The WW domain of IQGAP1 binds directly to the p110α catalytic subunit of PI 3-kinase.

IQGAP1 is a multi-domain cancer-associated protein that serves as a scaffold protein for multiple signaling pathways. Numerous binding partners have been found for the calponin homology, IQ and GAP-related domains in IQGAP1. Identification of a binding partner for its WW domain has proven elusive, however, even though a cell-penetrating peptide derived from this domain has marked anti-tumor activity. Here, using in vitro binding assays with human proteins and co-precipitation from human cells, we show that the WW domain of human IQGAP1 binds directly to the p110α catalytic subunit of phosphoinositide 3-kinase (PI3K). In contrast, the WW domain does not bind to ERK1/2, MEK1/2, or the p85α regulatory subunit of PI3K when p85α is expressed alone. However, the WW domain is able to bind to the p110α/p85α heterodimer when both subunits are co-expressed, as well as to the mutationally activated p110α/p65α heterodimer. We present a model of the structure of the IQGAP1 WW domain, and experimentally identify key residues in the hydrophobic core and beta strands of the WW domain that are required for binding to p110α. These findings contribute to a more precise understanding of IQGAP1-mediated scaffolding, and of how IQGAP1-derived therapeutic peptides might inhibit tumorigenesis.

Effect of magnitude and variability of energy of activation in multisite ultrasensitive biochemical processes.

Protein activity is often regulated by ligand binding or by post-translational modifications such as phosphorylation. Moreover, proteins that are regulated in this way often contain multiple ligand binding sites or modification sites, which can operate to create an ultrasensitive dose response. Here, we consider the contribution of the individual modification/binding sites to the activation process, and how their individual values affect the ultrasensitive behavior of the overall system. We use a generalized Monod-Wyman-Changeux (MWC) model that allows for variable conformational free energy contributions from distinct sites, and associate a so-called activation parameter to each site. Our analysis shows that the ultrasensitivity generally increases as the conformational free energy contribution from one or more sites is strengthened. Furthermore, ultrasensitivity depends on the mean of the activation parameters and not on their variability. In some cases, we find that the best way to maximize ultrasensitivity is to make the contribution from all sites as strong as possible. These results provide insights into the performance objectives of multiple modification/binding sites and thus help gain a greater understanding of signaling and its role in diseases.

Miles to go (mtgo) encodes FNDC3 proteins that interact with the chaperonin subunit CCT3 and are required for NMJ branching and growth in Drosophila.

Analysis of mutants that affect formation and function of the Drosophila larval neuromuscular junction (NMJ) has provided valuable insight into genes required for neuronal branching and synaptic growth. We report that NMJ development in Drosophila requires both the Drosophila ortholog of FNDC3 genes; CG42389 (herein referred to as miles to go; mtgo), and CCT3, which encodes a chaperonin complex subunit. Loss of mtgo function causes late pupal lethality with most animals unable to escape the pupal case, while rare escapers exhibit an ataxic gait and reduced lifespan. NMJs in mtgo mutant larvae have dramatically reduced branching and growth and fewer synaptic boutons compared with control animals. Mutant larvae show normal locomotion but display an abnormal self-righting response and chemosensory deficits that suggest additional functions of mtgo within the nervous system. The pharate lethality in mtgo mutants can be rescued by both low-level pan- and neuronal-, but not muscle-specific expression of a mtgo transgene, supporting a neuronal-intrinsic requirement for mtgo in NMJ development. Mtgo encodes three similar proteins whose domain structure is most closely related to the vertebrate intracellular cytosolic membrane-anchored fibronectin type-III domain-containing protein 3 (FNDC3) protein family. Mtgo physically and genetically interacts with Drosophila CCT3, which encodes a subunit of the TRiC/CCT chaperonin complex required for maturation of actin, tubulin and other substrates. Drosophila larvae heterozygous for a mutation in CCT3 that reduces binding between CCT3 and MTGO also show abnormal NMJ development similar to that observed in mtgo null mutants. Hence, the intracellular FNDC3-ortholog MTGO and CCT3 can form a macromolecular complex, and are both required for NMJ development in Drosophila.

