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Sample records for pip-5 kinase ialpha

  1. 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}.

  2. The lipid kinase PIP5K1C regulates pain signaling and sensitization

    PubMed Central

    Wright, Brittany D.; Loo, Lipin; Street, Sarah E.; Ma, Anqi; Taylor-Blake, Bonnie; Stashko, Michael A.; Jin, Jian; Janzen, William P.; Frye, Stephen V.; Zylka, Mark J.

    2014-01-01

    SUMMARY Numerous pain-producing (pronociceptive) receptors signal via phosphatidylinositol 4,5- bisphosphate (PIP2) hydrolysis. However, it is currently unknown which lipid kinases generate PIP2 in nociceptive dorsal root ganglia (DRG) neurons and if these kinases regulate pronociceptive receptor signaling. Here, we found that phosphatidylinositol 4-phosphate 5 kinase type 1C (PIP5K1C) is expressed at higher levels than any other PIP5K and, based on experiments with Pip5k1c+/− mice, generates at least half of all PIP2 in DRG neurons. Additionally, Pip5k1c haploinsufficiency reduces pronociceptive receptor signaling and TRPV1 sensitization in DRG neurons as well as thermal and mechanical hypersensitivity in mouse models of chronic pain. We identified a novel small molecule inhibitor of PIP5K1C (UNC3230) in a high-throughput screen. UNC3230 lowered PIP2 levels in DRG neurons and attenuated hypersensitivity when administered intrathecally or into the hindpaw. Our studies reveal that PIP5K1C regulates PIP2- dependent nociceptive signaling and suggest that PIP5K1C is a novel therapeutic target for chronic pain. PMID:24853942

  3. Development of a High-Throughput Screening Assay to Identify Inhibitors of the Lipid Kinase PIP5K1C.

    PubMed

    Wright, Brittany D; Simpson, Catherine; Stashko, Michael; Kireev, Dmitri; Hull-Ryde, Emily A; Zylka, Mark J; Janzen, William P

    2015-06-01

    Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) regulate a variety of cellular processes, including signaling through G protein-coupled receptors (GPCRs), endocytosis, exocytosis, and cell migration. These lipid kinases synthesize phosphatidylinositol 4,5-bisphosphate (PIP2) from phosphatidylinositol 4-phosphate [PI(4)P]. Because small-molecule inhibitors of these lipid kinases did not exist, molecular and genetic approaches were predominantly used to study PIP5K1 regulation of these cellular processes. Moreover, standard radioisotope-based lipid kinase assays cannot be easily adapted for high-throughput screening. Here, we report a novel, high-throughput, microfluidic mobility shift assay to identify inhibitors of PIP5K1C. This assay uses fluorescently labeled phosphatidylinositol 4-phosphate as the substrate and recombinant human PIP5K1C. Our assay exhibited high reproducibility, had a calculated adenosine triphosphate Michaelis constant (Km) of 15 µM, performed with z' values >0.7, and was used to screen a kinase-focused library of ~4700 compounds. From this screen, we identified several potent inhibitors of PIP5K1C, including UNC3230, a compound that we recently found can reduce nociceptive sensitization in animal models of chronic pain. This novel assay will allow continued drug discovery efforts for PIP5K1C and can be adapted easily to screen additional lipid kinases.

  4. Development of a high-throughput screening assay to identify inhibitors of the lipid kinase PIP5K1C

    PubMed Central

    Wright, Brittany D.; Simpson, Catherine; Stashko, Michael; Kireev, Dmitri; Hull-Ryde, Emily A.; Zylka, Mark J.; Janzen, William P.

    2015-01-01

    Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) regulate a variety of cellular processes including signaling through G protein-coupled receptors (GPCRs), endocytosis, exocytosis, and cell migration. These lipid kinases synthesize phosphatidylinositol 4,5-bisphosphate (PIP2) from phosphatidylinositol 4-phosphate [PI(4)P]. Since small molecule inhibitors of these lipid kinases did not exist, molecular and genetic approaches were predominantly used to study PIP5K1 regulation of these cellular processes. Moreover, standard radioisotope-based lipid kinase assays cannot be easily adapted for high-throughput screening. Here, we report a novel high-throughput microfluidic mobility shift assay to identify inhibitors of PIP5K1C. This assay utilizes fluorescently labeled phosphatidylinositol 4-phosphate as the substrate and recombinant human PIP5K1C. Our assay exhibited high reproducibility, had a calculated ATP Km of 15 µM, performed with z’ values >0.7, and was used to screen a kinase-focused library of ~4,700 compounds. From this screen, we identified several potent inhibitors of PIP5K1C, including UNC3230, a compound that we recently found can reduce nociceptive sensitization in animal models of chronic pain. This novel assay will allow continued drug discovery efforts for PIP5K1C and can be easily adapted to screen additional lipid kinases. PMID:25534829

  5. Expression and action of cyclic GMP-dependent protein kinase Ialpha in inflammatory hyperalgesia in rat spinal cord.

    PubMed

    Tao, Y X; Hassan, A; Haddad, E; Johns, R A

    2000-01-01

    Several lines of evidence have shown a role for the nitric oxide/cyclic guanosine monophosphate signaling pathway in the development of spinal hyperalgesia. However, the roles of effectors for cyclic guanosine monophosphate are not fully understood in the processing of pain in the spinal cord. The present study showed that cyclic guanosine monophosphate-dependent protein kinase Ialpha but not Ibeta was localized in the neuronal bodies and processes, and was distributed primarily in the superficial laminae of the spinal cord. Intrathecal administration of a selective inhibitor of cyclic guanosine monophosphate-dependent protein kinase Ialpha, Rp-8-[(4-chlorophenyl)thio]-cGMPS triethylamine, produced a significant antinociception demonstrated by the decrease in the number of flinches and shakes in the formalin test. This was accompanied by a marked reduction in formalin-induced c-fos expression in the spinal dorsal horn. Moreover, cyclic guanosine monophosphate-dependent protein kinase Ialpha protein expression was dramatically increased in the lumbar spinal cord 96 h after injection of formalin into a hindpaw, which occurred mainly in the superficial laminae on the ipsilateral side of a formalin-injected hindpaw. This up-regulation of cyclic guanosine monophosphate-dependent protein kinase Ialpha expression was completely blocked not only by a neuronal nitric oxide synthase inhibitor, 7-nitroindazole, and a soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, but also by an N-methyl-D-aspartate receptor antagonist, dizocilpine maleate (MK-801). The present results indicate that noxious stimulation not only initially activates but also later up-regulates cyclic guanosine monophosphate-dependent protein kinase Ialpha expression in the superficial laminae via an N-methyl-D-aspartate-nitric oxide-cyclic guanosine monophosphate signaling pathway, suggesting that cyclic guanosine monophosphate-dependent protein kinase Ialpha may play an

  6. PI4P5-kinase Ialpha is required for efficient HIV-1 entry and infection of T cells.

    PubMed

    Barrero-Villar, Marta; Barroso-González, Jonathan; Cabrero, J R; Gordón-Alonso, Mónica; Alvarez-Losada, Susana; Muñoz-Fernández, M A; Sánchez-Madrid, Francisco; Valenzuela-Fernández, Agustín

    2008-11-15

    HIV-1 envelope (Env) triggers membrane fusion between the virus and the target cell. The cellular mechanism underlying this process is not well known. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is known to be important for the late steps of the HIV-1 infection cycle by promoting Gag localization to the plasma membrane during viral assembly, but it has not been implicated in early stages of HIV-1 membrane-related events. In this study, we show that binding of the initial HIV-1 Env-gp120 protein induces PIP(2) production in permissive lymphocytes through the activation of phosphatidylinositol-4-phosphate 5-kinase (PI4P5-K) Ialpha. Overexpression of wild-type PI4P5-K Ialpha increased HIV-1 Env-mediated PIP(2) production and enhanced viral replication in primary lymphocytes and CEM T cells, whereas PIP(2) production and HIV-1 infection were both severely reduced in cells overexpressing the kinase-dead mutant D227A (D/A)-PI4P5-K Ialpha. Similar results were obtained with replicative and single-cycle HIV-1 particles. HIV-1 infection was also inhibited by knockdown of endogenous expression of PI4P5-K Ialpha. These data indicate that PI4P5-K Ialpha-mediated PIP(2) production is crucial for HIV-1 entry and the early steps of infection in permissive lymphocytes.

  7. Endosomal type Iγ PIP 5-kinase controls EGF receptor lysosomal sorting.

    PubMed

    Sun, Yue; Hedman, Andrew C; Tan, Xiaojun; Schill, Nicholas J; Anderson, Richard A

    2013-04-29

    Endosomal trafficking and degradation of epidermal growth factor receptor (EGFR) play an essential role in the control of its signaling. Phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P(2)) is an established regulator of endocytosis, whereas PtdIns3P modulates endosomal trafficking. However, we demonstrate here that type I gamma phosphatidylinositol phosphate 5-kinase i5 (PIPKIγi5), an enzyme that synthesizes PtdIns4,5P(2), controls endosome-to-lysosome sorting of EGFR. In this pathway, PIPKIγi5 interacts with sorting nexin 5 (SNX5), a protein that binds PtdIns4,5P(2) and other phosphoinositides. PIPKIγi5 and SNX5 localize to endosomes, and loss of either protein blocks EGFR sorting into intraluminal vesicles (ILVs) of the multivesicular body. Loss of ILV sorting greatly enhances and prolongs EGFR signaling. PIPKIγi5 and SNX5 prevent Hrs ubiquitination, and this facilitates the Hrs association with EGFR that is required for ILV sorting. These findings reveal that PIPKIγi5 and SNX5 form a signaling nexus that controls EGFR endosomal sorting, degradation, and signaling.

  8. (D)-Amino acid analogues of DT-2 as highly selective and superior inhibitors of cGMP-dependent protein kinase Ialpha.

    PubMed

    Nickl, Christian K; Raidas, Shiv Kumar; Zhao, Hong; Sausbier, Matthias; Ruth, Peter; Tegge, Werner; Brayden, Joseph E; Dostmann, Wolfgang R

    2010-03-01

    The cGMP-dependent protein kinase type I (PKG I) is an essential regulator of cellular function in blood vessels throughout the body. DT-2, a peptidic inhibitor of PKG, has played a central role in determining the molecular mechanisms of vascular control involving PKG and its signaling partners. Here, we report the development of (d)-amino acid DT-2 derivatives, namely the retro-inverso ri-(d)-DT-2 and the all (d)-amino acid analog, (d)-DT-2. Both peptide analogs were potent PKG Ialpha inhibitors with K(i) values of 5.5 nM (ri-(d)-DT-2) and 0.8 nM ((d)-DT-2) as determined using a hyperbolic mixed-type inhibition model. Also, both analogs were proteolytically stable in vivo, showed elevated selectivity, and displayed enhanced membrane translocation properties. Studies on isolated arteries from the resistance vasculature demonstrated that intraluminally perfused (d)-DT-2 significantly inhibited vasodilation induced by 8-Br-cGMP. Furthermore, in vivo application of (d)-DT-2 established a uniform translocation pattern in the resistance vasculature, with exception of the brain. Thus, (d)-DT-2 caused significant increases in mean arterial blood pressure in unrestrained, awake mice. Further, mesenteric arteries isolated from (d)-DT-2 treated animals showed a markedly reduced dilator response to 8-Br-cGMP in vitro. Our results clearly demonstrate that (d)-DT-2 is a superior inhibitor of PKG Ialpha and its application in vivo leads to sustained inhibition of PKG in vascular smooth muscle cells. The discovery of (d)-DT-2 may help our understanding of how blood vessels constrict and dilate and may also aid the development of new strategies and therapeutic agents targeted to the prevention and treatment of vascular disorders such as hypertension, stroke and coronary artery disease. Copyright 2009 Elsevier B.V. All rights reserved.

  9. The activation loop of PIP5K functions as a membrane sensor essential for lipid substrate processing

    PubMed Central

    Liu, Aizhuo; Sui, Dexin; Wu, Dianqing; Hu, Jian

    2016-01-01

    Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), a representative member of the phosphatidylinositol phosphate kinase (PIPK) family, is a major enzyme that biosynthesizes the signaling molecule PI(4,5)P2 (phosphatidylinositol 4,5-bisphosphate) in eukaryotic cells. The stringent specificity toward lipid substrates and the high sensitivity to the membrane environment strongly suggest a membrane-sensing mechanism, but the underlying structural basis is still largely unknown. We present a nuclear magnetic resonance (NMR) study on a peptide commensurate with a PIP5K’s activation loop, which has been reported to be a determinant of lipid substrate specificity and subcellular localization of PIP5K. Although the activation loop is severely disordered in the crystal structure of PIP5K, the NMR experiments showed that the largely unstructured peptide folded into an amphipathic helix upon its association with the 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) micellar surface. Systematic mutagenesis and functional assays further demonstrated the crucial roles of the amphipathic helix and its hydrophobic surface in kinase activity and membrane-sensing function, supporting a working model in which the activation loop is a critical structural module conferring a membrane-sensing mechanism on PIP5K. The activation loop, surprisingly functioning as a membrane sensor, represents a new paradigm of kinase regulation by the activation loop through protein-membrane interaction, which also lays a foundation on the regulation of PIP5K (and other PIPKs) by membrane lipids for future studies. PMID:28138522

  10. Resolution of structure of PIP5K1A reveals molecular mechanism for its regulation by dimerization and dishevelled

    PubMed Central

    Hu, Jian; Yuan, Qianying; Kang, Xue; Qin, Yuanbo; Li, Lin; Ha, Ya; Wu, Dianqing

    2015-01-01

    Type I phosphatidylinositol phosphate kinase (PIP5K1) phosphorylates the head group of phosphatidylinositol 4-phosphate (PtdIns4P) to generate PtdIns4,5P2, which plays important roles in a wide range of cellular functions including Wnt signalling. However, the lack of its structural information has hindered the understanding of its regulation. Here we report the crystal structure of the catalytic domain of zebrafish PIP5K1A at 3.3 Å resolution. This molecule forms a side-to-side dimer. Mutagenesis study of PIP5K1A reveals two adjacent interfaces for the dimerization and interaction with the DIX domain of the Wnt signalling molecule dishevelled. Although these interfaces are located distally to the catalytic/substrate-binding site, binding to these interfaces either through dimerization or the interaction with DIX stimulates PIP5K1 catalytic activity. DIX binding additionally enhances PIP5K1 substrate binding. Thus, this study elucidates regulatory mechanisms for this lipid kinase and provides a paradigm for the understanding of PIP5K1 regulation by their interacting molecules. PMID:26365782

  11. The role of PI3K/AKT-related PIP5K1α and the discovery of its selective inhibitor for treatment of advanced prostate cancer

    PubMed Central

    Semenas, Julius; Hedblom, Andreas; Miftakhova, Regina R.; Sarwar, Martuza; Larsson, Rikard; Shcherbina, Liliya; Johansson, Martin E.; Härkönen, Pirkko; Sterner, Olov; Persson, Jenny L.

    2014-01-01

    Nitrogen-containing heterocyclic compounds are an important class of molecules that are commonly used for the synthesis of candidate drugs. Phosphatidylinositol-4-phosphate 5-kinase-α (PIP5Kα) is a lipid kinase, similar to PI3K. However, the role of PIP5K1α in oncogenic processes and the development of inhibitors that selectively target PIP5K1α have not been reported. In the present study we report that overexpression of PIP5K1α is associated with poor prognosis in prostate cancer and correlates with an elevated level of the androgen receptor. Overexpression of PIP5K1α in PNT1A nonmalignant cells results in an increased AKT activity and an increased survival, as well as invasive malignant phenotype, whereas siRNA-mediated knockdown of PIP5K1α in aggressive PC-3 cells leads to a reduced AKT activity and an inhibition in tumor growth in xenograft mice. We further report a previously unidentified role for PIP5K1α as a druggable target for our newly developed compound ISA-2011B using a high-throughput KINOMEscan platform. ISA-2011B was discovered during our synthetic studies of C-1 indol-3-yl substituted 1,2,3,4-tetrahydroisoquinolines via a Pictet-Spengler approach. ISA-2011B significantly inhibits growth of tumor cells in xenograft mice, and we show that this is mediated by targeting PIP5K1α-associated PI3K/AKT and the downstream survival, proliferation, and invasion pathways. Further, siRNA-mediated knockdown of PIP5K1α exerts similar effects on PC3 cells as ISA-2011B treatment, significantly inhibiting AKT activity, increasing apoptosis and reducing invasion. Thus, PIP5K1α has high potential as a drug target, and compound ISA-2011B is interesting for further development of targeted cancer therapy. PMID:25071204

  12. The role of PI3K/AKT-related PIP5K1α and the discovery of its selective inhibitor for treatment of advanced prostate cancer.

    PubMed

    Semenas, Julius; Hedblom, Andreas; Miftakhova, Regina R; Sarwar, Martuza; Larsson, Rikard; Shcherbina, Liliya; Johansson, Martin E; Härkönen, Pirkko; Sterner, Olov; Persson, Jenny L

    2014-09-02

    Nitrogen-containing heterocyclic compounds are an important class of molecules that are commonly used for the synthesis of candidate drugs. Phosphatidylinositol-4-phosphate 5-kinase-α (PIP5Kα) is a lipid kinase, similar to PI3K. However, the role of PIP5K1α in oncogenic processes and the development of inhibitors that selectively target PIP5K1α have not been reported. In the present study we report that overexpression of PIP5K1α is associated with poor prognosis in prostate cancer and correlates with an elevated level of the androgen receptor. Overexpression of PIP5K1α in PNT1A nonmalignant cells results in an increased AKT activity and an increased survival, as well as invasive malignant phenotype, whereas siRNA-mediated knockdown of PIP5K1α in aggressive PC-3 cells leads to a reduced AKT activity and an inhibition in tumor growth in xenograft mice. We further report a previously unidentified role for PIP5K1α as a druggable target for our newly developed compound ISA-2011B using a high-throughput KINOMEscan platform. ISA-2011B was discovered during our synthetic studies of C-1 indol-3-yl substituted 1,2,3,4-tetrahydroisoquinolines via a Pictet-Spengler approach. ISA-2011B significantly inhibits growth of tumor cells in xenograft mice, and we show that this is mediated by targeting PIP5K1α-associated PI3K/AKT and the downstream survival, proliferation, and invasion pathways. Further, siRNA-mediated knockdown of PIP5K1α exerts similar effects on PC3 cells as ISA-2011B treatment, significantly inhibiting AKT activity, increasing apoptosis and reducing invasion. Thus, PIP5K1α has high potential as a drug target, and compound ISA-2011B is interesting for further development of targeted cancer therapy.

  13. Targeted suppression of AR-V7 using PIP5K1α inhibitor overcomes enzalutamide resistance in prostate cancer cells.

    PubMed

    Sarwar, Martuza; Semenas, Julius; Miftakhova, Regina; Simoulis, Athanasios; Robinson, Brian; Gjörloff Wingren, Anette; Mongan, Nigel P; Heery, David M; Johnsson, Heather; Abrahamsson, Per-Anders; Dizeyi, Nishtman; Luo, Jun; Persson, Jenny L

    2016-09-27

    One mechanism of resistance of prostate cancer (PCa) to enzalutamide (MDV3100) treatment is the increased expression of AR variants lacking the ligand binding-domain, the best characterized of which is AR-V7. We have previously reported that Phosphatidylinositol-4-phosphate 5-kinase alpha (PIP5Kα), is a lipid kinase that links to CDK1 and AR pathways. The discovery of PIP5Kα inhibitor highlight the potential of PIP5K1α as a drug target in PCa. In this study, we show that AR-V7 expression positively correlates with PIP5K1α in tumor specimens from PCa patients. Overexpression of AR-V7 increases PIP5K1α, promotes rapid growth of PCa in xenograft mice, whereas inhibition of PIP5K1α by its inhibitor ISA-2011B suppresses the growth and invasiveness of xenograft tumors overexpressing AR-V7. PIP5K1α is a key co-factor for both AR-V7 and AR, which are present as protein-protein complexes predominantly in the nucleus of PCa cells. In addition, PIP5K1α and CDK1 influence AR-V7 expression also through AKT-associated mechanism dependent on PTEN-status. ISA-2011B disrupts protein stabilization of AR-V7 which is dependent on PIP5K1α, leading to suppression of invasive growth of AR-V7-high tumors in xenograft mice. Our study suggests that combination of enzalutamide and PIP5K1α may have a significant impact on refining therapeutic strategies to circumvent resistance to antiandrogen therapies.

  14. Targeted suppression of AR-V7 using PIP5K1α inhibitor overcomes enzalutamide resistance in prostate cancer cells

    PubMed Central

    Sarwar, Martuza; Semenas, Julius; Miftakhova, Regina; Simoulis, Athanasios; Robinson, Brian; Wingren, Anette Gjörloff; Mongan, Nigel P.; Heery, David M.; Johnsson, Heather; Abrahamsson, Per-Anders; Dizeyi, Nishtman; Luo, Jun; Persson, Jenny L.

    2016-01-01

    One mechanism of resistance of prostate cancer (PCa) to enzalutamide (MDV3100) treatment is the increased expression of AR variants lacking the ligand binding-domain, the best characterized of which is AR-V7. We have previously reported that Phosphatidylinositol-4-phosphate 5-kinase alpha (PIP5Kα), is a lipid kinase that links to CDK1 and AR pathways. The discovery of PIP5Kα inhibitor highlight the potential of PIP5K1α as a drug target in PCa. In this study, we show that AR-V7 expression positively correlates with PIP5K1α in tumor specimens from PCa patients. Overexpression of AR-V7 increases PIP5K1α, promotes rapid growth of PCa in xenograft mice, whereas inhibition of PIP5K1α by its inhibitor ISA-2011B suppresses the growth and invasiveness of xenograft tumors overexpressing AR-V7. PIP5K1α is a key co-factor for both AR-V7 and AR, which are present as protein-protein complexes predominantly in the nucleus of PCa cells. In addition, PIP5K1α and CDK1 influence AR-V7 expression also through AKT-associated mechanism dependent on PTEN-status. ISA-2011B disrupts protein stabilization of AR-V7 which is dependent on PIP5K1α, leading to suppression of invasive growth of AR-V7-high tumors in xenograft mice. Our study suggests that combination of enzalutamide and PIP5K1α may have a significant impact on refining therapeutic strategies to circumvent resistance to antiandrogen therapies. PMID:27588408

  15. Identification of a conserved 8 aa insert in the PIP5K protein in the Saccharomycetaceae family of fungi and the molecular dynamics simulations and structural analysis to investigate its potential functional role.

    PubMed

    Khadka, Bijendra; Gupta, Radhey S

    2017-08-01

    Homologs of the phosphatidylinositol-4-phosphate-5-kinase (PIP5K), which controls a multitude of essential cellular functions, contain a 8 aa insert in a conserved region that is specific for the Saccharomycetaceae family of fungi. Using structures of human PIP4K proteins as templates, structural models were generated of the Saccharomyces cerevisiae and human PIP5K proteins. In the modeled S. cerevisiae PIP5K, the 8 aa insert forms a surface exposed loop, present on the same face of the protein as the activation loop of the kinase domain. Electrostatic potential analysis indicates that the residues from 8 aa conserved loop form a highly positively charged surface patch, which through electrostatic interaction with the anionic portions of phospholipid head groups, is expected to play a role in the membrane interaction of the yeast PIP5K. To unravel this prediction, molecular dynamics (MD) simulations were carried out to examine the binding interaction of PIP5K, either containing or lacking the conserved signature insert, with two different membrane lipid bilayers. The results from MD studies provide insights concerning the mechanistic of interaction of PIP5K with lipid bilayer, and support the contention that the identified 8 aa conserved insert in fungal PIP5K plays an important role in the binding of this protein with membrane surface. Proteins 2017; 85:1454-1467. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  16. [Association of (N251S)-PIP5K2A with schizophrenic disorders: a study of the Russian population of Siberia].

    PubMed

    Fedorenko, O Iu; Rudikov, E V; Gavrilova, V A; Boiarko, E G; Semke, A V; Ivanova, S A

    2013-01-01

    The phosphatidylinositol-4-phosphate-5-kinase type IIa (PIP5K2A) gene has been proposed as a putative susceptibility gene for schizophrenia on both positional and functional grounds. The association between the (N251S)-PIP5K2A (rs10828317) variant and schizophrenia was found in German and Dutch populations but was not replicated in several other populations. The purpose of the study was to examine whether the previously implicated (N251S)-PIP5K2A variant influences susceptibility to schizophrenia in the Russian population of Siberia. Authors studied 355 patients with schizophrenic disorders from the Russian population of Siberia. The control group consisted of 100 healthy. Results confirm the association of the (N251S)-PIP5K2A (rs10828317) polymorphism with schizophrenia (p=0.04, OR=2.48, 95%CI=1.19--5.17 for the CC genotype). The association can be explained by the inability of mutant kinase to activate the phosphatidylinositol-4,5-biphosphate dependent proteins, including neuronal KCNQ channels and glutamate EAAT3 transporters, which leads to the lack of dopaminergic and glutamatergic control in schizophrenic patients carriers of this mutation.

  17. Isolated regulatory domains of cGMP-dependent protein kinase Ialpha and Ibeta retain dimerization and native cGMP-binding properties and undergo isoform-specific conformational changes.

    PubMed

    Richie-Jannetta, Robyn; Busch, Jennifer L; Higgins, Kristin A; Corbin, Jackie D; Francis, Sharron H

    2006-03-17

    Molecular mechanisms that provide for cGMP activation of cGMP-dependent protein kinase (PKG) are unknown. PKGs are dimeric; each monomer contains a regulatory (R) and catalytic (C) domain. In this study, isolated recombinant R domains of PKGIalpha-(Delta349-670) and PKGIbeta-(Delta364-685) containing the dimerization and autoinhibitory subdomains and two allosteric cGMP-binding sites were expressed in Sf9 cells. Both R domains were dimers with elongated conformations (Stokes radii of 44 and 51 A, respectively, and frictional coefficients of 1.6 and 1.8, respectively). Exchange dissociation kinetics and K(D) values for cGMP were similar for each holoenzyme and its isolated R domain, indicating that under these conditions the C domain does not appreciably alter cGMP-binding functions of the R domain. As determined by gel filtration chromatography, cGMP binding caused elongation of the PKGIalpha-isolated R domain and contraction of the PKGIbeta-isolated R domain. Cyclic GMP-bound forms of the isoforms have similar physical dimensions that may reflect a common conformation of active isoforms. Elongation of the PKGIbeta holoenzyme associated with cGMP binding and PKG activation cannot be explained solely by conformational change in its R domain, but elongation of the PKGIalpha R domain may partially account for the elongation of wild type PKGIalpha associated with cGMP binding. The cGMP-induced conformational changes in the respective R domains are likely to be critical for kinase activation.

  18. Mutations in PIP5K3 Are Associated with François-Neetens Mouchetée Fleck Corneal Dystrophy

    PubMed Central

    Li, Shouling ; Tiab, Leila ; Jiao, Xiaodong ; Munier, Francis L. ; Zografos, Leonidas ; Frueh, Béatrice E. ; Sergeev, Yuri ; Smith, Janine ; Rubin, Benjamin ; Meallet, Mario A. ; Forster, Richard K. ; Hejtmancik, J. Fielding ; Schorderet, Daniel F. 

    2005-01-01

    François-Neetens fleck corneal dystrophy (CFD) is a rare, autosomal dominant corneal dystrophy characterized by numerous small white flecks scattered in all layers of the stroma. Linkage analysis localized CFD to a 24-cM (18-Mb) interval of chromosome 2q35 flanked by D2S2289 and D2S126 and containing PIP5K3. PIP5K3 is a member of the phosphoinositide 3-kinase family and regulates the sorting and traffic of peripheral endosomes that contain lysosomally directed fluid phase cargo, by controlling the morphogenesis and function of multivesicular bodies. Sequencing analysis disclosed missense, frameshift, and/or protein-truncating mutations in 8 of 10 families with CFD that were studied, including 2256delA, 2274delCT, 2709C→T (R851X), 3120C→T (Q988X), IVS19-1G→C, 3246G→T (E1030X), 3270C→T (R1038X), and 3466A→G (K1103R). The histological and clinical characteristics of patients with CFD are consistent with biochemical studies of PIP5K3 that indicate a role in endosomal sorting. PMID:15902656

  19. Thiazolidinediones inhibit REG I{alpha} gene transcription in gastrointestinal cancer cells

    SciTech Connect

    Yamauchi, Akiyo; Takahashi, Iwao; Takasawa, Shin; Nata, Koji; Noguchi, Naoya; Ikeda, Takayuki; Yoshikawa, Takeo; Shervani, Nausheen J.; Suzuki, Iwao; Uruno, Akira; Unno, Michiaki; Okamoto, Hiroshi Sugawara, Akira

    2009-02-13

    REG (Regenerating gene) I{alpha} protein functions as a growth factor for gastrointestinal cancer cells, and its mRNA expression is strongly associated with a poor prognosis in gastrointestinal cancer patients. We here demonstrated that PPAR{gamma}-agonist thiazolidinediones (TZDs) inhibited cell proliferation and REG I{alpha} protein/mRNA expression in gastrointestinal cancer cells. TZDs inhibited the REG I{alpha} gene promoter activity, via its cis-acting element which lacked PPAR response element and could not bind to PPAR{gamma}, in PPAR{gamma}-expressing gastrointestinal cancer cells. The inhibition was reversed by co-treatment with a specific PPAR{gamma}-antagonist GW9662. Although TZDs did not inhibit the REG I{alpha} gene promoter activity in PPAR{gamma}-non-expressing cells, PPAR{gamma} overexpression in the cells recovered their inhibitory effect. Taken together, TZDs inhibit REG I{alpha} gene transcription through a PPAR{gamma}-dependent pathway. The TZD-induced REG I{alpha} mRNA reduction was abolished by cycloheximide, indicating the necessity of novel protein(s) synthesis. TZDs may therefore be a candidate for novel anti-cancer drugs for patients with gastrointestinal cancer expressing both REG I{alpha} and PPAR{gamma}.

  20. PIP5K-dependent production of PIP2 sustains microtubule organization to establish polarized transport in the Drosophila oocyte.

    PubMed

    Gervais, Louis; Claret, Sandra; Januschke, Jens; Roth, Siegfried; Guichet, Antoine

    2008-12-01

    The attachment of the cytoskeleton to the plasma membrane is crucial in controlling the polarized transport of cell-fate-determining molecules. Attachment involves adaptor molecules, which have the capacity to bind to both the plasma membrane and elements of the cytoskeleton, such as microtubules and actin filaments. Using the Drosophila oocyte as a model system, we show that the type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K), Skittles, is necessary to sustain the organization of microtubules and actin cytoskeleton required for the asymmetric transport of oskar, bicoid and gurken mRNAs and thereby controls the establishment of cell polarity. We show that Skittles function is crucial to synthesize and maintain phosphatidylinositol 4,5 bisphosphate (PIP2) at the plasma membrane in the oocyte. Reduction of Skittles activity impairs activation at the plasma membrane of Moesin, a member of the ERM family known to link the plasma membrane to the actin-based cytoskeleton. Furthermore, we provide evidence that Skittles, by controlling the localization of Bazooka, Par-1 and Lgl, but not Lkb1, to the cell membrane, regulates PAR polarity proteins and the maintenance of specific cortical domains along the anteroposterior axis.

  1. Type I phosphatidylinositol 4-phosphate 5-kinase homo- and heterodimerization determines its membrane localization and activity.

    PubMed

    Lacalle, Rosa Ana; de Karam, Juan C; Martínez-Muñoz, Laura; Artetxe, Ibai; Peregil, Rosa M; Sot, Jesús; Rojas, Ana M; Goñi, Félix M; Mellado, Mario; Mañes, Santos

    2015-06-01

    Type I phosphatidylinositol 4-phosphate 5-kinases (PIP5KIs; α, β, and γ) are a family of isoenzymes that produce phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] using phosphatidylinositol 4-phosphate as substrate. Their structural homology with the class II lipid kinases [type II phosphatidylinositol 5-phosphate 4-kinase (PIP4KII)] suggests that PIP5KI dimerizes, although this has not been formally demonstrated. Neither the hypothetical structural dimerization determinants nor the functional consequences of dimerization have been studied. Here, we used Förster resonance energy transfer, coprecipitation, and ELISA to show that PIP5KIβ forms homo- and heterodimers with PIP5KIγ_i2 in vitro and in live human cells. Dimerization appears to be a general phenomenon for PIP5KI isoenzymes because PIP5KIβ/PIP5KIα heterodimers were also detected by mass spectrometry. Dimerization was independent of actin cytoskeleton remodeling and was also observed using purified proteins. Mutagenesis studies of PIP5KIβ located the dimerization motif at the N terminus, in a region homologous to that implicated in PIP4KII dimerization. PIP5KIβ mutants whose dimerization was impaired showed a severe decrease in PI(4,5)P2 production and plasma membrane delocalization, although their association to lipid monolayers was unaltered. Our results identify dimerization as an integral feature of PIP5K proteins and a central determinant of their enzyme activity.

  2. Phosphatidylinositol phosphate 5-kinase genes respond to phosphate deficiency for root hair elongation in Arabidopsis thaliana.

    PubMed

    Wada, Yukika; Kusano, Hiroaki; Tsuge, Tomohiko; Aoyama, Takashi

    2015-02-01

    Plants drastically alter their root system architecture to adapt to different underground growth conditions. During phosphate (Pi) deficiency, most plants including Arabidopsis thaliana enhance the development of lateral roots and root hairs, resulting in bushy and hairy roots. To elucidate the signal pathway specific for the root hair elongation response to Pi deficiency, we investigated the expression of type-B phosphatidylinositol phosphate 5-kinase (PIP5K) genes, as a quantitative factor for root hair elongation in Arabidopsis. At young seedling stages, the PIP5K3 and PIP5K4 genes responded to Pi deficiency in steady-state transcript levels via PHR1-binding sequences (P1BSs) in their upstream regions. Both pip5k3 and pip5k4 single mutants, which exhibit short-root-hair phenotypes, remained responsive to Pi deficiency for root hair elongation; however the pip5k3pip5k4 double mutant exhibited shorter root hairs than the single mutants, and lost responsiveness to Pi deficiency at young seedling stages. In the tactical complementation line in which modified PIP5K3 and PIP5K4 genes with base substitutions in their P1BSs were co-introduced into the double mutant, root hairs of young seedlings had normal lengths under Pi-sufficient conditions, but were not responsive to Pi deficiency. From these results, we conclude that a Pi-deficiency signal is transferred to the pathway for root hair elongation via the PIP5K genes.

