What Role Do Phosphatases Play in Signal Transduction Pathways

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FEBS J.
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Signalling by protein phosphatases and drug evolution: a systems-centred view

Abstract

Protein-modification cycles catalysed past opposing enzymes, such as kinases and phosphatases form the backbone of signalling networks. Whereas historically, kinases take been at the research forefront, a systems-centred approach reveals predominant roles of phosphatases in controlling the network response times and the spatiotemporal profiles of signalling activities. Emerging evidence suggests that phosphatase kinetics are critical for the network function and cell-fate decisions. Protein phosphatases can operate equally both immediate and delayed regulators of signal transduction, capable of attenuating or amplifying signalling. This versatility of phosphatase action emphasises the demand for systems biology approaches to encompass cellular signalling networks and predict the cellular outcomes of combinatorial drug interventions.

Keywords:

phosphatases, spatiotemporal dynamics, signalling cascades, systems biological science, drug discovery

Introduction

Multiple external cues including growth factors, cytokines and mechanical forces activate plasma membrane receptors such as receptor tyrosine kinases (RTKs), Thousand-protein coupled receptors and other receptor families. This creates spatiotemporal phosphorylation patterns which propagate through a wired network of signalling proteins and cascades. Frequently combined with intrinsic intracellular signalling, this tangled network non but transmits, but too processes and decodes the external data and gives rising to cellular response. For instance, signals from different receptors are integrated through common targets and pathway crosstalk, such as in cell cycle command [1, 2] or mTOR-mediated signalling [3]. Phosphatases play a vital function in cellular signalling by decision-making both the network dynamics and spatial localisation of phosphoproteins [iv, 5]. Although kinases have previously been in the limelight of scientific involvement, poly peptide tyrosine phosphatases (PTPs) and Ser/Thr phosphatases (PSPs) are becoming increasingly explored both as a enquiry topic and targets for drug development.

Phosphatases deed as both immediate and delayed negative regulators of protein phosphorylation, and this oftentimes results in attenuation or termination of bespeak transfer. Constitutive phosphatase activities shape the initial phosphorylation profiles of receptors, phosphorylated adaptors, Ser/Thr kinases and other signalling proteins, following transient activation by growth factors or other signals. These firsthand signalling responses develop very rapidly on the scale of second and minutes, and as we will see below phosphatases can play a dominant role in setting the spatiotemporal behaviour of protein phosphorylation in the cell. Induced phosphatase activities often create negative feedback loops, which accommodate cells to a more than permanent external stimulation, persisting on the timescale of hours. Interestingly, inhibition of PTPs by reactive oxygen species induced past activated RTKs, such equally epidermal growth factor receptor (EGFR) can create likewise positive feedback loops that facilitate the lateral propagation of EGFR phosphorylation at the plasma membrane [6]. This multifaceted part of phosphatases in the signalling control is illustrated by many examples, for instance, past the dual role of the SH2-domain containing poly peptide tyrosine phosphatase-ii (SHP2) in activation of the extracellular signal regulated kinase (ERK) which we discuss in detail beneath [seven–nine].

Compared to kinases, the progress towards agreement of the regulation of phosphatases was lagging behind due to technical challenges. For example, information technology is simply possible to assay the activeness of a given protein phosphatase
in vitro
if 1 already has the relevant substrate phosphorylated by a relevant poly peptide kinase. In fact, in the human being genome the numbers of dissimilar PTPs and RTKs are very similar, implying that versatility and specificity of the functions of these kinases and phosphatases tin can also be like [ten]. Although catalytic subunits of PSPs accept overlapping targets, the substrate specificity of PSPs is ofttimes achieved through their regulatory subunits [11, 12]. Different regulatory and scaffolding subunits recruit a catalytic subunit to specific sub-cellular locales where different targets reside. Individual ternary PSP complexes assembled in these locales have differential catalytic activities and endow a particular PTP with proper substrate specificities. In this review we focus on how substrate specificity is controlled for phosphatases of the PSP family.

Historically, kinases have been major drug targets for cancer and other diseases. All the same, versatility of phosphatase functions and their involvement in multiple feedback mechanism makes phosphatases attractive targets for time to come drug development. Nosotros will discuss how PSPs are advancing to the forefront of drug development. To demonstrate the potential of systems biology approaches in facilitating the selection of therapeutic targets, we develop a simplified mathematical model of the EGFR/SHP2 signalling pathway and explore
in silico
phosphatase-based therapies vs receptor inhibition. Both theoretical and experimental studies focusing on agreement roles of phosphatases in controlling the spatiotemporal dynamics of signalling networks will exist discussed. We will likewise show how phosphatase dynamics are regulated past the transcriptional machinery and how such transcriptional feedback loops control the entire signalling system in the context of mitogen-activated poly peptide kinase cascades.

Phosphatases shape temporal dynamics of signalling cascades

Betoken transduction via cascades of phosphorylation/dephosphorylation cycles is a hallmark of prison cell signalling. The highly conserved mitogen-activated protein kinase (MAPK) cascades, which have been extensively studied, control a range of important physiological processes, including proliferation, differentiation and apoptosis [thirteen, fourteen]. MAPK cascades consist of iii sequential levels, with phosphorylation and subsequent dephosphorylation catalysed by a kinase from a preceding level and a phosphatase at a given level, respectively.

