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Receptor-like cytoplasmic kinase (RLCK) subfamily VII members are involved in diverse biological processes, like reproduction, immunity, growth and development. Ubiquitination and proteasomal degradation of a RLCK VII member, BOTRYTIS-INDUCED KINASE1 (BIK1) play important roles in regulating immune signaling. It remains largely unknown whether most other RLCK VII members undergo ubiquitination and proteasomal degradation. Here, we select the 6-member RLCK VII-4 to examine the potential proteasomal degradation of its members. We find that three closely related RLCK VII-4 members, PBL38 (AvrPphB SUSCEPTIBLE1-LIKE38), PCRK1 (PTI-COMPROMISED RECEPTOR-LIKE CYTOPLASMIC KINASE1), and PCRK2 are under proteasomal control, while the other members in this group are not. Moreover, we demonstrate that PCRK2 undergoes ubiquitination and proteasomal in a kinase activity-dependent manner. However, the plasma membrane (PM) localization of PCRK2 is not required for its degradation. Our work suggests that many other RLCK VII members may undergo ubiquitination and proteasomal degradation to modulate their homeostasis and cellular functions.

The Hsp90 chaperone system interacts with a wide spectrum of client proteins, forming variable and dynamic multiprotein complexes that involve the intervention of cochaperone partners. Recent results suggest that the role of Hsp90 complexes is to establish interactions that suppress unwanted client activities, allow clients to be protected from degradation and respond to biochemical signals. Cryo-electron microscopy (cryoEM) provided the first key molecular picture of Hsp90 in complex with a kinase, Cdk4, and a cochaperone, Cdc37. Here, we use a combination of molecular dynamics (MD) simulations and advanced comparative analysis methods to elucidate key aspects of the functional dynamics of the complex, with different nucleotides bound at the N-terminal Domain of Hsp90. The results reveal that nucleotide-dependent structural modulations reverberate in a striking asymmetry of the dynamics of Hsp90 and identify specific patterns of long-range coordination between the nucleotide binding site, the client binding pocket, the cochaperone and the client. Our model establishes a direct atomic-resolution cross-talk between the ATP-binding site, the client region that is to be remodeled and the surfaces of the Cdc37-cochaperone.

Herein we report the discovery of 1-(5-(tert-butyl)isoxazol-3-yl)-3- (3-fluorophenyl)urea derivatives as new FLT3 inhibitors that are able to overcome the drug resistance mutations: the secondary D835Y and F691L mutations on the basis of the internal tandem duplications (ITD) mutation of FLT3 (FLT3-ITD/D835Y and FLT3-ITD/F691L, respectively). The most potent compound corresponds to 1-(5-(tert-butyl)isoxazol-3-yl)-3-(4-((6,7-dimethoxyquinolin-4-yl)oxy)-3- fluorophenyl)urea (4d), which showed IC50s (half maximal inhibitory concentrations) of 0.072 nM, 5.86 nM and 3.48 nM against FLT3-ITD, FLT3-ITD/F691L and FLT3-ITD/D835Y, respectively. Compound 4d also showed good selectivity for FLT3 in a kinase profiling assay. Collectively, 4d could be a good lead compound and deserves further in-depth studies.

Summary Protein kinases constitute a large class of signaling molecules frequently targeted in research and clinical uses. However, kinase inhibitors are notoriously non-specific, making it difficult to select an appropriate inhibitor for a given kinase. Available data from large-scale kinase inhibitor screens are often difficult to query. Here, we present KInhibition (https://kinhibition.fredhutch.org), an online portal that allows users to search publicly available datasets to find selective inhibitors for a chosen kinase or group of kinases. Compounds are sorted by a KInhibition Selectivity Score, calculated based on compounds' activity against the selected kinase(s) versus activity against all other kinases for which that compound has been profiled. The current version allows users to query four datasets, with a framework that can easily accommodate additional datasets. KInhibition represents a powerful platform through which researchers from broad areas of biology, chemistry, and pharmacology can easily interrogate large datasets to help guide their selection of kinase inhibitors., Graphical Abstract, Highlights • An easy-to-use tool that compiles datasets from kinase inhibitor screens • A KInhibition Selectivity Score quantifies each compound's selectivity • Users can select kinases, view compounds and selectivity, and download the results • KInhibition is broadly applicable across life science disciplines, Molecular Biology; Bioinformatics; Software Engineering