  3. Nuclear pool of phosphatidylinositol 4 phosphate 5 kinase 1α is modified by polySUMO-2 during apoptosis

    SciTech Connect

    Chakrabarti, Rajarshi; Bhowmick, Debajit; Bhargava, Varsha; Bhar, Kaushik; Siddhanta, Anirban

    2013-09-20

    Highlights: •Nuclear pool of PIP5K is SUMOylated. •Enhancement of SUMOylated nuclear PIP5K during apoptosis. •Nuclear PIP5K is modified by polySUMO-1 during apoptosis. •Nuclear PIP5K is modified by polySUMO-2 chain during apoptosis. -- Abstract: Phosphatidylinositol 4 phosphate 5 kinase 1α (PIP5K) is mainly localized in the cytosol and plasma membrane. Studies have also indicated its prominent association with nuclear speckles. The exact nature of this nuclear pool of PIP5K is not clear. Using biochemical and microscopic techniques, we have demonstrated that the nuclear pool of PIP5K is modified by SUMO-1 in HEK-293 cells stably expressing PIP5K. Moreover, this SUMOylated pool of PIP5K increased during apoptosis. PolySUMO-2 chain conjugated PIP5K was detected by pull-down experiment using affinity-tagged RNF4, a polySUMO-2 binding protein, during late apoptosis.

  4. Phosphatidylinositol 4-phosphate 5-kinases 1 and 2 are involved in the regulation of vacuole morphology during Arabidopsis thaliana pollen development.

    PubMed

    Ugalde, José-Manuel; Rodriguez-Furlán, Cecilia; Rycke, Riet De; Norambuena, Lorena; Friml, Jiří; León, Gabriel; Tejos, Ricardo

    2016-09-01

    The pollen grains arise after meiosis of pollen mother cells within the anthers. A series of complex structural changes follows, generating mature pollen grains capable of performing the double fertilization of the female megasporophyte. Several signaling molecules, including hormones and lipids, have been involved in the regulation and appropriate control of pollen development. Phosphatidylinositol 4-phophate 5-kinases (PIP5K), which catalyze the biosynthesis of the phosphoinositide PtdIns(4,5)P2, are important for tip polar growth of root hairs and pollen tubes, embryo development, vegetative plant growth, and responses to the environment. Here, we report a role of PIP5Ks during microgametogenesis. PIP5K1 and PIP5K2 are expressed during early stages of pollen development and their transcriptional activity respond to auxin in pollen grains. Early male gametophytic lethality to certain grade was observed in both pip5k1(-/-) and pip5k2(-/-) single mutants. The number of pip5k mutant alleles is directly related to the frequency of aborted pollen grains suggesting the two genes are involved in the same function. Indeed PIP5K1 and PIP5K2 are functionally redundant since homozygous double mutants did not render viable pollen grains. The loss of function of PIP5K1 and PIP5K2results in defects in vacuole morphology in pollen at the later stages and epidermal root cells. Our results show that PIP5K1, PIP5K2 and phosphoinositide signaling are important cues for early developmental stages and vacuole formation during microgametogenesis.

  5. Arabidopsis phosphatidylinositol 4-phosphate 5-kinase 2 contains a functional nuclear localization sequence and interacts with alpha-importins.

    PubMed

    Gerth, Katharina; Lin, Feng; Daamen, Franziska; Menzel, Wilhelm; Heinrich, Franziska; Heilmann, Mareike

    2017-09-26

    The Arabidopsis phosphoinositide kinase PIP5K2 has been implicated in the control of membrane trafficking and is important for development and growth. In addition to cytosolic functions of phosphoinositides, a nuclear phosphoinositide system has been proposed, but evidence for nuclear phosphoinositides in plants is limited. Fluorescence-tagged variants of PIP5K2 reside in the nucleus of Arabidopsis root meristem cells, in addition to reported plasma membrane localization. Here we report on the interaction of PIP5K2 with alpha-importins and characterize its nuclear localization sequences (NLSs). The PIP5K2 sequence contains four putative NLSs (NLSa-d) and only a PIP5K2 fragment containing NLSs is imported into nuclei of onion epidermis cells upon transient expression. PIP5K2 interacts physically with alpha-importin isoforms in cytosolic split-ubiquitin-based yeast-two-hybrid tests, in dot blot experiments, and in immuno-pull-downs. A 27-amino acid-fragment of PIP5K2 containing NLSc is necessary and sufficient to mediate the nuclear import of a large cargo fusion consisting of two mCherry markers fused to RubisCO large subunit. Substitution of basic residues in NLSc results in reduced import of PIP5K2 or other cargoes into plant nuclei. The data suggest that PIP5K2 is subject to active, alpha-importin-mediated nuclear import, consistent with a nuclear role for PIP5K2 in addition to its reported cytosolic functions. The detection of both substrate and product of PIP5K2 in plant nuclei according to reporter fluorescence and immunofluorescence further supports the notion of a nuclear phosphoinositide system in plants. Variants of PIP5K2 with reduced nuclear residence might serve as tools for the future functional study of plant nuclear phosphoinositides. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  6. Pip5 Transduction Peptides Direct High Efficiency Oligonucleotide-mediated Dystrophin Exon Skipping in Heart and Phenotypic Correction in mdx Mice

    PubMed Central

    Yin, HaiFang; Saleh, Amer F; Betts, Corinne; Camelliti, Patrizia; Seow, Yiqi; Ashraf, Shirin; Arzumanov, Andrey; Hammond, Suzan; Merritt, Thomas; Gait, Michael J; Wood, Matthew JA

    2011-01-01

    Induced splice modulation of pre-mRNAs shows promise to correct aberrant disease transcripts and restore functional protein and thus has therapeutic potential. Duchenne muscular dystrophy (DMD) results from mutations that disrupt the DMD gene open reading frame causing an absence of dystrophin protein. Antisense oligonucleotide (AO)-mediated exon skipping has been shown to restore functional dystrophin in mdx mice and DMD patients treated intramuscularly in two recent phase 1 clinical trials. Critical to the therapeutic success of AO-based treatment will be the ability to deliver AOs systemically to all affected tissues including the heart. Here, we report identification of a series of transduction peptides (Pip5) as AO conjugates for enhanced systemic and particularly cardiac delivery. One of the lead peptide-AO conjugates, Pip5e-AO, showed highly efficient exon skipping and dystrophin production in mdx mice with complete correction of the aberrant DMD transcript in heart, leading to >50% of the normal level of dystrophin in heart. Mechanistic studies indicated that the enhanced activity of Pip5e-phosphorodiamidate morpholino (PMO) is partly explained by more efficient nuclear delivery. Pip5 series derivatives therefore have significant potential for advancing the development of exon skipping therapies for DMD and may have application for enhanced cardiac delivery of other biotherapeutics. PMID:21505427

  7. Changes of MMP-1 and collagen type Ialpha1 by UVA, UVB and IRA are differentially regulated by Trx-1.

    PubMed

    Buechner, Nicole; Schroeder, Peter; Jakob, Sascha; Kunze, Kerstin; Maresch, Tanja; Calles, Christian; Krutmann, Jean; Haendeler, Judith

    2008-07-01

    Exposure of human skin to solar radiation, which includes ultraviolet (UV) radiation (UVA and UVB) visible light and infrared radiation, induces skin aging. The effects of light have been attributed to irradiation-induced reactive oxygen species (ROS) formation, but the specific signaling pathways are not well understood. Detrimental effects of solar radiation are dermal diseases and photoaging. Exposure of cultured human dermal fibroblasts to UVA, UVB or IRA increased ROS formation in vitro. One important redox regulator is the oxidoreductase thioredoxin-1 (Trx). Trx is ubiquitously expressed and has anti-oxidative and anti-apoptotic properties. Besides its function to reduce H(2)O(2), Trx binds to and regulates transcription factors. The aim of this study was to investigate whether Trx influences the regulation of MMP-1 and collagen Ialpha1 by UVA, UVB and IRA. We irradiated human dermal fibroblasts with UVA, UVB and IRA. UVA, UVB and IRA upregulated MMP-1 expression. Trx inhibited UVA-induced MMP-1 upregulation in a NFkappaB dependent manner. UVA, UVB and IRA reduced collagen Ialpha1 expression. Incubation with Trx inhibited the effects of UVB and IRA on collagen Ialpha1 expression. In conclusion, MMP-1 and collagen Ialpha1, which play important roles in aging processes, seems to be regulated by different transcriptional mechanisms and Trx can only influence distinct signaling pathways induced by UVA, UVB and probably IRA. Thus, Trx may serve as an important contributor to an "anti-aging therapeutic cocktail".

  8. Murine peptidoglycan recognition proteins PglyrpIalpha and PglyrpIbeta are encoded in the epidermal differentiation complex and are expressed in epidermal and hematopoietic tissues.

    PubMed

    Mathur, Punam; Murray, Beth; Crowell, Thomas; Gardner, Humphrey; Allaire, Normand; Hsu, Yen-Ming; Thill, Greg; Carulli, John P

    2004-06-01

    Peptidoglycan recognition proteins (PGRPs or PGLYRPs) are pattern recognition molecules that are found in insects and mammals and are critical for innate immune responses. PGRPs bind peptidoglycan, a ubiquitous component of bacterial cell walls, and are involved in killing bacteria, degrading peptidoglycan, and initiating host defense reactions. Relatively little is known about the four mammalian PGRPs. In this article, we report the sequences of mouse PglyrpIalpha and PglyrpIbeta and provide details of their expression in wild-type mouse tissues. PglyrpIalpha and PglyrpIbeta are encoded within the epidermal differentiation complex on mouse chromosome 3F. Both genes are expressed in epidermal and hematopoietic tissues. PglyrpIbeta is expressed in each of 16 tissues tested, while PglyrpIalpha expression is limited to fewer tissues, including the lung and spleen as well as several tissues of the digestive system. Both proteins are expressed in epithelial cells throughout the gut, and immunohistochemical staining shows expression in salivary glands, the squamous epithelium of the stomach, and the villi of the jejunum. Immunohistochemical staining further shows expression of both PglyrpIalpha and PglyrpIbeta in macrophages in the spleen. PglyrpIalpha is not expressed in resting RAW264.7 macrophage-like cells, but is induced by stimulation with lipopolysaccharide. PglyrpIbeta is constitutively expressed in RAW264.7 cells and is unaffected by lipopolysaccharide or peptidoglycan stimulation. Computational and experimental data suggest that these proteins are secreted. This work provides a step toward understanding the roles of PglyrpIalpha and PglyrpIbeta in host defense and chronic inflammatory conditions induced by bacteria or their components.

  9. Phosphatidylinositol 4-phosphate 5-kinase α contributes to Toll-like receptor 2-mediated immune responses in microglial cells stimulated with lipoteichoic acid.

    PubMed

    Nguyen, Tu Thi Ngoc; Seo, Eunjeong; Choi, Juyong; Le, Oanh Thi Tu; Kim, Ji Yun; Jou, Ilo; Lee, Sang Yoon

    2017-10-01

    Phosphatidylinositol 4,5-bisphosphate (PIP2) is an important lipid regulator of membrane signaling and remodeling processes. Accumulating evidence indicates a link between PIP2 metabolism and Toll-like receptor (TLR) signaling, a key transducer of immune responses such as inflammation, phagocytosis, and autophagy. Microglia are immune effector cells that serve as macrophages in the brain. Here, we examined the potential role of phosphatidylinositol 4-phosphate 5-kinase α (PIP5Kα), a PIP2-producing enzyme, in TLR2 signaling in microglial cells. Treatment of BV2 microglial cells with lipoteichoic acid (LTA), a TLR2 agonist, increased PIP5Kα expression in BV2 and primary microglial cells, but not in primary cultures from TLR2-deficient mice. PIP5Kα knockdown of BV2 cells with shRNA significantly suppressed LTA-induced activation of TLR2 downstream signaling, including the production of proinflammatory cytokines and phosphorylation of NF-κB, JNK, and p38 MAP kinase. Such suppression was reversed by complementation of PIP5Kα. PIP5Kα knockdown lowered PIP2 levels and impaired LTA-induced plasma membrane targeting of TIRAP, a PIP2-dependent adaptor required for TLR2 activation. Besides, PIP5Kα knockdown inhibited phagocytic uptake of E. coli particles and autophagy-related vesicle formation triggered by LTA. Taken together, these results support that PIP5Kα can positively mediate TLR2-associated immune responses through PIP2 production in microglial cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. Phosphatidylinositol 4-phosphate 5-kinase α is induced in ganglioside-stimulated brain astrocytes and contributes to inflammatory responses

    PubMed Central

    Kim, Bokyung; Yoon, Sarah; Kim, Yeon Joo; Liu, Tian; Woo, Joo Hong; Chwae, Yong-Joon; Joe, Eun-hye; Jou, Ilo

    2010-01-01

    In brain tissue, astrocytes play defensive roles in central nervous system integrity by mediating immune responses against pathological conditions. Type I phosphatidylinositol 4-phosphate 5-kinase α (PIP5Kα) that is responsible for production of phosphatidylinositol 4,5-bisphosphate (PI[4,5]P2) regulates many important cell functions at the cell surface. Here, we have examined whether PIP5Kα is associated with astrocyte inflammatory responses. Gangliosides are releasable from damaged cell membranes of neurons and capable of inducing inflammatory responses. We found that treatment of primary cultured astrocytes with gangliosides significantly enhanced PIP5Kα mRNA and protein expression levels. PI(4,5)P2 imaging using a fluorescent tubby (R332H) expression as a PI(4,5)P2-specific probe showed that ganglioside treatment increased PI(4,5)P2 level. Interestingly, microRNA-based PIP5Kα knockdown strongly reduced ganglioside-induced transcription of proinflammatory cytokines IL-1β and TNFα. PIP5Kα knockdown also suppressed ganglioside-induced phosphorylation and nuclear translocation of NF-κB and the degradation of IκB-α, indicating that PIP5Kα knockdown interfered with the ganglioside-activated NF-κB signaling. Together, these results suggest that PIP5Kα is a novel inflammatory mediator that undergoes upregulation and contributes to immune responses by facilitating NF-κB activation in ganglioside-stimulated astrocytes. PMID:20720456

  11. skittles, a Drosophila phosphatidylinositol 4-phosphate 5-kinase, is required for cell viability, germline development and bristle morphology, but not for neurotransmitter release.

    PubMed

    Hassan, B A; Prokopenko, S N; Breuer, S; Zhang, B; Paululat, A; Bellen, H J

    1998-12-01

    The phosphatidylinositol pathway is implicated in the regulation of numerous cellular functions and responses to extracellular signals. An important branching point in the pathway is the phosphorylation of phosphatidylinositol 4-phosphate by the phosphatidylinositol 4-phosphate 5-kinase (PIP5K) to generate the second messenger phosphatidylinositol 4,5-bis-phosphate (PIP2). PIP5K and PIP2 have been implicated in signal transduction, cytoskeletal regulation, DNA synthesis, and vesicular trafficking. We have cloned and generated mutations in a Drosophila PIP5K type I (skittles). Our analysis indicates that skittles is required for cell viability, germline development, and the proper structural development of sensory bristles. Surprisingly, we found no evidence for PIP5KI involvement in neural secretion.

  12. skittles, a Drosophila phosphatidylinositol 4-phosphate 5-kinase, is required for cell viability, germline development and bristle morphology, but not for neurotransmitter release.

    PubMed Central

    Hassan, B A; Prokopenko, S N; Breuer, S; Zhang, B; Paululat, A; Bellen, H J

    1998-01-01

    The phosphatidylinositol pathway is implicated in the regulation of numerous cellular functions and responses to extracellular signals. An important branching point in the pathway is the phosphorylation of phosphatidylinositol 4-phosphate by the phosphatidylinositol 4-phosphate 5-kinase (PIP5K) to generate the second messenger phosphatidylinositol 4,5-bis-phosphate (PIP2). PIP5K and PIP2 have been implicated in signal transduction, cytoskeletal regulation, DNA synthesis, and vesicular trafficking. We have cloned and generated mutations in a Drosophila PIP5K type I (skittles). Our analysis indicates that skittles is required for cell viability, germline development, and the proper structural development of sensory bristles. Surprisingly, we found no evidence for PIP5KI involvement in neural secretion. PMID:9832529

  13. Phosphatidylinositol 4-Phosphate 5-Kinase β Controls Recruitment of Lipid Rafts into the Immunological Synapse.

    PubMed

    Kallikourdis, Marinos; Trovato, Anna Elisa; Roselli, Giuliana; Muscolini, Michela; Porciello, Nicla; Tuosto, Loretta; Viola, Antonella

    2016-02-15

    Phosphatidylinositol 4,5-biphosphate (PIP2) is critical for T lymphocyte activation serving as a substrate for the generation of second messengers and the remodeling of actin cytoskeleton necessary for the clustering of lipid rafts, TCR, and costimulatory receptors toward the T:APC interface. Spatiotemporal analysis of PIP2 synthesis in T lymphocytes suggested that distinct isoforms of the main PIP2-generating enzyme, phosphatidylinositol 4-phosphate 5-kinase (PIP5K), play a differential role on the basis of their distinct localization. In this study, we analyze the contribution of PIP5Kβ to T cell activation and show that CD28 induces the recruitment of PIP5Kβ to the immunological synapse, where it regulates filamin A and lipid raft accumulation, as well as T cell activation, in a nonredundant manner. Finally, we found that Vav1 and the C-terminal 83 aa of PIP5Kβ are pivotal for the PIP5Kβ regulatory functions in response to CD28 stimulation. Copyright © 2016 by The American Association of Immunologists, Inc.

  14. Phosphatidylinositol 4-Phosphate 5-Kinases in the Regulation of T Cell Activation

    PubMed Central

    Porciello, Nicla; Kunkl, Martina; Viola, Antonella; Tuosto, Loretta

    2016-01-01

    Phosphatidylinositol 4,5-biphosphate kinases (PIP5Ks) are critical regulators of T cell activation being the main enzymes involved in the synthesis of phosphatidylinositol 4,5-biphosphate (PIP2). PIP2 is indeed a pivotal regulator of the actin cytoskeleton, thus controlling T cell polarization and migration, stable adhesion to antigen-presenting cells, spatial organization of the immunological synapse, and co-stimulation. Moreover, PIP2 also serves as a precursor for the second messengers inositol triphosphate, diacylglycerol, and phosphatidylinositol 3,4,5-triphosphate, which are essential for the activation of signaling pathways regulating cytokine production, cell cycle progression, survival, metabolism, and differentiation. Here, we discuss the impact of PIP5Ks on several T lymphocyte functions with a specific focus on the role of CD28 co-stimulation in PIP5K compartimentalization and activation. PMID:27242793

  15. Arabidopsis phosphatidylinositol monophosphate 5-kinase 2 is involved in root gravitropism through regulation of polar auxin transport by affecting the cycling of PIN proteins.

    PubMed

    Mei, Yu; Jia, Wen-Jing; Chu, Yu-Jia; Xue, Hong-Wei

    2012-03-01

    Phosphatidylinositol monophosphate 5-kinase (PIP5K) catalyzes the synthesis of PI-4,5-bisphosphate (PtdIns(4,5)P(2)) by phosphorylation of PI-4-phosphate at the 5 position of the inositol ring, and is involved in regulating multiple developmental processes and stress responses. We here report on the functional characterization of Arabidopsis PIP5K2, which is expressed during lateral root initiation and elongation, and whose expression is enhanced by exogenous auxin. The knockout mutant pip5k2 shows reduced lateral root formation, which could be recovered with exogenous auxin, and interestingly, delayed root gravity response that could not be recovered with exogenous auxin. Crossing with the DR5-GUS marker line and measurement of free IAA content confirmed the reduced auxin accumulation in pip5k2. In addition, analysis using the membrane-selective dye FM4-64 revealed the decelerated vesicle trafficking caused by PtdIns(4,5)P(2) reduction, which hence results in suppressed cycling of PIN proteins (PIN2 and 3), and delayed redistribution of PIN2 and auxin under gravistimulation in pip5k2 roots. On the contrary, PtdIns(4,5)P(2) significantly enhanced the vesicle trafficking and cycling of PIN proteins. These results demonstrate that PIP5K2 is involved in regulating lateral root formation and root gravity response, and reveal a critical role of PIP5K2/PtdIns(4,5)P(2) in root development through regulation of PIN proteins, providing direct evidence of crosstalk between the phosphatidylinositol signaling pathway and auxin response, and new insights into the control of polar auxin transport.

  16. ISA-2011B, a Phosphatidylinositol 4-Phosphate 5-Kinase α Inhibitor, Impairs CD28-Dependent Costimulatory and Pro-inflammatory Signals in Human T Lymphocytes

    PubMed Central

    Kunkl, Martina; Porciello, Nicla; Mastrogiovanni, Marta; Capuano, Cristina; Lucantoni, Federica; Moretti, Chiara; Persson, Jenny L.; Galandrini, Ricciarda; Buzzetti, Raffaella; Tuosto, Loretta

    2017-01-01

    Phosphatidylinositol 4,5-biphosphate (PIP2) is a membrane phospholipid that controls the activity of several proteins regulating cytoskeleton reorganization, cytokine gene expression, T cell survival, proliferation, and differentiation. Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) are the main enzymes involved in PIP2 biosynthesis by phosphorylating phosphatidylinositol 4-monophosphate (PI4P) at the D5 position of the inositol ring. In human T lymphocytes, we recently found that CD28 costimulatory molecule is pivotal for PIP2 turnover by recruiting and activating PIP5Kα. We also found that PIP5Kα is the main regulator of both CD28 costimulatory signals integrating those delivered by TCR as well as CD28 autonomous signals regulating the expression of pro-inflammatory genes. Given emerging studies linking alterations of PIP2 metabolism to immune-based diseases, PIP5Kα may represent a promising target to modulate immunity and inflammation. Herewith, we characterized a recently discovered inhibitor of PIP5Kα, ISA-2011B, for its inhibitory effects on T lymphocyte functions. We found that the inhibition of PIP5Kα lipid-kinase activity by ISA-2011B significantly impaired CD28 costimulatory signals necessary for TCR-mediated Ca2+ influx, NF-AT transcriptional activity, and IL-2 gene expression as well as CD28 autonomous signals regulating the activation of NF-κB and the transcription of pro-inflammatory cytokine and chemokine genes. Moreover, our data on the inhibitory effects of ISA-2011B on CD28-mediated upregulation of inflammatory cytokines related to Th17 cell phenotype in type 1 diabetes patients suggest ISA-2011B as a promising anti-inflammatory drug. PMID:28491063

  17. Phosphatidylinositol 4-phosphate 5-kinase α and Vav1 mutual cooperation in CD28-mediated actin remodeling and signaling functions.

    PubMed

    Muscolini, Michela; Camperio, Cristina; Porciello, Nicla; Caristi, Silvana; Capuano, Cristina; Viola, Antonella; Galandrini, Ricciarda; Tuosto, Loretta

    2015-02-01

    Phosphatidylinositol 4,5-biphosphate (PIP2) is a cell membrane phosphoinositide crucial for cell signaling and activation. Indeed, PIP2 is a pivotal source for second messenger generation and controlling the activity of several proteins regulating cytoskeleton reorganization. Despite its critical role in T cell activation, the molecular mechanisms regulating PIP2 turnover remain largely unknown. In human primary CD4(+) T lymphocytes, we have recently demonstrated that CD28 costimulatory receptor is crucial for regulating PIP2 turnover by allowing the recruitment and activation of the lipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5Kα). We also identified PIP5Kα as a key modulator of CD28 costimulatory signals leading to the efficient T cell activation. In this study, we extend these data by demonstrating that PIP5Kα recruitment and activation is essential for CD28-mediated cytoskeleton rearrangement necessary for organizing a complete signaling compartment leading to downstream signaling functions. We also identified Vav1 as the linker molecule that couples the C-terminal proline-rich motif of CD28 to the recruitment and activation of PIP5Kα, which in turn cooperates with Vav1 in regulating actin polymerization and CD28 signaling functions. Copyright © 2015 by The American Association of Immunologists, Inc.

  18. Association Study Indicates a Protective Role of Phosphatidylinositol-4-Phosphate-5-Kinase against Tardive Dyskinesia

    PubMed Central

    Fedorenko, Olga Yu; Lang, Florian; Toshchakova, Valentina A.; Boyarko, Evgenia G.; Semke, Arkadiy V.; Bokhan, Nikolay A.; Govorin, Nikolay V.; Aftanas, Lyubomir I.; Ivanova, Svetlana A.

    2015-01-01

    Background: Tardive dyskinesia is a disorder characterized by involuntary muscle movements that occur as a complication of long-term treatment with antipsychotic drugs. It has been suggested to be related to a malfunctioning of the indirect pathway of the motor part of the cortical-striatal-thalamic-cortical circuit, which may be caused by oxidative stress-induced neurotoxicity. Methods: The purpose of our study was to investigate the possible association between phosphatidylinositol-4-phosphate-5-kinase type IIa (PIP5K2A) function and tardive dyskinesia in 491 Caucasian patients with schizophrenia from 3 different psychiatric institutes in West Siberia. The Abnormal Involuntary Movement Scale was used to assess tardive dyskinesia. Individuals were genotyped for 3 single nucleotide polymorphisms in PIP5K2A gene: rs10828317, rs746203, and rs8341. Results: A significant association was established between the functional mutation N251S-polymorphism of the PIP5K2A gene (rs10828317) and tardive dyskinesia, while the other 2 examined nonfunctional single nucleotide polymorphisms were not related. Conclusions: We conclude from this association that PIP5K2A is possibly involved in a mechanism protecting against tardive dyskinesia-inducing neurotoxicity. This corresponds to our hypothesis that tardive dyskinesia is related to neurotoxicity at striatal indirect pathway medium-sized spiny neurons. PMID:25548108

  19. Protein Kinase D1 regulates focal adhesion dynamics and cell adhesion through Phosphatidylinositol-4-phosphate 5-kinase type-l γ

    PubMed Central

    Durand, Nisha; Bastea, Ligia I.; Long, Jason; Döppler, Heike; Ling, Kun; Storz, Peter

    2016-01-01

    Focal adhesions (FAs) are highly dynamic structures that are assembled and disassembled on a continuous basis. The balance between the two processes mediates various aspects of cell behavior, ranging from cell adhesion and spreading to directed cell migration. The turnover of FAs is regulated at multiple levels and involves a variety of signaling molecules and adaptor proteins. In the present study, we show that in response to integrin engagement, a subcellular pool of Protein Kinase D1 (PKD1) localizes to the FAs. PKD1 affects FAs by decreasing turnover and promoting maturation, resulting in enhanced cell adhesion. The effects of PKD1 are mediated through direct phosphorylation of FA-localized phosphatidylinositol-4-phosphate 5-kinase type-l γ (PIP5Klγ) at serine residue 448. This phosphorylation occurs in response to Fibronectin-RhoA signaling and leads to a decrease in PIP5Klγs’ lipid kinase activity and binding affinity for Talin. Our data reveal a novel function for PKD1 as a regulator of FA dynamics and by identifying PIP5Klγ as a novel PKD1 substrate provide mechanistic insight into this process. PMID:27775029

  20. Protein Kinase D1 regulates focal adhesion dynamics and cell adhesion through Phosphatidylinositol-4-phosphate 5-kinase type-l γ.

    PubMed

    Durand, Nisha; Bastea, Ligia I; Long, Jason; Döppler, Heike; Ling, Kun; Storz, Peter

    2016-10-24

    Focal adhesions (FAs) are highly dynamic structures that are assembled and disassembled on a continuous basis. The balance between the two processes mediates various aspects of cell behavior, ranging from cell adhesion and spreading to directed cell migration. The turnover of FAs is regulated at multiple levels and involves a variety of signaling molecules and adaptor proteins. In the present study, we show that in response to integrin engagement, a subcellular pool of Protein Kinase D1 (PKD1) localizes to the FAs. PKD1 affects FAs by decreasing turnover and promoting maturation, resulting in enhanced cell adhesion. The effects of PKD1 are mediated through direct phosphorylation of FA-localized phosphatidylinositol-4-phosphate 5-kinase type-l γ (PIP5Klγ) at serine residue 448. This phosphorylation occurs in response to Fibronectin-RhoA signaling and leads to a decrease in PIP5Klγs' lipid kinase activity and binding affinity for Talin. Our data reveal a novel function for PKD1 as a regulator of FA dynamics and by identifying PIP5Klγ as a novel PKD1 substrate provide mechanistic insight into this process.

  1. Identification of a stimulus-dependent DNase I hypersensitive site between the Ialpha and Calpha exons during immunoglobulin heavy chain class switch recombination.

    PubMed

    Ono, S J; Zhou, G; Tai, A K; Inaba, M; Kinoshita, K; Honjo, T

    2000-02-11

    The complete humoral response to foreign antigen depends upon two distinct recombination events within the heavy chain locus of immunoglobulin. The first recombination event takes place in what will become the antigen combining site of the antibody molecule, encoded by V, D and J segments. The second recombination event involves the looping-out of large spans of DNA which separate the various clusters of heavy chain exons which define the different immunoglobulin isotypes, or classes. While a great deal has been learned about the nature of the VDJ recombinase, very little is known about the nature of the class-switch recombinase. Using a cell system where class-switch recombination occurs primarily to the IgA locus, we have looked for stimulus-dependent changes in the chromatin structure of the IgA locus which might result from interactions between components of the recombinase and cis-elements within the region. We present evidence that strongly suggests that the class-switch recombinase interacts between the Ialpha and Calpha exons of IgA, just upstream of the highly reiterated DR1 and DR2 elements. However, although multiple potential SMAD-4 sites are located precisely within the DNase I hypersensitive site and 160 bp upstream of that site, we failed to detect any evidence of DNA/protein interactions near the hypersensitive site. Moreover, recombinant SMAD-3/4 proteins fail to interact with these sites with appreciable affinity in vitro. These data suggest that some other structural alteration at this site (e.g. RNA/DNA hybrid) may mediate the nuclease sensitivity.

  2. Novel PI(4)P 5-kinase homologue, Fab1p, essential for normal vacuole function and morphology in yeast.

    PubMed

    Yamamoto, A; DeWald, D B; Boronenkov, I V; Anderson, R A; Emr, S D; Koshland, D

    1995-05-01

    The FAB1 gene of budding yeast is predicted to encode a protein of 257 kDa that exhibits significant sequence homology to a human type II PI(4)P 5-kinase (PIP5K-II). The recently cloned human PIP5K-II specifically converts PI(4)P to PI(4,5)P2 (Boronenkov and Anderson, 1995). The region of highest similarity between Fab1p and PIP5K-II includes a predicted nucleotide binding motif, which is likely to correspond to the catalytic domain of the protein. Interestingly, neither PIP5K-II nor Fab1p exhibit significant homology with cloned PI 3-kinases or PI 4-kinases. fab1 mutations result in the formation of aploid and binucleate cells (hence the name FAB). In addition, loss of Fab1p function causes defects in vacuole function and morphology, cell surface integrity, and cell growth. Experiments with a temperature conditional fab1 mutant revealed that their vacuoles rapidly (within 30 min) enlarge to more than double the size upon shifting cells to the nonpermissive temperature. Additional experiments with the fab1 ts mutant together with results obtained with fab1 vps (vacuolar protein sorting defective) double mutants indicate that the nuclear division and cell surface integrity defects observed in fab1 mutants are secondary to the vacuole morphology defects. Based on these data, we propose that Fab1p is a PI(4)P 5-kinase and that the product of the Fab1p reaction, PIP2, functions as an important regulator of vacuole homeostasis perhaps by controlling membrane flux to and/or from the vacuole. Furthermore, a comparison of the phenotypes of fab1 mutants and other yeast mutants affecting PI metabolism suggests that phosphoinositides may serve as general regulators of several different membrane trafficking pathways.

  3. Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses.

    PubMed

    Barbosa, Inês C R; Shikata, Hiromasa; Zourelidou, Melina; Heilmann, Mareike; Heilmann, Ingo; Schwechheimer, Claus

    2016-12-15

    Polar transport of the phytohormone auxin through PIN-FORMED (PIN) auxin efflux carriers is essential for the spatiotemporal control of plant development. The Arabidopsis thaliana serine/threonine kinase D6 PROTEIN KINASE (D6PK) is polarly localized at the plasma membrane of many cells where it colocalizes with PINs and activates PIN-mediated auxin efflux. Here, we show that the association of D6PK with the basal plasma membrane and PINs is dependent on the phospholipid composition of the plasma membrane as well as on the phosphatidylinositol phosphate 5-kinases PIP5K1 and PIP5K2 in epidermis cells of the primary root. We further show that D6PK directly binds polyacidic phospholipids through a polybasic lysine-rich motif in the middle domain of the kinase. The lysine-rich motif is required for proper PIN3 phosphorylation and for auxin transport-dependent tropic growth. Polybasic motifs are also present at a conserved position in other D6PK-related kinases and required for membrane and phospholipid binding. Thus, phospholipid-dependent recruitment to membranes through polybasic motifs might not only be required for D6PK-mediated auxin transport but also other processes regulated by these, as yet, functionally uncharacterized kinases. © 2016. Published by The Company of Biologists Ltd.