Activity of signalling cascades such every bit the MAPK network can exist characterised by a number of key features, notably amplitude and duration of the signal output, both of which comport relevant physiological impact. Signal amplitude of MAPK activation exceeding a certain threshold was found as a requirement for the proliferation of fibroblasts [xv]. While on the other hand, the duration of MAPK activity in PC12 cells dictates whether the cells would proliferate or differentiate [xvi]. Moreover, rapid and transient MAPK activation in rat hepatocytes promotes the G1-South cell-cycle progression while prolonged MAPK activation inhibits this process [17]. By influencing unlike repertoires of target genes, the amplitude and duration of MAPK activation are disquisitional in determining prison cell responses [16–xix], and thus their quantitative description tin be used to proceeds insights into differential roles of the participating phosphatases and kinases in shaping the pour signalling outputs.

Theoretical analysis of signalling cascades without feedback loops has shown that the action of phosphatases outweigh that of kinases, exerting a dominant effect on the regulation of point duration [five]. On the other hand, kinases influence point amplitude rather than duration, although phosphatases can also contribute to the regulation of bespeak amplitude. This is particularly credible in weakly activated pathways where simply a modest proportion of the total kinase pool is phosphorylated. Under these conditions, point duration is entirely adamant by phosphatases, condign prolonged at ho-hum dephosphorylation rates. Interestingly, the position of a phosphatase within the cascade does not affect the extent to which it affects signal duration [v]. Mathematical studies on specific systems such as the ERK pathway have provided further back up to these predictions [twenty, 21]. In one such study utilising NRK fibroblasts [20], the cells were arrested in M
stage and ppERK concentrations were measured following stimulation with EGF in the presence of increasing doses of a MEK inhibitor [20]. Under these weather condition, increasing MEK inhibition resulted in a decreased top of a transient ERK activation, while having little effect on its elapsing. Still, applying a protein tyrosine phosphatase (PTP) inhibitor led to a broader ppERK tiptop, signifying a prolonged duration which is consistent with model predictions [xx]. These studies suggest that in signalling pathways such as the MAPK cascade, where point duration strongly determines cell fates, targeting phosphatases rather than kinases is a more than viable strategy to control prison cell responses.

Dual-specificity phosphatases (DUSP) as rapid feedback inhibitors

Equally mentioned above, MAPK pathway signalling has been implicated in the governing jail cell fate decisions. Diverse cellular events, such as proliferation, differentiation, migration and apoptosis all crave the proper functioning of MAPK cascades. A puzzling aspect has been of how i core module, such as the Ras/Raf/MEK/ERK pathway tin can elicit cell responses equally diametrically opposite as proliferation and differentiation. Information technology has emerged over time that the answer to this question is due to the fact that these pathways, which were traditionally thought to be linear cascades, are embedded in complex signalling networks of feedback interactions [14, 22, 23]. When a signal is relayed from the extracellular membrane via the MAPK-pathway into the nucleus, a networked pathway allows for boosted regulation by either integrating information from culling co-activated and suppressed pathways or by facilitating the self-regulation of the pathway past incorporating feedbacks. Although other classes of protein phosphatase, such as protein serine/threonine phosphatase 2A (PP2A) also take direct or indirect regulatory effects on the MAPK cascade, in this part of the review we will focus on how DUSPs elicit feedback control in the context of the Ras/Raf/MEK/ERK pathways.

In response to actress and intracellular signalling cues, cells induce regulatory feedbacks past two essentially unlike mechanisms. Either the activities of signal transducers are altered, or protein concentrations of these transducers are inverse. The activeness changes are more often than not accomplished postal service-translationally through altering modifications, such as phosphorylations that occur rapidly and at multiple levels of the pathway. For instance, ERK alone can phosphorylate and inactivate several upstream point transducers, including EGFR, SOS, Raf-1 and MEK [14]. Such feedback controls mediated by mail service-translational modification occur about immediately later on the initial signal has been triggered. Furthermore, protein concentrations can be changed by either increasing protein degradation, such as depletion of the EGF-receptor [24], or by triggering a rapid transcriptional response, which tin be induced on the time calibration of minutes. Many genes, which are strongly induced in this first transcriptional wave, are direct regulators of upstream signalling, showing that biological systems exploit both post-translational and transcriptional feedbacks. Even so, transcriptional feedbacks are inherently slower and more costly than post-translational ones, as it takes time and energy to induce substantial amounts of a nascent protein. Therefore, increasing the concentrations of feedback inhibitors has a delayed outcome on the signalling cascade and this inhibition is often sustained for longer periods. The increase of feedback inhibitor concentration allows the cellular organization, which is initially very sensitive to extracellular cues, to adapt to the new surroundings. This is done by adjusting the threshold required for signalling, by reducing the signalling sensitivity and by altering the dynamics of the response. The adaptation tin involve reducing the receptor affluence [24], expressing specific antagonists, such as Sprouty [25] and MIG-half-dozen [26] or inducing the expression of phosphatases, which dephosphorylate activating phosphorylation sites on signal transducers [27, 28]. Ane class of phosphatases, which is robustly induced upon activation of MAPK cascades, are dual-specificity phosphatases, reviewed in this journal edition past Steve Keyes. DUSPs are a subfamily of PTPs which bind MAPKs and dephosphorylate residues in their activation-loop leading to MAPK deactivation. Interestingly, DUSP activeness is additionally regulated by the substrates, and binding to MAPK increases DUSP activeness [29]. Boosted regulation is achieved through post-translational modifications, every bit many DUSPs are themselves substrates of MAPK [xxx] Therefore, inducible over-expression of DUSPs potently decreases MAPK-activity and is considered to be function of the cellular feedback mechanism.

