recruitment of downstream signaling components. How MELTP motifs are recognized is unclear. In this study, we report that Bub3, a 7-bladed β-propeller, is the MELTP reader. It contains an exceptionally well-conserved interface that docks the MELTP sequence on the side of the β-propeller in a previously unknown binding mode. Mutations targeting the Bub3 interface prevent kinetochore recruitment of the SAC kinase Bub1. Crucially, they also cause a checkpoint defect, showing that recognition of phosphorylated targets by Bub3 is required for checkpoint signaling. Our data provide the first detailed mechanistic insight into how phosphorylation promotes recruitment of checkpoint proteins to kinetochores. DOI: http://dx.doi.org/10.7554/eLife.01030.001

Mathematical model of the morphogenesis checkpoint in budding yeast

The morphogenesis checkpoint in budding yeast delays progression through the cell cycle in response to stimuli that prevent bud formation. Central to the checkpoint mechanism is Swe1 kinase: normally inactive, its activation halts cell cycle progression in G2. We propose a molecular network for Swe1 control, based on published observations of budding yeast and analogous control signals in fission yeast. The proposed Swe1 network is merged with a model of cyclin-dependent kinase regulation, converted into a set of differential equations and studied by numerical simulation. The simulations accurately reproduce the phenotypes of a dozen checkpoint mutants. Among other predictions, the model attributes a new role to Hsl1, a kinase known to play a role in Swe1 degradation: Hsl1 must also be indirectly responsible for potent inhibition of Swe1 activity. The model supports the idea that the morphogenesis checkpoint, like other checkpoints, raises the cell size threshold for progression from one phase of the cell cycle to the next.

Threshold‐controlled ubiquitination of the EGFR directs receptor fate

How the cell converts graded signals into threshold‐activated responses is a question of great biological relevance. Here, we uncover a nonlinear modality of epidermal growth factor receptor (EGFR)‐activated signal transduction, by demonstrating that the ubiquitination of the EGFR at the PM is threshold controlled. The ubiquitination threshold is mechanistically determined by the cooperative recruitment of the E3 ligase Cbl, in complex with Grb2, to the EGFR. This, in turn, is dependent on the simultaneous presence of two phosphotyrosines, pY1045 and either one of pY1068 or pY1086, on the same EGFR moiety. The dose–response curve of EGFR ubiquitination correlate precisely with the non‐clathrin endocytosis (NCE) mode of EGFR internalization. Finally, EGFR‐NCE mechanistically depends on EGFR ubiquitination, as the two events can be simultaneously re‐engineered on a phosphorylation/ubiquitination‐incompetent EGFR backbone. Since NCE controls the degradation of the EGFR, our findings have implications for how the cell responds to increasing levels of EGFR signalling, by varying the balance of receptor signalling and degradation/attenuation.

The influence of catalysis on mad2 activation dynamics.

Mad2 is a key component of the spindle assembly checkpoint, a safety device ensuring faithful sister chromatid separation in mitosis. The target of Mad2 is Cdc20, an activator of the anaphase-promoting complex/cyclosome (APC/C). Mad2 binding to Cdc20 is a complex reaction that entails the conformational conversion of Mad2 from an open (O-Mad2) to a closed (C-Mad2) conformer. Previously, it has been hypothesized that the conversion of O-Mad2 is accelerated by its conformational dimerization with C-Mad2. This hypothesis, known as the Mad2-template hypothesis, is based on the unproven assumption that the natural conversion of O-Mad2 required to bind Cdc20 is slow. Here, we provide evidence for this fundamental assumption and demonstrate that conformational dimerization of Mad2 accelerates the rate of Mad2 binding to Cdc20. On the basis of our measurements, we developed a set of rate equations that deliver excellent predictions of experimental binding curves under a variety of different conditions. Our results strongly suggest that the interaction of Mad2 with Cdc20 is rate limiting for activation of the spindle checkpoint. Conformational dimerization of Mad2 is essential to accelerate Cdc20 binding, but it does not modify the equilibrium of the Mad2:Cdc20 interaction, i.e., it is purely catalytic. These results surpass previously formulated objections to the Mad2-template model and predict that the release of Mad2 from Cdc20 is an energy-driven process.

Low duplicability and network fragility of cancer genes

We identified genomic and network properties of approximately 600 genes mutated in different cancer types. These genes tend not to duplicate but, unlike most human singletons, they encode central hubs of highly interconnected modules within the protein-protein interaction network (PIN). We find that cancer genes are fragile components of the human gene repertoire, sensitive to dosage modification. Furthermore, other nodes of the human PIN with similar properties are rare and probably enriched in candidate cancer genes.