  4. Mechanism of substrate specificity of phosphatidylinositol phosphate kinases

    PubMed Central

    Muftuoglu, Yagmur; Xue, Yi; Gao, Xiang; Wu, Dianqing; Ha, Ya

    2016-01-01

    The phosphatidylinositol phosphate kinase (PIPK) family of enzymes is primarily responsible for converting singly phosphorylated phosphatidylinositol derivatives to phosphatidylinositol bisphosphates. As such, these kinases are central to many signaling and membrane trafficking processes in the eukaryotic cell. The three types of phosphatidylinositol phosphate kinases are homologous in sequence but differ in catalytic activities and biological functions. Type I and type II kinases generate phosphatidylinositol 4,5-bisphosphate from phosphatidylinositol 4-phosphate and phosphatidylinositol 5-phosphate, respectively, whereas the type III kinase produces phosphatidylinositol 3,5-bisphosphate from phosphatidylinositol 3-phosphate. Based on crystallographic analysis of the zebrafish type I kinase PIP5Kα, we identified a structural motif unique to the kinase family that serves to recognize the monophosphate on the substrate. Our data indicate that the complex pattern of substrate recognition and phosphorylation results from the interplay between the monophosphate binding site and the specificity loop: the specificity loop functions to recognize different orientations of the inositol ring, whereas residues flanking the phosphate binding Arg244 determine whether phosphatidylinositol 3-phosphate is exclusively bound and phosphorylated at the 5-position. This work provides a thorough picture of how PIPKs achieve their exquisite substrate specificity. PMID:27439870

  5. ARC3, a chloroplast division factor, is a chimera of prokaryotic FtsZ and part of eukaryotic phosphatidylinositol-4-phosphate 5-kinase.

    PubMed

    Shimada, Hiroshi; Koizumi, Masato; Kuroki, Kouta; Mochizuki, Mariko; Fujimoto, Hitoshi; Ohta, Hiroyuki; Masuda, Tatsuru; Takamiya, Ken-ichiro

    2004-08-01

    The arc3 (accumulation and replication of chloroplast) mutant of Arabidopsis thaliana has a small number of abnormally large chloroplasts in the cell, suggesting that chloroplast division is arrested in the mutant and ARC3 has an important role in the initiation of chloroplast division. To elucidate the role of ARC3, first we identified the ARC3 gene, and determined the location of ARC3 protein during chloroplast division because the localization and spatial orientation of such division factors are vital for correct chloroplast division. Sequencing analysis showed that ARC3 was a fusion of the prokaryotic FtsZ and part of the eukaryotic phosphatidylinositol-4-phosphate 5-kinase (PIP5K) genes. The PIP5K-homologous region of ARC3 had no catalytic domain but a membrane-occupation-and-recognition-nexus (MORN) repeat motif. Immunofluorescence microscopy, Western blotting analysis and in vitro chloroplast import and protease protection assays revealed that ARC3 protein was soluble, and located on the outer surface of the chloroplast in a ring-like structure at the early stage of chloroplast division. Prokaryotes have one FtsZ as a gene for division but have no ARC3 counterparts, the chimera of FtsZ and PIP5K, suggesting that the ARC3 gene might have been generated from FtsZ as another division factor during the evolution of chloroplast by endosymbiosis.

  6. Oxidant sensor in the cGMP-binding pocket of PKGIα regulates nitroxyl-mediated kinase activity.

    PubMed

    Donzelli, Sonia; Goetz, Mara; Schmidt, Kjestine; Wolters, Markus; Stathopoulou, Konstantina; Diering, Simon; Prysyazhna, Oleksandra; Polat, Volkan; Scotcher, Jenna; Dees, Christian; Subramanian, Hariharan; Butt, Elke; Kamynina, Alisa; Schobesberger, Sophie; King, S Bruce; Nikolaev, Viacheslav O; de Wit, Cor; Leichert, Lars I; Feil, Robert; Eaton, Philip; Cuello, Friederike

    2017-08-30

    Despite the mechanisms for endogenous nitroxyl (HNO) production and action being incompletely understood, pharmacological donors show broad therapeutic promise and are in clinical trials. Mass spectrometry and site-directed mutagenesis showed that chemically distinct HNO donors 1-nitrosocyclohexyl acetate or Angeli's salt induced disulfides within cGMP-dependent protein kinase I-alpha (PKGIα), an interdisulfide between Cys42 of the two identical subunits of the kinase and a previously unobserved intradisulfide between Cys117 and Cys195 in the high affinity cGMP-binding site. Kinase activity was monitored in cells transfected with wildtype (WT), Cys42Ser or Cys117/195Ser PKGIα that cannot form the inter- or intradisulfide, respectively. HNO enhanced WT kinase activity, an effect significantly attenuated in inter- or intradisulfide-deficient PKGIα. To investigate whether the intradisulfide modulates cGMP binding, real-time imaging was performed in vascular smooth muscle cells expressing a FRET-biosensor comprising the cGMP-binding sites of PKGIα. HNO induced FRET changes similar to those elicited by an increase of cGMP, suggesting that intradisulfide formation is associated with activation of PKGIα. Intradisulfide formation in PKGIα correlated with enhanced HNO-mediated vasorelaxation in mesenteric arteries in vitro and arteriolar dilation in vivo in mice. HNO induces intradisulfide formation in PKGIα, inducing the same effect as cGMP binding, namely kinase activation and thus vasorelaxation.

  7. 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.

  8. Phylogenomics of phosphoinositide lipid kinases: perspectives on the evolution of second messenger signaling and drug discovery

    PubMed Central

    2011-01-01

    Background Phosphoinositide lipid kinases (PIKs) generate specific phosphorylated variants of phosatidylinositols (PtdIns) that are critical for second messenger signaling and cellular membrane remodeling. Mammals have 19 PIK isoforms spread across three major families: the PtIns 3-kinases (PI3Ks), PtdIns 4-kinases (PI4Ks), and PtdIns-P (PIP) kinases (PIPKs). Other eukaryotes have fewer yet varying PIK complements. PIKs are also an important, emerging class of drug targets for many therapeutic areas including cancer, inflammatory and metabolic diseases and host-pathogen interactions. Here, we report the genomic occurrences and evolutionary relationships or phylogenomics of all three PIK families across major eukaryotic groups and suggest potential ramifications for drug discovery. Results Our analyses reveal four core eukaryotic PIKs which are type III PIK4A and PIK4B, and at least one homolog each from PI3K (possibly PIK3C3 as the ancestor) and PIP5K families. We also applied evolutionary analyses to PIK disease ontology and drug discovery. Mutated PIK3CA are known to be oncogenic and several inhibitors are in anti-cancer clinical trials. We found conservation of activating mutations of PIK3CA in paralogous isoforms suggesting specific functional constraints on these residues. By mapping published compound inhibition data (IC50s) onto a phylogeny of PI3Ks, type II PI4Ks and distantly related, MTOR, ATM, ATR and PRKDC kinases, we also show that compound polypharmacology corresponds to kinase evolutionary relationships. Finally, we extended the rationale for drugs targeting PIKs of malarial Plasmodium falciparum, and the parasites, Leishmania sp. and Trypanosoma sp. by identifying those PIKs highly divergent from human homologs. Conclusion Our phylogenomic analysis of PIKs provides new insights into the evolution of second messenger signaling. We postulate two waves of PIK diversification, the first in metazoans with a subsequent expansion in cold

  9. Teaching resources. Protein kinases.

    PubMed

    Caplan, Avrom

    2005-02-22

    This Teaching Resource provides lecture notes and slides for a class covering the structure and function of protein kinases and is part of the course "Cell Signaling Systems: A Course for Graduate Students." The lecture begins with a discussion of the genomics and evolutionary relationships among kinases and then proceeds to describe the structure-function relationships of specific kinases, the molecular mechanisms underlying substrate specificity, and selected issues in regulation of kinase activity.

  10. 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

  11. Protein Kinases and Addiction

    PubMed Central

    Lee, Anna M.; Messing, Robert O.

    2011-01-01

    Although drugs of abuse have different chemical structures and interact with different protein targets, all appear to usurp common neuronal systems that regulate reward and motivation. Addiction is a complex disease that is thought to involve drug-induced changes in synaptic plasticity due to alterations in cell signaling, gene transcription, and protein synthesis. Recent evidence suggests that drugs of abuse interact with and change a common network of signaling pathways that include a subset of specific protein kinases. The best studied of these kinases are reviewed here and include extracellular signal-regulated kinase, cAMP-dependent protein kinase, cyclin-dependent protein kinase 5, protein kinase C, calcium/calmodulin-dependent protein kinase II, and Fyn tyrosine kinase. These kinases have been implicated in various aspects of drug addiction including acute drug effects, drug self-administration, withdrawal, reinforcement, sensitization, and tolerance. Identifying protein kinase substrates and signaling pathways that contribute to the addicted state may provide novel approaches for new pharma-cotherapies to treat drug addiction. PMID:18991950

  12. Phosphatidylinositol 4,5-Bisphosphate Influences PIN Polarization by Controlling Clathrin-Mediated Membrane Trafficking in Arabidopsis[C][W

    PubMed Central

    Ischebeck, Till; Werner, Stephanie; Krishnamoorthy, Praveen; Lerche, Jennifer; Meijón, Mónica; Stenzel, Irene; Löfke, Christian; Wiessner, Theresa; Im, Yang Ju; Perera, Imara Y.; Iven, Tim; Feussner, Ivo; Busch, Wolfgang; Boss, Wendy F.; Teichmann, Thomas; Hause, Bettina; Persson, Staffan; Heilmann, Ingo

    2013-01-01

    The functions of the minor phospholipid phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] during vegetative plant growth remain obscure. Here, we targeted two related phosphatidylinositol 4-phosphate 5-kinases (PI4P 5-kinases) PIP5K1 and PIP5K2, which are expressed ubiquitously in Arabidopsis thaliana. A pip5k1 pip5k2 double mutant with reduced PtdIns(4,5)P2 levels showed dwarf stature and phenotypes suggesting defects in auxin distribution. The roots of the pip5k1 pip5k2 double mutant had normal auxin levels but reduced auxin transport and altered distribution. Fluorescence-tagged auxin efflux carriers PIN-FORMED (PIN1)–green fluorescent protein (GFP) and PIN2-GFP displayed abnormal, partially apolar distribution. Furthermore, fewer brefeldin A–induced endosomal bodies decorated by PIN1-GFP or PIN2-GFP formed in pip5k1 pip5k2 mutants. Inducible overexpressor lines for PIP5K1 or PIP5K2 also exhibited phenotypes indicating misregulation of auxin-dependent processes, and immunolocalization showed reduced membrane association of PIN1 and PIN2. PIN cycling and polarization require clathrin-mediated endocytosis and labeled clathrin light chain also displayed altered localization patterns in the pip5k1 pip5k2 double mutant, consistent with a role for PtdIns(4,5)P2 in the regulation of clathrin-mediated endocytosis. Further biochemical tests on subcellular fractions enriched for clathrin-coated vesicles (CCVs) indicated that pip5k1 and pip5k2 mutants have reduced CCV-associated PI4P 5-kinase activity. Together, the data indicate an important role for PtdIns(4,5)P2 in the control of clathrin dynamics and in auxin distribution in Arabidopsis. PMID:24326589

  13. 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

  14. [Tyrosine kinase inhibitors].

    PubMed

    Robert, Jacques

    2011-11-01

    Membrane receptors with tyrosine kinase activity and cytoplasmic tyrosine kinases have emerged as important potential targets in oncology. Starting from basic structures such as anilino-quinazoline, numerous compounds have been synthesised, with the help of tyrosine kinase crystallography, which has allowed to optimise protein-ligand interactions. The catalytic domains of all kinases present similar three-dimensional structures, which explains that it may be difficult to identify molecules having a high specificity for a given tyrosine kinase. Some tyrosine kinase inhibitors are relatively specific for epidermal growth factor receptor (EGFR) such as géfitinib and erlotinib; other are mainly active against platelet-derived growth factor receptor (PDGFR) and the receptor KIT, such as imatinib or nilotinib, and other against vascular endothelial growth factor (VEGF) receptors involved in angiogenesis, such as sunitinib and sorafenib. The oral formulation of tyrosine kinase inhibitors is well accepted by the patients but may generate sometimes compliance problems requiring pharmacokinetic monitoring. This chemical family is in full expansion and several dozens of compounds have entered clinical trials.

  15. MAPKAP kinase-2; a novel protein kinase activated by mitogen-activated protein kinase.

    PubMed Central

    Stokoe, D; Campbell, D G; Nakielny, S; Hidaka, H; Leevers, S J; Marshall, C; Cohen, P

    1992-01-01

    A novel protein kinase, which was only active when phosphorylated by the mitogen-activated protein kinase (MAP kinase), has been purified 85,000-fold to homogeneity from rabbit skeletal muscle. This MAP kinase activated protein kinase, termed MAPKAP kinase-2, was distinguished from S6 kinase-II (MAPKAP kinase-1) by its response to inhibitors, lack of phosphorylation of S6 peptides and amino acid sequence. MAPKAP kinase-2 phosphorylated glycogen synthase at Ser7 and the equivalent serine (*) in the peptide KKPLNRTLS*VASLPGLamide whose sequence is similar to the N terminus of glycogen synthase. MAPKAP kinase-2 was resolved into two monomeric species of apparent molecular mass 60 and 53 kDa that had similar specific activities and substrate specificities. Peptide sequences of the 60 and 53 kDa species were identical, indicating that they are either closely related isoforms or derived from the same gene. MAP kinase activated the 60 and 53 kDa forms of MAPKAP kinase-2 by phosphorylating the first threonine residue in the sequence VPQTPLHTSR. Furthermore, Mono Q chromatography of extracts from rat phaeochromocytoma and skeletal muscle demonstrated that two MAP kinase isoforms (p42mapk and p44mapk) were the only enzymes in these cells that were capable of reactivating MAPKAP kinase-2. These results indicate that MAP kinase activates at least two distinct protein kinases, suggesting that it represents a point at which the growth factor-stimulated protein kinase cascade bifurcates. Images PMID:1327754

  16. Orphan kinases turn eccentric

    PubMed Central

    Mikolcevic, Petra; Rainer, Johannes; Geley, Stephan

    2012-01-01

    PCTAIRE kinases (PCTK) are a highly conserved, but poorly characterized, subgroup of cyclin-dependent kinases (CDK). They are characterized by a conserved catalytic domain flanked by N- and C-terminal extensions that are involved in cyclin binding. Vertebrate genomes contain three highly similar PCTAIRE kinases (PCTK1,2,3, a.k.a., CDK16,17,18), which are most abundant in post-mitotic cells in brain and testis. Consistent with this restricted expression pattern, PCTK1 (CDK16) has recently been shown to be essential for spermatogenesis. PCTAIREs are activated by cyclin Y (CCNY), a highly conserved single cyclin fold protein. By binding to N-myristoylated CCNY, CDK16 is targeted to the plasma membrane. Unlike conventional cyclin-CDK interactions, binding of CCNY to CDK16 not only requires the catalytic domain, but also domains within the N-terminal extension. Interestingly, phosphorylation within this domain blocks CCNY binding, providing a novel means of cyclin-CDK regulation. By using these functional characteristics, we analyzed “PCTAIRE” sequence containing protein kinase genes in genomes of various organisms and found that CCNY and CCNY-dependent kinases are restricted to eumetazoa and possibly evolved along with development of a central nervous system. Here, we focus on the structure and regulation of PCTAIREs and discuss their established functions. PMID:22895054

  17. Conserved herpesvirus protein kinases

    PubMed Central

    Gershburg, Edward; Pagano, Joseph S.

    2008-01-01

    Conserved herpesviral protein kinases (CHPKs) are a group of enzymes conserved throughout all subfamilies of Herpesviridae. Members of this group are serine/threonine protein kinases that are likely to play a conserved role in viral infection by interacting with common host cellular and viral factors; however along with a conserved role, individual kinases may have unique functions in the context of viral infection in such a way that they are only partially replaceable even by close homologues. Recent studies demonstrated that CHPKs are crucial for viral infection and suggested their involvement in regulation of numerous processes at various infection steps (primary infection, nuclear egress, tegumentation), although the mechanisms of this regulation remain unknown. Notwithstanding, recent advances in discovery of new CHPK targets, and studies of CHPK knockout phenotypes have raised their attractiveness as targets for antiviral therapy. A number of compounds have been shown to inhibit the activity of human cytomegalovirus (HCMV)-encoded UL97 protein kinase and exhibit a pronounced antiviral effect, although the same compounds are inactive against Epstein-Barr Virus (EBV)-encoded protein kinase BGLF4, illustrating the fact that low homology between the members of this group complicates development of compounds targeting the whole group, and suggesting that individualized, structure-based inhibitor design will be more effective. Determination of CHPK structures will greatly facilitate this task. PMID:17881303

  18. Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

    PubMed Central

    Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

    2005-01-01

    The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

  19. PAK family kinases

    PubMed Central

    Zhao, Zhuo-shen; Manser, Ed

    2012-01-01

    The p21-activated kinases (PAKs) are a family of Ser/Thr protein kinases that are represented by six genes in humans (PAK 1–6), and are found in all eukaryotes sequenced to date. Genetic and knockdown experiments in frogs, fish and mice indicate group I PAKs are widely expressed, required for multiple tissue development, and particularly important for immune and nervous system function in the adult. The group II PAKs (human PAKs 4–6) are more enigmatic, but their restriction to metazoans and presence at cell-cell junctions suggests these kinases emerged to regulate junctional signaling. Studies of protozoa and fungal PAKs show that they regulate cell shape and polarity through phosphorylation of multiple cytoskeletal proteins, including microtubule binding proteins, myosins and septins. This chapter discusses what we know about the regulation of PAKs and their physiological role in different model organisms, based primarily on gene knockout studies. PMID:23162738

  20. 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.

  1. Pantothenate - kinase associated neurodegeneration.

    PubMed

    Parmar, Alpana; Khare, Shruti; Srivastav, Vipul

    2012-04-01

    Neurodegeneration with brain iron accumulation is a group of disorders, the commonest of which is PKAN (Pantothenate kinase associated neurodegeneration). We present here, a case of 18 year old boy with progressive dementia, pyramidal and extrapyramidal involvement, dysarthria, seizures and myoclonus. The patient was diagnosed as PKAN (formerly Hallervorden Spatz disease) after "eye of tiger" appearance on neuro-imaging.

  2. 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.

  3. Kinase Inhibitors from Marine Sponges

    PubMed Central

    Skropeta, Danielle; Pastro, Natalie; Zivanovic, Ana

    2011-01-01

    Protein kinases play a critical role in cell regulation and their deregulation is a contributing factor in an increasing list of diseases including cancer. Marine sponges have yielded over 70 novel compounds to date that exhibit significant inhibitory activity towards a range of protein kinases. These compounds, which belong to diverse structural classes, are reviewed herein, and ordered based upon the kinase that they inhibit. Relevant synthetic studies on the marine natural product kinase inhibitors have also been included. PMID:22073013

  4. A Dibasic Amino Acid Pair Conserved in the Activation Loop Directs Plasma Membrane Localization and Is Necessary for Activity of Plant Type I/II Phosphatidylinositol Phosphate Kinase1[W

    PubMed Central

    Mikami, Koji; Saavedra, Laura; Hiwatashi, Yuji; Uji, Toshiki; Hasebe, Mitsuyasu; Sommarin, Marianne

    2010-01-01

    Phosphatidylinositol phosphate kinase (PIPK) is an enzyme involved in the regulation of cellular levels of phosphoinositides involved in various physiological processes, such as cytoskeletal organization, ion channel activation, and vesicle trafficking. In animals, research has focused on the modes of activation and function of PIPKs, providing an understanding of the importance of plasma membrane localization. However, it still remains unclear how this issue is regulated in plant PIPKs. Here, we demonstrate that the carboxyl-terminal catalytic domain, which contains the activation loop, is sufficient for plasma membrane localization of PpPIPK1, a type I/II B PIPK from the moss Physcomitrella patens. The importance of the carboxyl-terminal catalytic domain for plasma membrane localization was confirmed with Arabidopsis (Arabidopsis thaliana) AtPIP5K1. Our findings, in which substitution of a conserved dibasic amino acid pair in the activation loop of PpPIPK1 completely prevented plasma membrane targeting and abolished enzymatic activity, demonstrate its critical role in these processes. Placing our results in the context of studies of eukaryotic PIPKs led us to conclude that the function of the dibasic amino acid pair in the activation loop in type I/II PIPKs is plant specific. PMID:20427464

  5. Secondary kinase reactions catalyzed by yeast pyruvate kinase.

    PubMed

    Leblond, D J; Robinson, J L

    1976-06-07

    1. Yeast pyruvate kinase (EC 2.7.1.40) catalyzes, in addition to the primary, physiologically important reaction, three secondary kinase reactions, the ATP-dependent phosphorylations of fluoride (fluorokinase), hydroxylamine (hydroxylamine kinase) and glycolate (glycolate kinase). 2. These reactions are accelerated by fructose-1,6-bisphosphate, the allosteric activator of the primary reaction. Wth Mg2+ as the required divalent cation, none of these reactions are observed in the absence of fructose-biphosphate. With Mn2+, fructose-bisphosphate is required for the glycolate kinase reaction, but merely stimulates the other reactions. 3. The effect of other divalent cations and pH on three secondary kinase reactions was also examined. 4. Results are compared with those obtained from muscle pyruvate kinase and the implications of the results for the mechanism of the yeast enzyme are discussed.

  6. LIM-kinase1.

    PubMed

    Stanyon, C A; Bernard, O

    1999-01-01

    LIM-kinase1 (LIMK1) is a serine-only protein kinase that contains LIM and PDZ protein-protein interaction domains which is highly expressed in neurons. Overexpression of LIMK1 in cultured cells results in accumulation of filamentous (F-) actin. LIMK1 phosphorylates cofilin, an actin depolymerisation factor, which is then unable to bind and depolymerise F-actin. Rac-GTP enhances phosphorylation of LIMK1 and cofilin, which leads to accumulation of F-actin, while Rac-GDP and PMA reduce these effects. LIMK1 is therefore a key component of a signal transduction network that connects extracellular stimuli to changes in cytoskeletal structure. Control of cell morphology and mobility via LIMK1 activity may provide novel approaches to cancer therapy.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. Cyclin-dependent kinases

    PubMed Central

    2014-01-01

    Summary 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

  18. Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

    PubMed

    Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

    2016-07-29

    Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia*

    PubMed Central

    Roth Flach, Rachel J.; Danai, Laura V.; DiStefano, Marina T.; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B.; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K.; Bortell, Rita; Alonso, Laura C.; Czech, Michael P.

    2016-01-01

    Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo. After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. PMID:27226575

  20. 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

  1. A high-throughput radiometric kinase assay

    PubMed Central

    Duong-Ly, Krisna C.; Peterson, Jeffrey R.

    2016-01-01

    Aberrant kinase signaling has been implicated in a number of diseases. While kinases have become attractive drug targets, only a small fraction of human protein kinases have validated inhibitors. Screening libraries of compounds against a kinase or kinases of interest is routinely performed during kinase inhibitor development to identify promising scaffolds for a particular target and to identify kinase targets for compounds of interest. Screening of more focused compound libraries may also be conducted in the later stages of inhibitor development to improve potency and optimize selectivity. The dot blot kinase assay is a robust, high-throughput kinase assay that can be used to screen a number of small molecule compounds against one kinase of interest or several kinases. Here, a protocol for a dot blot kinase assay used for measuring insulin receptor kinase activity is presented. This protocol can be readily adapted for use with other protein kinases. PMID:26501904

  2. Aurora Kinases Throughout Plant Development.

    PubMed

    Weimer, Annika K; Demidov, Dmitri; Lermontova, Inna; Beeckman, Tom; Van Damme, Daniël

    2016-01-01

    Aurora kinases are evolutionarily conserved key mitotic determinants in all eukaryotes. Yeasts contain a single Aurora kinase, whereas multicellular eukaryotes have at least two functionally diverged members. The involvement of Aurora kinases in human cancers has provided an in-depth mechanistic understanding of their roles throughout cell division in animal and yeast models. By contrast, understanding Aurora kinase function in plants is only starting to emerge. Nevertheless, genetic, cell biological, and biochemical approaches have revealed functional diversification between the plant Aurora kinases and suggest a role in formative (asymmetric) divisions, chromatin modification, and genome stability. This review provides an overview of the accumulated knowledge on the function of plant Aurora kinases as well as some major challenges for the future.

  3. MAP kinase and pain

    PubMed Central

    Ji, Ru-Rong; Gereau, Robert W.; Malcangio, Marzia; Strichartz, Gary R.

    2008-01-01

    Mitogen-activated protein kinases (MAPKs) are important for intracellular signal transduction and play critical roles in regulating neural plasticity and inflammatory responses. The MAPK family consists of three major members: extracellular signal-regulated kinases (ERK), p38, and c-Jun N-terminal kinase (JNK), which represent three separate signaling pathways. Accumulating evidence shows that all three MAPK pathways contribute to pain sensitization after tissue and nerve injury via distinct molecular and cellular mechanisms. Activation (phosphorylation) of MAPKs under different persistent pain conditions results in the induction and maintenance of pain hypersensitivity via non-transcriptional and transcriptional regulation. In particular, ERK activation in spinal cord dorsal horn neurons by nociceptive activity, via multiple neurotransmitter receptors, and using different second messenger pathways plays a critical role in central sensitization by regulating the activity of glutamate receptors and potassium channels and inducing gene transcription. ERK activation in amygdala neurons is also required for inflammatory pain sensitization. After nerve injury, ERK, p38, and JNK are differentially activated in spinal glial cells (microglia vs astrocytes), leading to the synthesis of proinflammatory/pronociceptive mediators, thereby enhancing and prolonging pain. Inhibition of all three MAPK pathways has been shown to attenuate inflammatory and neuropathic pain in different animal models. Development of specific inhibitors for MAPK pathways to target neurons and glial cells may lead to new therapies for pain management. Although it is well documented that MAPK pathways can increase pain sensitivity via peripheral mechanisms, this review will focus on central mechanisms of MAPKs, especially ERK. PMID:19150373

  4. Acetate kinase and phosphotransacetylase.

    PubMed

    Ferry, James G

    2011-01-01

    Most of the methane produced in nature derives from the methyl group of acetate, the major end product of anaerobes decomposing complex plant material. The acetate is derived from the metabolic intermediate acetyl-CoA via the combined activities of phosphotransacetylase and acetate kinase. In Methanosarcina species, the enzymes function in the reverse direction to activate acetate to acetyl-CoA prior to cleavage into a methyl and carbonyl group of which the latter is oxidized providing electrons for reduction of the former to methane. Thus, phosphotransacetylase and acetate kinase have a central role in the conversion of complex organic matter to methane by anaerobic microbial food chains. Both enzymes have been purified from Methanosarcina thermophila and characterized. Both enzymes from M. thermophila have also been produced in Escherichia coli permitting crystal structures and amino acid variants, the kinetic and biochemical studies of which have lead to proposals for catalytic mechanisms. The high identity of both enzymes to paralogs in the domain Bacteria suggests ancient origins and common mechanisms. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Phosphatidylinositol 3'-kinase associates with an insulin receptor substrate-1 serine kinase distinct from its intrinsic serine kinase.

    PubMed Central

    Cengel, K A; Kason, R E; Freund, G G

    1998-01-01

    Serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been proposed as a counter-regulatory mechanism in insulin and cytokine signalling. Here we report that IRS-1 is phosphorylated by a wortmannin insensitive phosphatidylinositol 3'-kinase (PI 3-kinase)-associated serine kinase (PAS kinase) distinct from PI 3-kinase serine kinase. We found that PI 3-kinase immune complexes contain 5-fold more wortmannin-insensitive serine kinase activity than SH2-containing protein tyrosine phosphatase-2 (SHP2) and IRS-1 immune complexes. Affinity chromatography of cell lysates with a glutathione S-transferase fusion protein for the p85 subunit of PI 3-kinase showed that PAS kinase associated with the p85 subunit of PI 3-kinase. This interaction required unoccupied SH2 domain(s) but did not require the PI 3-kinase p110 subunit binding domain. In terms of function, PAS kinase phosphorylated IRS-1 and, after insulin stimulation, PAS kinase phosphorylated IRS-1 in PI 3-kinase-IRS-1 complexes. Phosphopeptide mapping showed that insulin-dependent in vivo sites of IRS-1 serine phosphorylation were comparable to those of PAS kinase phosphorylated IRS-1. More importantly, PAS kinase-dependent phosphorylation of IRS-1 reduced by 4-fold the ability of IRS-1 to act as an insulin receptor substrate. Taken together, these findings indicate that: (a) PAS kinase is distinct from the intrinsic serine kinase activity of PI 3-kinase, (b) PAS kinase associates with the p85 subunit of PI 3-kinase through SH2 domain interactions, and (c) PAS kinase is an IRS-1 serine kinase that can reduce the ability of IRS-1 to serve as an insulin receptor substrate. PMID:9761740

  6. Understanding the Polo Kinase machine.

    PubMed

    Archambault, V; Lépine, G; Kachaner, D

    2015-09-10

    The Polo Kinase is a central regulator of cell division required for several events of mitosis and cytokinesis. In addition to a kinase domain (KD), Polo-like kinases (Plks) comprise a Polo-Box domain (PBD), which mediates protein interactions with targets and regulators of Plks. In all organisms that contain Plks, one Plk family member fulfills several essential functions in the regulation of cell division, and here we refer to this conserved protein as Polo Kinase (Plk1 in humans). The PBD and the KD are capable of both cooperation and mutual inhibition in their functions. Crystal structures of the PBD, the KD and, recently, a PBD-KD complex have helped understanding the inner workings of the Polo Kinase. In parallel, an impressive array of molecular mechanisms has been found to mediate the regulation of the protein. Moreover, the targeting of Polo Kinase in the development of anti-cancer drugs has yielded several molecules with which to chemically modulate Polo Kinase to study its biological functions. Here we review our current understanding of the protein function and regulation of Polo Kinase as a fascinating molecular device in control of cell division.

  7. P21 activated kinases

    PubMed Central

    Rane, Chetan K; Minden, Audrey

    2014-01-01

    The p21 activated kinases (Paks) are well known effector proteins for the Rho GTPases Cdc42 and Rac. The Paks contain 6 members, which fall into 2 families of proteins. The first family consists of Paks 1, 2, and 3, and the second consists of Paks 4, 5, and 6. While some of the Paks are ubiquitously expressed, others have more restrictive tissue specificity. All of them are found in the nervous system. Studies using cell culture, transgenic mice, and knockout mice, have revealed important roles for the Paks in cytoskeletal organization and in many aspects of cell growth and development. This review discusses the basic structures of the Paks, and their roles in cell growth, development, and in cancer. PMID:24658305

  8. ERK kinases modulate the activation of PI3 kinase related kinases (PIKKs) in DNA damage response.

    PubMed

    Lin, Xiaozeng; Yan, Judy; Tang, Damu

    2013-12-01

    DNA damage response (DDR) is the critical surveillance mechanism in maintaining genome integrity. The mechanism activates checkpoints to prevent cell cycle progression in the presence of DNA lesions, and mediates lesion repair. DDR is coordinated by three apical PI3 kinase related kinases (PIKKs), including ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), and DNA-PKcs (the catalytic subunit of the DNA dependent protein kinase). These kinases are activated in response to specific DNA damage or lesions, resulting in checkpoint activation and DNA lesion repair. While it is clear that the pathways of ATM, ATR, and DNA-PK are the core components of DDR, there is accumulating evidence revealing the involvement of other cellular pathways in regulating DDR; this is in line with the concept that in addition to being a nuclear event DDR is also a cellular process. One of these pathways is the extracellular signal-regulated kinase (ERK) MAPK (mitogen-activated protein kinase) pathway. ERK is a converging point of multiple signal transduction pathways involved in cell proliferation, differentiation, and apoptosis. Adding to this list of pathways is the recent development of ERK in DDR. The ERK kinases (ERK1 and ERK2) contribute to the proper execution of DDR in terms of checkpoint activation and the repair of DNA lesions. This review summarizes the contributions of ERK to DDR with emphasis on the relationship of ERK kinases with the activation of ATM, ATR, and DNA-PKcs.

  9. NAK is an IkappaB kinase-activating kinase.

    PubMed

    Tojima, Y; Fujimoto, A; Delhase, M; Chen, Y; Hatakeyama, S; Nakayama, K; Kaneko, Y; Nimura, Y; Motoyama, N; Ikeda, K; Karin, M; Nakanishi, M

    2000-04-13

    Phosphorylation of IkappaB by the IkappaB kinase (IKK) complex is a critical step leading to IkappaB degradation and activation of transcription factor NF-kappaB. The IKK complex contains two catalytic subunits, IKKalpha and IKKbeta, the latter being indispensable for NF-kappaB activation by pro-inflammatory cytokines. Although IKK is activated by phosphorylation of the IKKbeta activation loop, the physiological IKK kinases that mediate responses to extracellular stimuli remain obscure. Here we describe an IKK-related kinase, named NAK (NF-kappaB-activating kinase), that can activate IKK through direct phosphorylation. NAK induces IkappaB degradation and NF-kappaB activity through IKKbeta. Endogenous NAK is activated by phorbol ester tumour promoters and growth factors, whereas catalytically inactive NAK specifically inhibits activation of NF-kappaB by protein kinase C-epsilon (PKCepsilon). Thus, NAK is an IKK kinase that may mediate IKK and NF-kappaB activation in response to growth factors that stimulate PKCepsilon activity.

  10. Neuronal migration and protein kinases

    PubMed Central

    Ohshima, Toshio

    2015-01-01

    The formation of the six-layered structure of the mammalian cortex via the inside-out pattern of neuronal migration is fundamental to neocortical functions. Extracellular cues such as Reelin induce intracellular signaling cascades through the protein phosphorylation. Migrating neurons also have intrinsic machineries to regulate cytoskeletal proteins and adhesion properties. Protein phosphorylation regulates these processes. Moreover, the balance between phosphorylation and dephosphorylation is modified by extracellular cues. Multipolar-bipolar transition, radial glia-guided locomotion and terminal translocation are critical steps of radial migration of cortical pyramidal neurons. Protein kinases such as Cyclin-dependent kinase 5 (Cdk5) and c-Jun N-terminal kinases (JNKs) involve these steps. In this review, I shall give an overview the roles of protein kinases in neuronal migration. PMID:25628530

  11. 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.