The pseudokinase group encompasses some 10% of protein kinases, but pseudokinases diverge from canonical kinases in key motifs. The two members of the small new kinase family 3 (NKF3) group are considered pseudokinases. These proteins, pseudopodium-enriched atypical kinase 1 (PEAK1, Sugen kinase 269, or SgK269) and pragmin (Sugen kinase 223 or SgK223), act as scaffolds in growth factor signaling pathways, and both contain a kinase fold with degraded kinase motifs at their C termini. These kinases may harbor regions that mediate oligomerization or control other aspects of signal transduction, but a lack of structural information has precluded detailed investigations into their functional roles. In this study, we determined the X-ray crystal structure of the PEAK1 pseudokinase domain to 2.3 Å resolution. The structure revealed that the PEAK1 kinase-like domain contains a closed nucleotide-binding cleft that in this conformation may deleteriously affect nucleotide binding. Moreover, we found that N- and C-terminal extensions create a highly unusual all α-helical split-dimerization region, termed here the split helical dimerization (SHED) region. Sequence conservation analysis suggested that this region facilitates a dimerization mode that is conserved between PEAK1 and pragmin. Finally, we observed structural similarities between the PEAK1 SHED region and the C-terminal extension of the Parkinson's disease-associated kinase PINK1. In summary, PEAK1's kinase cleft is occluded, and its newly identified SHED region may promote an unexpected dimerization mode. Similarities of PEAK1 with the active kinase PINK1 may reclassify the latter as a member of the new kinase family 3 group.

Kinase inhibitors are promising drugs to stabilize the endothelial barrier following inflammatory damage. However, our limited knowledge of how kinase signaling activates barrier-restorative pathways and the complexity of multi-target drugs have hindered drug discovery and repurposing efforts. Here, we apply a kinase regression approach that exploits drug polypharmacology to investigate endothelial barrier regulation. A screen of 28 kinase inhibitors identified multiple inhibitors that promote endothelial barrier integrity and revealed divergent barrier phenotypes for BCR-ABL drugs. Target deconvolution predicted 50 barrier-regulating kinases from diverse kinase families. Using gene knockdowns, we identified kinases with a role in endothelial barrier regulation and dissected different mechanisms of action of barrier-protective kinase inhibitors. These results demonstrate the importance of polypharmacology in the endothelial barrier phenotype of kinase inhibitors and provide promising new leads for barrier-strengthening therapies.

In this paper, we present the results of a ligand- and structure-based virtual screen targeting LRRK2, a kinase that has been implicated in Parkinson's disease. For the ligand-based virtual screen, the structures of 12 competitor compounds were used as queries for a variety of 2D and 3D searches. The structure-based virtual screen relied on homology models of LRRK2, as no X-ray structure is currently available in the public domain. From the virtual screening, 662 compounds were purchased, of which 35 showed IC50 values below 10μM in wild-type and/or mutant LRRK2 (a hit rate of 5.3%). Of these 35 hits, four were deemed to have potential for medicinal chemistry follow-up.

ALS-associated mutations in the kinase TBK1 with the same pathological outcome affect distinct signaling pathways.

Ryanodine Receptors are Ca2+ release channels expressed in the Endoplasmic and Sarcoplasmic Reticulum membranes. Gong et al [1] reported cryo-EM structures of the cardiac RyR2 complexed to Calmodulin, which can downregulate channel opening. Haji-Ghassemi et al [2] report crystal structures of an RyR2 domain with PKA, a kinase promoting channel opening.

