An allosteric interaction controls the activation mechanism of SHP2 tyrosine phosphatase

SHP2 is a protein tyrosine phosphatase (PTP) involved in multiple signaling pathways. Mutations of SHP2 can result in Noonan syndrome or pediatric malignancies. Inhibition of wild-type SHP2 represents a novel strategy against several cancers. SHP2 is activated by binding of a phosphopeptide to the N-SH2 domain of SHP2, thereby favoring dissociation of the N-SH2 domain and exposing the active site on the PTP domain. The conformational transitions controlling ligand affinity and PTP dissociation remain poorly understood. Using molecular simulations, we revealed an allosteric interaction restraining the N-SH2 domain into a SHP2-activating and a stabilizing state. Only ligands selecting for the activating N-SH2 conformation, depending on ligand sequence and binding mode, are effective activators. We validate the model of SHP2 activation by rationalizing modified basal activity and responsiveness to ligand stimulation of several N-SH2 variants. This study provides mechanistic insight into SHP2 activation and may open routes for SHP2 regulation

Discriminating between competing models for the allosteric regulation of oncogenic phosphatase SHP2 by characterizing its active state

The Src-homology 2 domain containing phosphatase 2 (SHP2) plays a critical role in crucial signaling pathways and is involved in oncogenesis and in developmental disorders. Its structure includes two SH2 domains (N-SH2 and C-SH2), and a protein tyrosine phosphatase (PTP) domain. Under basal conditions, SHP2 is auto-inhibited, with the N-SH2 domain blocking the PTP active site. Activation involves a rearrangement of the domains that makes the catalytic site accessible, coupled to the association between the SH2 domains and cognate proteins containing phosphotyrosines. Several aspects of this transition are debated and competing mechanistic models have been proposed. A crystallographic structure of SHP2 in an active state has been reported (PDB code 6crf), but several lines of evidence suggests that it is not fully representative of the conformations populated in solution. To clarify the structural rearrangements involved in SHP2 activation, enhanced sampling simulations of the autoinhibited and active states have been performed, for wild type SHP2 and its pathogenic E76K variant. Our results demonstrate that the crystallographic conformation of the active state is unstable in solution, and multiple interdomain arrangements are populated, thus allowing association to bisphosphorylated sequences. Contrary to a recent proposal, activation is coupled to the conformational changes of the N-SH2 binding site, which is significantly more accessible in the active sate, rather than to the structure of the central β-sheet of the domain. In this coupling, a previously undescribed role for the N-SH2 BG loop emerged

Targeting oncogenic Src homology 2 domain-containing phosphatase 2 (SHP2) by inhibiting its protein-protein interactions

We developed a new class of inhibitors of protein-protein interactions of the SHP2 phosphatase, which is pivotal in cell signaling and represents a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective for disease-associated SHP2 mutants. Considering that pathogenic lesions cause signaling hyperactivation due to increased levels of SHP2 association with cognate proteins, we developed peptide-based molecules with nanomolar affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation, and an affinity for pathogenic variants of SHP2 that is 2-20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool for investigating the role of protein-protein interactions in the function of SHP2

Structural characterization of the protein tyrosine phosphatase Shp2 in solution

Intracellular signalling cascades are mediated by a plethora of receptors, enzymes, adaptors and small molecules. The Protein Tyrosine Phosphatase (PTP) Shp2 is a highly conserved enzyme involved in a myriad of cellular processes including growth, differentiation and apoptosis. Shp2 is multi-domain protein composed of two SH2 domains in tandem, a PTP catalytic domain and a C-terminal tail containing multiple phosphorylation sites and a proline-rich region. The majority of biophysical research has utilised X-ray crystallography to study interactions and effects of mutations at the structural level. To gain a further understanding of Shp2 ligand binding and perturbations caused by disease relevant mutations, a structural investigation was performed with Nuclear Magnetic Resonance (NMR) spectroscopy and Small-angle X-ray Scattering (SAXS) in solution. The NMR signals from the backbone of both SH2 domains were assigned and residue-level interactions and differential SH2 domain specificities with peptides from the novel receptor G6b-B were delineated. In addition, the E76K point mutation that causes Noonan Syndrome and leukaemia was found to have increased conformational dynamics, the first experimental evidence of this phenomenon at the structural level