These feedback inhibitors are required to reply rapidly and with sufficiently high precision to changes in MAPK activity. Rapid turnover times are accomplished through fast protein and mRNA deposition rates, which are hallmarks of these feedback regulators [31]. This allows for a rapid transcriptional regulation of the protein, which in turn permits the accurate and reliable tuning of the signalling response.

Induced expression of phosphatases reduces the dose-response sensitivity and the signalling output, just can also fundamentally alter the dynamics of the response. For instance, NIH-3T3 cells evidence a rapid and sustained phosphorylation of the downstream ERK1/2 kinases post-obit stimulation with platelet-derived growth gene (PDGF) [32]. Interestingly, the pathway activation appears to be self-sustaining, every bit MAPK activity persists even when PDGF is washed out after the initial stimulus. Under these weather, ERK1/2 action is not linearly related to the PDGF input concentration. Incremental increasing PDGF concentrations does non lead to incremental increases in MAPK signalling, just rather results in a switch-like, all-or-nothing activation to a higher place a certain threshold, similar to an ultrasensitive activation. Although DUSP is induced by PDGF, this expression fails to dramatically bear on ERK1/ii activation dynamics [32]. Although postal service-peak MAPK action is reduced, the sustained MAPK activation dynamics still persists. Intriguingly, the behaviour of the organisation can change dramatically if cellular DUSP expression is substantially increased by pre-exposing NIH-3T3 cells to low PDGF concentrations. Re-stimulating these preconditioned NIH-3T3 with increasing PDGF concentrations dramatically changes the dose response sensitivity of the MAPK activity. The previously ultrasensitive system now displays a linear relationship between the PDGF input and the MAPK phosphorylation output. The transformation of a switch-like ultrasensitive response to a graded response illustrates flexibility and adaptability of the cellular signalling network, which in this case is mediated by an inducible phosphatase acting equally a feedback regulator [32].

Interestingly, DUSPs likewise shape the dynamics of mitogenic responses. This was elegantly demonstrated by utilising a cell line which expresses a rapidly inducible Ras isoform that harbours an oncogenic, activating mutation and constitutively stimulates the downstream Raf/MEK/ERK pathway [33]. Following expression of mutated Ras, ERK activity initially overshoots but afterwards 30 minutes rapidly reduces, resulting in a sharp activity peak. Importantly, after 30 minutes both the Ras input and ERK phosphorylation output obey a linear dose-response human relationship. A mathematical model of this arrangement showed that in social club to mimic this behaviour ERK activity has to react initially in an ultrasensitive manner, but this input-output relationship subsequently changes with DUSP expression. Thus, these finding confirm the results obtained in NIH-3T3 cells, further demonstrating that expression of DUSPs affects the amplitude, dose-response relationships and temporal dynamics of MAPK activation.

Interim in this way as rapid feedback regulator, DUSP tightly controls MAPK activeness. However, computational studies suggested that if DUSP-mediated feedback is too strong, it can also bring about oscillations [22, 34]. Such oscillations have been experimentally observed in the Fus3 MAPK pathway, responsible for regulating the mating-pheromone response, in Saccharomyces cerevisiae [35]. Strong correlation betwixt the oscillatory Fus3 activation peaks and periodic formation of additional mating projections suggests important physiological office of these oscillations. Experiments and mathematical modelling plant that transcriptional induction of the MAPK phosphatase Msg5 and the negative regulator of G poly peptide signalling Sst2 are required for maintenance of these oscillations [35].

Recent evidence further indicates that feedback control by DUSPs can shape the dynamics of the MAPK response differentially, depending on the cellular compartment [eighteen]. In MCF7 cells the MAPK pathway responds to Heregulin (HRG) treatment with a robust and sustained activation of ERK in the cytoplasm. Surprisingly, when ERK phosphorylation is monitored in the nuclear fraction, the sustained cytoplasmatic ERK indicate is translated into a transient response. This holds true even if phosphorylated ERK is normalised by the amount of total nuclear ERK, taking nuclear-cytoplasmatic shuttling into account. Intriguingly, knock-down of nuclear DUSPs by siRNAs is sufficient to transform HRG-induced ERK phosphorylation inside the nucleus form transient to sustained. Therefore, information technology appears that the difference between nuclear and cytoplasmatic ERK dynamics can exist due to the presence or higher expression and induction of specifically nuclear localised DUSPs.

Overall, it is becoming clear that the phosphatase-mediated deactivation of MAPK-pathways is used by the cell to control and regulate all aspects of signalling, would this be the elapsing, the amplitude or the localisation of the betoken. Thank you to this added control the organization can react with high adaptability and flexibility to irresolute and diverse environmental stimuli.