  12. CUL3 and protein kinases

    PubMed Central

    Metzger, Thibaud; Kleiss, Charlotte; Sumara, Izabela

    2013-01-01

    Posttranslational mechanisms drive fidelity of cellular processes. Phosphorylation and ubiquitination of substrates represent very common, covalent, posttranslational modifications and are often co-regulated. Phosphorylation may play a critical role both by directly regulating E3-ubiquitin ligases and/or by ensuring specificity of the ubiquitination substrate. Importantly, many kinases are not only critical regulatory components of these pathways but also represent themselves the direct ubiquitination substrates. Recent data suggest the role of CUL3-based ligases in both proteolytic and non-proteolytic regulation of protein kinases. Our own recent study identified the mitotic kinase PLK1 as a direct target of the CUL3 E3-ligase complex containing BTB-KELCH adaptor protein KLHL22.1 In this study, we aim at gaining mechanistic insights into CUL3-mediated regulation of the substrates, in particular protein kinases, by analyzing mechanisms of interaction between KLHL22 and PLK1. We find that kinase activity of PLK1 is redundant for its targeting for CUL3-ubiquitination. Moreover, CUL3/KLHL22 may contact 2 distinct motifs within PLK1 protein, consistent with the bivalent mode of substrate targeting found in other CUL3-based complexes. We discuss these findings in the context of the existing knowledge on other protein kinases and substrates targeted by CUL3-based E3-ligases. PMID:24067371

  13. Benzimidazole derivatives as kinase inhibitors.

    PubMed

    Garuti, Laura; Roberti, Marinella; Bottegoni, Giovanni

    2014-01-01

    Benzimidazole is a common kinase inhibitor scaffold and benzimidazole-based compounds interact with enzymes by multiple binding modes. In some cases, the benzimidazole acts as part of the hinge-binding motif, in others it has a scaffolding role without evidence for direct hinge binding. Several of these compounds are ATP-competitive inhibitors and show high selectivity by exploiting unique structural properties that distinguish one kinase from the majority of other kinases. However, the high specificity for a single target is not always sufficient. Thus another approach, called multi-target therapy, has been developed over the last few years. The simultaneous inhibition of various kinases may be useful because the disease is attacked at several relevant targets. Moreover, if a kinase becomes drug-resistant, a multitargeted drug can act on the other kinases. Some benzimidazole derivatives are multi-target inhibitors. In this article benzimidazole inhibitors are reported with their mechanisms of action, structure-activity relationship (SAR) and biological properties.

  14. Functional Tyrosine Kinase Inhibitor Profiling

    PubMed Central

    Arbiser, Jack L.; Govindarajan, Baskaran; Bai, Xianhe; Onda, Hiroaki; Kazlauskas, Andrius; Lim, So Dug; Amin, Mahul B.; Claesson-Welsh, Lena

    2002-01-01

    Tumors often exhibit activation of specific tyrosine kinases, which may allow targeting of therapy through inhibition of tyrosine kinase signaling. This strategy has been used successfully in the development of STI571 (gleevec), an inhibitor of bcr-abl tyrosine kinase that has been used successfully in the treatment of chronic myelogenous leukemia. STI571 also shows activity against c-kit and platelet-derived growth factor receptor-β (PDGFRβ) tyrosine kinase signaling, thus potentially expanding the number of tumors that may respond to it. We describe a simple and rapid method to assess functional activity of tyrosine kinase signaling that is broadly applicable to tumor types. As proof of principle, we have applied it to cells that serve as models of the autosomal-dominant tumor syndrome tuberous sclerosis (TS). We found that TS model cells derived from tuberin heterozygous mice and from a human renal angiomyolipoma are highly sensitive to PDGFR antagonists and that these cells express PDGFRβ. Given that PDGFRβ signaling is inhibited by STI571, we found that SV7tert human angiomyolipoma cells are sensitive to STI571. Thus, we describe a novel but simple method of determining the functional tyrosine kinase profile of a neoplastic cell and our results suggest that STI571 might be useful in the treatment of neoplasms commonly seen in patients with TS. PMID:12213705

  15. The protein kinase C family.

    PubMed

    Azzi, A; Boscoboinik, D; Hensey, C

    1992-09-15

    Protein kinase C represents a structurally homologous group of proteins similar in size, structure and mechanism of activation. They can modulate the biological function of proteins in a rapid and reversible manner. Protein kinase C participates in one of the major signal transduction systems triggered by the external stimulation of cells by various ligands including hormones, neurotransmitters and growth factors. Hydrolysis of membrane inositol phospholipids by phospholipase C or of phosphatidylcholine, generates sn-1,2-diacylglycerol, considered the physiological activator of this kinase. Other agents, such as arachidonic acid, participate in the activation of some of these proteins. Activation of protein kinase C by phorbol esters and related compounds is not physiological and may be responsible, at least in part, for their tumor-promoting activity. The cellular localization of the different calcium-activated protein kinases, their substrate and activator specificity are dissimilar and thus their role in signal transduction is unlike. A better understanding of the exact cellular function of the different protein kinase C isoenzymes requires the identification and characterization of their physiological substrates.

  16. TNF and MAP kinase signaling pathways

    PubMed Central

    Sabio, Guadalupe; Davis, Roger J.

    2014-01-01

    The binding of tumor necrosis factor α (TNFα) to cell surface receptors engages multiple signal transduction pathways, including three groups of mitogen-activated protein (MAP) kinases: extracellular-signal-regulated kinases (ERKs); the cJun NH2-terminal kinases (JNKs); and the p38 MAP kinases. These MAP kinase signalling pathways induce a secondary response by increasing the expression of several inflammatory cytokines (including TNFα) that contribute to the biological activity of TNFα. MAP kinases therefore function both upstream and down-stream of signalling by TNFα receptors. Here we review mechanisms that mediate these actions of MAP kinases during the response to TNFα. PMID:24647229

  17. Proteolytic susceptibility of creatine kinase isozymes and arginine kinase.

    PubMed

    Ercan, Altan; Grossman, Steven H

    2003-07-11

    The time course and dose-response to proteolysis of three dimeric isozymes of creatine kinase, CK-MM (muscle), CK-BB (brain), and CK-MB (heart) and the homologous monomer, arginine kinase were compared. Chymotrypsin and trypsin cause a rapid and significant loss of intact CK-BB, but limited hydrolysis of CK-MM. After 1h of hydrolysis by chymotrypsin, 80% of CK-MM is intact as judged by quantification of monomers after electrophoresis in sodium dodecyl sulfate. While 50% of the intact monomers of CK-MB remain under these conditions, no CK-BB monomers are detected. These results indicate that treatment with chymotrypsin leads to a CK-MB devoid of the B-subunit. When treated with trypsin for 1h, CK-MM is totally resistant to hydrolysis and all CK-BB is highly degraded. However, CK-MB exhibits approximately 90% intact monomers, indicating survival of intact B-subunit in CK-MB. This suggests that heterodimerization of a B-subunit with an M-subunit may have a protective effect against hydrolysis by trypsin. In view of the considerably larger number of potentially tryptic sensitive sites on the muscle isozyme, the resistance of CK-MM and susceptibility of CK-BB dimers to trypsin implies that differences in subunit tertiary structure are a factor in proteolysis of the homodimeric isozymes. Arginine kinase is rapidly degraded by trypsin, but is minimally affected by chymotrypsin. The finding that both a monomeric (arginine kinase) and dimeric (CK-BB) phosphagen kinase are highly susceptible to proteolysis by trypsin indicates that quaternary structure is not, in and of itself, an advantage in resistance to proteolysis. Since both arginine kinase and muscle creatine kinase are resistant to chymotryptic hydrolysis, it seems unlikely that in general, the increased packing density, which may result from dimerization can account for the stability of CK-MM towards trypsin.

  18. Overcoming Resistance to Inhibitors of the Akt Protein Kinase by Modulation of the Pim Kinase Pathway

    DTIC Science & Technology

    2014-10-01

    kinase . This grant proposal will explore the resistance to small molecule AKT protein kinase inhibitors mediated by the... molecule AKT protein kinase inhibitors is potentially mediated by the Pim-1 protein kinase , and that unique Pim protein kinase inhibitors that can in...application is essential for the development of this combined chemotherapeutic strategy. 15. SUBJECT TERMS Small Molecule AKT Inhibitors ,

  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. Discovering the first tyrosine kinase.

    PubMed

    Hunter, Tony

    2015-06-30

    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.

  1. WNK kinases and essential hypertension.

    PubMed

    Huang, Chou-Long; Kuo, Elizabeth; Toto, Robert D

    2008-03-01

    The present review summarizes recent literature and discusses the potential roles of WNKs in the pathogenesis of essential hypertension. WNKs (with-no-lysine [K]) are a recently discovered family of serine-threonine protein kinases with unusual protein kinase domains. The role of WNK kinases in the control of blood pressure was first revealed by the findings that mutations of two members, WNK1 and WNK4, cause Gordon's syndrome. Laboratory studies have revealed that WNK kinases play important roles in the regulation of sodium and potassium transport. Animal models have been created to unravel the pathophysiology of sodium transport disorders caused by mutations of the WNK4 gene. Potassium deficiency causes sodium retention and increases hypertension prevalence. The expression of WNK1 is upregulated by potassium deficiency, raising the possibility that WNK1 may contribute to salt-sensitive essential hypertension associated with potassium deficiency. Associations of polymorphisms of WNK genes with essential hypertension in the general population have been reported. Mutations of WNK1 and WNK4 cause hypertension at least partly by increasing renal sodium retention. The role of WNK kinases in salt-sensitive hypertension within general hypertension is suggested, but future work is required to firmly establish the connection.

  2. Endothelial Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 Is Critical for Lymphatic Vascular Development and Function

    PubMed Central

    Guo, Chang-An; Danai, Laura V.; Yawe, Joseph C.; Gujja, Sharvari; Edwards, Yvonne J. K.

    2016-01-01

    The molecular mechanisms underlying lymphatic vascular development and function are not well understood. Recent studies have suggested a role for endothelial cell (EC) mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) in developmental angiogenesis and atherosclerosis. Here, we show that constitutive loss of EC Map4k4 in mice causes postnatal lethality due to chylothorax, suggesting that Map4k4 is required for normal lymphatic vascular function. Mice constitutively lacking EC Map4k4 displayed dilated lymphatic capillaries, insufficient lymphatic valves, and impaired lymphatic flow; furthermore, primary ECs derived from these animals displayed enhanced proliferation compared with controls. Yeast 2-hybrid analyses identified the Ras GTPase-activating protein Rasa1, a known regulator of lymphatic development and lymphatic endothelial cell fate, as a direct interacting partner for Map4k4. Map4k4 silencing in ECs enhanced basal Ras and extracellular signal-regulated kinase (Erk) activities, and primary ECs lacking Map4k4 displayed enhanced lymphatic EC marker expression. Taken together, these results reveal that EC Map4k4 is critical for lymphatic vascular development by regulating EC quiescence and lymphatic EC fate. PMID:27044870

  3. Tyrosine Kinase Inhibitors in Lung Cancer

    PubMed Central

    Thomas, Anish; Rajan, Arun; Giaccone, Giuseppe

    2012-01-01

    SYNOPSIS ‘Driver mutations’ are essential for carcinogenesis as well as tumor progression as they confer a selective growth advantage to cancer cells. Identification of driver mutations in growth related protein kinases, especially tyrosine kinases have led to clinical development of an array of tyrosine kinase inhibitors in various malignancies, including lung cancer. Inhibition of epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinases have proven to be of meaningful clinical benefit, while inhibition of several other tyrosine kinases have been of limited clinical benefit, thus far. An improved understanding of tyrosine kinase biology has also led to faster drug development, identification of resistance mechanisms and ways to overcome resistance. In this review, we discuss the clinical data supporting the use and practical aspects of management of patients on epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinase inhibitors. PMID:22520981

  4. Differential regulation of rice mitogen activated protein kinase kinase (MKK) by abiotic stress.

    PubMed

    Kumar, Kundan; Rao, Kudupudi Prabhakara; Sharma, Pallavi; Sinha, Alok Krishna

    2008-10-01

    Mitogen activated protein kinase cascade plays a crucial role in various biotic and abiotic stresses, hormones, cell division and developmental processes. MAP kinase kinase being integral part of this cascade performs an important function of integrating upstream signals to mitogen activated protein kinase for further appropriate cellular responses. We here report cloning of five MAP kinase kinase members from Oryza sativa indica cultivar var. Pusa Basmati 1, namely MAP kinase kinases 1, 3, 4, 6 and 10-2. All these members, except MKK10-2 possess fully canonical motif structures of MAP kinase kinase. The deduced amino acid sequence showed changes at certain position within japonica and indica variety of rice. Analysis of transcript regulation by quantitative real time PCR revealed that these five members are differentially regulated by cold, heat, salinity and drought stresses. MAP kinase kinases 4 and 6 are strongly regulated by cold and salt stresses while MAP kinase kinase 1 is regulated by salt and drought stresses. MAP kinase kinase 10-2 is regulated only by cold stress. The study provides the indication of involvement of specific MAP kinase kinase in different abiotic stress signaling and also possible cross talks that exist during the signaling processes.

  5. [Kinase inhibitors against hematological malignancies].

    PubMed

    Tojo, Arinobu

    2014-06-01

    Dysregulation of protein phosphorylation, especially on tyrosine residues, plays a crucial role in development and progression of hematological malignancies. Since remarkable success in imatinib therapy of CML and Ph+ALL, extensive efforts have made to explore candidate molecular targets and next breakthrough drugs. Now that next generation ABL kinase inhibitors are available for CML, the therapeutic algorithm has been revolutionized. As for AML and lymphoid malignancies, many kinase inhibitors targeting FLT3, BTK and aurora-A are on early and late clinical trials, and a number of promising drugs including ibrutinib are picked up for further evaluation.

  6. Cyclic-GMP-dependent protein kinase inhibits the Ras/Mitogen-activated protein kinase pathway.

    PubMed

    Suhasini, M; Li, H; Lohmann, S M; Boss, G R; Pilz, R B

    1998-12-01

    Agents which increase the intracellular cyclic GMP (cGMP) concentration and cGMP analogs inhibit cell growth in several different cell types, but it is not known which of the intracellular target proteins of cGMP is (are) responsible for the growth-suppressive effects of cGMP. Using baby hamster kidney (BHK) cells, which are deficient in cGMP-dependent protein kinase (G-kinase), we show that 8-(4-chlorophenylthio)guanosine-3', 5'-cyclic monophosphate and 8-bromoguanosine-3',5'-cyclic monophosphate inhibit cell growth in cells stably transfected with a G-kinase Ibeta expression vector but not in untransfected cells or in cells transfected with a catalytically inactive G-kinase. We found that the cGMP analogs inhibited epidermal growth factor (EGF)-induced activation of mitogen-activated protein (MAP) kinase and nuclear translocation of MAP kinase in G-kinase-expressing cells but not in G-kinase-deficient cells. Ras activation by EGF was not impaired in G-kinase-expressing cells treated with cGMP analogs. We show that activation of G-kinase inhibited c-Raf kinase activation and that G-kinase phosphorylated c-Raf kinase on Ser43, both in vitro and in vivo; phosphorylation of c-Raf kinase on Ser43 uncouples the Ras-Raf kinase interaction. A mutant c-Raf kinase with an Ala substitution for Ser43 was insensitive to inhibition by cGMP and G-kinase, and expression of this mutant kinase protected cells from inhibition of EGF-induced MAP kinase activity by cGMP and G-kinase, suggesting that Ser43 in c-Raf is the major target for regulation by G-kinase. Similarly, B-Raf kinase was not inhibited by G-kinase; the Ser43 phosphorylation site of c-Raf is not conserved in B-Raf. Activation of G-kinase induced MAP kinase phosphatase 1 expression, but this occurred later than the inhibition of MAP kinase activation. Thus, in BHK cells, inhibition of cell growth by cGMP analogs is strictly dependent on G-kinase and G-kinase activation inhibits the Ras/MAP kinase pathway (i) by

  7. Evolutionary Ancestry of Eukaryotic Protein Kinases and Choline Kinases*

    PubMed Central

    Lai, Shenshen; Safaei, Javad

    2016-01-01

    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. PMID:26742849

  8. Characterization of PDZ-binding kinase, a mitotic kinase

    PubMed Central

    Gaudet, Suzanne; Branton, Daniel; Lue, Robert A.

    2000-01-01

    hDlg, the human homologue of the Drosophila Discs-large (Dlg) tumor suppressor protein, is known to interact with the tumor suppressor protein APC and the human papillomavirus E6 transforming protein. In a two-hybrid screen, we identified a 322-aa serine/threonine kinase that binds to the PDZ2 domain of hDlg. The mRNA for this PDZ-binding kinase, or PBK, is most abundant in placenta and absent from adult brain tissue. The protein sequence of PBK has all the characteristic protein kinase subdomains and a C-terminal PDZ-binding T/SXV motif. In vitro, PBK binds specifically to PDZ2 of hDlg through its C-terminal T/SXV motif. PBK and hDlg are phosphorylated at mitosis in HeLa cells, and the mitotic phosphorylation of PBK is required for its kinase activity. In vitro, cdc2/cyclin B phosphorylates PBK. This evidence shows how PBK could link hDlg or other PDZ-containing proteins to signal transduction pathways regulating the cell cycle or cellular proliferation. PMID:10779557

  9. Structure of the pseudokinase-kinase domains from protein kinase TYK2 reveals a mechanism for Janus kinase (JAK) autoinhibition.

    PubMed

    Lupardus, Patrick J; Ultsch, Mark; Wallweber, Heidi; Bir Kohli, Pawan; Johnson, Adam R; Eigenbrot, Charles

    2014-06-03

    Janus kinases (JAKs) are receptor-associated multidomain tyrosine kinases that act downstream of many cytokines and interferons. JAK kinase activity is regulated by the adjacent pseudokinase domain via an unknown mechanism. Here, we report the 2.8-Å structure of the two-domain pseudokinase-kinase module from the JAK family member TYK2 in its autoinhibited form. We find that the pseudokinase and kinase interact near the kinase active site and that most reported mutations in cancer-associated JAK alleles cluster in or near this interface. Mutation of residues near the TYK2 interface that are analogous to those in cancer-associated JAK alleles, including the V617F and "exon 12" JAK2 mutations, results in increased kinase activity in vitro. These data indicate that JAK pseudokinases are autoinhibitory domains that hold the kinase domain inactive until receptor dimerization stimulates transition to an active state.

  10. Kinase signalling in Huntington's disease.

    PubMed

    Bowles, Kathryn R; Jones, Lesley

    2014-01-01

    Alterations in numerous signal transduction pathways and aberrant activity of specific kinases have been identified in multiple cell and mouse models of Huntington's disease (HD), as well as in human HD brain. The balance and integration of a network of kinase signalling pathways is paramount for the regulation of a wide range of cellular and physiological processes, such as proliferation, differentiation, inflammation, neuronal plasticity and apoptosis. Unbalanced activity within these pathways provides a potential mechanism for many of the pathological phenotypes associated with HD, such as transcriptional dysregulation, inflammation and ultimately neurodegeneration. The characterisation of aberrant kinase signalling regulation in HD has been inconsistent and may be a result of failure to consider integration between multiple signalling pathways, as well as alterations that may occur over time with both age and disease progression. Collating the information about the effect of mHTT on signalling pathways demonstrates that it has wide ranging effects on multiple pro- and anti-apoptotic kinases, resulting in the dysregulation of numerous complex interactions within a dynamic network.

  11. Case report: pyruvate kinase deficiency.

    PubMed

    Rothman, J M

    1995-09-01

    Pyruvate kinase deficiency is a rare cause of congenital hemolytic anemia. Despite a paucity of reports, splenectomy resulted in successful outcomes for two siblings with this disorder. The sisters were diagnosed at birth with profound jaundice and congenital nonspherocytic hemolytic anemia.

  12. Genetics Home Reference: mevalonate kinase deficiency

    MedlinePlus

    ... shape, leading to a reduction of mevalonate kinase enzyme activity. Despite this shortage (deficiency) of mevalonate kinase activity, ... who have less than 1 percent of normal enzyme activity usually develop MVA. Learn more about the gene ...

  13. Degradation of Activated Protein Kinases by Ubiquitination

    PubMed Central

    Lu, Zhimin; Hunter, Tony

    2009-01-01

    Protein kinases are important regulators of intracellular signal transduction pathways and play critical roles in diverse cellular functions. Once a protein kinase is activated, its activity is subsequently downregulated through a variety of mechanisms. Accumulating evidence indicates that the activation of protein kinases commonly initiates their downregulation via the ubiquitin/proteasome pathway. Failure to regulate protein kinase activity or expression levels can cause human diseases. PMID:19489726

  14. Comprehensive assay of kinase catalytic activity reveals features of kinase inhibitor selectivity

    PubMed Central

    Anastassiadis, Theonie; Deacon, Sean W.; Devarajan, Karthik; Ma, Haiching; Peterson, Jeffrey R.

    2011-01-01

    Small-molecule protein kinase inhibitors are central tools for elucidating cellular signaling pathways and are promising therapeutic agents. Due to evolutionary conservation of the ATP-binding site, most kinase inhibitors that target this site promiscuously inhibit multiple kinases. Interpretation of experiments utilizing these compounds is confounded by a lack of data on the comprehensive kinase selectivity of most inhibitors. Here we profiled the activity of 178 commercially available kinase inhibitors against a panel of 300 recombinant protein kinases using a functional assay. Quantitative analysis revealed complex and often unexpected kinase-inhibitor interactions, with a wide spectrum of promiscuity. Many off-target interactions occur with seemingly unrelated kinases, revealing how large-scale profiling can be used to identify multi-targeted inhibitors of specific, diverse kinases. The results have significant implications for drug development and provide a resource for selecting compounds to elucidate kinase function and for interpreting the results of experiments that use them. PMID:22037377

  15. The receptor kinase family: primary structure of rhodopsin kinase reveals similarities to the beta-adrenergic receptor kinase.

    PubMed Central

    Lorenz, W; Inglese, J; Palczewski, K; Onorato, J J; Caron, M G; Lefkowitz, R J

    1991-01-01

    Light-dependent deactivation of rhodopsin as well as homologous desensitization of beta-adrenergic receptors involves receptor phosphorylation that is mediated by the highly specific protein kinases rhodopsin kinase (RK) and beta-adrenergic receptor kinase (beta ARK), respectively. We report here the cloning of a complementary DNA for RK. The deduced amino acid sequence shows a high degree of homology to beta ARK. In a phylogenetic tree constructed by comparing the catalytic domains of several protein kinases, RK and beta ARK are located on a branch close to, but separate from the cyclic nucleotide-dependent protein kinase and protein kinase C subfamilies. From the common structural features we conclude that both RK and beta ARK are members of a newly delineated gene family of guanine nucleotide-binding protein (G protein)-coupled receptor kinases that may function in diverse pathways to regulate the function of such receptors. Images PMID:1656454

  16. 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

  17. Aurora kinases: novel therapy targets in cancers.

    PubMed

    Tang, Anqun; Gao, Keyu; Chu, Laili; Zhang, Rui; Yang, Jing; Zheng, Junnian

    2017-01-29

    Aurora kinases, a family of serine/threonine kinases, consisting of Aurora A (AURKA), Aurora B (AURKB) and Aurora C (AURKC), are essential kinases for cell division via regulating mitosis especially the process of chromosomal segregation. Besides regulating mitosis, Aurora kinases have been implicated in regulating meiosis. The deletion of Aurora kinases could lead to failure of cell division and impair the embryonic development. Overexpression or gene amplification of Aurora kinases has been clarified in a number of cancers. And a growing number of studies have demonstrated that inhibition of Aurora kinases could potentiate the effect of chemotherapies. For the past decades, a series of Aurora kinases inhibitors (AKIs) developed effectively repress the progression and growth of many cancers both in vivo and in vitro, suggesting that Aurora kinases could be a novel therapeutic target. In this review, we'll first briefly present the structure, localization and physiological functions of Aurora kinases in mitosis, then describe the oncogenic role of Aurora kinases in tumorigenesis, we shall finally discuss the outcomes of AKIs combination with conventional therapy.

  18. Receptor tyrosine kinases in carcinogenesis.

    PubMed

    Zhang, Xiao-Ying; Zhang, Pei-Ying

    2016-11-01

    Receptor tyrosine kinases (RTKs) are cell surface glycoproteins with enzymatic activity involved in the regulation of various important functions. In all-important physiological functions including differentiation, cell-cell interactions, survival, proliferation, metabolism, migration and signaling these receptors are the key players of regulation. Additionally, mutations of RTKs or their overexpression have been described in many human cancers and are being explored as a novel avenue for a new therapeutic approach. Some of the deregulated RTKs observed to be significantly affected in cancers included vascular endothelial growth factor receptor, epidermal growth factor receptor, fibroblast growth factor receptor, RTK-like orphan receptor 1 (ROR1) and the platelet-derived growth factor receptor. These deregulated RTKs offer attractive possibilities for the new anticancer therapeutic approach involving specific targeting by monoclonal antibodies as well as kinase. The present review aimed to highlight recent perspectives of RTK ROR1 in cancer.

  19. 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.

  20. 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

  1. Nucleotide selectivity of antibiotic kinases.

    PubMed

    Shakya, Tushar; Wright, Gerard D

    2010-05-01

    Antibiotic kinases, which include aminoglycoside and macrolide phosphotransferases (APHs and MPHs), pose a serious threat to currently used antimicrobial therapies. These enzymes show structural and functional homology with Ser/Thr/Tyr kinases, which is suggestive of a common ancestor. Surprisingly, recent in vitro studies using purified antibiotic kinase enzymes have revealed that a number are able to utilize GTP as the antibiotic phospho donor, either preferentially or exclusively compared to ATP, the canonical phosphate donor in most biochemical reactions. To further explore this phenomenon, we examined three enzymes, APH(3')-IIIa, APH(2'')-Ib, and MPH(2')-I, using a competitive assay that mimics in vivo nucleotide triphosphate (NTP) concentrations and usage by each enzyme. Downstream analysis of reaction products by high-performance liquid chromatography enabled the determination of partitioning of phosphate flux from NTP donors to antibiotics. Using this ratio along with support from kinetic analysis and inhibitor studies, we find that under physiologic concentrations of NTPs, APH(3')-IIIa exclusively uses ATP, MPH(2')-I exclusively uses GTP, and APH(2'')-Ib is able to use both species with a preference for GTP. These differences reveal likely different pathways in antibiotic resistance enzyme evolution and can be exploited in selective inhibitor design to counteract resistance.

  2. The Role of Mitogen-Activated Protein Kinase-Activated Protein Kinases (MAPKAPKs) in Inflammation

    PubMed Central

    Moens, Ugo; Kostenko, Sergiy; Sveinbjørnsson, Baldur

    2013-01-01

    Mitogen-activated protein kinase (MAPK) pathways are implicated in several cellular processes including proliferation, differentiation, apoptosis, cell survival, cell motility, metabolism, stress response and inflammation. MAPK pathways transmit and convert a plethora of extracellular signals by three consecutive phosphorylation events involving a MAPK kinase kinase, a MAPK kinase, and a MAPK. In turn MAPKs phosphorylate substrates, including other protein kinases referred to as MAPK-activated protein kinases (MAPKAPKs). Eleven mammalian MAPKAPKs have been identified: ribosomal-S6-kinases (RSK1-4), mitogen- and stress-activated kinases (MSK1-2), MAPK-interacting kinases (MNK1-2), MAPKAPK-2 (MK2), MAPKAPK-3 (MK3), and MAPKAPK-5 (MK5). The role of these MAPKAPKs in inflammation will be reviewed. PMID:24705157

  3. Aurora kinase inhibitors as anticancer molecules.

    PubMed

    Katayama, Hiroshi; Sen, Subrata

    2010-01-01

    Aurora kinase family of serine/threonine kinases are important regulators of mitosis that are frequently over expressed in human cancers and have been implicated in oncogenic transformation including development of chromosomal instability in cancer cells. In humans, among the three members of the kinase family, Aurora-A, -B and -C, only Aurora-A and -B are expressed at detectable levels in all somatic cells undergoing mitotic cell division and have been characterized in greater detail for their involvement in cellular pathways relevant to the development of cancer associated phenotypes. Aurora-A and -B are being investigated as potential targets for anticancer therapy. Development of inhibitors against Aurora kinases as anticancer molecules gained attention because of the facts that aberrant expression of these kinases leads to chromosomal instability and derangement of multiple tumor suppressor and oncoprotein regulated pathways. Preclinical studies and early phase I and II clinical trials of multiple Aurora kinase inhibitors as targeted anticancer drugs have provided encouraging results. This article discusses functional involvement of Aurora kinase-A and -B in the regulation of cancer relevant cellular phenotypes together with findings on some of the better characterized Aurora kinase inhibitors in modulating the functional interactions of Aurora kinases. Future possibilities about developing next generation Aurora kinase inhibitors and their clinical utility as anticancer therapeutic drugs are also discussed.

  4. Protein kinase C-associated kinase can activate NFkappaB in both a kinase-dependent and a kinase-independent manner.

    PubMed

    Moran, Stewart T; Haider, Khaleda; Ow, Yongkai; Milton, Peter; Chen, Luojing; Pillai, Shiv

    2003-06-13

    Protein kinase C-associated kinase (PKK, also known as RIP4/DIK) activates NFkappaB when overexpressed in cell lines and is required for keratinocyte differentiation in vivo. However, very little is understood about the factors upstream of PKK or how PKK activates NFkappaB. Here we show that certain catalytically inactive mutants of PKK can activate NFkappaB, although to a lesser degree than wild type PKK. The deletion of specific domains of wild type PKK diminishes the ability of this enzyme to activate NFkappaB; the same deletions made on a catalytically inactive PKK background completely ablate NFkappaB activation. PKK may be phosphorylated by two specific mitogen-activated protein kinase kinase kinases, MEKK2 and MEKK3, and this interaction may in part be mediated through a critical activation loop residue, Thr184. Catalytically inactive PKK mutants that block phorbol ester-induced NFkappaB activation do not interfere with, but unexpectedly enhance, the activation of NFkappaB by these two mitogen-activated protein kinase kinase kinases. Taken together, these data indicate that PKK may function in both a kinase-dependent as well as a kinase-independent manner to activate NFkappaB.

  5. Kinases Involved in Both Autophagy and Mitosis.

    PubMed

    Li, Zhiyuan; Zhang, Xin

    2017-08-31

    Both mitosis and autophagy are highly regulated dynamic cellular processes and involve various phosphorylation events catalysed by kinases, which play vital roles in almost all physiological and pathological conditions. Mitosis is a key event during the cell cycle, in which the cell divides into two daughter cells. Autophagy is a process in which the cell digests its own cellular contents. Although autophagy regulation has mainly been studied in asynchronous cells, increasing evidence indicates that autophagy is in fact tightly regulated in mitosis. Here in this review, we will discuss kinases that were originally identified to be involved in only one of either mitosis or autophagy, but were later found to participate in both processes, such as CDKs (cyclin-dependent kinases), Aurora kinases, PLK-1 (polo-like kinase 1), BUB1 (budding uninhibited by benzimidazoles 1), MAPKs (mitogen-activated protein kinases), mTORC1 (mechanistic target of rapamycin complex 1), AMPK (AMP-activated protein kinase), PI3K (phosphoinositide-3 kinase) and protein kinase B (AKT). By focusing on kinases involved in both autophagy and mitosis, we will get a more comprehensive understanding about the reciprocal regulation between the two key cellular events, which will also shed light on their related therapeutic investigations.

  6. Aurora Kinase inhibitors as Anticancer Molecules

    PubMed Central

    Katayama, Hiroshi; Sen, Subrata

    2015-01-01

    Aurora kinase family of serine/threonine kinases are important regulators of mitosis that are frequently over expressed in human cancers and have been implicated in oncogenic transformation including development of chromosomal instability in cancer cells. In humans, among the three members of the kinase family, Aurora-A, -B and -C, only Aurora-A and -B are expressed in detectable levels in somatic cells undergoing mitotic cell division and have been characterized in greater detail for their involvement in cellular pathways relevant to the development of cancer associated phenotypes. Aurora-A and -B are being investigated as potential targets for anticancer therapy. Development of inhibitors against Aurora kinases as anticancer molecules gained attention because of the facts that aberrant expression of these kinases lead to chromosomal instability and derangement of multiple tumor suppressor and oncoprotein regulated pathways. Pre-clinical studies and early phase I and II clinical trials of multiple Aurora kinase inhibitors as targeted anticancer drugs have provided encouraging results. This article discusses functional involvement of Aurora kinase-A and -B in the regulation of cancer relevant cellular phenotypes together with findings on some of the better characterized Aurora kinase inhibitors in modulating the functional interactions of Aurora kinases. Future possibilities about developing next generation Aurora kinase inhibitors and their clinical utility as anticancer therapeutic drugs are also discussed. PMID:20863917

  7. Immunochemical characterization of rat brain protein kinase

    SciTech Connect

    Huang, K.P.; Huang, F.L.