Structural and evolutionary relationships among protein tyrosine phosphatase domains

With the current access to the whole genomes of various organisms and the completion of the first draft of the human genome, there is a strong need for a structure-function classification of protein families as an initial step in moving from DNA databases to a comprehensive understanding of human biology. As a result of the explosion in nucleic acid sequence information and the concurrent development of methods for high-throughput functional characterization of gene products, the genomic revolution also promises to provide a new paradigm for drug discovery, enabling the identification of molecular drug targets in a significant number of human diseases. This molecular view of diseases has contributed to the importance of combining primary sequence data with three-dimensional structure and has increased the awareness of computational homology modeling and its potential to elucidate protein function. In particular, when important proteins or novel therapeutic targets are identified—like the family of protein tyrosine phosphatases (PTPs) (reviewed in reference 53)—a structure-function classification of such protein families becomes an invaluable framework for further advances in biomedical science. Here, we present a comparative analysis of the structural relationships among vertebrate PTP domains and provide a comprehensive resource for sequence analysis of phosphotyrosine-specific PTPs

Regulation der Enzymaktivität der Proteintyrosinphosphatase SHP-1 durch monophosphorylierte SH2-Domänen-Liganden

In vielen intrazellulären Signaltransduktionskaskaden, die z.B. Zellproliferation, Zelldifferenzierung und Zelltod regulieren, hat die reversible Phosphorylierung von Tyrosinresten in Proteinen eine entscheidende Rolle. Proteintyrosinkinasen (PTKs), die z.B. nach extrazellulärer Stimulation durch Wachstumsfaktoren oder Cytokine aktiviert werden, führen intrazellulär zu einer Erhöhung des Phosphotyrosingehaltes. Die Tyrosinphosphorylierten Proteine können u.a. Moleküle binden, die Phosphotyrosin-spezifische Proteindomänen (SH2-, PTB-, C2 Domänen) enthalten, und somit das Signal weiterleiten. Proteintyrosinphosphatasen, die Gegenspieler der PTKs, katalysieren die hydrolytische Spaltung der Phosphatgruppe. Diese Reaktion mündet entweder in der Abschaltung oder der Aktivierung einer Signalkette. Eine Regulation dieser Kaskaden durch spezifische Inhibitoren und/oder Aktivatoren eröffnet die Möglichkeit, selektiv bestimmte physiologische und pathophysiologische Prozesse zu beeinflussen. Die pharmazeutische Forschung hat deshalb in den vergangenen Jahren großes Interesse an der Entwicklung spezifischer Inhibitoren für Proteintyrosinkinasen und –phosphatasen gezeigt. Einen interessanten Untersuchungsgegenstand in diesem Zusammenhang stellen die Phosphotyrosin-bindenden Domänen, insbesondere die Gruppe der SH2 (Src Homology 2)- Domänen, dar. Die cytosolische Proteintyrosinphosphatase SHP-1 enthält neben der katalytischen Domäne zwei SH2-Domänen. Das Enzym wird sowohl in hämatopoetischen als auch in epithelialen Zellen exprimiert und ist dort in verschiedene Signalwege involviert. Die Enzymaktivität von SHP-1 wird allosterisch über die SH2-Domänen, insbesondere die Nterminale SH2-Domäne, reguliert. Die Phosphatase kann u.a. effizient durch die Bindung eines Phosphotyrosin-haltigen Liganden an die N-SH2-Domäne aktiviert werden. Bisher ist jedoch nur unzureichend bekannt, welche strukturellen und konformationellen Determinanten eine effiziente Assoziation der Liganden bewirken und welche Aminosäuren nach Interaktion mit der Domäne die Aktivierung des Enzyms auslösen. Diese Informationen sind jedoch entscheidend für die Entwicklung von Molekülen, die die über die SH2-Domäne vermittelte Enzymaktivierung einschränken bzw. unterbinden. Mit Hilfe synthetischer Phosphopeptide sollen in dieser Arbeit die strukturellen Anforderungen für die Bindung an die N-SH2-Domäne sowie der entsprechende Aktivierungsmechanismus von SHP-1 detaillierter untersucht werden, um damit eine Grundlage für die Entwicklung von spezifischen SHP-1-Effektoren zu schaffen

