Enhancement of Tunability of MAPK Cascade Due to Coexistence of Processive and Distributive Phosphorylation Mechanisms

The processive phosphorylation mechanism becomes important when there is macromolecular crowding in the cytoplasm. Integrating the processive phosphorylation mechanism with the traditional distributive one, we propose a mixed dual-site phosphorylation (MDP) mechanism in a single-layer phosphorylation cycle. Further, we build a degree model by applying the MDP mechanism to a three-layer mitogen-activated protein kinase (MAPK) cascade. By bifurcation analysis, our study suggests that the crowded-environment-induced pseudoprocessive mechanism can qualitatively change the response of this biological network. By adjusting the degree of processivity in our model, we find that the MAPK cascade is able to switch between the ultrasensitivity, bistability, and oscillatory dynamical states. Sensitivity analysis shows that the theoretical results remain unchanged within a reasonably chosen variation of parameter perturbation. By scaling the reaction rates and also introducing new connections into the kinetic scheme, we further construct a proportion model of the MAPK cascade to validate our findings. Finally, it is illustrated that the spatial propagation of the activated MAPK signal can be improved (or attenuated) by increasing the degree of processivity of kinase (or phosphatase). Our research implies that the MDP mechanism makes the MAPK cascade become a flexible signal module, and the coexistence of processive and distributive phosphorylation mechanisms enhances the tunability of the MAPK cascade.     

Enhancement of Tunability of MAPK Cascade Due to Coexistence of Processive and Distributive Phosphorylation Mechanisms

The processive phosphorylation mechanism becomes important when there is macromolecular crowding in the cytoplasm. Integrating the processive phosphorylation mechanism with the traditional distributive one, we propose a mixed dual-site phosphorylation (MDP) mechanism in a single-layer phosphorylation cycle. Further, we build a degree model by applying the MDP mechanism to a three-layer mitogen-activated protein kinase (MAPK) cascade. By bifurcation analysis, our study suggests that the crowded-environment-induced pseudoprocessive mechanism can qualitatively change the response of this biological network. By adjusting the degree of processivity in our model, we find that the MAPK cascade is able to switch between the ultrasensitivity, bistability, and oscillatory dynamical states. Sensitivity analysis shows that the theoretical results remain unchanged within a reasonably chosen variation of parameter perturbation. By scaling the reaction rates and also introducing new connections into the kinetic scheme, we further construct a proportion model of the MAPK cascade to validate our findings. Finally, it is illustrated that the spatial propagation of the activated MAPK signal can be improved (or attenuated) by increasing the degree of processivity of kinase (or phosphatase). Our research implies that the MDP mechanism makes the MAPK cascade become a flexible signal module, and the coexistence of processive and distributive phosphorylation mechanisms enhances the tunability of the MAPK cascade.     

The Robustness of Proofreading to Crowding-Induced Pseudo-Processivity in the MAPK Pathway

Double phosphorylation of protein kinases is a common feature of signaling cascades. This motif may reduce cross-talk between signaling pathways because the second phosphorylation site allows for proofreading, especially when phosphorylation is distributive rather than processive. Recent studies suggest that phosphorylation can be pseudo-processive in the crowded cellular environment, since rebinding after the first phosphorylation is enhanced by slow diffusion. Here, we use a simple model with unsaturated reactants to show that specificity for one substrate over another drops as rebinding increases and pseudo-processive behavior becomes possible. However, this loss of specificity with increased rebinding is typically also observed if two distinct enzyme species are required for phosphorylation, i.e., when the system is necessarily distributive. Thus the loss of specificity is due to an intrinsic reduction in selectivity with increased rebinding, which benefits inefficient reactions, rather than pseudo-processivity itself. We also show that proofreading can remain effective when the intended signaling pathway exhibits high levels of rebinding-induced pseudo-processivity, unlike other proposed advantages of the dual phosphorylation motif.     

Intramolecular and intermolecular enzymatic modulation of ion channels in excised membrane patches.

