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Sample records for kinase cgamma leads

  1. Activation of mutant protein kinase C{gamma} leads to aberrant sequestration and impairment of its cellular function

    SciTech Connect

    Doran, Graeme; Davies, Kay E.; Talbot, Kevin

    2008-08-01

    Mutations in protein kinase C{gamma} (PKC{gamma}) cause the neurodegenerative disease spinocerebellar ataxia type 14 (SCA14). In this study, expression of an extensive panel of known SCA14-associated PKC{gamma} mutations as fusion proteins in cell culture led to the consistent formation of cytoplasmic aggregates in response to purinoceptor stimulation. Aggregates co-stained with antibodies to phosphorylated PKC{gamma} and the early endosome marker EEA1 but failed to redistribute to the cell membrane under conditions of oxidative stress. These studies suggest that Purkinje cell damage in SCA14 may result from a reduction of PKC{gamma} activity due its aberrant sequestration in the early endosome compartment.

  2. A collagen-related peptide regulates phospholipase Cgamma2 via phosphatidylinositol 3-kinase in human platelets.

    PubMed Central

    Pasquet, J M; Bobe, R; Gross, B; Gratacap, M P; Tomlinson, M G; Payrastre, B; Watson, S P

    1999-01-01

    The collagen receptor glycoprotein VI (GPVI) induces platelet activation through a similar pathway to that used by immune receptors. In the present study we have investigated the role of phosphatidylinositol 3-kinase (PI 3-kinase) in GPVI signalling. Our results show that collagen-related peptide ¿CRP: [GCP*(GPP*)(10)GCP*G](n); P*=hydroxyproline¿, which is selective to GPVI, induces formation of phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 3,4-bisphosphate [PI(3, 4)P(2)] in platelets. The increase in the two 3-phosphorylated lipids is inhibited completely by wortmannin and by LY294002, two structurally unrelated inhibitors of PI 3-kinase. The formation of inositol phosphates and phosphatidic acid (PA), two markers of phospholipase C (PLC) activation, by CRP are inhibited by between 50 and 85% in the presence of wortmannin and LY294002. This is associated with inhibition of elevation of intracellular Ca(2+) ([Ca(2+)](i)) and aggregation. Wortmannin and LY294002 also partially inhibit elevation of Ca(2+) by CRP in murine megakaryocytes. Microinjection of the pleckstrin-homology PH domain of Bruton's tyrosine kinase, which binds selectively to PI(3,4, 5)P(3), but not the R28C (Arg(28)-->Cys) mutant which binds to PI(3, 4,5)P(3) with low affinity, also inhibits elevation of [Ca(2+)](i) in megakaryocytes, suggesting that it is this lipid species which mediates the action of the PI 3-kinase pathway. Studies in platelets show that the action of wortmannin and LY294002 is not mediated through an alteration in tyrosine phosphorylation of PLCgamma2. These results demonstrate that PI 3-kinase is required for full activation of PLCgamma2 by GPVI in platelets and megakaryocytes. PMID:10432314

  3. Protein kinase C-gamma is present in adriamycin resistant HL-60 leukemia cells.

    PubMed

    Aquino, A; Warren, B S; Omichinski, J; Hartman, K D; Glazer, R I

    1990-01-30

    The isoform pattern of protein kinase C (PKC) was examined in wild-type and Adriamycin-resistant (HL-60/AR) HL-60 leukemia cells. Analyses were carried out by immunoblotting with mouse monoclonal antibodies against PKC-alpha and PKC-beta and a rabbit polyclonal antibody against the variable (V3) region of PKC-gamma. HL-60/AR cells contained an equivalent level of PKC-alpha and a lower amount of PKC-beta than HL-60 cells. In contrast, only HL-60/AR cells contained PKC-gamma. These results indicate that the regulation of this family of isoenzymes is altered in drug-resistant cells. PMID:2302237

  4. A role for Bruton's tyrosine kinase in B cell antigen receptor-mediated activation of phospholipase C-gamma 2

    PubMed Central

    1996-01-01

    Defects in the gene encoding Bruton's tyrosine kinase (Btk) result in a disease called X-linked agammaglobulinemia, in which there is a profound decrease of mature B cells due to a block in B cell development. Recent studies have shown that Btk is tyrosine phosphorylated and activated upon B cell antigen receptor (BCR) stimulation. To elucidate the functions of this kinase, we examined BCR signaling of DT40 B cells deficient in Btk. Tyrosine phosphorylation of phospholipase C (PLC)-gamma 2 upon receptor stimulation was significantly reduced in the mutant cells, leading to the loss of both BCR-coupled phosphatidylinositol hydrolysis and calcium mobilization. Pleckstrin homology and Src-homology 2 domains of Btk were required for PLC-gamma 2 activation. Since Syk is also required for the BCR-induced PLC-gamma 2 activation, our findings indicate that PLC-gamma 2 activation is regulated by Btk and Syk through their concerted actions. PMID:8691147

  5. Phospholipase C{gamma}1 stimulates transcriptional activation of the matrix metalloproteinase-3 gene via the protein kinase C/Raf/ERK cascade

    SciTech Connect

    Shin, Soon Young; Choi, Ha Young; Ahn, Bong-Hyun; Son, Sang Wook; Lee, Young Han . E-mail: younghan@hanyang.ac.kr

    2007-02-16

    The phospholipid hydrolase phospholipase C{gamma}1 (PLC{gamma}1) plays a major role in regulation of cell proliferation, development, and cell motility. Overexpression of PLC{gamma}1 is associated with tumor development, and it is overexpressed in some tumors. Matrix metalloproteinase-3 (MMP-3) is a protein involved in tumor invasion and metastasis. Here, we demonstrate that overexpression of PLC{gamma}1 stimulates MMP-3 expression at the transcriptional level via the PKC-mediated Raf/MEK1/ERK signaling cascade. We propose that modulation of PLC{gamma}1 activity might be of value in controlling the activity of MMPs, which are important regulators of invasion and metastasis in malignant tumors.

  6. The heptapeptide LSARLAF mediates platelet activation through phospholipase Cgamma2 independently of glycoprotein IIb-IIIa.

    PubMed Central

    Pearce, Andrew C; Wonerow, Peter; Marshall, Stuart J; Frampton, Jon; Gartner, T Kent; Watson, Steve P

    2004-01-01

    The seven-amino-acid peptide LSARLAF has been reported to activate platelets via the integrin GPIIb-IIIa (glycoprotein IIb-IIIa). Activation by LSARLAF is reinforced by release of ADP and thromboxanes, but the initiating event in the signalling cascade is not known. In the present study, we demonstrate that LSARLAF stimulates Src kinase-dependent tyrosine phosphorylation of many of the proteins in the GPIIb-IIIa cascade, including the tyrosine kinase Syk, the adapter SLP-76 (SH2-containing leucocyte phosphoprotein of 76 kDa) and PLCgamma2 (phospholipase Cgamma2). A critical role for PLCgamma2 in signalling by LSARLAF was demonstrated by abolition of aggregation in PLCgamma2-/- murine platelets to low concentrations of the peptide, although a partial recovery was seen with higher concentrations. In sharp contrast with the GPIIb-IIIa-regulated signalling cascade, aggregation was inhibited in murine platelets deficient in the adapter LAT (linker for activation of T-cells) and the Fc receptor gamma-chain. Aggregation was also partially inhibited by the cholesterol-lowering reagent, beta-methyl-cyclodextrin, at concentrations that disrupt membrane rafts, but do not interfere with signalling by GPIIb-IIIa. Furthermore, LSARLAF also stimulated tyrosine phosphorylation in GPIIb-deficient murine platelets, confirming that the integrin is not critical for activation of intracellular signalling pathways. LSARLAF also stimulated Ca2+ elevation in RBL-2H3 cells, which lack the platelet glycoproteins GPIIb, GPVI and GPIb. These results demonstrate that LSARLAF activates platelets through a PLCgamma2-dependent pathway that lies downstream of Src kinases and which is partially dependent on the Fc receptor gamma-chain, LAT and lipid rafts. The mechanism of cell activation by LSARLAF remains to be established, although the present results indicate that more than one surface glycoprotein may mediate this response. PMID:14558887

  7. Testis specific serine/threonine protein kinase 4 (TSSK4) leads to cell apoptosis relying on its kinase activity.

    PubMed

    Wang, Xiao-li; Wei, You-heng; Fu, Guo-long; Yu, Long

    2015-04-01

    Testis specific serine/threonine protein kinase 4 (TSSK4) belongs to the TSSK family, and its members play an important role in spermatogenesis and/or spermiogenesis. Mouse TSSK4 has been reported to be expressed exclusively in the testis and can maintain its kinase activity through autophosphorylation at Thr-197. However, its biological function remains poorly understood. Here we found that GFP-TSSK4-overexpressed HeLa cells showed apoptotic bodies, indicating TSSK4 can lead to apoptosis in vitro. Furthermore, TSSK4 induced apoptosis in different cell lines including HeLa, Cos-7 and H1299 tested by flow cytometry but not its kinase-dead mutant TSSK4-K54M. TSSK4 knockout mice showed increased testes weight and decreased apoptotic spermatogonia and spermatocytes at 21st day after birth tested by TUNEL technology. So TSSK4 was able to induce cell apoptosis in vitro depending on its kinase activity, which leads to abnormal testes weight and apoptosis, shedding light on its function in the process of spermatogenesis and/or spermiogenesis.

  8. Rational approaches towards lead optimization of kinase inhibitors: the issue of specificity.

    PubMed

    Badrinarayan, Preethi; Sastry, G Narahari

    2013-01-01

    Kinases are one of the most popular classes of drug targets as they are involved in signal transduction pathways, which are wired through a phosphotransfer cascade and elicit a number of important and essential physiological responses. Kinase specificity has emerged as one of the major issues to be addressed in drug discovery approaches. In most kinases the active site is the ATP binding site and finding suitable hits which maximize the affinity of binding has been traditionally important to obtain the type I inhibitors. While type I inhibitors have effective binding affinity more often than not they encounter side-effects usually associated with specificity. Therefore in recent times it has become indispensable to optimize specificity for developing effective kinase inhibitors. The review presents an overview of kinase drug discovery and the different strategies used to date for the design of kinase leads accounting for their success and failure. A number of strategies exploiting different aspects of kinases like allosteric site, size of the gatekeeper residue, DFG-loop, chemotype selectivity, non-covalent interactions, salt-bridge, solvation, etc. have been explored to circumvent the specificity problem in kinases. The probable hot-spots in kinases having a propensity to bring in specificity have been delineated with special emphasis on the design of type II inhibitors with increased specificity from existing type I using fragment tailoring approach. In this review we illustrate the current strategies by taking p38 MAP kinase as a model and expect that such strategies are general and can be extended to the other members of the kinase family. PMID:23260022

  9. Plasmodium falciparum Choline Kinase Inhibition Leads to a Major Decrease in Phosphatidylethanolamine Causing Parasite Death.

    PubMed

    Serrán-Aguilera, Lucía; Denton, Helen; Rubio-Ruiz, Belén; López-Gutiérrez, Borja; Entrena, Antonio; Izquierdo, Luis; Smith, Terry K; Conejo-García, Ana; Hurtado-Guerrero, Ramon

    2016-01-01

    Malaria is a life-threatening disease caused by different species of the protozoan parasite Plasmodium, with P. falciparum being the deadliest. Increasing parasitic resistance to existing antimalarials makes the necessity of novel avenues to treat this disease an urgent priority. The enzymes responsible for the synthesis of phosphatidylcholine and phosphatidylethanolamine are attractive drug targets to treat malaria as their selective inhibition leads to an arrest of the parasite's growth and cures malaria in a mouse model. We present here a detailed study that reveals a mode of action for two P. falciparum choline kinase inhibitors both in vitro and in vivo. The compounds present distinct binding modes to the choline/ethanolamine-binding site of P. falciparum choline kinase, reflecting different types of inhibition. Strikingly, these compounds primarily inhibit the ethanolamine kinase activity of the P. falciparum choline kinase, leading to a severe decrease in the phosphatidylethanolamine levels within P. falciparum, which explains the resulting growth phenotype and the parasites death. These studies provide an understanding of the mode of action, and act as a springboard for continued antimalarial development efforts selectively targeting P. falciparum choline kinase. PMID:27616047

  10. Plasmodium falciparum Choline Kinase Inhibition Leads to a Major Decrease in Phosphatidylethanolamine Causing Parasite Death

    PubMed Central

    Serrán-Aguilera, Lucía; Denton, Helen; Rubio-Ruiz, Belén; López-Gutiérrez, Borja; Entrena, Antonio; Izquierdo, Luis; Smith, Terry K.; Conejo-García, Ana; Hurtado-Guerrero, Ramon

    2016-01-01

    Malaria is a life-threatening disease caused by different species of the protozoan parasite Plasmodium, with P. falciparum being the deadliest. Increasing parasitic resistance to existing antimalarials makes the necessity of novel avenues to treat this disease an urgent priority. The enzymes responsible for the synthesis of phosphatidylcholine and phosphatidylethanolamine are attractive drug targets to treat malaria as their selective inhibition leads to an arrest of the parasite’s growth and cures malaria in a mouse model. We present here a detailed study that reveals a mode of action for two P. falciparum choline kinase inhibitors both in vitro and in vivo. The compounds present distinct binding modes to the choline/ethanolamine-binding site of P. falciparum choline kinase, reflecting different types of inhibition. Strikingly, these compounds primarily inhibit the ethanolamine kinase activity of the P. falciparum choline kinase, leading to a severe decrease in the phosphatidylethanolamine levels within P. falciparum, which explains the resulting growth phenotype and the parasites death. These studies provide an understanding of the mode of action, and act as a springboard for continued antimalarial development efforts selectively targeting P. falciparum choline kinase. PMID:27616047

  11. Mutation of the phospholipase C-gamma1-binding site of LAT affects both positive and negative thymocyte selection.

    PubMed

    Sommers, Connie L; Lee, Jan; Steiner, Kevin L; Gurson, Jordan M; Depersis, Corinne L; El-Khoury, Dalal; Fuller, Claudette L; Shores, Elizabeth W; Love, Paul E; Samelson, Lawrence E

    2005-04-01

    Linker for activation of T cells (LAT) is a scaffolding adaptor protein that is critical for T cell development and function. A mutation of LAT (Y136F) that disrupts phospholipase C-gamma1 activation and subsequent calcium influx causes a partial block in T cell development and leads to a severe lymphoproliferative disease in homozygous knock-in mice. One possible contribution to the fatal disease of LAT Y136F knock-in mice could be from autoreactive T cells generated in these mice because of altered thymocyte selection. To examine the impact of the LAT Y136F mutation on thymocyte positive and negative selection, we bred this mutation onto the HY T cell receptor (TCR) transgenic, recombination activating gene-2 knockout background. Female mice with this genotype showed a severe defect in positive selection, whereas male mice exhibited a phenotype resembling positive selection (i.e., development and survival of CD8(hi) HY TCR-specific T cells) instead of negative selection. These results support the hypothesis that in non-TCR transgenic, LAT Y136F knock-in mice, altered thymocyte selection leads to the survival and proliferation of autoreactive T cells that would otherwise be negatively selected in the thymus.

  12. Macrophage signalling upon mycobacterial infection: the MAP kinases lead the way.

    PubMed

    Schorey, Jeffrey S; Cooper, Andrea M

    2003-03-01

    Mycobacteria activate a series of macrophage signalling pathways upon engaging host cell receptors and during the invasion process. These signals initiate a cascade of events leading to the production of immune effector molecules including cytokines, chemokines and reactive nitrogen intermediates. This response by the macrophage is critical for the control of the mycobacterial infection and, not surprisingly, pathogenic mycobacteria have evolved mechanisms to limit this macrophage activation. Recent data has suggested that macrophages infected with pathogenic compared to non-pathogenic mycobacteria are restricted in their activation of the mitogen activated protein kinase (MAPK) pathways. Mitogen activated protein kinase activation in macrophages appears to play an important role in promoting antimycobacterial activity and in the production of various effector molecules following a mycobacterial infection. Therefore, the ability of pathogenic mycobacteria to limit MAPK activity is likely an important virulence mechanism and may be a potential therapeutic target.

  13. Leptin signalling and leptin-mediated activation of human platelets: importance of JAK2 and the phospholipases Cgamma2 and A2.

    PubMed

    Dellas, Claudia; Schäfer, Katrin; Rohm, Ilonka K; Lankeit, Mareike; Leifheit, Maren; Loskutoff, David J; Hasenfuss, Gerd; Konstantinides, Stavros V

    2007-11-01

    Leptin enhances agonist-induced platelet aggregation, and human platelets have been reported to express the leptin receptor. However, the pathways and mediators lying downstream of leptin binding to platelets remain, with few exceptions, unknown. In the present study, we sought to gain further insight into the possible role of leptin as a platelet agonist. Stimulation of platelets with leptin promoted thromboxane generation and activation of alpha(IIb)beta(3), as demonstrated by PAC-1 binding. Furthermore, it increased the adhesion to immobilised fibrinogen (p<0.001) and induced cytoskeletal rearrangement of both platelets and Meg01 cells. Leptin time- and dose-dependently phosphorylated the intracellular signalling molecules JAK2 and STAT3, although the importance of STAT3 for leptin-induced platelet activation remains to be determined. Important intracellular mediators and pathways activated by leptin downstream of JAK2 were found to include phosphatidylinositol-3 kinase, phospholipase Cgamma2 and protein kinase C, as well as the p38 MAP kinase-phospholipase A(2) axis. Accordingly, incubation with the specific inhibitors AG490, Ly294002, U73122, and SB203580 prevented leptin-mediated platelet activation. These results help delineate biologically relevant leptin signalling pathways in platelets and may improve our understanding of the mechanisms linking hyperleptinaemia to the increased thrombosis risk in human obesity. PMID:18000612

  14. Using existing drugs as leads for broad spectrum anthelmintics targeting protein kinases.

    PubMed

    Taylor, Christina M; Martin, John; Rao, Ramakrishna U; Powell, Kerrie; Abubucker, Sahar; Mitreva, Makedonka

    2013-02-01

    As one of the largest protein families, protein kinases (PKs) regulate nearly all processes within the cell and are considered important drug targets. Much research has been conducted on inhibitors for PKs, leading to a wealth of compounds that target PKs that have potential to be lead anthelmintic drugs. Identifying compounds that have already been developed to treat neglected tropical diseases is an attractive way to obtain lead compounds inexpensively that can be developed into much needed drugs, especially for use in developing countries. In this study, PKs from nematodes, hosts, and DrugBank were identified and classified into kinase families and subfamilies. Nematode proteins were placed into orthologous groups that span the phylum Nematoda. A minimal kinome for the phylum Nematoda was identified, and properties of the minimal kinome were explored. Orthologous groups from the minimal kinome were prioritized for experimental testing based on RNAi phenotype of the Caenorhabditis elegans ortholog, transcript expression over the life-cycle and anatomic expression patterns. Compounds linked to targets in DrugBank belonging to the same kinase families and subfamilies in the minimal nematode kinome were extracted. Thirty-five compounds were tested in the non-parasitic C. elegans and active compounds progressed to testing against nematode species with different modes of parasitism, the blood-feeding Haemonchus contortus and the filarial Brugia malayi. Eighteen compounds showed efficacy in C. elegans, and six compounds also showed efficacy in at least one of the parasitic species. Hypotheses regarding the pathway the compounds may target and their molecular mechanism for activity are discussed.

  15. Identification of “Preferred” Human Kinase Inhibitors for Sleeping Sickness Lead Discovery. Are Some Kinases Better than Others for Inhibitor Repurposing?

    PubMed Central

    2016-01-01

    A kinase-targeting cell-based high-throughput screen (HTS) against Trypanosoma brucei was recently reported, and this screening set included the Published Kinase Inhibitor Set (PKIS). From the PKIS was identified 53 compounds with pEC50 ≥ 6. Utilizing the published data available for the PKIS, a statistical analysis of these active antiparasitic compounds was performed, allowing identification of a set of human kinases having inhibitors that show a high likelihood for blocking T. brucei cellular proliferation in vitro. This observation was confirmed by testing other established inhibitors of these human kinases and by mining past screening campaigns at GlaxoSmithKline. Overall, although the parasite targets of action are not known, inhibitors of this set of human kinases displayed an enhanced hit rate relative to a random kinase-targeting HTS campaign, suggesting that repurposing efforts should focus primarily on inhibitors of these specific human kinases. We therefore term this statistical analysis-driven approach “preferred lead repurposing”. PMID:26998514

  16. Stimulation of neuropeptide Y gene expression by brain-derived neurotrophic factor requires both the phospholipase Cgamma and Shc binding sites on its receptor, TrkB.

    PubMed Central

    Williams, A G; Hargreaves, A C; Gunn-Moore, F J; Tavaré, J M

    1998-01-01

    In PC12 cells, it has been previously reported that nerve growth factor stimulates neuropeptide Y (NPY) gene expression. In the current study we examined the signalling pathways involved in this effect by transiently expressing in PC12 cells the receptor (TrkB) for the related neurotrophin, brain-derived neurotrophic factor (BDNF). BDNF caused a 3-fold induction of luciferase expression from a transiently co-transfected plasmid possessing the firefly luciferase gene under the control of the NPY promoter. This effect of BDNF was completely blocked by either a Y484F mutation in TrkB (which blocks high-affinity Shc binding to TrkB) or by a Y785F substitution [which blocks the binding, phosphorylation and activation of phospholipase Cgamma (PLCgamma)]. Activation of the NPY promoter by neurotrophin-3 in PC12 cells overexpressing TrkC was also completely blocked by a naturally occurring kinase insert which prevents the high-affinity binding of Shc and PLCgamma. NPY promoter activation by BDNF was blocked by PD98059, suggesting a role for mitogen-activated protein kinase (MAP kinase). Stimulation of NPY gene expression by PMA, but not by BDNF, was blocked by Ro-31-8220, a protein kinase C inhibitor, excluding a role for this serine/threonine protein kinase in the effect of BDNF. In addition, BDNF did not cause an elevation in cytosolic Ca2+ concentration. Taken together, our results suggest that stimulation of the NPY promoter by BDNF requires the simultaneous activation of two distinct pathways; one involves Shc and MAP kinase, and the other appears to be PLCgamma-independent but requires an intact tyrosine-785 on TrkB and so may involve an effector of TrkB signalling that remains to be identified. PMID:9677306

  17. Inhibition of Eimeria tenella CDK-related Kinase 2: From Target Identification to Lead Compounds

    PubMed Central

    Engels, Kristin; Beyer, Carsten; Fernández, Maria L. Suárez; Bender, Frank; Gaßel, Michael; Unden, Gottfried; Marhöfer, Richard J.; Mottram, Jeremy C.; Selzer, Paul M.

    2011-01-01

    Apicomplexan parasites encompass several human-pathogenic as well as animal-pathogenic protozoans like Plasmodium falciparum, Toxoplasma gondii, and Eimeria tenella. E. tenella is the causative agent of coccidiosis a disease of chickens, which causes tremendous economic losses to the world poultry industry. Considerable increase of drug resistance makes it necessary to develop and pursue new therapeutic strategies. Cyclin-dependent kinases (CDKs) are key molecules in the regulation of the cell cycle and are therefore prominent target proteins in parasitic diseases. Bioinformatic analysis revealed four potential CDK-like proteins of which one – E. tenella CDK-related kinase 2 (EtCRK2) – is already cloned, expressed and characterized.[1] Using the CDK specific inhibitor Flavopiridol in EtCRK2 enzyme assays and schizont maturation assays we could chemically validate CDK-like proteins as potential drug targets. An X-ray crystal structure of human CDK2 (HsCDK2) served as template to built protein models of EtCRK2 by comparative homology modeling. Structural differences in the ATP-binding site between EtCRK2 and HsCDK2 as well as chicken CDK3 have been addressed for the optimization of selective ATP-competitive inhibitors. Virtual screening and “wet-bench” high throughput screening campaigns on large compound libraries resulted in an initial set of hit compounds. These compounds were further analyzed and characterized leading to a set of four promising lead compounds inhibiting EtCRK2. PMID:20575139

  18. Wounding activates p38 map kinase and activation transcription factor 3 in leading keratinocytes.

    PubMed

    Harper, Erin G; Alvares, Stacy M; Carter, William G

    2005-08-01

    Quiescent epidermis anchors to laminin 5 in the basement membrane via integrin alpha6beta4. Wounding elevates expression of laminin 5, generating leading keratinocytes (LKs) that migrate via beta1 integrins. Laminin 5 was evaluated as a regulator of cell signaling, and mRNA and protein expression in LKs. An in vitro wound model was developed based on suspension and re-adhesion of quiescent human keratinocytes (HKs). DNA microarrays identified multiple mRNAs elevated 1.5 hours after suspension and re-adhesion including activation transcription factor 3 (ATF3). In vitro and in vivo, levels of ATF3 protein elevate in nuclei of LKs, but not in nuclei of the following cells, 2 hours after suspension or wounding but decline by 12-18 hours post injury. Significantly, null defects in laminin 5 or integrin beta4 that inhibit anchorage chronically elevate ATF3 in vivo. This suggests that adhesion to laminin 5, but not other ligands, suppresses activation. On suspension, ATF3 and other transcripts in the microarrays are elevated by phosphorylated p38 mitogen-activated protein kinase (P-p38), a stress kinase that regulates mRNA and cell motility. Inhibition of P-p38 with SB203580 prevents phosphorylation of ATF2, a transcription factor for ATF3 in LKs. Re-adhesion to laminin 5 via alpha6beta4 dephosphorylates P-p38 and suppresses ATF3 protein relative to cells in suspension. Thus, wounding of quiescent HKs disrupts laminin 5 adhesion to activate p38, generating mRNA transcripts that define LKs. Adhesion to deposits of laminin 5 via alpha6beta4 suppresses P-p38 and activation mRNAs including ATF3. Defects in laminin 5 and alpha6beta4 sustain P-p38 with probable pathological effects on transcription and migration.

  19. Inactivation of MARK4, an AMP-activated protein kinase (AMPK)-related kinase, leads to insulin hypersensitivity and resistance to diet-induced obesity.

    PubMed

    Sun, Chao; Tian, Liang; Nie, Jia; Zhang, Hai; Han, Xiao; Shi, Yuguang

    2012-11-01

    MARK4, also known as Par-1d/MarkL1, is a member of the AMP-activated protein kinase (AMPK)-related family of kinases, which are implicated in the regulation of dynamic biological functions, including glucose and energy homeostasis. However, the physiological function of MARK4 in mammals remains elusive. Here, we investigated a role for MARK4 in regulating energy homeostasis by generating mice with targeted inactivation of the mark4 gene. We show that MARK4 deficiency in mice caused hyperphagia, hyperactivity, and hypermetabolism, leading to protection from diet-induced obesity and its related metabolic complications through up-regulation of brown fat activity. Consequently, MARK4 deficiency mitigated insulin resistance associated with diet-induced obesity by dramatically enhancing insulin-stimulated AKT phosphorylation in major metabolic tissues. Ablation of MARK4 also significantly improved glucose homeostasis by up-regulating the activity and expression of AMPK kinase in key metabolic tissues. Taken together, these data identify a key role of MARK4 in energy metabolism, implicating the kinase as a novel drug target for the treatment of obesity and type 2 diabetes. PMID:22992738

  20. A novel calcium-dependent protein kinase inhibitor as a lead compound for treating cryptosporidiosis.

    PubMed

    Castellanos-Gonzalez, Alejandro; White, A Clinton; Ojo, Kayode K; Vidadala, Rama S R; Zhang, Zhongsheng; Reid, Molly C; Fox, Anna M W; Keyloun, Katelyn R; Rivas, Kasey; Irani, Ayesha; Dann, Sara M; Fan, Erkang; Maly, Dustin J; Van Voorhis, Wesley C

    2013-10-15

    Cryptosporidium parasites infect intestinal cells, causing cryptosporidiosis. Despite its high morbidity and association with stunting in the developing world, current therapies for cryptosporidiosis have limited efficacy. Calcium-dependent protein kinases (CDPKs) are essential enzymes in the biology of protozoan parasites. CDPK1 was cloned from the genome of Cryptosporidium parvum, and potent and specific inhibitors have been developed based on structural studies. In this study, we evaluated the anti-Cryptosporidium activity of a novel CDPK1 inhibitor, 1294, and demonstrated that 1294 significantly reduces parasite infection in vitro, with a half maximal effective concentration of 100 nM. Pharmacokinetic studies revealed that 1294 is well absorbed, with a half-life supporting daily administration. Oral therapy with 1294 eliminated Cryptosporidium parasites from 6 of 7 infected severe combined immunodeficiency-beige mice, and the parasites did not recur in these immunosuppressed mice. Mice treated with 1294 had less epithelial damage, corresponding to less apoptosis. Thus, 1294 is an important lead for the development of drugs for treatment of cryptosporidiosis.

  1. Doorway States As Principal Decay Pathway In 12C(12C,{gamma}) Radiative Capture

    SciTech Connect

    Jenkins, D.G.; Fulton, B.R.; Pearson, J.; Lister, C.J.; Carpenter, M.P.; Freeman, S.J.; Hammond, N.J.; Janssens, R.V.F.; Khoo, T.L.; Lauritsen, T.; Wuosmaa, A.H.; Fallon, P.; Goergen, A.; Macchiavelli, A.O.; McMahan, M.; Haas, F.

    2005-04-05

    The heavy-ion radiative capture reaction, 12C(12C,{gamma}), has been investigated at beam energies around 16 MeV. Three different experiments were performed. Capture cross-sections were obtained by measuring fused 24Mg residues using the FMA at ANL. These were found to significantly exceed values reported earlier. Subsequently, the decay pathways associated with radiative capture were studied in two separate measurements: one with the high-resolution Gammasphere array and a second with a high efficiency BGO array, where gamma rays were recorded in coincidence with 24Mg residues detected at the focal plane of the DRAGON recoil separator at TRIUMF. Both measurements indicate that a substantial fraction of the decay is mediated through high-lying doorway states, possibly associated with the long-predicted shape-isomeric band in 24Mg.

  2. Signalling pathway leading to an activation of mitogen-activated protein kinase by stimulating M3 muscarinic receptor.

    PubMed

    Kim, J Y; Yang, M S; Oh, C D; Kim, K T; Ha, M J; Kang, S S; Chun, J S

    1999-01-15

    The signalling pathway leading to an activation of mitogen-activated protein (MAP) kinase subtypes Erk-1 and -2 upon stimulation of muscarinic receptor with carbachol in human neuroblastoma SK-N-BE2(C) cells was investigated. Carbachol activated Erk-1/-2 by stimulating M3 muscarinic receptor, as determined by specific antagonists for individual muscarinic receptors. The activation of Erk-1/-2 by carbachol was blocked by the inhibition or down-regulation of protein kinase C (PKC). Among the multiple PKC isoforms expressed in SK-N-BE2(C) cells, only PKCepsilon was activated by the treatment of carbachol, and selective down-regulation of PKCepsilon was sufficient to block Erk-1/-2 activation. Carbachol treatment induced activation of the serine/threonine protein kinase Raf, and an inhibition of Raf blocked Erk-1/-2 activation. Ectopic expression of inhibitory small GTPase Ras, RasN17, blocked the carbachol-induced Raf activation without affecting the activation of PKCepsilon, while the inhibition of PKC blocked the Raf activation. Thus, these results suggest that carbachol-induced activation of PKCepsilon mediates Erk-1/-2 activation by a sequential activation of Ras, Raf and MAP kinase kinase.

  3. Mycobacterium tuberculosis Nucleoside Diphosphate Kinase Inactivates Small GTPases Leading to Evasion of Innate Immunity

    PubMed Central

    Sun, Jim; Singh, Vijender; Lau, Alice; Stokes, Richard W.; Obregón-Henao, Andrés; Orme, Ian M.; Wong, Dennis; Av-Gay, Yossef; Hmama, Zakaria

    2013-01-01

    Defining the mechanisms of Mycobacterium tuberculosis (Mtb) persistence in the host macrophage and identifying mycobacterial factors responsible for it are keys to better understand tuberculosis pathogenesis. The emerging picture from ongoing studies of macrophage deactivation by Mtb suggests that ingested bacilli secrete various virulence determinants that alter phagosome biogenesis, leading to arrest of Mtb vacuole interaction with late endosomes and lysosomes. While most studies focused on Mtb interference with various regulators of the endosomal compartment, little attention was paid to mechanisms by which Mtb neutralizes early macrophage responses such as the NADPH oxidase (NOX2) dependent oxidative burst. Here we applied an antisense strategy to knock down Mtb nucleoside diphosphate kinase (Ndk) and obtained a stable mutant (Mtb Ndk-AS) that displayed attenuated intracellular survival along with reduced persistence in the lungs of infected mice. At the molecular level, pull-down experiments showed that Ndk binds to and inactivates the small GTPase Rac1 in the macrophage. This resulted in the exclusion of the Rac1 binding partner p67phox from phagosomes containing Mtb or Ndk-coated latex beads. Exclusion of p67phox was associated with a defect of both NOX2 assembly and production of reactive oxygen species (ROS) in response to wild type Mtb. In contrast, Mtb Ndk-AS, which lost the capacity to disrupt Rac1-p67phox interaction, induced a strong ROS production. Given the established link between NOX2 activation and apoptosis, the proportion of Annexin V positive cells and levels of intracellular active caspase 3 were significantly higher in cells infected with Mtb Ndk-AS compared to wild type Mtb. Thus, knock down of Ndk converted Mtb into a pro-apoptotic mutant strain that has a phenotype of increased susceptibility to intracellular killing and reduced virulence in vivo. Taken together, our in vitro and in vivo data revealed that Ndk contributes significantly to

  4. The challenge of selecting protein kinase assays for lead discovery optimization

    PubMed Central

    Ma, Haiching; Deacon, Sean; Horiuchi, Kurumi

    2009-01-01

    Background Protein kinases represent one of the most promising groups of drug targets owing to their involvement in such pathological conditions as cancer, inflammatory diseases, neural disorders, and metabolism problems. In the last few years, numerous pharmaceutical and biotech companies have established kinase high-throughput screening (HTS) programs, and the reagent and service industries for kinase assay platforms, kits, and profiling services have begun to thrive. Objective The plethora of different assay formats available today poses a great challenge to scientists who want to select a technology that will be cost efficient, convenient to use, and have low false positive and false negative rates. Methods In the current review, we summarize the most commonly used kinase assay methods in the drug discovery process, present the advantages and disadvantages of each of these methods, and discuss the challenges of discovering kinase inhibitors by using these technologies. Conclusions The decision of selecting the assay formats for HTS or service platform for profiling should take into account not only the final goals of the screens but also the limitation of resources. PMID:19662101

  5. UV irradiation-induced apoptosis leads to activation of a 36-kDa myelin basic protein kinase in HL-60 cells

    SciTech Connect

    Lu, M.L.; Sato, Mitsuhiro; Cao, Boliang; Richie, J.P.

    1996-08-20

    UV irradiation induces apoptosis (or programmed cell death) in HL-60 promyelocytic leukemia cells within 3 h. UV-induced apoptosis is accompanied by activation of a 36-kDa myelin basic protein kinase (p36 MBP kinase). This kinase is also activated by okadaic acid and retinoic acid-induced apoptosis. Irrespective of the inducing agent, p36 MBP kinase activation is restricted to the subpopulation of cells actually undergoing apoptosis. Activation of p36 MBP kinase occurs in enucleated cytoplasts, indicating no requirements for a nucleus or fragmented DNA in signaling. We also demonstrate the activation of p36 kinase in tumor necrosis factor-{alpha}-and serum starvation-induced cell death using the human prostatic tumor cell line LNCap and NIH 3T3 fibroblasts, respectively. We postulate that p36 MBP kinase is a common component in diverse signaling pathways leading to apoptosis. 40 refs., 5 figs.

  6. Pervanadate activation of intracellular kinases leads to tyrosine phosphorylation and shedding of syndecan-1.

    PubMed Central

    Reiland, J; Ott, V L; Lebakken, C S; Yeaman, C; McCarthy, J; Rapraeger, A C

    1996-01-01

    Syndecan-1 is a transmembrane haparan sulphate proteoglycan that binds extracellular matrices and growth factors, making it a candidate to act between these regulatory molecules and intracellular signalling pathways. It has a highly conserved transmembrane/cytoplasmic domain that contains four conserved tyrosines. One of these is in a consensus sequence for tyrosine kinase phosphorylation. As an initial step to investigating whether or not phosphorylation of these tyrosines is part of a signal-transduction pathway, we have monitored the tyrosine phosphorylation of syndecan-1 by cytoplasmic tyrosine kinases in intact cells. Tyrosine phosphorylation of syndecan-1 is observed when NMuMG cells are treated with sodium orthovanadate or pervanadate, which have been shown to activate intracellular tyrosine kinases. Initial studies with sodium orthovanadate demonstrate a slow accumulation of phosphotyrosine on syndecan-1 over the course of several hours. Pervanadate, a more effective inhibitor of phosphatases, allows detection of phosphotyrosine on syndecan-1 within 5 min, with peak phosphorylation seen by 15 min. Concurrently, in a second process activated by pervanadate, syndecan-1 ectodomain is cleaved and released into the culture medium. Two phosphorylated fragments of syndecan-1 of apparent sizes 6 and 8 kDa remain with the cell after shedding of the ectodomain. The 8 kDa size class appears to be a highly phosphorylated form of the 6 kDa product, as it disappears if samples are dephosphorylated. These fragments contain the C-terminus of syndecan-1 and also retain at least a portion of the transmembrane domain, suggesting that they are produced by a cell surface cleavage event. Thus pervanadate treatment of cells results in two effects of syndecan-1: (i) phosphorylation of one or more of its tyrosines via the action of a cytoplasmic kinase(s) and (ii) cleavage and release of the ectodomain into the medium, producing a C-terminal fragment containing the transmembrane

  7. Fine particulate matter leads to reproductive impairment in male rats by overexpressing phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway.

    PubMed

    Cao, Xi-Ning; Yan, Chao; Liu, Dong-Yao; Peng, Jin-Pu; Chen, Jin-Jun; Zhou, Yue; Long, Chun-Lan; He, Da-Wei; Lin, Tao; Shen, Lian-Ju; Wei, Guang-Hui

    2015-09-17

    Maintenance of male reproductive function depends on normal sperm generation during which process Sertoli cells play a vital role. Studies found that fine particulate matter (PM) causes decreased male sperm quality, mechanism of which unestablished. We aim to investigate the definite mechanism of PM impairment on male reproduction. Male Sprague-Dawley rats were daily exposed to normal saline (NS) or PM2.5 with the doses of 9 mg/kg.b.w and 24 mg/kg.b.w. via intratracheal instillation for seven weeks. Reproductive function was tested by mating test and semen analysis after last exposure. Testes were collected to assess changes in histomorphology, and biomarkers including connexin 43 (Cx43), superoxide dismutase (SOD), phosphatidylinositol 3-kinase (PI3K) and phosphorylated protein kinase B (p-Akt). Male rats exposed to PM2.5 showed noticeable decreased fertility, significantly reduced sperm count, increased sperm abnormality rate and severe testicular damage in histomorphology. After PM2.5 exposure, the levels of Cx43 was significantly downregulated, and SOD was upregulated and downregulated significantly with different dose, respectively. Protein expression of PI3K and p-Akt dramatically enhanced, and the later one being located in Sertoli cells, the upward or declining trend was in dose dependent. PM2.5 exposure leads to oxidative stress impairment via PI3K/Akt signaling pathway on male reproduction in rats.

  8. Decay strength distributions in {sup 12}C({sup 12}C,{gamma}) radiative capture

    SciTech Connect

    Jenkins, D. G.; Fulton, B. R.; Marley, P.; Fox, S. P.; Glover, R.; Wadsworth, R.; Watson, D. L.; Courtin, S.; Haas, F.; Lebhertz, D.; Beck, C.; Papka, P.; Rousseau, M.; Sanchez i Zafra, A.; Hutcheon, D. A.; Davis, C.; Ottewell, D.; Pavan, M. M.; Pearson, J.; Ruiz, C.

    2007-10-15

    The heavy-ion radiative capture reaction, {sup 12}C({sup 12}C,{gamma}), has been investigated at energies both on- and off-resonance, with a particular focus on known resonances at E{sub c.m.}=6.0, 6.8, 7.5, and 8.0 MeV. Gamma rays detected in a BGO scintillator array were recorded in coincidence with {sup 24}Mg residues at the focal plane of the DRAGON recoil separator at TRIUMF. In this manner, the relative strength of all decay pathways through excited states up to the particle threshold could be examined for the first time. Isovector M1 transitions are found to be a important component of the radiative capture from the E{sub c.m.}=6.0 and 6.8 MeV resonances. Comparison with Monte Carlo simulations suggests that these resonances may have either J=0 or 2, with a preference for J=2. The higher energy resonances at E{sub c.m.}=7.5 and 8.0 MeV have a rather different decay pattern. The former is a clear candidate for a J=4 resonance, whereas the latter has a dominant J=4 character superposed on a J=2 resonant component underneath. The relationship between these resonances and the well-known quasimolecular resonances as well as resonances in breakup and electrofission of {sup 24}Mg into two {sup 12}C nuclei are discussed.

  9. Activation of H2O2-induced VSOR Cl- currents in HTC cells require phospholipase Cgamma1 phosphorylation and Ca2+ mobilisation.

    PubMed

    Varela, Diego; Simon, Felipe; Olivero, Pablo; Armisén, Ricardo; Leiva-Salcedo, Elías; Jørgensen, Finn; Sala, Francisco; Stutzin, Andrés

    2007-01-01

    Volume-sensitive outwardly rectifying (VSOR) Cl(-) channels participate in several physiological processes such as regulatory volume decrease, cell cycle regulation, proliferation and apoptosis. Recent evidence points to a significant role of hydrogen peroxide (H(2)O(2)) in VSOR Cl(-) channel activation. The aim of this study was to determine the signalling pathways responsible for H(2)O(2)-induced VSOR Cl(-) channel activation. In rat hepatoma (HTC) cells, H(2)O(2) elicited a transient increase in tyrosine phosphorylation of phospholipase Cgamma1 (PLCgamma1) that was blocked by PP2, a Src-family protein kinases inhibitor. Also, H(2)O(2) triggered an increase in cytosolic [Ca(2+)] that paralleled the time course of PLCgamma1 phosphorylation. The H(2)O(2)-induced [Ca(2+)](i) rise was prevented by the generic phospholipase C (PLC) inhibitor U73122 and the inositol 1,4,5-trisphosphate-receptor (IP(3)R) blocker 2-APB. In line with these results, manoeuvres that prevented PLCgamma1 activation and/or [Ca(2+)](i) rise, abolished H(2)O(2)-induced VSOR Cl(-) currents. Furthermore, in cells that overexpress a phosphorylation-defective dominant mutant of PLCgamma1, H(2)O(2) did not induce activation of VSOR Cl(-) currents. All these H(2)O(2)-induced effects were independent of extracellular Ca(2+). Our findings suggest that activation of PLCgamma1 and subsequent Ca(2+)(i) mobilisation mediate H(2)O(2)-induced VSOR Cl(-) currents, indicating that H(2)O(2) operates via redox-sensitive signalling pathways akin to those activated by osmotic challenges.

  10. Contrasting role of phospholipase C-{gamma}1 in the expression of immediate early genes induced by epidermal or platelet-derived growth factors

    SciTech Connect

    Liao Hongjun; Santos, Josue de los; Carpenter, Graham . E-mail: graham.carpenter@vanderbilt.edu

    2006-04-01

    While significant progress has been achieved in identifying the signal transduction elements that operate downstream of activated receptor tyrosine kinases, it remains unclear how different receptors utilize these signaling elements to achieve a common response. This study compares the capacity of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) to elicit the induction of immediate early gene (IEG) mRNAs in the presence or absence of phospholipase C-{gamma}1 (PLC-{gamma}1). The results show that while PDGF induction of nearly all IEG mRNAs is abrogated in plcg1 null cells, EGF induction of the same genes is variable in the null cells and exhibits three distinct responses. Five IEG mRNAs (Nup475, Cyr61, TF, Gly, TS7) are completely inducible by EGF in the presence or absence of PLC-{gamma}1, while three others (JE, KC, FIC) exhibit a stringent requirement for the presence of PLC-{gamma}1. The third type of response is exhibited by c-fos and COX-2. While these mRNAs are completely induced by EGF in the absence of PLC-{gamma}1, the time course of their accumulation is significantly delayed. No IEG was identified as completely inducible by EGF and PDGF in the absence of PLC-{gamma}1. Electrophoretic mobility shift assays (EMSA) demonstrate that PLC-{gamma}1 is necessary for nuclear extracts from PDGF-treated cells, but not EGF-treated cells, to interact with probes for AP-1 or NF-{kappa}B.

  11. Activation of H2O2-induced VSOR Cl- currents in HTC cells require phospholipase Cgamma1 phosphorylation and Ca2+ mobilisation.

    PubMed

    Varela, Diego; Simon, Felipe; Olivero, Pablo; Armisén, Ricardo; Leiva-Salcedo, Elías; Jørgensen, Finn; Sala, Francisco; Stutzin, Andrés

    2007-01-01

    Volume-sensitive outwardly rectifying (VSOR) Cl(-) channels participate in several physiological processes such as regulatory volume decrease, cell cycle regulation, proliferation and apoptosis. Recent evidence points to a significant role of hydrogen peroxide (H(2)O(2)) in VSOR Cl(-) channel activation. The aim of this study was to determine the signalling pathways responsible for H(2)O(2)-induced VSOR Cl(-) channel activation. In rat hepatoma (HTC) cells, H(2)O(2) elicited a transient increase in tyrosine phosphorylation of phospholipase Cgamma1 (PLCgamma1) that was blocked by PP2, a Src-family protein kinases inhibitor. Also, H(2)O(2) triggered an increase in cytosolic [Ca(2+)] that paralleled the time course of PLCgamma1 phosphorylation. The H(2)O(2)-induced [Ca(2+)](i) rise was prevented by the generic phospholipase C (PLC) inhibitor U73122 and the inositol 1,4,5-trisphosphate-receptor (IP(3)R) blocker 2-APB. In line with these results, manoeuvres that prevented PLCgamma1 activation and/or [Ca(2+)](i) rise, abolished H(2)O(2)-induced VSOR Cl(-) currents. Furthermore, in cells that overexpress a phosphorylation-defective dominant mutant of PLCgamma1, H(2)O(2) did not induce activation of VSOR Cl(-) currents. All these H(2)O(2)-induced effects were independent of extracellular Ca(2+). Our findings suggest that activation of PLCgamma1 and subsequent Ca(2+)(i) mobilisation mediate H(2)O(2)-induced VSOR Cl(-) currents, indicating that H(2)O(2) operates via redox-sensitive signalling pathways akin to those activated by osmotic challenges. PMID:17982259

  12. Homocysteine-NMDA receptor mediated activation of extracellular-signal regulated kinase leads to neuronal cell death

    PubMed Central

    Poddar, Ranjana; Paul, Surojit

    2009-01-01

    Hyper-homocysteinemia is an independent risk factor for stroke and neurological abnormalities. However the underlying cellular mechanisms by which elevated homocysteine can promote neuronal death is not clear. In the present study we have examined the role of NMDA receptor mediated activation of the extracellular-signal regulated mitogen activated protein (ERK MAP) kinase pathway in homocysteine-dependent neurotoxicity. The study demonstrates that in neurons L-homocysteine-induced cell death is mediated through activation of NMDA receptors. The study also shows that homocysteine-dependent NMDA receptor stimulation and resultant Ca2+ influx leads to rapid and sustained phosphorylation of ERK MAP kinase. Inhibition of ERK phosphorylation attenuates homocysteine mediated neuronal cell death thereby demonstrating that activation of ERK MAP kinase signaling pathway is an intermediate step that couples homocysteine mediated NMDA receptor stimulation to neuronal death. The findings also show that cAMP response-element binding protein (CREB), a pro-survival transcription factor and a downstream target of ERK, is only transiently activated following homocysteine exposure. The sustained activation of ERK but a transient activation of CREB together suggest that exposure to homocysteine initiates a feedback loop that shuts off CREB signaling without affecting ERK phosphorylation and thereby facilitates homocysteine mediated neurotoxicity. PMID:19508427

  13. Plesiomonas shigelloides Septic Shock Leading to Death of Postsplenectomy Patient with Pyruvate Kinase Deficiency and Hemochromatosis

    PubMed Central

    2016-01-01

    Although Plesiomonas shigelloides, a water-borne bacterium of the Enterobacteriaceae family, usually causes self-limiting gastroenteritis with diarrhea, several cases of sepsis have been reported. We report the case of a 43-year-old male patient with hemochromatosis, pyruvate kinase deficiency, and asplenia via splenectomy who developed septic shock caused by P. shigelloides complicated by respiratory failure, renal failure, liver failure, and disseminated intravascular coagulation. Early aggressive antimicrobial therapy and resuscitation measures were unsuccessful and the patient passed away. We kindly suggest clinicians to implement early diagnosis of septic shock, empirical coverage with antibiotics, and prompt volume resuscitation based on the high mortality rate of P. shigelloides bacteremia.

  14. Plesiomonas shigelloides Septic Shock Leading to Death of Postsplenectomy Patient with Pyruvate Kinase Deficiency and Hemochromatosis

    PubMed Central

    2016-01-01

    Although Plesiomonas shigelloides, a water-borne bacterium of the Enterobacteriaceae family, usually causes self-limiting gastroenteritis with diarrhea, several cases of sepsis have been reported. We report the case of a 43-year-old male patient with hemochromatosis, pyruvate kinase deficiency, and asplenia via splenectomy who developed septic shock caused by P. shigelloides complicated by respiratory failure, renal failure, liver failure, and disseminated intravascular coagulation. Early aggressive antimicrobial therapy and resuscitation measures were unsuccessful and the patient passed away. We kindly suggest clinicians to implement early diagnosis of septic shock, empirical coverage with antibiotics, and prompt volume resuscitation based on the high mortality rate of P. shigelloides bacteremia. PMID:27610253

  15. Plesiomonas shigelloides Septic Shock Leading to Death of Postsplenectomy Patient with Pyruvate Kinase Deficiency and Hemochromatosis.

    PubMed

    Samannodi, Mohammed; Zhao, Andrew; Nemshah, Yaser; Shiley, Kevin

    2016-01-01

    Although Plesiomonas shigelloides, a water-borne bacterium of the Enterobacteriaceae family, usually causes self-limiting gastroenteritis with diarrhea, several cases of sepsis have been reported. We report the case of a 43-year-old male patient with hemochromatosis, pyruvate kinase deficiency, and asplenia via splenectomy who developed septic shock caused by P. shigelloides complicated by respiratory failure, renal failure, liver failure, and disseminated intravascular coagulation. Early aggressive antimicrobial therapy and resuscitation measures were unsuccessful and the patient passed away. We kindly suggest clinicians to implement early diagnosis of septic shock, empirical coverage with antibiotics, and prompt volume resuscitation based on the high mortality rate of P. shigelloides bacteremia.

  16. Plesiomonas shigelloides Septic Shock Leading to Death of Postsplenectomy Patient with Pyruvate Kinase Deficiency and Hemochromatosis.

    PubMed

    Samannodi, Mohammed; Zhao, Andrew; Nemshah, Yaser; Shiley, Kevin

    2016-01-01

    Although Plesiomonas shigelloides, a water-borne bacterium of the Enterobacteriaceae family, usually causes self-limiting gastroenteritis with diarrhea, several cases of sepsis have been reported. We report the case of a 43-year-old male patient with hemochromatosis, pyruvate kinase deficiency, and asplenia via splenectomy who developed septic shock caused by P. shigelloides complicated by respiratory failure, renal failure, liver failure, and disseminated intravascular coagulation. Early aggressive antimicrobial therapy and resuscitation measures were unsuccessful and the patient passed away. We kindly suggest clinicians to implement early diagnosis of septic shock, empirical coverage with antibiotics, and prompt volume resuscitation based on the high mortality rate of P. shigelloides bacteremia. PMID:27610253

  17. Mutations in BCKD-kinase lead to a potentially treatable form of autism with epilepsy.

    PubMed

    Novarino, Gaia; El-Fishawy, Paul; Kayserili, Hulya; Meguid, Nagwa A; Scott, Eric M; Schroth, Jana; Silhavy, Jennifer L; Kara, Majdi; Khalil, Rehab O; Ben-Omran, Tawfeg; Ercan-Sencicek, A Gulhan; Hashish, Adel F; Sanders, Stephan J; Gupta, Abha R; Hashem, Hebatalla S; Matern, Dietrich; Gabriel, Stacey; Sweetman, Larry; Rahimi, Yasmeen; Harris, Robert A; State, Matthew W; Gleeson, Joseph G

    2012-10-19

    Autism spectrum disorders are a genetically heterogeneous constellation of syndromes characterized by impairments in reciprocal social interaction. Available somatic treatments have limited efficacy. We have identified inactivating mutations in the gene BCKDK (Branched Chain Ketoacid Dehydrogenase Kinase) in consanguineous families with autism, epilepsy, and intellectual disability. The encoded protein is responsible for phosphorylation-mediated inactivation of the E1α subunit of branched-chain ketoacid dehydrogenase (BCKDH). Patients with homozygous BCKDK mutations display reductions in BCKDK messenger RNA and protein, E1α phosphorylation, and plasma branched-chain amino acids. Bckdk knockout mice show abnormal brain amino acid profiles and neurobehavioral deficits that respond to dietary supplementation. Thus, autism presenting with intellectual disability and epilepsy caused by BCKDK mutations represents a potentially treatable syndrome.

  18. Translocation of mixed lineage kinase domain-like protein to plasma membrane leads to necrotic cell death

    PubMed Central

    Chen, Xin; Li, Wenjuan; Ren, Junming; Huang, Deli; He, Wan-ting; Song, Yunlong; Yang, Chao; Li, Wanyun; Zheng, Xinru; Chen, Pengda; Han, Jiahuai

    2014-01-01

    Mixed lineage kinase domain-like protein (MLKL) was identified to function downstream of receptor interacting protein 3 (RIP3) in tumor necrosis factor-α (TNF)-induced necrosis (also called necroptosis). However, how MLKL functions to mediate necroptosis is unknown. By reconstitution of MLKL function in MLKL-knockout cells, we showed that the N-terminus of MLKL is required for its function in necroptosis. The oligomerization of MLKL in TNF-treated cells is essential for necroptosis, as artificially forcing MLKL together by using the hormone-binding domain (HBD*) triggers necroptosis. Notably, forcing together the N-terminal domain (ND) but not the C-terminal kinase domain of MLKL causes necroptosis. Further deletion analysis showed that the four-α-helix bundle of MLKL (1-130 amino acids) is sufficient to trigger necroptosis. Both the HBD*-mediated and TNF-induced complexes of MLKL(ND) or MLKL are tetramers, and translocation of these complexes to lipid rafts of the plasma membrane precedes cell death. The homo-oligomerization is required for MLKL translocation and the signal sequence for plasma membrane location is located in the junction of the first and second α-helices of MLKL. The plasma membrane translocation of MLKL or MLKL(ND) leads to sodium influx, and depletion of sodium from the cell culture medium inhibits necroptosis. All of the above phenomena were not seen in apoptosis. Thus, the MLKL oligomerization leads to translocation of MLKL to lipid rafts of plasma membrane, and the plasma membrane MLKL complex acts either by itself or via other proteins to increase the sodium influx, which increases osmotic pressure, eventually leading to membrane rupture. PMID:24366341

  19. Inhibition of Rho Kinase (ROCK) Leads to Increased Cerebral Blood Flow and Stroke Protection

    PubMed Central

    Rikitake, Yoshiyuki; Kim, Hyung-Hwan; Huang, Zhihong; Seto, Minoru; Yano, Kazuo; Asano, Toshio; Moskowitz, Michael A.; Liao, James K.

    2009-01-01

    Background and Purpose Endothelium-derived nitric oxide (NO) plays a pivotal role in vascular protection. The Rho kinase (ROCK) inhibitor, hydroxyfasudil, prevents the downregulation of endothelial NO synthase (eNOS) under hypoxic conditions. However, it is unknown whether inhibition of ROCK can attenuate ischemia-induced endothelial dysfunction and tissue damage in vivo. Methods Human vascular endothelial cells were treated with increasing concentrations of hydroxyfasudil (0.1 to 100 μmol/L) and eNOS expression and activity were measured. To determine the physiological relevance of eNOS regulation by ROCK, we administered fasudil, which is metabolized to hydroxyfasudil in vivo, to mice for 2 days before subjecting them to middle cerebral artery occlusion. Cerebral blood flow, cerebral infarct size, and neurologic deficit were measured. Results In a concentration-dependent manner, hydroxyfasudil increased eNOS mRNA and protein expression, resulting in a 1.9- and 1.6-fold increase, respectively, at 10 μmol/L (P<0.05 for both). This correlated with a 1.5- and 2.3-fold increase in eNOS activity and NO production, respectively (P<0.05 for both). Fasudil increased cerebral blood flow to both ischemic and nonischemic brain areas, reduced cerebral infarct size by 33%, and improved neurologic deficit score by 37% (P<0.05). This correlated with inhibition of brain and vascular ROCK activity and increased eNOS expression and activity. Another ROCK inhibitor, Y-27632, also showed similar effects. The neuroprotective effects of fasudil were absent in eNOS-deficient mice. Conclusions These findings indicate that the neuroprotective effect of ROCK inhibition is mediated by endothelium-derived NO and suggest that ROCK may be an important therapeutic target for ischemic stroke. PMID:16141422

  20. Phosphorylation of TRPV1 by cyclin-dependent kinase 5 promotes TRPV1 surface localization, leading to inflammatory thermal hyperalgesia.

    PubMed

    Liu, Jiao; Du, Junxie; Yang, Yanrui; Wang, Yun

    2015-11-01

    Cyclin-dependent kinase 5 (Cdk5) is an important serine/threonine kinase that plays critical roles in many physiological processes. Recently, Cdk5 has been reported to phosphorylate TRPV1 at threonine 407 (Thr-407) in humans (Thr-406 in rats), which enhances the function of TRPV1 channel and promotes thermal hyperalgesia in the complete Freund's adjuvant (CFA)-induced inflammatory pain rats. However, the underlying mechanisms are still unknown. Here, we demonstrate that Cdk5 phosphorylates TRPV1 at Threonine 406 and promotes the surface localization of TRPV1, leading to inflammatory thermal hyperalgesia. The mutation of Thr-406 of TRPV1 to alanine reduced the interaction of TRPV1 with the cytoskeletal elements and decreased the binding of TRPV1 with the motor protein KIF13B, which led to reduced surface distribution of TRPV1. Disrupting the phosphorylation of TRPV1 at Thr-406 dramatically reduced the surface level of TRPV1 in HEK 293 cells after transient expression and the channel function in cultured dorsal root ganglion (DRG) neurons. Notably, intrathecal administration of the interfering peptide against the phosphorylation of Thr-406 alleviated heat hyperalgesia and reduced the surface level of TRPV1 in inflammatory pain rats. Together, these results demonstrate that Cdk5-mediated phosphorylation of TRPV1 at Thr-406 increases the surface level and the function of TRPV1, while the TAT-T406 peptide can effectively attenuate thermal hyperalgesia. Our studies provide a potential therapy for inflammatory pain.

  1. Elucidating the role of the TRPM7 alpha-kinase: TRPM7 kinase inactivation leads to magnesium deprivation resistance phenotype in mice.

    PubMed

    Ryazanova, Lillia V; Hu, Zhixian; Suzuki, Sayuri; Chubanov, Vladimir; Fleig, Andrea; Ryazanov, Alexey G

    2014-12-23

    TRPM7 is an unusual bi-functional protein containing an ion channel covalently linked to a protein kinase domain. TRPM7 is implicated in regulating cellular and systemic magnesium homeostasis. While the biophysical properties of TRPM7 ion channel and its function are relatively well characterized, the function of the TRPM7 enzymatically active kinase domain is not understood yet. To investigate the physiological role of TRPM7 kinase activity, we constructed mice carrying an inactive TRPM7 kinase. We found that these mice were resistant to dietary magnesium deprivation, surviving three times longer than wild type mice; also they displayed decreased chemically induced allergic reaction. Interestingly, mutant mice have lower magnesium bone content compared to wild type mice when fed regular diet; unlike wild type mice, mutant mice placed on magnesium-depleted diet did not alter their bone magnesium content. Furthermore, mouse embryonic fibroblasts isolated from TRPM7 kinase-dead animals exhibited increased resistance to magnesium deprivation and oxidative stress. Finally, electrophysiological data revealed that the activity of the kinase-dead TRPM7 channel was not significantly altered. Together, our results suggest that TRPM7 kinase is a sensor of magnesium status and provides coordination of cellular and systemic responses to magnesium deprivation.

  2. Loss of Cyclin-Dependent Kinase 5 from Parvalbumin Interneurons Leads to Hyperinhibition, Decreased Anxiety, and Memory Impairment

    PubMed Central

    Rudenko, Andrii; Seo, Jinsoo; Hu, Ji; Su, Susan C.; de Anda, Froylan Calderon; Durak, Omer; Ericsson, Maria; Carlén, Marie

    2015-01-01

    Perturbations in fast-spiking parvalbumin (PV) interneurons are hypothesized to be a major component of various neuropsychiatric disorders; however, the mechanisms regulating PV interneurons remain mostly unknown. Recently, cyclin-dependent kinase 5 (Cdk5) has been shown to function as a major regulator of synaptic plasticity. Here, we demonstrate that genetic ablation of Cdk5 in PV interneurons in mouse brain leads to an increase in GABAergic neurotransmission and impaired synaptic plasticity. PVCre;fCdk5 mice display a range of behavioral abnormalities, including decreased anxiety and memory impairment. Our results reveal a central role of Cdk5 expressed in PV interneurons in gating inhibitory neurotransmission and underscore the importance of such regulation during behavioral tasks. Our findings suggest that Cdk5 can be considered a promising therapeutic target in a variety of conditions attributed to inhibitory interneuronal dysfunction, such as epilepsy, anxiety disorders, and schizophrenia. PMID:25673832

  3. New Chemical Scaffolds for Human African Trypanosomiasis Lead Discovery from a Screen of Tyrosine Kinase Inhibitor Drugs

    PubMed Central

    Behera, Ranjan; Thomas, Sarah M.

    2014-01-01

    Human African trypanosomiasis (HAT) is caused by the protozoan Trypanosoma brucei. New drugs are needed to treat HAT because of undesirable side effects and difficulties in the administration of the antiquated drugs that are currently used. In human proliferative diseases, protein tyrosine kinase (PTK) inhibitors (PTKIs) have been developed into drugs (e.g., lapatinib and erlotinib) by optimization of a 4-anilinoquinazoline scaffold. Two sets of facts raise a possibility that drugs targeted against human PTKs could be “hits” for antitrypanosomal lead discoveries. First, trypanosome protein kinases bind some drugs, namely, lapatinib, CI-1033, and AEE788. Second, the pan-PTK inhibitor tyrphostin A47 blocks the endocytosis of transferrin and inhibits trypanosome replication. Following up on these concepts, we performed a focused screen of various PTKI drugs as possible antitrypanosomal hits. Lapatinib, CI-1033, erlotinib, axitinib, sunitinib, PKI-166, and AEE788 inhibited the replication of bloodstream T. brucei, with a 50% growth inhibitory concentration (GI50) between 1.3 μM and 2.5 μM. Imatinib had no effect (i.e., GI50 > 10 μM). To discover leads among the drugs, a mouse model of HAT was used in a proof-of-concept study. Orally administered lapatinib reduced parasitemia, extended the survival of all treated mice, and cured the trypanosomal infection in 25% of the mice. CI-1033 and AEE788 reduced parasitemia and extended the survival of the infected mice. On the strength of these data and noting their oral bioavailabilities, we propose that the 4-anilinoquinazoline and pyrrolopyrimidine scaffolds of lapatinib, CI-1033, and AEE788 are worth optimizing against T. brucei in medicinal chemistry campaigns (i.e., scaffold repurposing) to discover new drugs against HAT. PMID:24468788

  4. Pressure overload stimulated cardiac hypertrophy leads to a rapid decrease in the mRNA for creatine kinase

    SciTech Connect

    Boheler, K.; Popovich, B.; Dillmann, W.H.

    1987-05-01

    Cardiac hypertrophy (CH) leads to a decrease in creatine kinase (CK) enzymatic activity. To determine if the mRNA for CK also decreases with CH, they performed the following studies. Cardiac RNA was isolated from rats subjected to either abdominal aortic stenosis (AS) or sham surgery. Through Northern blot analysis, total cardiac RNA was quantitated with a CK specific /sup 32/P-labelled cDNA clone. At 3 and 8 days post-constriction, the mRNA for CK decreases by 54.6 +/- 7% and 65.3 +/- 18% respectively, whereas the heart weight increases by 19% and 37% relative to controls. Further studies indicate that CK mRNA also decreases by 41.8% in hypothyroid rats (Tx) but decreases by a total of 68.1% in Tx rats subjected to 8 days of AS. Pressure overload stimulated CH leads to a rapid decrease in CK mRNA in normal and Tx rats. This CK mRNA decrease may account for the decreased efficiency of contraction seen in CH.

  5. Structure-based lead discovery for protein kinase C zeta inhibitor design by exploiting kinase-inhibitor complex crystal structure data and potential therapeutics for preterm labour.

    PubMed

    Shao, Qing-Chun; Zhang, Cui-Juan; Li, Jie

    2014-10-14

    The protein kinase C (PKC) is a family of serine/threonine kinases with a broad range of cellular targets. Members of the PKC family participate at the diverse biological events involved in cellular proliferation, differentiation and survival. The PKC isoform zeta (PKCζ) is an atypical member that has recently been found to play an essential role in promoting human uterine contractility and thus been raised as a new target for treating preterm labour and other tocolytic diseases. In this study, an integrative protocol was described to graft hundreds of inhibitor ligands from their complex crystal structures with cognate kinases into the active pocket of PKCζ and, based on the modeled structures, to evaluate the binding strength of these inhibitors to the non-cognate PKCζ receptor by using a consensus scoring strategy. A total of 32 inhibitors with top score were compiled, and eight out of them were tested for inhibitory potency against PKCζ. Consequently, five compounds, i.e. CDK6 inhibitor fisetin, PIM1 inhibitor myricetin, CDK9 inhibitor flavopiridol and PknB inhibitor mitoxantrone as well as the promiscuous kinase inhibitor staurosporine showed high or moderate inhibitory activity on PKCζ, with IC50 values of 58 ± 9, 1.7 ± 0.4, 108 ± 17, 280 ± 47 and 0.019 ± 0.004 μM, respectively, while other three compounds, including two marketed drugs dasatinib and sunitinib as well as the Rho inhibitor fasudil, have not been detected to possess observable activity. Next, based on the modeled structure data we modified three flavonoid kinase inhibitors, i.e. fisetin, myricetin and flavopiridol, to generate a number of more potential molecular entities, two of which were found to have a moderately improved activity as compared to their parent compounds.

  6. Lead

    MedlinePlus

    ... Lead Share Facebook Twitter Google+ Pinterest Contact Us Lead Poisoning is Preventable If your home was built before ... of the RRP rule. Read more . Learn about Lead Poisoning Prevention Week . Report Uncertified Contractors and Environmental Violations ...

  7. How does cAMP/protein kinase A signaling lead to tumors in the adrenal cortex and other tissues?

    PubMed

    Almeida, Madson Q; Stratakis, Constantine A

    2011-04-10

    The overwhelming majority of benign lesions of the adrenal cortex leading to Cushing syndrome are linked to one or another abnormality of the cAMP signaling pathway. A small number of both massive macronodular adrenocortical disease and cortisol-producing adenomas harbor somatic GNAS mutations. Micronodular adrenocortical hyperplasias are either pigmented (the classic form being that of primary pigmented nodular adrenocortical disease) or non-pigmented; micronodular adrenocortical hyperplasias can be seen in the context of other conditions or isolated; for example, primary pigmented nodular adrenocortical disease usually occurs in the context of Carney complex, but isolated primary pigmented nodular adrenocortical disease has also been described. Both Carney complex and isolated primary pigmented nodular adrenocortical disease are caused by germline PRKAR1A mutations; somatic mutations of this gene that regulates cAMP-dependent protein kinase are also found in cortisol-producing adenomas, and abnormalities of PKA are present in most cases of massive macronodular adrenocortical disease. Micronodular adrenocortical hyperplasias and some cortisol-producing adenomas are associated with phosphodiesterase 11A and 8B defects, coded, respectively, by the PDE11A and PDE8B genes. Mouse models of Prkar1a deficiency also show that increased cAMP signaling leads to tumors in adrenal cortex and other tissues. In this review, we summarize all recent data from ours and other laboratories, supporting the view that Wnt-signaling acts as an important mediator of tumorigenicity induced by abnormal PRKAR1A function and aberrant cAMP signaling. PMID:21111774

  8. Lead

    MedlinePlus

    ... obvious symptoms, it frequently goes unrecognized. CDC’s Childhood Lead Poisoning Prevention Program is committed to the Healthy People ... Lead Levels Information for Parents Tips for preventing lead poisoning About Us Overview of CDC’s Childhood Lead Poisoning ...

  9. The Fc receptor gamma-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen.

    PubMed Central

    Poole, A; Gibbins, J M; Turner, M; van Vugt, M J; van de Winkel, J G; Saito, T; Tybulewicz, V L; Watson, S P

    1997-01-01

    Activation of mouse platelets by collagen is associated with tyrosine phosphorylation of multiple proteins including the Fc receptor gamma-chain, the tyrosine kinase Syk and phospholipase Cgamma2, suggesting that collagen signals in a manner similar to that of immune receptors. This hypothesis has been tested using platelets from mice lacking the Fc receptor gamma-chain or Syk. Tyrosine phosphorylation of Syk and phospholipase Cgamma2 by collagen stimulation is absent in mice lacking the Fc receptor gamma-chain. Tyrosine phosphorylation of phospholipase Cgamma2 by collagen stimulation is also absent in mice platelets which lack Syk, although phosphorylation of the Fc receptor gamma-chain is maintained. In contrast, tyrosine phosphorylation of platelet proteins by the G protein-coupled receptor agonist thrombin is maintained in mouse platelets deficient in Fc receptor gamma-chain or Syk. The absence of Fc receptor gamma-chain or Syk is accompanied by a loss of secretion and aggregation responses in collagen- but not thrombin-stimulated platelets. These observations provide the first direct evidence of an essential role for the immunoreceptor tyrosine-based activation motif (ITAM) in signalling by a non-immune receptor stimulus. PMID:9171347

  10. Fructose surges damage hepatic adenosyl-monophosphate-dependent kinase and lead to increased lipogenesis and hepatic insulin resistance.

    PubMed

    Gugliucci, Alejandro

    2016-08-01

    Fructose may be a key contributor to the biochemical alterations which promote the metabolic syndrome (MetS), non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2DM): (a) its consumption in all forms but especially in liquid form has much increased alongside with incidence of MetS conditions; (b) it is metabolized almost exclusively in the liver, where it stimulates de novo lipogenesis to drive hepatic triglyceride (TG) synthesis which (c) contributes to hepatic insulin resistance and NAFLD (Lustig et al., 2015; Weiss et al., 2013; Lim et al., 2010; Schwarzet al., 2015; Stanhope et al., 2009, 2013) [1-6]. The specifics of fructose metabolism and its main location in the liver serve to explain many of the possible mechanisms involved. It also opens questions, as the consequences of large increases in fructose flux to the liver may wreak havoc with the regulation of metabolism and would produce two opposite effects (inhibition and activation of AMP dependent kinase-AMPK) that would tend to cancel each other. We posit that (1) surges of fructose in the portal vein lead to increased unregulated flux to trioses accompanied by unavoidable methylglyoxal (MG) production, (2) the new, sudden flux exerts carbonyl stress on the three arginines on the γ subunits AMP binding site of AMPK, irreversible blocking some of the enzyme molecules to allosteric modulation, (3) this explains why, even when fructose quick phosphorylation increases AMP and should therefore activate AMPK, the effects of fructose are compatible with inactivation of AMPK, which then solves the apparent metabolic paradox. We put forward the hypothesis that fructose loads, via the increase in MG flux worsens the fructose-driven metabolic disturbances that lead to unrestricted de novo lipogenesis, fatty liver and hepatic insulin resistance. It does so via the silencing of AMPK. Our hypothesis is testable and if proven correct will shed some further light on fructose metabolism in the liver. It will

  11. Targeting Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MAPKAPK2, MK2): Medicinal Chemistry Efforts To Lead Small Molecule Inhibitors to Clinical Trials.

    PubMed

    Fiore, Mario; Forli, Stefano; Manetti, Fabrizio

    2016-04-28

    The p38/MAPK-activated kinase 2 (MK2) pathway is involved in a series of pathological conditions (inflammation diseases and metastasis) and in the resistance mechanism to antitumor agents. None of the p38 inhibitors entered advanced clinical trials because of their unwanted systemic side effects. For this reason, MK2 was identified as an alternative target to block the pathway but avoiding the side effects of p38 inhibition. However, ATP-competitive MK2 inhibitors suffered from low solubility, poor cell permeability, and scarce kinase selectivity. Fortunately, non-ATP-competitive inhibitors of MK2 have been already discovered that allowed circumventing the selectivity issue. These compounds showed the additional advantage to be effective at lower concentrations in comparison to the ATP-competitive inhibitors. Therefore, although the significant difficulties encountered during the development of these inhibitors, MK2 is still considered as an attractive target to treat inflammation and related diseases to prevent tumor metastasis and to increase tumor sensitivity to chemotherapeutics.