Mathematical modelling of shear stress signalling in endothelial cells.

In recent years it has become clear that cell signalling pathways are not simple linear chains of events as was once thought but frequently diverge, converge and employ positive and negative feedback. As a result signals show a complex pattern of spatial and temporal activity that is difficult to explain despite a wealth of experimental data. Systems Biology attempts to predict and understand the behaviour of complex systems by integrating information from diverse sources and principles drawn from a large number of different scientific disciplines. The signalling pathways regulating endothelial responses to shear stress have been extensively studied, since perturbed fluid flow contributes significantly to the development of heart disease. Shear stress activates many signals in endothelial cells, from ion influxes to protein phosphorylation and gene expression, and induces changes in endothelial morphology. Here a modelling and Systems Biology approach was taken to investigate and understand better the endothelial signal transduction networks that convert fluid flow stimulation into biochemical signals. A static signal transduction network was built from integrin cell surface receptors to activation of the tyrosine kinases focal adhesion kinase (FAK) and Src. Parameters for each reaction in this network were collected from the literature or, when necessary, estimated. To model how fluid flow initiates signalling in this network, the shear stress-induced calcium influx and the viscoelastic response of transmembrane receptors such as integrins to mechanical force were examined by means of mathematical modelling, using ordinary differential equations. These effects were used as primary activators of the shear stress response in endothelial cells, allowing quantitative analysis of the intracellular signal transduction flow which propagates from integrin to paxillin, FAK and Src activation. The magnitude and dependencies of each influence were examined individually and in conjunction with each other. The model was used to investigate the role and dynamic regulation of previously unstudied molecules in the network and the simulated results were compared against experimental data in order to validate hypotheses and increase our understanding of the molecular processes underlying the shear stress response

Epidermal growth factor receptor pathway phosphorylation dynamics

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2010.Cataloged from PDF version of thesis.Includes bibliographical references (p. 157-168).The epidermal growth factor receptor (EGFR, also known as ErbB 1) is a prototypical receptor tyrosine kinase (RTK) that activates multi-kinase phosphorylation cascades to regulate diverse cellular processes, including proliferation, migration and differentiation. ErbB 1 heterooligomerizes with three close homologues: ErbB2, ErbB3 and ErbB4. ErbB1-3 receptors are frequently mutated, overexpressed or activated by autocrine or paracrine ligand production in solid tumors and have been the target of extensive drug discovery efforts. Multiple small molecule kinase inhibitors and therapeutic antibodies against ErbB receptors are in clinical use or development. Despite their importance as RTKs, oncogenes and drug targets, regulation of ErbB receptors by the interplay of conformational change, phosphorylation, phosphatases and receptor trafficking remains poorly understood, and the impact of these dynamics on physiological activity and cellular responses to anti-ErbB drugs is largely unknown. This thesis investigates the dynamic opposition of kinases and phosphatases within the ErbB pathway. By standard biochemical analysis, ErbB receptors and downstream proteins appear to become phosphorylated and then dephosphorylated in approximately 30 minutes. However, pulse chase experiments where cells are exposed to ligand and then to small molecule kinase inhibitors reveal that individual proteins must in fact cycle rapidly between being phosphorylated and dephosphorylated in seconds. We construct a succession of differential equation-based models of varying biochemical resolution, each model appropriate for analyzing a different aspect of ErbB regulation, to help interpret the data and gain quantitative insight into receptor and drug biology. Rapid phosphorylation and dephosphorylation of receptors has important implications for the assembly dynamics of signalosomes. We find that signals are rapidly propagated through some downstream pathways but slowly through others, resulting in prolonged activation in the absence of upstream signal. We show that fast phosphorylation/dephosphorylation may provide cells with the flexibility necessary to rapidly detect and respond to changes in their extracellular environment. These fast dynamics also play a crucial role in determining the response to ErbB 1-targeting cancer therapies, which we find to vary significantly between drugs with different mechanisms of action. We show that treatment with one class of these drugs results in sustained signaling, instead of inhibition, and thus may actually promote tumor proliferation or invasion. Our work may help explain why certain drugs have been more effective in patients than others and suggests new approaches for evaluating biochemical signaling networks and targeted therapeutics.By Laura B. Kleiman.Ph.D