A calcium-activated potassium channel in posterior pituitary nerve terminals was modulated by phosphorylation and dephosphorylation. Nearly every patch of membrane containing this channel also contained both membrane bound protein phosphatase and membrane-bound protein kinase. By examining the statistical and kinetic nature of phosphorylation and dephosphorylation in excised patches, it was possible to evaluate two contrasting models for these enzymatic reactions. One of these models treated catalysis as an intermolecular process in which the enzyme and substrate are separate molecular species that diffuse and encounter one another during collisions. The second model treated catalysis as an intramolecular process in which the enzyme and substrate reside within a stable macromolecular complex. The study began with a Poisson analysis of the distribution of channel number in patches, and of the number of protein phosphatase-free and protein kinase-free patches. Subsequent kinetic analysis of dephosphorylation yielded an estimate of the mean number of protein phosphatase molecules per patch that was similar to the value obtained from Poisson analysis. Because these two estimates were independent predictions based on the intermolecular model, their agreement supported this model. Analysis of channel number in protein phosphatase-free patches and of the rarity of patches showing partial but incomplete rundown provided additional support for the intermolecular model over the intramolecular model. Furthermore, dephosphorylation exhibited monotonic kinetics with a rate well below the diffusion limit. Thus, several different lines of analysis support the intermolecular model for dephosphorylation, in which the protein phosphatase must encounter its substrate to effect catalysis. In contrast to the monotonic kinetics of dephosphorylation, the phosphorylation reaction exhibited sigmoidal kinetics, with a rate that depended on membrane potential. Voltage dependence is an unlikely property for a kinetic step involving encounters resulting from diffusion. Furthermore, the velocity of the phosphorylation reaction exceeded the diffusion limit, and this observation is inconsistent with the intermolecular model. Thus, both intermolecular and intramolecular enzymatic mechanisms operate in the modulation of the calcium-activated potassium channel of the posterior pituitary. These studies provide a functional characterization of the interactions between enzyme and substrate in intact patches of cell membrane.     

Molecular weight distribution of polymeric DNA-products, formed upon the action of matrix enzymes

Models of processive and distributive DNA synthesis and degradation catalysed by matrix enzymes were investigated. Distribution of polymer products dependent on the reaction model chosen, on the type of the matrix and on the enzyme-matrix initial concentration ratio were determined by methods of numerical modeling. Conditions were found where the scattering of the reaction polymer products was minimal. The homopolymer matrix choice of experimental condition may generate a distribution of product that obeys the Poisson distribution. Numerical investigations of polymerization (hydrolysis) processes showed that for a number of heteropolymer matrixes a distribution may exist with scattering much less than that for homopolymer matrixes. Conditions are found when processive and distributive models give different distributions of the reaction product.     

Accuracy of tRNA Charging and Codon: Anticodon Recognition; Relative Importance for Cellular Stability

Cellular homeostasis and the mechanisms which control homeostasis are important for understanding such fundamental processes as ageing and the origin of life. Several models have studied the importance of accurate protein synthesis for cellular stability, but these models have not considered the complexities of the translation process in any detail. Here we develop a new model which describes the interplay between aminoacyl-tRNA (aatRNA) synthetases, the cellular pool of charged tRNAs and the process of codon:anticodon recognition. We also take the processive character of the ribosomes into account. In common with previous work, our model predicts that the cellular translation apparatus can either be stable or deteriorate progressively with time. However, because our model explicitly describes different subreactions of the overall translation process, we are also able to assess the relative importance of accurate tRNA charging and codon:anticodon recognition for cellular stability. It appears that the tRNA charging by the aatRNA synthetases plays the key role in controlling the long-term stability of the cell. Ribosomal errors are less important because error-prone ribosomes, being processive, produce mainly inactive proteins which do not contribute to error propagation within the translation machinery.     

Predicting knot and catenane type of products of site-specific recombination on twist knot substrates

Site-specific recombination on supercoiled circular DNA molecules can yield a variety of knots and catenanes. Twist knots are some of the most common conformations of these products, and they can act as substrates for further rounds of site-specific recombination. They are also one of the simplest families of knots and catenanes. Yet, our systematic understanding of their implication in DNA and important cellular processes such as site-specific recombination is very limited. Here, we present a topological model of site-specific recombination characterizing all possible products of this reaction on twist knot substrates, extending the previous work of Buck and Flapan. We illustrate how to use our model to examine previously uncharacterized experimental data. We also show how our model can help determine the sequence of products in multiple rounds of processive recombination and distinguish between products of processive and distributive recombinations.This model studies generic site-specific recombination on arbitrary twist knot substrates, a subject for which there is limited global understanding. We also provide a systematic method of applying our model to a variety of different recombination systems.     