The protein p62/Sequestosome 1 (p62) has been described as a selective autophagy receptor and independently as a platform for pro-inflammatory and other intracellular signaling. How these seemingly disparate functional roles of p62 are coordinated has not been resolved. Here, we show that TAK1, a kinase involved in immune signaling, negatively regulates p62 action in autophagy. TAK1 reduces p62 localization to autophagosomes, dampening the autophagic degradation of both p62 and p62-directed autophagy substrates. TAK1 also relocalizes p62 into dynamic cytoplasmic bodies, a phenomenon that accompanies the stabilization of TAK1 complex components. On the other hand, p62 facilitates the assembly and activation of TAK1 complexes, suggesting a connection between p62's signaling functions and p62 body formation. Thus, TAK1 governs p62 action, switching it from an autophagy receptor to a signaling platform. This ability of TAK1 to disable p62 as an autophagy receptor may allow certain autophagic substrates to accumulate when needed for cellular functions.

Since quorum sensing (QS) is linked to the establishment of bacterial infection, its inactivation represents one of the newest strategies to fight bacterial pathogens. LsrK is a kinase playing a key role in the processing of autoinducer-2 (AI-2), a quorum-sensing mediator in gut enteric bacteria. Inhibition of LsrK might thus impair the quorum-sensing cascade and consequently reduce bacterial pathogenicity. Aiming for the development of a target-based assay for the discovery of LsrK inhibitors, we evaluated different assay set-ups based on ATP detection and optimized an automation-compatible method for the high-throughput screening of chemical libraries. The assay was then used to perform the screening of a 2000-compound library, which provided 12 active compounds with an IC50 &le, 10 &micro, M confirming the effectiveness and sensitivity of our assay. Follow-up studies on the positive hits led to the identification of two compounds, harpagoside and rosolic acid, active in a cell-based AI-2 QS interference assay, which are at the moment the most promising candidates for the development of a new class of antivirulence agents based on LsrK inhibition.

Drosophila provides an inexpensive and quantitative platform for measuring whole animal drug response. A complementary approach is virtual screening, where chemical libraries can be efficiently screened against protein target(s). Here, we present a unique discovery platform integrating structure-based modeling with Drosophila biology and organic synthesis. We demonstrate this platform by developing chemicals targeting a Drosophila model of Medullary Thyroid Cancer (MTC) characterized by a transformation network activated by oncogenic dRetM955T. Structural models for kinases relevant to MTC were generated for virtual screening to identify unique preliminary hits that suppressed dRetM955T-induced transformation. We then combined features from our hits with those of known inhibitors to create a ‘hybrid’ molecule with improved suppression of dRetM955T transformation. Our platform provides a framework to efficiently explore novel kinase inhibitors outside of explored inhibitor chemical space that are effective in inhibiting cancer networks while minimizing whole body toxicity., Author summary Effective and safe treatment of multigenic diseases often involves drugs that address multiple points along disease networks, i.e., polypharmacology. Polypharmacology is increasingly appreciated as a potentially desirable property of kinase drugs. However, most known drugs that interact with multiple targets have been identified as such by chance and most polypharmacological compounds are not chemically unique, resembling structures of known kinase inhibitors. The fruit fly Drosophila provides an inexpensive, rapid, quantitative, whole animal screening platform that has the potential to complement computational approaches. We present a chemical genetics approach that efficiently combines Drosophila with structural prediction and virtual screening, creating a unique discovery platform. We demonstrate the utility of our approach by developing useful small molecules targeting a kinase network in a Drosophila model of Medullary Thyroid Cancer (MTC) driven by oncogenic dRetM955T.