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Spatial separation of phosphatases and kinases can give rise to phosphoprotein gradients

Cells are 3-dimensional structures, and the spatial regulation of protein activities is important for many physiological processes, including cell division, motility and migration. In improver to their roles in temporal dynamics, phosphatases control the spatial behaviour of protein phosphorylation within the jail cell. When a protein phosphorylated at the plasma membrane spreads solely by diffusion, dephosphorylation mediated by a cytosolic phosphatase can effect in a steep slope of phosphorylation signal [4, 36]. This gradient is characterised past high concentrations of phosphorylated protein proximal to the membrane and low concentrations in the prison cell interior, with the disuse profile being almost exponential, if the phosphatase is far from saturation [37]. Interestingly, how fast the slope is terminated depends only on the diffusion coefficient and the apparent outset-lodge charge per unit constant of the phosphatase but not on the kinase. This result suggests that the localisation and catalytic activeness of phosphatases can play determinant roles in shaping spatial signalling gradients in cells. Experimental prove of such activity gradient is accumulating, which includes the pocket-size GTPase RAN [38], the yeast MAPK Fus3 [39], the phosphatase PTP1B [twoscore], aurora B kinase [41] and the yeast poly peptide kinase Pom1 [42]. Further piece of work that investigated the spatial signal propagation in simplified signalling pour models revealed like constraints to those found for the temporal responses. For activation signals to readily spread from the prison cell membrane into the cell interior, the Vmax/Km ratios for the phosphatases must be much smaller than such ratios for the kinases [43, 44].

Drug development targeting protein phosphatases

Kinases accept been major targets of drug discovery efforts [45]. This is partly considering kinases were idea to have dominant command over signalling systems, while phosphatases were considered lesss important analogue of the kinases with unclear involvement in cell fate decisions, mainly as consequence of phosphatases-specific technical challenges. Equally discussed earlier, this view is becoming obsolete since phosphatases tin impose significant influence in shaping the spatiotemporal dynamics of signalling pathways, thereby affecting cell fate decisions. Emerging systems biology approaches that combine mathematical modelling with quantitative experimentation can facilitate understanding of the network complexity and therapeutic target selection. Although the development of drugs targeting protein phosphatases is coming to the forefront, only a few of these drugs have progressed into clinical trials, and the degree of success of therapies targeting phosphatases is still to be determined. The main efforts focus on the treatment of diabetes, Alzheimer’s illness and cancer. Hither we summarise some of the strategies that take been used to target different classes of protein phosphatases and how systems biology tin exist used to develop better treatments that target phosphatases, for more detailed reviews please see [46–49].

Targeting protein tyrosine phosphatases

The poly peptide tyrosine phosphatases are characterised by the presence of the conserved sequence (H/V)C(10)5R(s/T) in the active site [50]. Out of more than than 100 PTPs, merely a few are considered to exist therapeutic targets [10]. For instance, although some PTPs behave as oncogenes, an RNAi screen against 107 PTPs has shown that in HeLa cells, knowck down of only 4 of these had a negative effect on prison cell survival, while knock out of 28 PTPs increased cell survival [51]. This screen shows that activation of some of these PTPs can potentially exist an antitumoural treatment. However the evolution of drugs that specifically target a item PTP is complicated past ii factors: (i) the high level of homology of the phosphatase domains of dissimilar PTPs and (two) the targeted sequences are highly charged, and many of the developed compounds cannot cross the membrane [47]. To increment the specificity, non-homologous neighbouring domains of the agile site are likewise targeted. In addition, the cell permeability for drugs tin can be increased by chemical manipulation [47].

Drugs targeting PTPs displaying oncogenic behaviour are in dissimilar phase of evolution. The proteins being targeted are PTP1B, SHP2, Cdc25, Cdc14, PRL-three and Eya1/3 [48]. Mutations of these proteins or changes in the level of expression seem to play a function in cancer and autoimmune diseases. For example, PTP1B is a negative regulator of the insulin receptor [52], and there is evidence that inhibition of this phosphatase increase sensitivity to insulin, making PTP1B a very attractive target for the treatment of obesity and diabetes [53]. Interestingly, orthovanadate was originally developed equally a drug to treat diabetes, long earlier it was known that information technology inhibits PTPs. PTP1B may also positively regulate HER2 [54], subsequently activating several proteins in the downstream EGF-signalling network, such as Src [54, 55] and p120RasGAP [46]. Therefore, it may besides be a potential therapeutic target for the treatment of breast cancer. PTP1B inhibitors have been adult using different approaches, the last generation are bidentate difluoromethylphosphonates designed to target the active site and a secondary substrate binding region close to the catalytic pocket [56]. These inhibitors bind PTP1B with higher affinity than other related PTPs and are being modified to increment their cell permeability [57] only have not gone into clinical trials yet. Ii PTP1B inhibitors, Ertiprotanib and Trodusquemine, have advanced into clinical trials for the treatment of obesity and diabetes, although the second stage clinical trial for Ertipotanib was discontinued due to lack of efficacy [48, 58]. Stage I clinical study of Trodusquemine is currently existence performed [59]. Another PTP that has been targeted is SHP2, a phosphatase that contains 2 SH-2 domains [60] and is considered a
bona fide
oncogene that regulates cell progression and migration by modulating Erk1/2 and FAK signaling [vii]. SHP2 is required for the full activation of ERK, and impaired SHP2 activity was establish responsible for the surprising phenomenon that activating EGFR mutations failed to fully induce ERK activation [61]. Activating mutations of SHP2 accept been identified in patients with unlike leukemias, solid tumourrs and in several germ line mutation syndromes such equally Noonan and Leopard syndromes [62]. Gain of role mutations in the N-SH2 domain impair the auto-inhibition of the PTP domain, and usually outcome in increased signaling from Ras, other oncogenes such as Src, and a general increase in the downstream betoken from different growth factor receptors [47]. Several SHP2 small molecule inhibitors have been produced and are in dissimilar phases of development (for a detailed review see [48]). One of the bigger issues in the evolution of these inhibitors is that SHP2 presents a high homology with SHP1, another PTP that acts as tumour suppressor. Thus, the SHP2 inhibitors should non inhibit SHP1, or they should have a higher affinity for SHP2 at the administration dose (Effigy 1). To date no SHP2 specific inhibitors have advanced to clinical trials, however a dual SHP1/ii and PTP1B inhibitor is currently in clinical trial in combination with interferon alpha treatment. This inhibitor seems to be well tolerated and augments immunological responses [63].