Synthese und biologische Wirksamkeit Abbau-stabilisierter Liganden der SH2-Domäne der Protein-Tyrosin-Phosphatase SHP-1

In Signaltransduktionsprozessen wird die Aktivität vieler Proteine hauptsächlich durch Phosphorylierung und Dephosphorylierung reguliert. Kinasen und Phosphatasen sind die Gegenspieler in diesem fein abgestimmten Regulationsprozess. Bezogen auf die Tyrosinphosphorylierung sind die konkreten Gegenspieler Proteintyrosinkinasen (PTKs) und Proteintyrosinphosphatasen (PTPs). Vertreter dieser Enzymfamilie sind an der Regulation zahlreicher biologischer Prozesse beteiligt. So ist die Proteintyrosinphosphatase SHP-1 ein negativer Regulator multipler Signaltransduktionprozesse. Sie gehört zur Gruppe der intrazellulären Phosphatasen und kommt vor allem in Zellen des Immunsystems, aber auch in Nervenzellen sowie in Spermien vor. Die SHP-1 spielt vor allem im Reifungsprozess dieser Zellen eine Rolle [1,2]. Die Aktivierung der SHP-1 erfolgt durch Bindung von Liganden an ihre N-terminale SH2-Domäne. Im ligandenfreien Zustand blockiert die SH2-Domäne das katalytische Zentrum der SHP-1. Die Bindung eines Liganden führt zu einer Konformationsänderung der SH2-Domäne und damit zur Freigabe der katalytischen Domäne. Für die physiologischen Funktionen ist die Bindung der SH2-Domäne an das ITIM-Motiv wesentlich [3]. Wegen ihrer entscheidenden Rolle in der Signaltransduktion sind Kinasen und Phosphatasen interessante Zielproteine für potentielle Arzneimittel. Dazu können entweder ihre aktiven Zentren gehemmt oder die Bindedomänen (SH2, PTB, IP3) durch aktivierende oder inaktivierende Liganden besetzt werden [4]. Voraussetzung für diese sogenannte Signaltransduktionstherapie ist die Internalisierung der Inhibitoren oder Liganden in die entsprechenden Zellen. Das kann mit Hilfe von untoxischen Penetrationshelfern, z.B. Zell-penetrierenden Peptiden, gelingen, setzt aber eine Stabilisierung der Inhibitoren oder Liganden gegen enzymatischen Abbau voraus. Im Falle der sehr selektiv wirksamen, aber leicht abbaubaren Peptide erfordert das eine Stabilisierung gegen Proteasen und möglichst auch gegen Phosphatasen. Dazu können entweder nichtproteinogene Aminosäuren in die Peptide eingebaut oder die Peptide zyklisiert werden [5, 6, 7]. Eine besonders effektive, aber synthetisch sehr anspruchsvolle Methode ist dabei die Rückgratzyklisierung. Abgeleitet vom Modelling eines angepassten Liganden für die SH2-Domäne der SHP-1 und einer angestrebten Phosphatasestabilität des Phosphotyrosin-Restes wurde von uns die Rückgratzyklisierung solcher Oktapeptiliganden versucht, bei denen die Lactambrücke am Phosphotyrosin beginn