  12. Phosphorylation and interaction of Myopodin by Integrin-Link Kinase Lead to Suppression of Cell Growth and Motility in Prostate Cancer Cells

    PubMed Central

    Yu, Yan-Ping; Luo, Jian-Hua

    2011-01-01

    Myopodin is a tumor suppressor gene that suppresses growth of prostate and urothelial carcinomas. However, the mechanism of myopodin tumor suppressor activity or signaling that leads to activation of myopodin remains unclear. In this report, we showed that the N-terminus of myopodin binds integrin-linked kinase (ILK) both in vivo and in vitro. An ILK interaction motif of 78 amino acids (amino acids 82–157) was identified in the N-terminus region of myopodin. Induction of ILK dependent kinase activity by integrin α7 led to phosphorylation of myopodin both in vivo and in vitro. Knocking down ILK dramatically reduced the inhibition of cell growth and motility mediated by myopodin. A mutant of myopodin lacking the ILK interaction motif is inactive in suppressing the growth and motility of PC3 cells. As a result, this study showed a novel and critical signaling pathway that leads to activation of myopodin. PMID:21643011

  13. Phorbol ester-induced activation of protein kinase C leads to increased formation of diacylglycerol in human neutrophils

    SciTech Connect

    Faellman, M.; Stendahl, O.; Andersson, T. )

    1989-03-01

    Human neutrophils stimulated with a phorbol ester (phorbol 12-myristate 13-acetate or phorbol 12,13-dibutyrate) responded with an increase in diacylglycerol, considered the natural activator of protein kinase C. The amounts of diacylglycerol formed were considerable, reaching 700-900% of basal after 20 minutes. In contrast, 4-{alpha}-phorbol 12-myristate 13-acetate did not induce any detectable formation of diacylglycerol. Simultaneously, phorbol 12-myristate 13-acetate exposure caused increased breakdown of both phosphatidylcholine and phosphatidylinositol 4,5-bisphosphate. These results suggest that once activated, protein kinase C can positively modulate its own activity by inducing additional formation of diacylglycerol from at least two different sources.

  14. Sequence, structure, and active site analyses of p38 MAP kinase: exploiting DFG-out conformation as a strategy to design new type II leads.

    PubMed

    Badrinarayan, Preethi; Sastry, G Narahari

    2011-01-24

    A new knowledge, structure, and sequence based strategy involving the effective exploitation of the DFG-out conformation is delineated. A comprehensive analysis of the structure, sequence, cocrystals, and active sites of p38 MAP kinase crystal structures present in Protein Data Bank (PDB) and the FDA approved MAP kinase drugs has been done, and the information is used for the design of type II leads. The 98 crystal structures, 138 cocrystals, and 31 FDA drugs comprise of 7 different sequences of 2 organisms viz., Homo sapiens and Mus musculus differing in sequence length, constituting both homo- and heterochains. Multiple sequence alignment with ClustalW showed >95% sequence similarity with highly conserved domains and a high propensity for mutations in the activation loop. The bound ligands were extracted, and their interactions with DFG in and out conformations were studied. These cocrystals and FDA drugs were fragmented on the basis of their binding interactions and their affinity to ATP and allosteric sites. The fragment library thus generated contains 106 fragments with overlapping drug fragments. A blue print constituting three main parts viz., head (ATP region), linker (DFG region), and tail (allosteric region) has thus been formulated and used to design 64 type II p38 MAP kinase inhibitors. The above strategy has been employed to design potent type II p38 MAP kinase inhibitors, which are shown to be very promising. PMID:21141877

  15. Diacylglycerol kinase zeta deficiency in a non-CD4+T cell compartment leads to increased peanut hypersensitivity

    PubMed Central

    Kulis, Mike; Wan, Chi-Keung; Gorentla, Balachandra K.; Burks, A. Wesley; Zhong, Xiao-Ping

    2011-01-01

    Summary Peanut sensitization in diacylglycerol kinase zeta (DGKζ) deficient mice led to elevated peanut-IgE levels and severe anaphylaxis. DGKζ deficient CD4+T cells did not account for the phenotype. Future studies will determine which immune lineage caused increased food hypersensitivity. PMID:21439625

  16. Thrombin activation of human platelets dissociates a complex containing gelsolin and actin from phosphatidylinositide-specific phospholipase Cgamma1.

    PubMed Central

    Baldassare, J J; Henderson, P A; Tarver, A; Fisher, G J

    1997-01-01

    We have examined the association of two cytoskeleton proteins, gelsolin and actin, with phosphatidylinositide-specific phospholipase Cgamma1 (PLCgamma1) in resting and thrombin-stimulated human platelets. In unstimulated platelets, gelsolin, actin and PLCgamma1 were immunoprecipitated as a complex by a polyclonal antibody to PLCgamma1. The association of gelsolin and actin was specific for PLCgamma1 because immunoprecipitates of PLCs beta2, beta3, gamma2 and delta1, which are also expressed in human platelets, did not contain detectable gelsolin or actin. Activation with thrombin resulted in platelet aggregation and the dissociation of gelsolin and actin from PLCgamma1. Inhibition of thrombin-induced platelet aggregation blocked the dissociation of gelsolin and actin from PLCgamma1. After stimulation with thrombin, PLCgamma1 activity in immunoprecipitates was increased 2-3-fold. This elevation in PLCgamma1 activity in response to thrombin activation was not observed when platelet aggregation was blocked. Although PLCgamma1 is tyrosine phosphorylated in response to many agonists, we could not detect, by Western analysis with anti-phosphotyrosine antibodies, tyrosine phosphorylation of PLCgamma1 immunoprecipitated from thrombin-stimulated platelets. These results demonstrate that PLCgamma1 is associated with gelsolin and actin in resting platelets, and that thrombin-induced platelet aggregation results in the dissociation of PLCgamma1 from gelsolin and actin, and the stimulation of PLCgamma1 activity. PMID:9164868

  17. Lead decreases cell survival, proliferation, and neuronal differentiation of primary cultured adult neural precursor cells through activation of the JNK and p38 MAP kinases.

    PubMed

    Engstrom, Anna; Wang, Hao; Xia, Zhengui

    2015-08-01

    Adult hippocampal neurogenesis is the process whereby adult neural precursor cells (aNPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate adult-born, functional neurons in the hippocampus. This process is modulated by various extracellular and intracellular stimuli, and the adult-born neurons have been implicated in hippocampus-dependent learning and memory. However, studies on how neurotoxic agents affect this process and the underlying mechanisms are limited. The goal of this study was to determine whether lead, a heavy metal, directly impairs critical processes in adult neurogenesis and to characterize the underlying signaling pathways using primary cultured SGZ-aNPCs isolated from adult mice. We report here that lead significantly increases apoptosis and inhibits proliferation in SGZ-aNPCs. In addition, lead significantly impairs spontaneous neuronal differentiation and maturation. Furthermore, we found that activation of the c-Jun NH2-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase signaling pathways are important for lead cytotoxicity. Our data suggest that lead can directly act on adult neural stem cells and impair critical processes in adult hippocampal neurogenesis, which may contribute to its neurotoxicity and adverse effects on cognition in adults.

  18. Recurrent BCAM-AKT2 fusion gene leads to a constitutively activated AKT2 fusion kinase in high-grade serous ovarian carcinoma

    PubMed Central

    Kannan, Kalpana; Coarfa, Cristian; Chao, Pei-Wen; Luo, Liming; Wang, Yan; Brinegar, Amy E.; Hawkins, Shannon M.; Milosavljevic, Aleksandar; Matzuk, Martin M.; Yen, Laising

    2015-01-01

    High-grade serous ovarian cancer (HGSC) is among the most lethal forms of cancer in women. Excessive genomic rearrangements, which are expected to create fusion oncogenes, are the hallmark of this cancer. Here we report a cancer-specific gene fusion between BCAM, a membrane adhesion molecule, and AKT2, a key kinase in the PI3K signaling pathway. This fusion is present in 7% of the 60 patient cancers tested, a significant frequency considering the highly heterogeneous nature of this malignancy. Further, we provide direct evidence that BCAM-AKT2 is translated into an in-frame fusion protein in the patient’s tumor. The resulting AKT2 fusion kinase is membrane-associated, constitutively phosphorylated, and activated as a functional kinase in cells. Unlike endogenous AKT2, whose activity is tightly regulated by external stimuli, BCAM-AKT2 escapes the regulation from external stimuli. Moreover, a BCAM-AKT2 fusion gene generated via chromosomal translocation using the CRISPR/Cas9 system leads to focus formation in both OVCAR8 and HEK-293T cell lines, suggesting that BCAM-AKT2 is oncogenic. Together, the results indicate that BCAM-AKT2 expression is a new mechanism of AKT2 kinase activation in HGSC. BCAM-AKT2 is the only fusion gene in HGSC that is proven to translate an aberrant yet functional kinase fusion protein with oncogenic properties. This recurrent genomic alteration is a potential therapeutic target and marker of a clinically relevant subtype for tailored therapy of HGSC. PMID:25733895

  19. Activation of the lutropin/choriogonadotropin receptor (LHR) in MA-10 cells leads to the tyrosine phosphorylation of the focal adhesion kinase (FAK) by a pathway that involves Src family kinases*

    PubMed Central

    Mizutani, Tetsuya; Shiraishi, Koji; Welsh, Toni; Ascoli, Mario

    2006-01-01

    We show that activation of the endogenous or recombinant LHR in mouse Leydig tumor cells (MA-10 cells) leads to the tyrosine phosphorylation of the focal adhesion kinase (FAK) and one of its substrates (paxillin). Using specific antibodies to the five tyrosine residues of FAK that become phosphorylated we show that activation of the LHR increases the phosphorylation of Tyr576 and Tyr577 but it does not affect the phosphorylation of Tyr397, Tyr861 or Tyr925. Because FAK is a prominent substrate for the Src family of tyrosine kinases (SFKs) we tested for their involvement in the LHR-mediated phosphorylation of FAK-Tyr576. Src is not detectable in MA-10 cells, but two other prominent members of this family (Fyn and Yes) are present. The LHR-mediated phosphorylation of FAK-Tyr576 is readily inhibited by PP2 (a pharmacological inhibitor of SFKs) and by dominant-negative mutants of SKFs. Moreover, activation of the LHR in MA-10 cells results in the stimulation of the activity of Fyn and Yes and overexpression of either of these two tyrosine kinases enhances the LHR-mediate phosphorylation of FAK-Tyr576. Studies involving activation of other G protein-coupled receptors, overexpression of the different Gα subunits, and the use of second messenger analogs suggest that the LHR-induced phosphorylation of FAK-Tyr576 in MA-10 cells is mediated by SFKs, and that this family of kinases is, in turn, independently or cooperatively activated by the LHR-induced stimulation of Gs and Gq/11-mediated pathways. PMID:16293639

  20. Chronic Lead Exposure and Mixed Factors of Gender×Age×Brain Regions Interactions on Dendrite Growth, Spine Maturity and NDR Kinase

    PubMed Central

    Xue, Weizhen; Yang, Qian-Qian; Wang, Shuang; Xu, Yi; Wang, Hui-Li

    2015-01-01

    NDR1/2 kinase is essential in dendrite morphology and spine formation, which is regulated by cellular Ca2+. Lead (Pb) is a potent blocker of L-type calcium channel and our recent work showed Pb exposure impairs dendritic spine outgrowth in hippocampal neurons in rats. But the sensitivity of Pb-induced spine maturity with mixed factors (gender×age×brain regions) remains unknown. This study aimed to systematically investigate the effect of Pb exposure on spine maturity in rat brain with three factors (gender×age×brain regions), as well as the NDR1/2 kinase expression. Sprague–Dawley rats were exposed to Pb from parturition to postnatal day 30, 60, 90, respectively. Golgi-Cox staining was used to examine spine maturity. Western blot assay was applied to measure protein expression and real-time fluorescence quantitative PCR assay was used to examine mRNA levels. The results showed chronic Pb exposure significantly decreased dendritic length and impaired spine maturity in both rat hippocampus and medial prefrontal cortex. The impairment of dendritic length induced by Pb exposure tended to adolescence > adulthood, hippocampus > medial prefrontal cortex and female > male. Pb exposure induced significant damage in spine maturity during adolescence and early adult while little damage during adult in male rat brain and female medial prefrontal cortex. Besides, there was sustained impairment from adolescence to adulthood in female hippocampus. Interestingly, impairment of spine maturity followed by Pb exposure was correlated with NDR1/2 kinase. The reduction of NDR1/2 kinase protein expression after Pb exposure was similar to the result of spine maturity. In addition, NDR2 and their substrate Rabin3 mRNA levels were significantly decreased by Pb exposure in developmental rat brain. Taken together, Pb exposure impaired dendrite growth and maturity which was subject to gender×age×brain regions effects and related to NDR1/2 signal expression. PMID:26368815

  1. Fragment Molecular Orbital Method Applied to Lead Optimization of Novel Interleukin-2 Inducible T-Cell Kinase (ITK) Inhibitors.

    PubMed

    Heifetz, Alexander; Trani, Giancarlo; Aldeghi, Matteo; MacKinnon, Colin H; McEwan, Paul A; Brookfield, Frederick A; Chudyk, Ewa I; Bodkin, Mike; Pei, Zhonghua; Burch, Jason D; Ortwine, Daniel F

    2016-05-12

    Inhibition of inducible T-cell kinase (ITK), a nonreceptor tyrosine kinase, may represent a novel treatment for allergic asthma. In our previous reports, we described the discovery of sulfonylpyridine (SAP), benzothiazole (BZT), indazole (IND), and tetrahydroindazole (THI) series as novel ITK inhibitors and how computational tools such as dihedral scans and docking were used to support this process. X-ray crystallography and modeling were applied to provide essential insight into ITK-ligand interactions. However, "visual inspection" traditionally used for the rationalization of protein-ligand affinity cannot always explain the full complexity of the molecular interactions. The fragment molecular orbital (FMO) quantum-mechanical (QM) method provides a complete list of the interactions formed between the ligand and protein that are often omitted from traditional structure-based descriptions. FMO methodology was successfully used as part of a rational structure-based drug design effort to improve the ITK potency of high-throughput screening hits, ultimately delivering ligands with potency in the subnanomolar range.

  2. Surface acidic amino acid of Pseudomonas/Halomonas chimeric nucleoside diphosphate kinase leads effective recovery from heat-denaturation.

    PubMed

    Tokunaga, Hiroko; Arakawa, Tsutomu; Tokunaga, Masao

    2013-07-01

    One of the hallmarks of halophilic properties is reversibility of thermal unfolding. A nucleoside diphosphate kinase (NDK) from a moderate halophile Halomonas sp. 593 (HaNDK) follows this behavior. His-tagged chimeric NDK (HisPaHaNDK) consisting of an N-terminal half of a non-halophilic Pseuodomonas aeruginosa NDK (PaNDK) and a Cterminal half of HaNDK loses this reversible property, indicating a critical role of the N-terminal portion of PaNDK in determining the reversibility of the chimeric protein. Various mutations were introduced at Arg45 and Lys61, based on the model NDK structure. It appears that having Glu at position 45 is critical in conferring the thermal reversibility to HisPa- HaNDK chimeric protein.

  3. Receptor-independent, direct membrane binding leads to cell surface lipid sorting and Syk kinase activation in dendritic cells

    PubMed Central

    Ng, Gilbert; Sharma, Karan; Ward, Sandra M.; Desrosiers, Melanie D.; Stephens, Leslie A.; Schoel, W. Michael; Li, Tonglei; Lowell, Clifford A.; Ling, Chang-Chun; Amrein, Matthias W.; Shi, Yan

    2008-01-01

    Summary Binding of particulate antigens by antigen presenting cells (APC) is a critical step in immune activation. Previously, we demonstrated that uric acid crystals are potent adjuvants, initiating a robust adaptive immune response. However, the mechanisms of activation are unknown. Using atomic force microscopy as a tool for real time single cell activation analysis, we report that uric acid crystals can directly engage cellular membranes, particularly the cholesterol components, with a force substantially stronger than protein based cellular contacts. Binding of particulate substances activates Syk kinase-dependent signaling in dendritic cells (DCs). These observations suggest a mechanism whereby immune cell activation can be triggered by solid structures via membrane lipid alteration without the requirement for specific cell surface receptors, and a testable hypothesis for crystal-associated arthropathies, inflammation and adjuvanticity. PMID:18993083

  4. Inhibition of Mer and Axl receptor tyrosine kinases leads to increased apoptosis and improved chemosensitivity in human neuroblastoma.

    PubMed

    Li, Yixin; Wang, Xiqian; Bi, Shaojie; Zhao, Kun; Yu, Chao

    2015-02-13

    Ectopic expression of Mer and Axl receptor tyrosine kinases (RTKs) are frequently found in various cancers as known to promote oncogenesis by activating antiapoptotic signaling pathways. However, the roles of these receptors in neuroblastoma remain unclear. We found Mer and Axl was co-expressed in neuroblastoma patient samples and cell lines. Ligand-dependent Mer or Axl activation led to an increase in phosphorylated ERK1/2, AKT and FAK indicating roles for these RTKs in multiple oncogenic processes. Furthermore, Mer and Axl knockdown led to apoptosis and inhibition of migration as well as a significant increase in chemosensitivity in response to cisplatin and vincristine treatment. Taken together, our results demonstrated that inhibition of Mer and Axl improved apoptotic response and chemosensitivity in neuroblastoma, providing new insights into development of novel therapeutic strategies by targeting these oncogenes.

  5. Mutations in TBCK, Encoding TBC1-Domain-Containing Kinase, Lead to a Recognizable Syndrome of Intellectual Disability and Hypotonia

    PubMed Central

    Bhoj, Elizabeth J.; Li, Dong; Harr, Margaret; Edvardson, Shimon; Elpeleg, Orly; Chisholm, Elizabeth; Juusola, Jane; Douglas, Ganka; Guillen Sacoto, Maria J.; Siquier-Pernet, Karine; Saadi, Abdelkrim; Bole-Feysot, Christine; Nitschke, Patrick; Narravula, Alekhya; Walke, Maria; Horner, Michele B.; Day-Salvatore, Debra-Lynn; Jayakar, Parul; Vergano, Samantha A. Schrier; Tarnopolsky, Mark A.; Hegde, Madhuri; Colleaux, Laurence; Crino, Peter; Hakonarson, Hakon

    2016-01-01

    Through an international multi-center collaboration, 13 individuals from nine unrelated families and affected by likely pathogenic biallelic variants in TBC1-domain-containing kinase (TBCK) were identified through whole-exome sequencing. All affected individuals were found to share a core phenotype of intellectual disability and hypotonia, and many had seizures and showed brain atrophy and white-matter changes on neuroimaging. Minor non-specific facial dysmorphism was also noted in some individuals, including multiple older children who developed coarse features similar to those of storage disorders. TBCK has been shown to regulate the mammalian target of rapamycin (mTOR) signaling pathway, which is also stimulated by exogenous leucine supplementation. TBCK was absent in cells from affected individuals, and decreased phosphorylation of phospho-ribosomal protein S6 was also observed, a finding suggestive of downregulation of mTOR signaling. Lastly, we demonstrated that activation of the mTOR pathway in response to L-leucine supplementation was retained, suggesting a possible avenue for directed therapies for this condition. PMID:27040691

  6. Identification and in vitro evaluation of new leads as selective and competitive glycogen synthase kinase-3β inhibitors through ligand and structure based drug design.

    PubMed

    Darshit, B S; Balaji, B; Rani, P; Ramanathan, M

    2014-09-01

    Glycogen synthase kinase-3β elicits multi-functional effects on intracellular signaling pathways, thereby making the kinase a therapeutic target in multiple pathologies. Hence, it is important to selectively inhibit GSK-3β over structurally and biologically similar targets, such as CDK5. The current study was designed to identify and evaluate novel ATP-competitive GSK-3β inhibitors. The study was designed to identify new leads by ligand based drug design, structure based drug design and in vitro evaluation. The best validated pharmacophore model (AADRRR) identified using LBDD was derived from a dataset of 135 molecules. There were 357 primary hits within the SPECS database using this pharmacophore model. A SBDD approach to the GSK-3β and CDK5 proteins was applied to all primary hits, and 5 selective inhibitors were identified for GSK-3β. GSK-3β and CDK5 in vitro kinase inhibition assays were performed with these molecules to confirm their selectivity for GSK-3β. The molecules showed IC50 values ranging from 0.825μM to 1.116μM and were 23- to 57-fold selective for GSK-3β. Of all the molecules, molecule 3 had the lowest IC50 value of 0.825μM. Our research identified molecules possessing benzothiophene, isoquinoline, thiazolidinedione imidazo-isoquinoline and quinazolinone scaffolds. Potency of these molecules may be due to H-bond interaction with backbone residues of Val135, Asp133 and side chain interaction with Tyr134. Selectivity over CDK5 may be due to side chain interactions with Asp200, backbone of Val61, ionic interaction with Lys60 and π-cationic interaction with Arg141. These selective molecules were also exhibited small atom hydrophobicity and H-bond interaction with water molecule.

  7. High CO2 Leads to Na,K-ATPase Endocytosis via c-Jun Amino-Terminal Kinase-Induced LMO7b Phosphorylation.

    PubMed

    Dada, Laura A; Trejo Bittar, Humberto E; Welch, Lynn C; Vagin, Olga; Deiss-Yehiely, Nimrod; Kelly, Aileen M; Baker, Mairead R; Capri, Joseph; Cohn, Whitaker; Whitelegge, Julian P; Vadász, István; Gruenbaum, Yosef; Sznajder, Jacob I

    2015-12-01

    The c-Jun amino-terminal kinase (JNK) plays a role in inflammation, proliferation, apoptosis, and cell adhesion and cell migration by phosphorylating paxillin and β-catenin. JNK phosphorylation downstream of AMP-activated protein kinase (AMPK) activation is required for high CO2 (hypercapnia)-induced Na,K-ATPase endocytosis in alveolar epithelial cells. Here, we provide evidence that during hypercapnia, JNK promotes the phosphorylation of LMO7b, a scaffolding protein, in vitro and in intact cells. LMO7b phosphorylation was blocked by exposing the cells to the JNK inhibitor SP600125 and by infecting cells with dominant-negative JNK or AMPK adenovirus. The knockdown of the endogenous LMO7b or overexpression of mutated LMO7b with alanine substitutions of five potential JNK phosphorylation sites (LMO7b-5SA) or only Ser-1295 rescued both LMO7b phosphorylation and the hypercapnia-induced Na,K-ATPase endocytosis. Moreover, high CO2 promoted the colocalization and interaction of LMO7b and the Na,K-ATPase α1 subunit at the plasma membrane, which were prevented by SP600125 or by transfecting cells with LMO7b-5SA. Collectively, our data suggest that hypercapnia leads to JNK-induced LMO7b phosphorylation at Ser-1295, which facilitates the interaction of LMO7b with Na,K-ATPase at the plasma membrane promoting the endocytosis of Na,K-ATPase in alveolar epithelial cells.

  8. High CO2 Leads to Na,K-ATPase Endocytosis via c-Jun Amino-Terminal Kinase-Induced LMO7b Phosphorylation

    PubMed Central

    Trejo Bittar, Humberto E.; Welch, Lynn C.; Vagin, Olga; Deiss-Yehiely, Nimrod; Kelly, Aileen M.; Baker, Mairead R.; Capri, Joseph; Cohn, Whitaker; Whitelegge, Julian P.; Vadász, István; Gruenbaum, Yosef; Sznajder, Jacob I.

    2015-01-01

    The c-Jun amino-terminal kinase (JNK) plays a role in inflammation, proliferation, apoptosis, and cell adhesion and cell migration by phosphorylating paxillin and β-catenin. JNK phosphorylation downstream of AMP-activated protein kinase (AMPK) activation is required for high CO2 (hypercapnia)-induced Na,K-ATPase endocytosis in alveolar epithelial cells. Here, we provide evidence that during hypercapnia, JNK promotes the phosphorylation of LMO7b, a scaffolding protein, in vitro and in intact cells. LMO7b phosphorylation was blocked by exposing the cells to the JNK inhibitor SP600125 and by infecting cells with dominant-negative JNK or AMPK adenovirus. The knockdown of the endogenous LMO7b or overexpression of mutated LMO7b with alanine substitutions of five potential JNK phosphorylation sites (LMO7b-5SA) or only Ser-1295 rescued both LMO7b phosphorylation and the hypercapnia-induced Na,K-ATPase endocytosis. Moreover, high CO2 promoted the colocalization and interaction of LMO7b and the Na,K-ATPase α1 subunit at the plasma membrane, which were prevented by SP600125 or by transfecting cells with LMO7b-5SA. Collectively, our data suggest that hypercapnia leads to JNK-induced LMO7b phosphorylation at Ser-1295, which facilitates the interaction of LMO7b with Na,K-ATPase at the plasma membrane promoting the endocytosis of Na,K-ATPase in alveolar epithelial cells. PMID:26370512

  9. Overexpression of Tyro3 receptor tyrosine kinase leads to the acquisition of taxol resistance in ovarian cancer cells.

    PubMed

    Lee, Chuhee

    2015-07-01

    The majority of patients with ovarian cancer are diagnosed at the advanced stages (III/IV) and their 5-year-survival rate is relatively low. One of the major causes of the poor prognosis of ovarian cancer is the development of resistance to first-line chemotherapy, including platinum and taxol. Therefore, improvements in current understanding of chemoresistance is required for the successful treatment of ovarian cancer. In the present study, taxol-resistant ovarian cancer cells, SKOV3/TR, were established by exposing parental SKOV3 cells to increasing concentrations of taxol. . Briefly, cells were treated with 1.5 nM (for 4 weeks), 3 nM (for 4 weeks), 6 nM (for 5 weeks), 12 nM (for 5 weeks) and 24 nM taxol (for 8 weeks) over 6 months. The SKOV3/TR cells were found to be smaller in size and rounder in shape compared with their parental cells. Cell viability and colony formation assays demonstrated an increase in the population doubling time of the SKOV3/TR cells, indicating a reduction in the proliferative capacity of these cells. Reverse transcription-polymerase chain reaction and western blot analysis revealed that, among the TAM receptor tyrosine kinases (RTKs), the mRNA and protein expression levels of Tyro3 RTK were increased, while those of Axl and Mer RTK were decreased in the SKOV3/TR cells. In addition, restoration of the level of Tyro3 by transfecting Tyro3-specific small interfering RNA into the SKOV3/TR cells reduced the proliferative capacity of the cells, indicating that upregulation of the expression of Tyro3 in SKOV3/TR cells may promote survival in the presence of taxol, which eventually resulted in the acquisition of resistance upon taxol treatment. The present study subsequently found that, in the SKOV3/TR cells, the level of intracellular reactive oxygen species (ROS) was elevated, and antioxidant treatment with N-acetyl cysteine (NAC) exerted more profound antiproliferative effects compared with the parental cells. The western blot analysis

  10. HIV-mediated phosphatidylinositol 3-kinase/serine-threonine kinase activation in APCs leads to programmed death-1 ligand upregulation and suppression of HIV-specific CD8 T cells.

    PubMed

    Muthumani, Karuppiah; Shedlock, Devon J; Choo, Daniel K; Fagone, Paolo; Kawalekar, Omkar U; Goodman, Jonathan; Bian, Chaoran B; Ramanathan, Aarti A; Atman, Parikh; Tebas, Pablo; Chattergoon, Michael A; Choo, Andrew Y; Weiner, David B

    2011-09-15

    Recent evidence demonstrates that HIV-1 infection leads to the attenuation of cellular immune responses, which has been correlated with the increased expression of programmed death (PD)-1 on virus-specific CD8(+) T cells. PD-1 is induced upon T cell activation, and its prolonged expression facilitates CD8(+) T cell inhibitory signals when bound to its B7 family ligands, PD-ligand (L)1/2, which are expressed on APCs. Importantly, early reports demonstrated that blockade of the PD-1/PD-L interaction by Abs may help to counter the development of immune exhaustion driven by HIV viral persistence. To better understand the regulation of the PD-1 pathway during HIV infection, we examined the ability of the virus to induce PD-L expression on macrophages and dendritic cells. We found a direct relationship between the infection of APCs and the expression of PD-L1 in which virus-mediated upregulation induced a state of nonresponsiveness in uninfected HIV-specific T cells. Furthermore, this exhaustion phenotype was revitalized by the blockade of PD-L1, after which T cells regained their capacity for proliferation and the secretion of proinflammatory cytokines IFN-γ, IL-2, and IL-12 upon restimulation. In addition, we identify a critical role for the PI3K/serine-threonine kinase signaling pathway in PD-L1 upregulation of APCs by HIV, because inhibition of these intracellular signal transducer enzymes significantly reduced PD-L1 induction by infection. These data identify a novel mechanism by which HIV exploits the immunosuppressive PD-1 pathway and suggest a new role for virus-infected cells in the local corruption of immune responses required for viral suppression.

  11. Reduction of brain barrier tight junctional proteins by lead exposure: role of activation of nonreceptor tyrosine kinase Src via chaperon GRP78.

    PubMed

    Song, Han; Zheng, Gang; Shen, Xue-Feng; Liu, Xin-Qin; Luo, Wen-Jing; Chen, Jing-Yuan

    2014-04-01

    Lead (Pb) has long been recognized as a neurodevelopmental toxin. Developing blood-brain barrier (BBB) is known to be a target of Pb neurotoxicity; however, the underlying mechanisms are still unclear. Recent evidence suggests that intracellular nonreceptor protein tyrosine kinase Src regulates tight junctional proteins (TJPs). This study was designed to investigate whether Pb acted on the Src-mediated cascade event leading to an altered TJP expression at BBB. Rats aged 20-22 days were exposed to Pb in drinking water (0, 100, 200, and 300 ppm Pb) for eight weeks. Electron microscopic and Western blot analyses revealed a severe leakage of BBB and significantly decreased expressions of TJP occludin and ZO-1. When cultured brain endothelial RBE4 cells were exposed to 10μM Pb for 24 h, expressions of phosphor-Src and an upstream regulator GRP78 were significantly increased by 6.42-fold and 8.29-fold (p < 0.01), respectively. Inactivation of Src pathway by a Src-specific inhibitor reversed Pb-induced downregulation of occludin, but not ZO-1; small interfering RNA knockdown of GRP78 attenuated Pb-induced Src phosphorylation and occludin reduction. Furthermore, Pb exposure caused redistribution of GRP78 from endoplasmic reticulum to cytosol and toward cell member. However, the data from immunoneutralization studies did not show the involvement of cell-surface GRP78 in regulating Src phosphorylation upon Pb exposure, suggesting that the cytosolic GRP78, rather than cell-surface GRP78, was responsible to Pb-induced Src activation and ensuing occludin reduction. Taken together, this study provides the evidence of a novel linkage of GRP78, Src activation to downregulation of occludin, and BBB disruption during Pb exposure.

  12. Establishment of a Structure–Activity Relationship of 1H-Imidazo[4,5-c]quinoline-Based Kinase Inhibitor NVP-BEZ235 as a Lead for African Sleeping Sickness

    PubMed Central

    2014-01-01

    Compound NVP-BEZ235 (1) is a potent inhibitor of human phospoinositide-3-kinases and mammalian target of rapamycin (mTOR) that also showed high inhibitory potency against Trypanosoma brucei cultures. With an eye toward using 1 as a starting point for anti-trypanosomal drug discovery, we report efforts to reduce host cell toxicity, to improve the physicochemical properties, and to improve the selectivity profile over human kinases. In this work, we have developed structure–activity relationships for analogues of 1 and have prepared analogues of 1 with improved solubility properties and good predicted central nervous system exposure. In this way, we have identified 4e, 9, 16e, and 16g as the most promising leads to date. We also report cell phenotype and phospholipidomic studies that suggest that these compounds exert their anti-trypanosomal effects, at least in part, by inhibition of lipid kinases. PMID:24805946

  13. New pyrazolo-[3,4-d]-pyrimidine derivative Src kinase inhibitors lead to cell cycle arrest and tumor growth reduction of human medulloblastoma cells

    PubMed Central

    Rossi, Alessandra; Schenone, Silvia; Angelucci, Adriano; Cozzi, Martina; Caracciolo, Valentina; Pentimalli, Francesca; Puca, Andrew; Pucci, Biagio; La Montagna, Raffaele; Bologna, Mauro; Botta, Maurizio; Giordano, Antonio

    2010-01-01

    Medulloblastoma is the most common malignant brain tumor in children, and despite improvements in the overall survival rate, it still lacks an effective treatment. Src plays an important role in cancer, and recently high Src activity was documented in medulloblastoma. In this report, we examined the effects of novel pyrazolo-[3,4-d]-pyrimidine derivative Src inhibitors in medulloblastoma. By MTS assay, we showed that the pyrimidine derivatives indicated as S7, S29, and SI163 greatly reduce the growth rate of medulloblastoma cells by inhibiting Src phosphorylation, compared with HT22 non-neoplastic nerve cells. These compounds also halt cells in the G2/M phase, and this effect likely occurs through the regulation of cdc2 and CDC25C phosphorylation, as shown by Western blot. Moreover, the exposure to pyrimidine derivatives induces apoptosis, assayed by the supravital propidium iodide assay, through modulation of the apoptotic proteins Bax and Bcl2, and inhibits tumor growth in vivo in a mouse model. Notably, S7, S29, and SI163 show major inhibitory effects on medulloblastoma cell growth compared with the chemotherapeutic agents cisplatin and etoposide. In conclusion, our results suggest that S7, S29, and SI163 could be novel attractive candidates for the treatment of medulloblastoma or tumors characterized by high Src activity.—Rossi, A., Schenone, S., Angelucci, A., Cozzi, M., Caracciolo, V., Pentimalli, F., Puca, A., Pucci, B., La Montagna, R., Bologna, M., Botta, M., Giordano, A. New pyrazolo-[3,4-d]-pyrimidine derivative Src kinase inhibitors lead to cell cycle arrest and tumor growth reduction of human medulloblastoma cells. PMID:20354138

  14. Sustained Receptor Stimulation Leads to Sequestration of Recycling Endosomes in a Classical Protein Kinase C- and Phospholipase D-dependent Manner*

    PubMed Central

    Idkowiak-Baldys, Jolanta; Baldys, Aleksander; Raymond, John R.; Hannun, Yusuf A.

    2009-01-01

    Considerable insight has been garnered on initial mechanisms of endocytosis of plasma membrane proteins and their subsequent trafficking through the endosomal compartment. It is also well established that ligand stimulation of many plasma membrane receptors leads to their internalization. However, stimulus-induced regulation of endosomal trafficking has not received much attention. In previous studies, we showed that sustained stimulation of protein kinase C (PKC) with phorbol esters led to sequestration of recycling endosomes in a juxtanuclear region. In this study, we investigated whether G-protein-coupled receptors that activate PKC exerted effects on endosomal trafficking. Stimulation of cells with serotonin (5-hydroxytryptamine (5-HT)) led to sequestration of the 5-HT receptor (5-HT2AR) into a Rab11-positive juxtanuclear compartment. This sequestration coincided with translocation of PKC as shown by confocal microscopy. Mechanistically the observed sequestration of 5-HT2AR was shown to require continuous PKC activity because it was inhibited by pretreatment with classical PKC inhibitor Gö6976 and could be reversed by posttreatment with this inhibitor. In addition, classical PKC autophosphorylation was necessary for receptor sequestration. Moreover inhibition of phospholipase D (PLD) activity and inhibition of PLD1 and PLD2 using dominant negative constructs also prevented this process. Functionally this sequestration did not affect receptor desensitization or resensitization as measured by intracellular calcium increase. However, the PKC- and PLD-dependent sequestration of receptors resulted in co-sequestration of other plasma membrane proteins and receptors as shown for epidermal growth factor receptor and protease activated receptor-1. This led to heterologous desensitization of those receptors and diverted their cellular fate by protecting them from agonist-induced degradation. Taken together, these results demonstrate a novel role for sustained receptor

  15. Targeting GD2 ganglioside and aurora A kinase as a dual strategy leading to cell death in cultures of human neuroblastoma cells.

    PubMed

    Horwacik, Irena; Durbas, Małgorzata; Boratyn, Elżbieta; Węgrzyn, Paulina; Rokita, Hanna

    2013-12-01

    The mechanism of the inhibitory effect of anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (mAb) on human neuroblastoma cells survival was studied in vitro. It was recently shown in IMR-32 cells that death induced by this antibody exhibited several characteristics typical of apoptosis. In this study we used cytotoxixity assays, qRT-PCR and immunoblotting to evaluate the response of several human neuroblastoma cell lines to the anti-GD2 14G2a mAb. We showed that the mAb decreases all three aurora kinases expression and phosphorylation in IMR-32 and LA-N-1 cells. Most importantly, we show, that MK-5108 specific aurora A kinase inhibitor decreases neuroblastoma cell survival, and when used in combination with the mAb, significantly potentiates cytotoxicity against IMR-32, CHP-134, and LA-N-5 neuroblastoma cells in vitro. It was shown that downregulation of aurora A kinase by the therapeutic antibody is associated with decreased levels of MYCN protein in cytoplasm, and induced expression of PHLDA1 and P53 proteins. PMID:23962557

  16. Tyrosine kinase gene rearrangements in epithelial malignancies.

    PubMed

    Shaw, Alice T; Hsu, Peggy P; Awad, Mark M; Engelman, Jeffrey A

    2013-11-01

    Chromosomal rearrangements that lead to oncogenic kinase activation are observed in many epithelial cancers. These cancers express activated fusion kinases that drive the initiation and progression of malignancy, and often have a considerable response to small-molecule kinase inhibitors, which validates these fusion kinases as 'druggable' targets. In this Review, we examine the aetiologic, pathogenic and clinical features that are associated with cancers harbouring oncogenic fusion kinases, including anaplastic lymphoma kinase (ALK), ROS1 and RET. We discuss the clinical outcomes with targeted therapies and explore strategies to discover additional kinases that are activated by chromosomal rearrangements in solid tumours.

  17. Tyrosine kinase gene rearrangements in epithelial malignancies

    PubMed Central

    Shaw, Alice T.; Hsu, Peggy P.; Awad, Mark M.; Engelman, Jeffrey A.

    2014-01-01

    Chromosomal rearrangements that lead to oncogenic kinase activation are observed in many epithelial cancers. These cancers express activated fusion kinases that drive the initiation and progression of malignancy, and often have a considerable response to small-molecule kinase inhibitors, which validates these fusion kinases as ‘druggable’ targets. In this Review, we examine the aetiologic, pathogenic and clinical features that are associated with cancers harbouring oncogenic fusion kinases, including anaplastic lymphoma kinase (ALK), ROS1 and RET. We discuss the clinical outcomes with targeted therapies and explore strategies to discover additional kinases that are activated by chromosomal rearrangements in solid tumours. PMID:24132104

  18. CD22 associates with protein tyrosine phosphatase 1C, Syk, and phospholipase C-gamma(1) upon B cell activation

    PubMed Central

    1996-01-01

    Cross-linking B cell antigen receptor (BCR) elicits early signal transduction events, including activation of protein tyrosine kinases, phosphorylation of receptor components, activation of phospholipase C- gamma (PLC-gamma), and increases in intracellular free Ca2+. In this article, we report that cross-linking the BCR led to a rapid translocation of cytosolic protein tyrosine phosphatase (PTP) 1C to the particulate fraction, where it became associated with a 140-150-kD tyrosyl-phosphorylated protein. Western blotting analysis identified this 140-150-kD protein to be CD22. The association of PTP-1C with CD22 was mediated by the NH2-terminal Src homology 2 (SH2) domain of PTP-1C. Complexes of either CD22/PTP-1C/Syk/PLC-gamma(1) could be isolated from B cells stimulated by BCR engagement or a mixture of hydrogen peroxidase and sodium orthovanadate, respectively. The binding of PLC- gamma(1) and Syk to tyrosyl-phosphorylated CD22 was mediated by the NH2- terminal SH2 domain of PLC-gamma(1) and the COOH-terminal SH2 domain of Syk, respectively. These observations suggest that tyrosyl- phosphorylated CD22 may downmodulate the activity of this complex by dephosphorylation of CD22, Syk, and/or PLC-gamma(1). Transient expression of CD22 and a null mutant of PTP-1C (PTP-1CM) in COS cells resulted in an increase in tyrosyl phosphorylation of CD22 and its interaction with PTP-1CM. By contrast, CD22 was not tyrosyl phosphorylated or associated with PTP-1CM in the presence of wild-type PTP-1C. These results suggest that tyrosyl-phosphorylated CD22 may be a substrate for PTP-1C regulates tyrosyl phosphorylation of CD22. PMID:8627166

  19. Affibody-mediated retention of the epidermal growth factor receptor in the secretory compartments leads to inhibition of phosphorylation in the kinase domain.

    PubMed

    Vernet, Erik; Lundberg, Emma; Friedman, Mikaela; Rigamonti, Nicolò; Klausing, Sandra; Nygren, Per-Ake; Gräslund, Torbjörn

    2009-09-01

    Abnormal activity of the epidermal growth factor receptor (EGFR) is associated with various cancer-related processes and motivates the search for strategies that can selectively block EGFR signalling. In this study, functional knockdown of EGFR was achieved through expression of an affibody construct, (ZEGFR:1907)(2-)KDEL, with high affinity for EGFR and extended with the amino acids KDEL to make it resident in the secretory compartments. Expression of (ZEGFR:1907)(2-)KDEL resulted in 80% reduction ofthe cell surface level of EGFR, and fluorescent staining for EGFR and the (ZEGFR:1907)(2-)KDEL construct showed overlapping intracellular localisation. Immunocapture of EGFR from cell lysates showed that an intracellular complex between EGFR and the affibody construct had been formed, further indicating aspecific interaction between the affibody construct and EGFR. Surface depletion of EGFR led to a dramatic decrease in the amount of kinase domain phosphorylated EGFR, coincident with a significant decrease in the proliferation rate. PMID:19552886

  20. Lack of IL-1 Receptor-Associated Kinase-4 Leads to Defective Th1 Cell Responses and Renders Mice Susceptible to Mycobacterial Infection.

    PubMed

    Marinho, Fábio V; Fahel, Júlia S; Scanga, Charles A; Gomes, Marco Tulio R; Guimarães, Gabriela; Carvalho, Gabrielle R M; Morales, Stefanny V; Báfica, André; Oliveira, Sergio Costa

    2016-09-01

    The Toll-like and IL-1 family receptors play critical roles in innate and adaptive immunity against intracellular pathogens. Although previous data demonstrated the importance of TLRs and IL-1R signaling events for the establishment of an effective immune response to mycobacteria, the possible function of the adaptor molecule IL-1R-associated kinase (IRAK)-4 against this pathogen has not been addressed. In this study, we determined the role of IRAK-4 in signaling pathways responsible for controlling mycobacterial infections. This kinase is important for the production of IL-12 and TNF-α by macrophages and dendritic cells exposed to mycobacteria. Moreover, Mycobacterium bovis-infected IRAK-4-knockout macrophages displayed impaired MAPK and NF-κB activation. IL-1β secretion and caspase-1 activation were also dependent on IRAK-4 signaling. Mice lacking IRAK-4 showed increased M. bovis burden in spleen, liver, and lungs and smaller liver granulomas during 60 d of infection compared with wild-type mice. Furthermore, 80% of IRAK-4(-/-) mice succumbed to virulent M. tuberculosis within 100 d following low-dose infection. This increased susceptibility to mycobacteria correlated with reduced IFN-γ/TNF-α recall responses by splenocytes, as well as fewer IL-12p70-producing APCs. Additionally, we observed that IRAK-4 is also important for the production of IFN-γ by CD4(+) T cells from infected mice. Finally, THP-1 cells treated with an IRAK-4 inhibitor and exposed to M. bovis showed reduced TNF-α and IL-12, suggesting that the results found in mice can be extended to humans. In summary, these data demonstrate that IRAK-4 is essential for innate and adaptive immunity and necessary for efficient control of mycobacterial infections.

  1. Ultra-deep sequencing leads to earlier and more sensitive detection of the tyrosine kinase inhibitor resistance mutation T315I in chronic myeloid leukemia

    PubMed Central

    Baer, Constance; Kern, Wolfgang; Koch, Sarah; Nadarajah, Niroshan; Schindela, Sonja; Meggendorfer, Manja; Haferlach, Claudia; Haferlach, Torsten

    2016-01-01

    Chronic myeloid leukemia cells acquire resistance to tyrosine kinase inhibitors through mutations in the ABL1 kinase domain. The T315I mutation mediates resistance to imatinib, dasatinib, nilotinib and bosutinib, whereas sensitivity to ponatinib remains. Mutation detection by conventional Sanger sequencing requires 10%–20% expansion of the mutated subclone. We studied the T315I mutation development by ultra-deep sequencing on the 454 XL+ platform (Roche) in comparison to Sanger sequencing. By ultra-deep sequencing, mutations were detected at loads of 1%–2%. We selected 40 patients who had failed first-line to third-line treatment (imatinib, dasatinib, nilotinib) and had high loads of the T315I mutation detected by Sanger sequencing. We confirmed T315I mutations by ultra-deep sequencing and investigated the mutation dynamics by backtracking earlier samples. In 20 of 40 patients, we identified the T315I three months (median) before Sanger sequencing detection limits were reached. To exclude sporadic low percentage mutation development without subsequent mutation outgrowth, we selected 42 patients without resistance mutations detected by Sanger sequencing but loss of major molecular response. Here, no mutation was detected by ultradeep sequencing. Additional non-T315I resistance mutations were found in 20 of 40 patients. Only 15% had two mutations per cell; the other cases showed multiple independently mutated clones and the T315I clone demonstrated a rapid outgrowth. In conclusion, T315I mutations could be detected earlier by ultra-deep sequencing compared to Sanger sequencing in a selected group of cases. Earlier mutation detection by ultra-deep sequencing might allow treatment to be changed before clonal increase of cells with the T315I mutation. PMID:27102501

  2. Phospholipase C-gamma1 is required for subculture-induced terminal differentiation of normal human oral keratinocytes.

    PubMed

    Oh, Ju-Eun; Kook, Joong-Ki; Park, Kyung-Hee; Lee, Gene; Seo, Byoung-Moo; Min, Byung-Moo

    2003-04-01

    Serial subculture of primary normal human oral keratinocytes (NHOKs) to the post-mitotic stage induces terminal differentiation, which is in part linked to elevated levels of phospholipase C (PLC)-gamma1. Therefore, PLC-gamma1 may be involved in the signal transduction system that leads to the calcium regulation of subculture-induced keratinocyte differentiation. To test this hypothesis, the expression of PLC-gamma1 in primary NHOKs was blocked by transfecting cells with the antisense PLC-gamma1 cDNA construct. These cells demonstrated dramatic reductions in PLC-gamma1 protein and in the differentiation markers involucrin and transglutaminase following calcium exposure and an increase (15-20%) in in vitro life span versus empty vector-transfected cells. In addition, we established the ability of antisense PLC-gamma1 to block the serial subculture-induced rise in intracellular calcium. Similar observations were made following treatment with the specific PLC inhibitor U73122. These results indicate that the terminal differentiation of NHOKs by serial subculture is associated with PLC-gamma1, which mediates calcium regulation by mobilizing intracellular calcium.

  3. Redox Regulation of Protein Kinases

    PubMed Central

    Truong, Thu H.; Carroll, Kate S.

    2015-01-01

    Protein kinases represent one of the largest families of genes found in eukaryotes. Kinases mediate distinct cellular processes ranging from proliferation, differentiation, survival, and apoptosis. Ligand-mediated activation of receptor kinases can lead to the production of endogenous H2O2 by membrane-bound NADPH oxidases. In turn, H2O2 can be utilized as a secondary messenger in signal transduction pathways. This review presents an overview of the molecular mechanisms involved in redox regulation of protein kinases and its effects on signaling cascades. In the first half, we will focus primarily on receptor tyrosine kinases (RTKs), whereas the latter will concentrate on downstream non-receptor kinases involved in relaying stimulant response. Select examples from the literature are used to highlight the functional role of H2O2 regarding kinase activity, as well as the components involved in H2O2 production and regulation during cellular signaling. In addition, studies demonstrating direct modulation of protein kinases by H2O2 through cysteine oxidation will be emphasized. Identification of these redox-sensitive residues may help uncover signaling mechanisms conserved within kinase subfamilies. In some cases, these residues can even be exploited as targets for the development of new therapeutics. Continued efforts in this field will further basic understanding of kinase redox regulation, and delineate the mechanisms involved in physiologic and pathological H2O2 responses. PMID:23639002

  4. Loss of Par-1a/MARK3/C-TAK1 Kinase Leads to Reduced Adiposity, Resistance to Hepatic Steatosis, and Defective Gluconeogenesis ▿

    PubMed Central

    Lennerz, Jochen K.; Hurov, Jonathan B.; White, Lynn S.; Lewandowski, Katherine T.; Prior, Julie L.; Planer, G. James; Gereau, Robert W.; Piwnica-Worms, David; Schmidt, Robert E.; Piwnica-Worms, Helen

    2010-01-01

    Par-1 is an evolutionarily conserved protein kinase required for polarity in worms, flies, frogs, and mammals. The mammalian Par-1 family consists of four members. Knockout studies of mice implicate Par-1b/MARK2/EMK in regulating fertility, immune homeostasis, learning, and memory as well as adiposity, insulin hypersensitivity, and glucose metabolism. Here, we report phenotypes of mice null for a second family member (Par-1a/MARK3/C-TAK1) that exhibit increased energy expenditure, reduced adiposity with unaltered glucose handling, and normal insulin sensitivity. Knockout mice were protected against high-fat diet-induced obesity and displayed attenuated weight gain, complete resistance to hepatic steatosis, and improved glucose handling with decreased insulin secretion. Overnight starvation led to complete hepatic glycogen depletion, associated hypoketotic hypoglycemia, increased hepatocellular autophagy, and increased glycogen synthase levels in Par-1a−/− but not in control or Par-1b−/− mice. The intercrossing of Par-1a−/− with Par-1b−/− mice revealed that at least one of the four alleles is necessary for embryonic survival. The severity of phenotypes followed a rank order, whereby the loss of one Par-1b allele in Par-1a−/− mice conveyed milder phenotypes than the loss of one Par-1a allele in Par-1b−/− mice. Thus, although Par-1a and Par-1b can compensate for one another during embryogenesis, their individual disruption gives rise to distinct metabolic phenotypes in adult mice. PMID:20733003

  5. Dectin-1-mediated Signaling Leads to Characteristic Gene Expressions and Cytokine Secretion via Spleen Tyrosine Kinase (Syk) in Rat Mast Cells*

    PubMed Central

    Kimura, Yukihiro; Chihara, Kazuyasu; Honjoh, Chisato; Takeuchi, Kenji; Yamauchi, Shota; Yoshiki, Hatsumi; Fujieda, Shigeharu; Sada, Kiyonao

    2014-01-01

    Dectin-1 recognizes β-glucan and plays important roles for the antifungal immunity through the activation of spleen tyrosine kinase (Syk) in dendritic cells or macrophages. Recently, expression of Dectin-1 was also identified in human and mouse mast cells, although its physiological roles were largely unknown. In this report, rat mast cell line RBL-2H3 was analyzed to investigate the molecular mechanism of Dectin-1-mediated activation and responses of mast cells. Treatment of cells with Dectin-1-specific agonist curdlan induced tyrosine phosphorylation of cellular proteins and the interaction of Dectin-1 with the Src homology 2 domain of Syk. These responses depended on tyrosine phosphorylation of the hemi-immunoreceptor tyrosine-based activation motif in the cytoplasmic tail of Dectin-1, whereas they were independent of the γ-subunit of high-affinity IgE receptor. DNA microarray and real-time PCR analyses showed that Dectin-1-mediated signaling stimulated gene expression of transcription factor Nfkbiz and inflammatory cytokines, such as monocyte chemoattractant protein-1, IL-3, IL-4, IL-13, and tumor necrosis factor (TNF)-α. The response was abrogated by pretreatment with Syk inhibitor R406. These results suggest that Syk is critical for Dectin-1-mediated activation of mast cells, although the signaling differs from that triggered by FcϵRI activation. In addition, these gene expressions induced by curdlan stimulation were specifically observed in mast cells, suggesting that Dectin-1-mediated signaling of mast cells offers new insight into the antifungal immunity. PMID:25246527

  6. The tetramethoxyflavone zapotin selectively activates protein kinase C epsilon, leading to its down-modulation accompanied by Bcl-2, c-Jun and c-Fos decrease.

    PubMed

    Toton, Ewa; Lisiak, Natalia; Rubis, Blazej; Budzianowski, Jaromir; Gruber, Peter; Hofmann, Johann; Rybczynska, Maria

    2012-05-01

    Zapotin, a tetramethoxyflavone, is a natural compound with a wide spectrum of activities in neoplastic cells. Protein kinase C epsilon (PKCε) has been shown to be oncogenic, with the ability to increase cell migration, invasion and survival of tumor cells. Here we report that zapotin inhibits cell proliferation. In wild-type HeLa cells with basal endogenous expression of PKCε, the IC(50) was found to be 17.9 ± 1.6 μM. In HeLa cells overexpressing doxycycline-inducible constitutively active PKCε (HeLaPKCεA/E), the IC(50) was 7.6 ± 1.3 μM, suggesting that PKCε enhances the anti-proliferative effect of zapotin. Moreover, we found that zapotin selectively activated PKCε in comparison with other PKC family members, but attenuated doxycycline-induced PKCε expression. As a result of zapotin treatment for 6, 12 and 24h, the doxycycline-induced levels of the two differently phosphorylated PKCε forms (87 kDa and 95 kDa) were decreased. Migration assays revealed that increasing concentrations of zapotin (from 3.5 to 15 μM) decreased migration of HeLaPKCεA/E cells. Furthermore, zapotin significantly increased the fraction of apoptotic cells in doxycycline-induced (HeLaPKCεA/E) cells after 24h and decreased the levels of Bcl-2, c-Jun, c-Fos. This was accompanied by a degradation of PARP-1. In summary, activation of PKCε and down-modulation of the induced PKCε level by zapotin were associated with decreased migration and increased apoptosis. These observations are consistent with the previously reported chemopreventive and chemotherapeutic action of zapotin.

  7. The tetramethoxyflavone zapotin selectively activates protein kinase C epsilon, leading to its down-modulation accompanied by Bcl-2, c-Jun and c-Fos decrease

    PubMed Central

    Toton, Ewa; Lisiak, Natalia; Rubis, Blazej; Budzianowski, Jaromir; Gruber, Peter; Hofmann, Johann; Rybczynska, Maria

    2012-01-01

    Zapotin, a tetramethoxyflavone, is a natural compound with a wide spectrum of activities in neoplastic cells. Protein kinase C epsilon (PKCε) has been shown to be oncogenic, with the ability to increase cell migration, invasion and survival of tumor cells. Here we report that zapotin inhibits cell proliferation. In wild-type HeLa cells with basal endogenous expression of PKCε, the IC50 was found to be 17.9 ± 1.6 μM. In HeLa cells overexpressing doxycycline-inducible constitutively active PKCε (HeLaPKCεA/E), the IC50 was 7.6 ± 1.3 μM, suggesting that PKCε enhances the anti-proliferative effect of zapotin. Moreover, we found that zapotin selectively activated PKCε in comparison with other PKC family members, but attenuated doxycycline-induced PKCε expression. As a result of zapotin treatment for 6, 12 and 24 h, the doxycycline-induced levels of the two differently phosphorylated PKCε forms (87 kDa and 95 kDa) were decreased. Migration assays revealed that increasing concentrations of zapotin (from 3.5 to 15 μM) decreased migration of HeLaPKCεA/E cells. Furthermore, zapotin significantly increased the fraction of apoptotic cells in doxycycline-induced (HeLaPKCεA/E) cells after 24 h and decreased the levels of Bcl-2, c-Jun, c-Fos. This was accompanied by a degradation of PARP-1. In summary, activation of PKCε and down-modulation of the induced PKCε level by zapotin were associated with decreased migration and increased apoptosis. These observations are consistent with the previously reported chemopreventive and chemotherapeutic action of zapotin. PMID:22381066

  8. Membrane Ig cross-linking regulates phosphatidylinositol 3-kinase in B lymphocytes.

    PubMed

    Gold, M R; Chan, V W; Turck, C W; DeFranco, A L

    1992-04-01

    Cross-linking of the B cell AgR results in activation of mature B cells and tolerization of immature B cells. The initial signaling events stimulated by membrane immunoglobulin (mIg) cross-linking are tyrosine phosphorylation of a number of proteins. Among the targets of mIg-induced tyrosine phosphorylation are the tyrosine kinases encoded by the lyn, blk, fyn, and syk genes, the mIg-associated proteins MB-1 and Ig-beta, phospholipase C-gamma 1 and -gamma 2, as well as many unidentified proteins. In this report we show that mIg cross-linking also regulates phosphatidylinositol 3-kinase (PtdIns 3-kinase), an enzyme that phosphorylates inositol phospholipids and plays a key role in mediating the effects of tyrosine kinases on growth control in fibroblasts. Cross-linking mIg on B lymphocytes greatly increased the amount of PtdIns 3-kinase activity which could be immunoprecipitated with anti-phosphotyrosine (anti-tyr(P) antibodies. This response was observed after mIg cross-linking in mIgM- and mIgG-bearing B cell lines and after cross-linking either mIgM or mIgD in murine splenic B cells. Thus, regulation of PtdIns 3-kinase is a common feature of signaling by several different isotypes of mIg. This response was rapid and peaked 2 to 3 min after the addition of anti-Ig antibodies. The anti-Ig-stimulated increase in PtdIns 3-kinase activity associated with anti-Tyr(P) immunoprecipitates could reflect increased tyrosine phosphorylation of PtdIns 3-kinase, increased activity of the enzyme, or both. In favor of the first possibility, the tyrosine kinase inhibitor herbimycin A blocked the increase in ant-Tyr(P)-immunoprecipitated PtdIns 3-kinase activity as well as the anti-Ig-induced tyrosine phosphorylation. Moreover, this response was not secondary to phospholipase C activation but rather seemed to be a direct consequence of mIg-induced tyrosine phosphorylation. Activation of the phosphoinositide pathway by a transfected M1 muscarinic acetylcholine receptor expressed in

  9. Structure based design of novel 6,5 heterobicyclic mitogen-activated protein kinase kinase (MEK) inhibitors leading to the discovery of imidazo[1,5-a] pyrazine G-479.

    PubMed

    Robarge, Kirk D; Lee, Wendy; Eigenbrot, Charles; Ultsch, Mark; Wiesmann, Christian; Heald, Robert; Price, Steve; Hewitt, Joanne; Jackson, Philip; Savy, Pascal; Burton, Brenda; Choo, Edna F; Pang, Jodie; Boggs, Jason; Yang, April; Yang, Xioaye; Baumgardner, Matthew

    2014-10-01

    Use of the tools of SBDD including crystallography led to the discovery of novel and potent 6,5 heterobicyclic MEKi's [J. Med. Chem.2012, 55, 4594]. The core change from a 5,6 heterobicycle to a 6,5 heterobicycle was driven by the desire for increased structural diversity and aided by the co-crystal structure of G-925 [J. Med. Chem.2012, 55, 4594]. The key design feature was the shift of the attachment of the five-membered heterocyclic ring towards the B ring while maintaining the key hydroxamate and anilino pharamcophoric elements in a remarkably similar position as in G-925. From modelling, changing the connection point of the five membered ring heterocycle placed the H-bond accepting nitrogen within a good distance and angle to the Ser212 [J. Med. Chem.2012, 55, 4594]. The resulting novel 6,5 benzoisothiazole MEKi G-155 exhibited improved potency versus aza-benzofurans G-925 and G-963 but was a potent inhibitor of cytochrome P450's 2C9 and 2C19. Lowering the logD by switching to the more polar imidazo[1,5-a] pyridine core significantly diminished 2C9/2C19 inhibition while retaining potency. The imidazo[1,5-a] pyridine G-868 exhibited increased potency versus the starting point for this work (aza-benzofuran G-925) leading to deprioritization of the azabenzofurans. The 6,5-imidazo[1,5-a] pyridine scaffold was further diversified by incorporating a nitrogen at the 7 position to give the imidazo[1,5-a] pyrazine scaffold. The introduction of the C7 nitrogen was driven by the desire to improve metabolic stability by blocking metabolism at the C7 and C8 positions (particularly the HLM stability). It was found that improving on G-868 (later renamed GDC-0623) required combining C7 nitrogen with a diol hydroxamate to give G-479. G-479 with polarity distributed throughout the molecule was improved over G-868 in many aspects.

  10. Human spleen tyrosine kinase p72Syk associates with the Src-family kinase p53/56Lyn and a 120-kDa phosphoprotein.

    PubMed Central

    Sidorenko, S P; Law, C L; Chandran, K A; Clark, E A

    1995-01-01

    The 72-kDa spleen tyrosine kinase (Syk) and Src-family kinase p53/56Lyn (Lyn) contribute to signaling via the B-cell antigen receptor complex. Here we show that Syk and Lyn from human B lymphocytes can interact directly. Syk and Lyn coimmunoprecipitated from mature and activated B-cell lines, and gel-purified Syk and Lyn reassociated in vitro, demonstrating their direct interaction. This Syk-Lyn interaction may be dependent on the stage of B-cell differentiation, since Syk-Lyn associations were not detected in pre-B and myeloma cell lines and Syk from an immature B-cell line did not reassociate with Lyn in vitro. Serine/threonine kinase activity was also associated with Syk. Crosslinking of cell surface IgM led to rapid activation of both tyrosine and serine/threonine protein kinase activities that resulted in phosphorylation in vitro of proteins coprecipitating with Syk--in particular, a serine/threonine phosphorylated protein 120 kDa in size (pp120). Several phosphoproteins, including one of 72 kDa and one of 120 kDa, coprecipitated with phospholipase C-gamma 1 (PLC gamma 1). Sequential immunoprecipitation identified the 72-kDa protein associated with PLC gamma 1 as Syk. The 120-kDa serine/threonine phosphorylated protein that coprecipitated with PLC gamma 1 resembled the Syk-associated pp120 by several criteria. Thus, pp120 may serve as a link between Syk and PLC gamma 1, coupling the B-cell antigen receptor to the phosphatidylinositol pathway. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:7831290

  11. Oncoprotein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2001-02-27

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  12. Inhibition of interleukin-1β-mediated interleukin-1 receptor-associated kinase 4 phosphorylation by zinc leads to repression of memory T helper type 17 response in humans.

    PubMed

    Lee, Hyunju; Kim, Bonah; Choi, Yeon Ho; Hwang, Yuri; Kim, Dong Hyun; Cho, Sunjung; Hong, Sung Jun; Lee, Won-Woo

    2015-12-01

    Zinc is an essential trace element that plays pivotal roles in multiple facets of the immune system. Besides its catalytic and structural roles, zinc also functions as an intracellular signalling molecule, and changes in zinc levels can cause both direct and indirect modulation of immune responses. Further, cytoplasmic levels of bioavailable zinc in immune cells are largely influenced by many extracellular stimuli. Here we provide evidence that zinc represses memory T helper type 17 responses in humans by inhibiting interleukin-1β (IL-1β)-mediated signal. In vitro zinc treatment of CD4(+) T cells in the presence of activated monocytes inhibited interferon-γ-producing cells and IL-17-producing cells, but not IL-4-producing cells. Of note, production of IL-17(+) cells from memory CD4(+) T cells, which is significantly up-regulated by lipopolysaccharide-stimulated monocytes, was preferentially repressed by zinc. Increased cytoplasmic zinc in T cells suppressed IL-1β signalling through repression of phosphorylation of IL-1 receptor-associated kinase 4 (IRAK4), so leading to an inhibitory effect on T helper type 17 responses facilitated by monocyte-derived IL-1β in humans. These findings suggest that extracellular zinc bioavailability may affect memory CD4(+) T-cell responses by modulating the zinc-mediated signalling pathway.

  13. Modification of the cysteine residues in IκBα kinase and NF-κB (p65) by xanthohumol leads to suppression of NF-κB–regulated gene products and potentiation of apoptosis in leukemia cells

    PubMed Central

    Harikumar, Kuzhuvelil B.; Kunnumakkara, Ajaikumar B.; Ahn, Kwang S.; Anand, Preetha; Krishnan, Sunil; Guha, Sushovan

    2009-01-01

    Xanthohumol (XN), a prenylated chalcone isolated from hop plant, exhibits anti-inflammatory, antiproliferative, and antiangiogenic properties through an undefined mechanism. Whether examined by intracellular esterase activity, phosphatidylserine externalization, DNA strand breaks, or caspase activation, we found that XN potentiated tumor necrosis factor–induced apoptosis in leukemia and myeloma cells. This enhancement of apoptosis correlated with down-regulation of nuclear factor-κB (NF-κB) survivin, bcl-xL, XIAP, cIAP1, cIAP2, cylin D1, and c-myc. XN down-regulated both constitutive and inducible NF-κB activation, inhibition of phosphorylation and degradation of IκBα, suppression of p65 nuclear translocation, and NF-κB–dependent reporter gene transcription. XN directly inhibited tumor necrosis factor-induced IκBα kinase (IKK) activation and a reducing agent abolished this inhibition, indicating the role of cysteine residue. XN had no effect on the IKK activity when cysteine residue 179 of IKK was mutated to alanine. XN also directly inhibited binding of p65 to DNA, a reducing agent reversed this effect, and mutation of cysteine residue 38 to serine of p65 abolished this effect. Thus, our results show that modification of cysteine residues of IKK and p65 by XN leads to inhibition of the NF-κB activation pathway, suppression of antiapoptotic gene products, and potentiation of apoptosis in leukemia cells. PMID:18952893

  14. Identification of anaplastic lymphoma kinase as a receptor for the growth factor pleiotrophin.

    PubMed

    Stoica, G E; Kuo, A; Aigner, A; Sunitha, I; Souttou, B; Malerczyk, C; Caughey, D J; Wen, D; Karavanov, A; Riegel, A T; Wellstein, A

    2001-05-18

    Pleiotrophin (PTN) is a secreted growth factor that induces neurite outgrowth and is mitogenic for fibroblasts, epithelial, and endothelial cells. During tumor growth PTN can serve as an angiogenic factor and drive tumor invasion and metastasis. To identify a receptor for PTN, we panned a phage display human cDNA library against immobilized PTN protein as a bait. From this we isolated a phage insert that was homologous to an amino acid sequence stretch in the extracellular domain (ECD) of the orphan receptor tyrosine kinase anaplastic lymphoma kinase (ALK). In parallel with PTN, ALK is highly expressed during perinatal development of the nervous system and down-modulated in the adult. Here we show in cell-free assays as well as in radioligand receptor binding studies in intact cells that PTN binds to the ALK ECD with an apparent Kd of 32 +/- 9 pm. This receptor binding is inhibited by an excess of PTN, by the ALK ECD, and by anti-PTN and anti-ECD antibodies. PTN added to ALK-expressing cells induces phosphorylation of both ALK and of the downstream effector molecules IRS-1, Shc, phospholipase C-gamma, and phosphatidylinositol 3-kinase. Furthermore, the growth stimulatory effect of PTN on different cell lines in culture coincides with the endogenous expression of ALK mRNA, and the effect of PTN is enhanced by ALK overexpression. From this we conclude that ALK is a receptor that transduces PTN-mediated signals and propose that the PTN-ALK axis can play a significant role during development and during disease processes.

  15. Intracellular signaling of the Ufo/Axl receptor tyrosine kinase is mediated mainly by a multi-substrate docking-site.

    PubMed

    Braunger, J; Schleithoff, L; Schulz, A S; Kessler, H; Lammers, R; Ullrich, A; Bartram, C R; Janssen, J W

    1997-06-01

    Ufo/Axl belongs to a new family of receptor tyrosine kinases with an extracellular structure similar to that of neural cell adhesion molecules. In order to elucidate intracellular signaling, the cytoplasmic moiety of Ufo/Axl was used to screen an expression library according to the CORT (cloning of receptor targets) method. Three putative Ufo substrates were identified: phospholipase Cgamma1 (PLCgamma), as well as p85alpha and p85beta subunits of phosphatidylinositol 3'-kinase (PI3-kinase). Subsequently, chimeric EGFR/Ufo receptors consisting of the extracellular domains of the epidermal growth factor receptor (EGFR) and the transmembrane and intracellular moiety of Ufo were engineered. Using different far-Western blot analyses and coimmunoprecipitation assays, receptor binding of PLCgamma and p85 proteins as well as GRB2, c-src and lck was examined in vitro and in vivo. Competitive inhibition of substrate binding and mutagenesis experiments with EGFR/Ufo constructs revealed C-terminal tyrosine 821 (EILpYVNMDEG) as a docking site for multiple effectors, namely PLCgamma, p85 proteins, GRB2, c-src and lck. Tyrosine 779 (DGLpYALMSRC) demonstrated an additional, but lower binding affinity for the p85 proteins in vitro. In addition, binding of PLCgamma occurred through tyrosine 866 (AGRpYVLCPST). Moreover, our in vivo data indicate that further direct or indirect binding sites for PLCgamma, GRB2, c-src and lck on the human Ufo receptor may exist.

  16. Protein kinase profiling assays: a technology review.

    PubMed

    Wang, Yuren; Ma, Haiching

    2015-11-01

    Protein kinases have become one of the most intensively pursued classes of drug targets for many diseases such as cancers and inflammatory diseases. Kinase profiling work seeks to understand general selectivity trends of lead compounds across the kinome, which help with target selection, compound prioritization, and potential implications in toxicity. Under the current drug discovery process, screening of compounds against comprehensive panels of kinases and their mutants has become the standard approach. Many screening assays and technologies which are compatible for high-throughput screening (HTS) against kinases have been extensively pursued and developed.

  17. Coordinate regulation of IkappaB kinases by mitogen-activated protein kinase kinase kinase 1 and NF-kappaB-inducing kinase.

    PubMed

    Nemoto, S; DiDonato, J A; Lin, A

    1998-12-01

    IkappaB kinases (IKKalpha and IKKbeta) are key components of the IKK complex that mediates activation of the transcription factor NF-kappaB in response to extracellular stimuli such as inflammatory cytokines, viral and bacterial infection, and UV irradiation. Although NF-kappaB-inducing kinase (NIK) interacts with and activates the IKKs, the upstream kinases for the IKKs still remain obscure. We identified mitogen-activated protein kinase kinase kinase 1 (MEKK1) as an immediate upstream kinase of the IKK complex. MEKK1 is activated by tumor necrosis factor alpha (TNF-alpha) and interleukin-1 and can potentiate the stimulatory effect of TNF-alpha on IKK and NF-kappaB activation. The dominant negative mutant of MEKK1, on the other hand, partially blocks activation of IKK by TNF-alpha. MEKK1 interacts with and stimulates the activities of both IKKalpha and IKKbeta in transfected HeLa and COS-1 cells and directly phosphorylates the IKKs in vitro. Furthermore, MEKK1 appears to act in parallel to NIK, leading to synergistic activation of the IKK complex. The formation of the MEKK1-IKK complex versus the NIK-IKK complex may provide a molecular basis for regulation of the IKK complex by various extracellular signals.

  18. Overexpression of kidney phosphatidylinositol 4-kinasebeta and phospholipase C(gamma1) proteins in two rodent models of polycystic kidney disease.

    PubMed

    Cuozzo, F P; Mishra, S; Jiang, J; Aukema, H M

    2002-05-21

    Our studies of renal phosphoinositide levels and metabolism in the pcy mouse with polycystic kidney disease (PKD) suggest that phosphatidylinositol kinase (PtdInsK) and phospholipase C (PLC) are elevated in this renal disorder. Therefore, the steady-state levels of select isoforms of these enzymes were examined in renal cytosolic and particulate (detergent-soluble) fractions in male and female normal and CD1-pcy/pcy (pcy) mice at 60, 120 and 180 days of age, and in male and female normal and diseased (Han:SPRD-cy) rats at 28 and 70 days of age. Disease-related increases in phosphatidylinositol 4-kinasebeta (PtdIns4Kbeta) and PLC(gamma1) levels were present in both models. PtdIns4Kbeta levels were higher by as much as 233% in pcy mice and by 95% in diseased Han:SPRD-cy rats compared to normals of the same age and gender. Steady-state levels of PLC(gamma1) were as much as 74% and 35% higher in pcy mice and diseased Han:SPRD-cy rats, respectively, compared to their controls. The consistency of these alterations in two accepted models of PKD indicates the importance of the phosphoinositide signalling pathway in the evolution of this disorder, and represents a potential site for therapeutic intervention. PMID:12009430

  19. Kinase inhibitor profiling reveals unexpected opportunities to inhibit disease-associated mutant kinases

    PubMed Central

    Duong-Ly, Krisna C.; Devarajan, Karthik; Liang, Shuguang; Horiuchi, Kurumi Y.; Wang, Yuren; Ma, Haiching; Peterson, Jeffrey R.

    2016-01-01

    Summary Small-molecule kinase inhibitors have typically been designed to inhibit wild-type kinases rather than the mutant forms that frequently arise in diseases such as cancer. Mutations can have serious clinical implications by increasing kinase catalytic activity or conferring therapeutic resistance. To identify opportunities to repurpose inhibitors against disease-associated mutant kinases, we conducted a large-scale functional screen of 183 known kinase inhibitors against 76 recombinant, mutant kinases. The results revealed lead compounds with activity against clinically important mutant kinases including ALK, LRRK2, RET, and EGFR as well as unexpected opportunities for repurposing FDA-approved kinase inhibitors as leads for additional indications. Furthermore, using T674I PDGFRα as an example, we show how single-dose screening data can provide predictive structure-activity data to guide subsequent inhibitor optimization. This study provides a resource for the development of inhibitors against numerous disease-associated mutant kinases and illustrates the potential of unbiased profiling as an approach to compound-centric inhibitor development. PMID:26776524

  20. Multifunctional Abl kinases in health and disease.

    PubMed

    Khatri, Aaditya; Wang, Jun; Pendergast, Ann Marie

    2016-01-01

    The Abelson tyrosine kinases were initially identified as drivers of leukemia in mice and humans. The Abl family kinases Abl1 and Abl2 regulate diverse cellular processes during development and normal homeostasis, and their functions are subverted during inflammation, cancer and other pathologies. Abl kinases can be activated by multiple stimuli leading to cytoskeletal reorganization required for cell morphogenesis, motility, adhesion and polarity. Depending on the cellular context, Abl kinases regulate cell survival and proliferation. Emerging data support important roles for Abl kinases in pathologies linked to inflammation. Among these are neurodegenerative diseases and inflammatory pathologies. Unexpectedly, Abl kinases have also been identified as important players in mammalian host cells during microbial pathogenesis. Thus, the use of Abl kinase inhibitors might prove to be effective in the treatment of pathologies beyond leukemia and solid tumors. In this Cell Science at a Glance article and in the accompanying poster, we highlight the emerging roles of Abl kinases in the regulation of cellular processes in normal cells and diverse pathologies ranging from cancer to microbial pathogenesis.

