ABSTRACT
Mitosis in early embryos often proceeds at a rapid pace, but how this pace is achieved is not understood. Here, we show that cyclin B3 is the dominant driver of rapid embryonic mitoses in the C. elegans embryo. Cyclins B1 and B2 support slow mitosis (NEBD to anaphase â¼600 s), but the presence of cyclin B3 dominantly drives the approximately threefold faster mitosis observed in wildtype. Multiple mitotic events are slowed down in cyclin B1 and B2-driven mitosis, and cyclin B3-associated Cdk1 H1 kinase activity is â¼25-fold more active than cyclin B1-associated Cdk1. Addition of cyclin B1 to fast cyclin B3-only mitosis introduces an â¼60-s delay between completion of chromosome alignment and anaphase onset; this delay, which is important for segregation fidelity, is dependent on inhibitory phosphorylation of the anaphase activator Cdc20. Thus, cyclin B3 dominance, coupled to a cyclin B1-dependent delay that acts via Cdc20 phosphorylation, sets the rapid pace and ensures mitotic fidelity in the early C. elegans embryo.
Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Cyclin B1 , Embryo, Nonmammalian , Mitosis , Animals , Caenorhabditis elegans/embryology , Caenorhabditis elegans/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans Proteins/genetics , Cyclin B1/metabolism , Cyclin B1/genetics , Embryo, Nonmammalian/metabolism , Phosphorylation , CDC2 Protein Kinase/metabolism , CDC2 Protein Kinase/genetics , Cyclin B/metabolism , Cyclin B/genetics , Cdc20 Proteins/metabolism , Cdc20 Proteins/genetics , Cyclin B2/metabolism , Cyclin B2/geneticsABSTRACT
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.
Subject(s)
Mitogen-Activated Protein Kinases , Saccharomyces cerevisiae , Animals , Humans , Saccharomyces cerevisiae/genetics , Mitogen-Activated Protein Kinases/genetics , Mitogen-Activated Protein Kinases/metabolism , Signal Transduction , Phosphorylation , Protein Kinases/metabolism , Mammals/metabolismABSTRACT
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.
ABSTRACT
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.
Subject(s)
Mitogen-Activated Protein Kinase 1 , Repressor Proteins , Zinc Finger Protein GLI1 , Animals , Binding Sites , Cells, Cultured , Conserved Sequence , Hedgehog Proteins/metabolism , Humans , MAP Kinase Signaling System , Mutation , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Phosphorylation , Protein Binding , Zinc Finger Protein GLI1/antagonists & inhibitors , Zinc Finger Protein GLI1/genetics , Zinc Finger Protein GLI1/metabolismABSTRACT
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.
Subject(s)
Cadmium/metabolism , DNA-Binding Proteins/metabolism , Hippo Signaling Pathway/physiology , Protein Serine-Threonine Kinases/metabolism , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , Zinc/metabolism , Cadmium/toxicity , Cell Line, Tumor , Gene Expression Regulation/genetics , HEK293 Cells , HeLa Cells , Homeostasis/genetics , Humans , Inactivation, Metabolic/physiology , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Stress, Physiological/physiology , Transcription, Genetic/genetics , Tumor Suppressor Proteins/genetics , Zinc/toxicity , Transcription Factor MTF-1ABSTRACT
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.
Subject(s)
Binding Sites/physiology , Energy Metabolism/physiology , Protein Processing, Post-Translational/physiology , Proteins , Signal Transduction/physiology , Ligands , Models, Biological , Phosphorylation/physiology , Protein Conformation , Protein Subunits , Proteins/chemistry , Proteins/metabolism , ThermodynamicsABSTRACT
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.
Subject(s)
Neoplasms , Humans , MAP Kinase Signaling System , Mutation , PhosphorylationABSTRACT
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.
Subject(s)
Adenosine Triphosphate , Phosphotransferases , Catalytic DomainABSTRACT
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.
Subject(s)
Chaperonin Containing TCP-1/metabolism , Drosophila Proteins/metabolism , Neuromuscular Junction/growth & development , Neuromuscular Junction/metabolism , Alleles , Animals , Axons/physiology , Chaperonin Containing TCP-1/genetics , Drosophila Proteins/genetics , Drosophila melanogaster , Larva , Mutation , Neuromuscular Junction/enzymology , Neuromuscular Junction/genetics , Neurons/metabolism , Presynaptic Terminals/metabolism , Synapses/metabolism , Synaptic TransmissionABSTRACT
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.
Subject(s)
Mitogen-Activated Protein Kinase 1/chemistry , Mitogen-Activated Protein Kinase 3/chemistry , ras GTPase-Activating Proteins/chemistry , Humans , MAP Kinase Kinase 1/chemistry , MAP Kinase Kinase 1/genetics , MAP Kinase Kinase 1/metabolism , MAP Kinase Kinase 2/chemistry , MAP Kinase Kinase 2/genetics , MAP Kinase Kinase 2/metabolism , Mitogen-Activated Protein Kinase 1/genetics , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Mitogen-Activated Protein Kinase 3/metabolism , Protein Binding , Protein Domains , ras GTPase-Activating Proteins/genetics , ras GTPase-Activating Proteins/metabolismABSTRACT
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.