The WW domain of the scaffolding protein IQGAP1 is neither necessary nor sufficient for binding to the MAPKs ERK1 and ERK2.

Mitogen-activated protein kinase (MAPK) scaffold proteins, such as IQ motif containing GTPase activating protein 1 (IQGAP1), are promising targets for novel therapies against cancer and other diseases. Such approaches require accurate information about which domains on the scaffold protein bind to the kinases in the MAPK cascade. Results from previous studies have suggested that the WW domain of IQGAP1 binds to the cancer-associated MAPKs ERK1 and ERK2, and that this domain might thus offer a new tool to selectively inhibit MAPK activation in cancer cells. The goal of this work was therefore to critically evaluate which IQGAP1 domains bind to ERK1/2. Here, using quantitative in vitro binding assays, we show that the IQ domain of IQGAP1 is both necessary and sufficient for binding to ERK1 and ERK2, as well as to the MAPK kinases MEK1 and MEK2. Furthermore, we show that the WW domain is not required for ERK-IQGAP1 binding, and contributes little or no binding energy to this interaction, challenging previous models of how WW-based peptides might inhibit tumorigenesis. Finally, we show that the ERK2-IQGAP1 interaction does not require ERK2 phosphorylation or catalytic activity and does not involve known docking recruitment sites on ERK2, and we obtain an estimate of the dissociation constant (Kd ) for this interaction of 8 µm These results prompt a re-evaluation of published findings and a refined model of IQGAP scaffolding.

CK2 activates kinesin via induction of a conformational change.

Kinesin is the canonical plus-end microtubule motor and has been the focus of intense study since its discovery in 1985. We previously demonstrated a time-dependent inactivation of kinesin in vitro that was fully reversible by the addition of purified casein kinase 2 (CK2) and showed that this inactivation/reactivation pathway was relevant in cells. Here we show that kinesin inactivation results from a conformational change that causes the neck linker to be positioned closer to the motor domain. Furthermore, we show that treatment of kinesin with CK2 prevents and reverses this repositioning. Finally, we demonstrate that CK2 treatment facilitates ADP dissociation from the motor, resulting in a nucleotide-free state that promotes microtubule binding. Thus, we propose that kinesin inactivation results from neck-linker repositioning and that CK2-mediated reactivation results from CK2's dual ability to reverse this repositioning and to promote ADP release.

Two hydrophobic residues can determine the specificity of mitogen-activated protein kinase docking interactions.

MAPKs bind to many of their upstream regulators and downstream substrates via a short docking motif (the D-site) on their binding partner. MAPKs that are in different families (e.g. ERK, JNK, and p38) can bind selectively to D-sites in their authentic substrates and regulators while discriminating against D-sites in other pathways. Here we demonstrate that the short hydrophobic region at the distal end of the D-site plays a critical role in determining the high selectivity of JNK MAPKs for docking sites in their cognate MAPK kinases. Changing just 1 or 2 key hydrophobic residues in this submotif is sufficient to turn a weak JNK-binding D-site into a strong one, or vice versa. These specificity-determining differences are also found in the D-sites of the ETS family transcription factors Elk-1 and Net. Moreover, swapping two hydrophobic residues between these D-sites switches the relative efficiency of Elk-1 and Net as substrates for ERK versus JNK, as predicted. These results provide new insights into docking specificity and suggest that this specificity can evolve rapidly by changes to just 1 or 2 amino acids.

Protein scaffolds can enhance the bistability of multisite phosphorylation systems.