SHP-ane and SHP-two are phosphatases that tin can play reverse roles in the regulation of signalling pathways. (A). In cancer, a SHP-2 activating mutation or SHP-ii stimulation past oncogenic signals results in the activation of oncogenic pathways such equally the RAS/ERK and SRC pathway. (B) Inhibitors that specifically target SHP-2 or accept higher affinity for SHP-2 than for SHP-1 can lead to inhibition of these pathways, shifting the balance towards tumour suppression.

Targeting protein serine/threonine phosphatases

As mentioned above, the protein Ser/Thr phosphatases (PSPs) include a variety of proteins with more than xxx catalytic subunits that interact with dissimilar regulatory and structural subunits. The PSPs actually consists of iv families; the phospho-protein phosphatases (PPP), metallo-dependent poly peptide phosphatases (PPM) and Asp-based enzymes. However of these families the PPPs are responsible for the bulk of serine and threonine dephosphorylation [64]. These proteins take been shown to play an important role in the regulation of different biological functions in close relation with tyrosine kinases [65]. The PSPs are key regulators of kinase activity and their functional deregulation has been observed in different pathologies such as cancer and Alzheimer’south illness. Out of many members of the PSP family, PP2A has recently become a target for drug development, specifically in the context of cancer therapy. Several isoforms of PP2A act as
bona fide
tumour suppressors which negatively regulate mitogenic signals [66], although this phosphatase is also deregulated in dissimilar types of cancer such as breast, lung and melanoma [67, 68]. Inhibition of PP2A is necessary for the transformation and tumour progression of dissimilar cancers. Both mutation and loss of expression of all PP2A subunits accept been described (encounter [49]). In addition to the deregulation of PP2A subunits, the PP2A inhibitory proteins SET and PHAP-i take as well been linked to different malignancies. For instance, SET is overexpressed in BCR/ABL-driven leukemias [69], and PHAP-1 has been related to the aberrant phosphorylation of Tau poly peptide in Alzheimer’s illness [70]. In light of this observation dissimilar drugs that restore normal PP2A action are beingness studied. For example, sodium selenate decreases the Tau protein phosphorylation levels in mice and is currently is nether intense written report [71]. In the context of cancer, the rationale for developing PP2A targeting drugs is that restoring the phosphatase enzymatic activeness would outcome in inhibition of the transforming signal caused by oncogene expression. And so far the best known activator of PP2A is FTY720, a structural analog of sphingosine that has been approved for the handling of multiple sclerosis [72]. In different fauna models of leukemia, FTY720 has been shown to increase the rate of survival with few toxic side effects [49, 73], indicating that PP2A activation treatment may be a safe strategy in cancer treatment. Although still in the early on stages, these studies demonstrate that targeting PSPs such equally PP2A is a potentially useful therapeutic strategy, all the same the circuitous spatiotemporal regulation evident within these phosphatase networks suggests that farther understanding volition be required to generate the sensitivity and specificity essential for therapeutic applications.

Regulatory subunits in the spatiotemporal control of phosphatases

While phosphatases, such equally DUSPs, ensure the spatiotemporal regulation of pathway activeness through their tight transcriptional control and internal localisation sequences, regulation of other phosphatases, such every bit the various family of PSPs, represents an entirely different prototype of control. Singled-out from the paradigm of a monomeric phosphatase, specificity and control of PSP action (with the notable exception of the monomeric PP2Cs from the PPM family), is mediated past formation of a multi-component circuitous containing a catalytic subunit and a regulatory subunit. In some cases, the assembly is facilitated past a scaffolding subunit, resulting in a trimeric circuitous [74].

While the substrate specificity of kinases has been established upon the basis of linear motif recognition surrounding the phosphorylated amino acid, sites of PSP directed dephosphorylation do not brandish significant sequence similarity [75]. Instead, substrate specificity is accomplished through docking of the phosphatase complex at a site distant to the de-phosphorylated amino acid [12, 75]. Consensus motifs for regulatory subunit docking sites accept been established for some prominent members of the PSP family, including PP1 and PP2B (Calcineurin), although not for PP2A, for which multiple interactions and mail-translational modifications play a role in directing its catalytic action [76].