  1. Two Kinase Family Dramas

    PubMed Central

    Leonard, Thomas A.; Hurley, James H.

    2007-01-01

    In this issue, Lietha and colleagues (2007) report the structure of focal adhesion kinase (FAK) and reveal how FAK maintains an autoinhibited state. Together with the structure of another tyrosine kinase, ZAP-70 (Deindl et al., 2007), this work highlights the diversity of mechanisms that nature has evolved within the kinase superfamily to regulate their activity through autoinhibition. PMID:17574014

  2. Targeting cancer with small-molecular-weight kinase inhibitors.

    PubMed

    Fabbro, Doriano; Cowan-Jacob, Sandra W; Möbitz, Henrik; Martiny-Baron, Georg

    2012-01-01

    Protein and lipid kinases fulfill essential roles in many signaling pathways that regulate normal cell functions. Deregulation of these kinase activities lead to a variety of pathologies ranging from cancer to inflammatory diseases, diabetes, infectious diseases, cardiovascular disorders, cell growth and survival. 518 protein kinases and about 20 lipid-modifying kinases are encoded by the human genome, and a much larger proportion of additional kinases are present in parasite, bacterial, fungal, and viral genomes that are susceptible to exploitation as drug targets. Since many human diseases result from overactivation of protein and lipid kinases due to mutations and/or overexpression, this enzyme class represents an important target for the pharmaceutical industry. Approximately one third of all protein targets under investigation in the pharmaceutical industry are protein or lipid kinases.The kinase inhibitors that have been launched, thus far, are mainly in oncology indications and are directed against a handful of protein and lipid kinases. With one exception, all of these registered kinase inhibitors are directed toward the ATP-site and display different selectivities, potencies, and pharmacokinetic properties. At present, about 150 kinase-targeted drugs are in clinical development and many more in various stages of preclinical development. Kinase inhibitor drugs that are in clinical trials target all stages of signal transduction from the receptor protein tyrosine kinases that initiate intracellular signaling, through second-messenger-dependent lipid and protein kinases, and protein kinases that regulate the cell cycle.This review provides an insight into protein and lipid kinase drug discovery with respect to achievements, binding modes of inhibitors, and novel avenues for the generation of second-generation kinase inhibitors to treat cancers.

  3. Where Will LEAD Lead?

    ERIC Educational Resources Information Center

    Wildman, Louis

    After setting forth eight assumptions concerning the education of educational administrators, findings about the Leadership in Educational Administration Development (LEAD) program are discussed. The analysis is based on the first-year applications, telephone conversations with staff at a majority of the project sites, and additional material…

  4. Palmitate-induced interleukin 6 production is mediated by protein kinase C and nuclear-factor kappaB activation and leads to glucose transporter 4 down-regulation in skeletal muscle cells.

    PubMed

    Jové, Mireia; Planavila, Anna; Laguna, Juan Carlos; Vázquez-Carrera, Manuel

    2005-07-01

    The mechanisms by which elevated levels of free fatty acids cause insulin resistance are not well understood. In addition, accumulating evidence suggests a link between inflammation and type 2 diabetes. Here, we report that exposure of C2C12 skeletal muscle cells to 0.5 mm palmitate results in increased mRNA levels (3.5-fold induction; P < 0.05) and secretion (control 375 +/- 57 vs. palmitate 1129 +/- 177 pg/ml; P < 0.001) of the proinflammatory cytokine IL-6. Palmitate increased nuclear factor-kappaB activation and coincubation of the cells with palmitate and the nuclear factor-kappaB inhibitor pyrrolidine dithiocarbamate prevented both IL-6 expression and secretion. Furthermore, incubation of palmitate-treated cells with calphostin C, a strong and specific inhibitor of protein kinase C, and phorbol myristate acetate, that down-regulates protein kinase C in long-term incubations, abolished induction of IL-6 production. Finally, exposure of skeletal muscle cells to palmitate caused a fall in the mRNA levels of glucose transporter 4 and insulin-stimulated glucose uptake, whereas in the presence of anti-IL-6 antibody, which neutralizes the biological activity of mouse IL-6 in cell culture, these reductions were prevented. These findings suggest that IL-6 may mediate several of the prodiabetic effects of palmitate.

  5. Functional analysis of a phosphatidic acid binding domain in human Raf-1 kinase: mutations in the phosphatidate binding domain lead to tail and trunk abnormalities in developing zebrafish embryos.

    PubMed

    Ghosh, Sujoy; Moore, Sean; Bell, Robert M; Dush, Michael

    2003-11-14

    Previously, we and others identified a 35-amino acid segment within human Raf-1 kinase that preferentially binds phosphatidic acid. The presence of phosphatidic acid was found to be necessary for the translocation of Raf-1 to the plasma membrane. We have now employed a combination of alanine-scanning and deletion mutagenesis to identify the critical amino acid residues in Raf-1 necessary for interaction with phosphatidic acid. Progressive mutations within a tetrapeptide motif (residues 398-401 of human Raf-1) reduced and finally eliminated binding of Raf-1 to phosphatidic acid. We then injected zebrafish embryos with RNA encoding wild-type Raf-1 kinase or a mutant version with triple alanine mutations in the tetrapeptide motif and followed the morphological fate of embryonic development. Embryos with mutant but not wild-type Raf-1 exhibited defects in posterior axis formation exemplified by bent trunk and tail structures. Molecular evidence for lack of signaling through mutated Raf-1 was obtained by aberrant in situ hybridization of the ntl (no tail) gene, which functions downstream of Raf-1. Our results demonstrate that a functional phosphatidate binding site is necessary for Raf-1 function in embryonic development.

  6. LEADING WITH LEADING INDICATORS

    SciTech Connect

    PREVETTE, S.S.

    2005-01-27

    This paper documents Fluor Hanford's use of Leading Indicators, management leadership, and statistical methodology in order to improve safe performance of work. By applying these methods, Fluor Hanford achieved a significant reduction in injury rates in 2003 and 2004, and the improvement continues today. The integration of data, leadership, and teamwork pays off with improved safety performance and credibility with the customer. The use of Statistical Process Control, Pareto Charts, and Systems Thinking and their effect on management decisions and employee involvement are discussed. Included are practical examples of choosing leading indicators. A statistically based color coded dashboard presentation system methodology is provided. These tools, management theories and methods, coupled with involved leadership and employee efforts, directly led to significant improvements in worker safety and health, and environmental protection and restoration at one of the nation's largest nuclear cleanup sites.

  7. Prokaryotic Diacylglycerol Kinase and Undecaprenol Kinase

    PubMed Central

    Van Horn, Wade D.; Sanders, Charles R.

    2013-01-01

    Prokaryotic diacylglycerol kinase (DAGK) and undecaprenol kinase (UDPK) are the lone members of a family of multispan membrane enzymes that are very small, lack relationships to any other family of proteins—including water soluble kinases, and that exhibit an unusual structure and active site architecture. Escherichia coli DAGK plays an important role in recycling diacylglycerol produced as a byproduct of biosynthesis of molecules located in the periplasmic space. UDPK seems to play an analogous role in Gram-positive bacteria, where its importance is evident by the fact that UDPK is essential for biofilm formation by the oral pathogen Streptococcus mutans. DAGK has also long served as a model system for studies of membrane protein biocatalysis, folding, stability, and structure. This review explores our current understanding of the microbial physiology, enzymology, structural biology, and folding of the prokaryotic diacylglycerol kinase family, which is based on over 40 years of studies. PMID:22224599

  8. Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes.

    PubMed

    Sundaresan, A; Risin, D; Pellis, N R

    2004-06-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

  9. Modeled Microgravity-Induced Protein Kinase C Isoform Expression in Human Lymphocytes

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    2003-01-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited both in microgravity and modeled microgravity (MMG) as reflected in diminished DNA synthess in peripheral blood lymphocytes and their locomotion through gelled type 1 collagen. Direct activation of Protein Kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 19 and MMG-culture. Human lymphocytes were cultured and harvested at 24, 48, 72 and 96 hours and serial samples assessed for locomotion using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta and -epsilon was assessed by RT-PCR, flow cytometry and immunoblotting. Results indicated that PKC isoforms delta and epsilon were down-regulated by more than 50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 19 controls. Events upstream of PKC such as phosphorylation of Phospholipase C(gamma) (PLC-gamma) in MMG, revealed accumulation of inactive enzyme. Depressed Ca++ -independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than, but after ligand-receptor interaction. Keywords: Signal transduction, locomotion, immunity

  10. Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes

    NASA Technical Reports Server (NTRS)

    Sundaresan, A.; Risin, D.; Pellis, N. R.

    2004-01-01

    In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

  11. Study of solar features causing GMSs with 250c'gamma' 'smaller than' H 'smaller than' 400'gamma' during the period 1999-2010

    NASA Astrophysics Data System (ADS)

    Kumar, Rajiv

    2016-07-01

    The effect of solar features on geospheric conditions leading to geomagnetic storms (GMSs) with planetary index,A P ≥ 20 and the range of horizontal component of the Earth's magnetic field H such that 250γ

  12. Varicella-Zoster Virus Open Reading Frame 66 Protein Kinase and Its Relationship to Alphaherpesvirus US3 Kinases

    PubMed Central

    Erazo, Angela

    2014-01-01

    The varicella-zoster virus (VZV) open reading frame (ORF) 66 encodes a basophilic kinase orthologous to the US3 protein kinases found in all alphaherpesviruses. This review summarizes current information on the ORF66 kinase, and outlines apparent differences from other US3 kinases, as well as some of the conserved functions. One critical difference is the VZV ORF66 kinase targeting of the major regulatory VZV IE62 protein to control its nuclear import and assembly into the VZV virion, which is so far unprecedented in the alphaherpesviruses. However, ORF66 targets some cellular targets which are also targeted by US3 kinases of other herpesviruses, including the histone deacetylase-1 and 2 proteins, pathways that lead to changes in actin dynamics, and the targeting of substrates of protein kinase A, including the nuclear matrix protein matrin 3. PMID:20186610

  13. The Secretory Pathway Kinases

    PubMed Central

    Sreelatha, Anju; Kinch, Lisa N.; Tagliabracci, Vincent S.

    2015-01-01

    Protein phosphorylation is a nearly universal post-translation modification involved in a plethora of cellular events. Even though phosphorylation of extracellular proteins had been observed, the identity of the kinases that phosphorylate secreted proteins remained a mystery until recently. Advances in genome sequencing and genetic studies have paved the way for the discovery of a new class of kinases that localize within the endoplasmic reticulum, Golgi apparatus and the extracellular space. These novel kinases phosphorylate proteins and proteoglycans in the secretory pathway and appear to regulate various extracellular processes. Mutations in these kinases cause human disease, thus underscoring the biological importance of phosphorylation within the secretory pathway. PMID:25862977

  14. The Effects of Early Life Lead Exposure on the Expression of Glycogen Synthase Kinase-3β and Insulin-like Growth Factor 1 Receptor in the Hippocampus of Mouse Pups.

    PubMed

    Li, Ning; Qiao, MingWu; Zhang, PingAn; Li, Xing; Li, Li; Yu, ZengLi

    2016-01-01

    The present study was undertaken to investigate the effects of maternal lead exposure on expression of GSK-3β and IGF1R in the hippocampus of mice offspring. Lead exposure initiated from beginning of gestation to weaning. Lead acetate administered in drinking solutions was dissolved in distilled deionized water at the concentrations of 0.1, 0.5, and 1%, respectively. On the 21st postnatal day, the Pb levels were determined by graphite furnace atomic absorption spectrometry. The expression of GSK-3β and IGF1R in hippocampus was examined by immunohistochemistry and Western blotting. The lead levels in blood and hippocampus of all lead exposure groups were significantly higher than those of the control group (P < 0.05). Compared with the control group, the expression of GSK-3β was increased in lead-exposed groups (P < 0.05), but the expression of IGF1R was decreased (P < 0.05). The high expression of GSK-3β and low expression of IGF1R in the hippocampus of pups may contribute to the neurotoxicity associated with maternal Pb exposure.

  15. Novel pathway in Bcr-Abl signal transduction involves Akt-independent, PLC-gamma1-driven activation of mTOR/p70S6-kinase pathway.

    PubMed

    Markova, B; Albers, C; Breitenbuecher, F; Melo, J V; Brümmendorf, T H; Heidel, F; Lipka, D; Duyster, J; Huber, C; Fischer, T

    2010-02-01

    In chronic myeloid leukemia, activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway is crucial for survival and proliferation of leukemic cells. Essential downstream molecules involve mammalian target of rapamycin (mTOR) and S6-kinase. Here, we present a comprehensive analysis of the molecular events involved in activation of these key signaling pathways. We provide evidence for a previously unrecognized phospholipase C-gamma1 (PLC-gamma1)-controlled mechanism of mTOR/p70S6-kinase activation, which operates in parallel to the classical Akt-dependent machinery. Short-term imatinib treatment of Bcr-Abl-positive cells caused dephosphorylation of p70S6-K and S6-protein without inactivation of Akt. Suppression of Akt activity alone did not affect phosphorylation of p70-S6K and S6. These results suggested the existence of an alternative mechanism for mTOR/p70S6-K activation. In Bcr-Abl-expressing cells, we detected strong PLC-gamma1 activation, which was suppressed by imatinib. Pharmacological inhibition and siRNA knockdown of PLC-gamma1 blocked p70S6-K and S6 phosphorylation. By inhibiting the Ca-signaling, CaMK and PKCs we demonstrated participation of these molecules in the pathway. Suppression of PLC-gamma1 led to inhibition of cell proliferation and enhanced apoptosis. The novel pathway proved to be essential for survival and proliferation of leukemic cells and almost complete cell death was observed upon combined PLC-gamma1 and Bcr-Abl inhibition. The pivotal role of PLC-gamma1 was further confirmed in a mouse leukemogenesis model.

  16. Multiparameter Lead Optimization to Give an Oral Checkpoint Kinase 1 (CHK1) Inhibitor Clinical Candidate: (R)-5-((4-((Morpholin-2-ylmethyl)amino)-5-(trifluoromethyl)pyridin-2-yl)amino)pyrazine-2-carbonitrile (CCT245737).

    PubMed

    Osborne, James D; Matthews, Thomas P; McHardy, Tatiana; Proisy, Nicolas; Cheung, Kwai-Ming J; Lainchbury, Michael; Brown, Nathan; Walton, Michael I; Eve, Paul D; Boxall, Katherine J; Hayes, Angela; Henley, Alan T; Valenti, Melanie R; De Haven Brandon, Alexis K; Box, Gary; Jamin, Yann; Robinson, Simon P; Westwood, Isaac M; van Montfort, Rob L M; Leonard, Philip M; Lamers, Marieke B A C; Reader, John C; Aherne, G Wynne; Raynaud, Florence I; Eccles, Suzanne A; Garrett, Michelle D; Collins, Ian

    2016-06-01

    Multiparameter optimization of a series of 5-((4-aminopyridin-2-yl)amino)pyrazine-2-carbonitriles resulted in the identification of a potent and selective oral CHK1 preclinical development candidate with in vivo efficacy as a potentiator of deoxyribonucleic acid (DNA) damaging chemotherapy and as a single agent. Cellular mechanism of action assays were used to give an integrated assessment of compound selectivity during optimization resulting in a highly CHK1 selective adenosine triphosphate (ATP) competitive inhibitor. A single substituent vector directed away from the CHK1 kinase active site was unexpectedly found to drive the selective cellular efficacy of the compounds. Both CHK1 potency and off-target human ether-a-go-go-related gene (hERG) ion channel inhibition were dependent on lipophilicity and basicity in this series. Optimization of CHK1 cellular potency and in vivo pharmacokinetic-pharmacodynamic (PK-PD) properties gave a compound with low predicted doses and exposures in humans which mitigated the residual weak in vitro hERG inhibition. PMID:27167172

  17. Lead Poisoning

    MedlinePlus

    ... Experiments Stories Lessons Topics Games Activities Lessons MENU Lead Poisoning Kids Homepage Topics Pollution Lead Poisoning What is ... you can avoid contact with it! Sources of Lead Poisoning HOUSE PAINTS: Before1950, lead-based paint was used ...

  18. Lead Test

    MedlinePlus

    ... to determine lead sources, educating family members about lead poisoning , and instituting follow-up testing to monitor the ... high levels of lead, see the article on Lead Poisoning . The Occupational Safety and Health Administration (OSHA) has ...

  19. Lead Poisoning

    MedlinePlus

    Lead Poisoning What is it and who is affected? Lead is a highly toxic substance, exposure to which ... and children can suffer from the effects of lead poisoning, but childhood lead poisoning is much more frequent. ...

  20. Asymmetric Tyrosine Kinase Arrangements in Activation or Autophosphorylation of Receptor Tyrosine Kinases

    SciTech Connect

    J Bae; J Schlessinger

    2011-12-31

    Receptor tyrosine kinases (RTKs) play important roles in the control of many cellular processes including cell proliferation, cell adhesion, angiogenesis, and apoptosis. Ligand-induced dimerization of RTKs leads to autophosphorylation and activation of RTKs. Structural studies have shown that while isolated ectodomains of several RTKs form symmetric dimers the isolated cytoplasmic kinase domains of epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR) form asymmetric dimers during their activation. Binding of one kinase molecule of EGFR to a second kinase molecule asymmetrically leads to stimulation of kinase activity and enhanced autophosphorylation. Furthermore, the structures of the kinase domain of FGFR1 and FGFR2 reveal the formation of asymmetric interfaces in the processes of autophosphorylation at their specific phosphotyrosine (pY) sites. Disruption of asymmetric dimer interface of EGFR leads to reduction in enzymatic activity and drastic reduction of autophosphorylation of FGFRs in ligandstimulated live cells. These studies demonstrate that asymmetric dimer formation is as a common phenomenon critical for activation and autophosphorylation of RTKs.

  1. LKB1, the multitasking tumour suppressor kinase.

    PubMed

    Marignani, P A

    2005-01-01

    Mutations in the lkb1 gene are found in Peutz-Jeghers syndrome (PJS), with loss of heterozygosity or somatic mutations at the lkb1 locus, suggesting the gene product, the serine/threonine kinase LKB1, may function as a tumour suppressor. Patients with PJS are at a greater risk of developing cancers of epithelial tissue origin. It is widely accepted that the presence of hamartomatous polyps in PJS does not in itself lead to the development of malignancy. The signalling mechanisms that lead to these PJS related malignancies are not well understood. However, it is evident from the recent literature that LKB1 is a multitasking kinase, with unlimited potential in orchestrating cell activity. Thus far, LKB1 has been found to play a role in chromatin remodelling, cell cycle arrest, Wnt signalling, cell polarity, and energy metabolism, all of which may require the tumour suppressor function of this kinase and/or its catalytic activity.

  2. Mevalonate kinase deficiency: current perspectives

    PubMed Central

    Favier, Leslie A; Schulert, Grant S

    2016-01-01

    Mevalonate kinase deficiency (MKD) is a recessively inherited autoinflammatory disorder with a spectrum of manifestations, including the well-defined clinical phenotypes of hyperimmunoglobulinemia D and periodic fever syndrome and mevalonic aciduria. Patients with MKD have recurrent attacks of hyperinflammation associated with fever, abdominal pain, arthralgias, and mucocutaneous lesions, and more severely affected patients also have dysmorphisms and central nervous system anomalies. MKD is caused by mutations in the gene encoding mevalonate kinase, with the degree of residual enzyme activity largely determining disease severity. Mevalonate kinase is essential for the biosynthesis of nonsterol isoprenoids, which mediate protein prenylation. Although the precise pathogenesis of MKD remains unclear, increasing evidence suggests that deficiency in protein prenylation leads to innate immune activation and systemic hyperinflammation. Given the emerging understanding of MKD as an autoinflammatory disorder, recent treatment approaches have largely focused on cytokine-directed biologic therapy. Herein, we review the current genetic and pathologic understanding of MKD, its various clinical phenotypes, and the evolving treatment approach for this multifaceted disorder. PMID:27499643

  3. let-7 Overexpression Leads to an Increased Fraction of Cells in G2/M, Direct Down-regulation of Cdc34, and Stabilization of Wee1 Kinase in Primary Fibroblasts*S⃞

    PubMed Central

    Legesse-Miller, Aster; Elemento, Olivier; Pfau, Sarah J.; Forman, Joshua J.; Tavazoie, Saeed; Coller, Hilary A.

    2009-01-01

    microRNAs play a critically important role in a wide array of biological processes including those implicated in cancer, neuro-degenerative and metabolic disorders, and viral infection. Although we have begun to understand microRNA biogenesis and function, experimental demonstration of their functional effects and the molecular mechanisms by which they function remains a challenge. Members of the let-7/miR-98 family play a critical role in cell cycle control with respect to differentiation and tumorigenesis. In this study, we show that exogenous addition of pre-let-7 in primary human fibroblasts results in a decrease in cell number and an increased fraction of cells in the G2/M cell cycle phase. Combining microarray techniques with DNA sequence analysis to identify potential let-7 targets, we discovered 838 genes with a let-7 binding site in their 3′-untranslated region that were down-regulated upon overexpression of let-7b. Among these genes is cdc34, the ubiquitin-conjugating enzyme of the Skp1/cullin/F-box (SCF) complex. Cdc34 protein levels are strongly down-regulated by let-7 overexpression. Reporter assays demonstrated direct regulation of the cdc34 3′-untranslated region by let-7. We hypothesized that low Cdc34 levels would result in decreased SCF activity, stabilization of the SCF target Wee1, and G2/M accumulation. Consistent with this hypothesis, small interfering RNA-mediated down-regulation of Wee1 reversed the G2/M phenotype induced by let-7 overexpression. We conclude that Cdc34 is a functional target of let-7 and that let-7 induces down-regulation of Cdc34, stabilization of the Wee1 kinase, and an increased fraction of cells in G2/M in primary fibroblasts. PMID:19126550

  4. P7170: A Novel Molecule with Unique Profile of mTORC1/C2 and Activin Receptor-like Kinase 1 Inhibition Leading to Antitumor and Antiangiogenic Activity.

    PubMed

    Jalota-Badhwar, Archana; Bhatia, Dimple R; Boreddy, Srinivas; Joshi, Asavari; Venkatraman, Magesh; Desai, Nikesh; Chaudhari, Sarika; Bose, Julie; Kolla, Lakshmi S; Deore, Vijaykumar; Yewalkar, Nilambari; Kumar, Sanjay; Sharma, Rajiv; Damre, Anagha; More, Avinash; Sharma, Somesh; Agarwal, Veena R

    2015-05-01

    The mTOR pathway is often upregulated in cancer and thus intensively pursued as a target to design novel anticancer therapies. Approved and emerging drugs targeting the mTOR pathway have positively affected the clinical landscape. Recently, activin receptor-like kinase 1 (ALK1), belonging to the TGFβ receptor family, has been reported as an emerging target for antiangiogenic cancer therapy. Here, we describe a novel orally efficacious compound, P7170, that inhibits mTORC1/mTORC2/ALK1 activity with a potent cell growth inhibition. In cell-based assays, P7170 strongly inhibited (IC50 < 10 nmol/L) the phosphorylation of p70S6K (T389) and pAKT (S473). In many cancer cell lines, such as prostate, ovarian, colon, and renal, P7170 treatment resulted in marked cell growth inhibition. Furthermore, it induced G1-S cell-cycle arrest and autophagy. In vitro HUVEC tube formation, in vivo Matrigel plug, and rat aorta ring assays demonstrated that P7170 exhibited significant antiangiogenic activity. In addition, ALK1 knockdown studies in HUVEC confirmed that the antiangiogenic activity of P7170 was primarily due to ALK1 inhibition. Strong inhibition of ALK1 in addition to mTORC1/mTORC2 differentiates P7170 in its mechanism of action in comparison with existing inhibitors. In vivo mouse xenograft studies revealed P7170 to exhibit a significant dose-dependent tumor growth inhibition in a broad range of human tumor types when administered orally at 10 to 20 mg/kg doses. The distinctive pharmacological profile with favorable pharmacokinetic parameters and in vivo efficacy makes P7170 an attractive candidate for clinical development. It is currently being tested in phase I clinical studies.

  5. Receptor Tyrosine Kinase and Tyrosine Kinase Inhibitors

    PubMed Central

    Mirshafiey, Abbas; Ghalamfarsa, Ghasem; Asghari, Babak

    2014-01-01

    Receptor tyrosine kinases (RTKs) are essential components of signal transduction pathways that mediate cell-to-cell communication and their function as relay points for signaling pathways. They have a key role in numerous processes that control cellular proliferation and differentiation, regulate cell growth and cellular metabolism, and promote cell survival and apoptosis. Recently, the role of RTKs including TCR, FLT-3, c-Kit, c-Fms, PDGFR, ephrin, neurotrophin receptor, and TAM receptor in autoimmune disorder, especially rheumatoid arthritis and multiple sclerosis has been suggested. In multiple sclerosis pathogenesis, RTKs and their tyrosine kinase enzymes are selective important targets for tyrosine kinase inhibitor (TKI) agents. TKIs, compete with the ATP binding site of the catalytic domain of several tyrosine kinases, and act as small molecules that have a favorable safety profile in disease treatment. Up to now, the efficacy of TKIs in numerous animal models of MS has been demonstrated, but application of these drugs in human diseases should be tested in future clinical trials. PMID:25337443

  6. Tyrosine Kinase Inhibition: An Approach to Drug Development

    NASA Astrophysics Data System (ADS)

    Levitzki, Alexander; Gazit, Aviv

    1995-03-01

    Protein tyrosine kinases (PTKs) regulate cell proliferation, cell differentiation, and signaling processes in the cells of the immune system. Uncontrolled signaling from receptor tyrosine kinases and intracellular tyrosine kinases can lead to inflammatory responses and to diseases such as cancer, atherosclerosis, and psoriasis. Thus, inhibitors that block the activity of tyrosine kinases and the signaling pathways they activate may provide a useful basis for drug development. This article summarizes recent progress in the development of PTK inhibitors and demonstrates their potential use in the treatment of disease.

  7. Non-ATP competitive protein kinase inhibitors.

    PubMed

    Garuti, L; Roberti, M; Bottegoni, G

    2010-01-01

    Protein kinases represent an attractive target in oncology drug discovery. Most of kinase inhibitors are ATP-competitive and are called type I inhibitors. The ATP-binding pocket is highly conserved among members of the kinase family and it is difficult to find selective agents. Moreover, the ATP-competitive inhibitors must compete with high intracellular ATP levels leading to a discrepancy between IC50s measured by biochemical versus cellular assays. The non-ATP competitive inhibitors, called type II and type III inhibitors, offer the possibility to overcome these problems. These inhibitors act by inducing a conformational shift in the target enzyme such that the kinase is no longer able to function. In the DFG-out form, the phenylalanine side chain moves to a new position. This movement creates a hydrophobic pocket available for occupation by the inhibitor. Some common features are present in these inhibitors. They contain a heterocyclic system that forms one or two hydrogen bonds with the kinase hinge residue. They also contain a hydrophobic moiety that occupies the pocket formed by the shift of phenylalanine from the DFG motif. Moreover, all the inhibitors bear a hydrogen bond donor-acceptor pair, usually urea or amide, that links the hinge-binding portion to the hydrophobic moiety and interacts with the allosteric site. Examples of non ATP-competitive inhibitors are available for various kinases. In this review small molecules capable of inducing the DFG-out conformation are reported, especially focusing on structural feature, SAR and biological properties.

  8. Kinase active Misshapen regulates Notch signaling in Drosophila melanogaster.

    PubMed

    Mishra, Abhinava K; Sachan, Nalani; Mutsuddi, Mousumi; Mukherjee, Ashim

    2015-11-15

    Notch signaling pathway represents a principal cellular communication system that plays a pivotal role during development of metazoans. Drosophila misshapen (msn) encodes a protein kinase, which is related to the budding yeast Ste20p (sterile 20 protein) kinase. In a genetic screen, using candidate gene approach to identify novel kinases involved in Notch signaling, we identified msn as a novel regulator of Notch signaling. Data presented here suggest that overexpression of kinase active form of Msn exhibits phenotypes similar to Notch loss-of-function condition and msn genetically interacts with components of Notch signaling pathway. Kinase active form of Msn associates with Notch receptor and regulate its signaling activity. We further show that kinase active Misshapen leads to accumulation of membrane-tethered form of Notch. Moreover, activated Msn also depletes Armadillo and DE-Cadherin from adherens junctions. Thus, this study provides a yet unknown mode of regulation of Notch signaling by Misshapen. PMID:26431585

  9. Lead Toxicity

    MedlinePlus

    ... homes. • Most people, especially children, who suffer from lead poisoning are exposed through lead-contaminated household dust or ... and six if they are at risk of lead poisoning (see: ). Who can I call to get more ...

  10. RAF protein-serine/threonine kinases: Structure and regulation

    SciTech Connect

    Roskoski, Robert

    2010-08-27

    Research highlights: {yields} The formation of unique side-to-side RAF dimers is required for full kinase activity. {yields} RAF kinase inhibitors block MEK activation in cells containing oncogenic B-RAF. {yields} RAF kinase inhibitors can lead to the paradoxical increase in RAF kinase activity. -- Abstract: A-RAF, B-RAF, and C-RAF are a family of three protein-serine/threonine kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, differentiation, proliferation, and transformation to the cancerous state. RAS mutations occur in 15-30% of all human cancers, and B-RAF mutations occur in 30-60% of melanomas, 30-50% of thyroid cancers, and 5-20% of colorectal cancers. Activation of the RAF kinases requires their interaction with RAS-GTP along with dephosphorylation and also phosphorylation by SRC family protein-tyrosine kinases and other protein-serine/threonine kinases. The formation of unique side-to-side RAF dimers is required for full kinase activity. RAF kinase inhibitors are effective in blocking MEK1/2 and ERK1/2 activation in cells containing the oncogenic B-RAF Val600Glu activating mutation. RAF kinase inhibitors lead to the paradoxical increase in RAF kinase activity in cells containing wild-type B-RAF and wild-type or activated mutant RAS. C-RAF plays a key role in this paradoxical increase in downstream MEK-ERK activation.

  11. KLIFS: a knowledge-based structural database to navigate kinase-ligand interaction space.

    PubMed

    van Linden, Oscar P J; Kooistra, Albert J; Leurs, Rob; de Esch, Iwan J P; de Graaf, Chris

    2014-01-23

    Protein kinases regulate the majority of signal transduction pathways in cells and have become important targets for the development of designer drugs. We present a systematic analysis of kinase-ligand interactions in all regions of the catalytic cleft of all 1252 human kinase-ligand cocrystal structures present in the Protein Data Bank (PDB). The kinase-ligand interaction fingerprints and structure database (KLIFS) contains a consistent alignment of 85 kinase ligand binding site residues that enables the identification of family specific interaction features and classification of ligands according to their binding modes. We illustrate how systematic mining of kinase-ligand interaction space gives new insights into how conserved and selective kinase interaction hot spots can accommodate the large diversity of chemical scaffolds in kinase ligands. These analyses lead to an improved understanding of the structural requirements of kinase binding that will be useful in ligand discovery and design studies.

  12. ATP-competitive inhibitors block protein kinase recruitment to the Hsp90-Cdc37 system.

    PubMed

    Polier, Sigrun; Samant, Rahul S; Clarke, Paul A; Workman, Paul; Prodromou, Chrisostomos; Pearl, Laurence H

    2013-05-01

    Protein kinase clients are recruited to the Hsp90 molecular chaperone system via Cdc37, which simultaneously binds Hsp90 and kinases and regulates the Hsp90 chaperone cycle. Pharmacological inhibition of Hsp90 in vivo results in degradation of kinase clients, with a therapeutic effect in dependent tumors. We show here that Cdc37 directly antagonizes ATP binding to client kinases, suggesting a role for the Hsp90-Cdc37 complex in controlling kinase activity. Unexpectedly, we find that Cdc37 binding to protein kinases is itself antagonized by ATP-competitive kinase inhibitors, including vemurafenib and lapatinib. In cancer cells, these inhibitors deprive oncogenic kinases such as B-Raf and ErbB2 of access to the Hsp90-Cdc37 complex, leading to their degradation. Our results suggest that at least part of the efficacy of ATP-competitive inhibitors of Hsp90-dependent kinases in tumor cells may be due to targeted chaperone deprivation.

  13. Lead Poisoning

    MedlinePlus

    ... can be found in all parts of our environment. Much of it comes from human activities such as mining and manufacturing. Lead used to be in paint; older houses may still have lead paint. You could be exposed to lead by Eating food or drinking water that contains lead. Water pipes in older homes ...

  14. Lead poisoning

    SciTech Connect

    Rekus, J.F.

    1992-08-01

    Construction workers who weld, cut or blast structural steel coated with lead-based paint are at significant risk of lead poisoning. Although technology to control these exposures may not have existed when the lead standard was promulgated, it is available today. Employers who do not take steps to protect their employees from lead exposure may be cited and fined severely for their failure.

  15. Validation of in vivo pharmacodynamic activity of a novel PDGF receptor tyrosine kinase inhibitor using immunohistochemistry and quantitative image analysis.

    PubMed

    D'Andrea, Michael R; Mei, Jay M; Tuman, Robert W; Galemmo, Robert A; Johnson, Dana L

    2005-08-01

    With the advent of agents directed against specific molecular targets in drug discovery, it has become imperative to show a compound's cellular impact on the intended biomolecule in vivo. The objective of the present study was to determine if we could develop an assay to validate the in vivo effects of a compound. Hence, we investigated the in vivo pharmacodynamic activity of JNJ-10198409, a relatively selective inhibitor of platelet-derived growth factor receptor tyrosine kinase (PDGF-RTK), in tumor tissues after administering the compound orally in a nude mouse xenograft model of human LoVo colon cancer. We developed a novel assay to quantify the in vivo anti-PDGF-RTK activity of the inhibitor in tumor tissue by determining the phosphorylation status of phospholipase Cgamma1 (PLCgamma1), a key downstream cellular molecule in the PDGF-RTK signaling cascade. We used two antibodies, one specific for the total (phosphorylated and unphosphorylated forms) PLCgamma1 (pan-PLCgamma1) and the other, specific for phosphorylated form of PLCgamma1 (ph-PLCgamma1) to immunohistochemically detect their expression in tumor tissues. Computer-assisted image analysis was then used to directly compare the ratio of ph-PLCgamma1 to pan-PLCgamma1 immunolabeling intensities in serial sections (5 mum) of tumors obtained from vehicle- and JNJ-10198409-treated tumor-bearing mice. Our data showed statistically significant, dose-dependent differences in the ph-PLC/pan-PLC ratio among the four treatment groups (vehicle, 25, 50, and 100 mg/kg b.i.d.). These results confirmed this compound's ability to suppress PDGF-RTK downstream signaling in tumor tissues in vivo. In addition to this specific application of this in vivo validation approach to those targets that use PLCgamma as a downstream signaling partner, these methods may also benefit other drug discovery targets. PMID:16093435

  16. Non-degradative Ubiquitination of Protein Kinases

    PubMed Central

    Ball, K. Aurelia; Johnson, Jeffrey R.; Lewinski, Mary K.; Guatelli, John; Verschueren, Erik; Krogan, Nevan J.; Jacobson, Matthew P.

    2016-01-01

    Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well. PMID:27253329

  17. Non-degradative Ubiquitination of Protein Kinases.

    PubMed

    Ball, K Aurelia; Johnson, Jeffrey R; Lewinski, Mary K; Guatelli, John; Verschueren, Erik; Krogan, Nevan J; Jacobson, Matthew P

    2016-06-01

    Growing evidence supports other regulatory roles for protein ubiquitination in addition to serving as a tag for proteasomal degradation. In contrast to other common post-translational modifications, such as phosphorylation, little is known about how non-degradative ubiquitination modulates protein structure, dynamics, and function. Due to the wealth of knowledge concerning protein kinase structure and regulation, we examined kinase ubiquitination using ubiquitin remnant immunoaffinity enrichment and quantitative mass spectrometry to identify ubiquitinated kinases and the sites of ubiquitination in Jurkat and HEK293 cells. We find that, unlike phosphorylation, ubiquitination most commonly occurs in structured domains, and on the kinase domain, ubiquitination is concentrated in regions known to be important for regulating activity. We hypothesized that ubiquitination, like other post-translational modifications, may alter the conformational equilibrium of the modified protein. We chose one human kinase, ZAP-70, to simulate using molecular dynamics with and without a monoubiquitin modification. In Jurkat cells, ZAP-70 is ubiquitinated at several sites that are not sensitive to proteasome inhibition and thus may have other regulatory roles. Our simulations show that ubiquitination influences the conformational ensemble of ZAP-70 in a site-dependent manner. When monoubiquitinated at K377, near the C-helix, the active conformation of the ZAP-70 C-helix is disrupted. In contrast, when monoubiquitinated at K476, near the kinase hinge region, an active-like ZAP-70 C-helix conformation is stabilized. These results lead to testable hypotheses that ubiquitination directly modulates kinase activity, and that ubiquitination is likely to alter structure, dynamics, and function in other protein classes as well.

  18. Lead poisoning and brain cell function

    SciTech Connect

    Goldstein, G.W. Kennedy Institute, Baltimore, MD )

    1990-11-01

    Exposure to excessive amounts of inorganic lead during the toddler years may produce lasting adverse effects upon brain function. Maximal ingestion of lead occurs at an age when major changes are occurring in the density of brain synaptic connections. The developmental reorganization of synapses is, in part, mediated by protein kinases, and these enzymes are particularly sensitive to stimulation by lead. By inappropriately activating specific protein kinases, lead poisoning may disrupt the development of neural networks without producing overt pathological alterations. The blood-brain barrier is another potential vulnerable site for the neurotoxic action of lead. protein kinases appear to regulate the development of brain capillaries and the expression of the blood-brain barrier properties. Stimulation of protein kinase by lead may disrupt barrier development and alter the precise regulation of the neuronal environment that is required for normal brain function. Together, these findings suggest that the sensitivity of protein kinases to lead may in part underlie the brain dysfunction observed in children poisoned by this toxicant.

  19. Lead poisoning.

    PubMed Central

    Landrigan, P J; Todd, A C

    1994-01-01

    Lead poisoning is the most common disease of environmental origin in the United States today. Adult lead poisoning results primarily from exposure by inhalation in the workplace. Pediatric lead poisoning results principally from the ingestion of lead from environmental media, including paint chips, dust, soil, drinking water, ceramics, and medications. Lead is toxic to many organ systems, among them developing erythrocytes, the kidneys, and the nervous system. Lead-induced toxicity to the central nervous system causes delayed development, diminished intelligence, and altered behavior. In young children, this effect has been demonstrated convincingly to occur at blood lead levels between 10 and 20 micrograms per dl. The Centers for Disease Control and Prevention has recommended that a blood lead level of 10 micrograms per dl or higher be considered evidence of increased lead absorption, and the National Academy of Sciences has concurred in that recommendation. Unresolved issues in need of further study include the frequency of screening young children for lead, the question of whether women should be offered screening for lead before conceiving a pregnancy, the role of x-ray fluorescence analysis in assessing lead in bone, and the appropriate legislative response of the United States government to lead-based paint abatement. PMID:7941534

  20. Kinase cascades regulating entry into apoptosis.

    PubMed Central

    Anderson, P

    1997-01-01

    All cells are constantly exposed to conflicting environment cues that signal cell survival or cell death. Survival signals are delivered by autocrine or paracrine factors that actively suppress a default death pathway. In addition to survival factor withdrawal, cell death can be triggered by environmental stresses such as heat, UV light, and hyperosmolarity or by dedicated death receptors (e.g., FAS/APO-1 and tumor necrosis factor [TNF] receptors) that are counterparts of growth factor or survival receptors at the cell surface. One of the ways that cells integrate conflicting exogenous stimuli is by phosphorylation (or dephosphorylation) of cellular constituents by interacting cascades of serine/threonine and tyrosine protein kinases (and phosphatases). Survival factors (e.g., growth factors and mitogens) activate receptor tyrosine kinases and selected mitogen-activated, cyclin-dependent, lipid-activated, nucleic acid-dependent, and cyclic AMP-dependent kinases to promote cell survival and proliferation, whereas environmental stress (or death factors such as FAS/APO-1 ligand and TNF-alpha) activates different members of these kinase families to inhibit cell growth and, under some circumstances, promote apoptotic cell death. Because individual kinase cascades can interact with one another, they are able to integrate conflicting exogenous stimuli and provide a link between cell surface receptors and the biochemical pathways leading to cell proliferation or cell death. PMID:9106363

  1. Lead poisoning

    MedlinePlus

    ... lead is still found in some modern faucets. Soil contaminated by decades of car exhaust or years ... house paint scrapings. Lead is more common in soil near highways and houses. Hobbies involving soldering, stained ...

  2. Salicylic acid activates a 48-kD MAP kinase in tobacco.

    PubMed

    Zhang, S; Klessig, D F

    1997-05-01

    The involvement of phosphorylation/dephosphorylation in the salicylic acid (SA) signal transduction pathway leading to pathogenesis-related gene induction has previously been demonstrated using kinase and phosphatase inhibitors. Here, we show that in tobacco suspension cells, SA induced a rapid and transient activation of a 48-kD kinase that uses myelin basic protein as a substrate. This kinase is called the p48 SIP kinase (for SA-Induced Protein kinase). Biologically active analogs of SA, which induce pathogenesis-related genes and enhanced resistance, also activated this kinase, whereas inactive analogs did not. Phosphorylation of a tyrosine residue(s) in the SIP kinase was associated with its activation. The SIP kinase was purified to homogeneity from SA-treated tobacco suspension culture cells. The purified SIP kinase is strongly phosphorylated on a tyrosine residue(s), and treatment with either protein tyrosine or serine/threonine phosphatases abolished its activity. Using primers corresponding to the sequences of internal tryptic peptides, we cloned the SIP kinase gene. Analysis of the SIP kinase sequence indicates that it belongs to the MAP kinase family and that it is distinct from the other plant MAP kinases previously implicated in stress responses, suggesting that different members of the MAP kinase family are activated by different stresses.

  3. The erbB-2 mitogenic signaling pathway: tyrosine phosphorylation of phospholipase C-gamma and GTPase-activating protein does not correlate with erbB-2 mitogenic potency.

    PubMed Central

    Fazioli, F; Kim, U H; Rhee, S G; Molloy, C J; Segatto, O; Di Fiore, P P

    1991-01-01

    The erbB-2 gene product, gp185erbB-2, unlike the structurally related epidermal growth factor (EGF) receptor (EGFR), exhibits constitutive kinase and transforming activity. We used a chimeric EGFR/erbB-2 expression vector to compare the mitogenic signaling pathway of the erbB-2 kinase with that of the EGFR, at similar levels of expression, in response to EGF stimulation. The EGFR/erbB-2 chimera was significantly more active in inducing DNA synthesis than the EGFR when either was expressed in NIH 3T3 cells. Analysis of biochemical pathways implicated in signal transduction by growth factor receptors indicated that both phospholipase C type gamma (PLC-gamma) and the p21ras GTPase-activating protein (GAP) are substrates for the erbB-2 kinase in NIH 3T3 fibroblasts. However, under conditions in which activation of the erbB-2 kinase induced DNA synthesis at least fivefold more efficiently than the EGFR, the levels of erbB-2- or EGFR-induced tyrosine phosphorylation of PLC-gamma and GAP were comparable. In addition, the stoichiometry of tyrosine phosphorylation of these putative substrates by erbB-2 appeared to be at least an order of magnitude lower than that induced by platelet-derived growth factor receptors at comparable levels of mitogenic potency. Thus, our results indicate that differences in tyrosine phosphorylation of PLC-gamma and GAP do not account for the differences in mitogenic activity of the erbB-2 kinase compared with either the EGFR or platelet-derived growth factor receptor in NIH 3T3 fibroblasts. Images PMID:1672440

  4. Feedback Regulation of Kinase Signaling Pathways by AREs and GREs

    PubMed Central

    Vlasova-St. Louis, Irina; Bohjanen, Paul R.

    2016-01-01

    In response to environmental signals, kinases phosphorylate numerous proteins, including RNA-binding proteins such as the AU-rich element (ARE) binding proteins, and the GU-rich element (GRE) binding proteins. Posttranslational modifications of these proteins lead to a significant changes in the abundance of target mRNAs, and affect gene expression during cellular activation, proliferation, and stress responses. In this review, we summarize the effect of phosphorylation on the function of ARE-binding proteins ZFP36 and ELAVL1 and the GRE-binding protein CELF1. The networks of target mRNAs that these proteins bind and regulate include transcripts encoding kinases and kinase signaling pathways (KSP) components. Thus, kinase signaling pathways are involved in feedback regulation, whereby kinases regulate RNA-binding proteins that subsequently regulate mRNA stability of ARE- or GRE-containing transcripts that encode components of KSP. PMID:26821046

  5. Phosphatidylinositol kinase from rabbit reticulocytes

    SciTech Connect

    Tuazon, P.T.; Heng, A.B.W.; Traugh, J.A.

    1986-05-01

    Phosphatidylinositol (PI) kinase was isolated from the postribosomal supernatant of rabbit reticulocytes. This activity was identified by the formation of a product that comigrated with phosphatidylinositol-4-phosphate (PIP) when purified PI was phosphorylated in the presence of (/sup 32/P)ATP and Mg/sup 2 +/. Three major peaks of PI kinase activity were resolved by chromatography on DEAE-cellulose. The first peak eluted at 50-100 mM NaCl together with several serine protein kinases, casein kinase (CK) I and protease activated kinase (PAK) I and II. The PI kinase was subsequently separated from the protein kinases by chromatography on phosphocellulose. The second peak eluted at 125-160 mM NaCl and contained another lipid kinase activity that produced a product which comigrated with phosphatidic acid on thin layer chromatography. The third peak, which eluted at 165-200 mM NaCl, partly comigrated with casein kinase (CK) II and an active protein kinase(s) which phosphorylated mixed histone and histone I. CK II and the histone kinase activities were also separated by chromatography on phosphocelluslose. The different forms of PI kinase were characterized and compared with respect to substrate and salt requirements.

  6. Microbial Protein-tyrosine Kinases*

    PubMed Central

    Chao, Joseph D.; Wong, Dennis; Av-Gay, Yossef

    2014-01-01

    Microbial ester kinases identified in the past 3 decades came as a surprise, as protein phosphorylation on Ser, Thr, and Tyr amino acids was thought to be unique to eukaryotes. Current analysis of available microbial genomes reveals that “eukaryote-like” protein kinases are prevalent in prokaryotes and can converge in the same signaling pathway with the classical microbial “two-component” systems. Most microbial tyrosine kinases lack the “eukaryotic” Hanks domain signature and are designated tyrosine kinases based upon their biochemical activity. These include the tyrosine kinases termed bacterial tyrosine kinases (BY-kinases), which are responsible for the majority of known bacterial tyrosine phosphorylation events. Although termed generally as bacterial tyrosine kinases, BY-kinases can be considered as one family belonging to the superfamily of prokaryotic protein-tyrosine kinases in bacteria. Other members of this superfamily include atypical “odd” tyrosine kinases with diverse mechanisms of protein phosphorylation and the “eukaryote-like” Hanks-type tyrosine kinases. Here, we discuss the distribution, phylogeny, and function of the various prokaryotic protein-tyrosine kinases, focusing on the recently discovered Mycobacterium tuberculosis PtkA and its relationship with other members of this diverse family of proteins. PMID:24554699

  7. Visualizing autophosphorylation in histidine kinases.

    PubMed

    Casino, Patricia; Miguel-Romero, Laura; Marina, Alberto

    2014-01-01

    Reversible protein phosphorylation is the most widespread regulatory mechanism in signal transduction. Autophosphorylation in a dimeric sensor histidine kinase is the first step in two-component signalling, the predominant signal-transduction device in bacteria. Despite being the most abundant sensor kinases in nature, the molecular bases of the histidine kinase autophosphorylation mechanism are still unknown. Furthermore, it has been demonstrated that autophosphorylation can occur in two directions, cis (intrasubunit) or trans (intersubunit) within the dimeric histidine kinase. Here, we present the crystal structure of the complete catalytic machinery of a chimeric histidine kinase. The structure shows an asymmetric histidine kinase dimer where one subunit is caught performing the autophosphorylation reaction. A structure-guided functional analysis on HK853 and EnvZ, two prototypical cis- and trans-phosphorylating histidine kinases, has allowed us to decipher the catalytic mechanism of histidine kinase autophosphorylation, which seems to be common independently of the reaction directionality.

  8. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Linn, Anning

    1996-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK.

  9. Plant 5-Methylthioribose Kinase

    PubMed Central

    Guranowski, Andrzej

    1983-01-01

    Activity of 5-methylthioribose kinase, the enzyme which catalyzes the ATP-dependent formation of 1-phospho-5-methylthioribose, has been revealed in the extracts from various higher plant species. Almost 2,000-fold-purified enzyme has been obtained from yellow lupin (Lupinus luteus L. cv Topaz) seed extract. Molecular weight of the native enzyme is 70,000 as judged by gel filtration. The lupin 5-methylthioribose kinase exhibits a strict requirement for divalent metal ions. Among the ions tested, only Mg2+ and Mn2+ acted as cofactors. The curve of kinase initial velocity versus pH reaches plateau at pH 10 to 10.5. The Km values calculated for 5-methylthioribose and ATP are 4.3 and 8.3 micromolar, respectively. Among nucleoside triphosphates tested as potential phosphate donors, only dATP could substitute in the reaction for ATP. 5-Isobutylthioribose, an analog of 5-methylthioribose, proved to be the γ-ATP-phosphate acceptor, too. The compound inhibits competitively synthesis of 1-phospho-5-methylthioribose (Ki = 1.4 micromolar). Lupin 5-methylthioribose kinase is completely and irreversibly inhibited by the antisulfhydryl reagent, p-hydroxymercuribenzoate. As in bacteria (Ferro, Barrett, Shapiro 1978 J Biol Chem 253: 6021-6025), the enzyme may be involved in a new, alternative pathway of methionine synthesis in plant tissues. PMID:16662931

  10. Leading Democratically

    ERIC Educational Resources Information Center

    Brookfield, Stephen

    2010-01-01

    Democracy is the most venerated of American ideas, the one for which wars are fought and people die. So most people would probably agree that leaders should be able to lead well in a democratic society. Yet, genuinely democratic leadership is a relative rarity. Leading democratically means viewing leadership as a function or process, rather than…

  11. Novel cinnoline-based inhibitors of LRRK2 kinase activity.

    PubMed

    Garofalo, Albert W; Adler, Marc; Aubele, Danielle L; Bowers, Simeon; Franzini, Maurizio; Goldbach, Erich; Lorentzen, Colin; Neitz, R Jeffrey; Probst, Gary D; Quinn, Kevin P; Santiago, Pam; Sham, Hing L; Tam, Danny; Truong, Anh P; Ye, Xiaocong M; Ren, Zhao

    2013-01-01

    Leucine rich repeat kinase 2 (LRRK2) has been implicated in the pathogenesis of Parkinson's disease (PD). Inhibition of LRRK2 kinase activity is a therapeutic approach that may lead to new treatments for PD. Herein we report the discovery of a series of cinnoline-3-carboxamides that are potent against both wild-type and mutant LRRK2 kinase activity in biochemical assays. These compounds are also shown to be potent inhibitors in a cellular assay and to have good to excellent CNS penetration. PMID:23219325

  12. Choline phosphate potentiates sphingosine-1-phosphate-induced Raf-1 kinase activation dependent of Ras--phosphatidylinositol-3-kinase pathway.

    PubMed

    Lee, Michael; Han, Sang Seop

    2002-04-01

    In NIH3T3 cells, sphingosine-1-phosphate (S1P) caused a significant increase of Raf-1 kinase activity as early as 2 min. Interestingly, choline phosphate (ChoP) produced synergistic increase of S1P-stimulated Raf-1 kinase activation in the presence of ATP while showing additive effect in the absence of ATP. However, Raf-1 kinase activation induced by S1P decreased in the presence of ATP when applied alone. The overexpression of N-terminal fragment of Raf-1 (RfI) to inhibit Raf--Ras interaction caused the inhibition of S1P-induced Raf-1 kinase activation. Also, wortmannin, phosphatidylinositol-3-kinase (PI3K) inhibitor, exhibited inhibitory effects on S1P-induced activation of Raf-1 kinase. In addition, we demonstrated that the chemical antioxidant, N-acetylcysteine attenuated Raf-1 activation induced by S1P, suggesting that H(2)O(2) may be required for the signalling pathway leading to Raf-1 activation. This H(2)O(2)-induced Raf-1 kinase activation was also blocked by inhibition of Ras--PI3K signalling pathway using alpha-hydroxyfarnesylphosphonic acid and wortmannin. Taken together, these results indicate that S1P-induced Raf-1 kinase activation is mediated by H(2)O(2) stimulation of Ras--PI3K pathway, and is enhanced by ChoP in the presence of ATP.

  13. LEAD STUDIES

    PubMed Central

    Aub, Joseph C.; Reznikoff, Paul; Smith, Dorothea E.

    1924-01-01

    It appears, from the investigations in other laboratories, that the anemia observed in cases of lead poisoning is due to destruction of blood rather than to diminished production of blood. The method of poisoning cells in vitro with lead was adopted in order to study this phenomenon, and distinct effects were thereby obtained, even when only 0.001 mg. of lead is added to approximately 5 billion washed red corpuscles. In order to obtain optimum results the usual dosage employed was ten times this or 0.01 mg. per 5 billion cells. The following changes were observed in cells so treated. 1. Such a marked increase in the resistance to hypotonic salt solution develops that complete hemolysis does not occur until the cells are exposed to a saline solution of 0.05 per cent. Untreated cells are completely hemolyzed in 0.25 or 0.225 per cent saline. 2. This reaction is quantitative and varies with the concentration of lead used. Under the conditions of our experiments this phenomenon seems to be unique. The effects of arsenic are very slight in comparison. 3. While from this reaction it may be concluded that lead increases cellular resistance, it also appears that it shortens the life of blood cells. This may be demonstrated by the much more rapid appearance of hemolysis than normal when the cells are merely allowed to stand in Ringer solution of any dilution. 4. In rabbits with acute lead poisoning these same phenomena may be noted in vivo. 5. Both phenomena may be changed in vitro by varying the time and temperature of the reaction and the concentration of lead, as Fici has already pointed out. 6. If normal cells stand in Ringer solution for 6 hours something diffuses into the solution which largely reduces the action of lead. After repeated washing these cells react with lead in the usual manner. 7. Small amounts of serum react with lead and eliminate its effects. Red blood cells, treated with a mixture of lead and blood serum, show normal hemolysis in hypotonic salt

  14. Designing novel kinases using evolutionary sequence analysis

    NASA Astrophysics Data System (ADS)

    Mody, Areez; Weiner, Joan; Iyer, Lakshman; Ramanathan, Sharad

    2006-03-01

    Cellular pathways with new functions are thought to arise from the duplication and divergence of proteins in existing pathways. The MAP kinase pathways in eukaryotes provide one example of this. These pathways consist of the MAP kinase proteins which are responsible for evoking the correct response to external stimuli. In the yeast Saccharomyces cerevisiae these pathways detect pheromones, osmolar stresses and nutrient levels, leading the cell into dramatic changes of morphology. Despite being homologous to each other, the MAP kinase proteins show specificity of function. We investigate the nature of the amino acid sequences conferring this specificity. To this end, we i) search the sequences of similar proteins in other Eukaryote species, ii) make a study of simple theoretical models exploring the constraints felt by these protein segments and iii) experimentally construct, a large suite of hybrid proteins made of segments taken from the homologous proteins. These are then expressed in Yeast cells to see what function they are able to perform. Particularly we also ask whether it is possible to design a new kinase protein possessing new function and specificity.

  15. Mycobacterium tuberculosis Serine/Threonine Protein Kinases

    PubMed Central

    PRISIC, SLADJANA; HUSSON, ROBERT N.

    2014-01-01

    The Mycobacterium tuberculosis genome encodes 11 serine/threonine protein kinases (STPKs). A similar number of two-component systems are also present, indicating that these two signal transduction mechanisms are both important in the adaptation of this bacterial pathogen to its environment. The M. tuberculosis phosphoproteome includes hundreds of Ser- and Thr-phosphorylated proteins that participate in all aspects of M. tuberculosis biology, supporting a critical role for the STPKs in regulating M. tuberculosis physiology. Nine of the STPKs are receptor type kinases, with an extracytoplasmic sensor domain and an intracellular kinase domain, indicating that these kinases transduce external signals. Two other STPKs are cytoplasmic and have regulatory domains that sense changes within the cell. Structural analysis of some of the STPKs has led to advances in our understanding of the mechanisms by which these STPKs are activated and regulated. Functional analysis has provided insights into the effects of phosphorylation on the activity of several proteins, but for most phosphoproteins the role of phosphorylation in regulating function is unknown. Major future challenges include characterizing the functional effects of phosphorylation for this large number of phosphoproteins, identifying the cognate STPKs for these phosphoproteins, and determining the signals that the STPKs sense. Ultimately, combining these STPK-regulated processes into larger, integrated regulatory networks will provide deeper insight into M. tuberculosis adaptive mechanisms that contribute to tuberculosis pathogenesis. Finally, the STPKs offer attractive targets for inhibitor development that may lead to new therapies for drug-susceptible and drug-resistant tuberculosis. PMID:25429354

  16. Oncoprotein protein kinase

    DOEpatents

    Karin, M.; Hibi, M.; Lin, A.

    1997-02-25

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE is disclosed. The polypeptide has serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences. The method of detection of JNK is also provided. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites. 44 figs.

  17. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2004-03-16

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  18. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2003-02-04

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  19. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1998-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  20. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1999-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  1. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; Davis, Roger; Derijard, Benoit

    2005-03-08

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  2. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  3. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  4. Oncoprotein protein kinase

    DOEpatents

    Davis, Roger; Derijard, Benoit; Karin, Michael; Hibi, Masahiko; Lin, Anning

    2005-01-25

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  5. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Lin, Anning

    1999-11-30

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD or 55 kD as determined by reducing SDS-PAGE, having serine and theonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  6. The molecular basis of targeting protein kinases in cancer therapeutics.

    PubMed

    Tsai, Chung-Jung; Nussinov, Ruth

    2013-08-01

    In this paper, we provide an overview of targeted anticancer therapies with small molecule kinase inhibitors. First, we discuss why a single constitutively active kinase emanating from a variety of aberrant genetic alterations is capable of transforming a normal cell, leading it to acquire the hallmarks of a cancer cell. To draw attention to the fact that kinase inhibition in targeted cancer therapeutics differs from conventional cytotoxic chemotherapy, we exploit a conceptual framework explaining why suppressed kinase activity will selectively kill only the so-called oncogene 'addicted' cancer cell, while sparing the healthy cell. Second, we introduce the protein kinase superfamily in light of its common active conformation with precisely positioned structural elements, and the diversified auto-inhibitory conformations among the kinase families. Understanding the detailed activation mechanism of individual kinases is essential to relate the observed oncogenic alterations to the elevated constitutively active state, to identify the mechanism of consequent drug resistance, and to guide the development of the next-generation inhibitors. To clarify the vital importance of structural guidelines in studies of oncogenesis, we explain how somatic mutations in EGFR result in kinase constitutive activation. Third, in addition to the common theme of secondary (acquired) mutations that prevent drug binding from blocking a signaling pathway which is hijacked by the aberrant activated kinase, we discuss scenarios of drug resistance and relapse by compensating lesions that bypass the inactivated pathway in a vertical or horizontal fashion. Collectively, these suggest that the future challenge of cancer therapy with small molecule kinase inhibitors will rely on the discovery of distinct combinations of optimized drugs to target individual subtypes of different cancers.

  7. Cyclin-dependent kinases.

    PubMed

    Malumbres, Marcos

    2014-01-01

    Cyclin-dependent kinases (CDKs) are protein kinases characterized by needing a separate subunit - a cyclin - that provides domains essential for enzymatic activity. CDKs play important roles in the control of cell division and modulate transcription in response to several extra- and intracellular cues. The evolutionary expansion of the CDK family in mammals led to the division of CDKs into three cell-cycle-related subfamilies (Cdk1, Cdk4 and Cdk5) and five transcriptional subfamilies (Cdk7, Cdk8, Cdk9, Cdk11 and Cdk20). Unlike the prototypical Cdc28 kinase of budding yeast, most of these CDKs bind one or a few cyclins, consistent with functional specialization during evolution. This review summarizes how, although CDKs are traditionally separated into cell-cycle or transcriptional CDKs, these activities are frequently combined in many family members. Not surprisingly, deregulation of this family of proteins is a hallmark of several diseases, including cancer, and drug-targeted inhibition of specific members has generated very encouraging results in clinical trials. PMID:25180339

  8. Ecotoxicology: Lead

    USGS Publications Warehouse

    Scheuhammer, A.M.; Beyer, W.N.; Schmitt, C.J.; Jorgensen, Sven Erik; Fath, Brian D.

    2008-01-01

    Lead (Pb) is a naturally occurring metallic element; trace concentrations are found in all environmental media and in all living things. However, certain human activities, especially base metal mining and smelting; combustion of leaded gasoline; the use of Pb in hunting, target shooting, and recreational angling; the use of Pb-based paints; and the uncontrolled disposal of Pb-containing products such as old vehicle batteries and electronic devices have resulted in increased environmental levels of Pb, and have created risks for Pb exposure and toxicity in invertebrates, fish, and wildlife in some ecosystems.

  9. Tetraethyl lead

    Integrated Risk Information System (IRIS)

    Tetraethyl lead ; CASRN 78 - 00 - 2 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  10. Effects of lead on vascular reactivity.

    PubMed Central

    Chai, S S; Webb, R C

    1988-01-01

    Considerable controversy exists concerning the possible role of lead in the etiology of human hypertension. In animal studies, there is convincing evidence that lead alters cardiovascular responsiveness; rats drinking water containing 100 ppm lead develop a chronic, significant 15 to 20 mm Hg elevation in systolic blood pressure. Pressor responsiveness to catecholamines is enhanced in animals chronically exposed to lead, and the responsiveness of isolated vascular smooth muscle to adrenergic agonists is increased in rats with lead-induced hypertension. Experimental evidence suggests that alterations in the cellular mechanisms that regulate intracellular calcium concentration may contribute to the abnormal vascular function in lead-induced hypertension. Recent work in our laboratory indicates that increased vascular reactivity in genetic hypertension is associated with altered activity of the protein kinase C branch of the calcium messenger system. Contractile responses to lead in rabbit mesenteric artery are potentiated by activators (phorbol esters) of this enzyme complex, and a selective inhibitor of protein kinase C inhibited contractions induced by lead. Based on these results, it is proposed that a cellular component of the action of lead to increase vascular reactivity may relate to the role of protein kinase C in smooth muscle contraction. PMID:3060355

  11. Characterization of Novel Src Family Kinase Inhibitors to Attenuate Microgliosis

    PubMed Central

    Manocha, Gunjan D.; Puig, Kendra L.; Austin, Susan A.; Seyb, Kathleen; Glicksman, Marcie A.; Combs, Colin K.

    2015-01-01

    Microgliosis is a major hallmark of Alzheimer’s disease (AD) brain pathology. Aβ peptide is hypothesized to act as a stimulus for microglia leading to activation of non-receptor tyrosine kinases and subsequent secretion of pro-inflammatory cytokines. Therefore, the signaling pathways mediating microglial activation may be important therapeutic targets of anti-inflammatory therapy for AD. Four novel compounds were chosen after high throughput screening kinase activity assays determined them as potential Lyn kinase inhibitors. Their kinase inhibitory and anti-inflammatory effect on Aβ-stimulated activation was assessed using the murine microglial cell line, BV2. Cells were treated with the compounds to determine effects on active, phosphorylated levels of Src family kinases, Src and Lyn, as well as MAP kinases ERK, JNK and p38. Only one compound, LDDN-0003499, produced a dose dependent decrease in basal levels of active, phosphorylated Src and Lyn in the BV2 cells. LDDN-0003499 treatment also attenuated the Aβ-stimulated increase in active, phosphorylated levels of Lyn/Src and TNFα and IL-6 secretion. This study identifies a novel small molecule Src family tyrosine kinase inhibitor with anti-inflammatory effects in response to Aβ stimulation of microglia. Further in vitro/in vivo characterization of LDDN-0003499 as well as structural modification may provide a new tool for attenuating microglial-mediated brain inflammatory conditions such as that occurring in AD. PMID:26161952

  12. Molecular basis of MAP kinase regulation

    PubMed Central

    Peti, Wolfgang; Page, Rebecca

    2013-01-01

    Mitogen-activated protein kinases (MAPKs; ERK1/2, p38, JNK, and ERK5) have evolved to transduce environmental and developmental signals (growth factors, stress) into adaptive and programmed responses (differentiation, inflammation, apoptosis). Almost 20 years ago, it was discovered that MAPKs contain a docking site in the C-terminal lobe that binds a conserved 13-16 amino acid sequence known as the D- or KIM-motif (kinase interaction motif). Recent crystal structures of MAPK:KIM-peptide complexes are leading to a precise understanding of how KIM sequences contribute to MAPK selectivity. In addition, new crystal and especially NMR studies are revealing how residues outside the canonical KIM motif interact with specific MAPKs and contribute further to MAPK selectivity and signaling pathway fidelity. In this review, we focus on these recent studies, with an emphasis on the use of NMR spectroscopy, isothermal titration calorimetry and small angle X-ray scattering to investigate these processes. PMID:24115095

  13. Map kinases in fungal pathogens.

    PubMed

    Xu, J R

    2000-12-01

    MAP kinases in eukaryotic cells are well known for transducing a variety of extracellular signals to regulate cell growth and differentiation. Recently, MAP kinases homologous to the yeast Fus3/Kss1 MAP kinases have been identified in several fungal pathogens and found to be important for appressorium formation, invasive hyphal growth, and fungal pathogenesis. This MAP kinase pathway also controls diverse growth or differentiation processes, including conidiation, conidial germination, and female fertility. MAP kinases homologous to yeast Slt2 and Hog1 have also been characterized in Candida albicans and Magnaporthe grisea. Mutants disrupted of the Slt2 homologues have weak cell walls, altered hyphal growth, and reduced virulence. The Hog1 homologues are dispensable for growth but are essential for regulating responses to hyperosmotic stress in C. albicans and M. grisea. Overall, recent studies have indicated that MAP kinase pathways may play important roles in regulating growth, differentiation, survival, and pathogenesis in fungal pathogens. PMID:11273677

  14. Detailed search for protein kinase(s) involved in plasma membrane H+-ATPase activity regulation of yeast cells.

    PubMed

    Pereira, Renata R; Castanheira, Diogo; Teixeira, Janaina A; Bouillet, Leoneide E M; Ribeiro, Erica M C; Trópia, Maria M J; Alvarez, Florencia; Correa, Lygia F M; Mota, Bruno E F; Conceição, Luis Eduardo F R; Castro, Ieso M; Brandão, Rogelio L

    2015-03-01

    This study displays a screening using yeast strains deficient in protein kinases known to exist in Saccharomyces cerevisiae. From 95 viable single mutants, 20 mutants appear to be affected in the glucose-induced extracellular acidification. The mutants that are unaffected in calcium signaling were tested for their sensitivity to hygromycin B. Furthermore, we verified whether the remaining mutants produced enzymes that are appropriately incorporated at plasma membrane. Finally, we measure the kinetic properties of the enzyme in purified plasma membranes from glucose-starved as well as glucose-fermenting cells. We confirmed the kinase Ptk2 involvement in H(+)-ATPase regulation (increase of affinity for ATP). However, the identification of the kinase(s) responsible for phosphorylation that leads to an increase in Vmax appears to be more complex. Complementary experiments were performed to check how those protein kinases could be related to the control of the plasma membrane H(+)-ATPase and/or the potential membrane. In summary, our results did not permit us to identify the protein kinase(s) involved in regulating the catalytic efficiency of the plasma membrane H(+)-ATPase. Therefore, our results indicate that the current regulatory model based on the phosphorylation of two different sites located in the C-terminus tail of the enzyme could be inappropriate.

  15. An X-ray structural study of pyruvate dehydrogenase kinase: A eukaryotic serine kinase with a prokaryotic histidine-kinase fold

    NASA Astrophysics Data System (ADS)

    Steussy, Calvin Nicklaus, Jr.

    2001-07-01

    Pyruvate Dehydrogenase Kinase is an enzyme that controls the flow of glucose through the eukaryotic cell and contributes to the pathology of diabetes mellitus. Early work on this kinase demonstrated that it has an amino acid sequence much like bacterial histidine kinases, but an activity similar to that of modern serine/threonine kinases. This project utilized the techniques of X-ray crystallography to determine molecular structure of pyruvate dehydrogenase kinase, isozyme 2. The structure was phased using selenium substituted for sulfur in methionine residues, and data at multiple wavelengths was collected at the National Synchrotron Light Source, Brookhaven National Laboratories. PDK 2 was found to fold into a two-domain monomer that forms a dimer through two beta sheets in the C-terminal domain. The N-terminal domain is an alpha-helical bundle while the C-terminal domain is an alpha/beta sandwich. The fold of the C-terminal domain is very similar to that of the prokaryotic histidine kinases, indicating that they share a common ancestor. The catalytic mechanism, however, has evolved to use general base catalysis to activate the serine substrate, rather than the direct nucleophilic attack by the imidazole sidechain used in the prokaryotic kinases. Thus, the structure of the protein echoes its prokaryotic ancestor, while the chemical mechanism has adapted to a serine substrate. The electrostatic surface of PDK2 leads to the suggestion that the lipoyl domain of the pyruvate dehydrogenase kinase, an important associated structure, may bind in the cleft formed between the N- and C-terminal domains. In addition, a network of hydrogen bonds directly connects the nucleotide binding pocket to the dimer interface, suggesting that there may be some interaction between dimer formation and ATP binding or ADP release.

  16. A conserved isoleucine maintains the inactive state of Bruton’s tyrosine kinase

    PubMed Central

    Boyken, Scott E.; Chopra, Nikita; Xie, Qian; Joseph, Raji E.; Wales, Thomas E.; Fulton, D. Bruce; Engen, John R.; Jernigan, Robert L.; Andreotti, Amy H.

    2014-01-01

    Despite high homology among non-receptor tyrosine kinases, different kinase families employ a diverse array of regulatory mechanisms. For example, the catalytic kinase domains of the Tec family kinases are inactive without assembly of the adjacent regulatory domains, whereas the Src kinase domains are autoinhibited by the assembly of similar adjacent regulatory domains. Using molecular dynamic simulations, biochemical assays, and biophysical approaches, we have uncovered an isoleucine residue in the kinase domain of the Tec family member Btk that, when mutated to the closely related leucine, leads to a shift in the conformational equilibrium of the kinase domain toward the active state. The single amino acid mutation results in measureable catalytic activity for the Btk kinase domain in the absence of the regulatory domains. We suggest this isoleucine side chain in the Tec family kinases acts as a ‘wedge’ that restricts the conformational space available to key regions in the kinase domain, preventing activation until the kinase domain associates with its regulatory subunits and overcomes the energetic barrier to activation imposed by the isoleucine side chain. PMID:25193673

  17. Adenylate kinase complements nucleoside diphosphate kinase deficiency in nucleotide metabolism.

    PubMed Central

    Lu, Q; Inouye, M

    1996-01-01

    Nucleoside diphosphate (NDP) kinase is a ubiquitous nonspecific enzyme that evidently is designed to catalyze in vivo ATP-dependent synthesis of ribo- and deoxyribonucleoside triphosphates from the corresponding diphosphates. Because Escherichia coli contains only one copy of ndk, the structural gene for this enzyme, we were surprised to find that ndk disruption yields bacteria that are still viable. These mutant cells contain a protein with a small amount NDP kinase activity. The protein responsible for this activity was purified and identified as adenylate kinase. This enzyme, also called myokinase, catalyzes the reversible ATP-dependent synthesis of ADP from AMP. We found that this enzyme from E. coli as well as from higher eukaryotes has a broad substrate specificity displaying dual enzymatic functions. Among the nucleoside monophosphate kinases tested, only adenylate kinase was found to have NDP kinase activity. To our knowledge, this is the first report of NDP kinase activity associated with adenylate kinase. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8650159

  18. T cell antigen receptor engagement stimulates c-raf phosphorylation and induces c-raf-associated kinase activity via a protein kinase C-dependent pathway.

    PubMed

    Siegel, J N; Klausner, R D; Rapp, U R; Samelson, L E

    1990-10-25

    The c-raf kinase has been shown to be activated following stimulation of several tyrosine kinase growth factor receptors. We examined changes in c-raf following engagement of the T cell receptor for antigen (TCR), a stimulus which activates both a non-receptor tyrosine kinase and protein kinase C (PKC). We found that activation of the T-cell receptor on the T cell hybridoma 2B4 causes a rapid and stoichiometric hyperphosphorylation of c-raf and an increase in c-raf-associated kinase activity. Phosphoamino acid analysis showed that the phosphorylation was entirely on serine residues. High-resolution phosphopeptide mapping showed the appearance of a single major new phosphopeptide with TCR stimulation. That phosphopeptide was shown to comigrate with the major new phosphopeptide induced in response to phorbol ester. When cells were depleted of PKC by pretreatment with high concentrations of phorbol ester, TCR stimulation was no longer capable of inducing c-raf-associated kinase activity. To determine whether activation of the tyrosine kinase alone would activate c-raf, we examined the 2B4 variant cell line FL.8. In response to Thy-1 stimulation, these cells activate the tyrosine kinase but not protein kinase C due to a deficiency in TCR eta chain expression. We found that in contrast to Thy-1 stimulation of 2B4 cells, stimulation of FL.8 cells does not lead to the induction of c-raf-associated kinase activity, although phorbol ester activates the kinase to an equivalent degree in both cells. We conclude that T cell receptor activation of c-raf occurs via phosphorylation by the serine/threonine kinase PKC. Activation of c-raf through PKC represents a mechanism distinct from that reported for tyrosine kinase growth factor receptors.