The phosphorylation of a substrate at multiple sites is a common protein modification that can give rise to important structural and electrostatic changes. Scaffold proteins can enhance protein phosphorylation by facilitating an interaction between a protein kinase enzyme and its target substrate. In this work we consider a simple mathematical model of a scaffold protein and show that under specific conditions, the presence of the scaffold can substantially raise the likelihood that the resulting system will exhibit bistable behavior. This phenomenon is especially pronounced when the enzymatic reactions have sufficiently large K(M), compared to the concentration of the target substrate. We also find for a closely related model that bistable systems tend to have a specific kinetic conformation. Using deficiency theory and other methods, we provide a number of necessary conditions for bistability, such as the presence of multiple phosphorylation sites and the dependence of the scaffold binding/unbinding rates on the number of phosphorylated sites.

Mitogen-activated protein kinase (MAPK) cascades-A yeast perspective.

Discovery of the class of protein kinase now dubbed a mitogen (or messenger)-activated protein kinase (MAPK) is an illustrative example of how disparate lines of investigation can converge and reveal an enzyme family universally conserved among eukaryotes, from single-celled microbes to humans. Moreover, elucidation of the circuitry controlling MAPK function defined a now overarching principle in enzyme regulation-the concept of an activation cascade mediated by sequential phosphorylation events. Particularly ground-breaking for this field of exploration were the contributions of genetic approaches conducted using several model organisms, but especially the budding yeast Saccharomyces cerevisiae. Notably, examination of how haploid yeast cells respond to their secreted peptide mating pheromones was crucial in pinpointing genes encoding MAPKs and their upstream activators. Fully contemporaneous biochemical analysis of the activities elicited upon stimulation of mammalian cells by insulin and other growth- and differentiation-inducing factors lead eventually to the demonstration that components homologous to those in yeast were involved. Continued studies of these pathways in yeast were integral to other foundational discoveries in MAPK signaling, including the roles of tethering, scaffolding and docking interactions.

ERK2 MAP kinase regulates SUFU binding by multisite phosphorylation of GLI1.

Crosstalk between the Hedgehog and MAPK signaling pathways occurs in several types of cancer and contributes to clinical resistance to Hedgehog pathway inhibitors. Here we show that MAP kinase-mediated phosphorylation weakens the binding of the GLI1 transcription factor to its negative regulator SUFU. ERK2 phosphorylates GLI1 on three evolutionarily conserved target sites (S102, S116, and S130) located near the high-affinity binding site for SUFU; these phosphorylations cooperate to weaken the affinity of GLI1-SUFU binding by over 25-fold. Phosphorylation of any one, or even any two, of the three sites does not result in the level of SUFU release seen when all three sites are phosphorylated. Tumor-derived mutations in R100 and S105, residues bordering S102, also diminish SUFU binding, collectively defining a novel evolutionarily conserved SUFU affinity-modulating region. In cultured mammalian cells, GLI1 variants containing phosphomimetic substitutions of S102, S116, and S130 displayed an increased ability to drive transcription. We conclude that multisite phosphorylation of GLI1 by ERK2 or other MAP kinases weakens GLI1-SUFU binding, thereby facilitating GLI1 activation and contributing to both physiological and pathological crosstalk.

The Hippo pathway kinases LATS1 and LATS2 attenuate cellular responses to heavy metals through phosphorylating MTF1.

Heavy metals are both integral parts of cells and environmental toxicants, and their deregulation is associated with severe cellular dysfunction and various diseases. Here we show that the Hippo pathway plays a critical role in regulating heavy metal homeostasis. Hippo signalling deficiency promotes the transcription of heavy metal response genes and protects cells from heavy metal-induced toxicity, a process independent of its classic downstream effectors YAP and TAZ. Mechanistically, the Hippo pathway kinase LATS phosphorylates and inhibits MTF1, an essential transcription factor in the heavy metal response, resulting in the loss of heavy metal response gene transcription and cellular protection. Moreover, LATS activity is inhibited following heavy metal treatment, where accumulated zinc directly binds and inhibits LATS. Together, our study reveals an interplay between the Hippo pathway and heavy metals, providing insights into this growth-related pathway in tissue homeostasis and stress response.