    1986-11-05

    Polyclonal antibodies against rat brain protein kinase C (the Ca/sup 2 +//phospholipid-dependent enzyme) were raised in goat. These antibodies can neutralize completely the kinase activity in purified enzyme preparation as well as that in the crude homogenate. Immunoblot analysis of the purified and the crude protein kinase C preparations revealed a major immunoreactive band of 80 kDa. The antibodies also recognize the same enzyme from other rat tissues. Neuronal tissues (cerebral cortex, cerebellum, hypothalamus, and retina) and lymphoid organs (thymus and spleen) were found to be enriched in protein kinase C, whereas lung, kidney, liver, heart, and skeletal muscle contained relatively low amounts of this kinase. Limited proteolysis of the purified rat brain protein kinase C with trypsin results in an initial degradation of the kinase into two major fragments of 48 and 38 kDa. Both fragments are recognized by the antibodies. However, further digestion of the 48-kDa fragment to 45 kDa and the 38-kDa fragment to 33 kDa causes a loss of the immunoreactivity. Upon incubation of the cerebellar extract with Ca/sup 2 +/, the 48-kDa fragment was also identified as a major proteolytic product of protein kinase C. Proteolytic degradation of protein kinase C converts the Ca/sup 2 +//phospholipid-dependent kinase to an independent form without causing a large impairment of the binding of (/sup 3/H)phorbol 12,13-dibutyrate. The two major proteolytic fragments were separated by ion exchange chromatography and one of them (45-48 kDa) was identified as a protein kinase and the other (33-38 kDa) as a phorbol ester-binding protein. These results demonstrate that rat brain protein kinase C is composed of two functionally distinct units, namely, a protein kinase and a Ca/sup 2 +/-independent/phospholipid-dependent phorbol ester-binding protein.

  8. Association of protein kinase Cmu with type II phosphatidylinositol 4-kinase and type I phosphatidylinositol-4-phosphate 5-kinase.

    PubMed

    Nishikawa, K; Toker, A; Wong, K; Marignani, P A; Johannes, F J; Cantley, L C

    1998-09-04

    Protein kinase Cmu (PKCmu), also named protein kinase D, is an unusual member of the PKC family that has a putative transmembrane domain and pleckstrin homology domain. This enzyme has a substrate specificity distinct from other PKC isoforms (Nishikawa, K., Toker, A., Johannes, F. J., Songyang, Z., and Cantley, L. C. (1997) J. Biol. Chem. 272, 952-960), and its mechanism of regulation is not yet clear. Here we show that PKCmu forms a complex in vivo with a phosphatidylinositol 4-kinase and a phosphatidylinositol-4-phosphate 5-kinase. A region of PKCmu between the amino-terminal transmembrane domain and the pleckstrin homology domain is shown to be involved in the association with the lipid kinases. Interestingly, a kinase-dead point mutant of PKCmu failed to associate with either lipid kinase activity, indicating that autophosphorylation may be required to expose the lipid kinase interaction domain. Furthermore, the subcellular distribution of the PKCmu-associated lipid kinases to the particulate fraction depends on the presence of the amino-terminal region of PKCmu including the predicted transmembrane region. These results suggest a novel model in which the non-catalytic region of PKCmu acts as a scaffold for assembly of enzymes involved in phosphoinositide synthesis at specific membrane locations.

  9. Protein kinase C-associated kinase (PKK), a novel membrane-associated, ankyrin repeat-containing protein kinase.

    PubMed

    Chen, L; Haider, K; Ponda, M; Cariappa, A; Rowitch, D; Pillai, S

    2001-06-15

    A novel murine membrane-associated protein kinase, PKK (protein kinase C-associated kinase), was cloned on the basis of its physical association with protein kinase Cbeta (PKCbeta). The regulated expression of PKK in mouse embryos is consistent with a role for this kinase in early embryogenesis. The human homolog of PKK has over 90% identity to its murine counterpart, has been localized to chromosome 21q22.3, and is identical to the PKCdelta-interacting kinase, DIK (Bahr, C., Rohwer, A., Stempka, L., Rincke, G., Marks, F., and Gschwendt, M. (2000) J. Biol. Chem. 275, 36350-36357). PKK comprises an N-terminal kinase domain and a C-terminal region containing 11 ankyrin repeats. PKK exhibits protein kinase activity in vitro and associates with cellular membranes. PKK exists in three discernible forms at steady state: an underphosphorylated form of 100 kDa; a soluble, cytosolic, phosphorylated form of 110 kDa; and a phosphorylated, detergent-insoluble form of 112 kDa. PKK is initially synthesized as an underphosphorylated soluble 100-kDa protein that is quantitatively converted to a detergent-soluble 110-kDa form. This conversion requires an active catalytic domain. Although PKK physically associates with PKCbeta, it does not phosphorylate this PKC isoform. However, PKK itself may be phosphorylated by PKCbeta. PKK represents a developmentally regulated protein kinase that can associate with membranes. The functional significance of its association with PKCbeta remains to be ascertained.

  10. MAP kinase activator from insulin-stimulated skeletal muscle is a protein threonine/tyrosine kinase.

    PubMed Central

    Nakielny, S; Cohen, P; Wu, J; Sturgill, T

    1992-01-01

    A 'MAP kinase activator' was purified several thousand-fold from insulin-stimulated rabbit skeletal muscle, which resembled the 'activator' from nerve growth factor-stimulated PC12 cells in that it could be inactivated by incubation with protein phosphatase 2A, but not by protein tyrosine phosphatases and its apparent molecular mass was 45-50 kDa. In the presence of MgATP, 'MAP kinase activator' converted the normal 'wild-type' 42 kDa MAP kinase from an inactive dephosphorylated form to the fully active diphosphorylated species. Phosphorylation occurred on the same threonine and tyrosine residues which are phosphorylated in vivo in response to growth factors or phorbol esters. A mutant MAP kinase produced by changing a lysine at the active centre to arginine was phosphorylated in an identical manner by the 'MAP kinase activator', but no activity was generated. The results demonstrate that 'MAP kinase activator' is a protein kinase (MAP kinase kinase) and not a protein that stimulates the autophosphorylation of MAP kinase. MAP kinase kinase is the first established example of a protein kinase that can phosphorylate an exogenous protein on threonine as well as tyrosine residues. Images PMID:1318193

  11. Using Bioluminescent Kinase Profiling Strips to Identify Kinase Inhibitor Selectivity and Promiscuity.

    PubMed

    Zegzouti, Hicham; Hennek, Jacquelyn; Goueli, Said A

    2016-01-01

    The advancement of a kinase inhibitor throughout drug discovery and development is predicated upon its selectivity towards the target of interest. Thus, profiling the compound against a broad panel of kinases is important for providing a better understanding of its activity and for obviating any off-target activities that can result in undesirable consequences. To assess the selectivity and potency of an inhibitor against multiple kinases, it is desirable to use a universal assay that can monitor the activity of all classes of kinases regardless of the nature of their substrates. The luminescent ADP-Glo kinase assay is a universal platform that measures kinase activity by quantifying the amount of the common kinase reaction product ADP. Here we present a method using standardized kinase profiling systems for inhibitor profiling studies based on ADP detection by luminescence. The kinase profiling systems are sets of kinases organized by family, presented in multi-tube strips containing eight enzymes, each with corresponding substrate strips, and standardized for optimal kinase activity. We show that using the kinase profiling strips we could quickly and easily generate multiple selectivity profiles using small or large kinase panels, and identify compound promiscuity within the kinome.

  12. 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

  13. Pathway illuminated: visualizing protein kinase C signaling.

    PubMed

    Violin, Jonathan D; Newton, Alexandra C

    2003-12-01

    Protein kinase C has been at the center of cell signaling since the discovery 25 years ago that it transduces signals that promote phospholipid hydrolysis. In recent years, the use of genetically encoded fluorescent reporters has enabled studies of the regulation of protein kinase C signaling in living cells. Advances in imaging techniques have unveiled unprecedented detail of the signal processing mechanics of protein kinase C, from the second messengers calcium and diacylglycerol that regulate protein kinase C activity, to the locations and kinetics of different protein kinase C isozymes, to the spatial and temporal dynamics of substrate phosphorylation by this key enzyme. This review discusses how fluorescence imaging studies have illuminated the fidelity with which protein kinase C transduces rapidly changing extracellular information into intracellular phosphorylation signals.

  14. The phosphoinositide 3-kinase pathway.

    PubMed

    Cantley, Lewis C

    2002-05-31

    Phosphorylated lipids are produced at cellular membranes during signaling events and contribute to the recruitment and activation of various signaling components. The role of phosphoinositide 3-kinase (PI3K), which catalyzes the production of phosphatidylinositol-3,4,5-trisphosphate, in cell survival pathways; the regulation of gene expression and cell metabolism; and cytoskeletal rearrangements are highlighted. The PI3K pathway is implicated in human diseases including diabetes and cancer, and understanding the intricacies of this pathway may provide new avenues for therapuetic intervention.

  15. How do kinases transfer phosphoryl groups?

    PubMed

    Matte, A; Tari, L W; Delbaere, L T

    1998-04-15

    Understanding how phosphoryl transfer is accomplished by kinases, a ubiquitous group of enzymes, is central to many biochemical processes. Qualitative analysis of the crystal structures of enzyme-substrate complexes of kinases reveals structural features of these enzymes important to phosphoryl transfer. Recently determined crystal structures which mimic the transition state complex have added new insight into the debate as to whether kinases use associative or dissociative mechanisms of catalysis.

  16. Spatial gradients in kinase cascade regulation.

    PubMed

    Kazmierczak, B; Lipniacki, T

    2010-11-01

    The spatiotemporal kinetics of proteins and other substrates regulate cell fate and signaling. In this study, we consider a reaction-diffusion model of interaction of membrane receptors with a two-step kinase cascade. The receptors activate the 'up-stream' kinase, which may diffuse over cell volume and activate the 'down-stream' kinase, which is also diffusing. Both kinase species and receptors are inactivated by uniformly distributed phosphatases. The positive feedback, key to the considered dynamics, arises since the up-stream kinase activates the receptors. Such a mutual interaction is characteristic for immune cell receptors. Based on the proposed model, we demonstrated that cell sensitivity (measured as a critical value of phosphatase activity at which cell maybe activated) increases with decreasing motility of receptor-interacting kinases and with increasing polarity of receptors distribution. These two effects are cooperating, the effect of receptors localisation close to one pole of the cell grows with the decreasing kinase diffusion and vanishes in the infinite diffusion limit. As the cell sensitivity increases with decreasing diffusion of receptor-interacting kinase, the overall activity of the down-stream kinase increases with its diffusion. In conclusion, the analysis of the proposed model shows that, for the fixed substrate interaction rates, spatial distribution of the surface receptors together with the motility of intracellular kinases control cell signalling and sensitivity to extracellular signals. The increase of the cell sensitivity can be achieved by (i) localisation of receptors in a small subdomain of the cell membrane, (ii) lowering the motility of receptor-interacting kinase, (iii) increasing the motility of down-stream kinases which distribute the signal over the whole cell.

  17. Phosphorylation and activation of p40 tyrosine kinase by casein kinase-1.

    PubMed

    Vila, J; Payne, D M; Zioncheck, T F; Harrison, M L; Itarte, E; Weber, M J

    1990-05-07

    Because examination of regulatory trans-phosphorylations can help elucidate the cellular functions of tyrosyl protein kinases, we have investigated the effects of phosphorylation by casein kinase-1 on the activity of the p40 tyrosyl protein kinase. We find that casein kinase-1 can phosphorylate the p40 tyrosyl kinase on serine and threonine residues, in part on a unique tryptic peptide. The phosphorylation induces a substantial increase in the tyrosyl protein kinase activity of p40, in contrast to most instances in which serine/threonine phosphorylation inhibits activity of tyrosyl protein kinases. These findings raise the possibility that p40 might be part of a protein phosphorylation network in which casein kinase-1 participates.

  18. Tyrosine kinases in inflammatory dermatologic disease

    PubMed Central

    Paniagua, Ricardo T.; Fiorentino, David; Chung, Lorinda; Robinson, William H.

    2010-01-01

    Tyrosine kinases are enzymes that catalyze the phosphorylation of tyrosine residues on protein substrates. They are key components of signaling pathways that drive an array of cellular responses including proliferation, differentiation, migration, and survival. Specific tyrosine kinases have recently been identified as critical to the pathogenesis of several autoimmune and inflammatory diseases. Small-molecule inhibitors of tyrosine kinases are emerging as a novel class of therapy that may provide benefit in certain patient subsets. In this review, we highlight tyrosine kinase signaling implicated in inflammatory dermatologic diseases, evaluate strategies aimed at inhibiting these aberrant signaling pathways, and discuss prospects for future drug development. PMID:20584561

  19. MST kinases in development and disease

    PubMed Central

    2015-01-01

    The mammalian MST kinase family, which is related to the Hippo kinase in Drosophila melanogaster, includes five related proteins: MST1 (also called STK4), MST2 (also called STK3), MST3 (also called STK24), MST4, and YSK1 (also called STK25 or SOK1). MST kinases are emerging as key signaling molecules that influence cell proliferation, organ size, cell migration, and cell polarity. Here we review the regulation and function of these kinases in normal physiology and pathologies, including cancer, endothelial malformations, and autoimmune disease. PMID:26370497

  20. Diacylglycerol kinases in membrane trafficking

    PubMed Central

    Xie, Shuwei; Naslavsky, Naava; Caplan, Steve

    2015-01-01

    Diacylglycerol kinases (DGKs) belong to a family of cytosolic kinases that regulate the phosphorylation of diacylglycerol (DAG), converting it into phosphatidic acid (PA). There are 10 known mammalian DGK isoforms, each with a different tissue distribution and substrate specificity. These differences allow regulation of cellular responses by fine-tuning the delicate balance of cellular DAG and PA. DGK isoforms are best characterized as mediators of signal transduction and immune function. However, since recent studies reveal that DAG and PA are also involved in the regulation of endocytic trafficking, it is therefore anticipated that DGKs also plays an important role in membrane trafficking. In this review, we summarize the literature discussing the role of DGK isoforms at different stages of endocytic trafficking, including endocytosis, exocytosis, endocytic recycling, and transport from/to the Golgi apparatus. Overall, these studies contribute to our understanding of the involvement of PA and DAG in endocytic trafficking, an area of research that is drawing increasing attention in recent years. PMID:27057419

  1. Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases

    PubMed Central

    Shiu, Shin-Han; Bleecker, Anthony B.

    2001-01-01

    Plant receptor-like kinases (RLKs) are proteins with a predicted signal sequence, single transmembrane region, and cytoplasmic kinase domain. Receptor-like kinases belong to a large gene family with at least 610 members that represent nearly 2.5% of Arabidopsis protein coding genes. We have categorized members of this family into subfamilies based on both the identity of the extracellular domains and the phylogenetic relationships between the kinase domains of subfamily members. Surprisingly, this structurally defined group of genes is monophyletic with respect to kinase domains when compared with the other eukaryotic kinase families. In an extended analysis, animal receptor kinases, Raf kinases, plant RLKs, and animal receptor tyrosine kinases form a well supported group sharing a common origin within the superfamily of serine/threonine/tyrosine kinases. Among animal kinase sequences, Drosophila Pelle and related cytoplasmic kinases fall within the plant RLK clade, which we now define as the RLK/Pelle family. A survey of expressed sequence tag records for land plants reveals that mosses, ferns, conifers, and flowering plants have similar percentages of expressed sequence tags representing RLK/Pelle homologs, suggesting that the size of this gene family may have been close to the present-day level before the diversification of land plant lineages. The distribution pattern of four RLK subfamilies on Arabidopsis chromosomes indicates that the expansion of this gene family is partly a consequence of duplication and reshuffling of the Arabidopsis genome and of the generation of tandem repeats. PMID:11526204

  2. Dominant Mutations of Drosophila Map Kinase Kinase and Their Activities in Drosophila and Yeast Map Kinase Cascades

    PubMed Central

    Lim, Y. M.; Tsuda, L.; Inoue, Y. H.; Irie, K.; Adachi-Yamada, T.; Hata, M.; Nishi, Y.; Matsumoto, K.; Nishida, Y.

    1997-01-01

    Eight alleles of Dsor1 encoding a Drosophila homologue of mitogen-activated protein (MAP) kinase kinase were obtained as dominant suppressors of the MAP kinase kinase kinase D-raf. These Dsor1 alleles themselves showed no obvious phenotypic consequences nor any effect on the viability of the flies, although they were highly sensitive to upstream signals and strongly interacted with gain-of-function mutations of upstream factors. They suppressed mutations for receptor tyrosine kinases (RTKs); torso (tor), sevenless (sev) and to a lesser extent Drosophila EGF receptor (DER). Furthermore, the Dsor1 alleles showed no significant interaction with gain-of-function mutations of DER. The observed difference in activity of the Dsor1 alleles among the RTK pathways suggests Dsor1 is one of the components of the pathway that regulates signal specificity. Expression of Dsor1 in budding yeast demonstrated that Dsor1 can activate yeast MAP kinase homologues if a proper activator of Dsor1 is coexpressed. Nucleotide sequencing of the Dsor1 mutant genes revealed that most of the mutations are associated with amino acid changes at highly conserved residues in the kinase domain. The results suggest that they function as suppressors due to increased reactivity to upstream factors. PMID:9136016

  3. Novel library of selenocompounds as kinase modulators.

    PubMed

    Plano, Daniel; Ibáñez, Elena; Calvo, Alfonso; Palop, Juan Antonio; Sanmartín, Carmen

    2011-07-27

    Although the causes of cancer lie in mutations or epigenic changes at the genetic level, their molecular manifestation is the dysfunction of biochemical pathways at the protein level. The 518 protein kinases encoded by the human genome play a central role in various diseases, a fact that has encouraged extensive investigations on their biological function and three dimensional structures. Selenium (Se) is an important nutritional trace element involved in different physiological functions with antioxidative, antitumoral and chemopreventive properties. The mechanisms of action for selenocompounds as anticancer agents are not fully understood, but kinase modulation seems to be a possible pathway. Various organosulfur compounds have shown antitumoral and kinase inhibition effects but, in many cases, the replacement of sulfur by selenium improves the antitumoral effect of compounds. Although Se atom possesses a larger atomic volume and nucleophilic character than sulfur, Se can also formed interactions with aminoacids of the catalytic centers of proteins. So, we propose a novel chemical library that includes organoselenium compounds as kinase modulators. In this study thirteen selenocompounds have been evaluated at a concentration of 3 or 10 µM in a 24 kinase panel using a Caliper LabChip 3000 Drug Discover Platform. Several receptor (EGFR, IGFR1, FGFR1…) and non-receptor (Abl) kinases have been selected, as well as serine/threonine/lipid kinases (AurA, Akt, CDKs, MAPKs…) implicated in main cancer pathways: cell cycle regulation, signal transduction, angiogenesis regulation among them. The obtained results showed that two compounds presented inhibition values higher than 50% in at least four kinases and seven derivatives selectively inhibited one or two kinases. Furthermore, three compounds selectively activated IGF-1R kinase with values ranging from -98% to -211%. In conclusion, we propose that the replacement of sulfur by selenium seems to be a potential and

  4. Protein kinase biochemistry and drug discovery.

    PubMed

    Schwartz, Phillip A; Murray, Brion W

    2011-12-01

    Protein kinases are fascinating biological catalysts with a rapidly expanding knowledge base, a growing appreciation in cell regulatory control, and an ascendant role in successful therapeutic intervention. To better understand protein kinases, the molecular underpinnings of phosphoryl group transfer, protein phosphorylation, and inhibitor interactions are examined. This analysis begins with a survey of phosphate group and phosphoprotein properties which provide context to the evolutionary selection of phosphorylation as a central mechanism for biological regulation of most cellular processes. Next, the kinetic and catalytic mechanisms of protein kinases are examined with respect to model aqueous systems to define the elements of catalysis. A brief structural biology overview further delves into the molecular basis of catalysis and regulation of catalytic activity. Concomitant with a prominent role in normal physiology, protein kinases have important roles in the disease state. To facilitate effective kinase drug discovery, classic and emerging approaches for characterizing kinase inhibitors are evaluated including biochemical assay design, inhibitor mechanism of action analysis, and proper kinetic treatment of irreversible inhibitors. As the resulting protein kinase inhibitors can modulate intended and unintended targets, profiling methods are discussed which can illuminate a more complete range of an inhibitor's biological activities to enable more meaningful cellular studies and more effective clinical studies. Taken as a whole, a wealth of protein kinase biochemistry knowledge is available, yet it is clear that a substantial extent of our understanding in this field remains to be discovered which should yield many new opportunities for therapeutic intervention.

  5. An atlas of human kinase regulation.

    PubMed

    Ochoa, David; Jonikas, Mindaugas; Lawrence, Robert T; El Debs, Bachir; Selkrig, Joel; Typas, Athanasios; Villén, Judit; Santos, Silvia Dm; Beltrao, Pedro

    2016-12-01

    The coordinated regulation of protein kinases is a rapid mechanism that integrates diverse cues and swiftly determines appropriate cellular responses. However, our understanding of cellular decision-making has been limited by the small number of simultaneously monitored phospho-regulatory events. Here, we have estimated changes in activity in 215 human kinases in 399 conditions derived from a large compilation of phosphopeptide quantifications. This atlas identifies commonly regulated kinases as those that are central in the signaling network and defines the logic relationships between kinase pairs. Co-regulation along the conditions predicts kinase-complex and kinase-substrate associations. Additionally, the kinase regulation profile acts as a molecular fingerprint to identify related and opposing signaling states. Using this atlas, we identified essential mediators of stem cell differentiation, modulators of Salmonella infection, and new targets of AKT1. This provides a global view of human phosphorylation-based signaling and the necessary context to better understand kinase-driven decision-making. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  6. Fetal anaemia due to pyruvate kinase deficiency.

    PubMed Central

    Gilsanz, F; Vega, M A; Gómez-Castillo, E; Ruiz-Balda, J A; Omeñaca, F

    1993-01-01

    Pyruvate kinase deficiency was diagnosed in an infant by umbilical vessel sampling at 30 weeks' gestation. Although three previous hydropic siblings had been stillborn or died in the neonatal period, this infant survived with transfusion dependent haemolytic anaemia. Prompt fetal diagnosis of pyruvate kinase deficiency is feasible and allows better management of hydrops fetalis due to this disorder. PMID:8285758

  7. Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes.

    PubMed

    Cestari, Igor; Stuart, Ken

    2015-05-26

    African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing.

  8. Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes

    PubMed Central

    Cestari, Igor; Stuart, Ken

    2015-01-01

    African trypanosomes evade clearance by host antibodies by periodically changing their variant surface glycoprotein (VSG) coat. They transcribe only one VSG gene at a time from 1 of about 20 telomeric expression sites (ESs). They undergo antigenic variation by switching transcription between telomeric ESs or by recombination of the VSG gene expressed. We show that the inositol phosphate (IP) pathway controls transcription of telomeric ESs and VSG antigenic switching in Trypanosoma brucei. Conditional knockdown of phosphatidylinositol 5-kinase (TbPIP5K) or phosphatidylinositol 5-phosphatase (TbPIP5Pase) or overexpression of phospholipase C (TbPLC) derepresses numerous silent ESs in T. brucei bloodstream forms. The derepression is specific to telomeric ESs, and it coincides with an increase in the number of colocalizing telomeric and RNA polymerase I foci in the nucleus. Monoallelic VSG transcription resumes after reexpression of TbPIP5K; however, most of the resultant cells switched the VSG gene expressed. TbPIP5K, TbPLC, their substrates, and products localize to the plasma membrane, whereas TbPIP5Pase localizes to the nucleus proximal to telomeres. TbPIP5Pase associates with repressor/activator protein 1 (TbRAP1), and their telomeric silencing function is altered by TbPIP5K knockdown. These results show that specific steps in the IP pathway control ES transcription and antigenic switching in T. brucei by epigenetic regulation of telomere silencing. PMID:25964327

  9. 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

  10. Discovery of Type II Inhibitors of TGFβ-Activated Kinase 1 (TAK1) and Mitogen-Activated Protein Kinase Kinase Kinase Kinase 2 (MAP4K2)

    DOE PAGES

    Tan, Li; Nomanbhoy, Tyzoon; Gurbani, Deepak; ...

    2014-07-17

    Here, we developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity against two mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as pharmacologically well interrogated kinases such as p38α (MAPK14) and ABL. Further investigation of the structure–activity relationship (SAR) resulted in the identification of potent dual TAK1 and MAP4K2 inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors such as 16more » and 17. Some of these inhibitors possess good pharmacokinetic properties that will enable their use in pharmacological studies in vivo. Lastly, a 2.4 Å cocrystal structure of TAK1 in complex with 1 confirms that the activation loop of TAK1 assumes the DFG-out conformation characteristic of type II inhibitors.« less

  11. Aurora Kinase Inhibitors: Current Status and Outlook.

    PubMed

    Bavetsias, Vassilios; Linardopoulos, Spiros

    2015-01-01

    The Aurora kinase family comprises of cell cycle-regulated serine/threonine kinases important for mitosis. Their activity and protein expression are cell cycle regulated, peaking during mitosis to orchestrate important mitotic processes including centrosome maturation, chromosome alignment, chromosome segregation, and cytokinesis. In humans, the Aurora kinase family consists of three members; Aurora-A, Aurora-B, and Aurora-C, which each share a conserved C-terminal catalytic domain but differ in their sub-cellular localization, substrate specificity, and function during mitosis. In addition, Aurora-A and Aurora-B have been found to be overexpressed in a wide variety of human tumors. These observations led to a number of programs among academic and pharmaceutical organizations to discovering small molecule Aurora kinase inhibitors as anti-cancer drugs. This review will summarize the known Aurora kinase inhibitors currently in the clinic, and discuss the current and future directions.

  12. WNK Kinases in Development and Disease.

    PubMed

    Rodan, Aylin R; Jenny, Andreas

    2017-01-01

    WNK (With-No-Lysine (K)) kinases are serine-threonine kinases characterized by an atypical placement of a catalytic lysine within the kinase domain. Mutations in human WNK1 or WNK4 cause an autosomal dominant syndrome of hypertension and hyperkalemia, reflecting the fact that WNK kinases are critical regulators of renal ion transport processes. Here, the role of WNKs in the regulation of ion transport processes in vertebrate and invertebrate renal function, cellular and organismal osmoregulation, and cell migration and cerebral edema will be reviewed, along with emerging literature demonstrating roles for WNKs in cardiovascular and neural development, Wnt signaling, and cancer. Conserved roles for these kinases across phyla are emphasized. © 2017 Elsevier Inc. All rights reserved.

  13. Aurora Kinase Inhibitors: Current Status and Outlook

    PubMed Central

    Bavetsias, Vassilios; Linardopoulos, Spiros

    2015-01-01

    The Aurora kinase family comprises of cell cycle-regulated serine/threonine kinases important for mitosis. Their activity and protein expression are cell cycle regulated, peaking during mitosis to orchestrate important mitotic processes including centrosome maturation, chromosome alignment, chromosome segregation, and cytokinesis. In humans, the Aurora kinase family consists of three members; Aurora-A, Aurora-B, and Aurora-C, which each share a conserved C-terminal catalytic domain but differ in their sub-cellular localization, substrate specificity, and function during mitosis. In addition, Aurora-A and Aurora-B have been found to be overexpressed in a wide variety of human tumors. These observations led to a number of programs among academic and pharmaceutical organizations to discovering small molecule Aurora kinase inhibitors as anti-cancer drugs. This review will summarize the known Aurora kinase inhibitors currently in the clinic, and discuss the current and future directions. PMID:26734566

  14. Protein Kinases in Zucchini (Characterization of Calcium-Requiring Plasma Membrane Kinases).

    PubMed Central

    Verhey, S. D.; Gaiser, J. C.; Lomax, T. L.

    1993-01-01

    Using an in situ phosphorylation assay with zucchini (Cucurbita pepo L. cv Dark Green) seedling tissue, we have identified numerous polypeptides that are capable of acting as protein kinases. Total protein preparations from different organs contain different kinase profiles, but all are within the range of 55 to 70 kD. At least four kinases are associated with highly purified plasma membranes from etiolated zucchini hypocotyls. The major phosphorylated polypeptides from plasma membranes range in apparent molecular mass from 58 to 68 kD. The plasma membrane kinases are activated by micromolar concentrations of calcium and phosphorylate serine, and, to a lesser extent, threonine residues. These characteristics are similar to those of a soluble calcium-dependent protein kinase that has been purified to homogeneity from soybean suspension cultures. Three of the zucchini plasma membrane kinases share antigenic epitopes with the soluble soybean kinase. The presence of kinase activity at different apparent molecular masses may be indicative of separate kinases with similar characteristics. The zucchini hypocotyl protein kinases are not removed from plasma membrane vesicles by 0.5 M NaCl/5 mM ethylenediaminetetraacetate or by detergent concentrations below the critical micelle concentration of two types of detergent. This indicates that the plasma membrane protein kinases are tightly associated with the membrane in zucchini seedlings. PMID:12231949

  15. KinasePhos: a web tool for identifying protein kinase-specific phosphorylation sites.

    PubMed

    Huang, Hsien-Da; Lee, Tzong-Yi; Tzeng, Shih-Wei; Horng, Jorng-Tzong

    2005-07-01

    KinasePhos is a novel web server for computationally identifying catalytic kinase-specific phosphorylation sites. The known phosphorylation sites from public domain data sources are categorized by their annotated protein kinases. Based on the profile hidden Markov model, computational models are learned from the kinase-specific groups of the phosphorylation sites. After evaluating the learned models, the model with highest accuracy was selected from each kinase-specific group, for use in a web-based prediction tool for identifying protein phosphorylation sites. Therefore, this work developed a kinase-specific phosphorylation site prediction tool with both high sensitivity and specificity. The prediction tool is freely available at http://KinasePhos.mbc.nctu.edu.tw/.

  16. Diacylglycerol kinase is phosphorylated in vivo upon stimulation of the epidermal growth factor receptor and serine/threonine kinases, including protein kinase C-epsilon.

    PubMed Central

    Schaap, D; van der Wal, J; van Blitterswijk, W J; van der Bend, R L; Ploegh, H L

    1993-01-01

    In signal transduction, diacylglycerol (DG) kinase attenuates levels of the second messenger DG by converting it to phosphatidic acid. A previously cloned full-length human 86 kDa DG kinase cDNA was expressed as fusion protein in Escherichia coli, to aid in the generation of DG-kinase-specific monoclonal antibodies suitable for immunoprecipitation experiments. To investigate whether phosphorylation of DG kinase is a possible mechanism for its regulation, COS-7 cells were transiently transfected with the DG kinase cDNA and phosphorylation of the expressed DG kinase was induced by various stimuli. Activation of both cyclic AMP-dependent protein kinase and protein kinase C (PKC) resulted in phosphorylation of DG kinase on serine residues in vivo, and both kinases induced this phosphorylation within the same tryptic phosphopeptide, suggesting that they may exert similar control over DG kinase. No phosphorylation was observed upon ionomycin treatment, intended to activate Ca2+/calmodulin-dependent kinases. Co-transfections of DG kinase with either PKC-alpha or PKC-epsilon cDNA revealed that both protein kinases, when stimulated, are able to phosphorylate DG kinase. For PKC-epsilon, DG kinase is the first in vivo substrate identified. Stimulation with epidermal growth factor (EGF) of COS-7 cells transfected with both DG kinase and EGF-receptor cDNA results mainly in phosphorylation of DG kinase on tyrosine. Since the EGF receptor has an intrinsic tyrosine kinase activity, this finding implies that DG kinase may be a direct substrate for the activated EGF receptor. Images Figure 2 Figure 3 Figure 4 PMID:7679574

  17. NDP kinase reactivity towards 3TC nucleotides.

    PubMed

    Kreimeyer, A; Schneider, B; Sarfati, R; Faraj, A; Sommadossi, J P; Veron, M; Deville-Bonne, D

    2001-05-01

    Nucleoside diphosphate (NDP) kinase is usually considered as the enzyme responsible for the last step of the cellular phosphorylation pathway leading to the synthesis of biologically active triphospho-derivatives of nucleoside analogs used in antiviral therapies and in particular in the treatment of AIDS. NDP kinase lacks specificity for the nucleobase and can use as substrate both ribo- or 2'-deoxyribonucleotides. However, only nucleoside analogs with a sugar moiety in the D-configuration (e.g. 3'-deoxy-3'-azidothymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (d4T)) have so far been analyzed as substrates of NDP kinase. In contrast, beta-L-2',3'-dideoxy-3'-thiacytidine (3TC), also called lamivudine, is a nucleoside analog that is now widely used in AIDS therapy and has a sugar moiety in the L-configuration. Using protein fluorescence to monitor the phosphotransfer between the enzyme and the nucleotide derivative at the presteady state, we have studied the reactivity of 3TC triphosphate and of other L-dideoxynucleotides with NDP kinase. We found that L-dideoxynucleoside triphosphates have a poor affinity for NDP kinase and that the catalytic efficiency of the phosphorylation of L-dideoxyderivatives is very low as compared with their D-enantiomers. We discuss these results using a computer model of 3TC diphosphate bound to the NDP kinase active site. NDP kinase may not seem to be the major enzyme phosphorylating 3TC-DP, in contrast to current opinion.

  18. Polo-like kinase-activating kinases: Aurora A, Aurora B and what else?

    PubMed

    Archambault, Vincent; Carmena, Mar

    2012-04-15

    The events of cell division are regulated by a complex interplay between kinases and phosphatases. Cyclin-dependent kinases (Cdks), polo-like kinases (Plks) and Aurora kinases play central roles in this process. Polo kinase (Plk1 in humans) regulates a wide range of events in mitosis and cytokinesis. To ensure the accuracy of these processes, polo activity itself is subject to complex regulation. Phosphorylation of polo in its T loop (or activation loop) increases its kinase activity several-fold. It has been shown that Aurora A kinase, with its co-factor Bora, activates Plk1 in G(2), and that this is essential for recovery from cell cycle arrest induced by DNA damage. In a recent article published in PLoS Biology, we report that Drosophila polo is activated by Aurora B kinase at centromeres, and that this is crucial for polo function in regulating chromosome dynamics in prometaphase. Our results suggest that this regulatory pathway is conserved in humans. Here, we propose a model for the collaboration between Aurora B and polo in the regulation of kinetochore attachment to microtubules in early mitosis. Moreover, we suggest that Aurora B could also function to activate Polo/Plk1 in cytokinesis. Finally, we discuss recent findings and open questions regarding the activation of polo and polo-like kinases by different kinases in mitosis, cytokinesis and other processes.