  19. Targeting Tyrosine Kinases and Autophagy in Prostate Cancer

    PubMed Central

    2010-01-01

    . Excessive autophagy, sometimes, could lead to type II programmed cell death. We demonstrated that autophagy blockade sensitizes prostate cancer cells toward Src tyrosine kinase inhibitor. Thus, a combination therapy based on Src tyrosine kinase inhibitor and autophagy modulator deserves further attention as a potential treatment for relapsed prostate cancer. PMID:21350583

  20. Conformation-specific inhibitors of Raf kinases.

    PubMed

    Wang, Xiaolun; Schleicher, Kristin

    2013-01-01

    Since the discovery linking B-Raf mutations to human tumors in 2002, significant advances in the development of Raf inhibitors have been made, leading to the recent approval of two Raf inhibitor drugs. This chapter includes a brief introduction to B-Raf as a validated target and focuses on the three different binding modes observed with Raf small-molecule inhibitors. These various binding modes lock the Raf kinase in different conformations that impact the toxicity profiles of the inhibitors. Possible solutions to mitigate the side effects caused by inhibitor-induced dimerization are also discussed.

  1. Focal adhesion kinase

    PubMed Central

    Stone, Rebecca L; Baggerly, Keith A; Armaiz-Pena, Guillermo N; Kang, Yu; Sanguino, Angela M; Thanapprapasr, Duangmani; Dalton, Heather J; Bottsford-Miller, Justin; Zand, Behrouz; Akbani, Rehan; Diao, Lixia; Nick, Alpa M; DeGeest, Koen; Lopez-Berestein, Gabriel; Coleman, Robert L; Lutgendorf, Susan; Sood, Anil K

    2014-01-01

    This investigation describes the clinical significance of phosphorylated focal adhesion kinase (FAK) at the major activating tyrosine site (Y397) in epithelial ovarian cancer (EOC) cells and tumor-associated endothelial cells. FAK gene amplification as a mechanism for FAK overexpression and the effects of FAK tyrosine kinase inhibitor VS-6062 on tumor growth, metastasis, and angiogenesis were examined. FAK and phospho-FAKY397 were quantified in tumor (FAK-T; pFAK-T) and tumor-associated endothelial (FAK-endo; pFAK-endo) cell compartments of EOCs using immunostaining and qRT-PCR. Associations between expression levels and clinical variables were evaluated. Data from The Cancer Genome Atlas were used to correlate FAK gene copy number and expression levels in EOC specimens. The in vitro and in vivo effects of VS-6062 were assayed in preclinical models. FAK-T and pFAK-T overexpression was significantly associated with advanced stage disease and increased microvessel density (MVD). High MVD was observed in tumors with elevated endothelial cell FAK (59%) and pFAK (44%). Survival was adversely affected by FAK-T overexpression (3.03 vs 2.06 y, P = 0.004), pFAK-T (2.83 vs 1.78 y, P < 0.001), and pFAK-endo (2.33 vs 2.17 y, P = 0.005). FAK gene copy number was increased in 34% of tumors and correlated with expression levels (P < 0.001). VS-6062 significantly blocked EOC and endothelial cell migration as well as endothelial cell tube formation in vitro. VS-6062 reduced mean tumor weight by 56% (P = 0.005), tumor MVD by 40% (P = 0.0001), and extraovarian metastasis (P < 0.01) in orthotopic EOC mouse models. FAK may be a unique therapeutic target in EOC given the dual anti-angiogenic and anti-metastatic potential of FAK inhibitors. PMID:24755674

  2. The role of phosphagen specificity loops in arginine kinase

    PubMed Central

    Azzi, Arezki; Clark, Shawn A.; Ellington, W. Ross; Chapman, Michael S.

    2004-01-01

    Phosphagen kinases catalyze the reversible transfer of a phosphate between ATP and guanidino substrates, a reaction that is central to cellular energy homeostasis. Members of this conserved family include creatine and arginine kinases and have similar reaction mechanisms, but they have distinct specificities for different guanidino substrates. There has not been a full structural rationalization of specificity, but two loops have been implicated repeatedly. A small domain loop is of length that complements the size of the guanidino substrate, and is located where it could mediate a lock-and-key mechanism. The second loop contacts the substrate with a valine in the methyl-substituted guanidinium of creatine, and with a glutamate in the unsubstituted arginine substrate, leading to the proposal of a discriminating hydrophobic/hydrophilic minipocket. In the present work, chimeric mutants were constructed with creatine kinase loop elements inserted into arginine kinase. Contrary to the prior rationalizations of specificity, most had measurable arginine kinase activity but no creatine kinase activity or enhanced phosphocreatine binding. Guided by structure, additional mutations were introduced in each loop, recovering arginine kinase activities as high as 15% and 64% of wild type, respectively, even though little activity would be expected in the constructs if the implicated sites had dominant roles in specificity. An atomic structure of the mismatched complex of arginine kinase with creatine and ADP indicates that specificity can also be mediated by an active site that allows substrate prealignment that is optimal for reactivity only with cognate substrates and not with close homologs that bind but do not react. PMID:14978299

  3. A Positive Selection for Nucleoside Kinases in E. coli

    PubMed Central

    Shelat, Nirav Y.; Parhi, Sidhartha; Ostermeier, Marc

    2016-01-01

    Engineering heterologous nucleoside kinases inside E. coli is a difficult process due to the integral role nucleosides play in cell division and transcription. Nucleoside analogs are used in many kinase screens that depend on cellular metabolization of the analogs. However, metabolic activation of these analogs can be toxic through disruptions of DNA replication and transcription because of the analogs’ structural similarities to native nucleosides. Furthermore, the activity of engineered kinases can be masked by endogenous kinases in the cytoplasm, which leads to more difficulties in assessing target activity. A positive selection method that can discern a heterologous kinases’ enzymatic activity without significantly influencing the cell’s normal metabolic systems would be beneficial. We have developed a means to select for a nucleoside kinase’s activity by transporting the kinase to the periplasmic space of an E. coli strain that has its PhoA alkaline phosphatase knocked out. Our proof-of-principle studies demonstrate that the herpes simplex virus thymidine kinase (HSV-TK) can be transported to the periplasmic space in functional form by attaching a tat-signal sequence to the N-terminus of the protein. HSV-TK phosphorylates the toxic nucleoside analog 3’-azido-3’-deoxythymidine (AZT), and this charged, monophosphate form of AZT cannot cross the inner membrane. The translocation of HSV-TK provides significant resistance to AZT when compared to bacteria lacking a periplasmic HSV-TK. However, resistance decreased dramatically above 40 μg/ml AZT. We propose that this threshold can be used to select for higher activity variants of HSV-TK and other nucleoside kinases in a manner that overcomes the efficiency and localization issues of previous selection schemes. Furthermore, our selection strategy should be a general strategy to select or evaluate nucleoside kinases that phosphorylate nucleosides such as prodrugs that would otherwise be toxic to E. coli

  4. Bivalent Inhibitors of Protein Kinases

    PubMed Central

    Gower, Carrie M.; Chang, Matthew E. K.; Maly, Dustin J.

    2015-01-01

    Protein kinases are key players in a large number of cellular signaling pathways. Dysregulated kinase activity has been implicated in a number of diseases, and members of this enzyme family are of therapeutic interest. However, due to the fact that most inhibitors interact with the highly conserved ATP-binding sites of kinases, it is a significant challenge to develop pharmacological agents that target only one of the greater than 500 kinases present in humans. A potential solution to this problem is the development of bisubstrate and bivalent kinase inhibitors, in which an active site-directed moiety is tethered to another ligand that targets a location outside of the ATP-binding cleft. Because kinase signaling specificity is modulated by regions outside of the ATP-binding site, strategies that exploit these interactions have the potential to provide reagents with high target selectivity. This review highlights examples of kinase interaction sites that can potentially be exploited by bisubstrate and bivalent inhibitors. Furthermore, an overview of efforts to target these interactions with bisubstrate and bivalent inhibitors is provided. Finally, several examples of the successful application of these reagents in a cellular setting are described. PMID:24564382

  5. Biochemical and Structural Insights into Doublecortin-like Kinase Domain 1.

    PubMed

    Patel, Onisha; Dai, Weiwen; Mentzel, Mareike; Griffin, Michael D W; Serindoux, Juliette; Gay, Yoann; Fischer, Stefanie; Sterle, Shoukat; Kropp, Ashleigh; Burns, Christopher J; Ernst, Matthias; Buchert, Michael; Lucet, Isabelle S

    2016-09-01

    Doublecortin-like kinase 1 (DCLK1) is a serine/threonine kinase that belongs to the family of microtubule-associated proteins. Originally identified for its role in neurogenesis, DCLK1 has recently been shown to regulate biological processes outside of the CNS. DCLK1 is among the 15 most common putative driver genes for gastric cancers and is highly mutated across various other human cancers. However, our present understanding of how DCLK1 dysfunction leads to tumorigenesis is limited. Here, we provide evidence that DCLK1 kinase activity negatively regulates microtubule polymerization. We present the crystal structure of the DCLK1 kinase domain at 1.7 Å resolution, providing detailed insight into the ATP-binding site that will serve as a framework for future drug design. This structure also allowed for the mapping of cancer-causing mutations within the kinase domain, suggesting that a loss of kinase function may contribute to tumorigenesis. PMID:27545623

  6. The Src family kinases: distinct functions of c-Src, Yes, and Fyn in the liver.

    PubMed

    Reinehr, Roland; Sommerfeld, Annika; Häussinger, Dieter

    2013-04-01

    The Src family kinases Yes, Fyn, and c-Src play a pivotal role in regulating diverse liver functions such as bile flow, proteolysis, apoptosis, and proliferation and are regulated by anisoosmotic cell volume changes, death receptor ligands, and bile acids. For example, cell swelling leads to an integrin-sensed and focal adhesion kinase-mediated activation of c-Src-triggering choleresis, proteolysis inhibition, regulatory volume decrease via p38MAPK and proliferation via the activation of the epidermal growth factor receptor and extracellular regulated kinases 1 and 2. In contrast, hepatocyte shrinkage generates an almost instantaneous oxidative stress response that triggers the activation of c-Jun N-terminal kinase and the Src family kinases Fyn and Yes. Whereas Fyn activation mediates cholestasis, Yes triggers CD95 activation and apoptosis. This review will discuss the role of Src family kinases in the regulation of liver function with emphasis on their role in osmo-signaling and bile acid signaling.

  7. Phosphorylation and activation of calcineurin by glycogen synthase (casein) kinase-1 and cyclic AMP-dependent protein kinase

    SciTech Connect

    Singh, T.J.; Wang, J.H.

    1986-05-01

    Calcineurin is a phosphoprotein phosphatase that is activated by divalent cations and further stimulated by calmodulin. In this study calcineurin is shown to be a substrate for both glycogen synthase (casein) kinase-1 (CK-1) and cyclic AMP-dependent protein kinase (A-kinase). Either kinase can catalyze the incorporation of 1.0-1.4 mol /sup 32/P/mol calcineurin. Analysis by SDS-PAGE revealed that only the ..cap alpha.. subunit is phosphorylated. Phosphorylation of calcineurin by either kinase leads to its activation. Using p-nitrophenyl phosphate as a substrate the authors observed a 2-3 fold activation of calcineurin by either Mn/sup 2 +/ or Ni/sup 2 +/ (in the presence or absence of calmodulin) after phosphorylation of calcineurin by either CK-1 or A-kinase. In the absence of Mn/sup 2 +/ or Ni/sup 2 +/ phosphorylated calcineurin, like the nonphosphorylated enzyme, showed very little activity. Ni/sup 2 +/ was a more potent activator of phosphorylated calcineurin compared to Mn/sup 2 +/. Higher levels of activation (5-8 fold) of calcineurin by calmodulin was observed when phosphorylated calcineurin was pretreated with Ni/sup 2 +/ before measurement of phosphatase activity. These results indicate that phosphorylation may be an important mechanism by which calcineurin activity is regulated by Ca/sup 2 +/.

  8. The early evolution of the phosphagen kinases--insights from choanoflagellate and poriferan arginine kinases.

    PubMed

    Conejo, Maria; Bertin, Matt; Pomponi, Shirley A; Ellington, W Ross

    2008-01-01

    Arginine kinase (AK) is a member of a large family of phosphoryl transfer enzymes called phosphagen (guanidino) kinases. AKs are present in certain protozoans, sponges, cnidarians, and both lophotrochozoan and ecdysozoan protostomes. Another phosphagen kinase, creatine kinase (CK), is found in sponges, cnidarians, and both deuterostome and protostome groups but does not appear to be present in protozoans. To probe the early evolution of phosphagen kinases, we have amplified the cDNAs for AKs from three choanoflagellates and from the hexactinellid sponge Aphrocallistes beatrix and the demosponges Suberites fuscus and Microciona prolifera. Phylogenetic analysis using maximum likelihood of these choanoflagellate and sponge AKs with other AK sequences revealed that the AK from the choanoflagellate Monosiga brevicollis clusters with the AK from the glass sponge Aphrocallistes and is part of a larger cluster containing AKs from the demosponges Suberites and Microciona as well as basal and protostome invertebrates. In contrast, AKs from Codonosiga gracilis and Monosiga ovata form a distinct cluster apart from all other AK sequences. tBLASTn searches of the recently released M. brevicollis genome database showed that this species has three unique AK genes-one virtually identical to the M. brevicollis cDNA and the other two showing great similarity to C. gracilis and M. ovata AKs. Three distinct AK genes are likely present in choanoflagellates. Two of these AKs display extensive similarity to both CKs and an AK from sponges. Previous work has shown CK evolved from an AK-like ancestor prior to the divergence of sponges. The present results provide evidence suggesting that the initial gene duplication event(s) leading to the CK lineage may have occurred before the divergence of the choanoflagellate and animal lineages. PMID:18064398

  9. c-Jun controls the efficiency of MAP kinase signaling by transcriptional repression of MAP kinase phosphatases

    SciTech Connect

    Sprowles, Amy; Wu Yimi; Kung, H.-J.; Wisdom, Ron . E-mail: ronald.wisdom@ucdmc.ucdavis.edu

    2005-08-15

    The mammalian JNK signaling pathway regulates the transcriptional response of cells to environmental stress, including UV irradiation. This signaling pathway is composed of a classical MAP kinase cascade; activation results in phosphorylation of the transcription factor substrates c-Jun and ATF2, and leads to changes in gene expression. The defining components of this pathway are conserved in the fission yeast S. pombe, where the genetic studies have shown that the ability of the JNK homolog Spc1 to be activated in response to UV irradiation is dependent on the presence of the transcription factor substrate Atf1. We have used genetic analysis to define the role of c-Jun in activation of the mammalian JNK signaling pathway. Our results show that optimal activation of JNK requires the presence of its transcription factor substrate c-Jun. Mutational analysis shows that the ability of c-Jun to support efficient activation of JNK requires the ability of Jun to bind DNA, suggesting a transcriptional mechanism. Consistent with this, we show that c-Jun represses the expression of several MAP kinase phosphatases. In the absence of c-Jun, the increased expression of MAP kinase phosphatases leads to impaired activation of the ERK, JNK, and p38 MAP kinases after pathway activation. The results show that one function of c-Jun is to regulate the efficiency of signaling by the ERK, p38, and JNK MAP kinases, a function that is likely to affect cellular responses to many different stimuli.

  10. Requirements for activation and RAFT localization of the T-lymphocyte kinase Rlk/Txk

    PubMed Central

    Chamorro, Mario; Czar, Michael J; Debnath, Jayanta; Cheng, Genhong; Lenardo, Michael J; Varmus, Harold E; Schwartzberg, Pamela L

    2001-01-01

    Background The Tec family kinases are implicated in signaling from lymphocyte antigen receptors and are activated following phosphorylation by Src kinases. For most Tec kinases, this activation requires an interaction between their pleckstrin homology (PH) domains and the products of phosphoinositide 3-Kinase, which localizes Tec kinases to membrane RAFTs. Rlk/Txk is a Tec related kinase expressed in T cells that lacks a pleckstrin homology domain, having instead a palmitoylated cysteine-string motif. To evaluate Rlk's function in T cell receptor signaling cascades, we examined the requirements for Rlk localization and activation by Src family kinases. Results We demonstrate that Rlk is also associated with RAFTs, despite its lack of a pleckstrin homology domain. Rlk RAFT association requires the cysteine-string motif and is independent of PI3 Kinase activity. We further demonstrate that Rlk can be phosphorylated and activated by Src kinases, leading to a decrease in its half-life. A specific tyrosine in the activation loop of Rlk, Y420, is required for phosphorylation and activation, as well as for decreased stability, but is not required for lipid RAFT association. Mutation of this tyrosine also prevents increased tyrosine phosphorylation of Rlk after stimulation of the T cell receptor, suggesting that Rlk is phosphorylated by Src family kinases in response to T cell receptor engagement. Conclusions Like the other related Tec kinases, Rlk is associated with lipid RAFTs and can be phosphorylated and activated by Src family kinases, supporting a role for Rlk in signaling downstream of Src kinases in T cell activation. PMID:11353545

  11. Kinases as Novel Therapeutic Targets in Asthma and Chronic Obstructive Pulmonary Disease.

    PubMed

    Barnes, Peter J

    2016-07-01

    Multiple kinases play a critical role in orchestrating the chronic inflammation and structural changes in the respiratory tract of patients with asthma and chronic obstructive pulmonary disease (COPD). Kinases activate signaling pathways that lead to contraction of airway smooth muscle and release of inflammatory mediators (such as cytokines, chemokines, growth factors) as well as cell migration, activation, and proliferation. For this reason there has been great interest in the development of kinase inhibitors as anti-inflammatory therapies, particular where corticosteroids are less effective, as in severe asthma and COPD. However, it has proven difficult to develop selective kinase inhibitors that are both effective and safe after oral administration and this has led to a search for inhaled kinase inhibitors, which would reduce systemic exposure. Although many kinases have been implicated in inflammation and remodeling of airway disease, very few classes of drug have reached the stage of clinical studies in these diseases. The most promising drugs are p38 MAP kinases, isoenzyme-selective PI3-kinases, Janus-activated kinases, and Syk-kinases, and inhaled formulations of these drugs are now in development. There has also been interest in developing inhibitors that block more than one kinase, because these drugs may be more effective and with less risk of losing efficacy with time. No kinase inhibitors are yet on the market for the treatment of airway diseases, but as kinase inhibitors are improved from other therapeutic areas there is hope that these drugs may eventually prove useful in treating refractory asthma and COPD. PMID:27363440

  12. Isolation of chloroplastic phosphoglycerate kinase

    SciTech Connect

    Macioszek, J.; Anderson, L.E. ); Anderson, J.B. )

    1990-09-01

    We report here a method for the isolation of high specific activity phosphoglycerate kinase (EC 2.7.2.3) from chloroplasts. The enzyme has been purified over 200-fold from pea (Pisum sativum L.) stromal extracts to apparent homogeneity with 23% recovery. Negative cooperativity is observed with the two enzyme phosphoglycerate kinase/glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) couple restored from the purified enzymes when NADPH is the reducing pyridine nucleotide, consistent with earlier results obtained with crude chloroplastic extracts. Michaelis Menten kinetics are observed when 3-phosphoglycerate is held constant and phosphoglycerate kinase is varied, which suggests that phosphoglycerate kinase-bound 1,3-bisphosphoglycerate may be the preferred substrate for glyceraldehyde-3-P dehydrogenase in the chloroplast.

  13. Protein Crystals of Raf Kinase

    NASA Technical Reports Server (NTRS)

    1995-01-01

    This image shows crystals of the protein raf kinase grown on Earth (photo a) and on USML-2 (photo b). The space-grown crystals are an order of magnitude larger. Principal Investigator: Dan Carter of New Century Pharmaceuticals

  14. The azaindole framework in the design of kinase inhibitors.

    PubMed

    Mérour, Jean-Yves; Buron, Frédéric; Plé, Karen; Bonnet, Pascal; Routier, Sylvain

    2014-01-01

    This review article illustrates the growing use of azaindole derivatives as kinase inhibitors and their contribution to drug discovery and innovation. The different protein kinases which have served as targets and the known molecules which have emerged from medicinal chemistry and Fragment-Based Drug Discovery (FBDD) programs are presented. The various synthetic routes used to access these compounds and the chemical pathways leading to their synthesis are also discussed. An analysis of their mode of binding based on X-ray crystallography data gives structural insights for the design of more potent and selective inhibitors. PMID:25460315

  15. PREX1 Protein Function Is Negatively Regulated Downstream of Receptor Tyrosine Kinase Activation by p21-activated Kinases (PAKs).

    PubMed

    Barrows, Douglas; He, John Z; Parsons, Ramon

    2016-09-16

    Downstream of receptor tyrosine kinase and G protein-coupled receptor (GPCR) stimulation, the phosphatidylinositol 3,4,5-trisphosphate (PIP3)-dependent Rac exchange factor (PREX) family of guanine nucleotide exchange factors (GEFs) activates Rho GTPases, leading to important roles for PREX proteins in numerous cellular processes and diseases, including cancer. PREX1 and PREX2 GEF activity is activated by the second messengers PIP3 and Gβγ, and further regulation of PREX GEF activity occurs by phosphorylation. Stimulation of receptor tyrosine kinases by neuregulin and insulin-like growth factor 1 (IGF1) leads to the phosphorylation of PREX1; however, the kinases that phosphorylate PREX1 downstream of these ligands are not known. We recently reported that the p21-activated kinases (PAKs), which are activated by GTP-bound Ras-related C3 botulinum toxin substrate 1 (Rac1), mediate the phosphorylation of PREX2 after insulin receptor activation. Here we show that certain phosphorylation events on PREX1 after insulin, neuregulin, and IGF1 treatment are PAK-dependent and lead to a reduction in PREX1 binding to PIP3 Like PREX2, PAK-mediated phosphorylation also negatively regulates PREX1 GEF activity. Furthermore, the onset of PREX1 phosphorylation was delayed compared with the phosphorylation of AKT, supporting a model of negative feedback downstream of PREX1 activation. We also found that the phosphorylation of PREX1 after isoproterenol and prostaglandin E2-mediated GPCR activation is partially PAK-dependent and likely also involves protein kinase A, which is known to reduce PREX1 function. Our data point to multiple mechanisms of PREX1 negative regulation by PAKs within receptor tyrosine kinase and GPCR-stimulated signaling pathways that have important roles in diseases such as diabetes and cancer. PMID:27481946

  16. Feature-similarity kinase classifier as a ligand engineering tool

    PubMed Central

    Maddipati, Sridhar; Fernández, Ariel

    2006-01-01

    Kinases have been often targeted in drug therapy aimed at blocking signaling pathways. However, the conservation of protein structure across homologs often leads to uncontrolled cross reactivity. On the other hand, sticky packing defects in proteins are typically not conserved across homologs, making them ligand-anchoring sites potentially important to enhance selectivity. Thus, we introduce a hierarchical clustering of PDB-reported kinases according to packing differences. This kinome partitioning is highly correlated with proximity relations arising from the pharmacological profiling of kinases. A variable packing sensitivity is observed for individual drugs, with highly promiscuous ligands being the most insensitive to packing differences. Our classifier enables a strategy to design selective inhibitors. PMID:17110166

  17. Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway.

    PubMed Central

    Khwaja, A; Rodriguez-Viciana, P; Wennström, S; Warne, P H; Downward, J

    1997-01-01

    Upon detachment from the extracellular matrix, epithelial cells enter into programmed cell death, a phenomenon known as anoikis, ensuring that they are unable to survive in an inappropriate location. Activated ras oncogenes protect cells from this form of apoptosis. The nature of the survival signals activated by integrin engagement and usurped by oncogenic Ras are unknown: here we show that in both cases phosphoinositide 3-OH kinase (PI 3-kinase), but not Raf, mediates this protection, acting through protein kinase B/Akt (PKB/Akt). Constitutively activated PI 3-kinase or PKB/Akt block anoikis, while inhibition of PI 3-kinase abrogates protection by Ras, but not PKB/Akt. Inhibition of either PI 3-kinase or PKB/Akt induces apoptosis in adherent epithelial cells. Attachment of cells to matrix leads to rapid elevation of the levels of PI 3-kinase lipid products and PKB/Akt activity, both of which remain high in Ras-transformed cells even in suspension. PI 3-kinase acting through PKB/Akt is therefore implicated as a key mediator of the aberrant survival of Ras-transformed epithelial cells in the absence of attachment, and mediates matrix-induced survival of normal epithelial cells. PMID:9184223

  18. Regulation of mouse gamete interaction by a sperm tyrosine kinase.

    PubMed

    Leyton, L; LeGuen, P; Bunch, D; Saling, P M

    1992-12-15

    A 95-kDa mouse sperm protein has been previously identified as a putative receptor involved in the sperm-egg interactions that lead to fertilization. The ligand for this receptor is the zona pellucida glycoprotein ZP3. This constituent of the oocyte-specific extracellular matrix mediates not only sperm binding to the zona but also triggers acrosomal exocytosis. The latter, also termed the acrosome reaction, is a key regulatory event upon which fertilization is absolutely dependent. Previously, we showed that the 95-kDa protein that binds ZP3 is a substrate for tyrosine kinase, and its phosphotyrosine content increases after sperm-zona pellucida binding. Here, we show the presence of protein tyrosine kinase activity in sperm plasma membranes and in electroeluted 95-kDa protein. The tyrosine kinase activity of the isolated protein is stimulated by solubilized zona pellucida and inhibited by tyrphostin RG-50864, a membrane-permeable tyrosine kinase inhibitor. Furthermore, tyrphostin inhibits zona-triggered acrosomal exocytosis in a dose-dependent manner. These findings indicate that the 95-kDa protein participates in a critical regulatory event of gamete interaction; moreover, our experiments suggest that sperm protein tyrosine kinase may be an excellent target for the control of fertility.

  19. Discovering the first tyrosine kinase

    PubMed Central

    Hunter, Tony

    2015-01-01

    In the middle of the 20th century, animal tumor viruses were heralded as possible models for understanding human cancer. By the mid-1970s, the molecular basis by which tumor viruses transform cells into a malignant state was beginning to emerge as the first viral genomic sequences were reported and the proteins encoded by their transforming genes were identified and characterized. This was a time of great excitement and rapid progress. In 1978, prompted by the discovery from Ray Erikson’s group that the Rous sarcoma virus (RSV) v-Src–transforming protein had an associated protein kinase activity specific for threonine, my group at the Salk Institute set out to determine whether the polyomavirus middle T-transforming protein had a similar kinase activity. Here, I describe the experiments that led to the identification of a kinase activity associated with middle T antigen and our serendipitous discovery that this activity was specific for tyrosine in vitro, and how this in turn led to the fortuitous observation that the v-Src–associated kinase activity was also specific for tyrosine. Our finding that v-Src increased the level of phosphotyrosine in cellular proteins in RSV-transformed cells confirmed that v-Src is a tyrosine kinase and transforms cells by phosphorylating proteins on tyrosine. My colleague Bart Sefton and I reported these findings in the March issue of PNAS in 1980. Remarkably, all of the experiments in this paper were accomplished in less than one month. PMID:26130799

  20. The discovery of novel vascular endothelial growth factor receptor tyrosine kinases inhibitors: pharmacophore modeling, virtual screening and docking studies.

    PubMed

    Yu, Hui; Wang, Zhanli; Zhang, Liangren; Zhang, Jufeng; Huang, Qian

    2007-03-01

    We have applied pharmacophore generation, database searching and docking methodologies to discover new structures for the design of vascular endothelial growth factor receptors, the tyrosine kinase insert domain-containing receptor kinase inhibitors. The chemical function based pharmacophore models were built for kinase insert domain-containing receptor kinase inhibitors from a set of 10 known inhibitors using the algorithm HipHop, which is implemented in the CATALYST software. The highest scoring HipHop model consists of four features: one hydrophobic, one hydrogen bond acceptor, one hydrogen bond donor and one ring aromatic function. Using the algorithm CatShape within CATALYST, the bound conformation of 4-amino-furo [2, 3-d] pyrimidine binding to kinase insert domain-containing receptor kinase was used to generate a shape query. A merged shape and hypothesis query that is in an appropriate alignment was then built. The combined shape and hypothesis model was used as a query to search Maybridge database for other potential lead compounds. A total of 39 compounds were retrieved as hits. The hits obtained were docked into kinase insert domain-containing receptor kinase active site. One novel potential lead was proposed based on CATALYST fit value, LigandFit docking scores, and examination of how the hit retain key interactions known to be required for kinase binding. This compound inhibited vascular endothelial growth factor stimulated kinase insert domain-containing receptor phosphorylation in human umbilical vein endothelial cells. PMID:17441906

  1. The discovery of novel vascular endothelial growth factor receptor tyrosine kinases inhibitors: pharmacophore modeling, virtual screening and docking studies.

    PubMed

    Yu, Hui; Wang, Zhanli; Zhang, Liangren; Zhang, Jufeng; Huang, Qian

    2007-03-01

    We have applied pharmacophore generation, database searching and docking methodologies to discover new structures for the design of vascular endothelial growth factor receptors, the tyrosine kinase insert domain-containing receptor kinase inhibitors. The chemical function based pharmacophore models were built for kinase insert domain-containing receptor kinase inhibitors from a set of 10 known inhibitors using the algorithm HipHop, which is implemented in the CATALYST software. The highest scoring HipHop model consists of four features: one hydrophobic, one hydrogen bond acceptor, one hydrogen bond donor and one ring aromatic function. Using the algorithm CatShape within CATALYST, the bound conformation of 4-amino-furo [2, 3-d] pyrimidine binding to kinase insert domain-containing receptor kinase was used to generate a shape query. A merged shape and hypothesis query that is in an appropriate alignment was then built. The combined shape and hypothesis model was used as a query to search Maybridge database for other potential lead compounds. A total of 39 compounds were retrieved as hits. The hits obtained were docked into kinase insert domain-containing receptor kinase active site. One novel potential lead was proposed based on CATALYST fit value, LigandFit docking scores, and examination of how the hit retain key interactions known to be required for kinase binding. This compound inhibited vascular endothelial growth factor stimulated kinase insert domain-containing receptor phosphorylation in human umbilical vein endothelial cells.

  2. Modelling the 2-kinase domain of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase on adenylate kinase.

    PubMed Central

    Bertrand, L; Vertommen, D; Depiereux, E; Hue, L; Rider, M H; Feytmans, E

    1997-01-01

    Simultaneous multiple alignment of available sequences of the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase revealed several segments of conserved residues in the 2-kinase domain. The sequence of the kinase domain was also compared with proteins of known three-dimensional structure. No similarity was found between the kinase domain of 6-phosphofructo-2-kinase and 6-phosphofructo-1-kinase. This questions the modelling of the 2-kinase domain on bacterial 6-phosphofructo-1-kinase that has previously been proposed [Bazan, Fletterick and Pilkis (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 9642-9646]. However, sequence similarities were found between the 2-kinase domain and several nucleotide-binding proteins, the most similar being adenylate kinase. A structural model of the 2-kinase domain based on adenylate kinase is proposed. It accommodates all the results of site-directed mutagenesis studies carried out to date on residues in the 2-kinase domain. It also allows residues potentially involved in catalysis and/or substrate binding to be predicted. PMID:9032445

  3. Bimodal lipid substrate dependence of phosphatidylinositol kinase.

    PubMed

    Ganong, B R

    1990-07-24

    Phosphatidylinositol (PI) kinase activity was solubilized from rat liver microsomes and partially purified by chromatography on hydroxyapatite and Reactive Green 19-Superose. Examination of the ATP dependence using a mixed micellar assay gave a Km of 120 microM. The dependence of reaction rate on PI was more complicated. PI kinase bound a large amount of Triton X-100, and as expected for a micelle-associated enzyme utilizing a micelle-associated lipid substrate, the reaction rate was dependent on the micellar mole fraction, PI/(PI + Triton X-100), with a Km of 0.02 (unitless). Activity showed an additional dependence on bulk PI concentration at high micelle dilution. These results demonstrated two kinetically distinguishable steps leading to formation of a productive PI/enzyme(/ATP) complex. The rate of the first step, which probably represents exchange of PI from the bulk micellar pool into enzyme-containing micelles, depends on bulk PI concentration. The rate of the second step, association of PI with enzyme within a single micelle, depends on the micellar mole fraction of PI. Depression of the apparent Vmax at low ionic strength suggested that electrostatic repulsion between negatively charged PI/Triton X-100 mixed micelles inhibits PI exchange, consistent with a model in which intermicellar PI exchange depends on micellar collisions.

  4. The FRK1 mitogen-activated protein kinase kinase kinase (MAPKKK) from Solanum chacoense is involved in embryo sac and pollen development.

    PubMed

    Lafleur, Edith; Kapfer, Christelle; Joly, Valentin; Liu, Yang; Tebbji, Faiza; Daigle, Caroline; Gray-Mitsumune, Madoka; Cappadocia, Mario; Nantel, André; Matton, Daniel P

    2015-04-01

    The fertilization-related kinase 1 (ScFRK1), a nuclear-localized mitogen-activated protein kinase kinase kinase (MAPKKK) from the wild potato species Solanum chacoense, belongs to a small group of pMEKKs that do not possess an extended N- or C-terminal regulatory domain. Initially selected based on its highly specific expression profile following fertilization, in situ expression analyses revealed that the ScFRK1 gene is also expressed early on during female gametophyte development in the integument and megaspore mother cell and, later, in the synergid and egg cells of the embryo sac. ScFRK1 mRNAs are also detected in pollen mother cells. Transgenic plants with lower or barely detectable levels of ScFRK1 mRNAs lead to the production of small fruits with severely reduced seed set, resulting from a concomitant decline in the number of normal embryo sacs produced. Megagametogenesis and microgametogenesis were affected, as megaspores did not progress beyond the functional megaspore (FG1) stage and the microspore collapsed around the first pollen mitosis. As for other mutants that affect embryo sac development, pollen tube guidance was severely affected in the ScFRK1 transgenic lines. Gametophyte to sporophyte communication was also affected, as observed from a marked change in the transcriptomic profiles of the sporophytic tissues of the ovule. The ScFRK1 MAPKKK is thus involved in a signalling cascade that regulates both male and female gamete development. PMID:25576576

  5. Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium

    SciTech Connect

    Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty; Lin, Yu-Hui; Amani, Mehrnaz; Allali-Hassani, Abdellah; Senisterra, Guillermo; Vedadi, Masoud; Tempel, Wolfram; Mackenzie, Farrell; Chau, Irene; Lourido, Sebastian; Sibley, L. David; Hui, Raymond

    2010-09-21

    Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium binding triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.

  6. A chemoproteomic method for identifying cellular targets of covalent kinase inhibitors

    PubMed Central

    Chen, Ying-Chu; Zhang, Chao

    2016-01-01

    Protein kinases are attractive drug targets for numerous human diseases including cancers, diabetes and neurodegeneration. A number of kinase inhibitors that covalently target a cysteine residue in their target kinases have recently entered use in the cancer clinic. Despite the advantages of covalent kinases inhibitors, their inherent reactivity can lead to non-specific binding to other cellular proteins and cause off- target effects in cells. It is thus essential to determine the identity of these off targets in order to fully account for the phenotype and to improve the selectivity and efficacy of covalent inhibitors. Herein we present a detailed protocol for a chemoproteomic method to enrich and identify cellular targets of covalent kinase inhibitors. PMID:27551330

  7. [Effects of RhoA/Rho-kinase in the regulation of penile erection].

    PubMed

    Xia, Chuan; Jiang, Rui

    2007-06-01

    The erectile response of the penis depends on a balance between vasoconstrictor agents, which cause cavernosal smooth muscle to contract limiting blood inflow, and vasodilators, which relax cavernosal smooth muscle leading to increased blood inflow and erection. This review emphasizes the role of the RhoA/Rho-kinase pathway in the cavernosal circulation. While it is widely held that the nitric oxide-cyclic GMP-protein kinase G(NO-cGMP-PKG) pathway mediates vasorelaxation and penile erection, the vasoconstrictor actions of endothelin ET-1 and NE are reported to be mediated by the RhoA/Rho-kinase pathway in the cavernosal circulation and NO relax cavernosal smooth by inhibition of Rho-kinase. The application of Rho-kinase inhibitor on the penile erection may represent a new and promising method of treatment for erectile dysfunction.

  8. Evolutionary Ancestry of Eukaryotic Protein Kinases and Choline Kinases.

    PubMed

    Lai, Shenshen; Safaei, Javad; Pelech, Steven

    2016-03-01

    The reversible phosphorylation of proteins catalyzed by protein kinases in eukaryotes supports an important role for eukaryotic protein kinases (ePKs) in the emergence of nucleated cells in the third superkingdom of life. Choline kinases (ChKs) could also be critical in the early evolution of eukaryotes, because of their function in the biosynthesis of phosphatidylcholine, which is unique to eukaryotic membranes. However, the genomic origins of ePKs and ChKs are unclear. The high degeneracy of protein sequences and broad expansion of ePK families have made this fundamental question difficult to answer. In this study, we identified two class-I aminoacyl-tRNA synthetases with high similarities to consensus amino acid sequences of human protein-serine/threonine kinases. Comparisons of primary and tertiary structures supported that ePKs and ChKs evolved from a common ancestor related to glutaminyl aminoacyl-tRNA synthetases, which may have been one of the key factors in the successful of emergence of ancient eukaryotic cells from bacterial colonies.

  9. Cyclin-Dependent Kinase Suppression by WEE1 Kinase Protects the Genome through Control of Replication Initiation and Nucleotide Consumption

    PubMed Central

    Beck, Halfdan; Nähse-Kumpf, Viola; Larsen, Marie Sofie Yoo; O'Hanlon, Karen A.; Patzke, Sebastian; Holmberg, Christian; Mejlvang, Jakob; Groth, Anja; Nielsen, Olaf

    2012-01-01

    Activation of oncogenes or inhibition of WEE1 kinase deregulates cyclin-dependent kinase (CDK) activity and leads to replication stress; however, the underlying mechanism is not understood. We now show that elevation of CDK activity by inhibition of WEE1 kinase rapidly increases initiation of replication. This leads to nucleotide shortage and reduces replication fork speed, which is followed by SLX4/MUS81-mediated DNA double-strand breakage. Fork speed is normalized and DNA double-strand break (DSB) formation is suppressed when CDT1, a key factor for replication initiation, is depleted. Furthermore, addition of nucleosides counteracts the effects of unscheduled CDK activity on fork speed and DNA DSB formation. Finally, we show that WEE1 regulates the ionizing radiation (IR)-induced S-phase checkpoint, consistent with its role in control of replication initiation. In conclusion, these results suggest that deregulated CDK activity, such as that occurring following inhibition of WEE1 kinase or activation of oncogenes, induces replication stress and loss of genomic integrity through increased firing of replication origins and subsequent nucleotide shortage. PMID:22907750

  10. KID, a Kinase Inhibitor Database project.

    PubMed

    Collin, O; Meijer, L

    1999-01-01

    The Kinase Inhibitor Database is a small specialized database dedicated to the gathering of information on protein kinase inhibitors. The database is accessible through the World Wide Web system and gives access to structural and bibliographic information on protein kinase inhibitors. The data in the database will be collected and submitted by researchers working in the kinase inhibitor field. The submitted data will be checked by the curator of the database before entry.

  11. PINCH proteins regulate cardiac contractility by modulating integrin-linked kinase-protein kinase B signaling.

    PubMed

    Meder, Benjamin; Huttner, Inken G; Sedaghat-Hamedani, Farbod; Just, Steffen; Dahme, Tillman; Frese, Karen S; Vogel, Britta; Köhler, Doreen; Kloos, Wanda; Rudloff, Jessica; Marquart, Sabine; Katus, Hugo A; Rottbauer, Wolfgang

    2011-08-01

    Integrin-linked kinase (ILK) is an essential component of the cardiac mechanical stretch sensor and is bound in a protein complex with parvin and PINCH proteins, the so-called ILK-PINCH-parvin (IPP) complex. We have recently shown that inactivation of ILK or β-parvin activity leads to heart failure in zebrafish via reduced protein kinase B (PKB/Akt) activation. Here, we show that PINCH proteins localize at sarcomeric Z disks and costameres in the zebrafish heart and skeletal muscle. To investigate the in vivo role of PINCH proteins for IPP complex stability and PKB signaling within the vertebrate heart, we inactivated PINCH1 and PINCH2 in zebrafish. Inactivation of either PINCH isoform independently leads to instability of ILK, loss of stretch-responsive anf and vegf expression, and progressive heart failure. The predominant cause of heart failure in PINCH morphants seems to be loss of PKB activity, since PKB phosphorylation at serine 473 is significantly reduced in PINCH-deficient hearts and overexpression of constitutively active PKB reconstitutes cardiac function in PINCH morphants. These findings highlight the essential function of PINCH proteins in controlling cardiac contractility by granting IPP/PKB-mediated signaling.

  12. Small molecule kinase inhibitor LRRK2-IN-1 demonstrates potent activity against colorectal and pancreatic cancer through inhibition of doublecortin-like kinase 1

    PubMed Central

    2014-01-01

    Background Doublecortin-like kinase 1 (DCLK1) is emerging as a tumor specific stem cell marker in colorectal and pancreatic cancer. Previous in vitro and in vivo studies have demonstrated the therapeutic effects of inhibiting DCLK1 with small interfering RNA (siRNA) as well as genetically targeting the DCLK1+ cell for deletion. However, the effects of inhibiting DCLK1 kinase activity have not been studied directly. Therefore, we assessed the effects of inhibiting DCLK1 kinase activity using the novel small molecule kinase inhibitor, LRRK2-IN-1, which demonstrates significant affinity for DCLK1. Results Here we report that LRRK2-IN-1 demonstrates potent anti-cancer activity including inhibition of cancer cell proliferation, migration, and invasion as well as induction of apoptosis and cell cycle arrest. Additionally we found that it regulates stemness, epithelial-mesenchymal transition, and oncogenic targets on the molecular level. Moreover, we show that LRRK2-IN-1 suppresses DCLK1 kinase activity and downstream DCLK1 effector c-MYC, and demonstrate that DCLK1 kinase activity is a significant factor in resistance to LRRK2-IN-1. Conclusions Given DCLK1’s tumor stem cell marker status, a strong understanding of its biological role and interactions in gastrointestinal tumors may lead to discoveries that improve patient outcomes. The results of this study suggest that small molecule inhibitors of DCLK1 kinase should be further investigated as they may hold promise as anti-tumor stem cell drugs. PMID:24885928

  13. 1,25-Dihydroxyvitamin D3 activates Raf kinase and Raf perinuclear translocation via a protein kinase C-dependent pathway.

    PubMed

    Lissoos, T W; Beno, D W; Davis, B H

    1993-11-25

    1,25-Dihydroxyvitamin D3's (D3) potential mitogenic mechanism of action was pursued in cultured rat hepatic Ito cells, a fibrogenic effector cell which proliferates in vivo during liver injury and fibrogenesis. D3 stimulated Ito cell DNA synthesis and potentiated platelet-derived growth factor-induced mitogenesis. D3's enhancement of [3H]thymidine incorporation was associated with nuclear Egr expression. Recent studies have causally linked the activated proto-oncogene c-Raf with downstream Egr induction. The serine-threonine kinase Raf protein is phosphorylation-activated by a large array of agonists including plasma membrane and cytoplasmic tyrosine kinases but has not previously been associated with the steroid superfamily of mediators. To consider potential prenuclear acute pathways of D3-induced stimulation, the activation of Raf was examined following D3 exposure. D3 induced Raf activation as assessed via (a) enhanced Raf phosphorylation following in vivo 32P labeling, (b) enhanced kinase function utilizing exogenous histone 1 protein as substrate, and (c) the shift in Raf physical localization changing from a diffuse cytoplasmic distribution to a perinuclear domain. A similar activation of Raf kinase was found in 3T3 cells exposed to D3 with enhanced histone phosphorylation detectable within 1 min following stimulation. The proximal cascade leading to Raf kinase activation may involve a protein kinase activity was severely attenuated by stimulated kinase activity was severely attenuated by previous phorbol ester treatment for 20 h or staurosporine pretreatment.

  14. Kinase requirements in human cells: V. Synthetic lethal interactions between p53 and the protein kinases SGK2 and PAK3.

    PubMed

    Baldwin, Amy; Grueneberg, Dorre A; Hellner, Karin; Sawyer, Jacqueline; Grace, Miranda; Li, Wenliang; Harlow, Ed; Munger, Karl

    2010-07-13

    Cervical carcinomas are initiated through a series of well-defined stages that rely on the expression of human papillomavirus (HPV) oncogenes. A panel of 100 small hairpin RNAs that target essential kinases in many tumor types was used to study the stepwise appearance of kinase requirements during cervical tumor development. Twenty-six kinases were commonly required in three cell lines derived from frank carcinomas, and each kinase requirement was traced to the specific stage in which the requirement emerged. Six kinases became required following HPV-induced immortalization, and the requirement for two kinases, SGK2 and PAK3, was mapped to the inactivation of p53 in primary human epithelial cells. Loss of the p53 tumor suppressor in other primary epithelial cells also induced dependence on SGK2 and PAK3. Hence, SGK2 and PAK3 provide important cellular functions following p53 inactivation, fulfilling the classical definition of synthetic lethality; loss of p53, SGK2, or PAK3 alone has little effect on cell viability, whereas loss of p53 together with either SGK2 or PAK3 loss leads to cell death. Whereas tumor suppressor gene mutations are not directly druggable, other proteins or pathways that become obligatory to cell viability following tumor suppressor loss provide theoretical targets for tumor suppressor-specific drug discovery efforts. The kinases SGK2 and PAK3 may thus represent such targets for p53-specific drug development.

  15. Models for the activation pathway of epidermal growth factor receptor protein-tyrosine kinase

    SciTech Connect

    Campion, S.R.; Niyogi, S.K. )

    1991-03-15

    Activation of the epidermal growth factor (EGF) receptor's intrinsic protein-tyrosine kinase activity, which occurs upon formation of the receptor-ligand complex, is the critical regulatory event affecting the subsequent EGF-dependent cellular responses leading to DNA synthesis and cell proliferation. The molecular mechanism by which EGF-dependent activation of receptor kinase activity takes place is not clearly understood. In this study, the growth factor-dependent activation of the EGF receptor tyrosine kinase was examined in vitro using detergent-solubilized, partially purified GEF receptors from A5431 human epidermoid carcinoma cells. Evaluation of the cooperativity observed in the EGF-dependent activation of soluble receptor tyrosine kinase would suggest a mechanism requiring the binding of the EGF peptide to both ligand binding sites on a receptor dimer to induce full receptor kinase activity. Equations describing potential cooperative kinase activation pathways have been examined. The theoretical system which best simulates the allosteric regulation observed in the experimental kinase activation data is that describing multiple essential activation. In addition, studies using mutant analogs of the EGF peptide ligand appear to confirm the requirement for an essential conformational change in the receptor-ligand complex to activate the receptor kinase activity. Several mutant growth factor analogues are able to occupy the ligand binding sites on the receptor without inducing the fully active receptor conformation.

  16. Abscisic Acid Induces Mitogen-Activated Protein Kinase Activation in Barley Aleurone Protoplasts.

    PubMed

    Knetsch, MLW.; Wang, M.; Snaar-Jagalska, B. E.; Heimovaara-Dijkstra, S.

    1996-06-01

    Abscisic acid (ABA) induces a rapid and transient mitogen-activated protein (MAP) kinase activation in barley aleurone protoplasts. MAP kinase activity, measured as myelin basic protein phosphorylation by MAP kinase immunoprecipitates, increased after 1 min, peaked after 3 min, and decreased to basal levels after ~5 min of ABA treatment in vivo. Antibodies recognizing phosphorylated tyrosine residues precipitate with myelin basic protein kinase activity that has identical ABA activation characteristics and demonstrate that tyrosine phosphorylation of MAP kinase occurs during activation. The half-maximal concentration of ABA required for MAP kinase activation, 3 x 10-7 M, is very similar to that required for ABA-induced rab16 gene expression. The tyrosine phosphatase inhibitor phenylarsine oxide can completely block ABA-induced MAP kinase activation and rab16 gene expression. These results lead us to conclude that ABA activates MAP kinase via a tyrosine phosphatase and that these steps are a prerequisite for ABA induction of rab16 gene expression.

  17. Abscisic Acid Induces Mitogen-Activated Protein Kinase Activation in Barley Aleurone Protoplasts.

    PubMed Central

    Knetsch, MLW.; Wang, M.; Snaar-Jagalska, B. E.; Heimovaara-Dijkstra, S.

    1996-01-01

    Abscisic acid (ABA) induces a rapid and transient mitogen-activated protein (MAP) kinase activation in barley aleurone protoplasts. MAP kinase activity, measured as myelin basic protein phosphorylation by MAP kinase immunoprecipitates, increased after 1 min, peaked after 3 min, and decreased to basal levels after ~5 min of ABA treatment in vivo. Antibodies recognizing phosphorylated tyrosine residues precipitate with myelin basic protein kinase activity that has identical ABA activation characteristics and demonstrate that tyrosine phosphorylation of MAP kinase occurs during activation. The half-maximal concentration of ABA required for MAP kinase activation, 3 x 10-7 M, is very similar to that required for ABA-induced rab16 gene expression. The tyrosine phosphatase inhibitor phenylarsine oxide can completely block ABA-induced MAP kinase activation and rab16 gene expression. These results lead us to conclude that ABA activates MAP kinase via a tyrosine phosphatase and that these steps are a prerequisite for ABA induction of rab16 gene expression. PMID:12239411

  18. Phosphatidylinositol 3-kinase, protein kinase B and ribosomal S6 kinases in the stimulation of thyroid epithelial cell proliferation by cAMP and growth factors in the presence of insulin.

    PubMed

    Coulonval, K; Vandeput, F; Stein, R C; Kozma, S C; Lamy, F; Dumont, J E

    2000-06-01

    The proliferation of most normal cells depends on the co-operation of several growth factors and hormones, each with a specific role, but the key events involved in the action of each necessary stimulant remain largely uncharacterized. In the present study, the pathways involved in the mechanism(s) of co-operation have been investigated in primary cultures of dog thyroid epithelial cells. In this physiologically relevant system, thyroid stimulating hormone (TSH) acting through cAMP, epidermal growth factor (EGF) and phorbol esters (such as PMA) induce DNA synthesis. Their effect requires stimulation of the insulin-like growth factor-1 (IGF-1) receptor by either IGF-1 or insulin, which are not themselves mitogenic agents. In contrast, hepatocyte growth factor (HGF) is itself fully mitogenic. The results of the study demonstrate that cAMP, EGF, HGF and PMA stimulate p70 ribosomal S6 kinase (p70 S6 kinase). However, insulin/IGF-1 also stimulate p70 S6 kinase. Thus stimulation of p70 S6 kinase might be necessary, but is certainly not sufficient, for the induction of DNA synthesis and is not specific for any stimulated pathway. In contrast, phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase B (PKB) activation by insulin and HGF is strong and sustained, whereas it is weak and transient with EGF and absent in the presence of TSH or PMA. These findings suggest that: (i) stimulation of PI 3-kinases and/or PKB is not involved in the cAMP-dependent pathways leading to thyrocyte proliferation, or in the action of PMA, (ii) the stimulation of the PI 3-kinase/PKB pathway may account for the permissive action of insulin/IGF-1 in the proliferation of these cells, and (iii) the stimulation of this pathway by HGF may explain why this agent does not require insulin or IGF-1 for its mitogenic action. PMID:10816429

  19. Assessing Kinase Activity in Plants with In-Gel Kinase Assays.

    PubMed

    Wang, Pengcheng; Zhu, Jian-Kang

    2016-01-01

    The in-gel protein kinase assay is a powerful method to measure the protein phosphorylation activity of specific protein kinases. Any protein substrate can be embedded in polyacrylamide gels where they can be phosphorylated by protein kinases that are separated in the gel under denaturing conditions and then renatured. The kinase activity can be visualized in situ in the gels by autoradiography. This method has been used to compare the activities of protein kinases in parallel samples or to identify their potential substrates. Here, we describe in detail an in-gel kinase assay to measure the activity of some protein kinases in plants.

  20. (Na+ + K+)-ATPase Is a Target for Phosphoinositide 3-Kinase/Protein Kinase B and Protein Kinase C Pathways Triggered by Albumin*

    PubMed Central

    Peruchetti, Diogo B.; Pinheiro, Ana Acacia S.; Landgraf, Sharon S.; Wengert, Mira; Takiya, Christina M.; Guggino, William B.; Caruso-Neves, Celso

    2011-01-01

    In recent decades, evidence has confirmed the crucial role of albumin in the progression of renal disease. However, the possible role of signaling pathways triggered by physiologic concentrations of albumin in the modulation of proximal tubule (PT) sodium reabsorption has not been considered. In the present work, we have shown that a physiologic concentration of albumin increases the expression of the α1 subunit of (Na+ + K+)-ATPase in LLC-PK1 cells leading to an increase in enzyme activity. This process involves the sequential activation of PI3K/protein kinase B and protein kinase C pathways promoting inhibition of protein kinase A. This integrative network is inhibited when albumin concentration is increased, similar to renal disease, leading to a decrease in the α1 subunit of (Na+ + K+)-ATPase expression. Together, the results indicate that variation in albumin concentration in PT cells has an important effect on PT sodium reabsorption and, consequently, on renal sodium excretion. PMID:22057272

  1. Lead Poisoning Prevention Tips

    MedlinePlus

    ... CDC.gov . Lead Home Calendar of Events National Lead Poisoning Prevention Week Archived Materials CDC's Childhood Lead Poisoning Prevention Program Advisory Committee (ACCLPP) Current Activities Blood ...

  2. Lead - nutritional considerations

    MedlinePlus

    Lead poisoning - nutritional considerations; Toxic metal - nutritional considerations ... utensils . Old paint poses the greatest danger for lead poisoning , especially in young children. Tap water from lead ...

  3. Src kinase regulation by phosphorylation and dephosphorylation

    SciTech Connect

    Roskoski, Robert . E-mail: biocrr@lsuhsc.edu

    2005-05-27

    Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTP{alpha}, PTP{epsilon}, and PTP{lambda}. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined.

  4. Protein Kinase A: A Master Kinase of Granulosa Cell Differentiation

    PubMed Central

    Puri, Pawan; Little-Ihrig, Lynda; Chandran, Uma; Law, Nathan C.; Hunzicker-Dunn, Mary; Zeleznik, Anthony J.

    2016-01-01

    Activation of protein kinase A (PKA) by follicle stimulating hormone (FSH) transduces the signal that drives differentiation of ovarian granulosa cells (GCs). An unresolved question is whether PKA is sufficient to initiate the complex program of GC responses to FSH. We compared signaling pathways and gene expression profiles of GCs stimulated with FSH or expressing PKA-CQR, a constitutively active mutant of PKA. Both FSH and PKA-CQR stimulated the phosphorylation of proteins known to be involved in GC differentiation including CREB, ß-catenin, AKT, p42/44 MAPK, GAB2, GSK-3ß, FOXO1, and YAP. In contrast, FSH stimulated the phosphorylation of p38 MAP kinase but PKA-CQR did not. Microarray analysis revealed that 85% of transcripts that were up-regulated by FSH were increased to a comparable extent by PKA-CQR and of the transcripts that were down-regulated by FSH, 76% were also down-regulated by PKA-CQR. Transcripts regulated similarly by FSH and PKA-CQR are involved in steroidogenesis and differentiation, while transcripts more robustly up-regulated by PKA-CQR are involved in ovulation. Thus, PKA, under the conditions of our experimental approach appears to function as a master upstream kinase that is sufficient to initiate the complex pattern of intracellular signaling pathway and gene expression profiles that accompany GC differentiation. PMID:27324437

  5. Lead levels - blood

    MedlinePlus

    Blood lead levels ... is used to screen people at risk for lead poisoning. This may include industrial workers and children ... also used to measure how well treatment for lead poisoning is working. Lead is common in the ...

  6. Dynamic Clustering of the Bacterial Sensory Kinase BaeS.

    PubMed

    Koler, Moriah; Frank, Vered; Amartely, Hadar; Friedler, Assaf; Vaknin, Ady

    2016-01-01

    Several bacterial sensory-kinase receptors form clusters on the cell membrane. However, the dynamics of sensory-kinase clustering are largely unclear. Using measurements of fluorescence anisotropy and time-lapse imaging of Escherichia coli cells, we demonstrate that copper ions trigger self-association of BaeS receptors and lead to rapid formation of clusters, which can be reversibly dispersed by a metal chelator. Copper ions did not trigger self-association of other fluorescently tagged sensory kinases, and other divalent metal ions could not elicit self-association of BaeS. The histidine residues in the BaeS periplasmic domain are essential for copper binding in vitro and are important for the copper-induced BaeS responses in vivo. BaeS clustering was triggered also under conditions that directly triggered BaeS-dependent transcriptional responses. Thus, clustering of sensory kinase receptors can be dynamic and context dependent and can be triggered by specific environmental cues. PMID:26950881

  7. Dynamic Clustering of the Bacterial Sensory Kinase BaeS

    PubMed Central

    Koler, Moriah; Frank, Vered; Amartely, Hadar; Friedler, Assaf; Vaknin, Ady

    2016-01-01

    Several bacterial sensory-kinase receptors form clusters on the cell membrane. However, the dynamics of sensory-kinase clustering are largely unclear. Using measurements of fluorescence anisotropy and time-lapse imaging of Escherichia coli cells, we demonstrate that copper ions trigger self-association of BaeS receptors and lead to rapid formation of clusters, which can be reversibly dispersed by a metal chelator. Copper ions did not trigger self-association of other fluorescently tagged sensory kinases, and other divalent metal ions could not elicit self-association of BaeS. The histidine residues in the BaeS periplasmic domain are essential for copper binding in vitro and are important for the copper-induced BaeS responses in vivo. BaeS clustering was triggered also under conditions that directly triggered BaeS-dependent transcriptional responses. Thus, clustering of sensory kinase receptors can be dynamic and context dependent and can be triggered by specific environmental cues. PMID:26950881

  8. Protein kinase D activity controls endothelial nitric oxide synthesis.

    PubMed

    Aicart-Ramos, Clara; Sánchez-Ruiloba, Lucía; Gómez-Parrizas, Mónica; Zaragoza, Carlos; Iglesias, Teresa; Rodríguez-Crespo, Ignacio

    2014-08-01

    Vascular endothelial growth factor (VEGF) regulates key functions of the endothelium, such as angiogenesis or vessel repair in processes involving endothelial nitric oxide synthase (eNOS) activation. One of the effector kinases that become activated in endothelial cells upon VEGF treatment is protein kinase D (PKD). Here, we show that PKD phosphorylates eNOS, leading to its activation and a concomitant increase in NO synthesis. Using mass spectrometry, we show that the purified active kinase specifically phosphorylates recombinant eNOS on Ser1179. Treatment of endothelial cells with VEGF or phorbol 12,13-dibutyrate (PDBu) activates PKD and increases eNOS Ser1179 phosphorylation. In addition, pharmacological inhibition of PKD and gene silencing of both PKD1 and PKD2 abrogate VEGF signaling, resulting in a clear diminished migration of endothelial cells in a wound healing assay. Finally, inhibition of PKD in mice results in an almost complete disappearance of the VEGF-induced vasodilatation, as monitored through determination of the diameter of the carotid artery. Hence, our data indicate that PKD is a new regulatory kinase of eNOS in endothelial cells whose activity orchestrates mammalian vascular tone. PMID:24928905

  9. Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters.

    PubMed

    Bermingham, Daniel P; Blakely, Randy D

    2016-10-01

    Modulation of neurotransmission by the monoamines dopamine (DA), norepinephrine (NE), and serotonin (5-HT) is critical for normal nervous system function. Precise temporal and spatial control of this signaling in mediated in large part by the actions of monoamine transporters (DAT, NET, and SERT, respectively). These transporters act to recapture their respective neurotransmitters after release, and disruption of clearance and reuptake has significant effects on physiology and behavior and has been linked to a number of neuropsychiatric disorders. To ensure adequate and dynamic control of these transporters, multiple modes of control have evolved to regulate their activity and trafficking. Central to many of these modes of control are the actions of protein kinases, whose actions can be direct or indirectly mediated by kinase-modulated protein interactions. Here, we summarize the current state of our understanding of how protein kinases regulate monoamine transporters through changes in activity, trafficking, phosphorylation state, and interacting partners. We highlight genetic, biochemical, and pharmacological evidence for kinase-linked control of DAT, NET, and SERT and, where applicable, provide evidence for endogenous activators of these pathways. We hope our discussion can lead to a more nuanced and integrated understanding of how neurotransmitter transporters are controlled and may contribute to disorders that feature perturbed monoamine signaling, with an ultimate goal of developing better therapeutic strategies. PMID:27591044

  10. Design and combinatorial synthesis of a novel kinase-focused library using click chemistry-based fragment assembly.

    PubMed

    Irie, Takayuki; Fujii, Ikuo; Sawa, Masaaki

    2012-01-01

    Fragment-based lead discovery is a new approach for lead generation that has emerged in the past decade. Because the initial fragments identified in the fragment screening typically show weak binding affinity, an intensive medicinal chemistry effort would be required to grow initial fragments into a potential lead compound. Here we demonstrate a kinase focused evolved fragment (KFEF) library, constructed by click chemistry-based fragment assembly, that is a valuable source of kinase inhibitors. This combinatorial assembly of two fragments, kinase-privileged alkyne fragments and diversified azide fragments, by two cycloaddition reactions shows a unique potential for the one-step synthesis of structurally diverse evolved fragments. The screening of this triazole-based KFEF library allowed the rapid identification of potent lead candidates for FLT3 and GSK3β kinase.

  11. Where Will LEAD Lead? An Update on My LEAD Research.

    ERIC Educational Resources Information Center

    Wildman, Louis

    Issues in the future of a federal collaborative program, Leadership in Educational Administration Development (LEAD), are discussed in this paper. Problems attributed to LEAD are its antagonistic posture to educational administration programs in higher education and political conflicts of interest. Methodology involved analysis of successful LEAD…

  12. Potential lead exposures from lead crystal decanters.

    PubMed Central

    Appel, B R; Kahlon, J K; Ferguson, J; Quattrone, A J; Book, S A

    1992-01-01

    We measured the concentrations of lead leached into 4% acetic acid, white port, and a synthetic alcoholic beverage that were stored in lead crystal decanters for 1-, 2-, and 10-day periods at room temperature. In decanters from 14 different manufacturers, measured lead concentrations ranged from 100 to 1800 micrograms/L. The pH of the leaching medium is probably the dominant factor determining the extent of lead leached, with greater leaching occurring at lower pH values. The consumption of alcoholic beverages stored in lead crystal decanters is judged to pose a hazard. PMID:1456345

  13. Endocytosis of Receptor Tyrosine Kinases

    PubMed Central

    Goh, Lai Kuan

    2013-01-01

    Endocytosis is the major regulator of signaling from receptor tyrosine kinases (RTKs). The canonical model of RTK endocytosis involves rapid internalization of an RTK activated by ligand binding at the cell surface and subsequent sorting of internalized ligand-RTK complexes to lysosomes for degradation. Activation of the intrinsic tyrosine kinase activity of RTKs results in autophosphorylation, which is mechanistically coupled to the recruitment of adaptor proteins and conjugation of ubiquitin to RTKs. Ubiquitination serves to mediate interactions of RTKs with sorting machineries both at the cell surface and on endosomes. The pathways and kinetics of RTK endocytic trafficking, molecular mechanisms underlying sorting processes, and examples of deviations from the standard trafficking itinerary in the RTK family are discussed in this work. PMID:23637288

  14. Lead in petrol. The isotopic lead experiment

    SciTech Connect

    Facchetti, S. )

    1989-10-01

    Many studies were dedicated to the evaluation of the impact of automotive lead on the environment and to the assessment of its absorption in the human population. They can be subdivided into two groups, those based on changes of air and blood lead concentrations and those based on changes of air and blood lead isotopic compositions. According to various authors, 50-66% of the lead added to petrol is mobilized in the atmosphere, while most of the remainder adheres to the walls of the exhaust system from which it is expelled by mechanical and thermal shocks in the forms of easily sedimented particles. The fraction directly emitted by engine exhaust fumes is found in the form of fine particles, which can be transferred a long way from the emitting sources. However important the contribution of petrol lead to the total airborne lead may be, our knowledge does not permit a straightforward calculation of the percentage of petrol lead in total blood lead, which of course can also originate from other sources (e.g., industrial, natural). To evaluate this percentage in 1973, the idea of the Isotopic Lead Experiment (ILE project) was conceived to label, on a regional scale, petrol with a nonradioactive lead of an isotopic composition sufficiently different from that of background lead and sufficiently stable in time. This Account summarizes the main results obtained by the ILE project.

  15. Oncoprotein protein kinase antibody kit

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    2008-12-23

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  16. Bone lead, hypertension, and lead nephropathy

    SciTech Connect

    Wedeen, R.P.

    1988-06-01

    There is considerable clinical evidence that excessive lead absorption causes renal failure with hypertension and predisposes individuals to hypertension even in the absence of detectable renal failure. Recent analyses of transiliac bone biopsies indicate that unsuspected elevated bone leads may reflect the cause (or contributing cause) of end-stage renal disease in 5% of the European dialysis population. In these patients, bone lead levels were four times higher than in unexposed cadavers (6 micrograms/g wet weight) and approximated levels found in lead workers (30 micrograms/g). At present, the most reliable index of the body lead burden is the CaNa2 EDTA lead mobilization test. In vivo tibial X-ray-induced X-ray fluorescence (XRF) is a more practical noninvasive technique for assessing bone lead, which should find widespread application as a diagnostic tool and for epidemiologic studies.

  17. Antihypertensive action of 2-hydroxyoleic acid in SHRs via modulation of the protein kinase A pathway and Rho kinase.

    PubMed

    Alemany, Regina; Vögler, Oliver; Terés, Silvia; Egea, Carolina; Baamonde, Carmela; Barceló, Francisca; Delgado, Carlos; Jakobs, Karl H; Escribá, Pablo V

    2006-08-01

    Olive oil consumption leads to high monounsaturated fatty acid intake, especially oleic acid, and has been associated with a reduced risk of hypertension. However, the molecular mechanisms and contribution of its different components to lower blood pressure (BP) require further evaluation. Here, we examined whether a synthetic, non-beta-oxidation-metabolizable derivative of oleic acid, 2-hydroxyoleic acid (2-OHOA), can normalize BP in adult spontaneously hypertensive rats (SHRs) and whether its antihypertensive action involves cAMP-dependent protein kinase A (PKA) and Rho kinase, two major regulators of vascular smooth muscle contraction. Oral administration of 2-OHOA to SHRs induced sustained systolic BP decreases in a time-dependent (1-7 days) and dose-dependent (100-900 mg/kg every 12 h) manner. After 7 days of treatment with 2-OHOA (600 mg/kg), the systolic BP of SHRs was similar to that of normotensive Wistar Kyoto rats, returning to its initial hypertensive level after withdrawal of 2-OHOA. This treatment strongly increased the protein expression of the catalytic and regulatory RIalpha and RIIalpha PKA subunits as well as PKA activity in aortas from SHRs. Consistently, administration of the PKA inhibitor 8-bromo adenosine-3',5'-cyclic monophosphorothioate, Rp isomer, to 2-OHOA-treated SHRs induced a pronounced reversal (up to 59%) of the antihypertensive effect of 2-OHOA. Additionally, 2-OHOA completely reversed the pathological overexpression of aortic Rho kinase found in SHRs, suppressing the vasoconstrictory Rho kinase pathway.

  18. Evolution of breast cancer therapeutics: Breast tumour kinase's role in breast cancer and hope for breast tumour kinase targeted therapy.

    PubMed

    Hussain, Haroon A; Harvey, Amanda J

    2014-08-10

    There have been significant improvements in the detection and treatment of breast cancer in recent decades. However, there is still a need to develop more effective therapeutic techniques that are patient specific with reduced toxicity leading to further increases in patients' overall survival; the ongoing progress in understanding recurrence, resistant and spread also needs to be maintained. Better understanding of breast cancer pathology, molecular biology and progression as well as identification of some of the underlying factors involved in breast cancer tumourgenesis and metastasis has led to the identification of novel therapeutic targets. Over a number of years interest has risen in breast tumour kinase (Brk) also known as protein tyrosine kinase 6; the research field has grown and Brk has been described as a desirable therapeutic target in relation to tyrosine kinase inhibition as well as disruption of its kinase independent activity. This review will outline the current "state of play" with respect to targeted therapy for breast cancer, as well as discussing Brk's role in the processes underlying tumour development and metastasis and its potential as a therapeutic target in breast cancer.

  19. FMLP activates Ras and Raf in human neutrophils. Potential role in activation of MAP kinase.

    PubMed Central

    Worthen, G S; Avdi, N; Buhl, A M; Suzuki, N; Johnson, G L

    1994-01-01

    Chemoattractants bind to seven transmembrane-spanning, G-protein-linked receptors on polymorphonuclear leukocytes (neutrophils) and induce a variety of functional responses, including activation of microtubule-associated protein (MAP) kinase. Although the pathways by which MAP kinases are activated in neutrophils are unknown, we hypothesized that activation of the Ras/Raf pathway leading to activation of MAP/ERK kinase (MEK) would be induced by the chemoattractant f-met-leu-phe. Human neutrophils exposed to 10 nM FMLP for 30 s exhibited an MAP kinase kinase activity coeluting with MEK-1. Immunoprecipitation of Raf-1 kinase after stimulation with FMLP revealed an activity that phosphorylated MEK, was detectable at 30 s, and peaked at 2-3 min. Immunoprecipitation of Ras from both intact neutrophils labeled with [32P]orthophosphate and electropermeabilized neutrophils incubated with [32P]GTP was used to determine that FMLP treatment was associated with activation of Ras. Activation of both Ras and Raf was inhibited by treatment of neutrophils with pertussis toxin, indicating predominant linkage to the Gi2 protein. Although phorbol esters activated Raf, activation induced by FMLP appeared independent of protein kinase C, further suggesting that Gi2 was linked to Ras and Raf independent of phospholipase C and protein kinase C. Dibutyryl cAMP, which inhibits many neutrophil functional responses, blocked the activation of Raf by FMLP, suggesting that interruption of the Raf/MAP kinase pathway influences neutrophil responses to chemoattractants. These data suggest that Gi2-mediated receptor regulation of the Ras/Raf/MAP kinase pathway is a primary response to chemoattractants. Images PMID:8040337

  20. Differential regulation of Jun N-terminal kinase and p38MAP kinase by Galpha12.

    PubMed

    Dermott, Jonathan M; Ha, Ji Hee; Lee, Chang Ho; Dhanasekaran, N

    2004-01-01

    Based on the findings that the overexpression of the wild-type Galpha(12) (Galpha(12)WT) result in the oncogenic transformation of NIH3T3 cells in a serum-dependent manner, a model system has been established in which the mitogenic and subsequent cell transformation pathways activated by Galpha(12) can be turned on or off by the addition or removal of serum. Using this model system, our previous studies have shown that the stimulation of Galpha(12)WT or the expression of an activated mutant of Galpha(12) (Galpha(12)QL) leads to increased cell proliferation and subsequent oncogenic transformation of NIH3T3 cells, as well as persistent activation of Jun N-terminal kinases (JNKs). In the present studies, we show that the stimulation of Galpha(12)WT or the expression of Galpha(12)QL results in a potent inhibition of p38MAPK, and that the mechanism by which Galpha(12) inhibits p38MAPK activity involves the dual specificity kinases upstream of p38MAPK. The results indicate that Galpha(12) attenuates the activation of MKK3 and MKK4, which are known to stimulate only p38MAPK or p38MAPK and JNK, respectively. The results also suggest that Galpha(12) activates JNKs specifically through the stimulation of the JNK-specific upstream kinase MKK7. These findings demonstrate for the first time that Galpha(12) differentially regulates JNK and p38MAPK by specifically activating MKK7, while inhibiting MKK3 and MKK4 in NIH3T3 cells. Since the stimulation of p38MAPK is often associated with apoptotic responses, our findings suggest that Galpha(12) stimulates cell proliferation and neoplastic transformation of NIH3T3 cells by attenuating p38MAPK-associated apoptotic responses, while activating the mitogenic responses through the stimulation of ERK- and JNK-mediated signaling pathways. PMID:14712227

  1. Lead poisoning in children.

    PubMed

    Dapul, Heda; Laraque, Danielle

    2014-08-01

    There is no safe lead level in children. Primary prevention is the most effective way to bring about the complete removal of lead from the environment and eliminate lead poisoning as a public health concern. The National Lead Information Center can be reached via the Internet at www.epa.gov/lead and www.hud.gov/lead, or via phone at 1-800-424-LEAD (5323).