Motivation Identifying the kinase–substrate relationships is vital to understanding the phosphorylation events and various biological processes, especially signal transductions. Although large amount of phosphorylation sites have been detected, unfortunately, it is rarely known which kinases activate those sites. Despite distinct computational approaches have been proposed to predict the kinase–substrate interactions, the prediction accuracy still needs to be improved. Results In this paper, we propose a novel probabilistic model named as PhosD to predict kinase–substrate relationships based on protein domains with the assumption that kinase–substrate interactions are accomplished with kinase–domain interactions. By further taking into account protein–protein interactions, our PhosD outperforms other popular approaches on several benchmark datasets with higher precision. In addition, some of our predicted kinase–substrate relationships are validated by signaling pathways, indicating the predictive power of our approach. Furthermore, we notice that given a kinase, the more substrates are known for the kinase the more accurate its predicted substrates will be, and the domains involved in kinase–substrate interactions are found to be more conserved across proteins phosphorylated by multiple kinases. These findings can help develop more efficient computational approaches in the future. Availability and Implementation The data and results are available at http://comp-sysbio.org/phosd Supplementary information Supplementary data are available at Bioinformatics online.

Tuberous sclerosis complex (TSC) is a rare multi-system disorder, primary manifestations of which are benign tumors and lesions in various organs of the body, including the brain. TSC patients often suffer from epilepsy, mental retardation, and autism spectrum disorder (ASD). Therefore, TSC serves as a model of epilepsy, ASD, and tumorigenesis. TSC is caused by the lack of functional Tsc1-Tsc2 complex, which serves as a major cellular inhibitor of mammalian Target of Rapamycin Complex 1 (mTORC1). mTORC1 is a kinase controlling most of anabolic processes in eukaryotic cells. Consequently, mTORC1 inhibitors, such as rapamycin, serve as experimental or already approved drugs for several TSC symptoms. However, rapalogs, although quite effective, need to be administered chronically and likely for a lifetime, since therapy discontinuation results in tumor regrowth and epilepsy recurrence. Recent studies revealed that metabolism and excitability (in the case of neurons) of cells lacking Tsc1-Tsc2 complex are changed, and these features may potentially be used to treat some of TSC symptoms. In this review, we first provide basic facts about TSC and its molecular background, to next discuss the newest findings in TSC cell biology that can be used to improve existing therapies of TSC and other diseases linked to mTORC1 hyperactivation. © 2016 IUBMB Life, 68(12):955-962, 2016.

Summary ATP-competitive kinase inhibitors often bind several kinases due to the high conservation of the ATP binding pocket. Through clustering analysis of a large kinome profiling dataset, we found a cluster of eight promiscuous kinases that on average bind more than five times more kinase inhibitors than the other 398 kinases in the dataset. To understand the structural basis of promiscuous inhibitor binding, we determined the co-crystal structure of the receptor tyrosine kinase DDR1 with the type I inhibitors dasatinib and VX-680. Surprisingly, we find that DDR1 binds these type I inhibitors in an inactive conformation typically reserved for type II inhibitors. Our computational and biochemical studies show that DDR1 is unusually stable in this inactive conformation, giving a mechanistic explanation for inhibitor promiscuity. This phenotypic clustering analysis provides a strategy to obtain functional insights not available by sequence comparison alone.

The articles highlighted this week focus on signaling events that control appetite and weight; the effects of Hippo signaling on cell function during cardiac repair; a mechanism that drives cancer drug addiction; inactivation of a kinase complex by structural rearrangement; and the mechanism by which a noncoding viral RNA inhibits host cell apoptosis.

This week’s articles highlight a compound that may be useful for treating cryptosporidiosis and a kinase complex that links cell division to cellular metabolism.

Summary Computational characterization of differential kinase activity from phosphoproteomics datasets is critical for correctly inferring cellular circuitry and how signaling cascades are altered in drug treatment and/or disease. Kinase-Substrate Enrichment Analysis (KSEA) offers a powerful approach to estimating changes in a kinase’s activity based on the collective phosphorylation changes of its identified substrates. However, KSEA has been limited to programmers who are able to implement the algorithms. Thus, to make it accessible to the larger scientific community, we present a web-based application of this method: the KSEA App. Overall, we expect that this tool will offer a quick and user-friendly way of generating kinase activity estimates from high-throughput phosphoproteomics datasets. Availability and implementation the KSEA App is a free online tool: casecpb.shinyapps.io/ksea/. The source code is on GitHub: github.com/casecpb/KSEA/. The application is also available as the R package ‘KSEAapp’ on CRAN: CRAN.R-project.org/package=KSEAapp/. Supplementary information Supplementary data are available at Bioinformatics online.