Subject(s)
Activating Transcription Factor 2/chemistry , JNK Mitogen-Activated Protein Kinases/chemistry , MAP Kinase Kinase 4/chemistry , MAP Kinase Kinase 6/chemistry , Mitogen-Activated Protein Kinase 1/chemistry , Activating Transcription Factor 2/genetics , Amino Acid Sequence , Animals , Binding Sites , Gene Expression Regulation , Humans , Hydrophobic and Hydrophilic Interactions , Isoenzymes/chemistry , Isoenzymes/genetics , JNK Mitogen-Activated Protein Kinases/genetics , MAP Kinase Kinase 4/genetics , MAP Kinase Kinase 6/genetics , Mice , Mitogen-Activated Protein Kinase 1/genetics , Molecular Docking Simulation , Molecular Sequence Data , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Sequence Alignment , Signal TransductionABSTRACT
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.
Subject(s)
Casein Kinase II/chemistry , Casein Kinase II/metabolism , Kinesins/chemistry , Kinesins/metabolism , Signal Transduction/physiology , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Animals , Enzyme Activation/physiology , Humans , Microtubules/physiology , Models, Molecular , Protein Structure, TertiaryABSTRACT
Specific docking interactions between mitogen-activated protein kinases (MAPKs), their regulators, and their downstream substrates, are crucial for efficient and accurate signal transmission. To identify novel substrates of the c-Jun N-terminal kinase (JNK) family of MAPKs, we searched the human genome for proteins that contained (1), a predicted JNK-docking site (D-site); and (2), a cluster of putative JNK target phosphosites located close to the D-site. Here we describe a novel JNK substrate that emerged from this analysis, the functionally uncharacterized protein smoothelin-like 2 (SMTNL2). SMTNL2 protein bound with high-affinity to multiple MAPKs including JNK1-3 and ERK2; furthermore, the identity of conserved amino acids in the predicted docking site (residues 180-193) was necessary for this high-affinity binding. In addition, purified full-length SMTNL2 protein was phosphorylated by JNK1-3 in vitro, and this required the integrity of the D-site. Using mass spectrometry and mutagenesis, we identified four D-site-dependent phosphoacceptor sites in close proximity to the docking site, at S217, S241, T236 and T239. A short peptide comprised of the SMTNL2 D-site inhibited JNK-mediated phosphorylation of the ATF2 transcription factor, showing that SMTNL2 can compete with other substrates for JNK binding. Moreover, when transfected into HEK293 cells, SMTNL2 was phosphorylated by endogenous JNK in a D-site dependent manner, on the same residues identified in vitro. SMTNL2 protein was expressed in many mammalian tissues, with a notably high expression in skeletal muscle. Consistent with the hypothesis that SMTNL2 has a function in skeletal muscle, SMTNL2 protein expression was strongly induced during the transition from myoblasts to myotubes in differentiating C2C12 cells.
Subject(s)
JNK Mitogen-Activated Protein Kinases/metabolism , Phosphoproteins/metabolism , Amino Acid Sequence , Animals , Binding Sites , Cell Line , HEK293 Cells , Humans , JNK Mitogen-Activated Protein Kinases/chemistry , Mice , Molecular Sequence Data , Myoblasts/cytology , Myoblasts/metabolism , Phosphoproteins/chemistry , Phosphorylation , Substrate SpecificityABSTRACT
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.
Subject(s)
Models, Biological , Proteins/chemistry , Proteins/metabolism , Binding Sites , Computational Biology , Computer Simulation , Kinetics , Linear Models , Phosphorylation , Protein Kinases/metabolism , Protein Processing, Post-Translational , Protein StabilityABSTRACT
Kinesin-1 is a plus-end microtubule-based motor, and defects in kinesin-based transport are linked to diseases including neurodegeneration. Kinesin can auto-inhibit via a head-tail interaction, but is believed to be active otherwise. Here we report a tail-independent inactivation of kinesin, reversible by the disease-relevant signalling protein, casein kinase 2 (CK2). The majority of initially active kinesin (native or tail-less) loses its ability to interact with microtubules in vitro, and CK2 reverses this inactivation (approximately fourfold) without altering kinesin's single motor properties. This activation pathway does not require motor phosphorylation, and is independent of head-tail auto-inhibition. In cultured mammalian cells, reducing CK2 expression, but not its kinase activity, decreases the force required to stall lipid droplet transport, consistent with a decreased number of active kinesin motors. Our results provide the first direct evidence of a protein kinase upregulating kinesin-based transport, and suggest a novel pathway for regulating the activity of cargo-bound kinesin.