  2. DYRK1A kinase inhibitors with emphasis on cancer.

    PubMed

    Ionescu, A; Dufrasne, F; Gelbcke, M; Jabin, I; Kiss, R; Lamoral-Theys, D

    2012-11-01

    Various types of cancers (including gliomas, melanomas, and esophageal, pancreas and non-small-cell lung cancers) display intrinsic resistance to pro-apoptotic stimuli, such as conventional chemotherapy and radiotherapy, and/or the activation of a multidrug resistance phenotype, which are major barriers to effective treatment and lead to poor patient prognosis. The DYRK1A kinase is directly implicated in the resistance of cancer cells to pro-apoptotic stimuli and drives several pathways that enhance proliferation, migration, and the reduction of cell death, leading to very aggressive biological behavior in cancer cell populations. The DYRK1A kinase is also implicated in neurological diseases and in neoangiogenic processes. Thus, the DYRK1A kinase is of great interest for both cancer and neuroscience research. During the last decade, numerous compounds that inhibit DYRK1A have been synthesized. The present review discusses the available molecules known to interfere with DYRK1A activity and the implications of DYRK1A in cancer and other diseases and serves as a rational analysis for researchers who aim to improve the anti-DYRK1A activity of currently available compounds. PMID:23016545

  3. FES/FER kinase signaling in hematopoietic cells and leukemias.

    PubMed

    Craig, Andrew W B

    2012-01-01

    FES and FES-related (FER) comprise a unique subfamily of protein-tyrosine kinases (PTKs) that signal downstream of several classes of receptors involved in regulating hematopoietic cell development, survival, migration, and inflammatory mediator release. Activated alleles of FES are potent inducers of myeloid differentiation, however FES-deficient mice have only subtle differences in hematopoiesis. This may reflect overlapping function of other kinases such as FER. Studies of FES- and FER-deficient mice have revealed more prominent roles in regulating the activation of mature innate immune cells, including macrophages and mast cells. Recently, new insights into regulation of FES/FER kinases has emerged with the characterization of their N-terminal phospholipid-binding and membrane targeting FER/CIP4 homology-Bin/Amphyphysin/Rvs (F-BAR) and F-BAR extension (FX) domains. The F-BAR/FX domains regulate subcellular localization and FES/FER kinase activation. FES kinase activity is also enhanced upon ligand binding to its SH2 domain, which may lead to further phosphorylation of the same ligand, or other ligand-associated proteins. In mast cells, SH2 ligands of FES/FER include KIT receptor PTK, and the high affinity IgE receptor (FceRI) that trigger rapid activation of FES/FER and signaling to regulators of the actin cytoskeleton and membrane trafficking. Recently, FES/FER have also been implicated in growth and survival signaling in leukemias driven by oncogenic KIT and FLT3 receptors. With further definition of their roles in immune cells and their progenitors, FES/FER may emerge as relevant therapeutic targets in inflammatory diseases and leukemias.

  4. Mitogen-activated Protein Kinase Kinase Kinase 1 Protects against Nickel-induced Acute Lung Injury

    PubMed Central

    Mongan, Maureen; Tan, Zongqing; Chen, Liang; Peng, Zhimin; Dietsch, Maggie; Su, Bing; Leikauf, George; Xia, Ying

    2008-01-01

    Nickel compounds are environmental and occupational hazards that pose serious health problems and are causative factors of acute lung injury. The c-jun N-terminal kinases (JNKs) are regulated through a mitogen-activated protein (MAP) 3 kinase-MAP2 kinase cascade and have been implicated in nickel toxicity. In this study, we used genetically modified cells and mice to investigate the involvement of two upstream MAP3Ks, MAP3K1 and 2, in nickel-induced JNK activation and acute lung injury. In mouse embryonic fibroblasts, levels of JNK activation and cytotoxicity induced by nickel were similar in the Map3k2-null and wild-type cells but were much lower in the Map3k1/Map3k2 double-null cells. Conversely, the levels of JNK activation and cytotoxicity were unexpectedly much higher in the Map3k1-null cells. In adult mouse tissue, MAP3K1 was widely distributed but was abundantly expressed in the bronchiole epithelium of the lung. Accordingly, MAP3K1 ablation in mice resulted in severe nickel-induced acute lung injury and reduced survival. Based on these findings, we propose a role for MAP3K1 in reducing JNK activation and protecting the mice from nickel-induced acute lung injury. PMID:18467339

  5. Lead and the Romans

    ERIC Educational Resources Information Center

    Reddy, Aravind; Braun, Charles L.

    2010-01-01

    Lead poisoning has been a problem since early history and continues into modern times. An appealing characteristic of lead is that many lead salts are sweet. In the absence of cane and beet sugars, early Romans used "sugar of lead" (lead acetate) to sweeten desserts, fruits, and sour wine. People most at risk would have been those who consumed the…

  6. Feasibility of using molecular docking-based virtual screening for searching dual target kinase inhibitors.

    PubMed

    Zhou, Shunye; Li, Youyong; Hou, Tingjun

    2013-04-22

    Multitarget agents have been extensively explored for solving limited efficacies, poor safety, and resistant profiles of an individual target. Theoretical approaches for searching and designing multitarget agents are critically useful. Here, the performance of molecular docking to search dual-target inhibitors for four kinase pairs (CDK2-GSK3B, EGFR-Src, Lck-Src, and Lck-VEGFR2) was assessed. First, the representative structures for each kinase target were chosen by structural clustering of available crystal structures. Next, the performance of molecular docking to distinguish inhibitors from noninhibitors for each individual kinase target was evaluated. The results show that molecular docking-based virtual screening illustrates good capability to find known inhibitors for individual targets, but the prediction accuracy is structurally dependent. Finally, the performance of molecular docking to identify the dual-target kinase inhibitors for four kinase pairs was evaluated. The analyses show that molecular docking successfully filters out most noninhibitors and achieves promising performance for identifying dual-kinase inhibitors for CDK2-GSK3B and Lck-VEGFR2. But a high false-positive rate leads to low enrichment of true dual-target inhibitors in the final list. This study suggests that molecular docking serves as a useful tool in searching inhibitors against dual or even multiple kinase targets, but integration with other virtual screening tools is necessary for achieving better predictions.

  7. Biochemical and functional characterization of novel NADH kinase in the enteric protozoan parasite Entamoeba histolytica.

    PubMed

    Jeelani, Ghulam; Husain, Afzal; Sato, Dan; Soga, Tomoyoshi; Suematsu, Makoto; Nozaki, Tomoyoshi

    2013-02-01

    NAD(H) kinase catalyzes the phosphorylation of NAD(H) to form NADP(H) using ATP or inorganic polyphosphate as a phosphoryl donor. While the enzyme is conserved throughout prokaryotes and eukaryotes, remarkable differences in kinetic parameters including substrate preference, cation dependence, and physiological roles exist among the organisms. In the present study, we biochemically characterized NAD(H) kinase from the anaerobic/microaerophilic fermentative protozoan parasite Entamoeba histolytica, which lacks the conventional mitochondria capable of oxidative phosphorylation, leading to ATP. The kinetic properties of E. histolytica NAD(H) kinase recombinantly produced in Escherichia coli showed remarkable differences from those in bacteria and higher eukaryotes. Entamoeba NAD(H) kinase preferred NADH to NAD+ as the phosphoryl acceptor, utilized nucleoside triphosphates including ATP, GTP and deoxyATP, but not nucleoside di-, mono-phosphates, or inorganic polyphosphates, as the phosphoryl donor. To further understand the physiological roles in E. histolytica, we generated a stable transformant overexpressing NAD(H) kinase. Overexpression of NAD(H) kinase resulted in a 1.6-2 fold increase in the NADPH and NADP+ concentrations, a 40% reduction of the intracellular concentration of reactive oxygen species, and also led to increased tolerance toward hydrogen peroxide. These data, together with the essentially of NAD(H) kinase gene, underscore its significance as an NADP(H)-producing enzyme in this organism, and should help in designing of drugs targeting this enzyme.

  8. Selective inhibition of the kinase DYRK1A by targeting its folding process.

    PubMed

    Kii, Isao; Sumida, Yuto; Goto, Toshiyasu; Sonamoto, Rie; Okuno, Yukiko; Yoshida, Suguru; Kato-Sumida, Tomoe; Koike, Yuka; Abe, Minako; Nonaka, Yosuke; Ikura, Teikichi; Ito, Nobutoshi; Shibuya, Hiroshi; Hosoya, Takamitsu; Hagiwara, Masatoshi

    2016-01-01

    Autophosphorylation of amino-acid residues is part of the folding process of various protein kinases. Conventional chemical screening of mature kinases has missed inhibitors that selectively interfere with the folding process. Here we report a cell-based assay that evaluates inhibition of a kinase at a transitional state during the folding process and identify a folding intermediate-selective inhibitor of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), which we refer to as FINDY. FINDY suppresses intramolecular autophosphorylation of Ser97 in DYRK1A in cultured cells, leading to its degradation, but does not inhibit substrate phosphorylation catalysed by the mature kinase. FINDY also suppresses Ser97 autophosphorylation of recombinant DYRK1A, suggesting direct inhibition, and shows high selectivity for DYRK1A over other DYRK family members. In addition, FINDY rescues DYRK1A-induced developmental malformations in Xenopus laevis embryos. Our study demonstrates that transitional folding intermediates of protein kinases can be targeted by small molecules, and paves the way for developing novel types of kinase inhibitors. PMID:27102360

  9. Investigation of potential glycogen synthase kinase 3 inhibitors using pharmacophore mapping and virtual screening.

    PubMed

    Dessalew, Nigus; Bharatam, Prasad V

    2006-09-01

    Glycogen synthase kinase-3 is a serine/threonine kinase that has attracted significant drug discovery attention in recent years. To investigate the identification of new potential glycogen synthase kinase-3 inhibitors, a pharmacophore mapping study was carried out using a set of 21 structurally diverse glycogen synthase kinase-3 inhibitors. A hypothesis containing four features: two hydrophobic, one hydrogen bond donor and another hydrogen bond acceptor was found to be the best from the 10 common feature hypotheses produced by HipHop module of Catalyst. The best hypothesis has a high cost of 156.592 and higher best fit values were obtained for the 21 inhibitors using this best hypothesis than the other HipHop hypotheses. The best hypothesis was then used to screen electronically the NCI2000 database. The hits obtained were docked into glycogen synthase kinase-3beta active site. A total of five novel potential leads were proposed after: (i) visual examination of how well they dock into the glycogen synthase kinase-3beta-binding site, (ii) comparative analysis of their FlexX, G-Score, PMF-Score, ChemScore and D-Scores values, (iii) comparison of their best fit value with the known inhibitors and (iv) examination of the how the hits retain interactions with the important amino acid residues of glycogen synthase kinase-3beta-binding site. PMID:17062013

  10. Design of substrate-based BCR-ABL kinase inhibitors using the cyclotide scaffold

    PubMed Central

    Huang, Yen-Hua; Henriques, Sónia T.; Wang, Conan K.; Thorstholm, Louise; Daly, Norelle L.; Kaas, Quentin; Craik, David J.

    2015-01-01

    The constitutively active tyrosine kinase BCR-ABL is the underlying cause of chronic myeloid leukemia (CML). Current CML treatments rely on the long-term use of tyrosine kinase inhibitors (TKIs), which target the ATP binding site of BCR-ABL. Over the course of treatment, 20–30% of CML patients develop TKI resistance, which is commonly attributed to point mutations in the drug-binding region. We design a new class of peptide inhibitors that target the substrate-binding site of BCR-ABL by grafting sequences derived from abltide, the optimal substrate of Abl kinase, onto a cell-penetrating cyclotide MCoTI-II. Three grafted cyclotides show significant Abl kinase inhibition in vitro in the low micromolar range using a novel kinase inhibition assay. Our work also demonstrates that a reengineered MCoTI-II with abltide sequences grafted in both loop 1 and 6 inhibits the activity of [T315I]Abl in vitro, a mutant Abl kinase harboring the “gatekeeper” mutation which is notorious for being multidrug resistant. Results from serum stability and cell internalization studies confirm that the MCoTI-II scaffold provides enzymatic stability and cell-penetrating properties to the lead molecules. Taken together, our study highlights that reengineered cyclotides incorporating abltide-derived sequences are promising substrate-competitive inhibitors for Abl kinase and the T315I mutant. PMID:26264857

  11. Selective inhibition of the kinase DYRK1A by targeting its folding process

    PubMed Central

    Kii, Isao; Sumida, Yuto; Goto, Toshiyasu; Sonamoto, Rie; Okuno, Yukiko; Yoshida, Suguru; Kato-Sumida, Tomoe; Koike, Yuka; Abe, Minako; Nonaka, Yosuke; Ikura, Teikichi; Ito, Nobutoshi; Shibuya, Hiroshi; Hosoya, Takamitsu; Hagiwara, Masatoshi

    2016-01-01

    Autophosphorylation of amino-acid residues is part of the folding process of various protein kinases. Conventional chemical screening of mature kinases has missed inhibitors that selectively interfere with the folding process. Here we report a cell-based assay that evaluates inhibition of a kinase at a transitional state during the folding process and identify a folding intermediate-selective inhibitor of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), which we refer to as FINDY. FINDY suppresses intramolecular autophosphorylation of Ser97 in DYRK1A in cultured cells, leading to its degradation, but does not inhibit substrate phosphorylation catalysed by the mature kinase. FINDY also suppresses Ser97 autophosphorylation of recombinant DYRK1A, suggesting direct inhibition, and shows high selectivity for DYRK1A over other DYRK family members. In addition, FINDY rescues DYRK1A-induced developmental malformations in Xenopus laevis embryos. Our study demonstrates that transitional folding intermediates of protein kinases can be targeted by small molecules, and paves the way for developing novel types of kinase inhibitors. PMID:27102360

  12. Investigation of potential glycogen synthase kinase 3 inhibitors using pharmacophore mapping and virtual screening.

    PubMed

    Dessalew, Nigus; Bharatam, Prasad V

    2006-09-01

    Glycogen synthase kinase-3 is a serine/threonine kinase that has attracted significant drug discovery attention in recent years. To investigate the identification of new potential glycogen synthase kinase-3 inhibitors, a pharmacophore mapping study was carried out using a set of 21 structurally diverse glycogen synthase kinase-3 inhibitors. A hypothesis containing four features: two hydrophobic, one hydrogen bond donor and another hydrogen bond acceptor was found to be the best from the 10 common feature hypotheses produced by HipHop module of Catalyst. The best hypothesis has a high cost of 156.592 and higher best fit values were obtained for the 21 inhibitors using this best hypothesis than the other HipHop hypotheses. The best hypothesis was then used to screen electronically the NCI2000 database. The hits obtained were docked into glycogen synthase kinase-3beta active site. A total of five novel potential leads were proposed after: (i) visual examination of how well they dock into the glycogen synthase kinase-3beta-binding site, (ii) comparative analysis of their FlexX, G-Score, PMF-Score, ChemScore and D-Scores values, (iii) comparison of their best fit value with the known inhibitors and (iv) examination of the how the hits retain interactions with the important amino acid residues of glycogen synthase kinase-3beta-binding site.

  13. Protein Kinase C δ: a Gatekeeper of Immune Homeostasis.

    PubMed

    Salzer, Elisabeth; Santos-Valente, Elisangela; Keller, Bärbel; Warnatz, Klaus; Boztug, Kaan

    2016-10-01

    Human autoimmune disorders present in various forms and are associated with a life-long burden of high morbidity and mortality. Many different circumstances lead to the loss of immune tolerance and often the origin is suspected to be multifactorial. Recently, patients with autosomal recessive mutations in PRKCD encoding protein kinase c delta (PKCδ) have been identified, representing a monogenic prototype for one of the most prominent forms of humoral systemic autoimmune diseases, systemic lupus erythematosus (SLE). PKCδ is a signaling kinase with multiple downstream target proteins and with functions in various signaling pathways. Interestingly, mouse models have indicated a special role of the ubiquitously expressed protein in the control of B-cell tolerance revealed by the severe autoimmunity in Prkcd (-/-) knockout mice as the major phenotype. As such, the study of PKCδ deficiency in humans has tremendous potential in enhancing our knowledge on the mechanisms of B-cell tolerance. PMID:27541826

  14. Protein Kinase C δ: a Gatekeeper of Immune Homeostasis.

    PubMed

    Salzer, Elisabeth; Santos-Valente, Elisangela; Keller, Bärbel; Warnatz, Klaus; Boztug, Kaan

    2016-10-01

    Human autoimmune disorders present in various forms and are associated with a life-long burden of high morbidity and mortality. Many different circumstances lead to the loss of immune tolerance and often the origin is suspected to be multifactorial. Recently, patients with autosomal recessive mutations in PRKCD encoding protein kinase c delta (PKCδ) have been identified, representing a monogenic prototype for one of the most prominent forms of humoral systemic autoimmune diseases, systemic lupus erythematosus (SLE). PKCδ is a signaling kinase with multiple downstream target proteins and with functions in various signaling pathways. Interestingly, mouse models have indicated a special role of the ubiquitously expressed protein in the control of B-cell tolerance revealed by the severe autoimmunity in Prkcd (-/-) knockout mice as the major phenotype. As such, the study of PKCδ deficiency in humans has tremendous potential in enhancing our knowledge on the mechanisms of B-cell tolerance.

  15. In-situ coupling between kinase activities and protein dynamics within single focal adhesions

    PubMed Central

    Wu, Yiqian; Zhang, Kaiwen; Seong, Jihye; Fan, Jason; Chien, Shu; Wang, Yingxiao; Lu, Shaoying

    2016-01-01

    The dynamic activation of oncogenic kinases and regulation of focal adhesions (FAs) are crucial molecular events modulating cell adhesion in cancer metastasis. However, it remains unclear how these events are temporally coordinated at single FA sites. Therefore, we targeted fluorescence resonance energy transfer (FRET)-based biosensors toward subcellular FAs to report local molecular events during cancer cell adhesion. Employing single FA tracking and cross-correlation analysis, we quantified the dynamic coupling characteristics between biochemical kinase activities and structural FA within single FAs. We show that kinase activations and FA assembly are strongly and sequentially correlated, with the concurrent FA assembly and Src activation leading focal adhesion kinase (FAK) activation by 42.6 ± 12.6 sec. Strikingly, the temporal coupling between kinase activation and individual FA assembly reflects the fate of FAs at later stages. The FAs with a tight coupling tend to grow and mature, while the less coupled FAs likely disassemble. During FA disassembly, however, kinase activations lead the disassembly, with FAK being activated earlier than Src. Therefore, by integrating subcellularly targeted FRET biosensors and computational analysis, our study reveals intricate interplays between Src and FAK in regulating the dynamic life of single FAs in cancer cells. PMID:27383747

  16. In-situ coupling between kinase activities and protein dynamics within single focal adhesions.

    PubMed

    Wu, Yiqian; Zhang, Kaiwen; Seong, Jihye; Fan, Jason; Chien, Shu; Wang, Yingxiao; Lu, Shaoying

    2016-01-01

    The dynamic activation of oncogenic kinases and regulation of focal adhesions (FAs) are crucial molecular events modulating cell adhesion in cancer metastasis. However, it remains unclear how these events are temporally coordinated at single FA sites. Therefore, we targeted fluorescence resonance energy transfer (FRET)-based biosensors toward subcellular FAs to report local molecular events during cancer cell adhesion. Employing single FA tracking and cross-correlation analysis, we quantified the dynamic coupling characteristics between biochemical kinase activities and structural FA within single FAs. We show that kinase activations and FA assembly are strongly and sequentially correlated, with the concurrent FA assembly and Src activation leading focal adhesion kinase (FAK) activation by 42.6 ± 12.6 sec. Strikingly, the temporal coupling between kinase activation and individual FA assembly reflects the fate of FAs at later stages. The FAs with a tight coupling tend to grow and mature, while the less coupled FAs likely disassemble. During FA disassembly, however, kinase activations lead the disassembly, with FAK being activated earlier than Src. Therefore, by integrating subcellularly targeted FRET biosensors and computational analysis, our study reveals intricate interplays between Src and FAK in regulating the dynamic life of single FAs in cancer cells. PMID:27383747

  17. Receptor Tyrosine Kinases in Drosophila Development

    PubMed Central

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

    Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases. PMID:23732470

  18. Protein kinase D enzymes – novel kinase targets in pancreatic cancer

    PubMed Central

    Liou, Geou-Yarh; Storz, Peter

    2016-01-01

    Summary Pancreatic ductal adenocarcinoma (PDA) is characterized by advanced stage desmoplastic tumors with a high prevalence of genetic abnormalities. Occurrence of PDA is linked to activating Kras mutations and aberrant epidermal growth factor receptor signaling, leading to additional activation of wildtype Kras. Since Kras is difficult to target, there is a constant need to identify novel targets acting downstream of this molecule in driving the formation or progression of PDA. Recently, it was shown that Protein kinase D enzymes not only are increasingly expressed in PDA, but also causatively linked to the development and progression of this cancer. They act downstream of both mutant Kras and growth factors and therefore may represent ideal novel targets. PMID:26174103

  19. Regulation of Sphingolipid Biosynthesis by the Morphogenesis Checkpoint Kinase Swe1*

    PubMed Central

    Chauhan, Neha; Han, Gongshe; Somashekarappa, Niranjanakumari; Gable, Kenneth; Dunn, Teresa; Kohlwein, Sepp D.

    2016-01-01

    Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticulum-localized Orm family proteins. Defective SL synthesis in Saccharomyces cerevisiae leads to increased phosphorylation and inhibition of Orm proteins by the kinase Ypk1. Here we present evidence that the yeast morphogenesis checkpoint kinase, Swe1, regulates SL biosynthesis independent of the Ypk1 pathway. Deletion of the Swe1 kinase renders mutant cells sensitive to serine palmitoyltransferase inhibition due to impaired sphingoid long-chain base synthesis. Based on these data and previous results, we suggest that Swe1 kinase perceives alterations in SL homeostasis, activates SL synthesis, and may thus represent the missing regulatory link that controls the SL rheostat during the cell cycle. PMID:26634277

  20. The role and targeting of Aurora kinases in head and neck cancer

    PubMed Central

    Mehra, Ranee; Serebriiskii, Ilya G.; Burtness, Barbara; Astsaturov, Igor; Golemis, Erica A.

    2014-01-01

    Summary Controlled activation of the Aurora kinases regulates mitotic progression in normal cells. Overexpression and hyperactivation of the Aurora-A and -B kinases play a leading role in tumorigenesis, inducing aneuploidy and genomic instability. In squamous cell carcinomas of the head and neck (SCCHN), overexpression of Aurora-A is associated with decreased survival, and reduction of Aurora-A and -B expression inhibits SCCHN cell growth and increases apoptosis. In this article, we provide a basic overview of the biological functions of Aurora kinases in normal cells and in cancer, and review both small studies and high throughput datasets that implicate Aurora-A, particularly, in the pathogenesis of SCCHN. Early phase clinical trials are beginning to evaluate the activity of small molecule inhibitors of the Aurora kinases. We summarize the state of current trials evaluating Aurora inhibitors in SCCHN, and discuss rational directions for future drug combination trials and biomarkers for use with Aurora-inhibiting agents. PMID:23993387

  1. Discovery of Bivalent Kinase Inhibitors via Enzyme-Templated Fragment Elaboration

    PubMed Central

    2015-01-01

    We have employed novel fragment-based screening methodology to discover bivalent kinase inhibitors with improved selectivity. Starting from a low molecular weight promiscuous kinase inhibitor, we appended a thiol for subsequent reaction with a library of acrylamide electrophiles. Enzyme-templated screening was performed to identify acrylamides that assemble into bivalent inhibitors of c-Src kinase. Upon identification of acrylamide fragments that improve the binding affinity of our lead thiol, we characterized the resulting bivalent inhibitors and identified a series of kinase inhibitors with improved potency and selectivity compared to the thiol-containing precursor. Provided that protein can be prepared free of endogenous reactive cysteines, our methodology is general and could be applied to nearly any enzyme of interest. PMID:26286460

  2. Protein Kinase D family kinases: roads start to segregate.

    PubMed

    Wille, Christoph; Seufferlein, Thomas; Eiseler, Tim

    2014-01-01

    Highly invasive pancreatic tumors are often recognized in late stages due to a lack of clear symptoms and pose major challenges for treatment and disease management. Broad-band Protein Kinase D (PKD) inhibitors have recently been proposed as additional treatment option for this disease. PKDs are implicated in the control of cancer cell motility, angiogenesis, proliferation and metastasis. In particular, PKD2 expression is elevated in pancreatic cancer, whereas PKD1 expression is comparably lower. In our recent study we report that both kinases control PDAC cell invasive properties in an isoform-specific, but opposing manner. PKD1 selectively mediates anti-migratory/anti-invasive features by preferential regulation of the actin-regulatory Cofilin-phosphatase Slingshot1L (SSH1L). PKD2, on the other hand enhances invasion and angiogenesis of PDAC cells in 3D-ECM cultures and chorioallantois tumor models by stimulating expression and secretion of matrix-metalloproteinase 7 and 9 (MMP7/9). MMP9 also enhances PKD2-mediated tumor angiogenesis releasing extracellular matrix-bound VEGF-A. We thus suggest high PKD2 expression and loss of PKD1 may be beneficial for tumor cells to enhance their matrix-invading abilities. In our recent study we demonstrate for the first time PKD1 and 2 isoform-selective effects on pancreatic cancer cell invasion, in-vitro and in-vivo, defining isoform-specific regulation of PKDs as a major future issue. PMID:24847910

  3. Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and stress-activated protein kinase pathways that converge on Elk-1.

    PubMed Central

    Zinck, R; Cahill, M A; Kracht, M; Sachsenmaier, C; Hipskind, R A; Nordheim, A

    1995-01-01

    Inhibitors of protein synthesis, such as anisomycin and cycloheximide, lead to superinduction of immediate-early genes. We demonstrate that these two drugs activate intracellular signaling pathways involving both the mitogen-activated protein kinase (MAPK) and stress-activated protein kinase (SAPK) cascades. The activation of either pathway correlates with phosphorylation of the c-fos regulatory transcription factor Elk-1. In HeLa cells, anisomycin stabilizes c-fos mRNA when protein synthesis is inhibited to only 50%. Under these conditions, anisomycin, in contrast to cycloheximide, rapidly induces kinase activation and efficient Elk-1 phosphorylation. However, full inhibition of translation by either drug leads to prolonged activation of SAPK activity, while MAPK induction is transient. This correlates with prolonged Elk-1 phosphorylation and c-fos transcription. Elk-1 induction and c-fos activation are also observed in KB cells, in which anisomycin strongly induces SAPKs but not MAPKs. Purified p54 SAPK alpha efficiently phosphorylates the Elk-1 C-terminal domain in vitro and comigrates with anisomycin-activated kinases in in-gel kinase assays. Thus, Elk-1 provides a potential convergence point for the MAPK and SAPK signaling pathways. The activation of signal cascades and control of transcription factor function therefore represent prominent processes in immediate-early gene superinduction. PMID:7651411

  4. Pivotal Role of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 in Inflammatory Pulmonary Diseases

    PubMed Central

    Qian, Feng; Deng, Jing; Wang, Gang; Ye, Richard D.; Christman, John W.

    2016-01-01

    Mitogen-activated protein kinase (MAPK)-activated protein kinase (MK2) is exclusively regulated by p38 MAPK in vivo. Upon activation of p38 MAPK, MK2 binds with p38 MAPK, leading to phosphorylation of TTP, Hsp27, Akt and Cdc25 that are involved in regulation of various essential cellular functions. In this review, we discuss current knowledge about molecular mechanisms of MK2 in regulation of TNF-α production, NADPH oxidase activation, neutrophil migration, and DNA-damage-induced cell cycle arrest which are involved in the molecular pathogenesis of acute lung injury, pulmonary fibrosis, and non-small-cell lung cancer. Collectively current and emerging new information indicate that developing MK2 inhibitors and blocking MK2-mediated signal pathways is a potential therapeutic strategy for treatment of inflammatory and fibrotic lung diseases and lung cancer. PMID:26119506

  5. Tec family kinases in inflammation and disease.

    PubMed

    Horwood, Nicole J; Urbaniak, Ania M; Danks, Lynett

    2012-04-01

    Over the last decade, the Tec family of nonreceptor tyrosine kinases (Btk, Tec, Bmx, Itk, and Rlk) have been shown to play a key role in inflammation and bone destruction. Bruton's tyrosine kinase (Btk) has been the most widely studied due to the critical role of this kinase in B-cell development and recent evidence showing that blocking Btk signaling is effective in ameliorating lymphoma progression and experimental arthritis. This review will examine the role of TFK in myeloid cell function and the potential of targeting these kinases as a therapeutic intervention in autoimmune disorders such as rheumatoid arthritis. PMID:22449071

  6. Inhibition of the mitogen-activated protein kinase pathway triggers B16 melanoma cell differentiation.

    PubMed

    Englaro, W; Bertolotto, C; Buscà, R; Brunet, A; Pagès, G; Ortonne, J P; Ballotti, R

    1998-04-17

    In B16 melanoma cells, mitogen-activated protein (MAP) kinases are activated during cAMP-induced melanogenesis (Englaro, W., Rezzonico, R., Durand-Clément, M., Lallemand, D., Ortonne, J. P., and Ballotti, R. (1995) J. Biol. Chem. 270, 24315-24320). To establish the role of the MAP kinases in melanogenesis, we studied the effects of a specific MAP kinase kinase (MEK) inhibitor PD 98059 on different melanogenic parameters. We showed that PD 98059 inhibits the activation of MAP kinase extracellular signal-regulated kinase 1 by cAMP, but does not impair the effects of cAMP either on the morphological differentiation, characterized by an increase in dendrite outgrowth, or on the up-regulation of tyrosinase that is the key enzyme in melanogenesis. On the contrary, PD 98059 promotes by itself cell dendricity and increases the tyrosinase amount and activity. Moreover, down-regulation of the MAP kinase pathway by PD 98059, or with dominant negative mutants of p21(ras) and MEK, triggers a stimulation of the tyrosinase promoter activity and enhances the effect of cAMP on this parameter. Conversely, activation of the MAP kinase pathway, using constitutive active mutants of p21(ras) and MEK, leads to an inhibition of basal and cAMP-induced tyrosinase gene transcription. These results demonstrate that the MAP kinase pathway activation is not required for cAMP-induced melanogenesis. Furthermore, the inhibition of this pathway induces B16 melanoma cell differentiation, while a sustained activation impairs the melanogenic effect of cAMP-elevating agents. PMID:9545341

  7. Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases.

    PubMed

    Chopra, Nikita; Wales, Thomas E; Joseph, Raji E; Boyken, Scott E; Engen, John R; Jernigan, Robert L; Andreotti, Amy H

    2016-03-01

    Bruton's tyrosine kinase (Btk) is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA). Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues. In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix. We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site. PMID:27010561

  8. Dynamic Allostery Mediated by a Conserved Tryptophan in the Tec Family Kinases

    PubMed Central

    Chopra, Nikita; Wales, Thomas E.; Joseph, Raji E.; Boyken, Scott E.; Engen, John R.; Jernigan, Robert L.; Andreotti, Amy H.

    2016-01-01

    Bruton’s tyrosine kinase (Btk) is a Tec family non-receptor tyrosine kinase that plays a critical role in immune signaling and is associated with the immunological disorder X-linked agammaglobulinemia (XLA). Our previous findings showed that the Tec kinases are allosterically activated by the adjacent N-terminal linker. A single tryptophan residue in the N-terminal 17-residue linker mediates allosteric activation, and its mutation to alanine leads to the complete loss of activity. Guided by hydrogen/deuterium exchange mass spectrometry results, we have employed Molecular Dynamics simulations, Principal Component Analysis, Community Analysis and measures of node centrality to understand the details of how a single tryptophan mediates allostery in Btk. A specific tryptophan side chain rotamer promotes the functional dynamic allostery by inducing coordinated motions that spread across the kinase domain. Either a shift in the rotamer population, or a loss of the tryptophan side chain by mutation, drastically changes the coordinated motions and dynamically isolates catalytically important regions of the kinase domain. This work also identifies a new set of residues in the Btk kinase domain with high node centrality values indicating their importance in transmission of dynamics essential for kinase activation. Structurally, these node residues appear in both lobes of the kinase domain. In the N-lobe, high centrality residues wrap around the ATP binding pocket connecting previously described Catalytic-spine residues. In the C-lobe, two high centrality node residues connect the base of the R- and C-spines on the αF-helix. We suggest that the bridging residues that connect the catalytic and regulatory architecture within the kinase domain may be a crucial element in transmitting information about regulatory spine assembly to the catalytic machinery of the catalytic spine and active site. PMID:27010561

  9. Kinase-impaired BRAF mutations in lung cancer confer sensitivity to dasatinib.

    PubMed

    Sen, Banibrata; Peng, Shaohua; Tang, Ximing; Erickson, Heidi S; Galindo, Hector; Mazumdar, Tuhina; Stewart, David J; Wistuba, Ignacio; Johnson, Faye M

    2012-05-30

    During a clinical trial of the tyrosine kinase inhibitor dasatinib for advanced non-small cell lung cancer (NSCLC), one patient responded dramatically and remains cancer-free 4 years later. A comprehensive analysis of his tumor revealed a previously undescribed, kinase-inactivating BRAF mutation ((Y472C)BRAF); no inactivating BRAF mutations were found in the nonresponding tumors taken from other patients. Cells transfected with (Y472C)BRAF exhibited CRAF, MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase), and ERK (extracellular signal-regulated kinase) activation-characteristics identical to signaling changes that occur with previously known kinase-inactivating BRAF mutants. Dasatinib selectively induced senescence in NSCLC cells with inactivating BRAF mutations. Transfection of other NSCLC cells with these BRAF mutations also increased these cells' dasatinib sensitivity, whereas transfection with an activating BRAF mutation led to their increased dasatinib resistance. The sensitivity induced by (Y472C)BRAF was reversed by the introduction of a BRAF mutation that impairs RAF dimerization. Dasatinib inhibited CRAF modestly, but concurrently induced RAF dimerization, resulting in ERK activation in NSCLC cells with kinase-inactivating BRAF mutations. The sensitivity of NSCLC with kinase-impaired BRAF to dasatinib suggested synthetic lethality of BRAF and an unknown dasatinib target. Inhibiting BRAF in NSCLC cells expressing wild-type BRAF likewise enhanced these cells' dasatinib sensitivity. Thus, the patient's BRAF mutation was likely responsible for his tumor's marked response to dasatinib, suggesting that tumors bearing kinase-impaired BRAF mutations may be exquisitely sensitive to dasatinib. Moreover, the potential synthetic lethality of combination therapy including dasatinib and BRAF inhibitors may lead to additional therapeutic options against cancers with wild-type BRAF. PMID:22649091

  10. Sensitive kinase assay linked with phosphoproteomics for identifying direct kinase substrates.

    PubMed

    Xue, Liang; Wang, Wen-Horng; Iliuk, Anton; Hu, Lianghai; Galan, Jacob A; Yu, Shuai; Hans, Michael; Geahlen, Robert L; Tao, W Andy

    2012-04-10

    Our understanding of the molecular control of many disease pathologies requires the identification of direct substrates targeted by specific protein kinases. Here we describe an integrated proteomic strategy, termed kinase assay linked with phosphoproteomics, which combines a sensitive kinase reaction with endogenous kinase-dependent phosphoproteomics to identify direct substrates of protein kinases. The unique in vitro kinase reaction is carried out in a highly efficient manner using a pool of peptides derived directly from cellular kinase substrates and then dephosphorylated as substrate candidates. The resulting newly phosphorylated peptides are then isolated and identified by mass spectrometry. A further comparison of these in vitro phosphorylated peptides with phosphopeptides derived from endogenous proteins isolated from cells in which the kinase is either active or inhibited reveals new candidate protein substrates. The kinase assay linked with phosphoproteomics strategy was applied to identify unique substrates of spleen tyrosine kinase (Syk), a protein-tyrosine kinase with duel properties of an oncogene and a tumor suppressor in distinctive cell types. We identified 64 and 23 direct substrates of Syk specific to B cells and breast cancer cells, respectively. Both known and unique substrates, including multiple centrosomal substrates for Syk, were identified, supporting a unique mechanism that Syk negatively affects cell division through its centrosomal kinase activity. PMID:22451900

  11. Lead Surveillance Program

    NASA Technical Reports Server (NTRS)

    1993-01-01

    Background on lead exposure is presented including forms of lead, sources, hematologic effects, neurologic effects, endocrine effects, renal effects, and reproductive and developmental effects. The purpose of the Lead Surveillance Program at LeRC is outlined, and the specifics of the Medical Surveillance Program for Lead Exposure at LeRC are discussed.

  12. A novel viral thymidylate kinase with dual kinase activity.

    PubMed

    Guevara-Hernandez, Eduardo; Arvizu-Flores, Aldo A; Lugo-Sanchez, Maria E; Velazquez-Contreras, Enrique F; Castillo-Yañez, Francisco J; Brieba, Luis G; Sotelo-Mundo, Rogerio R

    2015-10-01

    Nucleotide phosphorylation is a key step in DNA replication and viral infections, since suitable levels of nucleotide triphosphates pool are required for this process. Deoxythymidine monophosphate (dTMP) is produced either by de novo or salvage pathways, which is further phosphorylated to deoxythymidine triphosphate (dTTP). Thymidyne monophosphate kinase (TMK) is the enzyme in the junction of both pathways, which phosphorylates dTMP to yield deoxythymidine diphosphate (dTDP) using adenosine triphosphate (ATP) as a phosphate donor. White spot syndrome virus (WSSV) genome contains an open reading frame (ORF454) that encodes a thymidine kinase and TMK domains in a single polypeptide. We overexpressed the TMK ORF454 domain (TMKwssv) and its specific activity was measured with dTMP and dTDP as phosphate acceptors. We found that TMKwssv can phosphorylate dTMP to yield dTDP and also is able to use dTDP as a substrate to produce dTTP. Kinetic parameters K M and k cat were calculated for dTMP (110 μM, 3.6 s(-1)), dTDP (251 μM, 0.9 s(-1)) and ATP (92 μM, 3.2 s(-1)) substrates, and TMKwssv showed a sequential ordered bi-bi reaction mechanism. The binding constants K d for dTMP (1.9 μM) and dTDP (10 μM) to TMKwssv were determined by Isothermal Titration Calorimetry. The affinity of the nucleotidic analog stavudine monophosphate was in the same order of magnitude (K d 3.6 μM) to the canonical substrate dTMP. These results suggest that nucleotide analogues such as stavudine could be a suitable antiviral strategy for the WSSV-associated disease.

  13. Cyclin-dependent kinases in C. elegans

    PubMed Central

    Boxem, Mike

    2006-01-01

    Cell division is an inherent part of organismal development, and defects in this process can lead to developmental abnormalities as well as cancerous growth. In past decades, much of the basic cell-cycle machinery has been identified, and a major challenge in coming years will be to understand the complex interplay between cell division and multicellular development. Inevitably, this requires the use of more complex multicellular model systems. The small nematode Caenorhabditis elegans is an excellent model system to study the regulation of cell division in a multicellular organism, and is poised to make important contributions to this field. The past decade has already seen a surge in cell-cycle research in C. elegans, yielding information on the function of many basic cell-cycle regulators, and making inroads into the developmental control of cell division. This review focuses on the in vivo roles of cyclin-dependent kinases in C. elegans, and highlights novel findings implicating CDKs in coupling development to cell-cycle progression. PMID:16759361

  14. Measuring the Activity of Leucine-Rich Repeat Kinase 2: A Kinase Involved in Parkinson's Disease

    PubMed Central

    Lee, Byoung Dae; Li, Xiaojie; Dawson, Ted M.; Dawson, Valina L.

    2015-01-01

    Mutations in the LRRK2 (Leucine-Rich Repeat Kinase 2) gene are the most common cause of autosomal dominant Parkinson's disease. LRRK2 has multiple functional domains including a kinase domain. The kinase activity of LRRK2 is implicated in the pathogenesis of Parkinson's disease. Developing an assay to understand the mechanisms of LRRK2 kinase activity is important for the development of pharmacologic and therapeutic applications. Here, we describe how to measure in vitro LRRK2 kinase activity and its inhibition. PMID:21960214

  15. Pyridopyrimidine analogues as novel adenosine kinase inhibitors.

    PubMed

    Zheng, G Z; Lee, C; Pratt, J K; Perner, R J; Jiang, M Q; Gomtsyan, A; Matulenko, M A; Mao, Y; Koenig, J R; Kim, K H; Muchmore, S; Yu, H; Kohlhaas, K; Alexander, K M; McGaraughty, S; Chu, K L; Wismer, C T; Mikusa, J; Jarvis, M F; Marsh, K; Kowaluk, E A; Bhagwat, S S; Stewart, A O

    2001-08-20

    A novel series of pyridopyrimidine analogues 9 was identified as potent adenosine kinase inhibitors based on the SAR and computational studies. Substitution of the C7 position of the pyridopyrimidino core with C2' substituted pyridino moiety increased the in vivo potency and enhanced oral bioavailability of these adenosine kinase inhibitors.

  16. Genetics Home Reference: pyruvate kinase deficiency

    MedlinePlus

    ... National (UK) Information Centre for Metabolic Diseases National Organization for Rare Disorders (NORD): Pyruvate Kinase Deficiency Genetic Testing Registry (1 link) Pyruvate kinase deficiency of red cells Scientific articles on PubMed (1 link) PubMed OMIM (1 link) ...

  17. Sequential Transphosphorylation of the BRI1/BAK1 Receptor Kinase Pair Regulates Early Events of the Brassinosteriod Signaling Pathway Promoting Plant Growth and Development

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Brassinosteroids (BRs) regulate multiple aspects of plant growth and development through a signal transduction pathway that is initiated by BR binding to the transmembrane receptor kinase BRI1. Activated BRI1 heterodimerizes with a second receptor kinase, BAK1, leading to enhanced signaling output. ...

  18. Recent inventions on receptor tyrosine kinase RET modulation.

    PubMed

    Jurvansuu, Jaana M; Goldman, Adrian

    2008-01-01

    Rearranged during transfection, RET, is a receptor tyrosine kinase expressed in neural crest derived cell lineages. RET is activated by dimerisation facilitated by its binding to the heterodimeric complex formed by Glial cell-derived neurotrophic factor (GDNF) -family ligand (GFL) and GNDF-family receptor (GFR). Both GDNFs and their co-receptors are a small protein family of four members. RET kinase mediated signaling can lead to survival, cell growth, differentiation, and migration. Pharmaceutically RET is of interest due to its involvement in several disease conditions. Oncogenic RET activation by mutations or rearragements predisposes to cancers like multiple endocrine neoplasia type 2 (A and B) and medullary thyroid carcinoma. Loss-of-function mutations in RET are a strong susceptibility factor for Hirschsprung disease, which is characterized by lack of ganglion cells in gastrointestinal tract. All the GFLs promote neuronal survival and GDNF is one of the most potent neurotrophic factors for dopaminergic neurons. Therefore, the neuroprotective capacity of RET activation to override the apoptotic program in neurodegenerative diseases, like in dying midbrain dopaminergic neurons in Parkinson's disease, is of great interest. This article reviews the recent international patents on modulation of RET kinase activity by small-molecule and peptide-based agonists and antagonists.

  19. Eph-mediated tyrosine phosphorylation of citron kinase controls abscission.

    PubMed

    Jungas, Thomas; Perchey, Renaud T; Fawal, Mohamad; Callot, Caroline; Froment, Carine; Burlet-Schiltz, Odile; Besson, Arnaud; Davy, Alice

    2016-08-29

    Cytokinesis is the last step of cell division, culminating in the physical separation of daughter cells at the end of mitosis. Cytokinesis is a tightly regulated process that until recently was mostly viewed as a cell-autonomous event. Here, we investigated the role of Ephrin/Eph signaling, a well-known local cell-to-cell communication pathway, in cell division. We show that activation of Eph signaling in vitro leads to multinucleation and polyploidy, and we demonstrate that this is caused by alteration of the ultimate step of cytokinesis, abscission. Control of abscission requires Eph kinase activity, and Src and citron kinase (CitK) are downstream effectors in the Eph-induced signal transduction cascade. CitK is phosphorylated on tyrosines in neural progenitors in vivo, and Src kinase directly phosphorylates CitK. We have identified the specific tyrosine residues of CitK that are phosphorylated and show that tyrosine phosphorylation of CitK impairs cytokinesis. Finally, we show that, similar to CitK, Ephrin/Eph signaling controls neuronal ploidy in the developing neocortex. Our study indicates that CitK integrates intracellular and extracellular signals provided by the local environment to coordinate completion of cytokinesis. PMID:27551053

  20. Targeting lung cancer through inhibition of checkpoint kinases

    PubMed Central

    Syljuåsen, Randi G.; Hasvold, Grete; Hauge, Sissel; Helland, Åslaug

    2015-01-01

    Inhibitors of checkpoint kinases ATR, Chk1, and Wee1 are currently being tested in preclinical and clinical trials. Here, we review the basic principles behind the use of such inhibitors as anticancer agents, and particularly discuss their potential for treatment of lung cancer. As lung cancer is one of the most deadly cancers, new treatment strategies are highly needed. We discuss how checkpoint kinase inhibition in principle can lead to selective killing of lung cancer cells while sparing the surrounding normal tissues. Several features of lung cancer may potentially be exploited for targeting through inhibition of checkpoint kinases, including mutated p53, low ERCC1 levels, amplified Myc, tumor hypoxia and presence of lung cancer stem cells. Synergistic effects have also been reported between inhibitors of ATR/Chk1/Wee1 and conventional lung cancer treatments, such as gemcitabine, cisplatin, or radiation. Altogether, inhibitors of ATR, Chk1, and Wee1 are emerging as new cancer treatment agents, likely to be useful in lung cancer treatment. However, as lung tumors are very diverse, the inhibitors are unlikely to be effective in all patients, and more work is needed to determine how such inhibitors can be utilized in the most optimal ways. PMID:25774168

  1. Protein kinase C mechanisms that contribute to cardiac remodelling

    PubMed Central

    Newton, Alexandra C.; Antal, Corina E.; Steinberg, Susan F.

    2016-01-01

    Protein phosphorylation is a highly-regulated and reversible process that is precisely controlled by the actions of protein kinases and protein phosphatases. Factors that tip the balance of protein phosphorylation lead to changes in a wide range of cellular responses, including cell proliferation, differentiation and survival. The protein kinase C (PKC) family of serine/threonine kinases sits at nodal points in many signal transduction pathways; PKC enzymes have been the focus of considerable attention since they contribute to both normal physiological responses as well as maladaptive pathological responses that drive a wide range of clinical disorders. This review provides a background on the mechanisms that regulate individual PKC isoenzymes followed by a discussion of recent insights into their role in the pathogenesis of diseases such as cancer. We then provide an overview on the role of individual PKC isoenzymes in the regulation of cardiac contractility and pathophysiological growth responses, with a focus on the PKC-dependent mechanisms that regulate pump function and/or contribute to the pathogenesis of heart failure. PMID:27433023

  2. Synthetic sulfoglycolipids targeting the serine-threonine protein kinase Akt.

    PubMed

    Costa, Barbara; Dangate, Milind; Vetro, Maria; Donvito, Giulia; Gabrielli, Luca; Amigoni, Loredana; Cassinelli, Giuliana; Lanzi, Cinzia; Ceriani, Michela; De Gioia, Luca; Filippi, Giulia; Cipolla, Laura; Zaffaroni, Nadia; Perego, Paola; Colombo, Diego

    2016-08-15

    The serine-threonine protein kinase Akt, also known as protein kinase B, is a key component of the phosphoinositide 3-kinase (PI3K)-Akt-mTOR axis. Deregulated activation of this pathway is frequent in human tumors and Akt-dependent signaling appears to be critical in cell survival. PI3K activation generates 3-phosphorylated phosphatidylinositols that bind Akt pleckstrin homology (PH) domain. The blockage of Akt PH domain/phosphoinositides interaction represents a promising approach to interfere with the oncogenic potential of over-activated Akt. In the present study, phosphatidyl inositol mimics based on a β-glucoside scaffold have been synthesized as Akt inhibitors. The compounds possessed one or two lipophilic moieties of different length at the anomeric position of glucose, and an acidic or basic group at C-6. Docking studies, ELISA Akt inhibition assays, and cellular assays on different cell models highlighted 1-O-octadecanoyl-2-O-β-d-sulfoquinovopyranosyl-sn-glycerol as the best Akt inhibitor among the synthesized compounds, which could be considered as a lead for further optimization in the design of Akt inhibitors.

  3. Regulation of cAMP-dependent Protein Kinases

    PubMed Central

    Diskar, Mandy; Zenn, Hans-Michael; Kaupisch, Alexandra; Kaufholz, Melanie; Brockmeyer, Stefanie; Sohmen, Daniel; Berrera, Marco; Zaccolo, Manuela; Boshart, Michael; Herberg, Friedrich W.; Prinz, Anke

    2010-01-01

    cAMP-dependent protein kinases are reversibly complexed with any of the four isoforms of regulatory (R) subunits, which contain either a substrate or a pseudosubstrate autoinhibitory domain. The human protein kinase X (PrKX) is an exemption as it is inhibited only by pseudosubstrate inhibitors, i.e. RIα or RIβ but not by substrate inhibitors RIIα or RIIβ. Detailed examination of the capacity of five PrKX-like kinases ranging from human to protozoa (Trypanosoma brucei) to form holoenzymes with human R subunits in living cells shows that this preference for pseudosubstrate inhibitors is evolutionarily conserved. To elucidate the molecular basis of this inhibitory pattern, we applied bioluminescence resonance energy transfer and surface plasmon resonance in combination with site-directed mutagenesis. We observed that the conserved αH-αI loop residue Arg-283 in PrKX is crucial for its RI over RII preference, as a R283L mutant was able to form a holoenzyme complex with wild type RII subunits. Changing the corresponding αH-αI loop residue in PKA Cα (L277R), significantly destabilized holoenzyme complexes in vitro, as cAMP-mediated holoenzyme activation was facilitated by a factor of 2–4, and lead to a decreased affinity of the mutant C subunit for R subunits, significantly affecting RII containing holoenzymes. PMID:20819953

  4. Terreic Acid, a Quinone Epoxide Inhibitor of Bruton's Tyrosine Kinase

    NASA Astrophysics Data System (ADS)

    Kawakami, Yuko; Hartman, Stephen E.; Kinoshita, Eiji; Suzuki, Hidefumi; Kitaura, Jiro; Yao, Libo; Inagaki, Naoki; Franco, Alessandra; Hata, Daisuke; Maeda-Yamamoto, Mari; Fukamachi, Hiromi; Nagai, Hiroichi; Kawakami, Toshiaki

    1999-03-01

    Bruton's tyrosine kinase (Btk) plays pivotal roles in mast cell activation as well as in B cell development. Btk mutations lead to severe impairments in proinflammatory cytokine production induced by cross-linking of high-affinity IgE receptor on mast cells. By using an in vitro assay to measure the activity that blocks the interaction between protein kinase C and the pleckstrin homology domain of Btk, terreic acid (TA) was identified and characterized in this study. This quinone epoxide specifically inhibited the enzymatic activity of Btk in mast cells and cell-free assays. TA faithfully recapitulated the phenotypic defects of btk mutant mast cells in high-affinity IgE receptor-stimulated wild-type mast cells without affecting the enzymatic activities and expressions of many other signaling molecules, including those of protein kinase C. Therefore, this study confirmed the important roles of Btk in mast cell functions and showed the usefulness of TA in probing into the functions of Btk in mast cells and other immune cell systems. Another insight obtained from this study is that the screening method used to identify TA is a useful approach to finding more efficacious Btk inhibitors.

  5. MAP4K Family Kinases in Immunity and Inflammation.

    PubMed

    Chuang, Huai-Chia; Wang, Xiaohong; Tan, Tse-Hua

    2016-01-01

    MAP kinase kinase kinase kinases (MAP4Ks) belong to the mammalian Ste20-like family of serine/threonine kinases. MAP4Ks including MAP4K1/HPK1, MAP4K2/GCK, MAP4K3/GLK, MAP4K4/HGK, MAP4K5/KHS, and MAP4K6/MINK have been reported to induce JNK activation through activating the MAP3K-MAP2K cascade. The physiological roles of MAP4Ks in immunity and inflammation are largely unknown until recent studies using biochemical approaches and knockout mice. Surprisingly, JNK is not the major target of MAP4Ks in immune cells; MAP4Ks regulate immune responses through novel targets. HPK1 inhibits T-cell receptor (TCR) signaling and B-cell receptor signaling via inducing phosphorylation/ubiquitination of SLP-76 and BLNK, respectively. GLK activates TCR signaling through phosphorylating/activating PKCθ. T-cell-mediated immune responses and Th17-mediated experimental autoimmune diseases are enhanced in HPK1 knockout mice but ameliorated in GLK knockout mice. Consistently, HPK1 levels are decreased in peripheral blood mononuclear cells and T cells from patients with psoriatic arthritis and systemic lupus erythematosus (SLE), respectively. Moreover, GLK levels are increased in T cells from patients with SLE, rheumatoid arthritis, or adult-onset Still's disease; the percentages of GLK-overexpression T cells are correlated with the disease activity. In addition, HGK phosphorylates and induces TRAF2 protein degradation, leading to negative regulation of IL-6 production in resting T cells. Loss of HGK in T cells results in spontaneous systemic inflammation and type 2 diabetes in mice. HGK is also involved in cancer cell migration. To date, the phenotypes of knockout mice for GCK, KHS, and MINK have not been reported; the roles of these three MAP4Ks in immune cell signaling are discussed in this review. Taken together, MAP4K family kinases play diverse roles in immune cell signaling, immune responses, and inflammation. PMID:26791862

  6. Selective regulation of MAP kinase signaling by an endomembrane phosphatidylinositol 4-kinase.

    PubMed

    Cappell, Steven D; Dohlman, Henrik G

    2011-04-29

    Multiple MAP kinase pathways share components yet initiate distinct biological processes. Signaling fidelity can be maintained by scaffold proteins and restriction of signaling complexes to discreet subcellular locations. For example, the yeast MAP kinase scaffold Ste5 binds to phospholipids produced at the plasma membrane and promotes selective MAP kinase activation. Here we show that Pik1, a phosphatidylinositol 4-kinase that localizes primarily to the Golgi, also regulates MAP kinase specificity but does so independently of Ste5. Pik1 is required for full activation of the MAP kinases Fus3 and Hog1 and represses activation of Kss1. Further, we show by genetic epistasis analysis that Pik1 likely regulates Ste11 and Ste50, components shared by all three MAP kinase pathways, through their interaction with the scaffold protein Opy2. These findings reveal a new regulator of signaling specificity functioning at endomembranes rather than at the plasma membrane. PMID:21388955

  7. Intermolecular and Intramolecular Interactions Regulate Catalytic Activity of Myotonic Dystrophy Kinase-Related Cdc42-Binding Kinase α

    PubMed Central

    Tan, Ivan; Seow, Kah Tong; Lim, Louis; Leung, Thomas

    2001-01-01

    Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) is a Cdc42-binding serine/threonine kinase with multiple functional domains. We had previously shown MRCKα to be implicated in Cdc42-mediated peripheral actin formation and neurite outgrowth in HeLa and PC12 cells, respectively. Here we demonstrate that native MRCK exists in high-molecular-weight complexes. We further show that the three independent coiled-coil (CC) domains and the N-terminal region preceding the kinase domain are responsible for intermolecular interactions leading to MRCKα multimerization. N terminus-mediated dimerization and consequent transautophosphorylation are critical processes regulating MRCKα catalytic activities. A region containing the two distal CC domains (CC2 and CC3; residues 658 to 930) was found to interact intramolecularly with the kinase domain and negatively regulates its activity. Its deletion also resulted in an active kinase, confirming a negative autoregulatory role. We provide evidence that the N terminus-mediated dimerization and activation of MRCK and the negative autoregulatory kinase–distal CC interaction are two mutually exclusive events that tightly regulate the catalytic state of the kinase. Disruption of this interaction by a mutant kinase domain resulted in increased kinase activity. MRCK kinase activity was also elevated when cells were treated with phorbol ester, which can interact directly with a cysteine-rich domain next to the distal CC domain. We therefore suggest that binding of phorbol ester to MRCK releases its autoinhibition, allowing N-terminal dimerization and subsequent kinase activation. PMID:11283256

  8. Tyrosine Kinase Inhibitors and Pregnancy

    PubMed Central

    Abruzzese, Elisabetta; Trawinska, Malgorzata Monika; Perrotti, Alessio Pio; De Fabritiis, Paolo

    2014-01-01

    The management of patients with chronic myeloid leukemia (CML) during pregnancy has become recently a matter of continuous debate. The introduction of the Tyrosine Kinase Inhibitors (TKIs) in clinical practice has dramatically changed the prognosis of CML patients; in fact, patients diagnosed in chronic phase can reasonably expect many years of excellent disease control and good quality of life, as well as a normal life expectancy, including the necessity to address issues relating to fertility and pregnancy. Physicians are frequently being asked for advice regarding the need for, and/or the appropriateness of, stopping treatment in order to conceive. In this report, we will review the data published in terms of fertility, conception, pregnancy, pregnancy outcome and illness control for TKI treated CML patients, as well as how to manage a planned and/or unplanned pregnancy. PMID:24804001

  9. Training volume, androgen use and serum creatine kinase activity.

    PubMed

    Häkkinen, K; Alén, M

    1989-09-01

    Serum creatine kinase (CK) activities were investigated in elite male strength athletes (n = 20) during normal weight training and bodybuilding training (one training session per day), during high volume strength training (two sessions per day) and during strength training (one session per day) with the use of high dose synthetic androgens (five athletes in each subgroup). The findings demonstrated that the increase in serum CK was highest in the subgroup using androgens. These results suggest that strength training with the use of androgenic steroids leads to higher serum CK activities than normal strength training.

  10. Calcium-Oxidant Signaling Network Regulates AMP-activated Protein Kinase (AMPK) Activation upon Matrix Deprivation*

    PubMed Central

    Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

    2016-01-01

    The AMP-activated protein kinase (AMPK) has recently been implicated in anoikis resistance. However, the molecular mechanisms that activate AMPK upon matrix detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, whereas re-attachment to the matrix leads to its dephosphorylation and inactivation. Because matrix detachment leads to loss of integrin signaling, we investigated whether integrin signaling negatively regulates AMPK activation. However, modulation of focal adhesion kinase or Src, the major downstream components of integrin signaling, failed to cause a corresponding change in AMPK signaling. Further investigations revealed that the upstream AMPK kinases liver kinase B1 (LKB1) and Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) contribute to AMPK activation upon detachment. In LKB1-deficient cells, we found AMPK activation to be predominantly dependent on CaMKKβ. We observed no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment was not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signaling, and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that endoplasmic reticulum calcium release-induced store-operated calcium entry contributes to intracellular calcium increase, leading to reactive oxygen species production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. Thus, the Ca2+/reactive oxygen species-triggered LKB1/CaMKK-AMPK signaling cascade may provide a quick, adaptable switch to promote survival of metastasizing cancer cells. PMID:27226623

  11. Optochemical Activation of Kinase Function in Live Cells

    PubMed Central

    Karginov, Andrei V.; Hahn, Klaus M.; Deiters, Alexander

    2015-01-01

    Summary Manipulation of protein kinase activity is widely used to dissect signaling pathways controlling physiological and pathological processes. Common methods often cannot provide the desired spatial and temporal resolution in control of kinase activity. Regulation of kinase activity by photocaged kinase inhibitors has been successfully used to achieve tight temporal and local control, but inhibitors are limited to inactivation of kinases, and often do not provide the desired specificity. Here we report detailed methods for light-mediated activation of kinases in living cells using engineered rapamycin-regulated kinases (RapR-kinases) in conjunction with a photocaged analog of rapamycin. PMID:24718793

  12. Lead and tap water

    MedlinePlus

    Water contaminated with lead ... The Environmental Protection Agency (EPA) monitors drinking water in the United States. It requires water suppliers to produce annual water quality reports. These reports include information about lead amounts, and they ...

  13. VOLUMETRIC LEAD ASSAY

    SciTech Connect

    M.A. Ebadian, Ph.D.; S.K. Dua; David Roelant; Sachin Kumar

    2001-01-01

    This report describes a system for handling and radioassay of lead, consisting of a robot, a conveyor, and a gamma spectrometer. The report also presents a cost-benefit analysis of options: radioassay and recycling lead vs. disposal as waste.

  14. Exposures to lead.

    PubMed

    Callan, Anna C; Hinwood, Andrea L

    2011-01-01

    The Pacific Basin Consortium for Environment and Health hosted a workshop on Exposures to Lead. Speakers from Australia and the United States of America addressed current research knowledge on lead exposures and health effects in children, risk assessment and communication issues in dealing with lead exposure sources, different methods for assessing exposure, and the variety of scenarios where lead still remains a pollutant of concern. Mining continues to be a source of lead for many communities, and approaches to reducing exposures in these settings present particular challenges. A Perth Declaration for the Global Reduction of Childhood Lead Exposure was signed by participants of the meeting and is aimed at increasing attention to the need to continue to assess lead in the environment and to develop strategies to reduce lead in the environment and exposure by communities. PMID:21714377

  15. Exposures to lead.

    PubMed

    Callan, Anna C; Hinwood, Andrea L

    2011-01-01

    The Pacific Basin Consortium for Environment and Health hosted a workshop on Exposures to Lead. Speakers from Australia and the United States of America addressed current research knowledge on lead exposures and health effects in children, risk assessment and communication issues in dealing with lead exposure sources, different methods for assessing exposure, and the variety of scenarios where lead still remains a pollutant of concern. Mining continues to be a source of lead for many communities, and approaches to reducing exposures in these settings present particular challenges. A Perth Declaration for the Global Reduction of Childhood Lead Exposure was signed by participants of the meeting and is aimed at increasing attention to the need to continue to assess lead in the environment and to develop strategies to reduce lead in the environment and exposure by communities.

  16. Pyp1 and Pyp2 PTPases dephosphorylate an osmosensing MAP kinase controlling cell size at division in fission yeast.

    PubMed

    Millar, J B; Buck, V; Wilkinson, M G

    1995-09-01

    Simultaneous inactivation of pyp1 and pyp2 PTPases in fission yeast leads to aberrant cell morphology and growth arrest. Spontaneous recessive mutations that bypass the requirement for pyp1 and pyp2 and reside in two complementation groups were isolated, sty1 and sty2. sty1- and sty2- mutant cells are substantially delayed in the timing of mitotic initiation. We have isolated the sty1 gene, which encodes a MAP kinase that is closely related to a subfamily of MAP kinases regulated by osmotic stress including Saccharomyces cervisiae HOG1 and human CSBP1. We find that sty2 is allelic to the wis1 MAP kinase kinase and that delta sty1 and delta wis1 cells are unable to grow in high osmolarity medium. Osmotic stress induces both tyrosine phosphorylation of Sty1 and a reduction in cell size at division. Pyp2 associates with and tyrosine dephosphorylates Sty1 in vitro. We find that wis1-dependent induction of pyp2 mRNA is responsible for tyrosine dephosphorylation of Sty1 in vivo on prolonged exposure to osmotic stress. We conclude that Pyp1 and Pyp2 are tyrosine-specific MAP kinase phosphatases that inactivate an osmoregulated MAP kinase, Sty1, which acts downstream of the Wis1 MAP kinase kinase to control cell size at division in fission yeast. PMID:7657164

  17. Cell cycle-dependent regulation of Aurora kinase B mRNA by the Microprocessor complex.

    PubMed

    Jung, Eunsun; Seong, Youngmo; Seo, Jae Hong; Kwon, Young-Soo; Song, Hoseok

    2014-03-28

    Aurora kinase B regulates the segregation of chromosomes and the spindle checkpoint during mitosis. In this study, we showed that the Microprocessor complex, which is responsible for the processing of the primary transcripts during the generation of microRNAs, destabilizes the mRNA of Aurora kinase B in human cells. The Microprocessor-mediated cleavage kept Aurora kinase B at a low level and prevented premature entrance into mitosis. The cleavage was reduced during mitosis leading to the accumulation of Aurora kinase B mRNA and protein. In addition to Aurora kinase B mRNA, the processing of other primary transcripts of miRNAs were also decreased during mitosis. We found that the cleavage was dependent on an RNA helicase, DDX5, and the association of DDX5 and DDX17 with the Microprocessor was reduced during mitosis. Thus, we propose a novel mechanism by which the Microprocessor complex regulates stability of Aurora kinase B mRNA and cell cycle progression.

  18. Different Protein Kinase C Isoforms Determine Growth Factor Specificity in Neuronal Cells

    PubMed Central

    Corbit, Kevin C.; Soh, Jae-Won; Yoshida, Keiko; Eves, Eva M.; Weinstein, I. Bernard; Rosner, Marsha Rich

    2000-01-01

    Although mitogenic and differentiating factors often activate a number of common signaling pathways, the mechanisms leading to their distinct cellular outcomes have not been elucidated. In a previous report, we demonstrated that mitogen-activated protein (MAP) kinase (ERK) activation by the neurogenic agents fibroblast growth factor (FGF) and nerve growth factor is dependent on protein kinase Cδ (PKCδ), whereas MAP kinase activation in response to the mitogen epidermal growth factor (EGF) is independent of PKCδ in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells. We now show that EGF activates MAP kinase through a PKCζ-dependent pathway involving phosphatidylinositol 3-kinase and PDK1 in H19-7 cells. PKCζ, like PKCδ, acts upstream of MEK, and PKCζ can potentiate Raf-1 activation by EGF. Inhibition of PKCζ also blocks EGF-induced DNA synthesis as monitored by bromodeoxyuridine incorporation in H19-7 cells. Finally, in embryonic rat brain hippocampal cell cultures, inhibitors of PKCζ or PKCδ suppress MAP kinase activation by EGF or FGF, respectively, indicating that these factors activate distinct signaling pathways in primary as well as immortalized neural cells. Taken together, these results implicate different PKC isoforms as determinants of growth factor signaling specificity within the same cell. Furthermore, these data provide a mechanism whereby different growth factors can differentially activate a common signaling intermediate and thereby generate biological diversity. PMID:10891480

  19. Vaccinia-Related Kinase 2 Modulates the Stress Response to Hypoxia Mediated by TAK1▿

    PubMed Central

    Blanco, Sandra; Santos, Claudio; Lazo, Pedro A.

    2007-01-01

    Hypoxia represents a major stress that requires an immediate cellular response in which different signaling pathways participate. Hypoxia induces an increase in the activity of TAK1, an atypical mitogen-activated protein kinase kinase kinase (MAPKKK), which responds to oxidative stress by triggering cascades leading to the activation of c-Jun N-terminal kinase (JNK). JNK activation by hypoxia requires assembly with the JIP1 scaffold protein, which might also interact with other intracellular proteins that are less well known but that might modulate MAPK signaling. We report that TAK1 is able to form a stable complex with JIP1 and thus regulate the activation of JNK, which in turn determines the cellular stress response to hypoxia. This activation of TAK1-JIP1-JNK is suppressed by vaccinia-related kinase 2 (VRK2). VRK2A is able to interact with TAK1 by its C-terminal region, forming stable complexes. The kinase activity of VRK2 is not necessary for this interaction or the downregulation of AP1-dependent transcription. Furthermore, reduction of the endogenous VRK2 level with short hairpin RNA can increase the response induced by hypoxia, suggesting that the intracellular levels of VRK2 can determine the magnitude of this stress response. PMID:17709393

  20. DNA-dependent protein kinase and checkpoint kinase 2 synergistically activate a latent population of p53 upon DNA damage.

    PubMed

    Jack, Melissa T; Woo, Richard A; Motoyama, Noboru; Takai, Hitoyuki; Lee, Patrick W K

    2004-04-01

    The role of the checkpoint kinase 2 (Chk2) as an upstream activator of p53 following DNA damage has been controversial. We have recently shown that Chk2 and the DNA-dependent protein kinase (DNA-PK) are both involved in DNA damage-induced apoptosis but not G(1) arrest in mouse embryo fibroblasts. Here we demonstrate that Chk2 is required to activate p53 in vitro as measured by its ability to bind its consensus DNA target sequence following DNA damage and is in fact the previously unidentified factor working synergistically with DNA-PK to activate p53. The gene mutated in ataxia telangiectasia is not involved in this p53 activation. Using wortmannin, serine 15 mutants of p53, DNA-PK null cells and Chk2 null cells, we demonstrate that DNA-PK and Chk2 act independently and sequentially on p53. Furthermore, the p53 target of these two kinases represents a latent (preexisting) population of p53. Taken together, the results from these studies are consistent with a model in which DNA damage causes an immediate and sequential modification of latent p53 by DNA-PK and Chk2, which under appropriate conditions can lead to apoptosis. PMID:14752107

  1. PHOTOEMISSION PROPERTIES OF LEAD.

    SciTech Connect

    SMEDLEY,J.; RAO,T.; WARREN,J.; SEKUTOWICZ,J.; LEFFERTS,R.; LIPSKI,A.

    2004-07-05

    In this paper we present a study of the photoemission properties of lead at several UV wavelengths, including a study of the damage threshold of electroplated lead under laser cleaning. A quantum efficiency in excess of 0.1% has been achieved for a laser cleaned, electroplated lead sample with a laser wavelength of 193 nm. Niobium cathodes have been measured for comparison, and lead is found to be a superior photoemitter for all measured wavelengths.

  2. Transplacental transport of lead

    SciTech Connect

    Goyer, R.A. )

    1990-11-01

    Neurotoxicity is the major health effect from exposure to lead for infants and young children, and there is current concern regarding possible toxic effects of lead on the child while in utero. there is no placental-fetal barrier to lead transport. Maternal and fetal blood lead levels are nearly identical, so lead passes through the placenta unencumbered. Lead has been measured in the fetal brain as early as the end of the first trimester (13 weeks). There is a similar rate of increase in brain size and lead content throughout pregnancy in the fetus of mothers in the general population, so concentration of lead probably does not differ greatly during gestation unless exposure of the mother changes. Cell-specific sensitivity to the toxic effects of lead, however, may be greater the younger the fetus. Lead toxicity to the nervous system is characterized by edema or swelling of the brain due to altered permeability of capillary endothelial cells. Experimental studies suggest that immature endothelial cells forming the capillaries of the developing brain are less resistant to the effects of lead, permitting fluid and cations including lead to reach newly formed components of the brain, particularly astrocytes and neurons. Also, the ability of astrocytes and neurons to sequester lead in the form of lead protein complexes occurs only in the later stages of fetal development, permitting lead in maturing brain cells to interact with vital subcellular organelles, particularly mitochondria, which are the major cellular energy source. Intracellular lead also affects binding sites for calcium which, in turn, may affect numerous cell functions including neurotransmitter release.

  3. Insulin-induced Drosophila S6 kinase activation requires phosphoinositide 3-kinase and protein kinase B.

    PubMed Central

    Lizcano, Jose M; Alrubaie, Saif; Kieloch, Agnieszka; Deak, Maria; Leevers, Sally J; Alessi, Dario R

    2003-01-01

    An important mechanism by which insulin regulates cell growth and protein synthesis is through activation of the p70 ribosomal S6 protein kinase (S6K). In mammalian cells, insulin-induced PI3K (phosphoinositide 3-kinase) activation, generates the lipid second messenger PtdIns(3,4,5) P (3), which is thought to play a key role in triggering the activation of S6K. Although the major components of the insulin-signalling pathway are conserved in Drosophila, recent studies suggested that S6K activation does not require PI3K in this system. To investigate further the role of dPI3K (Drosophila PI3K) in dS6K (Drosophila S6K) activation, we examined the effect of two structurally distinct PI3K inhibitors on insulin-induced dS6K activation in Kc167 and S2 Drosophila cell lines. We found that both inhibitors prevented insulin-stimulated phosphorylation and activation of dS6K. To investigate further the role of the dPI3K pathway in regulating dS6K activation, we also used dsRNAi (double-stranded RNA-mediated interference) to decrease expression of dPI3K and the PtdIns(3,4,5) P (3) phosphatase dPTEN ( Drosophila phosphatase and tensin homologue deleted on chromosome 10) in Kc167 and S2 cells. Knock-down of dPI3K prevented dS6K activation, whereas knock-down of dPTEN, which would be expected to increase PtdIns(3,4,5) P (3) levels, stimulated dS6K activity. Moreover, when the expression of the dPI3K target, dPKB (Drosophila protein kinase B), was decreased to undetectable levels, we found that insulin could no longer trigger dS6K activation. This observation provides the first direct demonstration that dPKB is required for insulin-stimulated dS6K activation. We also present evidence that the amino-acid-induced activation of dS6K in the absence of insulin, thought to be mediated by dTOR (Drosophila target of rapamycin), which is unaffected by the inhibition of dPI3K by wortmannin. The results of the present study support the view that, in Drosophila cells, dPI3K and dPKB, as well d

  4. TLR signalling affects sperm mitochondrial function and motility via phosphatidylinositol 3-kinase and glycogen synthase kinase-3α.

    PubMed

    Zhu, Xingxing; Shi, Dongyan; Li, Xiaoqian; Gong, Weijuan; Wu, Fengjiao; Guo, Xuejiang; Xiao, Hui; Liu, Lixin; Zhou, Hong

    2016-03-01

    Infection in male and female genital tracts can lead to infertility. The underlying mechanisms of this process remain unclear. Toll-like receptors (TLRs) recognize conserved structures and respond to pathogens by initiating signals that activate inflammatory gene transcription. Here, we demonstrate that TLR activation in sperm reduces sperm motility via signalling through myeloid differentiation factor 88 (MyD88), phosphatidylinositol 3-kinase (PI3K), and glycogen synthase kinase (GSK)-3α. Upon TLR activation, phosphorylated forms of PI3K and GSK3α were detected in the mitochondria, and the mitochondrial membrane potential was impaired in sperm. In addition, mitochondrial ATP levels were decreased after TLR agonist stimulation. Furthermore, blocking PI3K or GSK3α activation abrogated these effects and reversed the TLR-induced reduction in sperm motility. These results identify a previously unrecognized TLR signalling pathway that leads to dysfunctional sperm mitochondria, which reduce sperm motility. Our study reveals a novel mechanism by which pathogenic infection affects sperm motility and possibly leads to infertility.