This week’s papers describe therapies for myeloid cancers and a kinase to inhibit in order to prevent pathogenic bone destruction.

Long-term depression (LTD) is a reduction in the efficacy of neuronal synapses, but the molecular basis of LTD signaling and how these signals lead to phenotypic outcomes, such as the shrinkage of synaptic regions, is not clear. In a new report, Woolfrey et al. use chemically-induced LTD and a multitude of in vitro biochemical assays to provide evidence that synaptic removal of the scaffolding protein AKAP79/150 promotes LTD-induced spine shrinkage. The further identification of CaMKII, a kinase primarily associated with long-term potentiation (LTP), as a requirement for AKAP79/150 removal, uncovers unexpected interplay between different post-translational modifications and points to a new model of LTD.

DYRK1B is a kinase over-expressed in certain cancer cells (including colon, ovarian, pancreatic, etc.). Recent publications have demonstrated inhibition of DYRK1B could be an attractive target for cancer therapy. From a data-mining effort, the team has discovered analogues of pyrido[2,3-d]pyrimidines as potent enantio-selective inhibitors of DYRK1B. Cells treated with a tool compound from this series showed the same cellular effects as down regulation of DYRK1B with siRNA. Such effects are consistent with the proposed mechanism of action. Progress of the SAR study is presented.

Genetically encoded, FRET based biosensors have revolutionized cell signalling research in the past decade. They allow tracking dynamic activities of signalling molecules such as protein kinases, Rho GTPases or second messengers in real time on a subcellular level. This article gives an overview about design, components and employment of different types of FRET biosensors. Exemplarily, a kinase activity reporter to visualize protein kinase D activity at the Golgi complex is presented.

Background Kinases and phosphatases (KP) form complex self-regulating networks essential for cellular signal processing. In spite of having a wealth of data about interactions among KPs and their substrates, we have very limited models of the structures of the directed networks they form and consequently our ability to formulate hypotheses about how their structure determines the flow of information in these networks is restricted. Results We assembled and studied the largest bona fide kinase-phosphatase network (KP-Net) known to date for the yeast Saccharomyces cerevisiae. Application of the vertex sort (VS) algorithm on the KP-Net allowed us to elucidate its hierarchical structure in which nodes are sorted into top, core and bottom layers, forming a bow tie structure with a strongly connected core layer. Surprisingly, phosphatases tend to sort into the top layer, implying they are less regulated by phosphorylation than kinases. Superposition of the widest range of KP biological properties over the KP-Net hierarchy shows that core layer KPs: (i), receive the largest number of inputs; (ii), form bottlenecks implicated in multiple pathways and in decision-making; (iii), and are among the most regulated KPs both temporally and spatially. Moreover, top layer KPs are more abundant and less noisy than those in the bottom layer. Finally, we showed that the VS algorithm depends on node degrees without biasing the biological results of the sorted network. The VS algorithm is available as an R package (https://cran.r-project.org/web/packages/VertexSort/index.html). Conclusions The KP-Net model we propose possesses a bow tie hierarchical structure in which the top layer appears to ensure highest fidelity and the core layer appears to mediate signal integration and cell state-dependent signal interpretation. Our model of the yeast KP-Net provides both functional insight into its organization as we understand today and a framework for future investigation of information processing in yeast and eukaryotes in general. Electronic supplementary material The online version of this article (doi:10.1186/s12918-017-0418-0) contains supplementary material, which is available to authorized users.

Caspase-8 can initiate apoptosis, but it is also required for embryonic development and immune cell proliferation. While several non-apoptotic roles for caspase-8 have been proposed, recent work has indicated that the requirement for caspase-8 in development and proliferation is defined by suppression of RIPK3, a kinase that can trigger an alternative form of cell death called programmed necrosis. We will consider these recent findings, and how they can be reconciled with earlier work on the non-apoptotic roles of caspase-8.