A peptide model system for processive phosphorylation by Src family kinases

The Src homology 2 (SH2) and Src homology 3 (SH3) domains of Src family kinases are involved in substrate recognition in vivo. Many cellular substrates of Src kinases contain a large number of potential phosphorylation sites, and the SH2 and SH3 domains of Src are known to be required for phosphorylation of these substrates. In principle, Src could phosphorylate these substrates by either a processive mechanism, in which the enzyme remains bound to the peptide substrate during multiple phosphorylation events, or a nonprocessive (distributive) mechanism, where each phosphorylation requires a separate binding interaction between enzyme and substrate. Here we use a synthetic peptide system to demonstrate that Hck, a Src family kinase, can phosphorylate substrates containing an SH2 domain ligand by a processive mechanism. Hck catalyzes the phosphorylation of these sites in a defined order. Furthermore, we show that addition of an SH3 domain to a peptide can enhance its phosphorylation both by activating Hck and by increasing the affinity of the substrate. On the basis of our observations on the role of the SH2 and SH3 domains in substrate recognition, we present a model for substrate targeting in vivo.     

Unsupervised logic-based mechanism inference for network-driven biological processes

Modern analytical techniques enable researchers to collect data about cellular states, before and after perturbations. These states can be characterized using analytical techniques, but the inference of regulatory interactions that explain and predict changes in these states remains a challenge. Here we present a generalizable, unsupervised approach to generate parameter-free, logic-based models of cellular processes, described by multiple discrete states. Our algorithm employs a Hamming-distance based approach to formulate, test, and identify optimized logic rules that link two states. Our approach comprises two steps. First, a model with no prior knowledge except for the mapping between initial and attractor states is built. We then employ biological constraints to improve model fidelity. Our algorithm automatically recovers the relevant dynamics for the explored models and recapitulates key aspects of the biochemical species concentration dynamics in the original model. We present the advantages and limitations of our work and discuss how our approach could be used to infer logic-based mechanisms of signaling, gene-regulatory, or other input-output processes describable by the Boolean formalism.     

Effect of positive feedback loops on the robustness of oscillations in the network of cyclin-dependent kinases driving the mammalian cell cycle

The transitions between the G(1) , S, G(2) and M phases of the mammalian cell cycle are driven by a network of cyclin-dependent kinases (Cdks), whose sequential activation is regulated by intertwined negative and positive feedback loops. We previously proposed a detailed computational model for the Cdk network, and showed that this network is capable of temporal self-organization in the form of sustained oscillations, which govern ordered progression through the successive phases of the cell cycle [Gérard and Goldbeter (2009) Proc Natl Acad Sci USA106, 21643-21648]. We subsequently proposed a skeleton model for the cell cycle that retains the core regulatory mechanisms of the detailed model [Gérard and Goldbeter (2011) Interface Focus1, 24-35]. Here we extend this skeleton model by incorporating Cdk regulation through phosphorylation/dephosphorylation and by including the positive feedback loops that underlie the dynamics of the G(1) /S and G(2) /M transitions via phosphatase Cdc25 and via phosphatase Cdc25 and kinase Wee1, respectively. We determine the effects of these positive feedback loops and ultrasensitivity in phosphorylation/dephosphorylation on the dynamics of the Cdk network. The multiplicity of positive feedback loops as well as the existence of ultrasensitivity promote the occurrence of bistability and increase the amplitude of the oscillations in the various cyclin/Cdk complexes. By resorting to stochastic simulations, we further show that the presence of multiple, redundant positive feedback loops in the G(2) /M transition of the cell cycle markedly enhances the robustness of the Cdk oscillations with respect to molecular noise.     

Processive phosphorylation of p130Cas by Src depends on SH3-polyproline interactions