  5. Lead Poisoning in Schools.

    ERIC Educational Resources Information Center

    Guyaux, Susan

    1990-01-01

    Overexposure to lead can permanently impair a child's mental and physical development. This article discusses sources of lead paint, survey and testing methods, management and abatement plans, drinking water contamination, and associated federal standards. Although lead is present in soil and in art, theater, and vocational programs, no federal…

  6. Lead Poisoning (For Parents)

    MedlinePlus

    ... Story" 5 Things to Know About Zika & Pregnancy Lead Poisoning KidsHealth > For Parents > Lead Poisoning Print A A ... Family en español La intoxicación por plomo About Lead Poisoning If you have young kids, it's important to ...

  7. Learn about Lead

    MedlinePlus

    ... Determine if your family is at risk for lead poisoning with the Lead Poisoning Home Checklist (PDF) . Top of page What do I do if I think my child or I have been exposed to lead? Talk to your pediatrician, general physician, or local ...

  8. Lead Poisoning in Children.

    ERIC Educational Resources Information Center

    Boeckx, Roger L.

    1986-01-01

    Urban children are exposed to lead through the air they breathe, the water they drink, and the food and nonfood substances they ingest. The history, diagnosis, and treatment of lead poisoning in these children are discussed. Includes information on the toxicology of lead and the various risk classes. (JN)

  9. Lead Poisoning in Childhood.

    ERIC Educational Resources Information Center

    Pueschel, Siegfried M., Ed.; Linakis, James G., Ed.; Anderson, Angela C., Ed.

    The magnitude of childhood lead poisoning has been inexplicably neglected by modern medicine and by legislators. However, since the 1970s, increased attention has been focused on lead poisoning, and advances have been made in several areas, including understanding of the neurodevelopmental and behavioral ramifications of lead poisoning, and…

  10. Lead Poisoning in Children.

    ERIC Educational Resources Information Center

    Drummond, A. H., Jr.

    1981-01-01

    Early symptoms of lead poisoning in children are often overlooked. Lead poisoning has its greatest effects on the brain and nervous system. The obvious long-term solution to the lead poisoning problem is removal of harmful forms of the metal from the environment. (JN)

  11. Lead and children

    PubMed Central

    Abelsohn, Alan R.; Sanborn, Margaret

    2010-01-01

    Abstract OBJECTIVE To provide family physicians with a practical, evidence-based approach to screening for and preventing children’s exposure to lead. SOURCES OF INFORMATION MEDLINE was searched using terms relevant to lead exposure and poisoning. We reviewed English-language articles published in 2003 to 2008. Most cited studies provide level 2 or 3 evidence. MAIN MESSAGE Lead is a developmental neurotoxin. Children are most commonly exposed and they are most vulnerable. Lead exposure has been associated with many cognitive and motor deficits, as well as distractibility and other characteristics of attention deficit hyperactivity disorder. Although children’s blood lead levels have declined considerably over the past 3 decades with removal of lead from gasoline and paint, children can still be exposed to lead from lead paint in older homes, toys, and other sources. Because post-exposure treatment cannot reverse the cognitive effects of lead exposure, preventing lead exposure is essential. CONCLUSION Family physicians have an important role in screening for children at high risk of lead exposure, and in educating families to prevent the exposure of children to lead. PMID:20547517

  12. Lead poisoning: An overview

    NASA Technical Reports Server (NTRS)

    Gendel, Neil

    1993-01-01

    A problem that should be of great concern to all of us is the lead poisoning of children. First, I would like to present a short overview concerning the reasons everyone should care about lead poisoning, then discuss the history of lead poisoning, what is happening today across the country, and the future.

  13. Lead in Christmas lights.

    PubMed

    Laquatra, Joseph; Coyne, Lelia M; Pierce, Mark R

    2008-12-01

    A recent California proposition led to awareness that lead is a stabilizer in the Polyvinyl Chloride (PVC) jacketing that covers conductors in Christmas lights. The objective of this study is to examine the level of accessible lead in Christmas lights. Following U.S. Environmental Protection Agency (U.S. EPA) Lead Inspectors' procedures, researchers at Cornell University and in Nebraska conducted wipe samples and total lead content samples of newly purchased and older Christmas light sets. Samples were analyzed for lead content. Lead was present in varying amounts on all samples. The amount of lead from the Nebraska samples, normalized to length of strings, was independent of analyzing laboratory, analysis method, age of string, and repeat sampling, both immediately and after extended storage. A later analysis of these same strings by the Cornell team showed diminished quantities. Amounts of surface lead normalized to crude estimates of the area of light string indicated surface concentrations in excess of U.S. EPA clearance level for lead on window sills. Whether exposure to lead in Christmas lights affects blood lead levels in humans is unknown. No standards exist for lead content in this product, and no protocols exist for conducting tests on it. Therefore, consumers may wish to exercise caution to reduce possible exposure.

  14. Analysis of lead toxicity in human cells

    PubMed Central

    2012-01-01

    Background Lead is a metal with many recognized adverse health side effects, and yet the molecular processes underlying lead toxicity are still poorly understood. Quantifying the injurious effects of lead is also difficult because of the diagnostic limitations that exist when analyzing human blood and urine specimens for lead toxicity. Results We analyzed the deleterious impact of lead on human cells by measuring its effects on cytokine production and gene expression in peripheral blood mononuclear cells. Lead activates the secretion of the chemokine IL-8 and impacts mitogen-dependent activation by increasing the secretion of the proinflammatory cytokines IL-6 and TNF-α and of the chemokines IL-8 and MIP1-α in the presence of phytohemagglutinin. The recorded changes in gene expression affected major cellular functions, including metallothionein expression, and the expression of cellular metabolic enzymes and protein kinase activity. The expression of 31 genes remained elevated after the removal of lead from the testing medium thereby allowing for the measurement of adverse health effects of lead poisoning. These included thirteen metallothionein transcripts, three endothelial receptor B transcripts and a number of transcripts which encode cellular metabolic enzymes. Cellular responses to lead correlated with blood lead levels and were significantly altered in individuals with higher lead content resultantly affecting the nervous system, the negative regulation of transcription and the induction of apoptosis. In addition, we identified changes in gene expression in individuals with elevated zinc protoporphyrin blood levels and found that genes regulating the transmission of nerve impulses were affected in these individuals. The affected pathways were G-protein mediated signaling, gap junction signaling, synaptic long-term potentiation, neuropathic pain signaling as well as CREB signaling in neurons. Cellular responses to lead were altered in subjects with high

  15. Protein kinase Calpha and epsilon small-molecule targeted therapeutics: a new roadmap to two Holy Grails in drug discovery?

    PubMed

    O'Brian, Catherine A; Chu, Feng; Bornmann, William G; Maxwell, David S

    2006-02-01

    Protein kinase (PK)Calpha and epsilon are rational targets for cancer therapy. However, targeted experimental therapeutics that inhibit PKCalpha or epsilon are unavailable. The authors established recently that covalent modification of an active-site cysteine in human PKCepsilon, Cys452, by small molecules, for example 2-mercaptoethanolamine, is necessary and sufficient to render PKCepsilon kinase-dead. Cys452 is conserved in only eleven human protein kinase genes, including PKCalpha. Therefore, the design of small molecules that bind PKC active sites with an electrophile substituent positioned proximal to the Cys452 side chain may lead to targeted therapeutics that selectively inhibit PKCepsilon, PKCalpha or other PKC isozymes.

  16. Phosphatidylinositol 3-kinase association with the osteoclast cytoskeleton, and its involvement in osteoclast attachment and spreading.

    PubMed

    Lakkakorpi, P T; Wesolowski, G; Zimolo, Z; Rodan, G A; Rodan, S B

    1997-12-15

    Osteoclast activation involves attachment to the mineralized bone matrix and reorganization of the cytoskeleton, leading to polarization of the cell. Signaling molecules, PI3-kinase, rho A, and pp60c-src, were shown to be essential for osteoclastic bone resorption. In this study we have focused on the involvement of these signaling molecules in the early event of osteoclast activation: attachment, spreading, and organization of the cytoskeleton. Highly purified osteoclasts were fractionated into Triton X-100-soluble or cytosolic and Triton X-100-insoluble or cytoskeletal fractions, and the distribution of above-mentioned signaling molecules between the two fractions was examined. PI3-kinase, rho A, and pp60c-src all showed translocation to the cytoskeletal fraction upon osteoclast attachment to plastic. However, PI3-kinase and rho A, but not pp60c-src, showed further translocation of 2.4- and 3.2-fold, respectively, upon attachment of osteoclasts to bone. PI3-kinase translocation to the cytoskeleton was inhibited by either cytochalasin B or colchicine. Furthermore, treatment of osteoclasts with the PI3-kinase inhibitor wortmannin decreased its translocation, suggesting that PI3-kinase activity was needed for its translocation. Moreover, wortmannin inhibited osteoclast attachment to both bone and plastic and caused drastic changes in osteoclast morphology resulting in rounding of the cells, disappearance of F-actin structures or podosomes, and appearance of punctate or vesicular structures inside the cells. Osteoblastic MB1.8 cells and IC-21 macrophages did not show additional translocation of PI3-kinase or rho A upon attachment to bone or changes in attachment or morphology in response to wortmannin. Finally, PI3-kinase coimmunoprecipitated with alpha v beta 3 integrin from osteoclasts. PMID:9434625

  17. Cinnamic Acid Derivatives as Inhibitors of Oncogenic Protein Kinases--Structure, Mechanisms and Biomedical Effects.

    PubMed

    Mielecki, Marcin; Lesyng, Bogdan

    2016-01-01

    Cinnamic acid belongs to phenolic-acid class of polyphenols, one of the most abundant plant secondary metabolites. These substances are widely studied because of plethora of their biological activities. In particular, their inhibition of protein kinases contributes to the pleiotropic effects in the cell. Protein kinases are essential in controlling cell signaling networks. Selective targeting of oncogenic protein kinases increases clinical anticancer efficacy. Cinnamic acid and related compounds have inspired researchers in the design of numerous synthetic and semisynthetic inhibitors of oncogenic protein kinases for the past three decades. Interest in cinnamoyl-scaffold-containing compounds revived in recent years, which was stimulated by modern drug design and discovery methodologies such as in vitro and in silico HTS. This review presents cinnamic acid derivatives and analogs for which direct inhibition of protein kinases was identified. We also summarize significance of the above protein kinase families - validated or promising targets for anticancer therapies. The inhibition mode may vary from ATP-competitive, through bisubstrate-competitive and mixedcompetitive, to non-competitive one. Kinase selectivity is often correlated with subtle chemical modifications, and may also be steered by an additional non-cinnamoyl fragment of the inhibitor. Specific cinnamic acid congeners may synergize their effects in the cell by a wider range of activities, like suppression of additional enzymes, e.g. deubiquitinases, influencing the same signaling pathways (e.g. JAK2/STAT). Cinnamic acid, due to its biological and physicochemical properties, provides nature-inspired ideas leading to novel inhibitors of oncogenic protein kinases and related enzymes, capable to target a variety of cancer cells.

  18. PAK4 kinase activity and somatic mutation promote carcinoma cell motility and influence inhibitor sensitivity

    PubMed Central

    Whale, Andrew D.; Dart, Anna; Holt, Mark; Jones, Gareth E.; Wells, Claire M.

    2012-01-01

    Hepatocyte growth factor (HGF) and its receptor (c-Met) are associated with cancer cell motility and invasiveness. p21-activated kinase 4 (PAK4), a potential therapeutic target, is recruited to and activated by c-Met. In response, PAK4 phosphorylates LIM kinase 1 (LIMK1) in an HGF-dependent manner in metastatic prostate carcinoma cells. PAK4 overexpression is known to induce increased cell migration speed but the requirement for kinase activity has not been established. We have used a panel of PAK4 truncations and mutations in a combination of over-expression and RNAi rescue experiments to determine the requirement for PAK4 kinase activity during carcinoma cell motility downstream of HGF. We find that neither the kinase domain alone nor a PAK4 mutant unable to bind Cdc42 is able to fully rescue cell motility in a PAK4-deficient background. Nevertheless, we find that PAK4 kinase activity and associated LIMK1 activity are essential for carcinoma cell motility, highlighting PAK4 as a potential anti-metastatic therapeutic target. We also show here that overexpression of PAK4 harboring a somatic mutation, E329K, increased the HGF-driven motility of metastatic prostate carcinoma cells. E329 lies within the G-loop region of the kinase. Our data suggest E329K mutation leads to a modest increase in kinase activity conferring resistance to competitive ATP inhibitors in addition to promoting cell migration. The existence of such a mutation may have implications for the development of PAK4-specific competitive ATP inhibitors should PAK4 be further explored for clinical inhibition. PMID:22689056

  19. Expression of human tyrosine kinase-negative epidermal growth factor receptor amplifies signaling through endogenous murine epidermal growth factor receptor.

    PubMed

    Hack, N; Sue-A-Quan, A; Mills, G B; Skorecki, K L

    1993-12-15

    Recent findings have suggested that certain ligand-dependent responses to EGF may be propagated in a manner that is not dependent on the intrinsic tyrosine kinase activity of the epidermal growth factor receptor (EGF-R, Campos-Gonzalez, R., and Glenney, J. R., Jr. (1992) J. Biol. Chem. 267, 14535-14538) or, alternatively, that these responses may occur through the interaction of the human tyrosine kinase-deficient EGF-R with an as yet unidentified kinase (Selva, E., Raden, D. L., and Davis, R. J. (1993) J. Biol. Chem. 268, 2250-2254). These conclusions represent a significant departure from our current understanding of signal transduction by receptor tyrosine kinases. Therefore we examined the effect of expression of tyrosine kinase-negative human EGF receptor in murine NIH-3T3-2.2 cells on the EGF-dependent phosphorylation of mitogen-activated protein (MAP-2) kinase. In parental cells (NIH-3T3-2.2) that express low levels of endogenous murine EGF-R, there was no demonstrable EGF-dependent coupling to MAP-2 kinase. In NIH-3T3-2.2 cells transfected with tyrosine kinase-negative human EGF-R, there was unexpected EGF-dependent phosphorylation of MAP-2 kinase. Analysis of the tyrosine kinase-negative human EGF-R in these cells revealed significant tyrosine phosphorylation of the EGF-R. A low level of endogenous murine EGF-R present in these cells were also phosphorylated on tyrosine residues and displayed autokinase activity. Similar results were obtained using an unrelated cell line (B82L cells), in which EGF-dependent phosphorylation of MAP-2 kinase was previously attributed to signal propagation through a tyrosine kinase-negative human EGF-R (Campos-Gonzalez, R., and Glenney, J. R., Jr. (1992) J. Biol. Chem. 267, 14535-14538). Taken together, these results suggest that the tyrosine kinase-negative human EGF-R are able to amplify the response to activation of low levels of endogenous murine EGF-R, thus leading to EGF-dependent phosphorylation of MAP-2 kinase in cells

  20. Novel insights into the mechanisms of mitotic spindle assembly by NEK kinases

    PubMed Central

    Prosser, Suzanna L.; O'Regan, Laura; Fry, Andrew M.

    2016-01-01

    ABSTRACT The mitotic spindle is the apparatus upon which chromosomes are segregated during cell division. We have discovered new roles for two members of the NIMA-related kinase (NEK) family in different molecular processes of spindle assembly. Moreover, loss of these proteins leads to segregation errors that drive cancer progression. PMID:27314078

  1. Structure-Based Design of an Organoruthenium Phosphatidyl-inositol-3-Kinase Inhibitor Reveals a Switch Governing Lipid Kinase Potency and Selectivity

    SciTech Connect

    Xie,P.; Williams, D.; Atilla-Gokcumen, G.; Milk, L.; Xiao, M.; Smalley, K.; Herlyn, M.; Meggers, E.; Marmorstein, R.

    2008-01-01

    Mutations that constitutively activate the phosphatidyl-inositol-3-kinase (PI3K) signaling pathway, including alterations in PI3K, PTEN, and AKT, are found in a variety of human cancers, implicating the PI3K lipid kinase as an attractive target for the development of therapeutic agents to treat cancer and other related diseases. In this study, we report on the combination of a novel organometallic kinase inhibitor scaffold with structure-based design to develop a PI3K inhibitor, called E5E2, with an IC50 potency in the mid-low-nanomolar range and selectivity against a panel of protein kinases. We also show that E5E2 inhibits phospho-AKT in human melanoma cells and leads to growth inhibition. Consistent with a role for the PI3K pathway in tumor cell invasion, E5E2 treatment also inhibits the migration of melanoma cells in a 3D spheroid assay. The structure of the PI3K?/E5E2 complex reveals the molecular features that give rise to this potency and selectivity toward lipid kinases with implications for the design of a subsequent generation of PI3K-isoform-specific organometallic inhibitors.

  2. Withdrawal of Essential Amino Acids Increases Autophagy by a Pathway Involving Ca2+/Calmodulin-dependent Kinase Kinase-β (CaMKK-β)*

    PubMed Central

    Ghislat, Ghita; Patron, Maria; Rizzuto, Rosario; Knecht, Erwin

    2012-01-01

    Autophagy is the main lysosomal catabolic process that becomes activated under stress conditions, such as amino acid starvation and cytosolic Ca2+ upload. However, the molecular details on how both conditions control autophagy are still not fully understood. Here we link essential amino acid starvation and Ca2+ in a signaling pathway to activate autophagy. We show that withdrawal of essential amino acids leads to an increase in cytosolic Ca2+, arising from both extracellular medium and intracellular stores, which induces the activation of adenosine monophosphate-activated protein kinase (AMPK) via Ca2+/calmodulin-dependent kinase kinase-β (CaMKK-β). Furthermore, we show that autophagy induced by amino acid starvation requires AMPK, as this induction is attenuated in its absence. Subsequently, AMPK activates UNC-51-like kinase (ULK1), a mammalian autophagy-initiating kinase, through phosphorylation at Ser-555 in a process that requires CaMKK-β. Finally, the mammalian target of rapamycin complex C1 (mTORC1), a negative regulator of autophagy downstream of AMPK, is inhibited by amino acid starvation in a Ca2+-sensitive manner, and CaMKK-β appears to be important for mTORC1 inactivation, especially in the absence of extracellular Ca2+. All these results highlight that amino acid starvation regulates autophagy in part through an increase in cellular Ca2+ that activates a CaMKK-β-AMPK pathway and inhibits mTORC1, which results in ULK1 stimulation. PMID:23027865

  3. Characterization of some molecular mechanisms governing autoactivation of the catalytic domain of the anaplastic lymphoma kinase.

    PubMed

    Tartari, Carmen J; Gunby, Rosalind H; Coluccia, Addolorata M L; Sottocornola, Roberta; Cimbro, Barbara; Scapozza, Leonardo; Donella-Deana, Arianna; Pinna, Lorenzo A; Gambacorti-Passerini, Carlo

    2008-02-15

    NPM/ALK is an oncogenic fusion protein expressed in approximately 50% of anaplastic large cell lymphoma cases. It derives from the t(2;5)(p23;q35) chromosomal translocation that fuses the catalytic domain of the tyrosine kinase, anaplastic lymphoma kinase (ALK), with the dimerization domain of the ubiquitously expressed nucleophosmin (NPM) protein. Dimerization of the ALK kinase domain leads to its autophosphorylation and constitutive activation. Activated NPM/ALK stimulates downstream survival and proliferation signaling pathways leading to malignant transformation. Herein, we investigated the molecular mechanisms of autoactivation of the catalytic domain of ALK. Because kinases are typically regulated by autophosphorylation of their activation loops, we systematically mutated (Tyr --> Phe) three potential autophosphorylation sites contained in the "YXXXYY" motif of the ALK activation loop, and determined the effect of these mutations on the catalytic activity and biological function of NPM/ALK. We observed that mutation of both the second and third tyrosine residues (YFF mutant) did not affect the kinase activity or transforming ability of NPM/ALK. In contrast, mutation of the first and second (FFY), first and third (FYF), or all three (FFF) tyrosine residues impaired both kinase activity and transforming ability of NPM/ALK. Furthermore, a DFF mutant, in which the aspartic residue introduces a negative charge similar to a phosphorylated tyrosine, possessed catalytic activity similar to the YFF mutant. Together, our findings indicate that phosphorylation of the first tyrosine of the YXXXYY motif is necessary for the autoactivation of the ALK kinase domain and the transforming activity of NPM/ALK. PMID:18070884

  4. Soybean nodule autoregulation receptor kinase phosphorylates two kinase-associated protein phosphatases in vitro.

    PubMed

    Miyahara, Akira; Hirani, Tripty A; Oakes, Marie; Kereszt, Attila; Kobe, Bostjan; Djordjevic, Michael A; Gresshoff, Peter M

    2008-09-12

    The NARK (nodule autoregulation receptor kinase) gene, a negative regulator of cell proliferation in nodule primordia in several legumes, encodes a receptor kinase that consists of an extracellular leucine-rich repeat and an intracellular serine/threonine protein kinase domain. The putative catalytic domain of NARK was expressed and purified as a maltose-binding or a glutathione S-transferase fusion protein in Escherichia coli. The recombinant NARK proteins showed autophosphorylation activity in vitro. Several regions of the NARK kinase domain were shown by mass spectrometry to possess phosphoresidues. The kinase-inactive protein K724E failed to autophosphorylate, as did three other proteins corresponding to phenotypically detected mutants defective in whole plant autoregulation of nodulation. A wild-type NARK fusion protein transphosphorylated a kinase-inactive mutant NARK fusion protein, suggesting that it is capable of intermolecular autophosphorylation in vitro. In addition, Ser-861 and Thr-963 in the NARK kinase catalytic domain were identified as phosphorylation sites through site-directed mutagenesis. The genes coding for the kinase-associated protein phosphatases KAPP1 and KAPP2, two putative interacting components of NARK, were isolated. NARK kinase domain phosphorylated recombinant KAPP proteins in vitro. Autophosphorylated NARK kinase domain was, in turn, dephosphorylated by both KAPP1 and KAPP2. Our results suggest a model for signal transduction involving NARK in the control of nodule development.

  5. Law: toxic lead aftermath

    SciTech Connect

    Goldstein, E.A.

    1983-03-01

    The paper describes the events which began with an EPA proposal to weaken the lead-in-gas regulations. Because of the outcry from environmentalists and expert testimony from the medical community, the EPA reversed its policy and issued new standards which would reduce lead emissions between 1983 and 1990 by 34 percent (128,000 tons). Scientific evidence presented showed a clear reduction in blood lead levels from 1976-1980 which paralleled decreases of lead in gasoline. Results from lead poisoning clinics which linked chronic low lead exposures to decreased classroom performance and other learning disabilities were presented. Lawyers from several environmental groups took the agency to court on the related issue of attaining national ambient air quality standards for lead. (JMT)

  6. Lead in the environment

    USGS Publications Warehouse

    Pattee, O.H.; Pain, D.J.; Hoffman, David J.; Rattner, Barnett A.; Burton, G. Allen; Cairns, John=

    2003-01-01

    Anthropogenic uses of lead have probably altered its availability and environmental distribution more than any other toxic element. Consequently, lead concentrations in many living organisms may be approaching thresholds of toxicity for the adverse effects of lead. Such thresholds are difficult to define, as they vary with the chemical and physical form of lead, exposure regime, other elements present and also vary both within and between species. The technological capability to accurately quantify low lead concentrations has increased over the last decade, and physiological and behavioral effects have been measured in wildlife with tissue lead concentrations below those previously considered safe for humans.s.236 Consequently. lead criteria for the protection of wildlife and human health are frequently under review, and 'thresholds' of lead toxicity are being reconsidered. Proposed lead criteria for the protection of natural resources have been reviewed by Eisler. Uptake of lead by plants is limited by its generally low availability in soils and sediments, and toxicity may be limited by storage mechanisms and its apparently limited translocation within most plants. Lead does not generally accumulate within the foliar parts of plants, which limits its transfer to higher trophic levels. Although lead may concentrate in plant and animal tissues, no evidence of biomagnification exists. Acid deposition onto surface waters and soils with low buffering capacity may influence the availability of lead for uptake by plants and animals, and this may merit investigation at susceptible sites. The biological significance of chronic low-level lead exposure to wildlife is sometimes difficult to quantify. Animals living in urban environments or near point sources of lead emission are inevitably subject to greater exposure to lead and enhanced risk of lead poisoning. Increasingly strict controls on lead emissions in many countries have reduced exposure to lead from some sources

  7. Neonatal hyperbilirubinemia caused by pyruvate kinase deficiency.

    PubMed

    Hammer, S G; Lewan, R B

    1988-01-01

    We report an infant with neonatal hyperbilirubinemia due to pyruvate kinase deficiency. The initial approach involved rapid evaluation, phototherapy, and close monitoring of serum bilirubin levels. Follow-up included maintenance on folic acid, monitoring blood counts, and educating the parents about the course of pyruvate kinase deficiency, especially aplastic crisis. We suggest that the informed family practitioner can manage neonatal hyperbilirubinemia and pyruvate kinase deficiency with referrals at critical times to pediatric or surgical specialists. The practitioner must be able to recognize quickly the need for exchange transfusion for severe jaundice and for blood transfusions or splenectomy when significant anemia or aplastic crisis occurs.

  8. Functional analysis of anomeric sugar kinases.

    PubMed

    Conway, Louis P; Voglmeir, Josef

    2016-09-01

    Anomeric sugar kinases perform fundamental roles in the metabolism of carbohydrates. Under- or overexpression of these enzymes, or mutations causing functional impairments can give rise to diseases such as galactosaemia and so the study of this class of kinase is of critical importance. In addition, anomeric sugar kinases which are naturally promiscuous, or have been artificially made so, may find application in the synthesis of libraries of drug candidates (for example, antibiotics), and natural or unnatural oligosaccharides and glycoconjugates. In this review, we provide an overview of the biological functions of these enzymes, the tools which have been developed to investigate them, and the current frontiers in their study. PMID:27351442

  9. Ground-based experiments complement microgravity flight opportunities in the investigation of the effects of space flight on the immune response: is protein kinase C gravity sensitive?

    NASA Technical Reports Server (NTRS)

    Chapes, S. K.; Woods, K. M.; Armstrong, J. W.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    This manuscript briefly reviews ground-based and flight experiments, discusses how those experiments complement each other, and details how those experiments lead us to speculate about the gravity-sensitive nature of protein kinase C.

  10. Multiple signaling pathways leading to the activation of interferon regulatory factor 3.

    PubMed

    Servant, Marc J; Grandvaux, Nathalie; Hiscott, John

    2002-09-01

    Virus infection of susceptible cells activates multiple signaling pathways that orchestrate the activation of genes, such as cytokines, involved in the antiviral and innate immune response. Among the kinases induced are the mitogen-activated protein (MAP) kinases, Jun-amino terminal kinases (JNK) and p38, the IkappaB kinase (IKK) and DNA-PK. In addition, virus infection also activates an uncharacterized VAK responsible for the C-terminal phosphorylation and subsequent activation of interferon regulatory factor 3 (IRF-3). Virus-mediated activation of IRF-3 through VAK is dependent on viral entry and transcription, since replication deficient virus failed to induce IRF-3 activity. The pathways leading to VAK activation are not well characterized, but IRF-3 appears to represent a novel cellular detection pathway that recognizes viral nucleocapsid (N) structure. Recently, the range of inducers responsible for IRF-3 activation has increased. In addition to virus infection, recognition of bacterial infection mediated through lipopolysaccharide by Toll-like receptor 4 has also been reported. Furthermore, MAP kinase kinase kinase (MAP KKK)-related pathways and DNA-PK induce N-terminal phosphorylation of IRF-3. This review summarizes recent observations in the identification of novel signaling pathways leading to IRF-3 activation.

  11. Beta-agonist- and prostaglandin E1-induced translocation of the beta-adrenergic receptor kinase: evidence that the kinase may act on multiple adenylate cyclase-coupled receptors.

    PubMed Central

    Strasser, R H; Benovic, J L; Caron, M G; Lefkowitz, R J

    1986-01-01

    beta-Adrenergic receptor kinase (beta-AR kinase) is a cytosolic enzyme that phosphorylates the beta-adrenergic receptor only when it is occupied by an agonist [Benovic, J. Strasser, R. H., Caron, M. G. & Lefkowitz, R. J. (1986) Proc. Natl. Acad. Sci. USA 83, 2797-2801.] It may be crucially involved in the processes that lead to homologous or agonist-specific desensitization of the receptor. Stimulation of DDT1MF-2 hamster smooth muscle cells or S49 mouse lymphoma cells with a beta-agonist leads to translocation of 80-90% of the beta-AR kinase activity from the cytosol to the plasma membrane. The translocation process is quite rapid, is concurrent with receptor phosphorylation, and precedes receptor desensitization and sequestration. It is also transient, since much of the activity returns to the cytosol as the receptors become sequestered. Stimulation of beta-AR kinase translocation is a receptor-mediated event, since the beta-antagonist propranolol blocks the effect of agonist. In the kin- mutant of the S49 cells (lacks cAMP-dependent protein kinase), prostaglandin E1, which provokes homologous desensitization of its own receptor, is at least as effective as isoproterenol in promoting beta-AR kinase translocation to the plasma membrane. However, in the DDT1MF-2 cells, which contain alpha 1-adrenergic receptors coupled to phosphatidylinositol turnover, the alpha 1-agonist phenylephrine is ineffective. These results suggest that the first step in homologous desensitization of the beta-adrenergic receptor may be an agonist-promoted translocation of beta-AR kinase from cytosol to plasma membrane and that beta-AR kinase may represent a more general adenylate cyclase-coupled receptor kinase that participates in regulating the function of many such receptors. Images PMID:3018728

  12. Lead identification of novel and selective TYK2 inhibitors.

    PubMed

    Liang, Jun; Tsui, Vickie; Van Abbema, Anne; Bao, Liang; Barrett, Kathy; Beresini, Maureen; Berezhkovskiy, Leo; Blair, Wade S; Chang, Christine; Driscoll, James; Eigenbrot, Charles; Ghilardi, Nico; Gibbons, Paul; Halladay, Jason; Johnson, Adam; Kohli, Pawan Bir; Lai, Yingjie; Liimatta, Marya; Mantik, Priscilla; Menghrajani, Kapil; Murray, Jeremy; Sambrone, Amy; Xiao, Yisong; Shia, Steven; Shin, Young; Smith, Jan; Sohn, Sue; Stanley, Mark; Ultsch, Mark; Zhang, Birong; Wu, Lawren C; Magnuson, Steven

    2013-09-01

    A therapeutic rationale is proposed for the treatment of inflammatory diseases, such as psoriasis and inflammatory bowel diseases (IBD), by selective targeting of TYK2. Hit triage, following a high-throughput screen for TYK2 inhibitors, revealed pyridine 1 as a promising starting point for lead identification. Initial expansion of 3 separate regions of the molecule led to eventual identification of cyclopropyl amide 46, a potent lead analog with good kinase selectivity, physicochemical properties, and pharmacokinetic profile. Analysis of the binding modes of the series in TYK2 and JAK2 crystal structures revealed key interactions leading to good TYK2 potency and design options for future optimization of selectivity.

  13. Rac-1 and Raf-1 kinases, components of distinct signaling pathways, activate myotonic dystrophy protein kinase

    NASA Technical Reports Server (NTRS)

    Shimizu, M.; Wang, W.; Walch, E. T.; Dunne, P. W.; Epstein, H. F.

    2000-01-01

    Myotonic dystrophy protein kinase (DMPK) is a serine-threonine protein kinase encoded by the myotonic dystrophy (DM) locus on human chromosome 19q13.3. It is a close relative of other kinases that interact with members of the Rho family of small GTPases. We show here that the actin cytoskeleton-linked GTPase Rac-1 binds to DMPK, and coexpression of Rac-1 and DMPK activates its transphosphorylation activity in a GTP-sensitive manner. DMPK can also bind Raf-1 kinase, the Ras-activated molecule of the MAP kinase pathway. Purified Raf-1 kinase phosphorylates and activates DMPK. The interaction of DMPK with these distinct signals suggests that it may play a role as a nexus for cross-talk between their respective pathways and may partially explain the remarkable pleiotropy of DM.

  14. Lead-protein interactions as a basis for lead toxicity.

    PubMed

    Goering, P L

    1993-01-01

    The interaction of lead (Pb) with proteins may represent a fundamental mechanism by which Pb exerts toxicity. In this overview, various factors which influence the interaction of Pb with proteins will be discussed. Pb interacts with enzyme functional groups, and high-affinity metal-binding proteins, such as Pb-binding proteins and metallothioneins, can mediate this Pb-enzyme interaction. Many other factors influence Pb-protein interactions including ligand competition and binding affinities; protein folding and the nature of the metal-binding site; rates of protein synthesis and degradation; and intracellular localization of the ligand and metal. The remainder of this overview will focus on specific examples of important proteins known to be influenced by Pb or which hypothetically may be influenced by Pb. Gaps in knowledge and important research needs are emphasized. Many of the factors discussed play a role in the relative sensitivity of various enzymes in heme biosynthesis to Pb. Disruption of this critical pathway by Pb may result in neuropathologies and accumulation of neurotoxic heme precursors. High-affinity metal-binding proteins have been shown to play a role in mediating Pb inhibition of the octameric Zn-containing enzyme, ALA dehydratase. Knowledge of regional localization in brain and the postnatal ontogeny of the high-affinity metal-binding proteins may be pivotal in understanding Pb neurotoxicity. Other specific examples related to or potentially related to Pb toxicity which are discussed include nucleic acid binding proteins, calmodulin, protein kinase C, and carbonic anhydrase. These proteins will serve as models to understand some basic principles and differences in Pb-protein interactions.

  15. Lead Poison Detection

    NASA Technical Reports Server (NTRS)

    1976-01-01

    With NASA contracts, Whittaker Corporations Space Science division has developed an electro-optical instrument to mass screen for lead poisoning. Device is portable and detects protoporphyrin in whole blood. Free corpuscular porphyrins occur as an early effect of lead ingestion. Also detects lead in urine used to confirm blood tests. Test is inexpensive and can be applied by relatively unskilled personnel. Similar Whittaker fluorometry device called "drug screen" can measure morphine and quinine in urine much faster and cheaper than other methods.

  16. Contribution of PIP-5 kinase I{alpha} to raft-based Fc{gamma}RIIA signaling

    SciTech Connect

    Szymanska, Ewelina; Korzeniowski, Marek; Raynal, Patrick; Sobota, Andrzej; Kwiatkowska, Katarzyna

    2009-04-01

    Receptor Fc{gamma}IIA (Fc{gamma}RIIA) associates with plasma membrane rafts upon activation to trigger signaling cascades leading to actin polymerization. We examined whether compartmentalization of PI(4,5)P{sub 2} and PI(4,5)P{sub 2}-synthesizing PIP5-kinase I{alpha} to rafts contributes to Fc{gamma}RIIA signaling. A fraction of PIP5-kinase I{alpha} was detected in raft-originating detergent-resistant membranes (DRM) isolated from U937 monocytes and other cells. The DRM of U937 monocytes contained also a major fraction of PI(4,5)P{sub 2}. PIP5-kinase I{alpha} bound PI(4,5)P{sub 2}, and depletion of the lipid displaced PIP5-kinase I{alpha} from the DRM. Activation of Fc{gamma}RIIA in BHK transfectants led to recruitment of the kinase to the plasma membrane and enrichment of DRM in PI(4,5)P{sub 2}. Immunofluorescence studies revealed that in resting cells the kinase was associated with the plasma membrane, cytoplasmic vesicles and the nucleus. After Fc{gamma}RIIA activation, PIP5-kinase I{alpha} and PI(4,5)P{sub 2} co-localized transiently with the activated receptor at distinct cellular locations. Immunoelectron microscopy studies revealed that PIP5-kinase I{alpha} and PI(4,5)P{sub 2} were present at the edges of electron-dense assemblies containing activated Fc{gamma}RIIA in their core. The data suggest that activation of Fc{gamma}RIIA leads to membrane rafts coalescing into signaling platforms containing PIP5-kinase I{alpha} and PI(4,5)P{sub 2}.

  17. The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold*

    PubMed Central

    Gutmann, Sascha; Hinniger, Alexandra; Fendrich, Gabriele; Drückes, Peter; Antz, Sylvie; Mattes, Henri; Möbitz, Henrik; Ofner, Silvio; Schmiedeberg, Niko; Stojanovic, Aleksandar; Rieffel, Sebastien; Strauss, André; Troxler, Thomas; Glatthar, Ralf; Sparrer, Helmut

    2015-01-01

    Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors. PMID:25918157

  18. The Crystal Structure of Cancer Osaka Thyroid Kinase Reveals an Unexpected Kinase Domain Fold.

    PubMed

    Gutmann, Sascha; Hinniger, Alexandra; Fendrich, Gabriele; Drückes, Peter; Antz, Sylvie; Mattes, Henri; Möbitz, Henrik; Ofner, Silvio; Schmiedeberg, Niko; Stojanovic, Aleksandar; Rieffel, Sebastien; Strauss, André; Troxler, Thomas; Glatthar, Ralf; Sparrer, Helmut

    2015-06-12

    Macrophages are important cellular effectors in innate immune responses and play a major role in autoimmune diseases such as rheumatoid arthritis. Cancer Osaka thyroid (COT) kinase, also known as mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and tumor progression locus 2 (Tpl-2), is a serine-threonine (ST) kinase and is a key regulator in the production of pro-inflammatory cytokines in macrophages. Due to its pivotal role in immune biology, COT kinase has been identified as an attractive target for pharmaceutical research that is directed at the discovery of orally available, selective, and potent inhibitors for the treatment of autoimmune disorders and cancer. The production of monomeric, recombinant COT kinase has proven to be very difficult, and issues with solubility and stability of the enzyme have hampered the discovery and optimization of potent and selective inhibitors. We developed a protocol for the production of recombinant human COT kinase that yields pure and highly active enzyme in sufficient yields for biochemical and structural studies. The quality of the enzyme allowed us to establish a robust in vitro phosphorylation assay for the efficient biochemical characterization of COT kinase inhibitors and to determine the x-ray co-crystal structures of the COT kinase domain in complex with two ATP-binding site inhibitors. The structures presented in this study reveal two distinct ligand binding modes and a unique kinase domain architecture that has not been observed previously. The structurally versatile active site significantly impacts the design of potent, low molecular weight COT kinase inhibitors.

  19. Crystal structures of two aminoglycoside kinases bound with a eukaryotic protein kinase inhibitor.

    PubMed

    Fong, Desiree H; Xiong, Bing; Hwang, Jiyoung; Berghuis, Albert M

    2011-05-09

    Antibiotic resistance is recognized as a growing healthcare problem. To address this issue, one strategy is to thwart the causal mechanism using an adjuvant in partner with the antibiotic. Aminoglycosides are a class of clinically important antibiotics used for the treatment of serious infections. Their usefulness has been compromised predominantly due to drug inactivation by aminoglycoside-modifying enzymes, such as aminoglycoside phosphotransferases or kinases. These kinases are structurally homologous to eukaryotic Ser/Thr and Tyr protein kinases and it has been shown that some can be inhibited by select protein kinase inhibitors. The aminoglycoside kinase, APH(3')-IIIa, can be inhibited by CKI-7, an ATP-competitive inhibitor for the casein kinase 1. We have determined that CKI-7 is also a moderate inhibitor for the atypical APH(9)-Ia. Here we present the crystal structures of CKI-7-bound APH(3')-IIIa and APH(9)-Ia, the first structures of a eukaryotic protein kinase inhibitor in complex with bacterial kinases. CKI-7 binds to the nucleotide-binding pocket of the enzymes and its binding alters the conformation of the nucleotide-binding loop, the segment homologous to the glycine-rich loop in eukaryotic protein kinases. Comparison of these structures with the CKI-7-bound casein kinase 1 reveals features in the binding pockets that are distinct in the bacterial kinases and could be exploited for the design of a bacterial kinase specific inhibitor. Our results provide evidence that an inhibitor for a subset of APHs can be developed in order to curtail resistance to aminoglycosides.

  20. Nonnucleoside inhibitors of adenosine kinase.

    PubMed

    Gomtsyan, Arthur; Lee, Chih-Hung

    2004-01-01

    Adenosine (ADO) is an endogenous inhibitory neuromodulator that increases nociceptive thresholds in response to tissue trauma and inflammation. Adenosine kinase (AK) is a key intracellular enzyme regulating intra- and extracellular concentrations of ADO. AK inhibition selectively amplifies extracellular ADO levels at cell and tissue sites where accelerated release of ADO occurs. AK inhibitors have been shown to provide effective antinociceptive, antiinflammatory and anticonvulsant activity in animal models, thus suggesting their potential therapeutic utility for pain, inflammation, epilepsy and possibly other central and peripheral nervous system diseases associated with cellular trauma and inflammation. This beneficial outcome may potentially lack nonspecific effects associated with the systemic administration of ADO receptor agonists. Until recently all of the reported AK inhibitors contained adenosine-like structural motif. The present review will discuss design, synthesis and analgesic and antiinflammatory properties of the novel nonnucleoside AK inhibitors that do not have close structural resemblance with the natural substrate ADO. Two classes of the nonnucleoside AK inhibitors are built on pyridopyrimidine and alkynylpyrimidine cores.

  1. Lead induces increased water permeability in astrocytes expressing aquaporin 4.

    PubMed

    Gunnarson, E; Axehult, G; Baturina, G; Zelenin, S; Zelenina, M; Aperia, A

    2005-01-01

    The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes. There is now compelling evidence that AQP4 may contribute to an unfavorable course in brain edema. Acute lead intoxication is a condition that causes brain damage preceded by brain edema. Here we report that lead increases AQP4 water permeability (P(f)) in astrocytes. A rat astrocyte cell line that does not express aquaporin 4 was transiently transfected with aquaporin 4 tagged with green fluorescent protein (GFP). Using confocal laser scanning microscopy we measured water permeability in these cells and in AQP4-negative cells located on the same plate. AQP4-expressing astrocytes had a three-fold higher water permeability than astrocytes not expressing AQP4. Lead exposure induced a significant, 40%, increase in water permeability in astrocytes expressing AQP4, but had no effect on P(f) in astrocytes not expressing AQP4. The increase in water permeability persisted after lead washout, while treatment with a lead chelator, meso-2,3-dimercaptosuccinic acid, abolished the lead-induced increase in P(f). The effect of lead was attenuated in the presence of a calcium (Ca(2+))/calmodulin-dependent protein kinase II (CaMKII) inhibitor, but not in the presence of a protein kinase C inhibitor. In cells expressing AQP4 where the consensus site for CaMKII phosphorylation was mutated, lead failed to increase water permeability. Lead exposure also increased P(f) in rat astroglial cells in primary culture, which express endogenous AQP4. Lead had no effect on P(f) in astrocytes transfected with aquaporin 3. In situ hybridization studies on rat brain after oral lead intake for three days showed no change in distribution of AQP4 mRNA. It is suggested that lead-triggered stimulation of water transport in AQP4-expressing astrocytes may contribute to the pathology of acute lead intoxication.

  2. Cellular response to low dose radiation: Role of phosphatidylinositol-3 kinase like kinases

    SciTech Connect

    Balajee, A.S.; Meador, J.A.; Su, Y.

    2011-03-24

    It is increasingly realized that human exposure either to an acute low dose or multiple chronic low doses of low LET radiation has the potential to cause different types of cancer. Therefore, the central theme of research for DOE and NASA is focused on understanding the molecular mechanisms and pathways responsible for the cellular response to low dose radiation which would not only improve the accuracy of estimating health risks but also help in the development of predictive assays for low dose radiation risks associated with tissue degeneration and cancer. The working hypothesis for this proposal is that the cellular mechanisms in terms of DNA damage signaling, repair and cell cycle checkpoint regulation are different for low and high doses of low LET radiation and that the mode of action of phosphatidylinositol-3 kinase like kinases (PIKK: ATM, ATR and DNA-PK) determines the dose dependent cellular responses. The hypothesis will be tested at two levels: (I) Evaluation of the role of ATM, ATR and DNA-PK in cellular response to low and high doses of low LET radiation in simple in vitro human cell systems and (II) Determination of radiation responses in complex cell microenvironments such as human EpiDerm tissue constructs. Cellular responses to low and high doses of low LET radiation will be assessed from the view points of DNA damage signaling, DNA double strand break repair and cell cycle checkpoint regulation by analyzing the activities (i.e. post-translational modifications and kinetics of protein-protein interactions) of the key target proteins for PI-3 kinase like kinases both at the intra-cellular and molecular levels. The proteins chosen for this proposal are placed under three categories: (I) sensors/initiators include ATM ser1981, ATR, 53BP1, gamma-H2AX, MDC1, MRE11, Rad50 and Nbs1; (II) signal transducers include Chk1, Chk2, FANCD2 and SMC1; and (III) effectors include p53, CDC25A and CDC25C. The primary goal of this proposal is to elucidate the

  3. Lead and growth.

    PubMed

    Kaji, Masayuki; Nishi, Yoshikazu

    2006-01-01

    Lead is highly toxic to the human body and children are much more vulnerable to lead toxicity than adults. Many studies have revealed that relatively low levels of blood lead can adversely affect human health, especially childhood growth and development. Blood lead levels (BLL) of children and adults have been decreasing recently almost all over the world, but a safety level for blood lead does not exist, and lead exposure is still a serious health problem especially for fetuses and children. Maternal lead burden causes fetal lead exposure and increases the risk of abortions, prematurity, low birth weight, and some minor anomalies. Infant BLL are inversely associated with weight gain. A negative relationship between somatic growth and BLL in children has been revealed. It has been suggested that lead exposure causes decrease of gonadotropin secretion of adolescents and delay of pubertal development. Several studies have revealed that children who are exposed to cigarette smoke have higher BLL than children who are not. Children should be protected from cigarette smoke for the purpose of avoiding the risk of increased BLL which might adversely affect their intellectual development and physical growth.

  4. Toxicological properties of lead.

    PubMed Central

    Damstra, T

    1977-01-01

    The pathological effects of lead on the renal, nervous, reproductive, endocrine, and immune systems have been reviewed. Emphasis is placed on reported subclinical effects due to chronic, low-level lead exposure. The crucial issue of whether subtle behavioral, intellectual, and developmental impairment occurs in young children, as a result of lead-induced CNS damage is discussed in detail. This issue remains unresolved. Further studies are needed in order to determine the long-term health effects of continuous, low-level lead exposure. PMID:198205

  5. Acute lead arsenate poisoning.

    PubMed

    Tallis, G A

    1989-12-01

    Three cases of acute lead arsenate poisoning which occurred in South Australia during a 12 month interval are described. The case reports demonstrate a number of features of the characteristic clinical syndrome which may follow ingestion of lead arsenate. The recommended management is immediate gastric lavage and subsequent chelation therapy with calcium EDTA and dimercaprol. Early gastric lavage may prevent significant lead absorption. However, arsenic acid (produced in the stomach when lead arsenate reacts with hydrochloric acid) is relatively water soluble and prompt gastric lavage is unlikely to prevent extensive arsenic absorption. It remains controversial as to whether chelation with dimercaprol prevents arsenical neuropathy.

  6. Lead poisoning: case studies

    PubMed Central

    Gordon, J N; Taylor, A; Bennett, P N

    2002-01-01

    Early clinical features of lead toxicity are non-specific and an occupational history is particularly valuable. Lead in the body comprises 2% in the blood (t1/2 35 days) and 95% in bone and dentine (t1/2 20–30 years). Blood lead may remain elevated for years after cessation from long exposure, due to redistribution from bone. Blood lead concentration is the most widely used marker for inorganic lead exposure. Zinc protoporphyrin (ZPP) concentration in blood usefully reflects lead exposure over the prior 3 months. Symptomatic patients with blood lead concentration >2.4 µmol l−1 (50 µg dl−1) or in any event >3.8 µmol l−1 (80 µg dl−1) should receive sodium calciumedetate i.v., followed by succimer by mouth for 19 days. Asymptomatic patients with blood lead concentration >2.4 µmol l−1 (50 µg dl−1) may be treated with succimer alone. Sodium calciumedetate should be given with dimercaprol to treat lead encephalopathy. PMID:11994050

  7. Ocular Toxicity of Tyrosine Kinase Inhibitors

    PubMed Central

    Davis, Mary Elizabeth

    2016-01-01

    Purpose/Objectives To review common tyrosine kinase inhibitors, as well as their ocular side effects and management. Data Sources A comprehensive literature search was conducted using cINahl®, Pubmed, and cochrane databases for articles published since 2004 with the following search terms: ocular toxicities, tyrosine kinase inhibitors, ophthalmology, adverse events, eye, and vision. Data Synthesis Tyrosine kinase inhibitors can cause significant eye toxicity. Conclusions Given the prevalence of new tyrosine kinase inhibitor therapies and the complexity of possible pathogenesis of ocular pathology, oncology nurses can appreciate the occurrence of ocular toxicities and the role of nursing in the management of these problems. Implications for Nursing Knowledge of the risk factors and etiology of ocular toxicity of targeted cancer therapies can guide nursing assessment, enhance patient education, and improve care management. Including a review of eye symptoms and vision issues in nursing assessment can enhance early detection and treatment of ocular toxicity. PMID:26906134

  8. Characterization of protein kinases from Blepharisma intermedium.

    PubMed

    Beyer, J

    1975-12-01

    Three protein kinases (EC 2.7.1.37) were detected in Blepharisma and partially purified. The enzymes were most active with histone as substrate protein. The stability of the bond between phosphate and protein acceptor showed the characteristics of seryl- or threonylphosphate. Protein kinase I was solubilized by ultrasonication or freezing and thawing, while the enzymes II and III were readily solubilized by mild homogenization. Protein II and III were noticeably activated by cAMP and cGMP, while protein kinase I was inhibited by cAMP. Associated with protein kinase II and III activity was the ability to bind labeled cAMP. The following molecular weights were determined: 90000 for enzyme I, 280000 for enzyme II, and 95000 for enzyme III. Various apparent Michaelis constants were estimated.

  9. Genetics Home Reference: mevalonate kinase deficiency

    MedlinePlus

    ... cytoskeleton), gene activity (expression), and protein production and modification. Most MVK gene mutations that cause mevalonate kinase ... What are the different ways in which a genetic condition can be inherited? More about Inheriting Genetic ...

  10. Human cervical cancer cells use Ca2+ signalling, protein tyrosine phosphorylation and MAP kinase in regulatory volume decrease.

    PubMed

    Shen, M R; Chou, C Y; Browning, J A; Wilkins, R J; Ellory, J C

    2001-12-01

    1. This study was aimed at identifying the signalling pathways involved in the activation of volume-regulatory mechanisms of human cervical cancer cells. 2. Osmotic swelling of human cervical cancer cells induced a substantial increase in intracellular Ca2+ ([Ca2+]i) by the activation of Ca2+ entry across the cell membrane, as well as Ca2+ release from intracellular stores. This Ca2+ signalling was critical for the normal regulatory volume decrease (RVD) response. 3. The activation of swelling-activated ion and taurine transport was significantly inhibited by tyrosine kinase inhibitors (genistein and tyrphostin AG 1478) and potentiated by the tyrosine phosphatase inhibitor Na3VO4. However, the Src family of tyrosine kinases was not involved in regulation of the swelling-activated Cl- channel. 4. Cell swelling triggered mitogen-activated protein (MAP) kinase cascades leading to the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) and p38 kinase. The volume-responsive ERK1/ERK2 signalling pathway linked with the activation of K+ and Cl- channels, and taurine transport. However, the volume-regulatory mechanism was independent of the activation of p38 MAP kinase. 5. The phosphorylated ERK1/ERK2 expression following a hypotonic shock was up-regulated by protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and down-regulated by PKC inhibitor staurosporine. The response of ERK activation to hypotonicity also required Ca2+ entry and depended on tyrosine kinase and mitogen-activated/ERK-activating kinase (MEK) activity. 6. Considering the results overall, osmotic swelling promotes the activation of tyrosine kinase and ERK1/ERK2 and raises intracellular Ca2+, all of which play a crucial role in the volume-regulatory mechanism of human cervical cancer cells. PMID:11731569

  11. Effect of Narrow Spectrum Versus Selective Kinase Inhibitors on the Intestinal Proinflammatory Immune Response in Ulcerative Colitis

    PubMed Central

    Biancheri, Paolo; Foster, Martyn R.; Fyfe, Matthew C. T.; MacDonald, Thomas T.; Sirohi, Sameer; Solanke, Yemisi; Wood, Eleanor; Rowley, Adele; Webber, Steve

    2016-01-01

    Background: Kinases are key mediators of inflammation, highlighting the potential of kinase inhibitors as treatments for inflammatory disorders. Selective kinase inhibitors, however, have proved disappointing, particularly in the treatment of rheumatoid arthritis and inflammatory bowel disease. Consequently, to improve efficacy, attention has turned to multikinase inhibition. Methods: The activity of a narrow spectrum kinase inhibitor, TOP1210, has been compared with selective kinase inhibitors (BIRB-796, dasatinib and BAY-61-3606) in a range of kinase assays, inflammatory cell assays, and in inflamed biopsies from patients with ulcerative colitis (UC). Effects on recombinant P38α, Src, and Syk kinase activities were assessed using Z-lyte assays (Invitrogen, Paisley, United Kingdom). Anti-inflammatory effects were assessed by measurement of proinflammatory cytokine release from peripheral blood mononuclear cells, primary macrophages, HT29 cells, inflamed colonic UC biopsies, and myofibroblasts isolated from inflamed colonic UC mucosa. Results: TOP1210 potently inhibits P38α, Src, and Syk kinase activities. Similarly, TOP1210 demonstrates potent inhibitory activity against proinflammatory cytokine release in each of the cellular assays and the inflamed colonic UC biopsies and myofibroblasts isolated from inflamed colonic UC mucosa. Generally, the selective kinase inhibitors showed limited and weaker activity in the cellular assays compared with the broad inhibitory profile of TOP1210. However, combination of the selective inhibitors led to improved efficacy and potency in both cellular and UC biopsy assays. Conclusions: Targeted, multikinase inhibition with TOP1210 leads to a broad efficacy profile in both the innate and adaptive immune responses, with significant advantages over existing selective kinase approaches, and potentially offers a much improved therapeutic benefit in inflammatory bowel disease. PMID:27104822

  12. Protein kinase A alterations in endocrine tumors.

    PubMed

    Yu, B; Ragazzon, B; Rizk-Rabin, M; Bertherat, J

    2012-09-01

    Various molecular and cellular alterations of the cyclic adenosine monophosphate (cAMP) pathway have been observed in endocrine tumors. Since protein kinase A (PKA) is a central key component of the cAMP pathway, studies of the alterations of PKA subunits in endocrine tumors reveal new aspects of the mechanisms of cAMP pathway alterations in human diseases. So far, most alterations have been observed for the regulatory subunits, mainly PRKAR1A and to a lower extent, PRKAR2B. One of the best examples of such alteration today is the multiple neoplasia syndrome Carney complex (CNC). The most common endocrine gland manifestations of CNC are pituitary GH-secreting adenomas, thyroid tumors, testicular tumors, and ACTH-independent Cushing's syndrome due to primary pigmented nodular adrenocortical disease (PPNAD). Heterozygous germline inactivating mutations of the PKA regulatory subunit RIα gene (PRKAR1A) are observed in about two-third of CNC patients, and also in patients with isolated PPNAD. PRKAR1A is considered as a tumor suppressor gene. Interestingly, these mutations can also be observed as somatic alterations in sporadic endocrine tumors. More than 120 different PRKAR1A mutations have been found today. Most of them lead to an unstable mutant mRNA, which will be degraded by nonsense mediated mRNA decay. In vitro and in vivo functional studies are in progress to understand the mechanisms of endocrine tumor development due to PKA regulatory subunits inactivation. PRKAR1A mutations stimulate in most models PKA activity, mimicking in some way cAMP pathway constitutive activation. Cross-talks with other signaling pathways summarized in this review have been described and might participate in endocrine tumorigenesis. PMID:22752956

  13. Myosin, Transgelin, and Myosin Light Chain Kinase

    PubMed Central

    Léguillette, Renaud; Laviolette, Michel; Bergeron, Celine; Zitouni, Nedjma; Kogut, Paul; Solway, Julian; Kachmar, Linda; Hamid, Qutayba; Lauzon, Anne-Marie

    2009-01-01

    Rationale: Airway smooth muscle (SM) of patients with asthma exhibits a greater velocity of shortening (Vmax) than that of normal subjects, and this is thought to contribute to airway hyperresponsiveness. A greater Vmax can result from increased myosin activation. This has been reported in sensitized human airway SM and in models of asthma. A faster Vmax can also result from the expression of specific contractile proteins that promote faster cross-bridge cycling. This possibility has never been addressed in asthma. Objectives: We tested the hypothesis that the expression of genes coding for SM contractile proteins is altered in asthmatic airways and contributes to their increased Vmax. Methods: We quantified the expression of several genes that code for SM contractile proteins in mild allergic asthmatic and control human airway endobronchial biopsies. The function of these contractile proteins was tested using the in vitro motility assay. Measurements and Main Results: We observed an increased expression of the fast myosin heavy chain isoform, transgelin, and myosin light chain kinase in patients with asthma. Immunohistochemistry demonstrated the expression of these genes at the protein level. To address the functional significance of this overexpression, we purified tracheal myosin from the hyperresponsive Fisher rats, which also overexpress the fast myosin heavy chain isoform as compared with the normoresponsive Lewis rats, and found a faster rate of actin filament propulsion. Conversely, transgelin did not alter the rate of actin filament propulsion. Conclusions: Selective overexpression of airway smooth muscle genes in asthmatic airways leads to increased Vmax, thus contributing to the airway hyperresponsiveness observed in asthma. PMID:19011151

  14. Rapamycin induces mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) expression through activation of protein kinase B and mitogen-activated protein kinase kinase pathways.

    PubMed

    Rastogi, Ruchi; Jiang, Zhongliang; Ahmad, Nisar; Rosati, Rita; Liu, Yusen; Beuret, Laurent; Monks, Robert; Charron, Jean; Birnbaum, Morris J; Samavati, Lobelia

    2013-11-22

    Mitogen-activated protein kinase phosphatase-1 (MKP-1), also known as dual specificity phosphatase-1 (DUSP-1), plays a crucial role in the deactivation of MAPKs. Several drugs with immune-suppressive properties modulate MKP-1 expression as part of their mechanism of action. We investigated the effect of mTOR inhibition through rapamycin and a dual mTOR inhibitor (AZD2014) on MKP-1 expression. Low dose rapamycin led to a rapid activation of both AKT and ERK pathways with a subsequent increase in MKP-1 expression. Rapamycin treatment led to phosphorylation of CREB, transcription factor 1 (ATF1), and ATF2, three transcription factors that bind to the cyclic AMP-responsive elements on the Mkp-1 promoter. Inhibition of either the MEK/ERK or the AKT pathway attenuated rapamycin-mediated MKP-1 induction. AZD2014 did not activate AKT but activated the ERK pathway, leading to a moderate MKP-1 induction. Using bone marrow-derived macrophages (BMDMs) derived from wild-type (WT) mice or mice deficient in AKT1 and AKT2 isoforms or BMDM from targeted deficiency in MEK1 and MEK2, we show that rapamycin treatment led to an increased MKP1 expression in BMDM from WT but failed to do so in BMDMs lacking the AKT1 isoform or MEK1 and MEK2. Importantly, rapamycin pretreatment inhibited LPS-mediated p38 activation and decreased nitric oxide and IL-6 production. Our work provides a conceptual framework for the observed immune modulatory effect of mTOR inhibition.

  15. A Molecular Brake in the Kinase Hinge Region Regulates the Activity of Receptor Tyrosine Kinases

    SciTech Connect

    Chen,H.; Ma, J.; Li, W.; Eliseenkova, A.; Xu, C.; Neubert, T.; Miller, W.; Mohammadi, M.

    2007-01-01

    Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory 'molecular brake' mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly.

  16. Identifying Kinase Substrates via a Heavy ATP Kinase Assay and Quantitative Mass Spectrometry

    PubMed Central

    Müller, André C.; Giambruno, Roberto; Weißer, Juliane; Májek, Peter; Hofer, Alexandre; Bigenzahn, Johannes W.; Superti-Furga, Giulio; Jessen, Henning J.; Bennett, Keiryn L.

    2016-01-01

    Mass spectrometry-based in vitro kinase screens play an essential role in the discovery of kinase substrates, however, many suffer from biological and technical noise or necessitate genetically-altered enzyme-cofactor systems. We describe a method that combines stable γ-[18O2]-ATP with classical in vitro kinase assays within a contemporary quantitative proteomic workflow. Our approach improved detection of known substrates of the non-receptor tyrosine kinase ABL1; and identified potential, new in vitro substrates. PMID:27346722

  17. Twitchin kinase interacts with MAPKAP kinase 2 in Caenorhabditis elegans striated muscle

    PubMed Central

    Matsunaga, Yohei; Qadota, Hiroshi; Furukawa, Miho; Choe, Heejoo (Helen); Benian, Guy M.

    2015-01-01

    In Caenorhabditis elegans, twitchin is a giant polypeptide located in muscle A-bands. The protein kinase of twitchin is autoinhibited by 45 residues upstream (NL) and 60 residues downstream (CRD) of the kinase catalytic core. Molecular dynamics simulation on a twitchin fragment revealed that the NL is released by pulling force. However, it is unclear how the CRD is removed. To identify proteins that may remove the CRD, we performed a yeast two-hybrid screen using twitchin kinase as bait. One interactor is MAK-1, C. elegans orthologue of MAPKAP kinase 2. MAPKAP kinase 2 is phosphorylated and activated by p38 MAP kinase. We demonstrate that the CRD of twitchin is important for binding to MAK-1. mak-1 is expressed in nematode body wall muscle, and antibodies to MAK-1 localize between and around Z-disk analogues and to the edge of A-bands. Whereas unc-22 mutants are completely resistant, mak-1 mutants are partially resistant to nicotine. MAK-1 can phosphorylate twitchin NL-Kin-CRD in vitro. Genetic data suggest the involvement of two other mak-1 paralogues and two orthologues of p38 MAP kinase. These results suggest that MAK-1 is an activator of twitchin kinase and that the p38 MAP kinase pathway may be involved in the regulation of twitchin. PMID:25851606

  18. Kinase-interacting substrate screening is a novel method to identify kinase substrates

    PubMed Central

    Amano, Mutsuki; Hamaguchi, Tomonari; Shohag, Md. Hasanuzzaman; Kozawa, Kei; Kato, Katsuhiro; Zhang, Xinjian; Yura, Yoshimitsu; Matsuura, Yoshiharu; Kataoka, Chikako; Nishioka, Tomoki

    2015-01-01

    Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases. PMID:26101221

  19. Redundant kinase activation and resistance of EGFR-tyrosine kinase inhibitors

    PubMed Central

    Luo, Min; Fu, Li-Wu

    2014-01-01

    Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have shown dramatic effects against that tumors harboring EGFR activating mutations in the EGFR intracytoplasmic tyrosine kinase domain and resulted in cell apoptosis. Unfortunately, a number of patients ultimately developed resistance by multiple mechanisms. Thus, elucidation of the mechanism of resistance to EGFR-TKIs can provide strategies for blocking or reversing the situation. Recent studies suggested that redundant kinase activation plays pivotal roles in escaping from the effects of EGFR-TKIs. Herein, we aimed to characterize several molecular events involved in the resistance to EGFR-TKIs mediated by redundant kinase activation. PMID:25520855

  20. Integration of Apoptosis Signal-Regulating Kinase 1-Mediated Stress Signaling with the Akt/Protein Kinase B-IκB Kinase Cascade

    PubMed Central

    Puckett, Mary C.; Goldman, Erinn H.; Cockrell, Lisa M.; Huang, Bei; Kasinski, Andrea L.; Du, Yuhong; Wang, Cun-Yu; Lin, Anning; Ichijo, Hidenori; Khuri, Fadlo

    2013-01-01

    Cellular processes are tightly controlled through well-coordinated signaling networks that respond to conflicting cues, such as reactive oxygen species (ROS), endoplasmic reticulum (ER) stress signals, and survival factors to ensure proper cell function. We report here a direct interaction between inhibitor of κB kinase (IKK) and apoptosis signal-regulating kinase 1 (ASK1), unveiling a critical node at the junction of survival, inflammation, and stress signaling networks. IKK can be activated by growth factor stimulation or tumor necrosis factor alpha engagement. IKK forms a complex with and phosphorylates ASK1 at a sensor site, Ser967, leading to the recruitment of 14-3-3, counteracts stress signal-triggered ASK1 activation, and suppresses ASK1-mediated functions. An inhibitory role of IKK in JNK signaling has been previously reported to depend on NF-κB-mediated gene expression. Our data suggest that IKK has a dual role: a transcription-dependent and a transcription-independent action in controlling the ASK1-JNK axis, coupling IKK to ROS and ER stress response. Direct phosphorylation of ASK1 by IKK also defines a novel IKK phosphorylation motif. Because of the intimate involvement of ASK1 in diverse diseases, the IKK/ASK1 interface offers a promising target for therapeutic development. PMID:23530055

  1. Disease Phenotypes in a Mouse Model of RNA Toxicity Are Independent of Protein Kinase Cα and Protein Kinase

    PubMed Central

    Kim, Yun K.; Yadava, Ramesh S.; Mandal, Mahua; Mahadevan, Karunasai; Yu, Qing; Leitges, Michael; Mahadevan, Mani S.

    2016-01-01

    Myotonic dystrophy type 1(DM1) is the prototype for diseases caused by RNA toxicity. RNAs from the mutant allele contain an expanded (CUG)n tract within the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. The toxic RNAs affect the function of RNA binding proteins leading to sequestration of muscleblind-like (MBNL) proteins and increased levels of CELF1 (CUGBP, Elav-like family member 1). The mechanism for increased CELF1 is not very clear. One favored proposition is hyper-phosphorylation of CELF1 by Protein Kinase C alpha (PKCα) leading to increased CELF1 stability. However, most of the evidence supporting a role for PKC-α relies on pharmacological inhibition of PKC. To further investigate the role of PKCs in the pathogenesis of RNA toxicity, we generated transgenic mice with RNA toxicity that lacked both the PKCα and PKCβ isoforms. We find that these mice show similar disease progression as mice wildtype for the PKC isoforms. Additionally, the expression of CELF1 is also not affected by deficiency of PKCα and PKCβ in these RNA toxicity mice. These data suggest that disease phenotypes of these RNA toxicity mice are independent of PKCα and PKCβ. PMID:27657532

  2. Recovering lead from batteries

    NASA Astrophysics Data System (ADS)

    David Prengaman, R.

    1995-01-01

    Over the past 20 years, a significant number of processes have been developed to recover lead from scrap batteries. These processes recover lead via hydrometallurgical processing of the paste component of the battery followed by electrowinning. A number of pilot plant operations have been conducted, but thus far none of the processes have become operational.

  3. Leadership, Leaders, and Leading

    ERIC Educational Resources Information Center

    Dean, Peter J.

    2004-01-01

    Arguably, the most important need in society today is to create individuals who will take action positively and progressively to lead in the private organization, in the the public institution, or in the governmental agency. Leadership action should not be held hostage by the notion that only those in formal positions of leadership can lead.…

  4. Rapid Lead Screening Test

    MedlinePlus

    ... and treated earlier before the damaging effects of lead poisoning occur. U.S. Department of Health and Human Services ... exceed 10μg/dL, the threshold used to indicate lead poisoning. The American Academy of Pediatrics (AAP) estimates one ...

  5. Lead Poisoning in Children.

    ERIC Educational Resources Information Center

    Lin-Fu, Jane S.

    This publication is a guide to help social and health workers plan a preventive campaign against lead poisoning, a cause of mental retardation other neurological handicaps, and death among children. The main victims are 1- to 6-year-olds living in areas where deteriorating housing prevails. Among the causes of lead poisoning are: ingestion of…

  6. Supersonic Leading Edge Receptivity

    NASA Technical Reports Server (NTRS)

    Maslov, Anatoly A.

    1998-01-01

    This paper describes experimental studies of leading edge boundary layer receptivity for imposed stream disturbances. Studies were conducted in the supersonic T-325 facility at ITAM and include data for both sharp and blunt leading edges. The data are in agreement with existing theory and should provide guidance for the development of more complete theories and numerical computations of this phenomena.

  7. Lead toxicity: a review

    PubMed Central

    Ara, Anjum; Usmani, Jawed Ahmad

    2015-01-01

    Lead toxicity is an important environmental disease and its effects on the human body are devastating. There is almost no function in the human body which is not affected by lead toxicity. Though in countries like US and Canada the use of lead has been controlled up to a certain extent, it is still used vehemently in the developing countries. This is primarily because lead bears unique physical and chemical properties that make it suitable for a large number of applications for which humans have exploited its benefits from historical times and thus it has become a common environmental pollutant. Lead is highly persistent in the environment and because of its continuous use its levels rise in almost every country, posing serious threats. This article reviews the works listed in the literature with recent updates regarding the toxicity of lead. Focus is also on toxic effects of lead on the renal, reproductive and nervous system. Finally the techniques available for treating lead toxicity are presented with some recent updates. PMID:27486361

  8. Lead carbonate scintillator materials

    DOEpatents

    Derenzo, Stephen E.; Moses, William W.

    1991-01-01

    Improved radiation detectors containing lead carbonate or basic lead carbonate as the scintillator element are disclosed. Both of these scintillators have been found to provide a balance of good stopping power, high light yield and short decay constant that is superior to other known scintillator materials. The radiation detectors disclosed are favorably suited for use in general purpose detection and in medical uses.

  9. LEAD IN CANDLE EMISSIONS

    EPA Science Inventory

    The candle-using public should be made aware that the core of candle wicks may contain lead. Used as a stiffening agent to keep the wick out of the molten wax, lead can be emitted as particulate to the air and then deposited on indoor surfaces. To define the problem, 100 sets of ...

  10. Lead polluters get punished

    SciTech Connect

    Not Available

    1991-08-09

    The Environmental Protection Agency (EPA) and the Department of Justice last week cracked down on 36 US companies for polluting the environment with lead. EPA slapped fines totaling more than $10 million on 12 of the offending companies, and Justice filed 24 civil complaints. Hank Habicht, deputy administrator of the EPA, said that his agency's initiative comes after 8 months of intense - and presumably successful - efforts at locating and documenting lead pollution in the soil, air, and water supply. Most feared has been lead's ability to damage the intellectual development of children. This caused the agency, Habicht said, to look beyond the usual suspect - lead in the water supply - to lead-laced dirt in residential areas. Meanwhile, the Department of Justice is using the EPA contamination data as well. Twenty US attorneys have been assigned to pore over the federal environmental statutes, including the Clean Water Act and the Superfund Law, in order to file civil complaints.

  11. Discovery of selective 4-Amino-pyridopyrimidine inhibitors of MAP4K4 using fragment-based lead identification and optimization.

    PubMed

    Crawford, Terry D; Ndubaku, Chudi O; Chen, Huifen; Boggs, Jason W; Bravo, Brandon J; Delatorre, Kelly; Giannetti, Anthony M; Gould, Stephen E; Harris, Seth F; Magnuson, Steven R; McNamara, Erin; Murray, Lesley J; Nonomiya, Jim; Sambrone, Amy; Schmidt, Stephen; Smyczek, Tanya; Stanley, Mark; Vitorino, Philip; Wang, Lan; West, Kristina; Wu, Ping; Ye, Weilan

    2014-04-24

    Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is a serine/threonine kinase implicated in the regulation of many biological processes. A fragment-based lead discovery approach was used to generate potent and selective MAP4K4 inhibitors. The fragment hit pursued in this article had excellent ligand efficiency (LE), an important attribute for subsequent successful optimization into drug-like lead compounds. The optimization efforts eventually led us to focus on the pyridopyrimidine series, from which 6-(2-fluoropyridin-4-yl)pyrido[3,2-d]pyrimidin-4-amine (29) was identified. This compound had low nanomolar potency, excellent kinase selectivity, and good in vivo exposure, and demonstrated in vivo pharmacodynamic effects in a human tumor xenograft model.

  12. Lead toxicity: current concerns.

    PubMed Central

    Goyer, R A

    1993-01-01

    Over the 20-year period since the first issue of Environmental Health Perspectives was published, there has been considerable progress in the understanding of the potential toxicity of exposure to lead. Many of these advances have been reviewed in published symposia, conferences, and review papers in EHP. This brief review identifies major advances as well as a number of current concerns that present opportunities for prevention and intervention strategies. The major scientific advance has been the demonstration that blood lead (PbB) levels of 10-15 micrograms/dL in newborn and very young infants result in cognitive and behavioral deficits. Further support for this observation is being obtained by prospective or longitudinal studies presently in progress. The mechanism(s) for the central nervous system effects of lead is unclear but involve lead interactions within calcium-mediated intracellular messenger systems and neurotransmission. Effects of low-level lead exposure on blood pressure, particularly in adult men, may be related to the effect of lead on calcium-mediated control of vascular smooth muscle contraction and on the renin-angiotensin system. Reproductive effects of lead have long been suspected, but low-level effects have not been well studied. Whether lead is a carcinogen or its association with renal adenocarcinoma is a consequence of cystic nephropathy is uncertain. Major risk factors for lead toxicity in children in the United States include nutrition, particularly deficiencies of essential metals, calcium, iron, and zinc, and housing and socioeconomic status. A goal for the year 2000 is to reduce prevalence of blood lead levels exceeding 15 micrograms/dL. Images FIGURE 2. PMID:8354166

  13. Lead toxicity: Current concerns

    SciTech Connect

    Goyer, R.A. )

    1993-04-01

    Over the 20-year period since the first issue of Environmental Health Perspectives was published, there has been considerable progress in the understanding of the potential toxicity of exposure to lead. Many of these advances have been reviewed in published symposia, conferences, and review papers in EHP. This brief review identifies major advances as well as a number of current concerns that present opportunities for prevention and intervention strategies. The major scientific advance has been the demonstration that blood lead (PbB) levels of 10-15 micrograms/dL in newborn and very young infants result in cognitive and behavioral deficits. Further support for this observation is being obtained by prospective or longitudinal studies presently in progress. The mechanism(s) for the central nervous system effects of lead is unclear but involve lead interactions within calcium-mediated intracellular messenger systems and neurotransmission. Effects of low-level lead exposure on blood pressure, particularly in adult men, may be related to the effect of lead on calcium-mediated control of vascular smooth muscle contraction and on the renin-angiotensin system. Reproductive effects of lead have long been suspected, but low-level effects have not been well studied. Whether lead is a carcinogen or its association with renal adenocarcinoma is a consequence of cystic nephropathy is uncertain. Major risk factors for lead toxicity in children in the United States include nutrition, particularly deficiencies of essential metals, calcium, iron, and zinc, and housing and socioeconomic status. A goal for the year 2000 is to reduce prevalence of blood lead levels exceeding 15 micrograms/dL. 97 refs.