Numerous studies have demonstrated that specificity within the man PP2A network is achieved through differential assembly of heterotrimeric complexes from the genomic repertoire of two catalytic subunits (PP2aCα/β), two scaffolding subunits (PR65α/β) and at least 15 known regulatory subunits coming from four carve up gene families (Termed B, B′, B″ and B‴) [77]. Past exploiting this differential assembly mechanism PP2A exerts control over a wide array of cellular processes through the availability of a multitude of individual heterotrimeric complexes [78, 79]. Furthermore, post-translational modifications besides play a significant function in the temporal regulation of the PP2A associates which occur on both the catalytic and regulatory subunits [76]. While the incorporation of B and some B′ regulatory subunits is inhibited upon Src-mediated phosphorylation of PP2aC, methylation of PP2aC may be required for the incorporation of B and possibly B′ subunits [76]. Phosphorylation of regulatory subunits also contributes to this temporal regulation in a kinase and subunit specific way [76, 80]. A further layer of spatial regulation is added to these heterotrimeric complexes through a diversity of localisation sequences within the regulatory subunits, limiting the spatial sphere of PP2A activity to specific sub-cellular locales [76, 78].

A prime number example of a network regulated by PP2A in such a complex manner is that governing the activation of ERK post-obit growth factor stimulation. Within this network PP2A can human activity at multiple levels to promote either the activation or inhibition of ERK, depending upon the site of PP2aC recruitment, a process controlled by various regulatory subunits (Effigy ii). Upon growth gene stimulation PP2aC is recruited to the KSR1/Raf1/MEK circuitous through the B family unit member PR55α/δ, where it is required for Raf1 activation via dephosphorylation of the inhibitory Due south259
site [81] and also of 14-3-3 bounden sites within Raf1 and KSR1 [82]. However, PP2aC acts via B′ family fellow member PR61β/δ to straight inhibit ERK[83] and besides indirectly promotes tyrosine dephosphorylation of Shc through an united nations-identified regulatory subunit [84]. Additionally, PP2aC too inhibits Ras-contained ERK activation by de-phosphorylating c-Src upon interaction with an alternative B family unit member PR55γ [85].


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A schematic representation of sites of PP2A activeness within the network of ERK activation. Ligand-mediated activation of receptor tyrosine kinases (RTK) at the plasma membrane leads to activation of the classical Ras/Raf/MEK kinase pathway leading to phosphorylation of ERK (pathway components shown in white, activating phosphorylations indicated with a black arrow). Private heterotrimeric PP2A complexes (shown in grey) containing the catalytic subunit (PP2aC), scaffolding subunit (PR65) and differing regulatory subunits are spatially separated inside the network based upon specific interaction between network components and each regulatory subunit. The activating de-phosphorylation of network components by PP2aC is indicated by a grey arrow and inhibitory de-phosphorylation by a blunt ended gray pointer.

Multi-faceted regulatory and combinatorial associates mechanisms such as these present a meaning challenge for experimental characterisation of the global PP2A network, a vital stride when considering PP2A as a therapeutic target. Many studies have focused on private complexes and their specific dephosphorylation targets, providing all-encompassing data on how these complexes act in isolation [76–80], however petty is known about regulation of PP2A at the network level.

Recent studies utilising systems level approaches have begun to yield meaning advances in this field. At 1 level, mathematical modelling has allowed characterisation of specific PP2A heterotrimers, the affluence of which was too low to measure out experimentally [86, 87], whilst interactomics based studies are beginning to piece together the PP2A network as a whole [88]. A recent written report utilised mass spectrometry based interactomics to investigate the whole network of interactions occurring beyond PP2A catalytic, scaffolding and regulatory subunits [88]. This report confirmed the simultaneous existence of a big pool of heterogeneous heterotrimeric PP2A complexes and placed these into singled-out modules characterised by the presence of regulatory subunits linked to specific cellular processes. Intriguingly, this written report highlighted the underlying complexity of the PP2A network by hinting at higher social club complexes containing proteins not previously associated with this network. Additionally, information technology besides suggested the utilisation of PP2A regulatory subunits by other PSP families, demonstrating evolutionary divergence of the human PP2A network from that of lower eukaryotes.

While systems biological science approaches are just starting to unravel the circuitous interactions and modifications involved in regulation of the PP2A network, the building of network level poly peptide-poly peptide interaction networks such as this will lay the foundation for farther studies examining the dynamic behaviour of these systems.

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How can systems biological science accelerate drug development targeting protein phosphatases?

Regulation of protein phosphorylation is seminal for many biological processes, and deregulation of kinases that catalyse phosphorylation leads to the onset of dissimilar diseases. This notion prompted the development of agents that target tyrosine kinases (TKs) years before phosphatases were considered every bit therapeutic targets. Near of TK targeting drugs were developed to inhibit specific kinases and were initially used every bit unmarried agents, based on the idea of oncogene addiction. However apart from imatinib, nigh of the inhibitors that are used in the clinic accept failed as single agents and they are given in combination with other treatments. This is due to an inherent biological back-up where different TKs have overlapping targets, and inhibition of a unmarried kinase is not sufficient to restore the normal intracellular phosphoprotein levels. Another problem is unspecific inhibition of other kinases that reduces the benefits of inhibiting a particular kinase. Hopefully, the lessons learned from the evolution of TK inhibitors can speed up the evolution of drug targeting phosphatases.