Inhibition of rho kinase (ROCK) has been recognized as an important target for a number of diseases, including glaucoma. Herein we report SAR development around two hits from a kinase library that led to the discovery of the ROCK inhibitor compound 38. In vitro and in vivo analysis of this compound, including its effects in a monkey model of glaucoma will be discussed.

BTK has emerged as a promising target for treating B-cell malignancies and autoimmune diseases, and there has been a growing demand to identify selective BTK inhibitors efficiently. In this Letter, we have designed and synthesized a new fluorescent probe to screen compounds that preferentially bind to an unactivated state of BTK (BTK [U]). The fluorescence of the probe was turned on in the presence of BTK [U], and quenched by the addition of compounds which preferentially bind to BTK [U]. This unique fluorescent probe was successfully applied to the screening of a kinase focused compound library. The results suggest that this new method is a simple and easy-to-perform assay to screen inhibitors of BTK [U].

We performed Gaussian network model based normal mode analysis of 3-dimensional structures of multiple active and inactive forms of protein kinases. In 14 different kinases, a more number of residues (1095) show higher structural fluctuations in inactive states than those in active states (525), suggesting that, in general, mobility of inactive states is higher than active states. This statistically significant difference is consistent with higher crystallographic B-factors and conformational energies for inactive than active states, suggesting lower stability of inactive forms. Only a small number of inactive conformations with the DFG motif in the ``in'' state were found to have fluctuation magnitudes comparable to the active conformation. Therefore our study reports for the first time, intrinsic higher structural fluctuation for almost all inactive conformations compared to the active forms. Regions with higher fluctuations in the inactive states are often localized to the aC-helix, aG-helix and activation loop which are involved in the regulation and/or in structural transitions between active and inactive states. Further analysis of 476 kinase structures involved in interactions with another domain/protein showed that many of the regions with higher inactive-state fluctuation correspond to contact interfaces. We also performed extensive GNM analysis of (i) insulin receptor kinase bound to another protein and (ii) holo and apo forms of active and inactive conformations followed by multi-factor analysis of variance. We conclude that binding of small molecules or other domains/proteins reduce the extent of fluctuation irrespective of active or inactive forms. Finally, we show that the perceived fluctuations serve as a useful input to predict the functional state of a kinase.

The current status of docking procedures for predicting protein-protein interactions starting from their three-dimensional structure is assessed from a first major evaluation of blind predictions. This evaluation was performed as part of a communitywide experiment on Critical Assessment of PRedicted Interactions (CAPRI). Seven newly determined structures of protein-protein complexes were available as targets for this experiment. These were the complexes between a kinase and its protein substrate, between a T-cell receptor beta-chain and a superantigen, and five antigen-antibody complexes. For each target, the predictors were given the experimental structures of the free components, or of one free and one bound component in a random orientation. The structure of the complex was revealed only at the time of the evaluation. A total of 465 predictions submitted by 19 groups were evaluated. These groups used a wide range of algorithms and scoring functions, some of which were completely novel. The quality of the predicted interactions was evaluated by comparing residue-residue contacts and interface residues to those in the X-ray structures and by analyzing the fit of the ligand molecules (the smaller of the two proteins in the complex) or of interface residues only, in the predicted versus target complexes. A total of 14 groups produced predictions, ranking from acceptable to highly accurate for five of the seven targets. The use of available biochemical and biological information, and in one instance structural information, played a key role in achieving this result. It was essential for identifying the native binding modes for the five correctly predicted targets, including the kinase-substrate complex where the enzyme changes conformation on association. But it was also the cause for missing the correct solution for the two remaining unpredicted targets, which involve unexpected antigen-antibody binding modes. Overall, this analysis reveals genuine progress in docking procedures but also illustrates the remaining serious limitations and points out the need for better scoring functions and more effective ways for handling conformational flexibility.