Many in vivo substrates of Src family tyrosine kinases possess sequences conforming to Src homology 2 and 3 (SH2 and SH3) domain-binding motifs. One such substrate is p130Cas, a protein that is hyperphosphorylated in v-Src transformed cells. Cas contains a substrate domain consisting of 15 potential tyrosine phosphorylation sites, C- and N-terminal polyproline regions fitting the consensus sequence for SH3 domain ligands, and a YDYV motif that binds the Src SH2 domain when phosphorylated. In an effort to understand the mechanisms of processive phosphorylation, we have explored the regions of Cas necessary for interaction with Src using the yeast two-hybrid system. Mutations in the SH2 domain-binding region of Cas or the Src SH2 domain have little effect in Cas-Src complex formation or phosphorylation. However, disruption of the C-terminal polyproline region of Cas completely abolishes interaction between the two proteins and results in impaired phosphorylation of Cas. Kinetic analyses using purified proteins indicated that multisite phosphorylation of Cas by Src follows a processive rather than a distributive mechanism. Furthermore, the kinetic studies show that there are two properties of the polyproline region of Cas that are important in enhancing substrate phosphorylation. First, the C-terminal polyproline serves to activate Src kinases through the process of SH3 domain displacement. Second, this region aids in anchoring the kinase to Cas to facilitate processive phosphorylation of the substrate domain. The two processes combine to ensure phosphorylation of Cas with high efficiency.     

Expanding the role of Src and protein-tyrosine phosphatases balance in modulating osteoblast metabolism: Lessons from mice

The widespread nature of protein phosphorylation/dephosphorylation underscores its key role in cell signaling metabolism, growth and differentiation. Tyrosine phosphorylation of cytoplasmic proteins is a critical event in the regulation of intracellular signaling pathways activated by external stimuli. An adequate balance in protein phosphorylation is a major factor in the regulation of osteoclast and osteoblast activities involved in bone metabolism. However, although phosphorylation is widely recognized as an important regulatory pathway in skeletal development and maintenance, the mechanisms involved are not fully understood. Among the putative protein-tyrosine kinases (ptk) and protein-tyrosine phosphatases (ptp) involved in this phenomenon there is increasing evidence that Src and low molecular weight-ptps play a central role in a range of osteoblast activities, from adhesion to differentiation. A role for Src in bone metabolism was first demonstrated in Src-deficient mice and has since been confirmed using low molecular weight Src inhibitors in animal models of osteoporosis. Several studies have shown that Src is important for cellular proliferation, adhesion and motility. In contrast, few studies have assessed the importance of the ptk/ptp balance in driving osteoblast metabolism. In this review, we summarize our current knowledge of the functional importance of the ptk/ptp balance in osteoblast metabolism, and highlight directions for future research that should improve our understanding of these critical signaling molecules.     

A multi-approach and multi-scale platform to model CD4+ T cells responding to infections

Immune responses rely on a complex adaptive system in which the body and infections interact at multiple scales and in different compartments. We developed a modular model of CD4+ T cells, which uses four modeling approaches to integrate processes at three spatial scales in different tissues. In each cell, signal transduction and gene regulation are described by a logical model, metabolism by constraint-based models. Cell population dynamics are described by an agent-based model and systemic cytokine concentrations by ordinary differential equations. A Monte Carlo simulation algorithm allows information to flow efficiently between the four modules by separating the time scales. Such modularity improves computational performance and versatility and facilitates data integration. We validated our technology by reproducing known experimental results, including differentiation patterns of CD4+ T cells triggered by different combinations of cytokines, metabolic regulation by IL2 in these cells, and their response to influenza infection. In doing so, we added multi-scale insights to single-scale studies and demonstrated its predictive power by discovering switch-like and oscillatory behaviors of CD4+ T cells that arise from nonlinear dynamics interwoven across three scales. We identified the inflamed lymph node’s ability to retain naive CD4+ T cells as a key mechanism in generating these emergent behaviors. We envision our model and the generic framework encompassing it to serve as a tool for understanding cellular and molecular immunological problems through the lens of systems immunology.     

A strategy to quantitate global phosphorylation of bone matrix proteins

Current studies of protein phosphorylation focus primarily on the importance of specific phosphoproteins and their landscapes of phosphorylation in the regulation of different cellular functions. However, global changes in phosphorylation of extracellular matrix phosphoproteins measured “in bulk” are equally important. For example, correct global phosphorylation of different bone matrix proteins is critical to healthy tissue biomineralization. To study changes of bone matrix global phosphorylation, we developed a strategy that combines a procedure for in vitro phosphorylation/dephosphorylation of fully mineralized bone in addition to quantitation of the global phosphorylation levels of bone matrix proteins. For the first time, we show that it is possible to enzymatically phosphorylate/dephosphorylate fully mineralized bone originating from either cadaveric human donors or laboratory animals (mice). Using our strategy, we detected the difference in the global phosphorylation levels of matrix proteins isolated from wild-type and osteopontin knockout mice. We also observed that the global phosphorylation levels of matrix proteins isolated from human cortical bone were lower than those isolated from trabecular bone. The developed strategy has the potential to open new avenues for studies on the global phosphorylation of bone matrix proteins and their role in biomineralization as well for other tissues/cells and protein-based materials.     