  19. [Kinase-Glo luminescent kinase assay for in vitro determination of PKA activity].

    PubMed

    Zhang, Yuan; Wu, Yan-lin; Liu, Qian; Chen, Chen; Qin, Yuan-yuan; Wu, Gui-yan; Gao, Hua

    2012-07-01

    To establish a simple, convenient and harmless non-radioactive method of determining protein kinase A (PKA) activity in vitro. Human PKAα cDNA was amplified from total RNA of HEK293 cells using RT-PCR method and then cloned into pGEX-6p-1 vector. In vitro purified GST-PKAα protein was identified by Western blot analysis. Finally, a non-radioactive method, Kinase-Glo luminescent kinase assay, was employed to determine the kinase activity of purified GST-PKAα. After the optimization of the induction conditions, we purified GST-PKAα protein successfully. We then determined GST-PKAα activity using Kinase-Glo luminescent kinase assay. We also further confirmed the kinase activity of GST-PKAα using H-89, a specific PKA inhibitor, and determined its IC(50); value (35.2±3.97) nmol/L which is consistent with reported value. The non-radioactive Kinase-Glo luminescent kinase assay is a simple, convenient and harmless method of determining the kinase activity of PKA. This method is effective for pre-clinical high-throughput screening of PKA inhibitor, discovering novel target proteins of PKA and investigating PKA phosphorylation sites in target proteins.

  20. Human Gastric Cancer Kinase Profile and Prognostic Significance of MKK4 Kinase

    PubMed Central

    Wu, Chew-Wun; Li, Anna F.-Y.; Chi, Chin-Wen; Huang, Chen Lung; Shen, King-Han; Liu, Wing-Yiu; Lin, Wen-chang

    2000-01-01

    Alterations of protein tyrosine kinase are often associated with uncontrolled cell growth and tumor progression. Knowledge of the overall expression pattern of tyrosine kinases should prove beneficial in understanding the signaling pathways involved in gastric cancer oncogenesis and in providing possible biomarkers for gastric cancer progression. To establish a general tyrosine-kinase expression profile, degenerated polymerase chain reaction primers designed from the consensus catalytic kinase motifs were used to amplify protein tyrosine kinase molecules from gastric cancer tissues. We observed more than 50 tyrosine and serine/threonine kinases from matching pairs of gastric cancer tissue and normal mucosa. Based on this new kinase profile information, we selected the MKK4 gene for further immunohistochemical studies. Statistical analysis of MKK4 protein expression and clinicopathological features indicated that MKK4 kinase expression could serve as a significant prognostic factor for relapse-free survival and for overall survival. We demonstrated a simple and sensitive method for establishing protein tyrosine-kinase expression profiles of human gastric cancer tissues as well as for discovering novel and useful clinical biomarkers from such kinase expression profiles. PMID:10854223

  1. Differential AMP-activated Protein Kinase (AMPK) Recognition Mechanism of Ca2+/Calmodulin-dependent Protein Kinase Kinase Isoforms.

    PubMed

    Fujiwara, Yuya; Kawaguchi, Yoshinori; Fujimoto, Tomohito; Kanayama, Naoki; Magari, Masaki; Tokumitsu, Hiroshi

    2016-06-24

    Ca(2+)/calmodulin-dependent protein kinase kinase β (CaMKKβ) is a known activating kinase for AMP-activated protein kinase (AMPK). In vitro, CaMKKβ phosphorylates Thr(172) in the AMPKα subunit more efficiently than CaMKKα, with a lower Km (∼2 μm) for AMPK, whereas the CaMKIα phosphorylation efficiencies by both CaMKKs are indistinguishable. Here we found that subdomain VIII of CaMKK is involved in the discrimination of AMPK as a native substrate by measuring the activities of various CaMKKα/CaMKKβ chimera mutants. Site-directed mutagenesis analysis revealed that Leu(358) in CaMKKβ/Ile(322) in CaMKKα confer, at least in part, a distinct recognition of AMPK but not of CaMKIα.

  2. Pantothenate kinase-associated neurodegeneration.

    PubMed

    Hartig, Monika B; Prokisch, Holger; Meitinger, Thomas; Klopstock, Thomas

    2012-08-01

    Pantothenate kinase-associated neurodegeneration (PKAN) is a hereditary progressive disorder and the most frequent form of neurodegeneration with brain iron accumulation (NBIA). PKAN patients present with a progressive movement disorder, dysarthria, cognitive impairment and retinitis pigmentosa. In magnetic resonance imaging, PKAN patients exhibit the pathognonomic "eye of the tiger" sign in the globus pallidus which corresponds to iron accumulation and gliosis as shown in neuropathological examinations. The discovery of the disease causing mutations in PANK2 has linked the disorder to coenzyme A (CoA) metabolism. PANK2 is the only one out of four PANK genes encoding an isoform which localizes to mitochondria. At least two other NBIA genes (PLA2G6, C19orf12) encode proteins that share with PANK2 a mitochondrial localization and all are suggested to play a role in lipid homeostasis. With no causal therapy available for PKAN until now, only symptomatic treatment is possible. A multi-centre retrospective study with bilateral pallidal deep brain stimulation in patients with NBIA revealed a significant improvement of dystonia. Recently, studies in the PANK Drosophila model "fumble" revealed improvement by the compound pantethine which is hypothesized to feed an alternate CoA biosynthesis pathway. In addition, pilot studies with the iron chelator deferiprone that crosses the blood brain barrier showed a good safety profile and some indication of efficacy. An adequately powered randomized clinical trial will start in 2012. This review summarizes clinical presentation, neuropathology and pathogenesis of PKAN.

  3. CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity.

    PubMed

    Oh, Man-Ho; Wu, Xia; Kim, Hyoung Seok; Harper, Jeffrey F; Zielinski, Raymond E; Clouse, Steven D; Huber, Steven C

    2012-11-30

    Although calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases, autophosphorylation on tyrosine residues was observed for soybean CDPKβ and several Arabidopsis isoforms (AtCPK4 and AtCPK34). We identified Ser-8, Thr-17, Tyr-24 (in the kinase domain), Ser-304, and Ser-358 as autophosphorylation sites of His(6)-GmCDPKβ. Overall autophosphorylation increased kinase activity with synthetic peptides, but autophosphorylation of Tyr-24 appears to attenuate kinase activity based on studies with the Y24F directed mutant. While much remains to be done, it is clear that several CDPKs are dual-specificity kinases, which raises the possibility that phosphotyrosine signaling may play a role in Ca(2+)/CDPK-mediated processes. Published by Elsevier B.V.

  4. 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.

  5. Archaeal Shikimate Kinase, a New Member of the GHMP-Kinase Family

    PubMed Central

    Daugherty, Matthew; Vonstein, Veronika; Overbeek, Ross; Osterman, Andrei

    2001-01-01

    Shikimate kinase (EC 2.7.1.71) is a committed enzyme in the seven-step biosynthesis of chorismate, a major precursor of aromatic amino acids and many other aromatic compounds. Genes for all enzymes of the chorismate pathway except shikimate kinase are found in archaeal genomes by sequence homology to their bacterial counterparts. In this study, a conserved archaeal gene (gi|1500322 in Methanococcus jannaschii) was identified as the best candidate for the missing shikimate kinase gene by the analysis of chromosomal clustering of chorismate biosynthetic genes. The encoded hypothetical protein, with no sequence similarity to bacterial and eukaryotic shikimate kinases, is distantly related to homoserine kinases (EC 2.7.1.39) of the GHMP-kinase superfamily. The latter functionality in M. jannaschii is assigned to another gene (gi|1591748), in agreement with sequence similarity and chromosomal clustering analysis. Both archaeal proteins, overexpressed in Escherichia coli and purified to homogeneity, displayed activity of the predicted type, with steady-state kinetic parameters similar to those of the corresponding bacterial kinases: Km,shikimate = 414 ± 33 μM, Km,ATP = 48 ± 4 μM, and kcat = 57 ± 2 s−1 for the predicted shikimate kinase and Km,homoserine = 188 ± 37 μM, Km,ATP = 101 ± 7 μM, and kcat = 28 ± 1 s−1 for the homoserine kinase. No overlapping activity could be detected between shikimate kinase and homoserine kinase, both revealing a >1,000-fold preference for their own specific substrates. The case of archaeal shikimate kinase illustrates the efficacy of techniques based on reconstruction of metabolism from genomic data and analysis of gene clustering on chromosomes in finding missing genes. PMID:11114929

  6. Crystal Structures of Two Aminoglycoside Kinases Bound with a Eukaryotic Protein Kinase Inhibitor

    PubMed Central

    Hwang, Jiyoung; Berghuis, Albert M.

    2011-01-01

    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 eurkaryotic 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. PMID:21573013

  7. Dynamics driven allostery in protein kinases

    PubMed Central

    Kornev, Alexandr P.; Taylor, Susan S.

    2015-01-01

    Protein kinases have very dynamic structures and their functionality strongly depends on their dynamic state. Active kinases reveal a dynamic pattern with residues clustering into semirigid communities that move in µs-ms timescale. Previously detected hydrophobic spines serve as connectors between communities. Communities do not follow the traditional subdomain structure of the kinase core or its secondary structure elements. Instead they are organized around main functional units. Integration of the communities depends on the assembly of the hydrophobic spine and phosphorylation of the activation loop. Single mutations can significantly disrupt the dynamic infrastructure and thereby interfere with long distance allosteric signaling that propagates throughout the whole molecule. Dynamics is proposed to be the underlying mechanism for allosteric regulation in protein kinases. PMID:26481499

  8. 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

  9. Genetics Home Reference: pyruvate kinase deficiency

    MedlinePlus

    ... glucose is broken down to produce adenosine triphosphate (ATP), the cell's main energy source. PKLR gene mutations ... pyruvate kinase enzyme function, causing a shortage of ATP in red blood cells and increased levels of ...

  10. 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.

  11. 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

  12. 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

  13. Seeding collaborations to advance kinase science with the GSK Published Kinase Inhibitor Set (PKIS).

    PubMed

    Drewry, David H; Willson, Timothy M; Zuercher, William J

    2014-01-01

    To catalyze research on historically untargeted protein kinases, we created the PKIS, an annotated set of 367 small molecule kinase inhibitors. The set has been widely distributed to academic collaborators as an open access tool. It has been used to identify chemical starting points for development of chemical probes for orphan kinases and to investigate kinase signaling in high content phenotypic assays. Access to the set comes with few restrictions other than the requirement that assay results be released into the public domain for the benefit of the entire research community. Examples from the efforts of several collaborators are summarized.

  14. Novel protein kinase C inhibitors: alpha-terthiophene derivatives.

    PubMed

    Kim, D S; Ashendel, C L; Zhou, Q; Chang, C T; Lee, E S; Chang, C J

    1998-10-06

    A series of alpha-terthiophene derivatives were prepared and their protein kinase C inhibitory activity were evaluated. The aldehyde derivatives were most potent inhibitors (IC50 < 1 microM). alpha-Terthiophene monoaldehyde was inactive in the inhibitions of protein kinase A, mitogen activated protein kinase and protein tyrosine kinase.

  15. Glycolate kinase activity in human red cells.

    PubMed

    Fujii, S; Beutler, E

    1985-02-01

    Human red cells manifest glycolate kinase activity. This activity copurifies with pyruvate kinase and is decreased in the red cells of subjects with hereditary pyruvate kinase deficiency. Glycolate kinase activity was detected in the presence of FDP or glucose-1,6-P2. In the presence of 1 mmol/L FDP, the Km for adenosine triphosphate (ATP) was 0.28 mmol/L and a half maximum velocity for glycolate was obtained at 40 mmol/L. The pH optimum of the reaction was over 10.5 With 10 mumol/L FDP, 500 mumol/L glucose-1,6-P2, 2 mmol/L ATP, 5 mmol/L MgCl2, and 50 mmol/L glycolate at pH 7.5, glycolate kinase activity was calculated to be approximately 0.0013 U/mL RBC. In view of this low activity even in the presence of massive amounts of glycolate, the glycolate kinase reaction cannot account for the maintenance of the reported phosphoglycolate level in human red cells.

  16. Kinase Activity Profiling of Pneumococcal Pneumonia

    PubMed Central

    Hoogendijk, Arie J.; Diks, Sander H.; van der Poll, Tom; Peppelenbosch, Maikel P.; Wieland, Catharina W.

    2011-01-01

    Background Pneumonia represents a major health burden. Previous work demonstrated that although the induction of inflammation is important for adequate host defense against pneumonia, an inability to regulate the host's inflammatory response within the lung later during infection can be detrimental. Intracellular signaling pathways commonly rely on activation of kinases, and kinases play an essential role in the regulation of the inflammatory response of immune cells. Methodology/Principal Findings Pneumonia was induced in mice via intranasal instillation of Streptococcus (S.) pneumoniae. Kinomics peptide arrays, exhibiting 1024 specific consensus sequences for protein kinases, were used to produce a systems biology analysis of cellular kinase activity during the course of pneumonia. Several differences in kinase activity revealed by the arrays were validated in lung homogenates of individual mice using western blot. We identified cascades of activated kinases showing that chemotoxic stress and a T helper 1 response were induced during the course of pneumococcal pneumonia. In addition, our data point to a reduction in WNT activity in lungs of S. pneumoniae infected mice. Moreover, this study demonstrated a reduction in overall CDK activity implying alterations in cell cycle biology. Conclusions/Significance This study utilizes systems biology to provide insight into the signaling events occurring during lung infection with the common cause of community acquired pneumonia, and may assist in identifying novel therapeutic targets in the treatment of bacterial pneumonia. PMID:21483672

  17. Modular composition predicts kinase/substrate interactions

    PubMed Central

    2010-01-01

    Background Phosphorylation events direct the flow of signals and metabolites along cellular protein networks. Current annotations of kinase-substrate binding events are far from complete. In this study, we scanned the entire human protein sequences using the PROSITE domain annotation tool to identify patterns of domain composition in kinases and their substrates. We identified statistically enriched pairs of strings of domains (signature pairs) in kinase-substrate couples presented in the 2006 version of the PTM database. Results The signature pairs enriched in kinase - substrate binding interactions turned out to be highly specific to kinase subtypes. The resulting list of signature pairs predicted kinase-substrate interactions in validation dataset not used in learning with high statistical accuracy. Conclusions The method presented here produces predictions of protein phosphorylation events with high accuracy and mid-level coverage. Our method can be used in expanding the currently available drafts of cell signaling pathways and thus will be an important tool in the development of combination drug therapies targeting complex diseases. PMID:20579376

  18. UV activation of receptor tyrosine kinase activity.

    PubMed

    Coffer, P J; Burgering, B M; Peppelenbosch, M P; Bos, J L; Kruijer, W

    1995-08-03

    The exposure of mammalian cells to ultraviolet radiation (UV) may lead to DNA damage resulting in mutation and thus possibly cancer, while irradiation can further act as a potent tumor promoter. In addition UV induces p21ras-mediated signalling leading to activation of transcription factors such as AP-1 and NF-kappa B, as well as activation of the Src tyrosine kinase. This 'UV-response' has been well studied in mammalian cells and furthermore is conserved in yeast, however the most upstream components of this signal transduction pathway have remained elusive. Here we show that UV rapidly activates both the EGF receptor and insulin receptor, as shown by tyrosine phosphorylation of these receptors. We demonstrate that this activation is due to autophosphorylation as it only occurs in cells containing receptors with a functional kinase domain. We have further analysed the propagation of the UV-induced signal to downstream events such as, IRS-1 and Shc tyrosine phosphorylation, phosphatidylinositol 3-kinase activation, leukotriene synthesis, MAP kinase activation and gene induction all of which are activated by UV irradiation. Importantly, we demonstrate that in cells expressing a 'kinase-dead' receptor mutant the UV-response is inhibited, blocking leukotriene synthesis, MAP kinase activation and transcriptional induction. Furthermore, prior-stimulation of cells with UV appears to reduce further responsiveness to addition of growth factor suggesting a common signaling pathway. These data demonstrate a critical role for receptor-mediated events in regulating the response mammalian cells to UV exposure.

  19. Compartmentalization of mammalian pantothenate kinases.

    PubMed

    Alfonso-Pecchio, Adolfo; Garcia, Matthew; Leonardi, Roberta; Jackowski, Suzanne

    2012-01-01

    The pantothenate kinases (PanK) catalyze the first and the rate-limiting step in coenzyme A (CoA) biosynthesis and regulate the amount of CoA in tissues by differential isoform expression and allosteric interaction with metabolic ligands. The four human and mouse PanK proteins share a homologous carboxy-terminal catalytic domain, but differ in their amino-termini. These unique termini direct the isoforms to different subcellular compartments. PanK1α isoforms were exclusively nuclear, with preferential association with the granular component of the nucleolus during interphase. PanK1α also associated with the perichromosomal region in condensing chromosomes during mitosis. The PanK1β and PanK3 isoforms were cytosolic, with a portion of PanK1β associated with clathrin-associated vesicles and recycling endosomes. Human PanK2, known to associate with mitochondria, was specifically localized to the intermembrane space. Human PanK2 was also detected in the nucleus, and functional nuclear localization and export signals were identified and experimentally confirmed. Nuclear PanK2 trafficked from the nucleus to the mitochondria, but not in the other direction, and was absent from the nucleus during G2 phase of the cell cycle. The localization of human PanK2 in these two compartments was in sharp contrast to mouse PanK2, which was exclusively cytosolic. These data demonstrate that PanK isoforms are differentially compartmentalized allowing them to sense CoA homeostasis in different cellular compartments and enable interaction with regulatory ligands produced in these same locations.

  20. Protein Kinase Activity of Phosphoinositide 3-Kinase Regulates Cytokine-Dependent Cell Survival

    PubMed Central

    Green, Benjamin D.; Barry, Emma F.; Ma, Yuefang; Woodcock, Joanna; Fitter, Stephen; Zannettino, Andrew C. W.; Pitson, Stuart M.; Hughes, Timothy P.; Lopez, Angel F.; Shepherd, Peter R.; Wei, Andrew H.; Ekert, Paul G.; Guthridge, Mark A.

    2013-01-01

    The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K), promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML) cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3) and granulocyte macrophage colony stimulating factor (GM-CSF) receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting such pathways in

  1. Interaction of phospholipase D1 with a casein-kinase-2-like serine kinase.

    PubMed Central

    Ganley, I G; Walker, S J; Manifava, M; Li, D; Brown, H A; Ktistakis, N T

    2001-01-01

    Phospholipase D (PLD)1 was phosphorylated in vivo and by an associated kinase in vitro following immunoprecipitation. Both phosphorylation events were greatly reduced in a catalytically inactive point mutant in which the serine residue at position 911 was converted into alanine (S911A). The kinase could be enriched from detergent-extracted brain membranes and bind and phosphorylate PLD1 that was immunoprecipitated from COS-7 cells. Using in-gel kinase assays we determined that the size of the kinase is approximately 40 kDa and that PLD1 is more effective than S911A in binding the kinase. Preliminary analysis of the phosphorylation sites on PLD1 suggested that the kinase belongs to the casein kinase 2 (CK2) family. Consistent with this, we found that the kinase could utilize GTP, and could be inhibited by heparin and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Membrane fractions from Chinese hamster ovary (CHO) cell lines that inducibly express PLD1 contained an endogenous kinase activity that phosphorylated PLD1 using GTP and was inhibited by DRB. Direct evidence that the kinase is CK2 came from observations that immunoprecipitates using PLD1 antibodies contained immunoreactive CK2alpha, and immunoprecipitates using CK2alpha antibodies contained immunoreactive PLD1. Co-expression of PLD1 in COS-7 cells with the two recombinant CK2 subunits, alpha or beta, suggests that the association of PLD1 with the kinase is through the beta subunit. Supporting this, phosphorylation of PLD1 by purified recombinant CK2alpha was enhanced by purified recombinant CK2beta. Assays measuring PLD1 catalytic activity following phosphorylation by CK2 suggest that this phosphorylation event does not influence PLD1-mediated hydrolysis of phosphatidylcholine in vitro. PMID:11171116

  2. Ubiquitin-Mediated Degradation of Aurora Kinases.

    PubMed

    Lindon, Catherine; Grant, Rhys; Min, Mingwei

    2015-01-01

    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

  3. 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

  4. 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

  5. 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

  6. Crystal Structure of Cryptosporidium parvum Pyruvate Kinase

    PubMed Central

    Cook, William J.; Senkovich, Olga; Aleem, Khadijah; Chattopadhyay, Debasish

    2012-01-01

    Pyruvate kinase plays a critical role in cellular metabolism of glucose by serving as a major regulator of glycolysis. This tetrameric enzyme is allosterically regulated by different effector molecules, mainly phosphosugars. In response to binding of effector molecules and substrates, significant structural changes have been identified in various pyruvate kinase structures. Pyruvate kinase of Cryptosporidium parvum is exceptional among known enzymes of protozoan origin in that it exhibits no allosteric property in the presence of commonly known effector molecules. The crystal structure of pyruvate kinase from C. parvum has been solved by molecular replacement techniques and refined to 2.5 Å resolution. In the active site a glycerol molecule is located near the γ-phosphate site of ATP, and the protein structure displays a partially closed active site. However, unlike other structures where the active site is closed, the α6' helix in C. parvum pyruvate kinase unwinds and assumes an extended conformation. In the crystal structure a sulfate ion is found at a site that is occupied by a phosphate of the effector molecule in many pyruvate kinase structures. A new feature of the C. parvum pyruvate kinase structure is the presence of a disulfide bond cross-linking the two monomers in the asymmetric unit. The disulfide bond is formed between cysteine residue 26 in the short N-helix of one monomer with cysteine residue 312 in a long helix (residues 303–320) of the second monomer at the interface of these monomers. Both cysteine residues are unique to C. parvum, and the disulfide bond remained intact in a reduced environment. However, the significance of this bond, if any, remains unknown at this time. PMID:23056503

  7. Cadmium activates a mitogen-activated protein kinase gene and MBP kinases in rice.

    PubMed

    Yeh, Chuan-Ming; Hsiao, Lin-June; Huang, Hao-Jen

    2004-09-01

    Mitogen-activated protein kinase (MAPK) pathways are modules involved in the transduction of extracellular signals to intracellular targets in all eukaryotes. In plants, it has been evidenced that MAPKs play a role in the signaling of biotic and abiotic stresses, plant hormones, and cell cycle cues. However, the effect of heavy metals on plant MAPKs has not been well examined. The Northern blot analysis of OsMAPK mRNA levels has shown that only OsMAPK2, but not OsMAPK3 and OsMAPK4, expressed in suspension-cultured cells in response to 100-400 microM Cd treatments. The OsMAPK2 transcripts increased within 12 h upon 400 microM Cd treatment. In addition, we found that 42- and 50-kDa MBP kinases were significantly activated by Cd treatment in rice suspension-cultured cells. And 40-, 42-, 50- and 64-kDa MBP kinases were activated in rice roots. Furthermore, GSH inhibits Cd-induced 40-kDa MBP kinase activation. By immunoblot analysis and immunoprecipitation followed by in-gel kinase assay, we confirmed that Cd-activated 42-kDa MBP kinase is a MAP kinase. Our results suggest that a MAP kinase cascade may function in the Cd-signalling pathway in rice.

  8. CDPKs are dual-specificity protein kinases and tyrosine autophosphorylation attenuates kinase activity

    USDA-ARS?s Scientific Manuscript database

    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...

  9. Homo- and heterodimerization of ROCO kinases: LRRK2 kinase inhibition by the LRRK2 ROCO fragment.

    PubMed

    Klein, Christian L; Rovelli, Giorgio; Springer, Wolfdieter; Schall, Christoph; Gasser, Thomas; Kahle, Philipp J

    2009-11-01

    Mutations in the gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most common cause of autosomal-dominant familial and late-onset sporadic Parkinson's disease (PD). LRRK2 is a large multi-domain protein featuring a GTP-binding C-terminal of Ras of complex proteins (ROC) (ROCO) domain combination unique for the ROCO protein family, directly followed by a kinase domain. Dimerization is a well-established phenomenon among protein kinases. Here, we confirm LRRK2 self-interaction, and provide evidence for general homo- and heterodimerization potential among the ROCO kinase family (LRRK2, LRRK1, and death-associated protein kinase 1). The ROCO domain was critically, though not exclusively involved in dimerization, as a LRRK2 deletion mutant lacking the ROCO domain retained dimeric properties. GTP binding did not appear to influence ROCO(LRRK2) self-interaction. Interestingly, ROCO(LRRK2) fragments exerted an inhibitory effect on both wild-type and the elevated G2019S LRRK2 autophosphorylation activity. Insertion of PD mutations into ROCO(LRRK2) reduced self-interaction and led to a reduction of LRRK2 kinase inhibition. Collectively, these results suggest a functional link between ROCO interactions and kinase activity of wild-type and mutant LRRK2. Importantly, our finding of ROCO(LRRK2) fragment-mediated LRRK2 kinase inhibition offers a novel lead for drug design and thus might have important implications for new therapeutic avenues in PD.

  10. The ErbB Kinase Domain: Structural Perspectives into Kinase Activation and Inhibition

    PubMed Central

    Bose, Ron; Zhang, Xuewu

    2009-01-01

    Epidermal growth factor receptor (EGFR) and its family members, ErbB2, ErB3 and ErB4, are receptor tyrosine kinases which send signals into the cell to regulate many critical processes including development, tissue homeostasis, and tumorigenesis. Central to the signaling of these receptors is their intracellular kinase domain, which is activated by ligand-induced dimerization of the receptor and phosphorylates several tyrosine residues in the C-terminal tail. The phosphorylated tail then recruits other signaling molecules and relays the signal to downstream pathways. A model of the autoinhibition, activation and feedback inhibition mechanisms for the ErbB kinase domain has emerged from a number of recent structural studies. Meanwhile, recent clinical studies have revealed the relationship between specific ErbB kinase mutations and the responsiveness to kinase inhibitor drugs. We will review these regulation mechanisms of the ErbB kinase domain, and discuss the binding specificity of kinase inhibitors and the effects of kinase domain mutations found in cancer patients from a structural perspective. PMID:18761339

  11. Interaction of SNF1 Protein Kinase with Its Activating Kinase Sak1▿

    PubMed Central

    Liu, Yang; Xu, Xinjing; Carlson, Marian

    2011-01-01

    The Saccharomyces cerevisiae SNF1 protein kinase, a member of the SNF1/AMP-activated protein kinase (AMPK) family, is activated by three kinases, Sak1, Tos3, and Elm1, which phosphorylate the Snf1 catalytic subunit on Thr-210 in response to glucose limitation and other stresses. Sak1 is the primary Snf1-activating kinase and is associated with Snf1 in a complex. Here we examine the interaction of Sak1 with SNF1. We report that Sak1 coimmunopurifies with the Snf1 catalytic subunit from extracts of both glucose-replete and glucose-limited cultures and that interaction occurs independently of the phosphorylation state of Snf1 Thr-210, Snf1 catalytic activity, and other SNF1 subunits. Sak1 interacts with the Snf1 kinase domain, and nonconserved sequences C terminal to the Sak1 kinase domain mediate interaction with Snf1 and augment the phosphorylation and activation of Snf1. The Sak1 C terminus is modified in response to glucose depletion, dependent on SNF1 activity. Replacement of the C terminus of Elm1 (or Tos3) with that of Sak1 enhanced the ability of the Elm1 kinase domain to interact with and phosphorylate Snf1. These findings indicate that the C terminus of Sak1 confers its function as the primary Snf1-activating kinase and suggest that the physical association of Sak1 with SNF1 facilitates responses to environmental change. PMID:21216941

  12. [Isoenzymatic forms and distribution of adenylate kinase and creatine kinase in the bovine adrenal medulla].

    PubMed

    Miras-Portugal, M T; Orera, A; Millaruelo, A

    1982-01-01

    Adenylate kinase, creatine kinase as well as their substrate and product levels have been investigated in the adrenal medullary tissue. The concentration of adenine nucleotides and creatine + creatine phosphate are 12.6 +/- 0.4 and 6.9 +/- 0.4 mumol/g wet weight respectively. Adenylate kinase is mainly in the cytosol; only 4% was found in mitochondria. The cytosol enzyme presents a Km for AMP of 5 X 10(-4) M and a Ki for diadenosine pentaphosphate of 0.6 X 10(-6) M. In gel electrophoresis, only one band of adenylate kinase activity can be seen, and its mobility is different from that of the brain enzyme. Creatine kinase from adrenal medulla is mainly found in cytosol; only 3-4% was associated with mitochondria. The cytosolic enzyme is mainly the BB isozyme form.

  13. Relationship of CDK-activating kinase and RNA polymerase II CTD kinase TFIIH/TFIIK.

    PubMed

    Feaver, W J; Svejstrup, J Q; Henry, N L; Kornberg, R D

    1994-12-16

    KIN28, a member of the p34cdc2/CDC28 family of protein kinases, is identified as a subunit of yeast RNA polymerase transcription factor IIH (TFIIH) on the basis of sequence determination, immunological reactivity, and copurification. KIN28 is, moreover, one of three subunits of TFIIK, a subassembly of TFIIH with protein kinase activity directed toward the C-terminal repeat domain (CTD) of the largest subunit of RNA polymerase II. Itself a phosphoprotein, KIN28 interacts specifically with the two largest subunits of RNA polymerase II. Previous work of others points to two further associations: KIN28 interacts in vivo with the cyclin CCL1, and KIN28 and CCL1 are homologous to human MO15 and cyclin H, which form the cyclin-dependent kinase-activating kinase (CAK). We show that human CAK possesses the CTD kinase activity characteristic of TFIIH.

  14. AMP-activated protein kinase--an archetypal protein kinase cascade?

    PubMed

    Hardie, D G; MacKintosh, R W

    1992-10-01

    Mammalian AMP-activated protein kinase is the central component of a protein kinase cascade which inactivates three key enzymes involved in the synthesis or release of free fatty acids and cholesterol inside the cell. The kinase cascade is activated by elevation of AMP, and perhaps also by fatty acid and cholesterol metabolites. The system may fulfil a protective function, preventing damage caused by depletion of ATP or excessive intracellular release of free lipids, a type of stress response. Recent evidence suggests that it may have been in existence for at least a billion years, since a very similar protein kinase cascade is present in higher plants. This system therefore represents an early eukaryotic protein kinase cascade, which is unique in that it is regulated by intracellular metabolites rather than extracellular signals or cell cycle events.

  15. The Link between Protein Kinase CK2 and Atypical Kinase Rio1

    PubMed Central

    Kubiński, Konrad; Masłyk, Maciej

    2017-01-01

    The atypical kinase Rio1 is widespread in many organisms, ranging from Archaebacteria to humans, and is an essential factor in ribosome biogenesis. Little is known about the protein substrates of the enzyme and small-molecule inhibitors of the kinase. Protein kinase CK2 was the first interaction partner of Rio1, identified in yeast cells. The enzyme from various sources undergoes CK2-mediated phosphorylation at several sites and this modification regulates the activity of Rio1. The aim of this review is to present studies of the relationship between the two different kinases, with respect to CK2-mediated phosphorylation of Rio1, regulation of Rio1 activity, and similar susceptibility of the kinases to benzimidazole inhibitors. PMID:28178206

  16. KIDFamMap: a database of kinase-inhibitor-disease family maps for kinase inhibitor selectivity and binding mechanisms

    PubMed Central

    Chiu, Yi-Yuan; Lin, Chih-Ta; Huang, Jhang-Wei; Hsu, Kai-Cheng; Tseng, Jen-Hu; You, Syuan-Ren; Yang, Jinn-Moon

    2013-01-01

    Kinases play central roles in signaling pathways and are promising therapeutic targets for many diseases. Designing selective kinase inhibitors is an emergent and challenging task, because kinases share an evolutionary conserved ATP-binding site. KIDFamMap (http://gemdock.life.nctu.edu.tw/KIDFamMap/) is the first database to explore kinase-inhibitor families (KIFs) and kinase-inhibitor-disease (KID) relationships for kinase inhibitor selectivity and mechanisms. This database includes 1208 KIFs, 962 KIDs, 55 603 kinase-inhibitor interactions (KIIs), 35 788 kinase inhibitors, 399 human protein kinases, 339 diseases and 638 disease allelic variants. Here, a KIF can be defined as follows: (i) the kinases in the KIF with significant sequence similarity, (ii) the inhibitors in the KIF with significant topology similarity and (iii) the KIIs in the KIF with significant interaction similarity. The KIIs within a KIF are often conserved on some consensus KIDFamMap anchors, which represent conserved interactions between the kinase subsites and consensus moieties of their inhibitors. Our experimental results reveal that the members of a KIF often possess similar inhibition profiles. The KIDFamMap anchors can reflect kinase conformations types, kinase functions and kinase inhibitor selectivity. We believe that KIDFamMap provides biological insights into kinase inhibitor selectivity and binding mechanisms. PMID:23193279

  17. Regulation of heart muscle pyruvate dehydrogenase kinase

    PubMed Central

    Cooper, Ronald H.; Randle, Philip J.; Denton, Richard M.