  14. Methods to Purify and Assay Secretory Pathway Kinases.

    PubMed

    Tagliabracci, Vincent S; Wen, Jianzhong; Xiao, Junyu

    2016-01-01

    Members of the four-jointed and VLK families of secretory pathway kinases appear to be responsible for the phosphorylation of secreted proteins and proteoglycans. These enzymes have been implicated in many biological processes and mutations in several of these kinases cause human diseases. Here, we describe methods to purify and assay two members of the four-jointed family of secretory kinases: the Fam20C protein kinase and the Fam20B proteoglycan kinase. PMID:27632012

  15. Ubiquitin-Mediated Degradation of Aurora Kinases

    PubMed Central

    Lindon, Catherine; Grant, Rhys; Min, Mingwei

    2016-01-01

    The Aurora kinases are essential regulators of mitosis in eukaryotes. In somatic cell divisions of higher eukaryotes, the paralogs Aurora kinase A (AurA) and Aurora kinase B (AurB) play non-overlapping roles that depend on their distinct spatiotemporal activities. These mitotic roles of Aurora kinases depend on their interactions with different partners that direct them to different mitotic destinations and different substrates: AurB is a component of the chromosome passenger complex that orchestrates the tasks of chromosome segregation and cytokinesis, while AurA has many known binding partners and mitotic roles, including a well-characterized interaction with TPX2 that mediates its role in mitotic spindle assembly. Beyond the spatial control conferred by different binding partners, Aurora kinases are subject to temporal control of their activation and inactivation. Ubiquitin-mediated proteolysis is a critical route to irreversible inactivation of these kinases, which must occur for ordered transition from mitosis back to interphase. Both AurA and AurB undergo targeted proteolysis after anaphase onset as substrates of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase, even while they continue to regulate steps during mitotic exit. Temporal control of Aurora kinase destruction ensures that AurB remains active at the midbody during cytokinesis long after AurA activity has been largely eliminated from the cell. Differential destruction of Aurora kinases is achieved despite the fact that they are targeted at the same time and by the same ubiquitin ligase, making these substrates an interesting case study for investigating molecular determinants of ubiquitin-mediated proteolysis in higher eukaryotes. The prevalence of Aurora overexpression in cancers and their potential as therapeutic targets add importance to the task of understanding the molecular determinants of Aurora kinase stability. Here, we review what is known about ubiquitin-mediated targeting

  16. A membrane-anchored protein kinase involved in Brassica self-incompatibility signaling.

    PubMed

    Murase, Kohji; Shiba, Hiroshi; Iwano, Megumi; Che, Fang-Sik; Watanabe, Masao; Isogai, Akira; Takayama, Seiji

    2004-03-01

    Self-incompatibility (SI) response in Brassica is initiated by haplotype-specific interactions between the pollen-borne ligand S locus protein 11/SCR and its stigmatic S receptor kinase, SRK. This binding induces autophosphorylation of SRK, which is then thought to trigger a signaling cascade that leads to self-pollen rejection. A recessive mutation of the modifier (m) gene eliminates the SI response in stigma. Positional cloning of M has revealed that it encodes a membrane-anchored cytoplasmic serine/threonine protein kinase, designated M locus protein kinase (MLPK). Transient expression of MLPK restores the ability of mm papilla cells to reject self-pollen, suggesting that MLPK is a positive mediator of Brassica SI signaling.

  17. Systematic functional analysis of kinases in the fungal pathogen Cryptococcus neoformans

    PubMed Central

    Lee, Kyung-Tae; So, Yee-Seul; Yang, Dong-Hoon; Jung, Kwang-Woo; Choi, Jaeyoung; Lee, Dong-Gi; Kwon, Hyojeong; Jang, Juyeong; Wang, Li Li; Cha, Soohyun; Meyers, Gena Lee; Jeong, Eunji; Jin, Jae-Hyung; Lee, Yeonseon; Hong, Joohyeon; Bang, Soohyun; Ji, Je-Hyun; Park, Goun; Byun, Hyo-Jeong; Park, Sung Woo; Park, Young-Min; Adedoyin, Gloria; Kim, Taeyup; Averette, Anna F.; Choi, Jong-Soon; Heitman, Joseph; Cheong, Eunji; Lee, Yong-Hwan; Bahn, Yong-Sun

    2016-01-01

    Cryptococcus neoformans is the leading cause of death by fungal meningoencephalitis; however, treatment options remain limited. Here we report the construction of 264 signature-tagged gene-deletion strains for 129 putative kinases, and examine their phenotypic traits under 30 distinct in vitro growth conditions and in two different hosts (insect larvae and mice). Clustering analysis of in vitro phenotypic traits indicates that several of these kinases have roles in known signalling pathways, and identifies hitherto uncharacterized signalling cascades. Virulence assays in the insect and mouse models provide evidence of pathogenicity-related roles for 63 kinases involved in the following biological categories: growth and cell cycle, nutrient metabolism, stress response and adaptation, cell signalling, cell polarity and morphology, vacuole trafficking, transfer RNA (tRNA) modification and other functions. Our study provides insights into the pathobiological signalling circuitry of C. neoformans and identifies potential anticryptococcal or antifungal drug targets. PMID:27677328

  18. Inhibition of an Erythrocyte Tyrosine Kinase with Imatinib Prevents Plasmodium falciparum Egress and Terminates Parasitemia

    PubMed Central

    Kesely, Kristina R.; Pantaleo, Antonella; Turrini, Francesco M.; Olupot-Olupot, Peter

    2016-01-01

    With half of the world’s population at risk for malaria infection and with drug resistance on the rise, the search for mutation-resistant therapies has intensified. We report here a therapy for Plasmodium falciparum malaria that acts by inhibiting the phosphorylation of erythrocyte membrane band 3 by an erythrocyte tyrosine kinase. Because tyrosine phosphorylation of band 3 causes a destabilization of the erythrocyte membrane required for parasite egress, inhibition of the erythrocyte tyrosine kinase leads to parasite entrapment and termination of the infection. Moreover, because one of the kinase inhibitors to demonstrate antimalarial activity is imatinib, i.e. an FDA-approved drug authorized for use in children, translation of the therapy into the clinic will be facilitated. At a time when drug resistant strains of P. falciparum are emerging, a strategy that targets a host enzyme that cannot be mutated by the parasite should constitute a therapeutic mechanism that will retard evolution of resistance. PMID:27768734

  19. Molecular pathways: targeting the kinase effectors of RHO-family GTPases.

    PubMed

    Prudnikova, Tatiana Y; Rawat, Sonali J; Chernoff, Jonathan

    2015-01-01

    RHO GTPases, members of the RAS superfamily of small GTPases, are adhesion and growth factor-activated molecular switches that play important roles in tumor development and progression. When activated, RHO-family GTPases such as RAC1, CDC42, and RHOA, transmit signals by recruiting a variety of effector proteins, including the protein kinases PAK, ACK, MLK, MRCK, and ROCK. Genetically induced loss of RHO function impedes transformation by a number of oncogenic stimuli, leading to an interest in developing small-molecule inhibitors that either target RHO GTPases directly, or that target their downstream protein kinase effectors. Although inhibitors of RHO GTPases and their downstream signaling kinases have not yet been widely adopted for clinical use, their potential value as cancer therapeutics continues to facilitate pharmaceutical research and development and is a promising therapeutic strategy.

  20. Designing selective inhibitors for calcium-dependent protein kinases in apicomplexans.

    PubMed

    Hui, Raymond; El Bakkouri, Majida; Sibley, L David

    2015-07-01

    Apicomplexan parasites cause some of the most severe human diseases, including malaria (caused by Plasmodium), toxoplasmosis, and cryptosporidiosis. Treatments are limited by the lack of effective drugs and development of resistance to available agents. By exploiting novel features of protein kinases in these parasites, it may be possible to develop new treatments. We summarize here recent advances in identifying small molecule inhibitors against a novel family of plant-like, calcium-dependent kinases that are uniquely expanded in apicomplexan parasites. Analysis of the 3D structure, activation mechanism, and sensitivity to small molecules had identified several attractive chemical scaffolds that are potent and selective inhibitors of these parasite kinases. Further optimization of these leads may yield promising new drugs for treatment of these parasitic infections. PMID:26002073

  1. CaMKII and at least two unidentified kinases phosphorylate regulatory light chain in non-contracting cardiomyocytes.

    PubMed

    Eikemo, Hilde; Moltzau, Lise Román; Nguyen, Cam H T; Levy, Finn Olav; Skomedal, Tor; Osnes, Jan-Bjørn

    2016-08-12

    In cardiac tissue, regulatory light chain (RLC, myosin light chain 2) phosphorylation (Ser(15)) leads to modulation of muscle contraction through Ca(2+)-sensitization. To elucidate which kinases that are involved in the basal (diastolic phase) RLC phosphorylation, we studied non-contracting adult rat cardiomyocytes. RLC kinase activities in situ were unmasked by maximally inhibiting myosin light chain phosphatase (MLCP) by calyculin A in the absence and presence of various protein kinase inhibitors. Surprisingly MLCK did not contribute to the phosphorylation of RLC in the non-contracting cardiomyocytes. Two kinase activity groups were revealed by different sensitivities to staurosporine. The fraction with the highest sensitivity to staurosporine was inhibited by KN-93, a selective CaMKII inhibitor, producing a 23% ± 7% reduction in RLC phosphorylation. Calmodulin antagonism (W7) and reduction in Ca(2+) (EGTA) combined with low concentration of staurosporine caused a larger decrease in RLC phosphorylation than staurosporine alone. These data strongly suggest that in addition to CaMKII, there is another Ca(2+)/calmodulin-dependent kinase and a Ca(2+)/calmodulin-independent kinase phosphorylating RLC. Thus the RLC phosphorylation seems to be ensured by redundant kinase activities.

  2. Lead-free piezoceramics

    NASA Astrophysics Data System (ADS)

    Saito, Yasuyoshi; Takao, Hisaaki; Tani, Toshihiko; Nonoyama, Tatsuhiko; Takatori, Kazumasa; Homma, Takahiko; Nagaya, Toshiatsu; Nakamura, Masaya

    2004-11-01

    Lead has recently been expelled from many commercial applications and materials (for example, from solder, glass and pottery glaze) owing to concerns regarding its toxicity. Lead zirconium titanate (PZT) ceramics are high-performance piezoelectric materials, which are widely used in sensors, actuators and other electronic devices; they contain more than 60 weight per cent lead. Although there has been a concerted effort to develop lead-free piezoelectric ceramics, no effective alternative to PZT has yet been found. Here we report a lead-free piezoelectric ceramic with an electric-field-induced strain comparable to typical actuator-grade PZT. We achieved this through the combination of the discovery of a morphotropic phase boundary in an alkaline niobate-based perovskite solid solution, and the development of a processing route leading to highly <001> textured polycrystals. The ceramic exhibits a piezoelectric constant d33 (the induced charge per unit force applied in the same direction) of above 300picocoulombs per newton (pCN-1), and texturing the material leads to a peak d33 of 416pCN-1. The textured material also exhibits temperature-independent field-induced strain characteristics.

  3. Janus kinase (JAK) inhibitors in the treatment of inflammatory and neoplastic diseases.

    PubMed

    Roskoski, Robert

    2016-09-01

    The Janus kinase (JAK) family of non-receptor protein-tyrosine kinases consists of JAK1, JAK2, JAK3, and TYK2 (tyrosine kinase-2). Each of these proteins contains a JAK homology pseudokinase (JH2) domain that regulates the adjacent protein kinase domain (JH1). JAK1/2 and TYK2 are ubiquitously expressed whereas JAK3 is found predominantly in hematopoietic cells. The Janus kinase family is regulated by numerous cytokines including interleukins, interferons, and hormones such as erythropoietin, thrombopoietin, and growth hormone. Ligand binding to cytokine and hormone receptors leads to the activation of associated Janus kinases, which then mediate the phosphorylation of the receptors. The SH2 domain of STATs (signal transducers and activators of transcription) binds to the receptor phosphotyrosines thereby promoting STAT phosphorylation by the Janus kinases and consequent activation. STAT dimers are translocated to the nucleus where they participate in the regulation of the expression of thousands of proteins. JAK-STAT dysregulation results in autoimmune disorders such as rheumatoid arthritis, ulcerative colitis, and Crohn disease. JAK-STAT dysregulation also plays a role in the pathogenesis of myelofibrosis, polycythemia vera, and other myeloproliferative illnesses. An activating JAK2 V617F mutation occurs in 95% of people with polycythemia vera and in a lower percentage of people with other neoplasms. JAK1/3 signaling participates in the pathogenesis of inflammatory afflictions while JAK1/2 signaling participates in the development of several malignancies including leukemias and lymphomas as well as myeloproliferative neoplasms. Tofacitinib is a pan-JAK inhibitor that is approved by the FDA for the treatment of rheumatoid arthritis and ruxolitinib is a JAK1/2 inhibitor that is approved for the treatment of polycythemia vera and myelofibrosis. PMID:27473820

  4. Mediator kinase module and human tumorigenesis

    PubMed Central

    Clark, Alison D.; Oldenbroek, Marieke; Boyer, Thomas G.

    2016-01-01

    Mediator is a conserved multi-subunit signal processor through which regulatory informatiosn conveyed by gene-specific transcription factors is transduced to RNA Polymerase II (Pol II). In humans, MED13, MED12, CDK8 and Cyclin C (CycC) comprise a four-subunit “kinase” module that exists in variable association with a 26-subunit Mediator core. Genetic and biochemical studies have established the Mediator kinase module as a major ingress of developmental and oncogenic signaling through Mediator, and much of its function in signal-dependent gene regulation derives from its resident CDK8 kinase activity. For example, CDK8-targeted substrate phosphorylation impacts transcription factor half-life, Pol II activity and chromatin chemistry and functional status. Recent structural and biochemical studies have revealed a precise network of physical and functional subunit interactions required for proper kinase module activity. Accordingly, pathologic change in this activity through altered expression or mutation of constituent kinase module subunits can have profound consequences for altered signaling and tumor formation. Herein, we review the structural organization, biological function and oncogenic potential of the Mediator kinase module. We focus principally on tumor-associated alterations in kinase module subunits for which mechanistic relationships as opposed to strictly correlative associations are established. These considerations point to an emerging picture of the Mediator kinase module as an oncogenic unit, one in which pathogenic activation/deactivation through component change drives tumor formation through perturbation of signal-dependent gene regulation. It follows that therapeutic strategies to combat CDK8-driven tumors will involve targeted modulation of CDK8 activity or pharmacologic manipulation of dysregulated CDK8-dependent signaling pathways. PMID:26182352

  5. Molecular docking studies of banana flower flavonoids as insulin receptor tyrosine kinase activators as a cure for diabetes mellitus

    PubMed Central

    Ganugapati, Jayasree; Baldwa, Aashish; Lalani, Sarfaraz

    2012-01-01

    Diabetes mellitus is a metabolic disorder caused due to insulin deficiency. Banana flower is a rich source of flavonoids that exhibit anti diabetic activity. Insulin receptor is a tetramer that belongs to a family of receptor tyrosine kinases. It contains two alpha subunits that form the extracellular domain and two beta subunits that constitute the intracellular tyrosine kinase domain. Insulin binds to the extracellular region of the receptor and causes conformational changes that lead to the activation of the tyrosine kinase. This leads to autophosphorylation, a step that is crucial in insulin signaling pathway. Hence, compounds that augment insulin receptor tyrosine kinase activity would be useful in the treatment of diabetes mellitus. The 3D structure of IR tyrosine kinase was obtained from PDB database. The list of flavonoids found in banana flower was obtained from USDA database. The structures of the flavonoids were obtained from NCBI Pubchem. Docking analysis of the flavonoids was performed using Autodock 4.0 and Autodock Vina. The results indicate that few of the flavonoids may be potential activators of IR tyrosine kinase. PMID:22493522

  6. Molecular docking studies of banana flower flavonoids as insulin receptor tyrosine kinase activators as a cure for diabetes mellitus.

    PubMed

    Ganugapati, Jayasree; Baldwa, Aashish; Lalani, Sarfaraz

    2012-01-01

    Diabetes mellitus is a metabolic disorder caused due to insulin deficiency. Banana flower is a rich source of flavonoids that exhibit anti diabetic activity. Insulin receptor is a tetramer that belongs to a family of receptor tyrosine kinases. It contains two alpha subunits that form the extracellular domain and two beta subunits that constitute the intracellular tyrosine kinase domain. Insulin binds to the extracellular region of the receptor and causes conformational changes that lead to the activation of the tyrosine kinase. This leads to autophosphorylation, a step that is crucial in insulin signaling pathway. Hence, compounds that augment insulin receptor tyrosine kinase activity would be useful in the treatment of diabetes mellitus. The 3D structure of IR tyrosine kinase was obtained from PDB database. The list of flavonoids found in banana flower was obtained from USDA database. The structures of the flavonoids were obtained from NCBI Pubchem. Docking analysis of the flavonoids was performed using Autodock 4.0 and Autodock Vina. The results indicate that few of the flavonoids may be potential activators of IR tyrosine kinase.

  7. Epidermal Growth Factor Receptor Transactivation Is Required for Mitogen-Activated Protein Kinase Activation by Muscarinic Acetylcholine Receptors in HaCaT Keratinocytes

    PubMed Central

    Ockenga, Wymke; Kühne, Sina; Bocksberger, Simone; Banning, Antje; Tikkanen, Ritva

    2014-01-01

    Non-neuronal acetylcholine plays a substantial role in the human skin by influencing adhesion, migration, proliferation and differentiation of keratinocytes. These processes are regulated by the Mitogen-Activated Protein (MAP) kinase cascade. Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to activation of the MAP kinase extracellular signal regulated kinase, together with the protein kinase Akt. The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR), which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells. The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation. Furthermore, phosphorylation, ubiquitination and endocytosis of the EGF receptor after cholinergic transactivation are different from that induced by a direct stimulation with EGF, suggesting that ligands other than EGF itself mediate the cholinergic transactivation. PMID:25421240

  8. KinasePA: Phosphoproteomics data annotation using hypothesis driven kinase perturbation analysis

    PubMed Central

    Yang, Pengyi; Patrick, Ellis; Humphrey, Sean J.; Ghazanfar, Shila; James, David E.; Jothi, Raja; Yang, Jean Yee Hwa

    2016-01-01

    Mass spectrometry (MS)-based quantitative phosphoproteomics has become a key approach for proteome-wide profiling of phosphorylation in tissues and cells. Traditional experimental design often compares a single treatment with a control, whereas increasingly more experiments are designed to compare multiple treatments with respect to a control. To this end, the development of bioinformatic tools that can integrate multiple treatments and visualise kinases and substrates under combinatorial perturbations is vital for dissecting concordant and/or independent effects of each treatment. Here, we propose a hypothesis driven kinase perturbation analysis (KinasePA) to annotate and visualise kinases and their substrates that are perturbed by various combinatorial effects of treatments in phosphoproteomics experiments. We demonstrate the utility of KinasePA through its application to two large-scale phosphoproteomics datasets and show its effectiveness in dissecting kinases and substrates within signalling pathways driven by unique combinations of cellular stimuli and inhibitors. We implemented and incorporated KinasePA as part of the “directPA” R package available from the comprehensive R archive network (CRAN). Furthermore, KinasePA also has an interactive web interface that can be readily applied to annotate user provided phosphoproteomics data (http://kinasepa.pengyiyang.org). PMID:27145998

  9. Co-inhibition of polo-like kinase 1 and Aurora kinases promotes mitotic catastrophe

    PubMed Central

    Li, Jingjing; Hong, Myung Jin; Chow, Jeremy P.H.; Man, Wing Yu; Mak, Joyce P.Y.; Ma, Hoi Tang; Poon, Randy Y.C.

    2015-01-01

    Mitosis is choreographed by a number of protein kinases including polo-like kinases and Aurora kinases. As these kinases are frequently dysregulated in cancers, small-molecule inhibitors have been developed for targeted anticancer therapies. Given that PLK1 and Aurora kinases possess both unique functions as well as co-regulate multiple mitotic events, whether pharmacological inhibition of these kinases together can enhance mitotic catastrophe remains an outstanding issue to be determined. Using concentrations of inhibitors that did not induce severe mitotic defects on their own, we found that both the metaphase arrest and mitotic slippage induced by inhibitors targeting Aurora A and Aurora B (MK-5108 and Barasertib respectively) were enhanced by a PLK1 inhibitor (BI 2536). We found that PLK1 is overexpressed in cells from nasopharyngeal carcinoma, a highly invasive cancer with poor prognosis, in comparison to normal nasopharyngeal epithelial cells. Nasopharyngeal carcinoma cells were more sensitive to BI 2536 as a single agent and co-inhibition with Aurora kinases than normal cells. These observations underscore the mechanism and potential benefits of targeting PLK1 and Aurora kinases to induce mitotic catastrophe in cancer cells. PMID:25871386

  10. Co-inhibition of polo-like kinase 1 and Aurora kinases promotes mitotic catastrophe.

    PubMed

    Li, Jingjing; Hong, Myung Jin; Chow, Jeremy P H; Man, Wing Yu; Mak, Joyce P Y; Ma, Hoi Tang; Poon, Randy Y C

    2015-04-20

    Mitosis is choreographed by a number of protein kinases including polo-like kinases and Aurora kinases. As these kinases are frequently dysregulated in cancers, small-molecule inhibitors have been developed for targeted anticancer therapies. Given that PLK1 and Aurora kinases possess both unique functions as well as co-regulate multiple mitotic events, whether pharmacological inhibition of these kinases together can enhance mitotic catastrophe remains an outstanding issue to be determined. Using concentrations of inhibitors that did not induce severe mitotic defects on their own, we found that both the metaphase arrest and mitotic slippage induced by inhibitors targeting Aurora A and Aurora B (MK-5108 and Barasertib respectively) were enhanced by a PLK1 inhibitor (BI 2536). We found that PLK1 is overexpressed in cells from nasopharyngeal carcinoma, a highly invasive cancer with poor prognosis, in comparison to normal nasopharyngeal epithelial cells. Nasopharyngeal carcinoma cells were more sensitive to BI 2536 as a single agent and co-inhibition with Aurora kinases than normal cells. These observations underscore the mechanism and potential benefits of targeting PLK1 and Aurora kinases to induce mitotic catastrophe in cancer cells. PMID:25871386

  11. Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

    PubMed Central

    Mahajan, S; Fargnoli, J; Burkhardt, A L; Kut, S A; Saouaf, S J; Bolen, J B

    1995-01-01

    Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation. PMID:7565679

  12. Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

    PubMed

    Mahajan, S; Fargnoli, J; Burkhardt, A L; Kut, S A; Saouaf, S J; Bolen, J B

    1995-10-01

    Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation. PMID:7565679

  13. KinasePA: Phosphoproteomics data annotation using hypothesis driven kinase perturbation analysis.

    PubMed

    Yang, Pengyi; Patrick, Ellis; Humphrey, Sean J; Ghazanfar, Shila; James, David E; Jothi, Raja; Yang, Jean Yee Hwa

    2016-07-01

    Mass spectrometry (MS)-based quantitative phosphoproteomics has become a key approach for proteome-wide profiling of phosphorylation in tissues and cells. Traditional experimental design often compares a single treatment with a control, whereas increasingly more experiments are designed to compare multiple treatments with respect to a control. To this end, the development of bioinformatic tools that can integrate multiple treatments and visualise kinases and substrates under combinatorial perturbations is vital for dissecting concordant and/or independent effects of each treatment. Here, we propose a hypothesis driven kinase perturbation analysis (KinasePA) to annotate and visualise kinases and their substrates that are perturbed by various combinatorial effects of treatments in phosphoproteomics experiments. We demonstrate the utility of KinasePA through its application to two large-scale phosphoproteomics datasets and show its effectiveness in dissecting kinases and substrates within signalling pathways driven by unique combinations of cellular stimuli and inhibitors. We implemented and incorporated KinasePA as part of the "directPA" R package available from the comprehensive R archive network (CRAN). Furthermore, KinasePA also has an interactive web interface that can be readily applied to annotate user provided phosphoproteomics data (http://kinasepa.pengyiyang.org).

  14. CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases but we made the surprising observation that soybean CDPK' and several Arabidopsis isoforms (AtCPK4 and AtCPK34) could also autophosphorylate on tyrosine residues. In studies with His6-GmCDPK', we ide...

  15. The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility

    NASA Astrophysics Data System (ADS)

    Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I.; Hantschel, Oliver

    2014-11-01

    The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

  16. Metabolic Dysfunction Consistent with Premature Aging Results from Deletion of Pim Kinases

    PubMed Central

    Din, Shabana; Konstandin, Mathias H; Johnson, Bevan; Emathinger, Jacqueline; Völkers, Mirko; Toko, Haruhiro; Collins, Brett; Ormachea, Lucy; Samse, Kaitlen; Kubli, Dieter A; De La Torre, Andrea; Kraft, Andrew S; Gustafsson, Asa B; Kelly, Daniel P; Sussman, Mark A

    2014-01-01

    Rationale The senescent cardiac phenotype is accompanied by changes in mitochondrial function and biogenesis causing impairment in energy provision. The relationship between myocardial senescence and Pim kinases deserves attention since Pim-1 kinase is cardioprotective, in part, by preservation of mitochondrial integrity. Study of the pathological effects resulting from genetic deletion of all Pim kinase family members could provide important insight regarding cardiac mitochondrial biology and the aging phenotype. Objective Demonstrate myocardial senescence is promoted by loss of Pim leading to premature aging and aberrant mitochondrial function. Methods and Results Cardiac myocyte senescence was evident at three months of age in Pim Triple KnockOut (PTKO) mice, where all three isoforms of Pim kinase family members are genetically deleted. Cellular hypertrophic remodeling and fetal gene program activation was followed by heart failure at six months in PTKO mice. Metabolic dysfunction is an underlying cause of cardiac senescence and instigates a decline in cardiac function. Altered mitochondrial morphology is evident consequential to Pim deletion together with decreased ATP levels and increased phosphorylated AMPK, exposing an energy deficiency in PTKO mice. Expression of the genes encoding master regulators of mitochondrial biogenesis, PPARγ coactivator-1 (PGC-1) α and β were diminished in PTKO hearts, as were downstream targets included in mitochondrial energy transduction, including fatty acid oxidation. Reversal of the dysregulated metabolic phenotype was observed by overexpressing c-Myc, a downstream target of Pim kinases. Conclusion Pim kinases prevent premature cardiac aging and maintain a healthy pool of functional mitochondria leading to efficient cellular energetics. PMID:24916111

  17. Leading Causes of Blindness

    MedlinePlus

    ... Navigation Bar Home Current Issue Past Issues Feature: Vision Leading Causes of Blindness Past Issues / Summer 2008 ... of the lenses in your eyes. They affect vision and are very common in older people. More ...

  18. A Magnetic Bead-Based Protein Kinase Assay with Dual Detection Techniques

    PubMed Central

    Zhou, Guangchang; Sylvester, Juliesta E.; Wu, Ding; Veach, Darren R.; Kron, Stephen J.

    2010-01-01

    A novel magnetic bead-based protein kinase assay was developed using MALDI-TOF mass spectrometry (MALDI-TOF MS) and immuno-chemifluorescence as two independent detection techniques. Abltide substrate was immobilized onto magnetic beads via non-covalent biotin-streptavidin interactions. This non-covalent immobilization strategy facilitated peptide release and allowed MALDI-TOF MS analysis of substrate phosphorylation. The use of magnetic beads provided rapid sample handling and allowed secondary analysis by immuno-chemifluorescence to determine the degree of substrate phosphorylation. This dual detection technique was used to evaluate the inhibition of c-Abl kinase by imatinib and dasatinib. For each inhibitor, IC50 (half-maximal inhibitory concentration) values determined by these two different detection methods were consistent and close to values reported in the literature. The high-throughput potential of this new approach to kinase assays was preliminarily demonstrated by screening a chemical library consisting of 31 compounds against c-Abl kinase using a 96-well plate. In this proof-of-principle experiment, both MALDI-TOF MS and immuno-chemifluorescence were able to compare inhibitor potencies with consistent values. Dual detection may significantly enhance the reliability of chemical library screening and identify false positives and negatives. Formatted for 96-well plates and with high-throughput potential, this dual detection kinase assay may provide a rapid, reliable and inexpensive route to the discovery of small molecule drug leads. PMID:20807497

  19. Molecular Mechanism of Selectivity among G Protein-Coupled Receptor Kinase 2 Inhibitors

    SciTech Connect

    Thal, David M.; Yeow, Raymond Y.; Schoenau, Christian; Huber, Jochen; Tesmer, John J.G.

    2012-07-11

    G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-G{beta}{gamma} complex in the presence of two of these inhibitors. Comparison with the apoGRK2-G{beta}{gamma} structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.

  20. PAK4 promotes kinase-independent stabilization of RhoU to modulate cell adhesion

    PubMed Central

    Dart, Anna E.; Box, Gary M.; Court, William; Gale, Madeline E.; Brown, John P.; Pinder, Sarah E.; Eccles, Suzanne A.

    2015-01-01

    P21-activated kinase 4 (PAK4) is a Cdc42 effector protein thought to regulate cell adhesion disassembly in a kinase-dependent manner. We found that PAK4 expression is significantly higher in high-grade human breast cancer patient samples, whereas depletion of PAK4 modifies cell adhesion dynamics of breast cancer cells. Surprisingly, systematic analysis of PAK4 functionality revealed that PAK4-driven adhesion turnover is neither dependent on Cdc42 binding nor kinase activity. Rather, reduced expression of PAK4 leads to a concomitant loss of RhoU expression. We report that RhoU is targeted for ubiquitination by the Rab40A–Cullin 5 complex and demonstrate that PAK4 protects RhoU from ubiquitination in a kinase-independent manner. Overexpression of RhoU rescues the PAK4 depletion phenotype, whereas loss of RhoU expression reduces cell adhesion turnover and migration. These data support a new kinase-independent mechanism for PAK4 function, where an important role of PAK4 in cellular adhesions is to stabilize RhoU protein levels. Thus, PAK4 and RhoU cooperate to drive adhesion turnover and promote cell migration. PMID:26598620

  1. Central Role of Pyruvate Kinase in Carbon Co-catabolism of Mycobacterium tuberculosis.

    PubMed

    Noy, Tahel; Vergnolle, Olivia; Hartman, Travis E; Rhee, Kyu Y; Jacobs, William R; Berney, Michael; Blanchard, John S

    2016-03-25

    Mycobacterium tuberculosis (Mtb) displays a high degree of metabolic plasticity to adapt to challenging host environments. Genetic evidence suggests thatMtbrelies mainly on fatty acid catabolism in the host. However,Mtbalso maintains a functional glycolytic pathway and its role in the cellular metabolism ofMtbhas yet to be understood. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and theMtbgenome harbors one putative pyruvate kinase (pykA, Rv1617). Here we show thatpykAencodes an active pyruvate kinase that is allosterically activated by glucose 6-phosphate (Glc-6-P) and adenosine monophosphate (AMP). Deletion ofpykApreventsMtbgrowth in the presence of fermentable carbon sources and has a cidal effect in the presence of glucose that correlates with elevated levels of the toxic catabolite methylglyoxal. Growth attenuation was also observed in media containing a combination of short chain fatty acids and glucose and surprisingly, in media containing odd and even chain fatty acids alone. Untargeted high sensitivity metabolomics revealed that inactivation of pyruvate kinase leads to accumulation of phosphoenolpyruvate (P-enolpyruvate), citrate, and aconitate, which was consistent with allosteric inhibition of isocitrate dehydrogenase by P-enolpyruvate. This metabolic block could be relieved by addition of the α-ketoglutarate precursor glutamate. Taken together, our study identifies an essential role of pyruvate kinase in preventing metabolic block during carbon co-catabolism inMtb.

  2. Assays to measure the activation of membrane tyrosine kinase receptors: focus on cellular methods.

    PubMed

    Minor, Lisa K

    2003-09-01

    Many methods have been explored as means to measure the activation and inhibition of tyrosine kinase receptors, in vitro using the isolated kinase domain, and in living cells. Kinase activity has been measured in enzyme assays using a peptide substrate, but with different detection systems. These include the radioactive FlashPlate assay, the fluorescent resonance energy transfer (FRET) assay, the dissociation-enhance lanthanide fluorescence immunoassay (DELFIA) and other formats. These methods have successfully identified inhibitors of receptor activity. Cell-based assays have recently emerged to measure receptor activation and inhibition. When membrane tyrosine kinase receptors become activated, they increase their state of phosphorylation. This phosphorylation may lead to an increase in tyrosine kinase-specific activity. Methods have been developed that take advantage of these properties. These include measuring the ligand-stimulated total tyrosine phosphorylation of the receptor using a DELFIA or an ELISA assay, measuring ligand-stimulated enzyme activation of the receptor by quantifying enzyme activity, and dimerization of the activated receptor using bioluminescence resonance energy transfer (BRET). Although cell-based assays are still in their infancy, these techniques may prove a valuable addition to the receptor screening strategy.

  3. PRKX, a Novel cAMP-Dependent Protein Kinase Member, Plays an Important Role in Development.

    PubMed

    Huang, Sizhou; Li, Qian; Alberts, Ian; Li, Xiaohong

    2016-03-01

    The human protein kinase X gene (PRKX) and cAMP-dependent protein kinase (PKA) are both c-AMP-dependent serine/threonine protein kinases within the protein kinase AGC subgroup. Of all the protein kinases in this group, PRKX is the least studied. PRKX has been isolated from patients with chondrodysplasia punctate and is involved in numerous processes, including sexual differentiation and fertilization, normal kidney development and autosomal dominant polycystic kidney disease (ADPKD), blood maturation, neural development, and angiogenesis in vitro. Although the role of PRKX in development and disease has been reported recently, the underlying mechanism of PRKX activity is largely unknown. In addition, based on the expression pattern of PRKX and the extensive role of PKA in disease and development, PRKX might have additional crucial functions that have not been addressed in the literature. In this review, we summarize the characteristics and developmental functions of PRKX that have been reported by recent studies. In particular, we elucidate the structural and functional differences between PRKX and PKA, as well as the possible roles of PRKX in development and related diseases. Finally, we propose future studies that could lead to important discoveries of more PRKX functions and the underlying mechanisms involved. PMID:26252946

  4. Molecular characterization of WDCP, a novel fusion partner for the anaplastic lymphoma tyrosine kinase ALK

    PubMed Central

    YOKOYAMA, NORIKO; MILLER, W. TODD

    2015-01-01

    Anaplastic lymphoma kinase (ALK) is a member of the receptor tyrosine kinase superfamily. The ALK gene is a site of frequent mutation and chromosomal rearrangement in various types of human cancers. A novel chromosomal translocation was recently identified in human colorectal cancer between the ALK gene and chromosome 2, open reading frame 44 (C2orf44), a gene of unknown function. As a first step in understanding the oncogenic properties of this fusion protein, C2orf44 cDNA was cloned and the encoded protein was characterized, which was designated as WD repeat and coiled coil containing protein (WDCP). A C-terminal proline-rich segment in WDCP was shown to mediate binding to the Src homology 3 domain of the Src family kinase hematopoietic cell kinase (Hck). Co-expression with Hck lead to tyrosine phosphorylation of WDCP. Chromatographic fractionation of WDCP-containing lysates indicates that the protein exists as an oligomer in mammalian cells. These results suggest that, in the context of the ALK-C2orf44 gene fusion, WDCP imposes an oligomeric structure on ALK that results in constitutive kinase activation and signaling. PMID:25469238

  5. Chemical synthesis and biological validation of immobilized protein kinase inhibitory Leucettines.

    PubMed

    Burgy, Guillaume; Tahtouh, Tania; Durieu, Emilie; Foll-Josselin, Béatrice; Limanton, Emmanuelle; Meijer, Laurent; Carreaux, François; Bazureau, Jean-Pierre

    2013-04-01

    Leucettines, a family of marine sponge-derived 2-aminoimidazolone alkaloids, are potent inhibitors of DYRKs (dual-specificity, tyrosine phosphorylation regulated kinases) and CLKs (cdc2-like kinases). They constitute promising pharmacological leads for the treatment of several diseases, including Alzheimer's disease and Down syndrome. In order to investigate the scope of potential targets of Leucettine L41, a representative member of the chemical class, we designed an affinity chromatography strategy based on agarose-immobilized leucettines. A synthesis protocol for the attachment of a polyethylene (3 or 4 units) linker to L41 was first established. The linker attachment site on L41 was selected on the basis of the co-crystal structure of L41 with several kinases. L41 was then covalently bound to agarose beads through the primary amine located at the end of the linker. Control, kinase inactive Leucettine was also immobilized, as well as free linker devoid of ligand. Extracts of several mouse tissues revealed a complex pattern of interacting proteins, some of which probably resulting from non-specific, hydrophobic binding, while others representing bona fide Leucettine-interacting proteins. DYRK1A and GSK-3 (glycogen synthase kinase-3) were confirmed as interacting targets by Western blotting in various mouse tissues. The Leucettine affinity chromatography resin constitutes a powerful tool to purify and identify the targets of this new promising therapeutic class of molecules. PMID:23454515

  6. Central Role of Pyruvate Kinase in Carbon Co-catabolism of Mycobacterium tuberculosis.

    PubMed

    Noy, Tahel; Vergnolle, Olivia; Hartman, Travis E; Rhee, Kyu Y; Jacobs, William R; Berney, Michael; Blanchard, John S

    2016-03-25

    Mycobacterium tuberculosis (Mtb) displays a high degree of metabolic plasticity to adapt to challenging host environments. Genetic evidence suggests thatMtbrelies mainly on fatty acid catabolism in the host. However,Mtbalso maintains a functional glycolytic pathway and its role in the cellular metabolism ofMtbhas yet to be understood. Pyruvate kinase catalyzes the last and rate-limiting step in glycolysis and theMtbgenome harbors one putative pyruvate kinase (pykA, Rv1617). Here we show thatpykAencodes an active pyruvate kinase that is allosterically activated by glucose 6-phosphate (Glc-6-P) and adenosine monophosphate (AMP). Deletion ofpykApreventsMtbgrowth in the presence of fermentable carbon sources and has a cidal effect in the presence of glucose that correlates with elevated levels of the toxic catabolite methylglyoxal. Growth attenuation was also observed in media containing a combination of short chain fatty acids and glucose and surprisingly, in media containing odd and even chain fatty acids alone. Untargeted high sensitivity metabolomics revealed that inactivation of pyruvate kinase leads to accumulation of phosphoenolpyruvate (P-enolpyruvate), citrate, and aconitate, which was consistent with allosteric inhibition of isocitrate dehydrogenase by P-enolpyruvate. This metabolic block could be relieved by addition of the α-ketoglutarate precursor glutamate. Taken together, our study identifies an essential role of pyruvate kinase in preventing metabolic block during carbon co-catabolism inMtb. PMID:26858255

  7. Human lead absorption -- Texas.

    PubMed

    1997-09-19

    In December 1971, the City-County Health Department in El Paso, Texas, discovered that an ore smelter in El Paso was discharging large quantities of lead and other metallic wastes into the air. Between 1969 and 1971, this smelter had released 1,116 tons of lead, 560 tons of zinc, 12 tons of cadmium, and 1.2 tons of arsenic into the atmosphere through its stacks.

  8. Lead carbonate scintillator materials

    DOEpatents

    Derenzo, S.E.; Moses, W.W.

    1991-05-14

    Improved radiation detectors containing lead carbonate or basic lead carbonate as the scintillator element are disclosed. Both of these scintillators have been found to provide a balance of good stopping power, high light yield and short decay constant that is superior to other known scintillator materials. The radiation detectors disclosed are favorably suited for use in general purpose detection and in medical uses. 3 figures.

  9. Identification of two eukaryote-like serine/threonine kinases encoded by Chlamydia trachomatis serovar L2 and characterization of interacting partners of Pkn1.

    PubMed

    Verma, Anita; Maurelli, Anthony T

    2003-10-01

    Genome sequencing of C. trachomatis serovar D revealed the presence of three putative open reading frames (ORFs), CT145 (Pkn1), CT673 (Pkn5), and CT301 (PknD), encoding eukaryote-like serine/threonine kinases (Ser/Thr kinases). Two of these putative kinase genes, CT145 and CT301, were PCR amplified from serovar L2, cloned, and sequenced. Predicted translation products of the ORFs showed the presence of conserved kinase motifs at the N terminus of the proteins. CT145 and CT301 (encoding Pkn1 and PknD, respectively) were expressed in Escherichia coli as GST fusion proteins. In vitro kinase assays with Escherichia coli-derived glutathione S-transferase fusion proteins showed autophosphorylation of Pkn1 and PknD, indicating that they are functional kinases. Gene expression analysis of these kinase genes in Chlamydia by reverse transcriptase PCR indicated expression of these kinases at the early mid phase of the developmental cycle. Immunoprecipitated native chlamydial Pkn1 and PknD proteins also showed autophosphorylation in an in vitro kinase assay. Phosphoamino acid analysis by thin-layer chromatography confirmed that Pkn1 and PknD are phosphorylated on both serine and threonine residues. Interaction of Pkn1 and PknD with each other as well as interaction of Pkn1 with inclusion membrane protein G (IncG) was demonstrated by using a bacterial two-hybrid system. These interactions were further suggested by phosphorylation of the proteins in in vitro kinase assays. This report is the first description of the existence of functional Ser/Thr kinases in Chlamydia. The results of these findings should lead to a better understanding of how Chlamydia interact and interfere with host signaling pathways, since kinases represent potential mediators of the intimate host-pathogen interactions that are essential to the intracellular life cycle of Chlamydia.

  10. Identification of Novel Small Molecule Inhibitors of Oncogenic RET Kinase.

    PubMed

    Moccia, Marialuisa; Liu, Qingsong; Guida, Teresa; Federico, Giorgia; Brescia, Annalisa; Zhao, Zheng; Choi, Hwan Geun; Deng, Xianming; Tan, Li; Wang, Jinhua; Billaud, Marc; Gray, Nathanael S; Carlomagno, Francesca; Santoro, Massimo

    2015-01-01

    Oncogenic mutation of the RET receptor tyrosine kinase is observed in several human malignancies. Here, we describe three novel type II RET tyrosine kinase inhibitors (TKI), ALW-II-41-27, XMD15-44 and HG-6-63-01, that inhibit the cellular activity of oncogenic RET mutants at two digit nanomolar concentration. These three compounds shared a 3-trifluoromethyl-4-methylpiperazinephenyl pharmacophore that stabilizes the 'DFG-out' inactive conformation of RET activation loop. They blocked RET-mediated signaling and proliferation with an IC50 in the nM range in fibroblasts transformed by the RET/C634R and RET/M918T oncogenes. They also inhibited autophosphorylation of several additional oncogenic RET-derived point mutants and chimeric oncogenes. At a concentration of 10 nM, ALW-II-41-27, XMD15-44 and HG-6-63-01 inhibited RET kinase and signaling in human thyroid cancer cell lines carrying oncogenic RET alleles; they also inhibited proliferation of cancer, but not non-tumoral Nthy-ori-3-1, thyroid cells, with an IC50 in the nM range. The three compounds were capable of inhibiting the 'gatekeeper' V804M mutant which confers substantial resistance to established RET inhibitors. In conclusion, we have identified a type II TKI scaffold, shared by ALW-II-41-27, XMD15-44 and HG-6-63-01, that may be used as novel lead for the development of novel agents for the treatment of cancers harboring oncogenic activation of RET.

  11. Tyrosine kinase BMX phosphorylates phosphotyrosine-primed motif mediating the activation of multiple receptor tyrosine kinases.

    PubMed

    Chen, Sen; Jiang, Xinnong; Gewinner, Christina A; Asara, John M; Simon, Nicholas I; Cai, Changmeng; Cantley, Lewis C; Balk, Steven P

    2013-05-28

    The nonreceptor tyrosine kinase BMX (bone marrow tyrosine kinase gene on chromosome X) is abundant in various cell types and activated downstream of phosphatidylinositol-3 kinase (PI3K) and the kinase Src, but its substrates are unknown. Positional scanning peptide library screening revealed a marked preference for a priming phosphorylated tyrosine (pY) in the -1 position, indicating that BMX substrates may include multiple tyrosine kinases that are fully activated by pYpY sites in the kinase domain. BMX phosphorylated focal adhesion kinase (FAK) at Tyr⁵⁷⁷ subsequent to its Src-mediated phosphorylation at Tyr⁵⁷⁶. Loss of BMX by RNA interference or by genetic deletion in mouse embryonic fibroblasts (MEFs) markedly impaired FAK activity. Phosphorylation of the insulin receptor in the kinase domain at Tyr¹¹⁸⁹ and Tyr¹¹⁹⁰, as well as Tyr¹¹⁸⁵, and downstream phosphorylation of the kinase AKT at Thr³⁰⁸ were similarly impaired by BMX deficiency. However, insulin-induced phosphorylation of AKT at Ser⁴⁷³ was not impaired in Bmx knockout MEFs or liver tissue from Bmx knockout mice, which also showed increased insulin-stimulated glucose uptake, possibly because of decreased abundance of the phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase). Thus, by identifying the pYpY motif as a substrate for BMX, our findings suggest that BMX functions as a central regulator among multiple signaling pathways mediated by tyrosine kinases. PMID:23716717

  12. Magnesium Diboride Current Leads

    NASA Technical Reports Server (NTRS)

    Panek, John

    2010-01-01

    A recently discovered superconductor, magnesium diboride (MgB2), can be used to fabricate conducting leads used in cryogenic applications. Dis covered to be superconducting in 2001, MgB2 has the advantage of remaining superconducting at higher temperatures than the previously used material, NbTi. The purpose of these leads is to provide 2 A of electricity to motors located in a 1.3 K environment. The providing environment is a relatively warm 17 K. Requirements for these leads are to survive temperature fluctuations in the 5 K and 11 K heat sinks, and not conduct excessive heat into the 1.3 K environment. Test data showed that each lead in the assembly could conduct 5 A at 4 K, which, when scaled to 17 K, still provided more than the required 2 A. The lead assembly consists of 12 steelclad MgB2 wires, a tensioned Kevlar support, a thermal heat sink interface at 4 K, and base plates. The wires are soldered to heavy copper leads at the 17 K end, and to thin copper-clad NbTi leads at the 1.3 K end. The leads were designed, fabricated, and tested at the Forschungszentrum Karlsruhe - Institut foer Technische Physik before inclusion in Goddard's XRS (X-Ray Spectrometer) instrument onboard the Astro-E2 spacecraft. A key factor is that MgB2 remains superconducting up to 30 K, which means that it does not introduce joule heating as a resistive wire would. Because the required temperature ranges are 1.3-17 K, this provides a large margin of safety. Previous designs lost superconductivity at around 8 K. The disadvantage to MgB2 is that it is a brittle ceramic, and making thin wires from it is challenging. The solution was to encase the leads in thin steel tubes for strength. Previous designs were so brittle as to risk instrument survival. MgB2 leads can be used in any cryogenic application where small currents need to be conducted at below 30 K. Because previous designs would superconduct only at up to 8 K, this new design would be ideal for the 8-30 K range.

  13. Protein kinase activators alter glial cholesterol esterification

    SciTech Connect

    Jeng, I.; Dills, C.; Klemm, N.; Wu, C.

    1986-05-01

    Similar to nonneural tissues, the activity of glial acyl-CoA cholesterol acyltransferase is controlled by a phosphorylation and dephosphorylation mechanism. Manipulation of cyclic AMP content did not alter the cellular cholesterol esterification, suggesting that cyclic AMP is not a bioregulator in this case. Therefore, the authors tested the effect of phorbol-12-myristate 13-acetate (PMA) on cellular cholesterol esterification to determine the involvement of protein kinase C. PMA has a potent effect on cellular cholesterol esterification. PMA depresses cholesterol esterification initially, but cells recover from inhibition and the result was higher cholesterol esterification, suggesting dual effects of protein kinase C. Studies of other phorbol analogues and other protein kinase C activators such as merezein indicate the involvement of protein kinase C. Oleoyl-acetyl glycerol duplicates the effect of PMA. This observation is consistent with a diacyl-glycerol-protein kinase-dependent reaction. Calcium ionophore A23187 was ineffective in promoting the effect of PMA. They concluded that a calcium-independent and protein C-dependent pathway regulated glial cholesterol esterification.

  14. RTKdb: database of Receptor Tyrosine Kinase.

    PubMed

    Grassot, Julien; Mouchiroud, Guy; Perrière, Guy

    2003-01-01

    Receptor Tyrosine Kinases (RTK) are transmembrane receptors specifically found in metazoans. They represent an excellent model for studying evolution of cellular processes in metazoans because they encompass large families of modular proteins and belong to a major family of contingency generating molecules in eukaryotic cells: the protein kinases. Because tyrosine kinases have been under close scrutiny for many years in various species, they are associated with a wealth of information, mainly in mammals. Presently, most categories of RTK were identified in mammals, but in a near future other model species will be sequenced, and will bring us RTKs from other metazoan clades. Thus, collecting RTK sequences would provide a good starting point as a new model for comparative and evolutionary studies applying to multigene families. In this context, we are developing the Receptor Tyrosine Kinase database (RTKdb), which is the only database on tyrosine kinase receptors presently available. In this database, protein sequences from eight model metazoan species are organized under the format previously used for the HOVERGEN, HOBACGEN and NUREBASE systems. RTKdb can be accessed through the PBIL (Pôle Bioinformatique Lyonnais) World Wide Web server at http://pbil.univ-lyon1.fr/RTKdb/, or through the FamFetch graphical user interface available at the same address.

  15. Src Kinase Regulation in Progressively Invasive Cancer

    PubMed Central

    Xu, Weichen; Allbritton, Nancy; Lawrence, David S.

    2012-01-01

    Metastatic progression is a multistep process that involves tumor growth and survival, motility and invasion, and subsequent proliferation in an inappropriate environment. The Src protein tyrosine kinase has been implicated in many of the biochemical pathways that drive these behaviors. Although Src itself is only rarely mutated in human tumors, its aberrant activity has been noted in various cancers and suggested to serve as a barometer of metastatic potential. With these features in mind, we examined Src kinase regulation at the structural, enzymatic, and expression levels as a function of progressively invasive prostate cancer cell lines. Surprisingly, both total Src content and kinase activity decrease with increasing cell line aggressiveness, an observation that appears to be inconsistent with the well-documented role of Src in the signaling pathways that drive growth and invasion. However, we do observe a direct correlation between Src kinase specific activity (total Src kinase activity/total Src content) and metastatic aggressiveness, possibly suggesting that in highly aggressive cell lines, key signaling enzymes are globally recruited to drive the cancerous phenotype. In addition, although the expected enhanced phosphorylation of Src at Tyr-416 (activation site) is present in the most aggressive prostate cancer cell lines, unexpectedly high phosphorylation levels at the Tyr-527 inhibitory site are observed as well. The latter, rather than representative of inhibited enzyme, is more indicative of primed Src responsive to local phosphorylated binding partners. PMID:23145001

  16. Environmental lead in Mexico

    SciTech Connect

    Albert, L.A.; Badillo, F. )

    1991-01-01

    From the data presented here, it can be concluded that environmental exposure to lead is a particularly severe problem in Mexico. As has been shown, there are very important sources of exposure to this metal: (a) for rural populations who manufacture and/or utilize lead-glazed pottery, (b) for urban populations who are exposed to high air lead concentrations due to the continued use of lead fuel additives, (c) for workers of several industries, mainly those of batteries and pigments, (d) for consumers who routinely eat canned foods such as hot peppers and fruit products, and (e) for the general population living in the vicinity of smelters, refineries and other industries that emit lead. Therefore, in Mexico only those native populations living in very primitive communities, far away from all civilized life, could be expected to be free from this exposure. At the same time, and despite the relatively few data available, it can be stated that the exposure to lead of populations in Mexico could be approaching levels that might be highly hazardous, in particular for the neuropsychological health of children. Regarding the presence of lead in the environment, despite the fact that the available studies are not enough, it is evident that pollution by this metal is widespread and that there is a serious lack of studies for most regions of the country, including several that might be expected to be highly polluted. At the same time, it is evident that the official attention paid to the problem, either in regulations, support of further studies, or implementation of effective control measures has been far from the level needed according to the available data.

  17. Virtual screening filters for the design of type II p38 MAP kinase inhibitors: a fragment based library generation approach.

    PubMed

    Badrinarayan, Preethi; Sastry, G Narahari

    2012-04-01

    In this work, we introduce the development and application of a three-step scoring and filtering procedure for the design of type II p38 MAP kinase leads using allosteric fragments extracted from virtual screening hits. The design of the virtual screening filters is based on a thorough evaluation of docking methods, DFG-loop conformation, binding interactions and chemotype specificity of the 138 p38 MAP kinase inhibitors from Protein Data Bank bound to DFG-in and DFG-out conformations using Glide, GOLD and CDOCKER. A 40 ns molecular dynamics simulation with the apo, type I with DFG-in and type II with DFG-out forms was carried out to delineate the effects of structural variations on inhibitor binding. The designed docking-score and sub-structure filters were first tested on a dataset of 249 potent p38 MAP kinase inhibitors from seven diverse series and 18,842 kinase inhibitors from PDB, to gauge their capacity to discriminate between kinase and non-kinase inhibitors and likewise to selectively filter-in target-specific inhibitors. The designed filters were then applied in the virtual screening of a database of ten million (10⁷) compounds resulting in the identification of 100 hits. Based on their binding modes, 98 allosteric fragments were extracted from the hits and a fragment library was generated. New type II p38 MAP kinase leads were designed by tailoring the existing type I ATP site binders with allosteric fragments using a common urea linker. Target specific virtual screening filters can thus be easily developed for other kinases based on this strategy to retrieve target selective compounds. PMID:22306417

  18. Kinase-Dependent and -Independent Roles of EphA2 in the Regulation of Prostate Cancer Invasion and Metastasis

    PubMed Central

    Taddei, Maria Letizia; Parri, Matteo; Angelucci, Adriano; Onnis, Barbara; Bianchini, Francesca; Giannoni, Elisa; Raugei, Giovanni; Calorini, Lido; Rucci, Nadia; Teti, Anna; Bologna, Mauro; Chiarugi, Paola

    2009-01-01

    Ligand-activated Eph tyrosine kinases regulate cellular repulsion, morphology, adhesion, and motility. EphA2 kinase is frequently up-regulated in several different types of cancers, including prostate, breast, colon, and lung carcinomas, as well as in melanoma. The existing data do not clarify whether EphA2 receptor phosphorylation or its simple overexpression, which likely leads to Eph kinase-independent responses, plays a role in the progression of malignant prostate cancer. In this study, we address the role of EphA2 tyrosine phosphorylation in prostate carcinoma cell adhesion, motility, invasion, and formation of metastases. Tumor cells expressing kinase-deficient EphA2 mutants, as well as an EphA2 variant lacking the cytoplasmic domain, are defective in ephrinA1-mediated cell rounding, retraction fiber formation, de-adhesion from the extracellular matrix, RhoA and Rac1 GTPase regulation, three-dimensional matrix invasion, and in vivo metastasis, suggesting a key role for EphA2 kinase activity. Nevertheless, EphA2 regulation of cell motility and invasion, as well as the formation of bone and visceral tumor colonies, reveals a component of both EphA2 kinase-dependent and -independent features. These results uncover a differential requirement for EphA2 kinase activity in the regulation of prostate carcinoma metastasis outcome, suggesting that although the kinase activity of EphA2 is required for the regulation of cell adhesion and cytoskeletal rearrangement, some distinct kinase-dependent and -independent pathways likely cooperate to drive cancer cell migration, invasion, and metastasis outcome. PMID:19264906

  19. Structural and Spectroscopic Analysis of the Kinase Inhibitor Bosutinib and an Isomer of Bosutinib Binding to the Abl Tyrosine Kinase Domain

    PubMed Central

    Levinson, Nicholas M.; Boxer, Steven G.

    2012-01-01

    Chronic myeloid leukemia (CML) is caused by the kinase activity of the BCR-Abl fusion protein. The Abl inhibitors imatinib, nilotinib and dasatinib are currently used to treat CML, but resistance to these inhibitors is a significant clinical problem. The kinase inhibitor bosutinib has shown efficacy in clinical trials for imatinib-resistant CML, but its binding mode is unknown. We present the 2.4 Å structure of bosutinib bound to the kinase domain of Abl, which explains the inhibitor's activity against several imatinib-resistant mutants, and reveals that similar inhibitors that lack a nitrile moiety could be effective against the common T315I mutant. We also report that two distinct chemical compounds are currently being sold under the name “bosutinib”, and report spectroscopic and structural characterizations of both. We show that the fluorescence properties of these compounds allow inhibitor binding to be measured quantitatively, and that the infrared absorption of the nitrile group reveals a different electrostatic environment in the conserved ATP-binding sites of Abl and Src kinases. Exploiting such differences could lead to inhibitors with improved selectivity. PMID:22493660

  20. Rapid Discovery and Structure–Activity Relationships of Pyrazolopyrimidines That Potently Suppress Breast Cancer Cell Growth via SRC Kinase Inhibition with Exceptional Selectivity over ABL Kinase

    PubMed Central

    2016-01-01

    Novel pyrazolopyrimidines displaying high potency and selectivity toward SRC family kinases have been developed by combining ligand-based design and phenotypic screening in an iterative manner. Compounds were derived from the promiscuous kinase inhibitor PP1 to search for analogs that could potentially target a broad spectrum of kinases involved in cancer. Phenotypic screening against MCF7 mammary adenocarcinoma cells generated target-agnostic structure–activity relationships that biased subsequent designs toward breast cancer treatment rather than to a particular target. This strategy led to the discovery of two potent antiproliferative leads with phenotypically distinct anticancer mode of actions. Kinase profiling and further optimization resulted in eCF506, the first small molecule with subnanomolar IC50 for SRC that requires 3 orders of magnitude greater concentration to inhibit ABL. eCF506 exhibits excellent water solubility, an optimal DMPK profile and oral bioavailability, halts SRC-associated neuromast migration in zebrafish embryos without inducing life-threatening heart defects, and inhibits SRC phosphorylation in tumor xenografts in mice. PMID:27115835

  1. Regulation of mitogen-activated protein kinases by a calcium/calmodulin-dependent protein kinase cascade.

    PubMed Central

    Enslen, H; Tokumitsu, H; Stork, P J; Davis, R J; Soderling, T R

    1996-01-01

    Membrane depolarization of NG108 cells gives rapid (< 5 min) activation of Ca2+/calmodulin-dependent protein kinase IV (CaM-KIV), as well as activation of c-Jun N-terminal kinase (JNK). To investigate whether the Ca2+-dependent activation of mitogen-activated protein kinases (ERK, JNK, and p38) might be mediated by the CaM kinase cascade, we have transfected PC12 cells, which lack CaM-KIV, with constitutively active mutants of CaM kinase kinase and/or CaM-KIV (CaM-KKc and CaM-KIVc, respectively). In the absence of depolarization, CaM-KKc transfection had no effect on Elk-dependent transcription of a luciferase reporter gene, whereas CaM-KIVc alone or in combination with CaM-KKc gave 7- to 10-fold and 60- to 80-fold stimulations, respectively, which were blocked by mitogen-activated protein (MAP) kinase phosphatase cotransfection. When epitope-tagged constructs of MAP kinases were co-transfected with CaM-KKc plus CaM-KIVc, the immunoprecipitated MAP kinases were activated 2-fold (ERK-2) and 7- to 10-fold (JNK-1 and p38). The JNK and p38 pathways were further investigated using specific c-Jun or ATF2-dependent transcriptional assays. We found that c-Jun/ATF2-dependent transcriptions were enhanced 7- to 10-fold by CaM-KIVc and 20- to 30-fold by CaM-KKc plus CaM-KIVc. In the case of the Jun-dependent transcription, this effect was not due to direct phosphorylation of c-Jun by activated CaM-KIV, since transcription was blocked by a dominant-negative JNK and by two MAP kinase phosphatases. Mutation of the phosphorylation site (Thr196) in CaM-KIV, which mediates its activation by CaM-KIV kinase, prevented activation of Elk-1, c-Jun, and ATF2 by the CaM kinase cascade. These results establish a new Ca2+-dependent mechanism for regulating MAP kinase pathways and resultant transcription. Images Fig. 1 Fig. 3 Fig. 4 PMID:8855261

  2. Interaction between protein kinase C and protein kinase A can modulate transmitter release at the rat neuromuscular synapse.

    PubMed

    Santafé, M M; Garcia, N; Lanuza, M A; Tomàs, M; Tomàs, J

    2009-02-15

    We used intracellular recording to investigate the functional interaction between protein kinase C (PKC) and protein kinase A (PKA) signal transduction cascades in the control of transmitter release in the neuromuscular synapses from adult rats. Our results indicate that: 1) PKA and PKC are independently involved in asynchronous release. 2) Evoked acetylcholine (ACh) release is enhanced with the PKA agonist Sp-8-BrcAMP and the PKC agonist phorbol ester (PMA). 3) PKA has a constitutive role in promoting a component of normal evoked transmitter release because, when the kinase is inhibited with H-89, the release diminishes. However, the PKC inhibitor calphostin C (CaC) does not affect ACh release. 4) PKA regulates neurotransmission without PKC involvement because, after PMA or CaC modulation of the PKC activity, coupling to the ACh release of PKA can normally be stimulated with Sp-8-BrcAMP or inhibited with H-89. 5) After PKA inhibition with H-89, PKC stimulation with PMA (or inhibition with CaC) does not lead to any change in evoked ACh release. However, in PKA-stimulated preparations with Sp-8-BrcAMP, PKC becomes tonically active, thus potentiating a component of release that can now be blocked with CaC. In normal conditions, therefore, PKA was able to modulate ACh release independently of PKC activity, whereas PKA stimulation caused the PKC coupling to evoked release. In contrast, PKA inhibition prevent PKC stimulation (with the phorbol ester) and coupling to ACh output. There was therefore some dependence of PKC on PKA activity in the fine control of the neuromuscular synaptic functionalism and ACh release.

  3. Arabidopsis MAP kinase 4 regulates gene expression through transcription factor release in the nucleus.

    PubMed

    Qiu, Jin-Long; Fiil, Berthe Katrine; Petersen, Klaus; Nielsen, Henrik Bjørn; Botanga, Christopher J; Thorgrimsen, Stephan; Palma, Kristoffer; Suarez-Rodriguez, Maria Cristina; Sandbech-Clausen, Signe; Lichota, Jacek; Brodersen, Peter; Grasser, Klaus D; Mattsson, Ole; Glazebrook, Jane; Mundy, John; Petersen, Morten

    2008-08-20

    Plant and animal perception of microbes through pathogen surveillance proteins leads to MAP kinase signalling and the expression of defence genes. However, little is known about how plant MAP kinases regulate specific gene expression. We report that, in the absence of pathogens, Arabidopsis MAP kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further supported by the suppression of PAD3 expression in mpk4-wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation.

  4. MAP kinase pathways: The first twenty years

    PubMed Central

    Avruch, Joseph

    2007-01-01

    The MAP kinases, discovered approximately twenty years ago, together with their immediate upstream regulators, are among the most highly studied signal transduction molecules. This body of work has shaped many aspects of our present views of signal transduction by protein kinases. The effort expended in this area reflects the extensive participation of these regulatory modules in the control of cell fate decisions, i.e., proliferation, differentiation and death, across all eukaryotic phylla and in all tissues of metazoans. The discovery of these kinases is reviewed, followed by a discussion of some of the features of this signaling module that account for its broad impact on cell function and its enormous interest to many investigators. PMID:17229475

  5. Exploring the scaffold universe of kinase inhibitors.

    PubMed

    Hu, Ye; Bajorath, Jürgen

    2015-01-01

    The scaffold concept was applied to systematically determine, analyze, and compare core structures of kinase inhibitors. From publicly available inhibitors of the human kinome, scaffolds and cyclic skeletons were systematically extracted and organized taking activity data, structural relationships, and retrosynthetic criteria into account. Scaffold coverage varied greatly across the kinome, and many scaffolds representing compounds with different activity profiles were identified. The majority of kinase inhibitor scaffolds were involved in well-defined yet distinct structural relationships, which had different consequences on compound activity. Scaffolds exclusively representing highly potent compounds were identified as well as structurally analogous scaffolds with very different degrees of promiscuity. Scaffold relationships presented herein suggest a variety of hypotheses for inhibitor design. Our detailed organization of the kinase inhibitor scaffold universe with respect to different activity and structural criteria, all scaffolds, and the original compound data assembled for our analysis are made freely available.

  6. Crystal structure of human nicotinamide riboside kinase.

    PubMed

    Khan, Javed A; Xiang, Song; Tong, Liang

    2007-08-01

    Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD(+) as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 A resolution and in a ternary complex with ADP and tiazofurin at 2.7 A resolution. The active site is located in a groove between the central parallel beta sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations. PMID:17698003

  7. Crystal Structure of Human Nicotinamide Riboside Kinase

    SciTech Connect

    Khan,J.; Xiang, S.; Tong, L.

    2007-01-01

    Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD{sup +} as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 {angstrom} resolution and in a ternary complex with ADP and tiazofurin at 2.7 {angstrom} resolution. The active site is located in a groove between the central parallel {beta} sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.

  8. Structure of the human dimeric ATM kinase

    PubMed Central

    Lau, Wilson C. Y.; Li, Yinyin; Liu, Zhe; Gao, Yuanzhu; Zhang, Qinfen; Huen, Michael S. Y.

    2016-01-01

    ABSTRACT DNA-double strand breaks activate the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) to initiate DNA damage signal transduction. This activation process involves autophosphorylation and dissociation of inert ATM dimers into monomers that are catalytically active. Using single-particle electron microscopy (EM), we determined the structure of dimeric ATM in its resting state. The EM map could accommodate the crystal structure of the N-terminal truncated mammalian target of rapamycin (mTOR), a closely related enzyme of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family, allowing for the localization of the N- and the C-terminal regions of ATM. In the dimeric structure, the actives sites are buried, restricting the access of the substrates to these sites. The unanticipated domain organization of ATM provides a basis for understanding its mechanism of inhibition. PMID:27097373

  9. Structure of the human dimeric ATM kinase.

    PubMed

    Lau, Wilson C Y; Li, Yinyin; Liu, Zhe; Gao, Yuanzhu; Zhang, Qinfen; Huen, Michael S Y

    2016-01-01

    DNA-double strand breaks activate the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) to initiate DNA damage signal transduction. This activation process involves autophosphorylation and dissociation of inert ATM dimers into monomers that are catalytically active. Using single-particle electron microscopy (EM), we determined the structure of dimeric ATM in its resting state. The EM map could accommodate the crystal structure of the N-terminal truncated mammalian target of rapamycin (mTOR), a closely related enzyme of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family, allowing for the localization of the N- and the C-terminal regions of ATM. In the dimeric structure, the actives sites are buried, restricting the access of the substrates to these sites. The unanticipated domain organization of ATM provides a basis for understanding its mechanism of inhibition. PMID:27097373

  10. Protein Kinases and Parkinson’s Disease

    PubMed Central

    Mehdi, Syed Jafar; Rosas-Hernandez, Hector; Cuevas, Elvis; Lantz, Susan M.; Barger, Steven W.; Sarkar, Sumit; Paule, Merle G.; Ali, Syed F.; Imam, Syed Z.

    2016-01-01

    Currently, the lack of new drug candidates for the treatment of major neurological disorders such as Parkinson’s disease has intensified the search for drugs that can be repurposed or repositioned for such treatment. Typically, the search focuses on drugs that have been approved and are used clinically for other indications. Kinase inhibitors represent a family of popular molecules for the treatment and prevention of various cancers, and have emerged as strong candidates for such repurposing because numerous serine/threonine and tyrosine kinases have been implicated in the pathobiology of Parkinson’s disease. This review focuses on various kinase-dependent pathways associated with the expression of Parkinson’s disease pathology, and evaluates how inhibitors of these pathways might play a major role as effective therapeutic molecules. PMID:27657053

  11. Kinase signaling in the spindle checkpoint.

    PubMed

    Kang, Jungseog; Yu, Hongtao

    2009-06-01

    The spindle checkpoint is a cell cycle surveillance system that ensures the fidelity of chromosome segregation. In mitosis, it elicits the "wait anaphase" signal to inhibit the anaphase-promoting complex or cyclosome until all chromosomes achieve bipolar microtubule attachment and align at the metaphase plate. Because a single kinetochore unattached to microtubules activates the checkpoint, the wait anaphase signal is thought to be generated by this kinetochore and is then amplified and distributed throughout the cell to inhibit the anaphase-promoting complex/cyclosome. Several spindle checkpoint kinases participate in the generation and amplification of this signal. Recent studies have begun to reveal the activation mechanisms of these checkpoint kinases. Increasing evidence also indicates that the checkpoint kinases not only help to generate the wait anaphase signal but also actively correct kinetochore-microtubule attachment defects. PMID:19228686

  12. A monoclonal antibody distinguishes two types of phosphatidylinositol 4-kinase.

    PubMed Central

    Endemann, G C; Graziani, A; Cantley, L C

    1991-01-01

    A monoclonal antibody has been developed against the type II PtdIns 4-kinase from bovine brain. This antibody, 4C5G, causes greater than 90% inhibition of the type II PtdIns 4-kinase from bovine brain, rat brain and human erythrocytes. However, it fails to inhibit type III PtdIns 4-kinase from bovine brain or PtdIns 3-kinase from rat liver. These results suggest that type II and type III PtdIns 4-kinases are distinct gene products, and that 4C5G will be useful in studying the function of the type II PtdIns 4-kinase. PMID:1846531

  13. Measuring MAP kinase activity in immune complex assays.

    PubMed

    Cherkasova, Vera A

    2006-11-01

    I present an overview of published methods for measuring mitogen activated protein (MAP) kinase activity on endogenous associated substrates, exogenously added substrates as well as determination of activation loop phosphorylation as a read-out of kinase activity in vivo. Detailed procedures for these assays are given for two MAP kinases (MAPKs) Fus3 and Kss1 and compared with other published protocols, including the protocols for Hog1 and Mpk1 MAPKs. Measuring kinase activity in immune complex assays can serve as an approach for identification of potential substrates of protein kinases as well as for detecting other kinase-associated proteins. PMID:16890454

  14. Pim kinases in cancer: diagnostic, prognostic and treatment opportunities.

    PubMed

    Blanco-Aparicio, Carmen; Carnero, Amancio

    2013-03-01

    PIM proteins belong to a family of ser/thr kinases composed of 3 members, PIM1, PIM2 and PIM3, with greatly overlapping functions. PIM kinases are mainly responsible for cell cycle regulation, antiapoptotic activity and the homing and migration of receptor tyrosine kinases mediated via the JAK/STAT pathway. PIM kinases have been found to be upregulated in many hematological malignancies and solid tumors. Although these kinases have been described as weak oncogenes, they are heavily targeted for anticancer drug discovery. The present review summarizes the discoveries made to date regarding PIM kinases as driving oncogenes in the process of tumorigenesis and their validation as drug targets. PMID:23041228

  15. Pyruvate kinase M2 is a phosphotyrosine-binding protein

    SciTech Connect

    Christofk, H.R.; Vander Heiden, M.G.; Wu, N.; Asara, J.M.; Cantley, L.C.

    2008-06-03

    Growth factors stimulate cells to take up excess nutrients and to use them for anabolic processes. The biochemical mechanism by which this is accomplished is not fully understood but it is initiated by phosphorylation of signalling proteins on tyrosine residues. Using a novel proteomic screen for phosphotyrosine-binding proteins, we have made the observation that an enzyme involved in glycolysis, the human M2 (fetal) isoform of pyruvate kinase (PKM2), binds directly and selectively to tyrosine-phosphorylated peptides. We show that binding of phosphotyrosine peptides to PKM2 results in release of the allosteric activator fructose-1,6-bisphosphate, leading to inhibition of PKM2 enzymatic activity. We also provide evidence that this regulation of PKM2 by phosphotyrosine signalling diverts glucose metabolites from energy production to anabolic processes when cells are stimulated by certain growth factors. Collectively, our results indicate that expression of this phosphotyrosine-binding form of pyruvate kinase is critical for rapid growth in cancer cells.