This Podcast features an interview with Alfred Singer, Gil Katz, and Leonid Pobezinsky, authors of a Research Article that appears in the 26 August 2014 issue of Science Signaling , about a microRNA that plays a role in the survival of T cells. During development in the thymus and after maturation and migration into the periphery, T cells require signaling through both the T cell receptor (TCR) and the interleukin-7 receptor (IL-7R) to survive. However, because sustained IL-7R signaling kills T cells, TCR signaling periodically inhibits IL-7R signaling to enable T cell survival. Katz et al . have identified a microRNA that is induced by T cell receptor activation and inhibits IL-7R signaling by reducing the abundance of Jak1, a kinase that transduces signaling downstream of IL-7R.

BIG1 and BIG2, brefeldin A-inhibited guanine nucleotide-exchange factors (GEFs) that activate ADP-ribosylation factors (Arfs) by accelerating the replacement of bound GDP with GTP, contain A kinase...

The Doc toxin from bacteriophage P1 (of the phd-doc toxin-antitoxin system) has served as a model for the family of Doc toxins, many of which are harbored in the genomes of pathogens. We have shown previously that the mode of action of this toxin is distinct from the majority derived from toxin-antitoxin systems: it does not cleave RNA; in fact P1 Doc expression leads to mRNA stabilization. However, the molecular triggers that lead to translation arrest are not understood. The presence of a Fic domain, albeit slightly altered in length and at the catalytic site, provided a clue to the mechanism of P1 Doc action, as most proteins with this conserved domain inactivate GTPases through addition of an adenylyl group (also referred to as AMPylation). We demonstrated that P1 Doc added a single phosphate group to the essential translation elongation factor and GTPase, elongation factor (EF)-Tu. The phosphorylation site was at a highly conserved threonine, Thr-382, which was blocked when EF-Tu was treated with the antibiotic kirromycin. Therefore, we have established that Fic domain proteins can function as kinases. This distinct enzymatic activity exhibited by P1 Doc also solves the mystery of the degenerate Fic motif unique to the Doc family of toxins. Moreover, we have established that all characterized Fic domain proteins, even those that phosphorylate, target pivotal GTPases for inactivation through a post-translational modification at a single functionally critical acceptor site.

In adipose tissue, there is a delicate balance between storing and expending energy. In this issue, Shinoda et al. (2015) use phosphoproteomics to identify casein kinase 2 (CK2) as a suppressor of brown adipocyte formation, providing insights into how adipose tissue regulates its composition of white versus brown adipocytes.

The rat gastric GATA DNA-binding protein, GATA-6 (GATA-GT1), was stably expressed in CHO-K1 cells. The GATA-6 protein was localized in the nucleus but not in the cytoplasm. Interestingly, when cells were treated with dibutyryl cAMP, the GATA-6 protein was specifically degraded. Such a phenomenon was not observed in the presence of 5′-AMP or dibutyryl cGMP. The cellular level of the GATA-6 protein was restored upon removal of dibutyryl cAMP. Degradation was also induced by cholera toxin, which increased the cellular cAMP concentration, and was inhibited by a protein kinase A inhibitor. However, activators of protein kinase C did not have any effect. The degradation was inhibited by proteasome inhibitors (PSI (benzyloxycarbonyl-Ile-Glu(O-t-Bu)-Ala-leucinal) and MG115 (benzyloxycarbonyl-Leu-Leu-norvalinal)) but not by those of lysosomes and serine proteases. These results suggest that a kinase-mediated protein phosphorylation is the cellular signal for degradation of the GATA-6 protein. This finding constitutes a novel aspect of regulation by GATA DNA-binding proteins, which are essential for developmental processes and tissue-specific transcription.

Molecular interactions can be detected using single engineered ion channels as stochastic sensors. This approach is used to examine events between a kinase and an inhibitor peptide tethered to an engineered channel [1] showing agreement with macroscopic biochemical data, as well as a tantalizing surprise.