    1974-01-01

    1. The activity of pig heart pyruvate dehydrogenase kinase was assayed by the incorporation of [32P]phosphate from [γ-32P]ATP into the dehydrogenase complex. There was a very close correlation between this incorporation and the loss of pyruvate dehydrogenase activity with all preparations studied. 2. Nucleoside triphosphates other than ATP (at 100μm) and cyclic 3′:5′-nucleotides (at 10μm) had no significant effect on kinase activity. 3. The Km for thiamin pyrophosphate in the pyruvate dehydrogenase reaction was 0.76μm. Sodium pyrophosphate, adenylyl imidodiphosphate, ADP and GTP were competitive inhibitors against thiamin pyrophosphate in the dehydrogenase reaction. 4. The Km for ATP of the intrinsic kinase assayed in three preparations of pig heart pyruvate dehydrogenase was in the range 13.9–25.4μm. Inhibition by ADP and adenylyl imidodiphosphate was predominantly competitive, but there was nevertheless a definite non-competitive element. Thiamin pyrophosphate and sodium pyrophosphate were uncompetitive inhibitors against ATP. It is suggested that ADP and adenylyl imidodiphosphate inhibit the kinase mainly by binding to the ATP site and that the adenosine moiety may be involved in this binding. It is suggested that thiamin pyrophosphate, sodium pyrophosphate, adenylyl imidodiphosphate and ADP may inhibit the kinase by binding through pyrophosphate or imidodiphosphate moieties at some site other than the ATP site. It is not known whether this is the coenzyme-binding site in the pyruvate dehydrogenase reaction. 5. The Km for pyruvate in the pyruvate dehydrogenase reaction was 35.5μm. 2-Oxobutyrate and 3-hydroxypyruvate but not glyoxylate were also substrates; all three compounds inhibited pyruvate oxidation. 6. In preparations of pig heart pyruvate dehydrogenase free of thiamin pyrophosphate, pyruvate inhibited the kinase reaction at all concentrations in the range 25–500μm. The inhibition was uncompetitive. In the presence of thiamin pyrophosphate

  18. Myosin 3A Kinase Activity Is Regulated by Phosphorylation of the Kinase Domain Activation Loop*

    PubMed Central

    Quintero, Omar A.; Unrath, William C.; Stevens, Stanley M.; Manor, Uri; Kachar, Bechara; Yengo, Christopher M.

    2013-01-01

    Class III myosins are unique members of the myosin superfamily in that they contain both a motor and kinase domain. We have found that motor activity is decreased by autophosphorylation, although little is known about the regulation of the kinase domain. We demonstrate by mass spectrometry that Thr-178 and Thr-184 in the kinase domain activation loop and two threonines in the loop 2 region of the motor domain are autophosphorylated (Thr-908 and Thr-919). The kinase activity of MYO3A 2IQ with the phosphomimic (T184E) or phosphoblock (T184A) mutations demonstrates that kinase activity is reduced 30-fold as a result of the T184A mutation, although the Thr-178 site only had a minor impact on kinase activity. Interestingly, the actin-activated ATPase activity of MYO3A 2IQ is slightly reduced as a result of the T178A and T184A mutations suggesting coupling between motor and kinase domains. Full-length GFP-tagged T184A and T184E MYO3A constructs transfected into COS7 cells do not disrupt the ability of MYO3A to localize to filopodia structures. In addition, we demonstrate that T184E MYO3A reduces filopodia elongation in the presence of espin-1, whereas T184A enhances filopodia elongation in a similar fashion to kinase-dead MYO3A. Our results suggest that as MYO3A accumulates at the tips of actin protrusions, autophosphorylation of Thr-184 enhances kinase activity resulting in phosphorylation of the MYO3A motor and reducing motor activity. The differential regulation of the kinase and motor activities allows for MYO3A to precisely self-regulate its concentration in the actin bundle-based structures of cells. PMID:24214986

  19. 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

  20. Nucleoside kinases in adult Schistosoma mansoni: phosphorylation of pyrimidine nucleosides.

    PubMed

    Naguib, Fardos N M; El Kouni, Mahmoud H

    2014-01-01

    Competition studies and column chromatography demonstrated that adults Schistosoma mansoni contains three nucleoside kinases that can phosphorylate pyrimidine nucleosides; a non-specific deoxyriboside kinase (EC 2.7.1.145), a specific uridine kinase and a specific cytidine kinase. The non-specific deoxyriboside kinase can phosphorylate all naturally occurring pyrimidine and purine 2'-deoxyribosides. Uridine and cytidine kinases are specific for uridine and cytidine, respectively. Various nucleoside 5'-triphosphate act as phosphate donors for the three kinases albeit to different degrees. Nucleoside kinases are critical in the activation of potential anti-parasitic drugs which may be identified among the numerous available pyrimidine nucleoside analogues. The finding that S. mansoni have nucleoside kinases that differ from their host enzymes raises the possibilities that certain pyrimidine nucleoside analogues could be selectively toxic to schistosomes. Copyright © 2014 Elsevier B.V. All rights reserved.

  1. Functional analysis of anomeric sugar kinases.

    PubMed

    Conway, Louis P; Voglmeir, Josef

    2016-09-02

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. 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

  3. Ocular Toxicity of Tyrosine Kinase Inhibitors.

    PubMed

    Davis, Mary Elizabeth

    2016-03-01

    To review common tyrosine kinase inhibitors, as well as their ocular side effects and management.
. A comprehensive literature search was conducted using CINAHL®, PubMed, and Cochrane databases for articles published since 2004 with the following search terms. Tyrosine kinase inhibitors can cause significant eye toxicity.
. 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.
. 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.

  4. Tyrosine kinase inhibitors in preclinical development.

    PubMed

    Levitt, M L; Koty, P P

    1999-01-01

    Due to the limited efficacy of cytotoxic chemotherapy in the treatment of advanced malignancy and its excessive toxicity precluding its use in chemoprevention, new therapeutic and preventive strategies have been sought. One of the most interesting of these new approaches is the manipulation of signal transduction pathways. Among the approaches being considered to eventuate such a strategy is the inhibition of autophosphorylation, a critical first step in the signal transduction pathways of many cell surface receptor tyrosine kinases, as well as of non-receptor tyrosine kinases. This article is intended to review those tyrosine kinase inhibitors that are currently in preclinical development, for which there are data to support consideration for their use in chemoprevention or cancer treatment. We will focus upon those agents that have received attention in the past several years.

  5. 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

  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.

  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. Exploring the scaffold universe of kinase inhibitors.

    PubMed

    Hu, Ye; Bajorath, Jürgen

    2015-01-08

    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.

  9. Phosphorylation of the Kinase Interaction Motif in Mitogen-activated Protein (MAP) Kinase Phosphatase-4 Mediates Cross-talk between Protein Kinase A and MAP Kinase Signaling Pathways*

    PubMed Central

    Dickinson, Robin J.; Delavaine, Laurent; Cejudo-Marín, Rocío; Stewart, Graeme; Staples, Christopher J.; Didmon, Mark P.; Trinidad, Antonio Garcia; Alonso, Andrés; Pulido, Rafael; Keyse, Stephen M.

    2011-01-01

    MAP kinase phosphatase 4 (DUSP9/MKP-4) plays an essential role during placental development and is one of a subfamily of three closely related cytoplasmic dual-specificity MAPK phosphatases, which includes the ERK-specific enzymes DUSP6/MKP-3 and DUSP7/MKP-X. However, unlike DUSP6/MKP-3, DUSP9/MKP-4 also inactivates the p38α MAP kinase both in vitro and in vivo. Here we demonstrate that inactivation of both ERK1/2 and p38α by DUSP9/MKP-4 is mediated by a conserved arginine-rich kinase interaction motif located within the amino-terminal non-catalytic domain of the protein. Furthermore, DUSP9/MKP-4 is unique among these cytoplasmic MKPs in containing a conserved PKA consensus phosphorylation site 55RRXSer-58 immediately adjacent to the kinase interaction motif. DUSP9/MKP-4 is phosphorylated on Ser-58 by PKA in vitro, and phosphorylation abrogates the binding of DUSP9/MKP-4 to both ERK2 and p38α MAP kinases. In addition, although mutation of Ser-58 to either alanine or glutamic acid does not affect the intrinsic catalytic activity of DUSP9/MKP-4, phospho-mimetic (Ser-58 to Glu) substitution inhibits both the interaction of DUSP9/MKP-4 with ERK2 and p38α in vivo and its ability to dephosphorylate and inactivate these MAP kinases. Finally, the use of a phospho-specific antibody demonstrates that endogenous DUSP9/MKP-4 is phosphorylated on Ser-58 in response to the PKA agonist forskolin and is also modified in placental tissue. We conclude that DUSP9/MKP-4 is a bona fide target of PKA signaling and that attenuation of DUSP9/MKP-4 function can mediate cross-talk between the PKA pathway and MAPK signaling through both ERK1/2 and p38α in vivo. PMID:21908610

  10. PAS kinase: a nutrient sensing regulator of glucose homeostasis.

    PubMed

    DeMille, Desiree; Grose, Julianne H

    2013-11-01

    Per-Arnt-Sim (PAS) kinase (PASK, PASKIN, and PSK) is a member of the group of nutrient sensing protein kinases. These protein kinases sense the energy or nutrient status of the cell and regulate cellular metabolism appropriately. PAS kinase responds to glucose availability and regulates glucose homeostasis in yeast, mice, and man. Despite this pivotal role, the molecular mechanisms of PAS kinase regulation and function are largely unknown. This review focuses on what is known about PAS kinase, including its conservation from yeast to man, identified substrates, associated phenotypes and role in metabolic disease.

  11. 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.

  12. 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.

  13. Protein kinase activity of the insulin receptor.

    PubMed Central

    Gammeltoft, S; Van Obberghen, E

    1986-01-01

    The insulin receptor is an integral membrane glycoprotein (Mr approximately 300,000) composed of two alpha-subunits (Mr approximately 130,000) and two beta-subunits (Mr approximately 95,000) linked by disulphide bonds. This oligomeric structure divides the receptor into two functional domains such that alpha-subunits bind insulin and beta-subunits possess tyrosine kinase activity. The amino acid sequence deduced from cDNA of the single polypeptide chain precursor of human placental insulin receptor revealed that alpha- and beta-subunits consist of 735 and 620 residues, respectively. The alpha-subunit is hydrophilic, disulphide-bonded, glycosylated and probably extracellular. The beta-subunit consists of a short extracellular region which links the alpha-subunit through disulphide bridges, a hydrophobic transmembrane region and a longer cytoplasmic region which is structurally homologous with other tyrosine kinases like the src oncogene product and EGF receptor kinases. The cellular function of insulin receptors is dual: transmembrane signalling and endocytosis of hormone. The binding of insulin to its receptor on the cell membrane induces transfer of signal from extracellular to cytoplasmic receptor domains leading to activation of cell metabolism and growth. In addition, hormone-receptor complexes are internalized leading to intracellular proteolysis of insulin, whereas receptors are recycled to the membrane. These phenomena are kinetically well-characterized, but their molecular mechanisms remain obscure. Insulin receptor in different tissues and animal species are homologous in their structure and function, but show also significant differences regarding size of alpha-subunits, binding kinetics, insulin specificity and receptor-mediated degradation. We suggest that this heterogeneity of receptors may be linked to the diversity in insulin effects on metabolism and growth in various cell types. The purified insulin receptor phosphorylates its own beta-subunit and

  14. Expression of Plant Receptor Kinases in Yeast.

    PubMed

    Barberini, María Laura; Muschietti, Jorge P

    2017-01-01

    The budding yeast Saccharomyces cerevisiae is a useful system to express recombinant proteins and analyze protein-protein interaction. Membrane-spanning proteins like plant receptor kinases find their way to the plasma membrane when expressed in yeast and seem to retain their structure and function. Here, we describe a general yeast DNA transformation procedure based on lithium acetate, salmon sperm DNA, and polyethylene glycol used to express recombinant proteins. Yeast cells expressing plant receptor kinases can be used for in vivo and in vitro studies of receptor function.

  15. Screening of kinase inhibitors targeting BRAF for regulating autophagy based on kinase pathways.

    PubMed

    Zhang, Yingmei; Xue, Dongbo; Wang, Xiaochun; Lu, Ming; Gao, Bo; Qiao, Xin

    2014-01-01

    The aim of this study was to identify agents that regulate autophagy. A total of 544 differentially expressed genes were screened from the intersection set of GSE2435 and GSE31040, which was obtained from the Gene Expression Omnibus database and 19 differentially expressed kinases were selected according to a 'protein kinase database'. Gene ontology‑biological process (GO-BP) enrichment analysis revealed that the 19 kinases were mainly associated with phosphorylation. The protein-protein interaction network exhibited 30 differentially expressed genes that interacted with BRAF, and GO-BP enrichment analysis showed the function of these genes were mainly involved in cell death and apoptosis. The kinase-kinase inhibitor regulatory network identified16 kinase inhibitors that specifically inhibited BRAF. Previous studies indicated that sorafenib is capable of regulating autophagy and regorafenib has also been reported; however, there have been no studies regarding the regulation of autophagy by afatinib, selumetinib, PD318088, axitinib, TAK-733, GDC-0980, GSK2126458, PLX-4720, AS703026, trametinib, GDC-0941 and PF-04217903. Thus, these kinase inhibitors are potential targets for further study on the regulation of autophagy in the future.

  16. 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

  17. Musk Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop.

    PubMed

    Camurdanoglu, B Z; Hrovat, C; Dürnberger, G; Madalinski, M; Mechtler, K; Herbst, R

    2016-09-26

    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.

  18. Type-II Kinase Inhibitor Docking, Screening, and Profiling Using Modified Structures of Active Kinase States

    PubMed Central

    Kufareva, Irina; Abagyan, Ruben

    2009-01-01

    Type-II kinase inhibitors represent a class of chemicals that trap their target kinases in an inactive, so-called DFG-out, state, occupying a hydrophobic pocket adjacent to the ATP binding site. These compounds are often more specific than those targeting active, DFG-in, kinase conformations. Unfortunately, the discovery of novel type-II scaffolds presents a considerable challenge, partly because the lack of compatible kinase structures makes structure-based methods inapplicable. We present a computational protocol for converting multiple available DFG-in structures of various kinases (∼70% of mammalian structural kinome) into accurate and specific models of their type-II-bound state. The models, described as Deletion-Of-Loop asp-PHe-gly-IN (DOLPHIN) kinase models, demonstrate exceptional performance in various inhibitor discovery applications, including compound pose prediction, screening, and in silico activity profiling. Given the abundance of the DFG-in structures, the presented approach opens possibilities for kinome-wide discovery of specific molecules targeting inactive kinase states. PMID:19053777

  19. CMP kinase from Escherichia coli is structurally related to other nucleoside monophosphate kinases.

    PubMed

    Bucurenci, N; Sakamoto, H; Briozzo, P; Palibroda, N; Serina, L; Sarfati, R S; Labesse, G; Briand, G; Danchin, A; Bărzu, O; Gilles, A M

    1996-02-02

    CMP kinase from Escherichia coli is a monomeric protein of 225 amino acid residues. The protein exhibits little overall sequence similarities with other known NMP kinases. However, residues involved in binding of substrates and/or in catalysis were found conserved, and sequence comparison suggested conservation of the global fold found in adenylate kinases or in several CMP/UMP kinases. The enzyme was purified to homogeneity, crystallized, and analyzed for its structural and catalytic properties. The crystals belong to the hexagonal space group P6(3), have unit cell parameters a = b = 82.3 A and c = 60.7 A, and diffract x-rays to a 1.9 A resolution. The bacterial enzyme exhibits a fluorescence emission spectrum with maximum at 328 nm upon excitation at 295 nm, which suggests that the single tryptophan residue (Trp30) is located in a hydrophobic environment. Substrate specificity studies showed that CMP kinase from E. coli is active with ATP, dATP, or GTP as donors and with CMP, dCMP, and arabinofuranosyl-CMP as acceptors. This is in contrast with CMP/UMP kinase from Dictyostelium discoideum, an enzyme active on CMP or UMP but much less active on the corresponding deoxynucleotides. Binding of CMP enhanced the affinity of E. coli CMP kinase for ATP or ADP, a particularity never described in this family of proteins that might explain inhibition of enzyme activity by excess of nucleoside monophosphate.

  20. 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.

  1. Identification and characterization of plant Haspin kinase as a histone H3 threonine kinase.

    PubMed

    Kurihara, Daisuke; Matsunaga, Sachihiro; Omura, Tomohiro; Higashiyama, Tetsuya; Fukui, Kiichi

    2011-04-28

    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. 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. 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.

  2. CZK3, a MAP kinase kinase kinase homolog in Cercospora zeae-maydis, regulates cercosporin biosynthesis, fungal development, and pathogenesis.

    PubMed

    Shim, Won-Bo; Dunkle, Larry D

    2003-09-01

    The fungus Cercospora zeae-maydis causes gray leaf spot of maize and produces cercosporin, a photosensitizing perylenequinone with toxic activity against a broad spectrum of organisms. However, little is known about the biosynthetic pathway or factors that regulate cercosporin production. Analysis of a cDNA subtraction library comprised of genes that are up-regulated during cercosporin synthesis revealed a sequence highly similar to mitogen-activated protein (MAP) kinases in other fungi. Sequencing and conceptual translation of the full-length genomic sequence indicated that the gene, which we designated CZK3, contains a 4,119-bp open reading frame devoid of introns and encodes a 1,373-amino acid sequence that is highly similar to Wis4, a MAP kinase kinase kinase in Schizosaccharomyces pombe. Targeted disruption of CZK3 suppressed expression of genes predicted to participate in cercosporin biosynthesis and abolished cercosporin production. The disrupted mutants grew faster on agar media than the wild type but were deficient in conidiation and elicited only small chlorotic spots on inoculated maize leaves compared with rectangular necrotic lesions incited by the wild type. Complementation of disruptants with the CZK3 open reading frame and flanking sequences restored wild-type levels of conidiation, growth rate, and virulence as well as the ability to produce cercosporin. The results suggest that cercosporin is a virulence factor in C. zeae-maydis during maize pathogenesis, but the pleiotropic effects of CZK3 disruption precluded definitive conclusions.

  3. A semisynthetic Eph receptor tyrosine kinase provides insight into ligand-induced kinase activation

    PubMed Central

    Singla, Nikhil; Erdjument-Bromage, Hediye; Himanen, Juha P.; Muir, Tom W.; Nikolov, Dimitar B.

    2011-01-01

    SUMMARY We have developed a methodology for generating milligram amounts of functional Eph tyrosine kinase receptor using the protein engineering approach of expressed protein ligation. Stimulation with ligand induces efficient autophosphorylation of the semisynthetic Eph construct. The in vitro phosphorylation of key Eph tyrosine residues upon ligand-induced activation was monitored via time-resolved, quantitative phosphoproteomics, suggesting a precise and unique order of phosphorylation of the Eph tyrosines in the kinase activation process. To our knowledge, this work represents the first reported semisynthesis of a receptor tyrosine kinase and provides a potentially general method for producing single-pass membrane proteins for structural and biochemical characterization. PMID:21439481

  4. 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.

  5. 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

  6. 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

  7. 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

  8. Tyrosine kinase inhibitors in veterinary medicine.

    PubMed

    London, Cheryl A

    2009-08-01

    Substantial progress in the field of molecular biology has permitted the identification of key abnormalities in cancer cells involving cell proteins that regulate signal transduction, cell survival, and cell proliferation. Such abnormalities often involve a class of proteins called tyrosine kinases that act to phosphorylate other proteins in the cell, tightly regulating a variety of cellular processes. A variety of small molecule inhibitors that target specific tyrosine kinases (known as tyrosine kinase inhibitors [TKIs]) have now been approved for the treatment of human cancer, and it is likely many more will become available in the near future. In some instances these inhibitors have exhibited significant clinical efficacy, and it is likely their biologic activity will be further enhanced as combination regimens with standard treatment modalities are explored. Although TKIs have been used extensively in humans, their application to cancers in dogs and cats is relatively recent. The TKIs Palladia (toceranib), Kinavet (masitinib), and Gleevec (imatinib) have been successfully used in dogs, and more recently Gleevec in cats. This article will review the biology of tyrosine kinase dysfunction in human and animal cancers, and the application of specific TKIs to veterinary cancer patients.

  9. 3-phosphoglycerate kinase from Hydrogenomonas facilis.

    NASA Technical Reports Server (NTRS)

    Mcfadden, B. A.; Schuster, E.

    1972-01-01

    Description of studies of the kinetics of heat inactivation of phosphoglycerate kinase in the soluble fraction from Hydrogenomonas facilis, its extensive purification, and inhibition by adenosine monophosphate (AMP). No evidence was found for an enzyme which catalyzes adenosine-triphosphate-dependent conversion of 3-phosphoglycerate to 1,3-diphosphoglycerate, AMP, and phosphate.

  10. Real-time protein kinase assay.

    PubMed

    Sun, Hongye; Low, Karen E; Woo, Sam; Noble, Richard L; Graham, Ronald J; Connaughton, Sonia S; Gee, Melissa A; Lee, Linda G

    2005-04-01

    We report a novel, real-time fluorogenic kinase assay. The peptide substrates are synthesized with a fluorescent dye and a hydrocarbon tail. The substrate self-assembles into micelles, increasing the local concentration of the dye and quenching its fluorescence. Upon phosphorylation, the fluorescence intensity increases 4-6-fold due to micelle reorganization. Both dynamic light scattering data and cryoelectron microscope images show that the size and the shape of the phosphopeptide micelles are significantly different from micelles of substrate peptide. The system provides a robust fluorescence increase in a real-time protein kinase assay. Unlike other fluorogenic systems, the fluorophore may be distant from the serine, threonine, or tyrosine that is phosphorylated. Assays for several kinases, including PKA, PKC, p38, MAPKAP K2, akt, Erk1, and src-family kinases, have been developed. IC(50) values of inhibitors for PKC betaII determined with this technology are consistent with published values. The utility of this assay to high-throughput screening was demonstrated with Sigma's LOPAC library, a collection of 640 compounds with known biological activities, and satisfactory results were obtained.

  11. Monoclonal Antibodies Against Xenopus Greatwall Kinase

    PubMed Central

    Wang, Ling; Fisher, Laura A.; Wahl, James K.

    2011-01-01

    Mitosis is known to be regulated by protein kinases, including MPF, Plk1, Aurora kinases, and so on, which become active in M-phase and phosphorylate a wide range of substrates to control multiple aspects of mitotic entry, progression, and exit. Mechanistic investigations of these kinases not only provide key insights into cell cycle regulation, but also hold great promise for cancer therapy. Recent studies, largely in Xenopus, characterized a new mitotic kinase named Greatwall (Gwl) that plays essential roles in both mitotic entry and maintenance. In this study, we generated a panel of mouse monoclonal antibodies (MAbs) specific for Xenopus Gwl and characterized these antibodies for their utility in immunoblotting, immunoprecipitation, and immunodepletion in Xenopus egg extracts. Importantly, we generated an MAb that is capable of neutralizing endogenous Gwl. The addition of this antibody into M-phase extracts results in loss of mitotic phosphorylation of Gwl, Plk1, and Cdk1 substrates. These results illustrate a new tool to study loss-of-function of Gwl, and support its essential role in mitosis. Finally, we demonstrated the usefulness of the MAb against human Gwl/MASTL. PMID:22008075

  12. 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.

  13. AGCVIII Kinases: at the crossroads of cellular signaling

    USDA-ARS?s Scientific Manuscript database

    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...

  14. Chlamydia trachomatis tarp is phosphorylated by src family tyrosine kinases.

    PubMed

    Jewett, Travis J; Dooley, Cheryl A; Mead, David J; Hackstadt, Ted

    2008-06-27

    The translocated actin recruiting phosphoprotein (Tarp) is injected into the cytosol shortly after Chlamydia trachomatis attachment to a target cell and subsequently phosphorylated by an unidentified tyrosine kinase. A role for Tarp phosphorylation in bacterial entry is unknown. In this study, recombinant C. trachomatis Tarp was employed to identify the host cell kinase(s) required for phosphorylation. Each tyrosine rich repeat of L2 Tarp harbors a sequence similar to a Src and Abl kinase consensus target. Furthermore, purified p60-src, Yes, Fyn, and Abl kinases were able to phosphorylate Tarp. Mutagenesis of potential tyrosines within a single tyrosine rich repeat peptide indicated that both Src and Abl kinases phosphorylate the same residues suggesting that C. trachomatis Tarp may serve as a substrate for multiple host cell kinases. Surprisingly, chemical inhibition of Src and Abl kinases prevented Tarp phosphorylation in culture and had no measurable effect on bacterial entry into host cells.

  15. Kinase activity ranking using phosphoproteomics data (KARP) quantifies the contribution of protein kinases to the regulation of cell viability.

    PubMed

    Wilkes, Edmund H; Casado, Pedro; Rajeeve, Vinothini; Cutillas, Pedro R

    2017-09-01

    Cell survival is regulated by a signaling network driven by the activity of protein kinases; however, determining the contribution that each kinase in the network makes to such regulation remains challenging. Here, we report a computational approach that uses mass spectrometry-based phosphoproteomics data to rank protein kinases based on their contribution to cell regulation. We found that the scores returned by this algorithm, which we have termed kinase activity ranking using phosphoproteomics data (KARP), were a quantitative measure of the contribution that individual kinases make to the signaling output. Application of KARP to the analysis of eight hematological cell lines revealed that cyclin-dependent kinase (CDK) 1/2, casein kinase (CK) 2, extracellular signal-related kinase (ERK), and p21-activated kinase (PAK) were the most frequently highly ranked kinases in these cell models. The patterns of kinase activation were cell-line specific yet showed a significant association with cell viability as a function of kinase inhibitor treatment. Thus, our study exemplifies KARP as an untargeted approach to empirically and systematically identify regulatory kinases within signaling networks. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Elm1 kinase activates the spindle position checkpoint kinase Kin4.

    PubMed

    Caydasi, Ayse Koca; Kurtulmus, Bahtiyar; Orrico, Maria I L; Hofmann, Astrid; Ibrahim, Bashar; Pereira, Gislene

    2010-09-20

    Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck-associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4.

  17. 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.

  18. Elm1 kinase activates the spindle position checkpoint kinase Kin4

    PubMed Central

    Caydasi, Ayse Koca; Kurtulmus, Bahtiyar; Orrico, Maria I.L.; Hofmann, Astrid; Ibrahim, Bashar

    2010-01-01

    Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck–associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4. PMID:20855503

  19. Toxoplasma gondii calcium-dependent protein kinase 1 is a target for selective kinase inhibitors

    PubMed Central

    Ojo, Kayode K; Larson, Eric T; Keyloun, Katelyn R; Castaneda, Lisa J; DeRocher, Amy E; Inampudi, Krishna K; Kim, Jessica E; Arakaki, Tracy L; Murphy, Ryan C; Zhang, Li; Napuli, Alberto J; Maly, Dustin J; Verlinde, Christophe LMJ; Buckner, Frederick S; Parsons, Marilyn; Hol, Wim GJ; Merritt, Ethan A; Van Voorhis, Wesley C

    2010-01-01

    New drugs are needed to treat toxoplasmosis. Toxoplasma gondii calcium-dependent protein kinases (TgCDPKs) are attractive targets because they are absent in mammals. We show that TgCDPK1 is inhibited by low nanomolar levels of bumped kinase inhibitors (BKIs), compounds designed to be inactive against mammalian kinases. Cocrystal structures of TgCDPK1 with BKIs confirm that the structural basis for selectivity is due to the unique glycine gatekeeper residue in the ATP-binding site at residue 128. We show that BKIs interfere with an early step in T. gondii infection of human cells in culture. Furthermore, we show that TgCDPK1 is the in vivo target of BKIs because T. gondii cells expressing a glycine to methionine gatekeeper mutant enzyme show significantly decreased sensitivity to this class of selective kinase inhibitors. Thus, design of selective TgCDPK1 inhibitors with low host toxicity may be achievable. PMID:20436472

  20. 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.

  1. Protein Kinases in Mammary Gland Development and Carcinogenesis

    DTIC Science & Technology

    1999-09-01

    differ among CaM kinase family members include their subcellular localization , regulation by autophosphorylation, and regulation by other proteins. In...addition, CaM kinases have unique amino- and carboxyl- terminal domains that contribute to kinase-specific differences in subcellular localization ...chromosomal localization of Punc, a calcium/calmodulin-dependent protein kinase, (Submitted). 14. Hennings, H., Glick, A., Lowry, D., Krsmanovic, L

  2. 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.

  3. 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.

  4. The Role of HAP Kinases in Breast Cancer.

    DTIC Science & Technology

    1997-09-01

    center kinase ( GCK ) and receptor interacting protein (RIP). GCK and RIP, in turn, associate with mitogen-activated protein Icinase-kinase-kinases...MAP3Ks) upstream of the SAPKs and p38s. We have succeeded in generating an MCF7 breast carcinoma cell line which stably expresses GCK under the control

  5. The Roles of Protein Kinases in Learning and Memory

    ERIC Educational Resources Information Center

    Giese, Karl Peter; Mizuno, Keiko

    2013-01-01

    In the adult mammalian brain, more than 250 protein kinases are expressed, but only a few of these kinases are currently known to enable learning and memory. Based on this information it appears that learning and memory-related kinases either impact on synaptic transmission by altering ion channel properties or ion channel density, or regulate…

  6. Problem-Solving Test: "In Vitro" Protein Kinase A Reaction

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

    Phosphorylation of proteins by protein kinases is an important mechanism in the regulation of protein activity. Among hundreds of protein kinases present in human cells, PKA, the first kinase discovered, belongs to the most important and best characterized group of these enzymes. The author presents an experiment that analyzes the "in vitro"…

  7. Reusable amperometric biosensor for measuring protein tyrosine kinase activity.

    PubMed

    Wang, Chung-Liang; Wei, Lan-Yi; Yuan, Chiun-Jye; Hwang, Kuo Chu

    2012-01-17

    This work presents a simple, low-cost and reusable label-free method for detecting protein tyrosine kinase activity using a tyrosinase-based amperometric biosensor (tyrosine kinase biosensor). This method is based on the observation that phosphorylation can block the tyrosinase-catalyzed oxidation of tyrosine or tyrosyl residue in peptides. Therefore, the activity of p60c-src protein tyrosine kinase (Src) on the developed tyrosine kinase biosensor could be quickly determined when its specific peptide substrate, p60c-src substrate I, was used. The tyrosine kinase biosensor was highly sensitive to the activity of Src with a linear dynamic range of 1.9-237.6 U/mL and the lowest detection limit of 0.23 U/mL. Interestingly, the tyrosine kinase activity can be measured using the developed tyrosine kinase biosensor repetitively without regeneration. The inhibitory effect of various kinase inhibitors on the Src activity could be determined on the tyrosine kinase biosensor. Src-specific inhibitors, PP2 and Src inhibitor I, effectively suppressed Src activity, whereas PD153035, an inhibitor of the epidermal growth factor receptor, was ineffective. Staurosporine, a universal kinase inhibitor, inhibited Src activity in an ATP concentration-dependent manner. These results suggests that the activities of tyrosine kinases and their behaviors toward various reagents can be effectively measured using the developed tyrosine kinase biosensor.

  8. Heart failure-specific changes in protein kinase signalling.

    PubMed

    Lorenz, Kristina; Stathopoulou, Konstantina; Schmid, Evelyn; Eder, Petra; Cuello, Friederike

    2014-06-01

    Among the myriad of molecular alterations occurring in heart failure development, aggravation of the disease is often attributed to global or local changes in protein kinase activity, thus making protein kinases attractive targets for therapeutic intervention. Since protein kinases do not only have maladaptive roles, but also contribute to the physiological integrity of cells, it is a challenging task to circumvent undesired inhibition of protein kinase activity. Identification of posttranslational modifications and/or protein-protein interactions that are exclusively apparent under pathophysiological conditions provides exciting information for alternative non-kinase inhibitory treatment strategies that eliminate maladaptive functions of a protein kinase, but preserve the beneficial ones. Here, we focus on the disease-specific regulation of a number of protein kinases, namely, Ca(2+)/calmodulin-dependent protein kinase II isoform δ (CaMKIIδ), G protein-coupled receptor kinase 2 (GRK2), extracellular signal-regulated kinase 1 and 2 (ERK1/2), protein kinase D (PKD) and protein kinase C isoform β2 (PKCβ2), which are embedded in complex signal transduction pathways implicated in heart failure development, and discuss potential avenues for novel treatment strategies to combat heart disease.

  9. Mnk kinase pathway: Cellular functions and biological outcomes

    PubMed Central

    Joshi, Sonali; Platanias, Leonidas C

    2014-01-01

    The mitogen-activated protein kinase (MAPK) interacting protein kinases 1 and 2 (Mnk1 and Mnk2) play important roles in controlling signals involved in mRNA translation. In addition to the MAPKs (p38 or Erk), multiple studies suggest that the Mnk kinases can be regulated by other known kinases such as Pak2 and/or other unidentified kinases by phosphorylation of residues distinct from the sites phosphorylated by the MAPKs. Several studies have established multiple Mnk protein targets, including PSF, heterogenous nuclear ribonucleoprotein A1, Sprouty 2 and have lead to the identification of distinct biological functions and substrate specificity for the Mnk kinases. In this review we discuss the pathways regulating the Mnk kinases, their known substrates as well as the functional consequences of engagement of pathways controlled by Mnk kinases. These kinases play an important role in mRNA translation via their regulation of eukaryotic initiation factor 4E (eIF4E) and their functions have important implications in tumor biology as well as the regulation of drug resistance to anti-oncogenic therapies. Other studies have identified a role for the Mnk kinases in cap-independent mRNA translation, suggesting that the Mnk kinases can exert important functional effects independently of the phosphorylation of eIF4E. The role of Mnk kinases in inflammation and inflammation-induced malignancies is also discussed. PMID:25225600

  10. The Roles of Protein Kinases in Learning and Memory

    ERIC Educational Resources Information Center

    Giese, Karl Peter; Mizuno, Keiko

    2013-01-01

    In the adult mammalian brain, more than 250 protein kinases are expressed, but only a few of these kinases are currently known to enable learning and memory. Based on this information it appears that learning and memory-related kinases either impact on synaptic transmission by altering ion channel properties or ion channel density, or regulate…

  11. Problem-Solving Test: "In Vitro" Protein Kinase A Reaction

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

    Phosphorylation of proteins by protein kinases is an important mechanism in the regulation of protein activity. Among hundreds of protein kinases present in human cells, PKA, the first kinase discovered, belongs to the most important and best characterized group of these enzymes. The author presents an experiment that analyzes the "in vitro"…

  12. Unwinding the Wnt action of casein kinase 1.

    PubMed

    Yim, Daniel G R; Virshup, David M

    2013-06-01

    The casein kinase 1 (CK1) family, a major intracellular serine/threonine kinase, is implicated in multiple pathways; however, understanding its regulation has proven challenging. A recent study published in Science identifying allosteric activation of CK1 by the DEAD-box RNA helicase DDX3 expands our understanding of the control of this abundant kinase family.