Signal transduction: turning a switch into a rheostat.

MAP kinase cascades are inherently switch-like, but, during yeast mating, MAPK signaling is graded. A new study suggests that the Ste5 scaffold protein is responsible for making this switch less switch-like.

Oscillatory phosphorylation of yeast Fus3 MAP kinase controls periodic gene expression and morphogenesis.

Signal-transduction networks can display complex dynamic behavior such as oscillations in the activity of key components [1-6], but it is often unclear whether such dynamic complexity is actually important for the network's regulatory functions [7, 8]. Here, we found that the mitogen-activated protein kinase (MAPK) Fus3, a key regulator of the yeast mating-pheromone response, undergoes sustained oscillations in its phosphorylation and activation state during continuous pheromone exposure. These MAPK activity oscillations led to corresponding oscillations in mating-gene expression. Oscillations in MAPK activity and gene expression required the negative regulator of G protein signaling Sst2 and partially required the MAPK phosphatase Msg5. Peaks in Fus3 activation correlated with periodic rounds of cell morphogenesis, with each peak preceding the formation of an additional mating projection. Preventing projection formation did not eliminate MAPK oscillation, but preventing MAPK oscillation blocked the formation of additional projections. A mathematical model was developed that reproduced several features of the observed oscillatory dynamics. These observations demonstrate a role for MAPK activity oscillation in driving a periodic downstream response and explain how the pheromone signaling pathway, previously thought to desensitize after 1-3 hr, controls morphology changes that continue for a much longer time.

Computational prediction and experimental verification of new MAP kinase docking sites and substrates including Gli transcription factors.

In order to fully understand protein kinase networks, new methods are needed to identify regulators and substrates of kinases, especially for weakly expressed proteins. Here we have developed a hybrid computational search algorithm that combines machine learning and expert knowledge to identify kinase docking sites, and used this algorithm to search the human genome for novel MAP kinase substrates and regulators focused on the JNK family of MAP kinases. Predictions were tested by peptide array followed by rigorous biochemical verification with in vitro binding and kinase assays on wild-type and mutant proteins. Using this procedure, we found new 'D-site' class docking sites in previously known JNK substrates (hnRNP-K, PPM1J/PP2Czeta), as well as new JNK-interacting proteins (MLL4, NEIL1). Finally, we identified new D-site-dependent MAPK substrates, including the hedgehog-regulated transcription factors Gli1 and Gli3, suggesting that a direct connection between MAP kinase and hedgehog signaling may occur at the level of these key regulators. These results demonstrate that a genome-wide search for MAP kinase docking sites can be used to find new docking sites and substrates.

A combination of multisite phosphorylation and substrate sequestration produces switchlike responses.

The phosphorylation of a protein on multiple sites has been proposed to promote the switchlike regulation of protein activity. Recent theoretical work, however, indicates that multisite phosphorylation, by itself, is less effective at creating switchlike responses than had been previously thought. The phosphorylation of a protein often alters its spatial localization, or its association with other proteins, and this sequestration can alter the accessibility of the substrate to the relevant kinases and phosphatases. Sequestration thus has the potential to interact with multisite phosphorylation to modulate ultrasensitivity and threshold. Here, using simple ordinary differential equations to represent phosphorylation, dephosphorylation, and binding/sequestration, we demonstrate that the combination of multisite phosphorylation and regulated substrate sequestration can produce a response that is both a good threshold and a good switch. Several strategies are explored, including both stronger and weaker sequestration with successive phosphorylations, as well as combinations that are more elaborate. In some strategies, such as when phosphorylation and dephosphorylation are segregated, a near-optimal switch is possible, where the effective Hill number equals the number of phosphorylation sites.

A scalable and integrative system for pathway bioinformatics and systems biology.