The presence of a kinase with the activation characteristics of protein kinase C (PKC) was demonstrated in supernatants of homogenates of the cestode Hymenolepis diminuta . Maximal phosphorylation activity was observed in diluted supernatants in the presence of Ca 2+ , phosphatidylserine and β-phorbol-12-myristate-13-acetate (PMA), whereas α-PMA was inactive. PKC-like activation in tissue slice extracts was abrogated by prior treatment of tissue slices with staurosporine. Specific activity of H. diminuta PKC was low compared with that of mammalian brain extracts but similar to that of other invertebrates. The present study demonstrates the presence of a kinase activity in H. diminuta that possesses activation and inhibition characteristics similar to the α-, β-, and γ-isomers of mammalian PKC.

In response to stress or injury the heart undergoes a pathological remodeling process, associated with hypertrophy, cardiomyocyte death and fibrosis, that ultimately causes cardiac dysfunction and heart failure. It has become increasingly clear that signaling events associated with these pathological cardiac remodeling events are regulated by scaffolding and anchoring proteins, which allow coordination of pathological signals in space and time. A-kinase anchoring proteins (AKAPs) constitute a family of functionally related proteins that organize multiprotein signaling complexes that tether the cAMP-dependent protein kinase (PKA) as well as other signaling enzymes to ensure integration and processing of multiple signaling pathways. This review will discuss the role of AKAPs in the cardiac response to stress. Particular emphasis will be given to the adaptative process associated with cardiac hypoxia as well as the remodeling events linked to cardiac hypertrophy and heart failure. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

Herein we describe the SAR of a novel series of 6-aryl-2-amino-triazolopyridines as potent and selective PI3Kγ inhibitors. The 6-aryl-triazolopyridine core was identified by chemoproteomic screening of a kinase focused library. Rapid chemical expansion around a bi-functional core identified the key features required for PI3Kγ activity and selectivity. The series was optimized to afford 43 (CZC19945), a potent PI3Kγ inhibitor with high oral bioavailability and selectivity over PI3Kα and PI3Kδ. Modification to the core afforded 53 (CZC24832) which showed increased selectivity over the entire kinome in particular over PI3Kβ.

Phosphoproteomic networks mediated by protein kinases are the key drivers of proliferative and survival signals underlying human cancers, and as such a number of kinases have been the subject of intensive drug discovery efforts. A key question that must be answered during clinical development is whether a kinase inhibitor is effectively inhibiting its appropriate target kinase and pathway in the tumor. Reverse-phase protein arrays (RPMAs) offer the ability to analyze behavior of entire signaling networks in response to drug treatment and thus have promise as a technology for monitoring cellular response to kinase inhibitors. We have shown that it is possible to use RPMAs to detect phosphorylation changes in key multiple signaling pathway proteins in response to targeted inhibitors of EGFR, MEK, and PI3 kinase.

Oncogenic kinase activity and the resulting aberrant growth and survival signaling are a common driving force of cancer. Accordingly, many successful molecularly targeted anticancer therapeutics are directed at inhibiting kinase activity. To assess kinase activity in minute patient samples, we have developed an immunocapture-based in vitro kinase assay on an integrated polydimethylsiloxane microfluidics platform that can reproducibly measure kinase activity from as few as 3,000 cells. For this platform, we adopted the standard radiometric 32P-ATP–labeled phosphate transfer assay. Implementation on a microfluidic device required us to develop methods for repeated trapping and mixing of solid-phase affinity microbeads. We also developed a solid-state beta-particle camera imbedded directly below the microfluidic device for real-time quantitative detection of the signal from this and other microfluidic radiobioassays. We show that the resulting integrated device can measure ABL kinase activity from BCR-ABL–positive leukemia patient samples. The low sample input requirement of the device creates new potential for direct kinase activity experimentation and diagnostics on patient blood, bone marrow, and needle biopsy samples. Cancer Res; 70(21); 8299–308. ©2010 AACR.