The utilize of mathematical models can help in the identification of appropriate targets and predict the efficiency, course of treatment and drug combinations that can have a therapeutic effect [45]. Although the use of mathematical models is still very limited in drug development there are already examples that show how systems biology tin can exist useful for drug evolution and clinical application [89–91]. For case, a mathematical model of the PI3K/AKT pathway was used in combination with clinical data to identify new biomarkers that tin can help to determine which patients would benefit from treatment with PI3K inhibitors and receptor TK inhibitors [92]. Similar approaches have been applied to predict the drug inhibition profile for the NFκB pathway [93], or to place optimal therapeutic targets for the activation of p53 [94].

The circuitous nature of phosphatase biology makes it extremely difficult to predict astray furnishings and determine which patients will do good from treatment with particular pharmacological agents. Furthermore as mentioned, one of the challenges for the development of agents that target PTPs is their selectivity hindered by the high homology amongst the members of the PTP family. The use of mathematical models can help to place “therapeutic windows” that will inhibit a given PTP without causing deleterious inhibition of other PTPs. For instance, inhibitors with a college affinity for SHP2 than for SHP1 could exist used at the doses that only affect SHP2. Furthermore, it is also likely that there will be synergistic effects upon the combination of phosphatase targeting drugs and TK inhibitors. The identification of these synergistic drug combinations may also be predicted using mathematical models.

Phosphatases-targeted therapies are suggested by a computational model of the EGFR pathway

Although efforts in targeting protein phosphatases for therapeutic purposes are already underway, this attempt might be significantly accelerated if guided by mathematical modelling and systems approaches to cell signalling. These methods tin can help in the identification of suitable targets, prediction of potential drug treatments and the efficiency of combined therapies. Every bit an illustrative example, we use a simplified mathematical model that incorporates the EGFR/ERK pathway and the tyrosine phosphatase SHP2 (the EGFR/SHP2 system) to explore alternative therapeutic strategies to inhibit ERK activation mediated by over-expressed EGFR. Our model predicts that within certain cancer cell contexts suppression of ERK activation by targeting the phosphatase SHP2 tin be more constructive than targeting the receptor.

SHP2 was reported to a take dual regulatory role [61]. Information technology negatively regulates phosphorylation of RTKs (due east.g., EGFR and insulin receptor) and adaptor proteins (e.g., insulin receptor substrate (IRS) and Grb2-associated folder i (GAB1) [nine]). Yet, SHP2 has strong positive effect on Ras activation, facilitating the full activation of the extracellular signal regulated kinase (ERK). This positive effect is related to the plasma membrane recruitment of SHP2 through the binding to phosphorylated tyrosine residues on the GAB1 and IRS scaffolds. SHP2 then afterward dephosphorylates multiple docking sites, involved in the binding and membrane recruitment of the GTPase-activating protein for Ras (RasGAP), enhancing Ras activity [seven, 8]. To business relationship for the activity of SHP2 on the downstream Raf-i/MEK/ERK cascade, we constructed an ODE-based model that extends our previously established EGRF network model [xix] to describe the SHP2/Ras/ERK pathway. The reactions involved in the model are illustrated in the scheme presented in
Figure three
(all charge per unit equations and parameter values are given in Supplementary Tables S1, S2 and S3). Briefly, in the EGFR/SHP2/Ras/ERK model, signal transduction is initiated by EGF binding to the extracellular domain of the monomeric EGFR (designated every bit R in the kinetic scheme, reaction i,
Effigy three). This causes dimerization and autophosphorylation of EGFR (reactions 2, 3 and 25), which is subsequently dephosphorylated by several phosphatases (reactions four and 26). To business relationship for the combinatorial complication of phosphorylation of different sites on EGFR and the fact that SHP2 specifically dephosphorylates the sites involved in RasGAP binding, we use an gauge ODE description by considering 2 split up forms of phosphorylated EGFR, designated RP1
and RP2
(Effigy 3) (see [95–97] for more rigorous approaches to reduce combinatorial complexity of bespeak transduction networks). We presume that bounden of proteins to these ii different tyrosine residues is statistically independent. The RP2
form mediates RasGAP-bounden and is dephosphorylated by active SHP2 that has bound to RP1
(reactions five–8 and 27). The adaptor proteins Shc and Grb2 demark competitively to the RP1
form, and Grb2 too bind to phosphorylated Shc (reactions 13–18). The Grb2-SOS complexes that have leap to EGFR or the EGFR-Shc complex catalyse the conversion of RasGDP to RasGTP (reaction 28), whereas the reverse transition is catalysed by RasGAP jump to the RP2
grade of EGFR (reaction 29). Activated Ras afterward turns on the Raf-MEK-ERK pour (reactions thirty–39).