  16. Identification of Ski as a target for Aurora A kinase

    PubMed Central

    Mosquera, Jocelyn; Armisen, Ricardo; Zhao, Hong Ling; Rojas, Diego A.; Maldonado, Edio; Tapia, Julio C; Colombo, Alicia; Hayman, Michael J; Marcelain, Katherine

    2011-01-01

    Ski is a negative regulator of the transforming growth factor-β and other signalling pathways. The absence of SKI in mouse fibroblasts leads to chromosome segregation defects and genomic instability, suggesting a role for Ski during mitosis. At this stage, Ski is phosphorylated but to date little is known about the kinases involved in this process. Here, we show that Aurora A kinase is able to phosphorylate Ski in vitro. In vivo, Aurora A and Ski co-localized at the centrosomes and co-immunoprecipitated. Conversely, a C-terminal truncation mutant of Ski (SkiΔ491–728) lacking a coiled-coil domain, displayed decreased centrosomal localization. This mutant no longer co-immunoprecipitated with Aurora-A in vivo, but was still phosphorylated in vitro, indicating that the Ski-Aurora A interaction takes place at the centrosomes. These data identify Ski as a novel target of Aurora A and contribute to an understanding of the role of these proteins in the mitotic process. PMID:21600873

  17. Development of Novel Adenosine Monophosphate-Activated Protein Kinase Activators

    PubMed Central

    Guh, Jih-Hwa; Chang, Wei-Ling; Yang, Jian; Lee, Su-Lin; Wei, Shuo; Wang, Dasheng; Kulp, Samuel K.; Chen, Ching-Shih

    2010-01-01

    In light of the unique ability of thiazolidinediones to mediate peroxisome proliferator-activated receptor (PPAR)γ-independent activation of adenosine monophosphate-activated protein kinase (AMPK) and suppression of interleukin (IL)-6 production, we conducted a screening of an in-house, thiazolidinedione-based focused compound library to identify novel agents with these dual pharmacological activities. Cell-based assays pertinent to the activation status of AMPK and mammalian homolog of target of rapamycin (i.e., phosphorylation of AMPK and p70 ribosomal protein S6 kinase, respectively), and IL-6/IL-6 receptor signaling (i.e., IL-6 production and signal transducer and activator of transcription 3 phosphorylation, respectively) in lipopolysaccharide (LPS)-stimulated THP-1 human macrophages were used to screen this compound library, which led to the identification of compound 53 (N-{4-[3-(1-Methylcyclohexylmethyl)-2,4-dioxo-thiazolidin-5-ylidene-methyl]-phenyl}-4-nitro-3-trifluoromethyl-benzenesulfonamide) as the lead agent. Evidence indicates that this drug-induced suppression of LPS-stimulated IL-6 production was attributable to AMPK activation. Furthermore, compound 53-mediated AMPK activation was demonstrated in C-26 colon adenocarcinoma cells, indicating that it is not a cell line-specific event. PMID:20170185

  18. Signaling Network Map of Endothelial TEK Tyrosine Kinase.

    PubMed

    Khan, Aafaque Ahmad; Sandhya, Varot K; Singh, Priyata; Parthasarathy, Deepak; Kumar, Awinav; Advani, Jayshree; Gattu, Rudrappa; Ranjit, Dhanya V; Vaidyanathan, Rama; Mathur, Premendu Prakash; Prasad, T S Keshava; Mac Gabhann, F; Pandey, Akhilesh; Raju, Rajesh; Gowda, Harsha

    2014-01-01

    TEK tyrosine kinase is primarily expressed on endothelial cells and is most commonly referred to as TIE2. TIE2 is a receptor tyrosine kinase modulated by its ligands, angiopoietins, to regulate the development and remodeling of vascular system. It is also one of the critical pathways associated with tumor angiogenesis and familial venous malformations. Apart from the vascular system, TIE2 signaling is also associated with postnatal hematopoiesis. Despite the involvement of TIE2-angiopoietin system in several diseases, the downstream molecular events of TIE2-angiopoietin signaling are not reported in any pathway repository. Therefore, carrying out a detailed review of published literature, we have documented molecular signaling events mediated by TIE2 in response to angiopoietins and developed a network map of TIE2 signaling. The pathway information is freely available to the scientific community through NetPath, a manually curated resource of signaling pathways. We hope that this pathway resource will provide an in-depth view of TIE2-angiopoietin signaling and will lead to identification of potential therapeutic targets for TIE2-angiopoietin associated disorders.

  19. The 2010 patent landscape for spleen tyrosine kinase inhibitors.

    PubMed

    Moretto, Alessandro F; Dehnhardt, Christoph; Kaila, Neelu; Papaioannou, Nikolaos; Thorarensen, Atli

    2012-05-01

    Discovery of small molecular inhibitors for treatment of rheumatoid arthritis is a major ongoing effort within the pharmaceutical industry. Spleen tyrosine kinase (SYK) is one of leading small molecular targets with regard to clinical development primarlly due to efforts by Rigel and Portola. In this review, we provide a comprehensive overview of the SYK patent landscape. The patent literature we evaluated relates to any organization that has filed applications that imply that SYK is the intended target. The interest in SYK was initiated in the early 2000's with many organizations, including several large pharmaceutical companies, and has been active for years. In general, the structural theme of most of the compounds in these applications is a traditional ATP competitive inhibitor with each organization having a different hinge binding element. In general, the attachment to the hinge is an aryl amine that is decorated with a solubilizing group. The other substituents are broadly variable across the numerous companies indicating that SYK has significant flexibility in its interactions in that portion of the kinase. This overview of the SYK patent literature and the learnings of the inhibitors' substitution patterns would be an important reference for anyone working in this area.

  20. LAMMER kinase contributes to genome stability in Ustilago maydis.

    PubMed

    de Sena-Tomás, Carmen; Sutherland, Jeanette H; Milisavljevic, Mira; Nikolic, Dragana B; Pérez-Martín, José; Kojic, Milorad; Holloman, William K

    2015-09-01

    Here we report identification of the lkh1 gene encoding a LAMMER kinase homolog (Lkh1) from a screen for DNA repair-deficient mutants in Ustilago maydis. The mutant allele isolated results from a mutation at glutamine codon 488 to a stop codon that would be predicted to lead to truncation of the carboxy-terminal kinase domain of the protein. This mutant (lkh1(Q488*)) is highly sensitive to ultraviolet light, methyl methanesulfonate, and hydroxyurea. In contrast, a null mutant (lkh1Δ) deleted of the entire lkh1 gene has a less severe phenotype. No epistasis was observed when an lkh1(Q488*)rad51Δ double mutant was tested for genotoxin sensitivity. However, overexpressing the gene for Rad51, its regulator Brh2, or the Brh2 regulator Dss1 partially restored genotoxin resistance of the lkh1Δ and lkh1(Q488*) mutants. Deletion of lkh1 in a chk1Δ mutant enabled these double mutant cells to continue to cycle when challenged with hydroxyurea. lkh1Δ and lkh1(Q488*) mutants were able to complete the meiotic process but exhibited reduced heteroallelic recombination and aberrant chromosome segregation. The observations suggest that Lkh1 serves in some aspect of cell cycle regulation after DNA damage or replication stress and that it also contributes to proper chromosome segregation in meiosis.

  1. X-Ray Crystal Structure of Bone Marrow Kinase in the X Chromosome: A Tec Family Kinase

    SciTech Connect

    Muckelbauer, Jodi; Sack, John S.; Ahmed, Nazia; Burke, James; Chang, ChiehYing Y.; Gao, Mian; Tino, Joseph; Xie, Dianlin; Tebben, Andrew J.

    2012-06-27

    Bone marrow kinase in the X chromosome, a member of the Tec family of tyrosine kinases, plays a role in both monocyte/macrophage trafficking as well as cytokine secretion. Although the structures of Tec family kinases Bruton's tyrosine kinase and IL-2-inducible T-cell kinase are known, the crystal structures of other Tec family kinases have remained elusive. We report the X-ray crystal structures of bone marrow kinase in the X chromosome in complex with dasatinib at 2.4 {angstrom} resolution and PP2 at 1.9 {angstrom} resolution. The bone marrow kinase in the X chromosome structures reveal a typical kinase protein fold; with well-ordered protein conformation that includes an open/extended activation loop and a stabilized DFG-motif rendering the kinase in an inactive conformation. Dasatinib and PP2 bind to bone marrow kinase in the X chromosome in the ATP binding pocket and display similar binding modes to that observed in other Tec and Src protein kinases. The bone marrow kinase in the X chromosome structures identify conformational elements of the DFG-motif that could potentially be utilized to design potent and/or selective bone marrow kinase in the X chromosome inhibitors.

  2. Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

    SciTech Connect

    Grose, C.; Jackson, W. ); Traugh, J.A. )

    1989-09-01

    Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing ({gamma}-{sup 32}P)ATP. The same glycoprotein was phosphorylated when ({sup 32}P)GTP was substituted for ({sup 32}P)ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.

  3. Lead zirconate titanate ceramics

    SciTech Connect

    Walker, B.E. Jr.

    1986-12-02

    This patent describes a lead zirconate titanate (PZT) piezoelectric ceramic composition which, based on total composition weight, consists essentially of a solid solution of lead zirconate and lead titanate in a PbZrO/sub 3/:PbTiO/sub 3/ ratio from about 0.505:0.495 to about 0.54:0.46; a halide salt selected from the group consisting of fluorides and chlorides of alkali metal and alkaline earth elements and mixtures thereof except for francium and radium in an amount from about 0.5 to 2 weight percent; and an oxide selected from the group consisting of magnesium, barium, scandium, aluminum, lanthanum, praesodynium, neodymium, samarium, and mixtures thereof in an amount from about 0.5 to about 6 weight percent, the relative amount of oxide being from about 1 to about 4 times that of the halide.

  4. Leading healthcare in complexity.

    PubMed

    Cohn, Jeffrey

    2014-12-01

    Healthcare institutions and providers are in complexity. Networks of interconnections from relationships and technology create conditions in which interdependencies and non-linear dynamics lead to surprising, unpredictable outcomes. Previous effective approaches to leadership, focusing on top-down bureaucratic methods, are no longer effective. Leading in complexity requires leaders to accept the complexity, create an adaptive space in which innovation and creativity can flourish and then integrate the successful practices that emerge into the formal organizational structure. Several methods for doing adaptive space work will be discussed. Readers will be able to contrast traditional leadership approaches with leading in complexity. They will learn new behaviours that are required of complexity leaders, along with challenges they will face, often from other leaders within the organization. PMID:25815410

  5. Leading healthcare in complexity.

    PubMed

    Cohn, Jeffrey

    2014-12-01

    Healthcare institutions and providers are in complexity. Networks of interconnections from relationships and technology create conditions in which interdependencies and non-linear dynamics lead to surprising, unpredictable outcomes. Previous effective approaches to leadership, focusing on top-down bureaucratic methods, are no longer effective. Leading in complexity requires leaders to accept the complexity, create an adaptive space in which innovation and creativity can flourish and then integrate the successful practices that emerge into the formal organizational structure. Several methods for doing adaptive space work will be discussed. Readers will be able to contrast traditional leadership approaches with leading in complexity. They will learn new behaviours that are required of complexity leaders, along with challenges they will face, often from other leaders within the organization.

  6. Muscarine enhances soluble amyloid precursor protein secretion in human neuroblastoma SH-SY5Y by a pathway dependent on protein kinase C(alpha), src-tyrosine kinase and extracellular signal-regulated kinase but not phospholipase C.

    PubMed

    Canet-Aviles, Rosa-Maria; Anderton, Mark; Hooper, Nigel M; Turner, Anthony J; Vaughan, Peter F T

    2002-06-15

    The signalling pathways by which muscarine and epidermal growth factor (EGF) regulate the secretion of the alpha-secretase cleavage product (sAPPalpha) of the amyloid precursor protein (APP) were examined in the human neuroblastoma SH-SY5Y. Using specific inhibitors it was found that over 80% of sAPPalpha secretion, enhanced by muscarine, occurred via the extracellular signal-regulated kinase (ERK1/2) member of the mitogen-activated protein kinase (MAPK) family and was dependent on protein kinase Calpha (PKCalpha) and a member of the Src family of non-receptor tyrosine kinases (Src-TK). In contrast the stimulation of sAPPalpha secretion by EGF was not affected by inhibitors of PKC nor Src-TK but was dependent on ERK1/2. In addition muscarine-enhanced sAPPalpha secretion and ERK1/2 activation were inhibited 60 and 80%, respectively, by micromolar concentrations of the phosphatidylinositol 3 kinase (PI-3K) inhibitor wortmannin. In comparison wortmannin decreased EGF stimulation of sAPPalpha secretion and ERK 1/2 activation by approximately 40%. Unexpectedly, U73122, an inhibitor of phosphoinositide-specific phospholipase C, did not inhibit muscarine enhancement of sAPPalpha secretion. These data are discussed in relation to a pathway for the enhancement of sAPPalpha secretion by muscarine which involves the activation of a Src-TK by G-protein beta/gamma-subunits leading to activation of PKCalpha, and ERK1/2 by a mechanism not involving phospholipase C. PMID:12191495

  7. Cloning and expression of the heterodimeric deoxyguanosine kinase/deoxyadenosine kinase of Lactobacillus acidophilus R-26.

    PubMed

    Ma, G T; Hong, Y S; Ives, D H

    1995-03-24

    Two uniquely paired deoxynucleoside kinases, deoxycytidine kinase/deoxyadenosine kinase (dCK/dAK) and deoxyguanosine kinase/deoxyadenosine kinase (dGK/dAK) are required, together with thymidine kinase (TK), for deoxynucleotide synthesis in Lactobacillus acidophilus R-26. Using polymerase chain reaction-generated probes based on N-terminal amino acid sequences, we have cloned tandem genes for 25- and 26-kDa polypeptides, whose derived amino acid sequences and size correspond to wild-type Lactobacillus enzyme subunits. Expression in Escherichia coli uses a single endogenous promoter and yields active dGK/dAK (approximately 3% of extracted protein) closely resembling wild-type dGK/dAK in specificity, kinetics, heterotropic activation, and end product inhibition. Alignment of cloned genes reveals 65% identity in their DNA sequences and 61% identity in derived amino acid sequences. Comparison with herpes-viral TKs reveals three conserved regions: glycine- and arginine-rich ATP-binding motifs and a D/E-R-S/H motif at the putative TK deoxynucleoside site. Greater homology, however, is seen upon multiple alignment of dGK with mammalian deoxycytidine kinases, yielding the consensus sequence-D/E-R-S-I/V-Y-x-D-.dGK also shares a sequence (-Y-D-P-T-I/L-E-D-S/Y-Y-) required for GTP hydrolysis by p21ras.

  8. Musk Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop

    PubMed Central

    Camurdanoglu, B. Z.; Hrovat, C.; Dürnberger, G.; Madalinski, M.; Mechtler, K.; Herbst, R.

    2016-01-01

    The neuromuscular junction (NMJ) forms when a motor neuron contacts a muscle fibre. A reciprocal exchange of signals initiates a cascade of signalling events that result in pre- and postsynaptic differentiation. At the centre of these signalling events stands muscle specific kinase (MuSK). MuSK activation, kinase activity and subsequent downstream signalling are crucial for NMJ formation as well as maintenance. Therefore MuSK kinase activity is tightly regulated to ensure proper NMJ development. We have identified a novel serine phosphorylation site at position 751 in MuSK that is increasingly phosphorylated upon agrin stimulation. S751 is also phosphorylated in muscle tissue and its phosphorylation depends on MuSK kinase activity. A phosphomimetic mutant of S751 increases MuSK kinase activity in response to non-saturating agrin concentrations . In addition, basal MuSK and AChR phosphorylation as well as AChR cluster size are increased. We believe that the phosphorylation of S751 provides a novel mechanism to relief the autoinhibition of the MuSK activation loop. Such a lower autoinhibition could foster or stabilize MuSK kinase activation, especially during stages when no or low level of agrin are present. Phosphorylation of S751 might therefore represent a novel mechanism to modulate MuSK kinase activity during prepatterning or NMJ maintenance. PMID:27666825

  9. Contribution of indazolinone tautomers to kinase activity.

    PubMed

    Vasudevan, Anil; Verzal, Mary K; Villamil, Clara I; Stewart, Kent D; Abad-Zapatero, Cele; Oie, Tetsuro; Djuric, Stevan W

    2012-07-15

    The design and synthesis of indazolinone containing kinase inhibitors are reported. Regioisomers that showed profound potency variation in previously-reported isoindolinone and aminoindazole systems were surprisingly found to have similar potencies in the case of the indazolinone chemical series. An interpretation using differential hinge hydrogen bonding and tautomeric equilibrium of indazolinone ring system is supported by quantum mechanics calculations. The equipotent inhibition of a representative kinase (KDR) by regioisomeric indazolinones 4 and 5 is clear evidence that in case of the indazolinone hinge, both tautomers are equally favored, and should be considered in design of inhibitors.

  10. Cell signaling by receptor-tyrosine kinases

    PubMed Central

    Lemmon, Mark A.; Schlessinger, Joseph

    2010-01-01

    Recent structural studies of receptor tyrosine kinases (RTKs) have revealed unexpected diversity in the mechanisms of their activation by growth factor ligands. Strategies for inducing dimerization by ligand binding are surprisingly diverse, as are mechanisms that couple this event to activation of the intracellular tyrosine kinase domains. As our understanding of these details becomes increasingly sophisticated, it provides an important context for therapeutically countering the effects of pathogenic RTK mutations in cancer and other diseases. Much remains to be learned, however, about the complex signaling networks downstream from RTKs and how alterations in these networks are translated into cellular responses. PMID:20602996

  11. Tyrosine phosphorylation is a mandatory proximal step in radiation-induced activation of the protein kinase C signaling pathway in human B-lymphocyte precursors.

    PubMed Central

    Uckun, F M; Schieven, G L; Tuel-Ahlgren, L M; Dibirdik, I; Myers, D E; Ledbetter, J A; Song, C W

    1993-01-01

    Ionizing radiation triggers a signal in human B-lymphocyte precursors that is intimately linked to an active protein-tyrosine kinase regulatory pathway. We show that in B-lymphocyte precursors, irradiation with gamma-rays leads to (i) stimulation of phosphatidylinositol turnover; (ii) downstream activation by covalent modification of multiple serine-specific protein kinases, including protein kinase C; and (iii) activation of nuclear factor kappa B. All of the radiation-induced signals were effectively prevented by the protein-tyrosine kinase inhibitors genistein and herbimycin A. Thus, tyrosine phosphorylation is an important and perhaps mandatory proximal step in the activation of the protein kinase C signaling cascade in human B-lymphocyte precursors. Our report expands current knowledge of the radiation-induced signaling cascade by clarifying the chronological sequence of biochemical events that follow irradiation. Images PMID:8419931

  12. Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei

    NASA Technical Reports Server (NTRS)

    Li, H.; Roux, S. J.

    1992-01-01

    Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.

  13. Identification and characterization of plant Haspin kinase as a histone H3 threonine kinase

    PubMed Central

    2011-01-01

    Background Haspin kinases are mitotic kinases that are well-conserved from yeast to human. Human Haspin is a histone H3 Thr3 kinase that has important roles in chromosome cohesion during mitosis. Moreover, phosphorylation of histone H3 at Thr3 by Haspin in fission yeast, Xenopus, and human is required for accumulation of Aurora B on the centromere, and the subsequent activation of Aurora B kinase activity for accurate chromosome alignment and segregation. Although extensive analyses of Haspin have been carried out in yeast and animals, the function of Haspin in organogenesis remains unclear. Results Here, we identified a Haspin kinase, designated AtHaspin, in Arabidopsis thaliana. The purified AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. Live imaging of AtHaspin-tdTomato and GFP-α-tubulin in BY-2 cells showed that AtHaspin-tdTomato localized on chromosomes during prometaphase and metaphase, and around the cell plate during cytokinesis. This localization of AtHaspin overlapped with that of phosphorylated Thr3 and Thr11 of histone H3 in BY-2 cells. AtHaspin-GFP driven by the native promoter was expressed in root meristems, shoot meristems, floral meristems, and throughout the whole embryo at stages of high cell division. Overexpression of a kinase domain mutant of AtHaspin decreased the size of the root meristem, which delayed root growth. Conclusions Our results indicated that the Haspin kinase is a histone H3 threonine kinase in A. thaliana. AtHaspin phosphorylated histone H3 at both Thr3 and Thr11 in vitro. The expression and dominant-negative analysis showed that AtHaspin may have a role in mitotic cell division during plant growth. Further analysis of coordinated mechanisms involving Haspin and Aurora kinases will shed new light on the regulation of chromosome segregation in cell division during plant growth and development. PMID:21527018

  14. Contribution of casein kinase 2 and spleen tyrosine kinase to CFTR trafficking and protein kinase A-induced activity.

    PubMed

    Luz, Simão; Kongsuphol, Patthara; Mendes, Ana Isabel; Romeiras, Francisco; Sousa, Marisa; Schreiber, Rainer; Matos, Paulo; Jordan, Peter; Mehta, Anil; Amaral, Margarida D; Kunzelmann, Karl; Farinha, Carlos M

    2011-11-01

    Previously, the pleiotropic "master kinase" casein kinase 2 (CK2) was shown to interact with CFTR, the protein responsible for cystic fibrosis (CF). Moreover, CK2 inhibition abolished CFTR conductance in cell-attached membrane patches, native epithelial ducts, and Xenopus oocytes. CFTR possesses two CK2 phosphorylation sites (S422 and T1471), with unclear impact on its processing and trafficking. Here, we investigated the effects of mutating these CK2 sites on CFTR abundance, maturation, and degradation coupled to effects on ion channel activity and surface expression. We report that CK2 inhibition significantly decreased processing of wild-type (wt) CFTR, with no effect on F508del CFTR. Eliminating phosphorylation at S422 and T1471 revealed antagonistic roles in CFTR trafficking: S422 activation versus T1471 inhibition, as evidenced by a severe trafficking defect for the T1471D mutant. Notably, mutation of Y512, a consensus sequence for the spleen tyrosine kinase (SYK) possibly acting in a CK2 context adjacent to the common CF-causing defect F508del, had a strong effect on both maturation and CFTR currents, allowing the identification of this kinase as a novel regulator of CFTR. These results reinforce the importance of CK2 and the S422 and T1471 residues for regulation of CFTR and uncover a novel regulation of CFTR by SYK, a recognized controller of inflammation.

  15. Role of Protein Kinase C, PI3-kinase and Tyrosine Kinase in Activation of MAP Kinase by Glucose and Agonists of G-protein Coupled Receptors in INS-1 Cells

    PubMed Central

    Böcker, Dietmar

    2001-01-01

    MAP (mitogen-activated protein) kinase (also called Erk 1/2) plays a crucial role in cell proliferation and differentiation. Its impact on secretory events is less well established. The interplay of protein kinase C (PKC), PI3-kinase nd cellular tyrosine kinase with MAP kinase activity using inhibitors and compounds such as glucose, phorbol 12-myristate 13-acetate (PMA) and agonists of G-protein coupled receptors like gastrin releasing peptide (GRP), oxytocin (OT) and glucose-dependent insulinotropic peptide (GIP) was investigated in INS-1 cells, an insulin secreting cell line. MAP kinase activity was determined by using a peptide derived from the EGF receptor as a MAP kinase substrate and [ P 32 ]ATP. Glucose as well as GRP, OT and GIP exhibited a time-dependent increase in MAP kinase activity with a maximum at time point 2.5 min. All further experiments were performed using 2.5 min incubations. The flavone PD 098059 is known to bind to the inactive forms of MEK1 (MAPK/ERK-Kinase) thus preventing activation by upstream activators. 20 μM PD 098059 ( IC 50 =51 μM) inhibited MAP kinase stimulated by either glucose, GRP, OT, GIP or PMA. Inhibiton (“downregulation”) of PKC by a long term (22h) pretreatment with 1 μM PMA did not influence MAP kinase activity when augmented by either of the above mentioned compound. To investigate whether PI3-kinase and cellular tyrosine kinase are involved in G-protein mediated effects on MAP kinase, inhibitors were used: 100 nM wortmannin (PI3-kinase inhibitor) reduced the effects of GRP, OT and GIP but not that of PMA; 100 μM genistein (tyrosine kinase inhibitor) inhibited the stimulatory effect of either above mentioned compound on MAP kinase activation. Inhibition of MAP kinase by 20 μM PD 098059 did not influence insulin secretion modulated by either compound (glucose, GRP, OT or GIP). [ H 3 ]Thymidine incorporation, however, was severely inhibited by PD 098059. Thus MAP kinase is important for INS-1 cell proliferation but

  16. Fluid-flow-induced mesenchymal stem cell migration: role of focal adhesion kinase and RhoA kinase sensors.

    PubMed

    Riehl, Brandon D; Lee, Jeong Soon; Ha, Ligyeom; Lim, Jung Yul

    2015-03-01

    The study of mesenchymal stem cell (MSC) migration under flow conditions with investigation of the underlying molecular mechanism could lead to a better understanding and outcome in stem-cell-based cell therapy and regenerative medicine. We used peer-reviewed open source software to develop methods for efficiently and accurately tracking, measuring and processing cell migration as well as morphology. Using these tools, we investigated MSC migration under flow-induced shear and tested the molecular mechanism with stable knockdown of focal adhesion kinase (FAK) and RhoA kinase (ROCK). Under steady flow, MSCs migrated following the flow direction in a shear stress magnitude-dependent manner, as assessed by root mean square displacement and mean square displacement, motility coefficient and confinement ratio. Silencing FAK in MSCs suppressed morphology adaptation capability and reduced cellular motility for both static and flow conditions. Interestingly, ROCK silencing significantly increased migration tendency especially under flow. Blocking ROCK, which is known to reduce cytoskeletal tension, may lower the resistance to skeletal remodelling during the flow-induced migration. Our data thus propose a potentially differential role of focal adhesion and cytoskeletal tension signalling elements in MSC migration under flow shear.

  17. Compartmentalization role of A-kinase anchoring proteins (AKAPs) in mediating protein kinase A (PKA) signaling and cardiomyocyte hypertrophy.

    PubMed

    Rababa'h, Abeer; Singh, Sonal; Suryavanshi, Santosh V; Altarabsheh, Salah Eldien; Deo, Salil V; McConnell, Bradley K

    2014-12-24

    The Beta-adrenergic receptors (β-ARs) stimulation enhances contractility through protein kinase-A (PKA) substrate phosphorylation. This PKA signaling is conferred in part by PKA binding to A-kinase anchoring proteins (AKAPs). AKAPs coordinate multi-protein signaling networks that are targeted to specific intracellular locations, resulting in the localization of enzyme activity and transmitting intracellular actions of neurotransmitters and hormones to its target substrates. In particular, mAKAP (muscle-selective AKAP) has been shown to be present on the nuclear envelope of cardiomyocytes with various proteins including: PKA-regulatory subunit (RIIα), phosphodiesterase-4D3, protein phosphatase-2A, and ryanodine receptor (RyR2). Therefore, through the coordination of spatial-temporal signaling of proteins and enzymes, mAKAP controls cyclic-adenosine monophosphate (cAMP) levels very tightly and functions as a regulator of PKA-mediated substrate phosphorylation leading to changes in calcium availability and myofilament calcium sensitivity. The goal of this review is to elucidate the critical compartmentalization role of mAKAP in mediating PKA signaling and regulating cardiomyocyte hypertrophy by acting as a scaffolding protein. Based on our literature search and studying the structure-function relationship between AKAP scaffolding protein and its binding partners, we propose possible explanations for the mechanism by which mAKAP promotes cardiac hypertrophy.

  18. Lead and compounds (inorganic)

    Integrated Risk Information System (IRIS)

    Lead and compounds ( inorganic ) ; CASRN 7439 - 92 - 1 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for

  19. Girls Leading Outward

    ERIC Educational Resources Information Center

    Hamed, Heather; Reyes, Jazmin; Moceri, Dominic C.; Morana, Laura; Elias, Maurice J.

    2011-01-01

    The authors describe a program implemented in Red Bank Middle School in New Jersey to help at-risk, minority middle school girls realize their leadership potential. The GLO (Girls Leading Outward) program was developed by the Developing Safe and Civil Schools Project at Rutgers University and is facilitated by university students. Selected middle…

  20. Lead Poisoning in Children.

    ERIC Educational Resources Information Center

    Lin-Fu, Jane S.

    Designed as a public information pamphlet, the text discusses the problem of lead poisoning in children. The preventable nature of the problem is stressed as well as needed action on the part of the public, physicians and other health workers, and the legislators. The pamphlet emphasizes that each of these areas is essential in preventing death or…

  1. Change, Lead, Succeed

    ERIC Educational Resources Information Center

    Munger, Linda; von Frank, Valerie

    2010-01-01

    Redefine leadership in your school, and create capacity through school leadership teams that successfully coordinate professional learning. "Change, Lead, Succeed" shows school leaders and teachers in leadership roles what they need to know to effectively create a culture for change. Find out what distinguishes a school leadership team from other…

  2. Leading through Conflict

    ERIC Educational Resources Information Center

    Gerzon, Mark

    2006-01-01

    This article talks about leading significant learning opportunities through conflict of ideas in a school system. Catalyzing school change can turn emotional differences of opinion into learning opportunities. Leaders who want to deal effectively with these challenging, often tense situations need to be more than good managers. They need to be…

  3. Leading for Engagement

    ERIC Educational Resources Information Center

    Yazzie-Mintz, Ethan

    2010-01-01

    As the dropout problem has grown--and as increasing numbers of students have started to see dropping out as a viable option for expressing their disaffection with school--practitioners, policymakers, and researchers have looked more closely at the factors that lead students to disengage from school and have attempted to find ways to create…

  4. Lead Thickness Measurements

    SciTech Connect

    Rucinski, R.; /Fermilab

    1998-02-16

    The preshower lead thickness applied to the outside of D-Zero's superconducting solenoid vacuum shell was measured at the time of application. This engineering documents those thickness measurements. The lead was ordered in sheets 0.09375-inch and 0.0625-inch thick. The tolerance on thickness was specified to be +/- 0.003-inch. The sheets all were within that thickness tolerance. The nomenclature for each sheet was designated 1T, 1B, 2T, 2B where the numeral designates it's location in the wrap and 'T' or 'B' is short for 'top' or 'bottom' half of the solenoid. Micrometer measurements were taken at six locations around the perimeter of each sheet. The width,length, and weight of each piece was then measured. Using an assumed pure lead density of 0.40974 lb/in{sup 3}, an average sheet thickness was calculated and compared to the perimeter thickness measurements. In every case, the calculated average thickness was a few mils thinner than the perimeter measurements. The ratio was constant, 0.98. This discrepancy is likely due to the assumed pure lead density. It is not felt that the perimeter is thicker than the center regions. The data suggests that the physical thickness of the sheets is uniform to +/- 0.0015-inch.

  5. ALL AGES LEAD MODEL

    EPA Science Inventory

    The Integrated Exposure Uptake Biokinetic (IEUBK) Model for Lead in Children (version 0.99d) was released in March 1994, and has been widely accepted in the risk assessment community as a tool for implementing the site specific risk assessment process when the issue is childhood...

  6. Leading by Learning

    ERIC Educational Resources Information Center

    Brookhart, Susan M.; Moss, Connie M.

    2013-01-01

    A lot has changed in the principalship since the principal was the head teacher in a school. Current principals are building administrators and that is likely to continue, the authors posit. Nonetheless, they report their study focusing on leadership's role in formative assessment concluded that in order to lead learning the principal must…

  7. Detecting disulfide-bound complexes and the oxidative regulation of cyclic nucleotide-dependent protein kinases by H2O2.

    PubMed

    Burgoyne, Joseph R; Eaton, Philip

    2013-01-01

    Hydrogen peroxide regulates intracellular signaling by oxidatively converting susceptible cysteine thiols to a modified state, which includes the formation of intermolecular disulfides. This type of oxidative modification can occur within the cAMP- and cGMP-dependent protein kinases often referred to as PKA and PKG, which have important roles in regulating cardiac contractility and systemic blood pressure. Both kinases are stimulated through conical pathways that elevate their respective cyclic nucleotides leading to direct kinase stimulation. However, PKA and PKG can also be functionally modulated independently of cyclic nucleotide stimulation through direct cysteine thiol oxidation leading to intermolecular disulfide formation. In the case of PKG, the formation of an intermolecular disulfide between two parallel dimeric subunits leads to enhanced kinase affinity for substrate. For PKA, the formation of two intermolecular disulfides between antiparallel dimeric regulatory RI subunits increases the affinity of this kinase for its binding partners, the A-kinase anchoring proteins, leading to increased PKA localization to its substrates. In this chapter, we describe the methods for detecting intermolecular disulfide-bound proteins and monitoring PKA and PKG oxidation within biological samples. PMID:23849862

  8. Dual activators of Protein Kinase R (PKR) and Protein Kinase R Like Kinase (PERK) Identify Common and Divergent Catalytic Targets

    PubMed Central

    Ming, Jie; Sun, Hong; Cao, Peng; Fusco, Dahlene N.; Chung, Raymond T.; Chorev, Michael; Jin, Qi; Aktas, Bertal H.

    2013-01-01

    Chemical genetics has evolved into a powerful tool for studying gene function in normal- and patho-biology. PKR and PERK, two eukaryotic translation initiation factor 2 alpha (eIF2α) kinases, play critical roles in maintenance of cellular hemostasis, metabolic stability, and anti-viral defenses. Both kinases interact with and phosphorylate additional substrates including tumor suppressor p53 and nuclear protein 90. Loss of function of both kinases has been studied by reverse genetics and recently identified inhibitors. In contrast, activating probes for studying the role of catalytic activity of these kinases are not available. We identified a 3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5,7-dihydroxy-4H-chromen-4-one (DHBDC) as specific dual activator of PKR and PERK by screening a chemical library of 20,000 small molecules in a dual luciferase surrogate eIF2α phosphorylation assay. We present here extensive biological characterization and preliminary structure-activity relationship of DHBDC, which phosphorylate eIF2α by activating PKR and PERK but no other eIF2α kinases. These agents also activate downstream effectors of eIF2α phosphorylation; inducing CHOP and suppressing cyclin D1 expression and inhibiting cancer cell proliferation, all in a manner dependent on PKR and PERK. Consistent with the role of eIF2α phosphorylation in viral infection, DHBDC inhibits proliferation of human hepatitis C virus. Finally, DHBDC induces phosphorylation of Ikβα, and activates NF-κB pathway. Surprisingly, activation of NF-κB pathway is dependent on PERK but independent of PKR activity. These data indicate that DHBDC is an invaluable probe for elucidating the role of PKR and PERK in normal- and patho-biology. PMID:23784735

  9. Mechanisms of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance and Strategies to Overcome Resistance in Lung Adenocarcinoma

    PubMed Central

    Chang, Yoon Soo; Choi, Chang-Min

    2016-01-01

    Somatic mutations that lead to hyperactivation of epidermal growth factor receptor (EGFR) signaling are detected in approximately 50% of lung adenocarcinoma in people from the Far East population and tyrosine kinase inhibitors are now the standard first line treatment for advanced disease. They have led to a doubling of progression-free survival and an increase in overall survival by more than 2 years. However, emergence of resistant clones has become the primary cause for treatment failure, and has created a new challenge in the daily management of patients with EGFR mutations. Identification of mechanisms leading to inhibitor resistance has led to new therapeutic modalities, some of which have now been adapted for patients with unsuccessful tyrosine kinase inhibitor treatment. In this review, we describe mechanisms of tyrosine kinase inhibitor resistance and the available strategies to overcoming resistance. PMID:27790276

  10. Discovery and Optimization of Macrocyclic Quinoxaline-pyrrolo-dihydropiperidinones as Potent Pim-1/2 Kinase Inhibitors.

    PubMed

    Cee, Victor J; Chavez, Frank; Herberich, Bradley; Lanman, Brian A; Pettus, Liping H; Reed, Anthony B; Wu, Bin; Wurz, Ryan P; Andrews, Kristin L; Chen, Jie; Hickman, Dean; Laszlo, Jimmy; Lee, Matthew R; Guerrero, Nadia; Mattson, Bethany K; Nguyen, Yen; Mohr, Christopher; Rex, Karen; Sastri, Christine E; Wang, Paul; Wu, Qiong; Wu, Tian; Xu, Yang; Zhou, Yihong; Winston, Jeffrey T; Lipford, J Russell; Tasker, Andrew S; Wang, Hui-Ling

    2016-04-14

    The identification of Pim-1/2 kinase overexpression in B-cell malignancies suggests that Pim kinase inhibitors will have utility in the treatment of lymphoma, leukemia, and multiple myeloma. Starting from a moderately potent quinoxaline-dihydropyrrolopiperidinone lead, we recognized the potential for macrocyclization and developed a series of 13-membered macrocycles. The structure-activity relationships of the macrocyclic linker were systematically explored, leading to the identification of 9c as a potent, subnanomolar inhibitor of Pim-1 and -2. This molecule also potently inhibited Pim kinase activity in KMS-12-BM, a multiple myeloma cell line with relatively high endogenous levels of Pim-1/2, both in vitro (pBAD IC50 = 25 nM) and in vivo (pBAD EC50 = 30 nM, unbound), and a 100 mg/kg daily dose was found to completely arrest the growth of KMS-12-BM xenografts in mice. PMID:27096050

  11. Discovery and Optimization of Macrocyclic Quinoxaline-pyrrolo-dihydropiperidinones as Potent Pim-1/2 Kinase Inhibitors.

    PubMed

    Cee, Victor J; Chavez, Frank; Herberich, Bradley; Lanman, Brian A; Pettus, Liping H; Reed, Anthony B; Wu, Bin; Wurz, Ryan P; Andrews, Kristin L; Chen, Jie; Hickman, Dean; Laszlo, Jimmy; Lee, Matthew R; Guerrero, Nadia; Mattson, Bethany K; Nguyen, Yen; Mohr, Christopher; Rex, Karen; Sastri, Christine E; Wang, Paul; Wu, Qiong; Wu, Tian; Xu, Yang; Zhou, Yihong; Winston, Jeffrey T; Lipford, J Russell; Tasker, Andrew S; Wang, Hui-Ling

    2016-04-14

    The identification of Pim-1/2 kinase overexpression in B-cell malignancies suggests that Pim kinase inhibitors will have utility in the treatment of lymphoma, leukemia, and multiple myeloma. Starting from a moderately potent quinoxaline-dihydropyrrolopiperidinone lead, we recognized the potential for macrocyclization and developed a series of 13-membered macrocycles. The structure-activity relationships of the macrocyclic linker were systematically explored, leading to the identification of 9c as a potent, subnanomolar inhibitor of Pim-1 and -2. This molecule also potently inhibited Pim kinase activity in KMS-12-BM, a multiple myeloma cell line with relatively high endogenous levels of Pim-1/2, both in vitro (pBAD IC50 = 25 nM) and in vivo (pBAD EC50 = 30 nM, unbound), and a 100 mg/kg daily dose was found to completely arrest the growth of KMS-12-BM xenografts in mice.

  12. Protein kinase cascades in the regulation of cardiac hypertrophy

    PubMed Central

    Dorn, Gerald W.; Force, Thomas

    2005-01-01

    In broad terms, there are 3 types of cardiac hypertrophy: normal growth, growth induced by physical conditioning (i.e., physiologic hypertrophy), and growth induced by pathologic stimuli. Recent evidence suggests that normal and exercise-induced cardiac growth are regulated in large part by the growth hormone/IGF axis via signaling through the PI3K/Akt pathway. In contrast, pathological or reactive cardiac growth is triggered by autocrine and paracrine neurohormonal factors released during biomechanical stress that signal through the Gq/phospholipase C pathway, leading to an increase in cytosolic calcium and activation of PKC. Here we review recent developments in the area of these cardiotrophic kinases, highlighting the utility of animal models that are helping to identify molecular targets in the human condition. PMID:15765134

  13. Phosphoinositide kinase signaling controls ER-PM cross-talk

    PubMed Central

    Omnus, Deike J.; Manford, Andrew G.; Bader, Jakob M.; Emr, Scott D.; Stefan, Christopher J.

    2016-01-01

    Membrane lipid dynamics must be precisely regulated for normal cellular function, and disruptions in lipid homeostasis are linked to the progression of several diseases. However, little is known about the sensory mechanisms for detecting membrane composition and how lipid metabolism is regulated in response to membrane stress. We find that phosphoinositide (PI) kinase signaling controls a conserved PDK-TORC2-Akt signaling cascade as part of a homeostasis network that allows the endoplasmic reticulum (ER) to modulate essential responses, including Ca2+-regulated lipid biogenesis, upon plasma membrane (PM) stress. Furthermore, loss of ER-PM junctions impairs this protective response, leading to PM integrity defects upon heat stress. Thus PI kinase–mediated ER-PM cross-talk comprises a regulatory system that ensures cellular integrity under membrane stress conditions. PMID:26864629

  14. Recent developments in receptor tyrosine kinases targeted anticancer therapy

    PubMed Central

    Raval, Samir H.; Singh, Ratn D.; Joshi, Dilip V.; Patel, Hitesh B.; Mody, Shailesh K.

    2016-01-01

    Novel concepts and understanding of receptors lead to discoveries and optimization of many small molecules and antibodies as anti-cancerous drugs. Receptor tyrosine kinases (RTKs) are such a promising class of receptors under the investigation in past three decades. RTKs are one of the essential mediators of cell signaling mechanism for various cellular processes. Transformations such as overexpression, dysregulation, or mutations of RTKs may result into malignancy, and thus are an important target for anticancer therapy. Numerous subfamilies of RTKs, such as epidermal growth factor receptor, vascular endothelial growth factor receptor, fibroblast growth factor receptors, insulin-like growth factor receptor, and hepatocyte growth factor receptor, have been being investigated in recent years as target for anticancer therapy. The present review focuses several small molecules drugs as well as monoclonal antibodies targeting aforesaid subfamilies either approved or under investigation to treat the various cancers. PMID:27051190

  15. AGCVIII Kinases: at the crossroads of cellular signaling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    AGCVIII kinases regulate diverse developmental and cellular processes in plants. As putative mediators of secondary messengers, AGCVIII kinases potentially integrate developmental and environmental cues into specific cellular responses through substrate phosphorylation. Here we discuss the functiona...

  16. Novel bone-targeted Src tyrosine kinase inhibitor drug discovery.

    PubMed

    Shakespeare, William C; Metcalf, Chester A; Wang, Yihan; Sundaramoorthi, Raji; Keenan, Terence; Weigele, Manfred; Bohacek, Regine S; Dalgarno, David C; Sawyer, Tomi K

    2003-09-01

    Bone-targeted Src tyrosine kinase (STK) inhibitors have recently been developed for the treatment of osteoporosis and cancer-related bone diseases. The concept of bone targeting derives from bisphosphonates, and from the evolution of such molecules in terms of therapeutic efficacy for the treatment of bone disorders. Interestingly, some of the earliest bisphosphonates were recognized for their ability to inhibit calcium carbonate precipitation (scaling) by virtue of their affinity to chelate calcium. This chelating property was subsequently exploited in the development of bisphosphonate analogs as inhibitors of the bone-resorbing cells known as osteoclasts, giving rise to breakthrough medicines, such as Fosamax (for the treatment of osteoporosis) and Zometa (for the treatment of osteoporosis and bone metastases). Relative to these milestone achievements, there is a tremendous opportunity to explore beyond the limited chemical space (functional group diversity) of such bisphosphonates to design novel bone-targeting moieties, which may be used to develop other classes of promising small-molecule drugs affecting different biological pathways. Here, we review studies focused on bone-targeted inhibitors of STK, a key enzyme in osteoclast-dependent bone resorption. Two strategies are described relative to bone-targeted STK inhibitor drug discovery: (i) the development of novel Src homology (SH)-2 inhibitors incorporating non-hydrolyzable phosphotyrosine mimics and exhibiting molecular recognition and bone-targeting properties, leading to the in vivo-effective lead compound AP-22408; and (ii) the development of novel ATP-based Src kinase inhibitors incorporating bone-targeting moieties, leading to the in vivo-effective lead compound AP-23236. In summary, AP-22408 and AP-23236, which differ mechanistically by virtue of blocking Src-dependent non-catalytic or catalytic activities in osteoclasts, exemplify ARIAD Pharmaceuticals' structure-based design of novel bone

  17. How do kinases contribute to tonicity-dependent regulation of the transcription factor NFAT5?

    PubMed Central

    Zhou, Xiaoming

    2016-01-01

    NFAT5 plays a critical role in maintaining the renal functions. Its dis-regulation in the kidney leads to or is associated with certain renal diseases or disorders, most notably the urinary concentration defect. Hypertonicity, which the kidney medulla is normally exposed to, activates NFAT5 through phosphorylation of a signaling molecule or NFAT5 itself. Hypotonicity inhibits NFAT5 through a similar mechanism. More than a dozen of protein and lipid kinases have been identified to contribute to tonicity-dependent regulation of NFAT5. Hypertonicity activates NFAT5 by increasing its nuclear localization and transactivating activity in the early phase and protein abundance in the late phase. The known mechanism for inhibition of NFAT5 by hypotonicity is a decrease of nuclear NFAT5. The present article reviews the effect of each kinase on NFAT5 nuclear localization, transactivation and protein abundance, and the relationship among these kinases, if known. Cyclosporine A and tacrolimus suppress immune reactions by inhibiting the phosphatase calcineurin-dependent activation of NFAT1. It is hoped that this review would stimulate the interest to seek explanations from the NFAT5 regulatory pathways for certain clinical presentations and to explore novel therapeutic approaches based on the pathways. On the basic science front, this review raises two interesting questions. The first one is how these kinases can specifically signal to NFAT5 in the context of hypertonicity or hypotonicity, because they also regulate other cellular activities and even opposite activities in some cases. The second one is why these many kinases, some of which might have redundant functions, are needed to regulate NFAT5 activity. This review reiterates the concept of signaling through cooperation. Cells need these kinases working in a coordinated way to provide the signaling specificity that is lacking in the individual one. Redundancy in regulation of NFAT5 is a critical strategy for cells to

  18. Structural Basis for Selective Small Molecule Kinase Inhibition of Activated c-Met

    SciTech Connect

    Rickert, Keith W.; Patel, Sangita B.; Allison, Timothy J.; Byrne, Noel J.; Darke, Paul L.; Ford, Rachael E.; Guerin, David J.; Hall, Dawn L.; Kornienko, Maria; Lu, Jun; Munshi, Sanjeev K.; Reid, John C.; Shipman, Jennifer M.; Stanton, Elizabeth F.; Wilson, Kevin J.; Young, Jonathon R.; Soisson, Stephen M.; Lumb, Kevin J.

    2012-03-15

    The receptor tyrosine kinase c-Met is implicated in oncogenesis and is the target for several small molecule and biologic agents in clinical trials for the treatment of cancer. Binding of the hepatocyte growth factor to the cell surface receptor of c-Met induces activation via autophosphorylation of the kinase domain. Here we describe the structural basis of c-Met activation upon autophosphorylation and the selective small molecule inhibiton of autophosphorylated c-Met. MK-2461 is a potent c-Met inhibitor that is selective for the phosphorylated state of the enzyme. Compound 1 is an MK-2461 analog with a 20-fold enthalpy-driven preference for the autophosphorylated over unphosphorylated c-Met kinase domain. The crystal structure of the unbound kinase domain phosphorylated at Tyr-1234 and Tyr-1235 shows that activation loop phosphorylation leads to the ejection and disorder of the activation loop and rearrangement of helix {alpha}C and the G loop to generate a viable active site. Helix {alpha}C adopts a orientation different from that seen in activation loop mutants. The crystal structure of the complex formed by the autophosphorylated c-Met kinase domain and compound 1 reveals a significant induced fit conformational change of the G loop and ordering of the activation loop, explaining the selectivity of compound 1 for the autophosphorylated state. The results highlight the role of structural plasticity within the kinase domain in imparting the specificity of ligand binding and provide the framework for structure-guided design of activated c-Met inhibitors.

  19. The role of phosphoinositide-3 kinase and PTEN in cardiovascular physiology and disease.

    PubMed

    Oudit, Gavin Y; Sun, Hui; Kerfant, Benoit-Gilles; Crackower, Michael A; Penninger, Josef M; Backx, Peter H

    2004-08-01

    Phosphoinositide-3 kinases (PI3Ks) are a family of evolutionary conserved lipid kinases that mediate many cellular responses in both physiologic and pathophysiologic states. Class I PI3K can be activated by either receptor tyrosine kinase (RTK)/cytokine receptor activation (class I(A)) or G-protein-coupled receptors (GPCR) (class I(B)). Once activated PI3Ks generate phosphatidylinositols (PtdIns) (3,4,5)P(3) leading to the recruitment and activation of Akt/protein kinase B (PKB), PDK1 and monomeric G-proteins (e.g. Rac-GTPases), which then activate a range of downstream targets including glycogen synthase kinase-3beta (GSK-3beta), mammalian target of rapamycin (mTOR), p70S6 kinase, endothelial nitric oxide synthase (eNOS) and several anti-apoptotic effectors. Class I(A) (PI3Kalpha, beta and delta) and class I(B) (PI3Kgamma) PI3Ks mediate distinct phenotypes in the heart and under negative control by the 3'-lipid phosphatase, phosphatase and tensin homolog on chromosome ten (PTEN) which dephosphorylate PtdIns(3,4,5)P(3) into PtdIns(4,5)P(2). PI3Kalpha, gamma and PTEN are expressed in cardiomyocytes, fibroblasts, endothelial cells and vascular smooth muscle cells where they modulate cell survival/apoptosis, hypertrophy, contractility, metabolism and mechanotransduction. Several transgenic and knockout models support a fundamental role of PI3K/PTEN signaling in the regulation of myocardial contractility and hypertrophy. Consequently the PI3K/PTEN signaling pathways are involved in a wide variety of diseases including cardiac hypertrophy, heart failure, preconditioning and hypertension. In this review, we discuss the biochemistry and molecular biology of PI3K (class I isoforms) and PTEN and their critical role in cardiovascular physiology and diseases.

  20. Divergent and convergent roles for kinases and phosphatases in neurofilament dynamics.

    PubMed

    Lee, Sangmook; Pant, Harish C; Shea, Thomas B

    2014-09-15

    C-terminal neurofilament phosphorylation mediates cation-dependent self-association leading to neurofilament incorporation into the stationary axonal cytoskeleton. Multiple kinases phosphorylate the C-terminal domains of the heavy neurofilament subunit (NF-H), including cyclin-dependent protein kinase 5 (CDK5), mitogen-activated protein kinases (MAPKs), casein kinase 1 and 2 (CK1 and CK2) and glycogen synthase kinase 3β (GSK3β). The respective contributions of these kinases have been confounded because they phosphorylate multiple substrates in addition to neurofilaments and display extensive interaction. Herein, differentiated NB2a/d1 cells were transfected with constructs expressing GFP-tagged NF-H, isolated NF-H sidearms and NF-H lacking the distal-most 187 amino acids. Cultures were treated with roscovitine, PD98059, Li(+), D4476, tetrabromobenzotriazole and calyculin, which are active against CDK5, MKK1 (also known as MAP2K1), GSK3β, CK1, CK2 and protein phosphatase 1 (PP1), respectively. Sequential phosphorylation by CDK5 and GSK3β mediated the neurofilament-neurofilament associations. The MAPK pathway (i.e. MKK1 to ERK1/2) was found to downregulate GSK3β, and CK1 activated PP1, both of which promoted axonal transport and restricted neurofilament-neurofilament associations to axonal neurites. The MAPK pathway and CDK5, but not CK1 and GSK3β, inhibited neurofilament proteolysis. These findings indicate that phosphorylation of neurofilaments by the proline-directed MAPK pathway and CDK5 counterbalance the impact of phosphorylation of neurofilaments by the non-proline-directed CK1 and GSK3β.

  1. CAST AWAY, a membrane-associated receptor-like kinase, inhibits organ abscission in Arabidopsis.

    PubMed

    Burr, Christian A; Leslie, Michelle E; Orlowski, Sara K; Chen, Iris; Wright, Catherine E; Daniels, Mark J; Liljegren, Sarah J

    2011-08-01

    Receptor-like kinase-mediated cell signaling pathways play fundamental roles in many aspects of plant growth and development. A pair of Arabidopsis (Arabidopsis thaliana) leucine-rich repeat receptor-like kinases (LRR-RLKs), HAESA (HAE) and HAESA-LIKE2 (HSL2), have been shown to activate the cell separation process that leads to organ abscission. Another pair of LRR-RLKs, EVERSHED (EVR) and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE1, act as inhibitors of abscission, potentially by modulating HAE/HSL2 activity. Cycling of these RLKs to and from the cell surface may be regulated by NEVERSHED (NEV), a membrane trafficking regulator that is essential for organ abscission. We report here the characterization of CAST AWAY (CST), a receptor-like cytoplasmic kinase that acts as a spatial inhibitor of cell separation. Disruption of CST suppresses the abscission defects of nev mutant flowers and restores the discrete identity of the trans-Golgi network in nev abscission zones. After organ shedding, enlarged abscission zones with obscured boundaries are found in nev cst flowers. We show that CST is a dual-specificity kinase in vitro and that myristoylation at its amino terminus promotes association with the plasma membrane. Using the bimolecular fluorescence complementation assay, we have detected interactions of CST with HAE and EVR at the plasma membrane of Arabidopsis protoplasts and hypothesize that CST negatively regulates cell separation signaling directly and indirectly. A model integrating the potential roles of receptor-like kinase signaling and membrane trafficking during organ separation is presented.

  2. Genetics Home Reference: phosphoglycerate kinase deficiency

    MedlinePlus

    ... the expand/collapse boxes. Download PDF Open All Close All Description Phosphoglycerate kinase deficiency is a genetic disorder that affects the body's ability to break down the simple sugar glucose, which is the primary energy source for most cells. Researchers have described two major ...

  3. Adhesion-related kinase induction of migration requires phosphatidylinositol-3-kinase and ras stimulation of rac activity in immortalized gonadotropin-releasing hormone neuronal cells.

    PubMed

    Nielsen-Preiss, Sheila M; Allen, Melissa P; Xu, Mei; Linseman, Daniel A; Pawlowski, John E; Bouchard, R J; Varnum, Brian C; Heidenreich, Kim A; Wierman, Margaret E

    2007-06-01

    GnRH neurons migrate into the hypothalamus during development. Although migratory defects may result in disordered activation of the reproductive axis and lead to delayed or absent sexual maturation, specific factors regulating GnRH neuronal migration remain largely unknown. The receptor tyrosine kinase, adhesion-related kinase (Ark) (also known as Axl, UFO, and Tyro7), has been implicated in the migration of GnRH neuronal cells. Binding of its ligand, growth arrest-specific gene 6 (Gas6), promotes cytoskeletal remodeling and migration of NLT GnRH neuronal cells via Rac and p38 MAPK. Here, we examined the Axl effectors proximal to Rac in the signaling pathway. Gas6/Axl-induced lamellipodia formation and migration were blocked after phosphatidylinositol-3-kinase (PI3K) inhibition in GnRH neuronal cells. The p85 subunit of PI3K coimmunoprecipitated with Axl and was phosphorylated in a Gas6-sensitive manner. In addition, PI3K inhibition in GnRH neuronal cells diminished Gas6-induced Rac activation. Exogenous expression of a dominant-negative form of Ras also decreased GnRH neuronal lamellipodia formation, migration, and Rac activation. PI3K inhibition blocked Ras in addition to Rac activation and migration. In contrast, pharmacological blockade of the phospholipase C gamma effectors, protein kinase C or calcium/calmodulin protein kinase II, had no effect on Gas6/Axl signaling to promote Rac activation or stimulate cytoskeletal reorganization and migration. Together, these data show that the PI3K-Ras pathway is a major mediator of Axl actions upstream of Rac to induce GnRH neuronal cell migration. PMID:17332061

  4. Kinase detection with gallium nitride based high electron mobility transistors

    PubMed Central

    Makowski, Matthew S.; Bryan, Isaac; Sitar, Zlatko; Arellano, Consuelo; Xie, Jinqiao; Collazo, Ramon; Ivanisevic, Albena

    2013-01-01

    A label-free kinase detection system was fabricated by the adsorption of gold nanoparticles functionalized with kinase inhibitor onto AlGaN/GaN high electron mobility transistors (HEMTs). The HEMTs were operated near threshold voltage due to the greatest sensitivity in this operational region. The Au NP/HEMT biosensor system electrically detected 1 pM SRC kinase in ionic solutions. These results are pertinent to drug development applications associated with kinase sensing. PMID:23918992

  5. Kinase detection with gallium nitride based high electron mobility transistors.

    PubMed

    Makowski, Matthew S; Bryan, Isaac; Sitar, Zlatko; Arellano, Consuelo; Xie, Jinqiao; Collazo, Ramon; Ivanisevic, Albena

    2013-07-01

    A label-free kinase detection system was fabricated by the adsorption of gold nanoparticles functionalized with kinase inhibitor onto AlGaN/GaN high electron mobility transistors (HEMTs). The HEMTs were operated near threshold voltage due to the greatest sensitivity in this operational region. The Au NP/HEMT biosensor system electrically detected 1 pM SRC kinase in ionic solutions. These results are pertinent to drug development applications associated with kinase sensing.

  6. Kinase detection with gallium nitride based high electron mobility transistors

    NASA Astrophysics Data System (ADS)

    Makowski, Matthew S.; Bryan, Isaac; Sitar, Zlatko; Arellano, Consuelo; Xie, Jinqiao; Collazo, Ramon; Ivanisevic, Albena

    2013-07-01

    A label-free kinase detection system was fabricated by the adsorption of gold nanoparticles functionalized with kinase inhibitor onto AlGaN/GaN high electron mobility transistors (HEMTs). The HEMTs were operated near threshold voltage due to the greatest sensitivity in this operational region. The Au NP/HEMT biosensor system electrically detected 1 pM SRC kinase in ionic solutions. These results are pertinent to drug development applications associated with kinase sensing.

  7. Kinase activity profiling of gram-negative pneumonia.

    PubMed

    Hoogendijk, Arie J; Diks, Sander H; Peppelenbosch, Maikel P; Van Der Poll, Tom; Wieland, Catharina W

    2011-01-01

    Pneumonia is a severe disease with high morbidity and mortality. A major causative pathogen is the Gram-negative bacterium Klebsiella (K.) pneumoniae. Kinases play an integral role in the transduction of intracellular signaling cascades and regulate a diverse array of biological processes essential to immune cells. The current study explored signal transduction events during murine Gram-negative pneumonia using a systems biology approach. Kinase activity arrays enable the analysis of 1,024 consensus sequences of protein kinase substrates. Using a kinase activity array on whole lung lysates, cellular kinase activities were determined in a mouse model of K. pneumoniae pneumonia. Notable kinase activities also were validated with phospho-specific Western blots. On the basis of the profiling data, mitogen-activated protein kinase (MAPK) signaling via p42 mitogen-activated protein kinase (p42) and p38 mitogen-activated protein kinase (p38) and transforming growth factor β (TGFβ) activity were reduced during infection, whereas v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homolog (avian) (SRC) activity generally was enhanced. AKT signaling was represented in both metabolic and inflammatory (mitogen-activated protein kinase kinase 2 [MKK], apoptosis signal-regulating kinase/mitogen-activated protein kinase kinase kinase 5 [ASK] and v-raf murine sarcoma viral oncogene homolog B1 [b-RAF]) context. This study reaffirms the importance of classic inflammation pathways, such as MAPK and TGFβ signaling and reveals less known involvement of glycogen synthase kinase 3β (GSK-3β), AKT and SRC signaling cassettes in pneumonia.

  8. LEAD SEVERING CONTRIVANCE

    DOEpatents

    Widmaier, W.

    1958-04-01

    A means for breaking an electrical circuit within an electronic tube during the process of manufacture is described. Frequently such circuits must be employed for gettering or vapor coating purposes, however, since an external pair of corector pins having no use after manufacture, is undesirable, this invention permits the use of existing leads to form a temporary circuit during manufacture, and severing it thereafter. One portion of the temporary circuit, made from a springy material such as tungsten, is spot welded to a fusable member. To cut the circuit an external radiant heat source melts the fusable member, allowing the tensed tungsten spring to contract and break the circuit. This inexpensive arrangement is particularly useful when the tube has a great many external leads crowded into the tube base.

  9. Mitogen-Activated Protein Kinase Kinase 3 Is Required for Regulation during Dark-Light Transition.

    PubMed

    Lee, Horim

    2015-07-01

    Plant growth and development are coordinately orchestrated by environmental cues and phytohormones. Light acts as a key environmental factor for fundamental plant growth and physiology through photosensory phytochromes and underlying molecular mechanisms. Although phytochromes are known to possess serine/threonine protein kinase activities, whether they trigger a signal transduction pathway via an intracellular protein kinase network remains unknown. In analyses of mitogen-activated protein kinase kinase (MAPKK, also called MKK) mutants, the mkk3 mutant has shown both a hypersensitive response in plant hormone gibberellin (GA) and a less sensitive response in red light signaling. Surprisingly, light-induced MAPK activation in wild-type (WT) seedlings and constitutive MAPK phosphorylation in dark-grown mkk3 mutant seedlings have also been found, respectively. Therefore, this study suggests that MKK3 acts in negative regulation in darkness and in light-induced MAPK activation during dark-light transition. PMID:26082029

  10. Pyrrolopyridine inhibitors of mitogen-activated protein kinase-activated protein kinase 2 (MK-2).

    PubMed

    Anderson, David R; Meyers, Marvin J; Vernier, William F; Mahoney, Matthew W; Kurumbail, Ravi G; Caspers, Nicole; Poda, Gennadiy I; Schindler, John F; Reitz, David B; Mourey, Robert J

    2007-05-31

    A new class of potent kinase inhibitors selective for mitogen-activated protein kinase-activated protein kinase 2 (MAPKAP-K2 or MK-2) for the treatment of rheumatoid arthritis has been prepared and evaluated. These inhibitors have IC50 values as low as 10 nM against the target and have good selectivity profiles against a number of kinases including CDK2, ERK, JNK, and p38. These MK-2 inhibitors have been shown to suppress TNFalpha production in U397 cells and to be efficacious in an acute inflammation model. The structure-activity relationships of this series, the selectivity for MK-2 and their activity in both in vitro and in vivo models are discussed. The observed selectivity is discussed with the aid of an MK-2/inhibitor crystal structure.

  11. Crystal Structure of the Protein Kinase Domain of Yeast AMP-Activated Protein Kinase Snf1

    SciTech Connect

    Rudolph,M.; Amodeo, G.; Bai, Y.; Tong, L.

    2005-01-01

    AMP-activated protein kinase (AMPK) is a master metabolic regulator, and is an important target for drug development against diabetes, obesity, and other diseases. AMPK is a hetero-trimeric enzyme, with a catalytic ({alpha}) subunit, and two regulatory ({beta} and {gamma}) subunits. Here we report the crystal structure at 2.2 Angstrom resolution of the protein kinase domain (KD) of the catalytic subunit of yeast AMPK (commonly known as SNF1). The Snf1-KD structure shares strong similarity to other protein kinases, with a small N-terminal lobe and a large C-terminal lobe. Two negative surface patches in the structure may be important for the recognition of the substrates of this kinase.

  12. The crystal structure of choline kinase reveals a eukaryotic protein kinase fold

    SciTech Connect

    Peisach, D.; Gee, P.; Kent, K.; Xu, Z.

    2010-03-08

    Choline kinase catalyzes the ATP-dependent phosphorylation of choline, the first committed step in the CDP-choline pathway for the biosynthesis of phosphatidylcholine. The 2.0 {angstrom} crystal structure of a choline kinase from C. elegans (CKA-2) reveals that the enzyme is a homodimeric protein with each monomer organized into a two-domain fold. The structure is remarkably similar to those of protein kinases and aminoglycoside phosphotransferases, despite no significant similarity in amino acid sequence. Comparisons to the structures of other kinases suggest that ATP binds to CKA-2 in a pocket formed by highly conserved and catalytically important residues. In addition, a choline binding site is proposed to be near the ATP binding pocket and formed by several structurally flexible loops.

  13. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies.

    PubMed

    Domanova, Westa; Krycer, James; Chaudhuri, Rima; Yang, Pengyi; Vafaee, Fatemeh; Fazakerley, Daniel; Humphrey, Sean; James, David; Kuncic, Zdenka

    2016-01-01

    In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds), making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs) is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE) in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same linear consensus motif

  14. Unraveling Kinase Activation Dynamics Using Kinase-Substrate Relationships from Temporal Large-Scale Phosphoproteomics Studies

    PubMed Central

    Chaudhuri, Rima; Yang, Pengyi; Vafaee, Fatemeh; Fazakerley, Daniel; Humphrey, Sean; James, David; Kuncic, Zdenka

    2016-01-01

    In response to stimuli, biological processes are tightly controlled by dynamic cellular signaling mechanisms. Reversible protein phosphorylation occurs on rapid time-scales (milliseconds to seconds), making it an ideal carrier of these signals. Advances in mass spectrometry-based proteomics have led to the identification of many tens of thousands of phosphorylation sites, yet for the majority of these the kinase is unknown and the underlying network topology of signaling networks therefore remains obscured. Identifying kinase substrate relationships (KSRs) is therefore an important goal in cell signaling research. Existing consensus sequence motif based prediction algorithms do not consider the biological context of KSRs, and are therefore insensitive to many other mechanisms guiding kinase-substrate recognition in cellular contexts. Here, we use temporal information to identify biologically relevant KSRs from Large-scale In Vivo Experiments (KSR-LIVE) in a data-dependent and automated fashion. First, we used available phosphorylation databases to construct a repository of existing experimentally-predicted KSRs. For each kinase in this database, we used time-resolved phosphoproteomics data to examine how its substrates changed in phosphorylation over time. Although substrates for a particular kinase clustered together, they often exhibited a different temporal pattern to the phosphorylation of the kinase. Therefore, although phosphorylation regulates kinase activity, our findings imply that substrate phosphorylation likely serve as a better proxy for kinase activity than kinase phosphorylation. KSR-LIVE can thereby infer which kinases are regulated within a biological context. Moreover, KSR-LIVE can also be used to automatically generate positive training sets for the subsequent prediction of novel KSRs using machine learning approaches. We demonstrate that this approach can distinguish between Akt and Rps6kb1, two kinases that share the same linear consensus motif

  15. Activation of stress-activated MAP protein kinases up-regulates expression of transgenes driven by the cytomegalovirus immediate/early promoter.

    PubMed Central

    Bruening, W; Giasson, B; Mushynski, W; Durham, H D

    1998-01-01

    The immediate/early promoter/enhancer of cytomegalovirus (CMV promoter) is one of the most commonly used promoters for expression of transgenes in eukaryotic cells. In practice, the CMV promoter is often thought of as a constitutively active unregulated promoter. However, we have observed that transcription from the CMV promoter can be up-regulated by a variety of environmental stresses. Many forms of cellular stress stimulate MAP kinase signalling pathways, resulting in activation of stress-activated protein kinases [SAPKs, also called Jun N-terminal kinases (JNKs)] and p38 kinases. We have found that the same conditions that lead to activation of SAPK/JNKs and p38 kinases can also dramatically increase expression from the CMV promoter. Inhibitors of p38 kinases abolished basal transcription from the CMV promoter and completely blocked stress-induced up-regulation of the CMV promoter. Overexpression of a dominant negative JNK kinase had no effect on basal transcription, but significantly reduced up-regulation caused by stress. These results have grave implications for use of the CMV promoter. If the CMV promoter can be up-regulated by cellular stresses, inadvertent activation of the stress kinase pathways may complicate, if not invalidate, the interpretation of a wide range of experiments. PMID:9421504

  16. Protein Kinase B (PknB) of Mycobacterium tuberculosis Is Essential for Growth of the Pathogen in Vitro as well as for Survival within the Host*

    PubMed Central

    Chawla, Yogesh; Upadhyay, Sandeep; Khan, Shazia; Nagarajan, Sathya Narayanan; Forti, Francesca; Nandicoori, Vinay Kumar

    2014-01-01

    The Mycobacterium tuberculosis protein kinase B (PknB) comprises an intracellular kinase domain, connected through a transmembrane domain to an extracellular region that contains four PASTA domains. The present study describes the comprehensive analysis of different domains of PknB in the context of viability in avirulent and virulent mycobacteria. We find stringent regulation of PknB expression necessary for cell survival, with depletion or overexpression of PknB leading to cell death. Although PknB-mediated kinase activity is essential for cell survival, active kinase lacking the transmembrane or extracellular domain fails to complement conditional mutants not expressing PknB. By creating chimeric kinases, we find that the intracellular kinase domain has unique functions in the virulent strain, which cannot be substituted by other kinases. Interestingly, we find that although the presence of the C-terminal PASTA domain is dispensable in the avirulent M. smegmatis, all four PASTA domains are essential in M. tuberculosis. The differential behavior of PknB vis-à-vis the number of essential PASTA domains and the specificity of kinase domain functions suggest that PknB-mediated growth and signaling events differ in virulent compared with avirulent mycobacteria. Mouse infection studies performed to determine the role of PknB in mediating pathogen survival in the host demonstrate that PknB is not only critical for growth of the pathogen in vitro but is also essential for the survival of the pathogen in the host. PMID:24706757

  17. Quantitative network mapping of the human kinome interactome reveals new clues for rational kinase inhibitor discovery and individualized cancer therapy.

    PubMed

    Cheng, Feixiong; Jia, Peilin; Wang, Quan; Zhao, Zhongming

    2014-06-15

    The human kinome is gaining importance through its promising cancer therapeutic targets, yet no general model to address the kinase inhibitor resistance has emerged. Here, we constructed a systems biology-based framework to catalogue the human kinome, including 538 kinase genes, in the broader context of the human interactome. Specifically, we constructed three networks: a kinase-substrate interaction network containing 7,346 pairs connecting 379 kinases to 36,576 phosphorylation sites in 1,961 substrates, a protein-protein interaction network (PPIN) containing 92,699 pairs, and an atomic resolution PPIN containing 4,278 pairs. We identified the conserved regulatory phosphorylation motifs (e.g., Ser/Thr-Pro) using a sequence logo analysis. We found the typical anticancer target selection strategy that uses network hubs as drug targets, might lead to a high adverse drug reaction risk. Furthermore, we found the distinct network centrality of kinases creates a high anticancer drug resistance risk by feedback or crosstalk mechanisms within cellular networks. This notion is supported by the systematic network and pathway analyses that anticancer drug resistance genes are significantly enriched as hubs and heavily participate in multiple signaling pathways. Collectively, this comprehensive human kinome interactome map sheds light on anticancer drug resistance mechanisms and provides an innovative resource for rational kinase inhibitor design.

  18. Drosophila Wee1 kinase rescues fission yeast from mitotic catastrophe and phosphorylates Drosophila Cdc2 in vitro.

    PubMed Central

    Campbell, S D; Sprenger, F; Edgar, B A; O'Farrell, P H

    1995-01-01

    Cdc2 kinase activity is required for triggering entry into mitosis in all known eukaryotes. Elaborate mechanisms have evolved for regulating Cdc2 activity so that mitosis occurs in a timely manner, when preparations for its execution are complete. In Schizosaccharomyces pombe, Wee1 and a related Mik1 kinase are Cdc2-inhibitory kinases that are required for preventing premature activation of the mitotic program. To identify Cdc2-inhibitory kinases in Drosophila, we screened for cDNA clones that rescue S. pombe wee1- mik1- mutants from lethal mitotic catastrophe. One of the genes identified in this screen, Drosophila wee1 (Dwee1), encodes a new Wee1 homologue. Dwee1 kinase is closely related to human and Xenopus Wee1 homologues, and can inhibit Cdc2 activity by phosphorylating a critical tyrosine residue. Dwee1 mRNA is maternally provided to embryos, and is zygotically expressed during the postblastoderm divisions of embryogenesis. Expression remains high in the proliferating cells of the central nervous system well after cells in the rest of the embryo have ceased dividing. The loss of zygotically expressed Dwee1 does not lead to mitotic catastrophe during postblastoderm cycles 14 to 16. This result may indicate that maternally provided Dwee1 is sufficient for regulating Cdc2 during embryogenesis, or it may reflect the presence of a redundant Cdc2 inhibitory kinase, as in fission yeast. Images PMID:8573790

  19. Endothelial NOS-dependent activation of c-Jun NH(2)- terminal kinase by oxidized low-density lipoprotein

    NASA Technical Reports Server (NTRS)

    Go, Y. M.; Levonen, A. L.; Moellering, D.; Ramachandran, A.; Patel, R. P.; Jo, H.; Darley-Usmar, V. M.

    2001-01-01

    Oxidized low-density lipoprotein (oxLDL) is known to activate a number of signal transduction pathways in endothelial cells. Among these are the c-Jun NH(2)-terminal kinase (JNK), also known as stress-activated protein kinase, and extracellular signal-regulated kinase (ERK). These mitogen-activated protein kinases (MAP kinase) determine cell survival in response to environmental stress. Interestingly, JNK signaling involves redox-sensitive mechanisms and is activated by reactive oxygen and nitrogen species derived from both NADPH oxidases, nitric oxide synthases (NOS), peroxides, and oxidized low-density lipoprotein (oxLDL). The role of endothelial NOS (eNOS) in the activation of JNK in response to oxLDL has not been examined. Herein, we show that on exposure of endothelial cells to oxLDL, both ERK and JNK are activated through independent signal transduction pathways. A key role of eNOS activation through a phosphatidylinositol-3-kinase-dependent mechanism leading to phosphorylation of eNOS is demonstrated for oxLDL-dependent activation of JNK. Moreover, we show that activation of ERK by oxLDL is critical in protection against the cytotoxicity of oxLDL.

  20. MDS1, a dosage suppressor of an mck1 mutant, encodes a putative yeast homolog of glycogen synthase kinase 3.

    PubMed Central

    Puziss, J W; Hardy, T A; Johnson, R B; Roach, P J; Hieter, P

    1994-01-01

    The yeast gene MCK1 encodes a serine/threonine protein kinase that is thought to function in regulating kinetochore activity and entry into meiosis. Disruption of MCK1 confers a cold-sensitive phenotype, a temperature-sensitive phenotype, and sensitivity to the microtubule-destabilizing drug benomyl and leads to loss of chromosomes during growth on benomyl. A dosage suppression selection was used to identify genes that, when present at high copy number, could suppress the cold-sensitive phenotype of mck1::HIS3 mutant cells. Several unique classes of clones were identified, and one of these, designated MDS1, has been characterized in some detail. Nucleotide sequence data reveal that MDS1 encodes a serine/threonine protein kinase that is highly homologous to the shaggy/zw3 kinase in Drosophila melanogaster and its functional homolog, glycogen synthase kinase 3, in rats. The presence of MDS1 in high copy number rescues both the cold-sensitive and the temperature-sensitive phenotypes, but not the benomyl-sensitive phenotype, associated with the disruption of MCK1. Analysis of strains harboring an mds1 null mutation demonstrates that MDS1 is not essential during normal vegetative growth but appears to be required for meiosis. Finally, in vitro experiments indicate that the proteins encoded by both MCK1 and MDS1 possess protein kinase activity with substrate specificity similar to that of mammalian glycogen synthase kinase 3. Images PMID:8264650