MOTIVATION: Progress in systems biology depends on developing scalable informatics tools to predictively model, visualize, and flexibly store information about complex biological systems. Scalability of these tools, as well as their ability to integrate within larger frameworks of evolving tools, is critical to address the multi-scale and size complexity of biological systems. RESULTS: Using current software technology, such as self-generation of database and object code from UML schemas, facilitates rapid updating of a scalable expert assistance system for modeling biological pathways. Distribution of key components along with connectivity to external data sources and analysis tools is achieved via a web service interface. AVAILABILITY: All sigmoid modeling software components and supplementary information are available through: http://www.igb.uci.edu/servers/sb.html.

Cancer Mutations: Molecular MEKanisms.

MEK, a central component of the Ras/MAPK cascade, is mutated in human tumors and developmental disorders. Recent studies are beginning to dissect the mechanisms that make these MEK mutants hyperactive.

G-protein signaling: a new branch in an old pathway.

A recent study provides evidence for a new branch of the yeast mating pathway in which a G-protein alpha subunit directly activates phosphatidylinositol 3-kinase at endosomes.

A conserved protein interaction network involving the yeast MAP kinases Fus3 and Kss1.

The Saccharomyces cerevisiae mitogen-activated protein kinases (MAPKs) Fus3 and Kss1 bind to multiple regulators and substrates. We show that mutations in a conserved docking site in these MAPKs (the CD/7m region) disrupt binding to an important subset of their binding partners, including the Ste7 MAPK kinase, the Ste5 adaptor/scaffold protein, and the Dig1 and Dig2 transcriptional repressors. Supporting the possibility that Ste5 and Ste7 bind to the same region of the MAPKs, they partially competed for Fus3 binding. In vivo, some of the MAPK mutants displayed reduced Ste7-dependent phosphorylation, and all of them exhibited multiple defects in mating and pheromone response. The Kss1 mutants were also defective in Kss1-imposed repression of Ste12. We conclude that MAPKs contain a structurally and functionally conserved docking site that mediates an overall positively acting network of interactions with cognate docking sites on their regulators and substrates. Key features of this interaction network appear to have been conserved from yeast to humans.

Pseudokinases: Flipping the ATP for AMPylation.

The crystal structure of SelO, a pseudokinase previously presumed to be inactive, reveals an ATP cofactor sitting in the active site in a flipped orientation compared with canonical kinases, leading to the discovery of an unexpected catalytic activity for this ancient enzyme.

Mitogen-activated protein kinases with distinct requirements for Ste5 scaffolding influence signaling specificity in Saccharomyces cerevisiae.

Scaffold proteins are believed to enhance specificity in cell signaling when different pathways share common components. The prototype scaffold Ste5 binds to multiple components of the Saccharomyces cerevisiae mating pheromone response pathway, thereby conducting the mating signal to the Fus3 mitogen-activated protein kinase (MAPK). Some of the kinases that Ste5 binds to, however, are also shared with other pathways. Thus, it has been presumed that Ste5 prevents its bound kinases from transgressing into other pathways and protects them from intrusions from those pathways. Here we found that Fus3MAPK required Ste5 scaffolding to receive legitimate signals from the mating pathway as well as misdirected signals leaking from other pathways. Furthermore, increasing the cellular concentration of active Ste5 enhanced the channeling of inappropriate stimuli to Fus3. This aberrant signal crossover resulted in the erroneous induction of cell cycle arrest and mating. In contrast to Fus3, the Kss1 MAPK did not require Ste5 scaffolding to receive either authentic or leaking signals. Furthermore, the Ste11 kinase, once activated via Ste5, was able to signal to Kss1 independently of Ste5 scaffolding. These results argue that Ste5 does not act as a barrier that actively prevents signal crossover to Fus3 and that Ste5 may not effectively sequester its activated kinases away from other pathways. Rather, we suggest that specificity in this network is promoted by the selective activation of Ste5 and the distinct requirements of the MAPKs for Ste5 scaffolding.