An external file that holds a picture, illustration, etc.
Object name is nihms358402f3.jpg

Kinetic scheme of the EGFR/SHP2 signalling mediated by adapter and target proteins. Numbering of individual steps is capricious.

Although this dynamic model is not comprehensive, it can be exploited to compare alternative therapies that perturb distinct classes of targets. Many cancer cell types have elevated expression of EGFR [61]. Inhibiting EGFR using an EGFR inhibitor, such as gefitinib seems to exist a preferred treatment to suppress the ERK pathway activity. Still, many patients showreduced gefitinib sensitivity, and new treatments that tin can overcome gefitinib resistance are required. Using a mathematical model, we compare two therapies that target either SHP2 (a phosphatase-based therapy) or EGFR (a kinase-based therapy).

This model shows that in normal cells, characterised past depression physiological EGFR levels, EGF stimulation induces a transient response of active Ras (Ras GTP) and ERK (doubly phosphorylated ERK, designated ppERK in
Fig. 3), encounter
Effigy 4A,B, which is consistent with the experimental observations. In cancer cells, EGFR overexpression often leads to sustained RasGTP and ppERK responses, which is also reflected by the model predictions (Figure 4C,D) [98]. Importantly, predictive simulations carried out using the model suggest that when the SHP2 level is too high in cancer cells, inhibition of SHP2 ameliorate suppresses active ERK and RasGTP levels compared to EGFR inhibitors (Figure 4C, D). Additionally, the model predicts that combining the 2 inhibitors in a dually-targeted therapy further decreases RasGTP and ppERK, thereby enhancing the efficiency of the handling (Figure 4C, D). For comparison purposes, the model causeless that both EGFR and SHP2 inhibitors reduce the concentrations of SHP2 and EGFR past 40% of their pre-handling levels. Interestingly, increasing the dosage of both inhibitors not only further suppresses the Ras/EGFR pathway, but too increases the efficacy of SHP2-based therapy over the EGFR-based therapy (information not shown).


An external file that holds a picture, illustration, etc.
Object name is nihms358402f4.jpg

A comparing of ii molecularly targeted therapies for the EGFR/SHP2 signalling system as illustrated in Figure 4. Fourth dimension-course concentrations of active RasGTP
(A)
and double-phosphorylated ERK
(B)
in response to EGF stimulation for normal cell with low EGFR level (100nM). Time-course concentrations of RasGTP
(C)
and double-phosphorylated ERK
(D)
in response to EGF stimulation in cancer cells, characterised past upwardly-regulated levels of EGFR (800nM). Time-course data is also included for the presence of an EGFR inhibitor (red), SHP2 inhibitor (blue) and combined treatment of both EGFR and SHP2 inhibitors (dashed imperial). Details of model equations and parameter values are given in the Supplementary Materials.

These simulations demonstrate that nether certain conditions, targeting SHP2 tin be a more viable strategy in suppressing ERK activation than targeting a tyrosine kinase receptor. This also highlights the emerging concept that the pattern of signal transduction therapies requires understanding of the underlying mechanisms that command aberrant signalling patterns and pathological cell traits. As we accept demonstrated, systems biology approaches tin reveal these hidden regulatory patterns and bring fresh avenues to drug discovery.

Concluding Remarks

The perception of phosphatases as enzymes whose part is solely to counteract kinases in linear signalling pipelines from receptors to target genes, is replaced by an emerging concept of a tangled kinase/phosphatase network that is tightly regulated through a multitude of negative and positive controls by feed-forrad and feedback loops. Phosphorylation and dephosphorylation of multiple tyrosine, serine and threonine residues on point transducers results in dramatic changes to their activities, leading to specific alterations in cellular phenotypes. This complex, combinatorial nature of cellular signalling highlights the need for systems biology methods to sympathise the roles of phosphatases in shaping the signalling dynamics and their targeting in drug development.

We are beginning to rationalise how the intricate network circuitries can determine the spatiotemporal signalling kinetics to precisely translate them into specific biological responses. We show that phosphatases can human activity as both immediate and delayed controllers of betoken processing and how the effects of this regulation can be negative or, surprisingly, positive in amplifying cellular responses. In addition, mathematical models take shown that phosphatase and non kinase activities predominantly command the response time of distinct signalling processes and phosphorylation/dephosphorylation cascades. The prominent role of phosphatases in shaping the spatial profiles of signalling activities within a cell has been recently revealed by both computational and experimental studies.

Systems biology models emerge as a novel tool to accelerate drug development. Every bit phosphatase targeting is coming to the forefront of drug development, there is the need to assess the systems consequences of drug-induced changes in phosphatase activities. Here we illustrate how computational modelling can assistance us predict the outcomes of drug therapies targeting unlike cellular processes. Further development of systems-level approaches will facilitate the selection of proper treatments for specific pathological conditions.

Supplementary Material

Supp Table S1-S3

Acknowledgments

We thank Walter Kolch for stimulating discussions and reading the manuscript. This work was supported by Science Foundation Ireland under Grant No. 06/CE/B1129 and NIH grant GM059570. We apologise that we could non cite many pertinent contributions to the field considering of space limitations.

Footnotes

Supplementary Material

A pdf supplementary file containing supplementary tables is available online.

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What Role Do Phosphatases Play in Signal Transduction Pathways

Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3368988/

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