Subject(s)
Casein Kinase II/metabolism , Kinesins/metabolism , Microtubules/metabolism , Animals , COS Cells , Cell Line , Chlorocebus aethiops , Kinesins/chemistry , Lipid Metabolism , Phosphorylation , RNA Interference , RNA, Small InterferingABSTRACT
BACKGROUND: Cells sense chemical spatial gradients and respond by polarizing internal components. This process can be disrupted by gradient noise caused by fluctuations in chemical concentration. RESULTS: We investigated how external gradient noise affects spatial sensing and response focusing on noise-filtering and the resultant tradeoffs. First, using a coarse-grained mathematical model of gradient-sensing and cell polarity, we characterized three negative consequences of noise: Inhibition of the extent of polarization, degradation of directional accuracy, and production of a noisy output polarization. Next, we explored filtering strategies and discovered that a combination of positive feedback, multiple signaling stages, and time-averaging produced good results. There was an important tradeoff, however, because filtering resulted in slower polarization. Simulations demonstrated that a two-stage filter-amplifier resulted in a balanced outcome. Then, we analyzed the effect of noise on a mechanistic model of yeast cell polarization in response to gradients of mating pheromone. This analysis showed that yeast cells likely also combine the above three filtering mechanisms into a filter-amplifier structure to achieve impressive spatial-noise tolerance, but with the consequence of a slow response time. Further investigation of the amplifier architecture revealed two positive feedback loops, a fast inner and a slow outer, both of which contributed to noise-tolerant polarization. This model also made specific predictions about how orientation performance depended upon the ratio between the gradient slope (signal) and the noise variance. To test these predictions, we performed microfluidics experiments measuring the ability of yeast cells to orient to shallow gradients of mating pheromone. The results of these experiments agreed well with the modeling predictions, demonstrating that yeast cells can sense gradients shallower than 0.1% µm-1, approximately a single receptor-ligand molecule difference between front and back, on par with motile eukaryotic cells. CONCLUSIONS: Spatial noise impedes the extent, accuracy, and smoothness of cell polarization. A combined filtering strategy implemented by a filter-amplifier architecture with slow dynamics was effective. Modeling and experimental data suggest that yeast cells employ these elaborate mechanisms to filter gradient noise resulting in a slow but relatively accurate polarization response.
Subject(s)
Cell Polarity , Models, Biological , Yeasts/cytology , Computer Simulation , Microfluidic Analytical Techniques , Pheromones/pharmacology , Signal Transduction , Yeasts/drug effects , Yeasts/metabolismABSTRACT
BMP is thought to induce hESC differentiation toward multiple lineages including mesoderm and trophoblast. The BMP-induced trophoblast phenotype is a long-standing paradox in stem cell biology. Here we readdressed BMP function in hESCs and mouse epiblast-derived cells. We found that BMP4 cooperates with FGF2 (via ERK) to induce mesoderm and to inhibit endoderm differentiation. These conditions induced cells with high levels of BRACHYURY (BRA) that coexpressed CDX2. BRA was necessary for and preceded CDX2 expression; both genes were essential for expression not only of mesodermal genes but also of trophoblast-associated genes. Maximal expression of the latter was seen in the absence of FGF but these cells coexpressed mesodermal genes and moreover they differed in cell surface and epigenetic properties from placental trophoblast. We conclude that BMP induces human and mouse pluripotent stem cells primarily to form mesoderm, rather than trophoblast, acting through BRA and CDX2.
Subject(s)
Bone Morphogenetic Protein 4/pharmacology , Cell Differentiation/drug effects , Cell Lineage/drug effects , Embryonic Stem Cells/cytology , Fetal Proteins/metabolism , Homeodomain Proteins/metabolism , Pluripotent Stem Cells/cytology , T-Box Domain Proteins/metabolism , Animals , CDX2 Transcription Factor , Chromones/pharmacology , Embryo, Mammalian/cytology , Embryo, Mammalian/drug effects , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Fetal Proteins/genetics , Fibroblast Growth Factor 2/pharmacology , Gene Expression Regulation, Developmental/drug effects , Gene Regulatory Networks/genetics , Glycoprotein Hormones, alpha Subunit/genetics , Glycoprotein Hormones, alpha Subunit/metabolism , Homeodomain Proteins/genetics , Humans , Keratin-7/genetics , Keratin-7/metabolism , Mesoderm/cytology , Mesoderm/drug effects , Mesoderm/metabolism , Mice , Morpholines/pharmacology , Neuropeptides/genetics , Neuropeptides/metabolism , Pluripotent Stem Cells/drug effects , Pluripotent Stem Cells/metabolism , Signal Transduction/drug effects , T-Box Domain Proteins/genetics , Transcription Factors/genetics , Transcription Factors/metabolism , Trophoblasts/cytology , Trophoblasts/drug effects , Trophoblasts/metabolismABSTRACT
In a recent study, the MAP kinase module involved in many human cancers has been reconstructed in yeast, in order to tinker with its behavior.
Subject(s)
Extracellular Signal-Regulated MAP Kinases/metabolism , MAP Kinase Kinase Kinases/metabolism , MAP Kinase Signaling System , Mitogen-Activated Protein Kinases/metabolism , Phosphorylation , Synthetic Biology , Yeasts/genetics , Yeasts/metabolismABSTRACT
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.