Signaling switches and bistability arising from multisite phosphorylation in protein kinase cascades

Mitogen-activated protein kinase (MAPK) cascades can operate as bistable switches residing in either of two different stable states. MAPK cascades are often embedded in positive feedback loops, which are considered to be a prerequisite for bistable behavior. Here we demonstrate that in the absence of any imposed feedback regulation, bistability and hysteresis can arise solely from a distributive kinetic mechanism of the two-site MAPK phosphorylation and dephosphorylation. Importantly, the reported kinetic properties of the kinase (MEK) and phosphatase (MKP3) of extracellular signal-regulated kinase (ERK) fulfill the essential requirements for generating a bistable switch at a single MAPK cascade level. Likewise, a cycle where multisite phosphorylations are performed by different kinases, but dephosphorylation reactions are catalyzed by the same phosphatase, can also exhibit bistability and hysteresis. Hence, bistability induced by multisite covalent modification may be a widespread mechanism of the control of protein activity.     

Analysis of the phosphoproteome of Chlamydomonas reinhardtii provides new insights into various cellular pathways

The unicellular flagellated green alga Chlamydomonas reinhardtii has emerged as a model organism for the study of a variety of cellular processes. Posttranslational control via protein phosphorylation plays a key role in signal transduction, regulation of gene expression, and control of metabolism. Thus, analysis of the phosphoproteome of C. reinhardtii can significantly enhance our understanding of various regulatory pathways. In this study, we have grown C. reinhardtii cultures in the presence of an inhibitor of Ser/Thr phosphatases to increase the phosphoprotein pool. Phosphopeptides from these cells were enriched by immobilized metal-ion affinity chromatography and analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS-MS as well as neutral-loss-triggered MS-MS-MS spectra. In this way, we were able to identify 360 phosphopeptides from 328 different phosphoproteins of C. reinhardtii, thus providing new insights into a variety of cellular processes, including metabolic and signaling pathways. Comparative analysis of the phosphoproteome also yielded new functional information on proteins controlled by redox regulation (thioredoxin target proteins) and proteins of the chloroplast 70S ribosome, the centriole, and especially the flagella, for which 32 phosphoproteins were identified. The high yield of phosphoproteins of the latter correlates well with the presence of several flagellar kinases and indicates that phosphorylation/dephosphorylation represents one of the key regulatory mechanisms of eukaryotic cilia. Our data also provide new insights into certain cilium-related mammalian diseases.     

Regulation of the water channel aquaporin-2 by posttranslational modification

The cellular functions of many eukaryotic membrane proteins, including the vasopressin-regulated water channel aquaporin-2 (AQP2), are regulated by posttranslational modifications. In this article, we discuss the experimental discoveries that have advanced our understanding of how posttranslational modifications affect AQP2 function, especially as they relate to the role of AQP2 in the kidney. We review the most recent data demonstrating that glycosylation and, in particular, phosphorylation and ubiquitination are mechanisms that regulate AQP2 activity, subcellular sorting and distribution, degradation, and protein interactions. From a clinical perspective, posttranslational modification resulting in protein misrouting or degradation may explain certain forms of nephrogenic diabetes insipidus. In addition to providing major insight into the function and dynamics of renal AQP2 regulation, the analysis of AQP2 posttranslational modification may provide general clues as to the role of posttranslational modification for regulation of other membrane proteins.     

Processive phosphorylation: mechanism and biological importance

Recent proteomic data indicate that a majority of the phosphorylated proteins in a eucaryotic cell contain multiple sites of phosphorylation. In many signaling events, a single kinase phosphorylates multiple sites on a target protein. Processive phosphorylation occurs when a protein kinase binds once to a substrate and phosphorylates all of the available sites before dissociating. In this review, we discuss examples of processive phosphorylation by serine/threonine kinases and tyrosine kinases. We describe current experimental approaches for distinguishing processive from non-processive phosphorylation. Finally, we contrast the biological situations that are suited to regulation by processive and non-processive phosphorylation.