  13. Mitogen-activated protein kinase cascades in Vitis vinifera

    PubMed Central

    Çakır, Birsen; Kılıçkaya, Ozan

    2015-01-01

    Protein phosphorylation is one of the most important mechanisms to control cellular functions in response to external and endogenous signals. Mitogen-activated protein kinases (MAPK) are universal signaling molecules in eukaryotes that mediate the intracellular transmission of extracellular signals resulting in the induction of appropriate cellular responses. MAPK cascades are composed of four protein kinase modules: MAPKKK kinases (MAPKKKKs), MAPKK kinases (MAPKKKs), MAPK kinases (MAPKKs), and MAPKs. In plants, MAPKs are activated in response to abiotic stresses, wounding, and hormones, and during plant pathogen interactions and cell division. In this report, we performed a complete inventory of MAPK cascades genes in Vitis vinifera, the whole genome of which has been sequenced. By comparison with MAPK, MAPK kinases, MAPK kinase kinases and MAPK kinase kinase kinase kinase members of Arabidopsis thaliana, we revealed the existence of 14 MAPKs, 5 MAPKKs, 62 MAPKKKs, and 7 MAPKKKKs in Vitis vinifera. We identified orthologs of V. vinifera putative MAPKs in different species, and ESTs corresponding to members of MAPK cascades in various tissues. This work represents the first complete inventory of MAPK cascades in V. vinifera and could help elucidate the biological and physiological functions of these proteins in V. vinifera. PMID:26257761

  14. Computational analysis of ABL kinase mutations allows predicting drug sensitivity against selective kinase inhibitors.

    PubMed

    Kamasani, Swapna; Akula, Sravani; Sivan, Sree Kanth; Manga, Vijjulatha; Duyster, Justus; Vudem, Dashavantha Reddy; Kancha, Rama Krishna

    2017-05-01

    The ABL kinase inhibitor imatinib has been used as front-line therapy for Philadelphia-positive chronic myeloid leukemia. However, a significant proportion of imatinib-treated patients relapse due to occurrence of mutations in the ABL kinase domain. Although inhibitor sensitivity for a set of mutations was reported, the role of less frequent ABL kinase mutations in drug sensitivity/resistance is not known. Moreover, recent reports indicate distinct resistance profiles for second-generation ABL inhibitors. We thus employed a computational approach to predict drug sensitivity of 234 point mutations that were reported in chronic myeloid leukemia patients. Initial validation analysis of our approach using a panel of previously studied frequent mutations indicated that the computational data generated in this study correlated well with the published experimental/clinical data. In addition, we present drug sensitivity profiles for remaining point mutations by computational docking analysis using imatinib as well as next generation ABL inhibitors nilotinib, dasatinib, bosutinib, axitinib, and ponatinib. Our results indicate distinct drug sensitivity profiles for ABL mutants toward kinase inhibitors. In addition, drug sensitivity profiles of a set of compound mutations in ABL kinase were also presented in this study. Thus, our large scale computational study provides comprehensive sensitivity/resistance profiles of ABL mutations toward specific kinase inhibitors.

  15. Comprehensive kinase profile of pacritinib, a nonmyelosuppressive Janus kinase 2 inhibitor

    PubMed Central

    Singer, Jack W; Al-Fayoumi, Suliman; Ma, Haiching; Komrokji, Rami S; Mesa, Ruben; Verstovsek, Srdan

    2016-01-01

    Pacritinib, potent inhibitor of Janus kinase 2 (JAK2), JAK2V617F, and fms-like receptor tyrosine kinase 3, is in Phase III development in myelofibrosis. Among type 1 inhibitors, pacritinib shows a lack of myelosuppression at doses that both inhibit JAK2/signal transducer and activator of transcription 3 pathway and demonstrate clinical efficacy. To elucidate these mechanisms and identify other disease targets, a kinome analysis screened 439 recombinant kinases at 100 nM pacritinib concentration. For kinases with >50% inhibition, pacritinib was titrated from 1 to 100 nM. JAK2, JAK2V617F, FLT3, colony-stimulating factor 1 receptor, and interleukin-1 receptor-associated kinase 1 achieved half-maximal inhibitory concentrations <50 nM. Pacritinib did not inhibit JAK1 (82% control at 100 nM). Lack of myelosuppression may stem from inhibiting JAK2 without affecting JAK1 and reducing hematopoietic inhibitory cytokines by suppressing interleukin-1 receptor-associated kinase 1 or colony-stimulating factor 1 receptor. The pacritinib kinome suggests therapeutic utility in acute myeloid leukemia, myelodysplastic syndrome, chronic myelomonocytic leukemia, solid tumors, and inflammatory conditions. PMID:27574472

  16. A Novel Mode of Protein Kinase Inhibition Exploiting Hydrophobic Motifs of Autoinhibited Kinases

    PubMed Central

    Eathiraj, Sudharshan; Palma, Rocio; Hirschi, Marscha; Volckova, Erika; Nakuci, Enkeleda; Castro, Jennifer; Chen, Chang-Rung; Chan, Thomas C. K.; France, Dennis S.; Ashwell, Mark A.

    2011-01-01

    Protein kinase inhibitors with enhanced selectivity can be designed by optimizing binding interactions with less conserved inactive conformations because such inhibitors will be less likely to compete with ATP for binding and therefore may be less impacted by high intracellular concentrations of ATP. Analysis of the ATP-binding cleft in a number of inactive protein kinases, particularly in the autoinhibited conformation, led to the identification of a previously undisclosed non-polar region in this cleft. This ATP-incompatible hydrophobic region is distinct from the previously characterized hydrophobic allosteric back pocket, as well as the main pocket. Generalized hypothetical models of inactive kinases were constructed and, for the work described here, we selected the fibroblast growth factor receptor (FGFR) tyrosine kinase family as a case study. Initial optimization of a FGFR2 inhibitor identified from a library of commercial compounds was guided using structural information from the model. We describe the inhibitory characteristics of this compound in biophysical, biochemical, and cell-based assays, and have characterized the binding mode using x-ray crystallographic studies. The results demonstrate, as expected, that these inhibitors prevent activation of the autoinhibited conformation, retain full inhibitory potency in the presence of physiological concentrations of ATP, and have favorable inhibitory activity in cancer cells. Given the widespread regulation of kinases by autoinhibitory mechanisms, the approach described herein provides a new paradigm for the discovery of inhibitors by targeting inactive conformations of protein kinases. PMID:21454610

  17. Atypical protein kinase Clambda binds and regulates p70 S6 kinase.

    PubMed Central

    Akimoto, K; Nakaya, M; Yamanaka, T; Tanaka, J; Matsuda, S; Weng, Q P; Avruch, J; Ohno, S

    1998-01-01

    p70 S6 kinase (p70 S6K) has been implicated in the regulation of cell cycle progression. However, the mechanism of its activation is not fully understood. In the present work, evidence is provided that an atypical protein kinase C (PKC) isotype, PKClambda, is indispensable, but not sufficient, for the activation of p70 S6K. Both the regulatory and kinase domains of PKClambda associate directly with p70 S6K. Overexpression of the kinase domain without kinase activity or the regulatory domain of PKClambda results in the suppression of the serum-induced activation of p70 S6K. In addition, two types of dominant-negative mutants of PKClambda, as well as a kinase-deficient mutant of p70 S6K, suppress serum-induced DNA synthesis and E2F activation. The overexpresion of the active form of PKClambda, however, fails to activate p70 S6K. These results suggest that PKClambda is a mediator in the regulation of p70 S6K activity and plays an important role in cell cycle progression. PMID:9761742

  18. Kinase impact assessment in the landscape of fusion genes that retain kinase domains: a pan-cancer study.

    PubMed

    Kim, Pora; Jia, Peilin; Zhao, Zhongming

    2016-12-24

    Assessing the impact of kinase in gene fusion is essential for both identifying driver fusion genes (FGs) and developing molecular targeted therapies. Kinase domain retention is a crucial factor in kinase fusion genes (KFGs), but such a systematic investigation has not been done yet. To this end, we analyzed kinase domain retention (KDR) status in chimeric protein sequences of 914 KFGs covering 312 kinases across 13 major cancer types. Based on 171 kinase domain-retained KFGs including 101 kinases, we studied their recurrence, kinase groups, fusion partners, exon-based expression depth, short DNA motifs around the break points and networks. Our results, such as more KDR than 5'-kinase fusion genes, combinatorial effects between 3'-KDR kinases and their 5'-partners and a signal transduction-specific DNA sequence motif in the break point intronic sequences, supported positive selection on 3'-kinase fusion genes in cancer. We introduced a degree-of-frequency (DoF) score to measure the possible number of KFGs of a kinase. Interestingly, kinases with high DoF scores tended to undergo strong gene expression alteration at the break points. Furthermore, our KDR gene fusion network analysis revealed six of the seven kinases with the highest DoF scores (ALK, BRAF, MET, NTRK1, NTRK3 and RET) were all observed in thyroid carcinoma. Finally, we summarized common features of 'effective' (highly recurrent) kinases in gene fusions such as expression alteration at break point, redundant usage in multiple cancer types and 3'-location tendency. Collectively, our findings are useful for prioritizing driver kinases and FGs and provided insights into KFGs' clinical implications. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  19. A retroviral-derived peptide phosphorylates protein kinase D/protein kinase Cmu involving phospholipase C and protein kinase C.

    PubMed

    Luangwedchakarn, Voravich; Day, Noorbibi K; Hitchcock, Remi; Brown, Pam G; Lerner, Danica L; Rucker, Rajivi P; Cianciolo, George J; Good, Robert A; Haraguchi, Soichi

    2003-05-01

    CKS-17, a synthetic peptide representing a unique amino acid motif which is highly conserved in retroviral transmembrane proteins and other immunoregulatory proteins, induces selective immunomodulatory functions, both in vitro and in vivo, and activates intracellular signaling molecules such as cAMP and extracellular signal-regulated kinases. In the present study, using Jurkat T-cells, we report that CKS-17 phosphorylates protein kinase D (PKD)/protein kinase C (PKC) mu. Total cell extracts from CKS-17-stimulated Jurkat cells were immunoblotted with an anti-phospho-PKCmu antibody. The results show that CKS-17 significantly phosphorylates PKD/PKCmu in a dose- and time-dependent manner. Treatment of cells with the PKC inhibitors GF 109203X and Ro 31-8220, which do not act directly on PKD/PKCmu, attenuates CKS-17-induced phosphorylation of PKD/PKCmu. In contrast, the selective protein kinase A inhibitor H-89 does not reverse the action of CKS-17. Furthermore, a phospholipase C (PLC) selective inhibitor, U-73122, completely blocks the phosphorylation of PKD/PKCmu by CKS-17 while a negative control U-73343 does not. In addition, substitution of lysine for arginine residues in the CKS-17 sequence completely abrogates the ability of CKS-17 to phosphorylate PKD/PKCmu. These results clearly indicate that CKS-17 phosphorylates PKD/PKCmu through a PLC- and PKC-dependent mechanism and that arginine residues play an essential role in this activity of CKS-17, presenting a novel modality of the retroviral peptide CKS-17 and molecular interaction of this compound with target cells.

  20. Effects of butyltins on mitogen-activated-protein kinase kinase kinase and Ras activity in human natural killer cells.

    PubMed

    Celada, Lindsay J; Whalen, Margaret M

    2014-09-01

    Butyltins (BTs) contaminate the environment and are found in human blood. BTs, tributyltin (TBT) and dibutyltin (DBT) diminish the cytotoxic function and levels of key proteins of human natural killer (NK) cells. NK cells are an initial immune defense against tumors, virally infected cells and antibody-coated cells and thus critical to human health. The signaling pathways that regulate NK cell functions include mitogen-activated protein kinases (MAPKs). Studies have shown that exposure to BTs leads to activation of specific MAPKs and MAPK kinases (MAP2Ks) in human NK cells. MAP2K kinases (MAP3Ks) are upstream activators of MAP2Ks, which then activate MAPKs. The current study examined if BT-induced activation of MAP3Ks was responsible for MAP2K and thus, MAPK activation. This study examines the effects of TBT and DBT on the total levels of two MAP3Ks, c-Raf and ASK1, as well as activating and inhibitory phosphorylation sites on these MAP3Ks. In addition, the immediate upstream activator of c-Raf, Ras, was examined for BT-induced alterations. Our results show significant activation of the MAP3K, c-Raf, in human NK cells within 10 min of TBT exposure and the MAP3K, ASK1, after 1 h exposures to TBT. In addition, our results suggest that both TBT and DBT affect the regulation of c-Raf.

  1. Elevated levels of serum creatine kinase induced by hyponatraemia.

    PubMed

    Goldenberg, I; Jonas, M; Thaler, M; Grossman, E

    1997-08-01

    Elevated serum creatine kinase levels are one of the major criteria for the diagnosis of myocardial injury. Noncardiac causes such as muscular and brain damage may also be associated with elevated serum creatine kinase levels. Hyponatremia may induce increased serum creatine kinase in association with rhabdomyolysis or with hypothyroidism. A patient is described where three episodes of hyponatraemia not associated with rhabdomyolysis or hypothyroidism induced transient elevations of serum creatine kinase levels. The association between hyponatraemia and elevated creatine kinase levels should be emphasized to prevent erroneous diagnosis of myocardial injury.

  2. 3-aminopyrazolopyrazine derivatives as spleen tyrosine kinase inhibitors.

    PubMed

    Shen, Jiayi; Li, Xiaokai; Zhang, Zhang; Luo, Jingfeng; Long, Huoyou; Tu, Zhengchao; Zhou, Xiaoping; Ding, Ke; Lu, Xiaoyun

    2016-11-01

    Spleen tyrosine kinase is a new promising target for drug discovery to treat human cancer and inflammatory disorders. A series of pyrazolopyrazine-3-amine and pyrazolopyrimidine-3-amine derivatives was designed and synthesized as new spleen tyrosine kinase inhibitors. The efforts yielded compound 6h with promising spleen tyrosine kinase inhibition in both enzymatic and B-lymphoma cell proliferation assays. Additionally, compound 6h dose dependently inhibited the activation of spleen tyrosine kinase signal in human B-cell lymphoma cells. Compound 6h might serve as a lead for further development of new spleen tyrosine kinase inhibitors. © 2016 John Wiley & Sons A/S.

  3. The nuts and bolts of AGC protein kinases.

    PubMed

    Pearce, Laura R; Komander, David; Alessi, Dario R

    2010-01-01

    The AGC kinase subfamily of protein kinases contains 60 members, including PKA, PKG and PKC. The family comprises some intensely examined protein kinases (such as Akt, S6K, RSK, MSK, PDK1 and GRK) as well as many less well-studied enzymes (such as SGK, NDR, LATS, CRIK, SGK494, PRKX, PRKY and MAST). Research has shed new light onto the architecture and regulatory mechanisms of these kinases. In addition, AGC kinases mediate diverse and important cellular functions, and their mutation and/or dysregulation contributes to the pathogenesis of many human diseases, including cancer and diabetes.

  4. Setting up a kinase discovery and development project.

    PubMed

    Bollag, Gideon

    2012-01-01

    Discovery of novel kinase inhibitors has matured rapidly over the last decade. Paramount to the successful development of kinase inhibitors is appropriate selectivity for validated targets. Many different approaches have been applied over the years, with varied results. There are currently thirteen different small molecule protein kinase inhibitors on the marketplace. Interestingly, a majority of these compounds lack precise selectivity for specific targets. This will change in the coming years, as technology for achieving improved selectivity becomes more widely applied. This chapter will focus on some of the critical considerations in setting up a kinase discovery and development project, citing examples particularly targeting the Raf kinases.

  5. Activation of S6 kinase in human neutrophils by calcium pyrophosphate dihydrate crystals: protein kinase C-dependent and phosphatidylinositol-3-kinase-independent pathways.

    PubMed Central

    Tudan, C; Jackson, J K; Charlton, L; Pelech, S L; Sahl, B; Burt, H M

    1998-01-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown previously to be a central enzyme in crystal-induced neutrophil activation. Since activation of the 70 kDa S6 kinase (p70S6K) has been shown to be dependent on PI 3-kinase activation in mammalian cells, and since the former is a key enzyme in the transmission of signals to the cell nucleus, activation of p70(S6K) was investigated in crystal-stimulated neutrophils. Cytosolic fractions from calcium pyrophosphate dihydrate (CPPD)-crystal-activated neutrophils were separated by Mono Q chromatography and analysed for phosphotransferase activity using a range of substrates and probed by Western analysis using antibodies to p70(S6K) and mitogen-activated protein kinase (MAP kinase). CPPD crystals induced a robust, transient activation (peak activity at 2 min) of p70(S6K) that was fully inhibited by pretreatment with rapamycin. This is the first report of the activation of p70(S6K) in neutrophil signal transduction pathways induced by an agonist. This crystal-induced activation of p70(S6K) could also be inhibited by a protein kinase C (PKC) inhibitor (Compound 3), but not by the PI 3-kinase inhibitor wortmannin. CPPD crystals also activated the ERK1 and ERK2 forms of MAP kinase (wortmannin insensitive), PKC (Compound 3 sensitive) and protein kinase B (wortmannin sensitive) in neutrophils. These data suggest that activation of p70(S6K) may proceed through a PI 3-kinase- and protein kinase B-independent but PKC-dependent pathway in crystal-activated neutrophils. PMID:9531494

  6. Activation of fat cell adenylate cyclase by protein kinase C

    SciTech Connect

    Naghshineh, S.; Noguchi, M.; Huang, K.P.; Londos, C.

    1986-05-01

    Purified protein kinase C (C-kinase) from guinea pig pancreas and rat brain stimulated adenylate cyclase activity in purified rat adipocyte membranes. Cyclase stimulation occurred over 100 to 1000 mU/ml of C-kinase activity, required greater than 10 ..mu..M calcium, proceeded without a lag, was not readily reversible, and required no exogenous phospholipid. Moreover, C-kinase inhibitors, such as chlorpromazine and palmitoyl carnitine, inhibited selectively adenylate cyclase which was activated by C-kinase and calcium. Depending on assay conditions, 10 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) either enhanced or was required for kinase action on cyclase. Also, TPA plus calcium promoted the quantitative association of C-kinase with membranes. Adenylate cyclase activation by C-kinase was seen both in the presence and absence of exogenous GTP, indicating that the kinase effect does not result from an action on the GTP-binding, inhibitory regulatory component (N/sub i/) of the cyclase system. Moreover, the kinase effect was seen in the presence of non-phosphorylating ATP analogs, such as AppNHp and AppCH/sub 2/p, suggesting that the effects of C-kinase described herein may result from association with, rather than phosphorylation of, adenylate cyclase.

  7. [LIM kinases and their roles in the nervous system].

    PubMed

    Zhang, Ru-yi; Ding, Yue-min; Zhang, Li-huang

    2014-01-01

    LIM kinase-1 (LIMK1) and LIM kinase-2 (LIMK2) are kinases that have serine/threonine and tyrosine dual specificity. Although they show significant structural similarity, LIMK1 and LIMK2 have different expression patterns, subcellular localization, and functions. Activation of LIM kinases regulates the downstream of Rho GTPases, and influences the architecture of the actin cytoskeleton by regulating the activity of cofilin. Recent studies have shown that LIM kinases play important roles in the nervous system. For example, development of the central nervous system is reliant upon the presence of LIMK1, and deletion of Limk1 gene is involved in the development of the human genetic disorder Williams syndrome. Therefore, it is of vital physiological significance to investigate the neuronal function of LIM kinases. In this review, we outline the structure, phosphorylation regulation and neuronal function of LIM kinases, so as to provide new ideas for the treatment of these neurological diseases.

  8. Phosphoproteomic Analysis of Aurora Kinase Inhibition in Monopolar Cytokinesis.

    PubMed

    Polat, Ayse Nur; Karayel, Özge; Giese, Sven H; Harmanda, Büşra; Sanal, Erdem; Hu, Chi-Kuo; Renard, Bernhard Y; Özlü, Nurhan

    2015-09-04

    Cytokinesis is the last step of the cell cycle that requires coordinated activities of the microtubule cytoskeleton, actin cytoskeleton, and membrane compartments. Aurora B kinase is one of the master regulatory kinases that orchestrate multiple events during cytokinesis. To reveal targets of the Aurora B kinase, we combined quantitative mass spectrometry with chemical genetics. Using the quantitative proteomic approach, SILAC (stable isotope labeling with amino acids in cell culture), we analyzed the phosphoproteome of monopolar cytokinesis upon VX680- or AZD1152-mediated aurora kinase inhibition. In total, our analysis quantified over 20 000 phosphopeptides in response to the Aurora-B kinase inhibition; 246 unique phosphopeptides were significantly down-regulated and 74 were up-regulated. Our data provide a broad analysis of downstream effectors of Aurora kinase and offer insights into how Aurora kinase regulates cytokinesis.

  9. Understanding and exploiting substrate recognition by protein kinases.

    PubMed

    Turk, Benjamin E

    2008-02-01

    Protein kinases play a virtually universal role in cellular regulation and are emerging as an important class of new drug targets, yet the cellular functions of most human kinases largely remain obscure. Aspects of substrate recognition common to all kinases in the ATP nucleotide binding site have been exploited in the generation of analog-specific mutants for exploring kinase function and discovering novel protein substrates. Likewise, understanding interactions with the protein substrate, which differ substantially between kinases, can also help to identify substrates and to produce tools for studying kinase pathways, including fluorescent biosensors. Principles of kinase substrate recognition are particularly valuable in guiding bioinformatics and phosphoproteomics approaches that impact our understanding of signaling pathways and networks on a global scale.

  10. Ror receptor tyrosine kinases: orphans no more

    PubMed Central

    Green, Jennifer L.; Kuntz, Steven G.; Sternberg, Paul W.

    2015-01-01

    Ror proteins are a conserved family of tyrosine kinase receptors that function in developmental processes, including skeletal and neuronal development, cell movement, and cell polarity. While Ror (receptor tyrosine kinase-like orphan receptor) proteins were originally named because the associated ligand and signaling pathway were unknown, recent studies in multiple species now establish that Ror proteins are Wnt receptors. Depending on the cellular context, Ror proteins can either activate or repress transcription of Wnt target genes and can modulate Wnt signaling by sequestering Wnt ligands. New evidence implicates Ror proteins in planar cell polarity (PCP), an alternative Wnt pathway. Here, we review the progress made in understanding these mysterious proteins and in particular we focus on their function as Wnt receptors. PMID:18848778

  11. WNK Kinases, Renal Ion Transport and Hypertension

    PubMed Central

    San-Cristobal, Pedro; de los Heros, Paola; Ponce-Coria, José; Moreno, Erika; Gamba, Gerardo

    2008-01-01

    Two members of a recently discovered family of protein kinases are the cause of an inherited disease known as pseudohypoaldosteronism type II (PHAII). These patients exhibit arterial hypertension together with hyperkalemia and metabolic acidosis. This is a mirror image of Gitelman disease that is due to inactivating mutations of the SLC12A3 gene that encodes the thiazide-sensitive Na+: Cl− cotransporter. The uncovered genes causing PHAII encode for serine/threonine kinases known as WNK1 and WNK4. Physiological and biochemical studies have revealed that WNK1 and WNK4 modulate the activity of several transport pathways of the aldosterone-sensitive distal nephron, thus increasing our understanding of how diverse renal ion transport proteins are coordinated to regulate normal blood pressure levels. Observations discussed in the present work place WNK1 and WNK4 as genes involved in the genesis of essential hypertension and as potential targets for the development of antihypertensive drugs. PMID:18547946

  12. Targeting checkpoint kinase 1 in cancer therapeutics.

    PubMed

    Tse, Archie N; Carvajal, Richard; Schwartz, Gary K

    2007-04-01

    Progression through the cell cycle is monitored by surveillance mechanisms known as cell cycle checkpoints. Our knowledge of the biochemical nature of checkpoint regulation during an unperturbed cell cycle and following DNA damage has expanded tremendously over the past decade. We now know that dysfunction in cell cycle checkpoints leads to genomic instability and contributes to tumor progression, and most agents used for cancer therapy, such as cytotoxic chemotherapy and ionizing radiation, also activate cell cycle checkpoints. Understanding how checkpoints are regulated is therefore important from the points of view of both tumorigenesis and cancer treatment. In this review, we present an overview of the molecular hierarchy of the checkpoint signaling network and the emerging role of checkpoint targets, especially checkpoint kinase 1, in cancer therapy. Further, we discuss the results of recent clinical trials involving the nonspecific checkpoint kinase 1 inhibitor, UCN-01, and the challenges we face with this new therapeutic approach.

  13. Phosphatidylinositol 4-kinases: Function, structure, and inhibition

    SciTech Connect

    Boura, Evzen Nencka, Radim

    2015-10-01

    The phosphatidylinositol 4-kinases (PI4Ks) synthesize phosphatidylinositol 4-phosphate (PI4P), a key member of the phosphoinositide family. PI4P defines the membranes of Golgi and trans-Golgi network (TGN) and regulates trafficking to and from the Golgi. Humans have two type II PI4Ks (α and β) and two type III enzymes (α and β). Recently, the crystal structures were solved for both type II and type III kinase revealing atomic details of their function. Importantly, the type III PI4Ks are hijacked by +RNA viruses to create so-called membranous web, an extensively phosphorylated and modified membrane system dedicated to their replication. Therefore, selective and potent inhibitors of PI4Ks have been developed as potential antiviral agents. Here we focus on the structure and function of PI4Ks and their potential in human medicine.

  14. MAP kinases phosphorylate rice WRKY45.

    PubMed

    Ueno, Yoshihisa; Yoshida, Riichiro; Kishi-Kaboshi, Mitsuko; Matsushita, Akane; Jiang, Chang-Jie; Goto, Shingo; Takahashi, Akira; Hirochika, Hirohiko; Takatsuji, Hiroshi

    2013-06-01

    WRKY45 transcription factor is a central regulator of disease resistance mediated by the salicylic acid (SA) signaling pathway in rice. SA-activated WRKY45 protein induces the accumulation of its own mRNA. However, the mechanism underlying this regulation is still unknown. Here, we report three lines of evidence showing that a mitogen-activated protein kinase (MAPK) cascade is involved in this regulation. An inhibitor of MAPK kinase (MAPKK) suppressed the increase in WRKY45 transcript level in response to SA. Two MAPKs, OsMPK4 and OsMPK6, phosphorylated WRKY45 protein in vitro. The activity of OsMPK6 was rapidly upregulated by SA treatment in rice cells. These results suggest that WRKY45 is regulated by MAPK-dependent phosphorylation in the SA pathway.

  15. Protein Kinase C Isozyme in Mammary Carcinogenesis.

    DTIC Science & Technology

    1996-10-01

    11 A B Clone 72 Clone 34 AKAP 86 Clone 35H CInn 72 lonn 4 P 95 lone 351 9. 5J 4.4- 688 431 Clone 45 Clone 35F Clone 64 Annexln 1 Clone 45 Clone 35F...purified antibodies directed against a variety of PKC substrates and an A-kinase anchoring protein, AKAP 95. (B) PolyA+ mRNAs isolated from confluent cell

  16. Molecular Imaging of the ATM Kinase Activity

    SciTech Connect

    Williams, Terence M.; Nyati, Shyam; Ross, Brian D.; Rehemtulla, Alnawaz

    2013-08-01

    Purpose: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks. ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and noninvasively measures ATM kinase activity in living cells and subjects. Methods and Materials: Using the split luciferase technology, we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence. Results: Treatment of ATMR-expressing cells with ATM inhibitors resulted in a dose-dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, whereas a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and small interfering (si)RNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models that correlated in a time-dependent fashion with changes in Chk2 activity. Conclusions: We describe the development and validation of a novel, specific, noninvasive bioluminescent reporter that enables monitoring of ATM activity in real time, in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in preclinical models.

  17. Nuclear glycogen and glycogen synthase kinase 3.

    PubMed

    Ragano-Caracciolo, M; Berlin, W K; Miller, M W; Hanover, J A

    1998-08-19

    Glycogen is the principal storage form of glucose in animal cells. It accumulates in electron-dense cytoplasmic granules and is synthesized by glycogen synthase (GS), the rate-limiting enzyme of glycogen deposition. Glycogen synthase kinase-3 (GSK-3) is a protein kinase that phosphorylates GS. Two nearly identical forms of GSK-3 exist: GSK-3 alpha and GSK-3 beta. Both are constitutively active in resting cells and their activity can be modulated by hormones and growth factors. GSK-3 is implicated in the regulation of many physiological responses in mammalian cells by phosphorylating substrates including neuronal cell adhesion molecule, neurofilaments, synapsin I, and tau. Recent observations point to functions for glycogen and glycogen metabolism in the nucleus. GSK-3 phosphorylates several transcription factors, and we have recently shown that it modifies the major nuclear pore protein p62. It also regulates PK1, a protein kinase required for maintaining the interphase state and for DNA replication in cycling Xenopus egg extracts. Recently, glycogen was shown to be required for nuclear reformation in vitro using ovulated Xenopus laevis egg lysates. Because neither glycogen nor GSK-3 has been localized to the nuclear envelope or intranuclear sites, glycogen and GSK-3 activites were measured in rat liver nuclei and nuclear reformation extracts. Significant quantities of glycogen-like material co-purified with the rat-liver nuclear envelope. GSK-3 is also highly enriched in the glycogen pellet of egg extracts of Xenopus that is required for nuclear assembly in vitro. Based on the finding that enzymes of glycogen metabolism copurify with glycogen, we propose that glycogen may serve a structural role as a scaffold for nuclear assembly and sequestration of critical kinases and phosphatases in the nucleus. Copyright 1998 Academic Press.

  18. Approach to asymptomatic creatine kinase elevation

    PubMed Central

    MOGHADAM-KIA, SIAMAK; ODDIS, CHESTER V.; AGGARWAL, ROHIT

    2016-01-01

    How to manage a patient who has an elevated serum creatine kinase (CK) level but no or insignificant muscle-related signs and symptoms is a clinical conundrum. The authors provide a systematic approach, including repeat testing after a period of rest, defining higher thresholds over which pursuing a diagnosis is worthwhile, and evaluating for a variety of nonneuromuscular causes. They also outline a workup for neuromuscular causes. PMID:26760521

  19. Sustained oscillations in the MAP kinase cascade.

    PubMed

    Hell, Juliette; Rendall, Alan D

    2016-10-29

    The MAP kinase cascade is a network of enzymatic reactions arranged in layers. In each layer occurs a multiple futile cycle of phosphorylations. The fully phosphorylated substrate then serves as an enzyme for the layer below. This paper focusses on the existence of parameters for which Hopf bifurcations occur and generate periodic orbits. Furthermore it is explained how geometric singular perturbation theory allows to generalize results from simple models to more complex ones. Copyright © 2016. Published by Elsevier Inc.

  20. Cell cycle dependent regulation of deoxycytidine kinase, deoxyguanosine kinase, and cytosolic 5'-nucleotidase I activity in MOLT-4 cells.

    PubMed

    Fyrberg, A; Mirzaee, S; Lotfi, K

    2006-01-01

    Activation of nucleoside analogues is dependent on kinases and 5'-nucleotidases and the balance between the activity of these enzymes. The purpose of this study was to analyze deoxycytidine kinase, deoxyguanosine kinase, and 4 different 5'-nucleotidases during cell cycle progression in MOLT-4 cells. The activity of both kinases was cell cycle dependent and increased during proliferation while the activity of cytosolic 5'-nucleotidase I decreased. We could show that the kinase activity was higher than the total nucleotidase activity, which was unchanged or decreased during cell cycle progression. These data may be important in designing modern combination therapy with nucleoside analogues.

  1. Classifying kinase conformations using a machine learning approach.

    PubMed

    McSkimming, Daniel Ian; Rasheed, Khaled; Kannan, Natarajan

    2017-02-02

    Signaling proteins such as protein kinases adopt a diverse array of conformations to respond to regulatory signals in signaling pathways. Perhaps the most fundamental conformational change of a kinase is the transition between active and inactive states, and defining the conformational features associated with kinase activation is critical for selectively targeting abnormally regulated kinases in diseases. While manual examination of crystal structures have led to the identification of key structural features associated with kinase activation, the large number of kinase crystal structures (~3,500) and extensive conformational diversity displayed by the protein kinase superfamily poses unique challenges in fully defining the conformational features associated with kinase activation. Although some computational approaches have been proposed, they are typically based on a small subset of crystal structures using measurements biased towards the active site geometry. We utilize an unbiased informatics based machine learning approach to classify all eukaryotic protein kinase conformations deposited in the PDB. We show that the orientation of the activation segment, measured by φ, ψ, χ1, and pseudo-dihedral angles more accurately classify kinase crystal conformations than existing methods. We show that the formation of the K-E salt bridge is statistically dependent upon the activation segment orientation and identify evolutionary differences between the activation segment conformation of tyrosine and serine/threonine kinases. We provide evidence that our method can identify conformational changes associated with the binding of allosteric regulatory proteins, and show that the greatest variation in inactive structures comes from kinase group and family specific side chain orientations. We have provided the first comprehensive machine learning based classification of protein kinase active/inactive conformations, taking into account more structures and measurements than

  2. Prediction of 492 human protein kinase substrate specificities.

    PubMed

    Safaei, Javad; Maňuch, Ján; Gupta, Arvind; Stacho, Ladislav; Pelech, Steven

    2011-10-14

    Complex intracellular signaling networks monitor diverse environmental inputs to evoke appropriate and coordinated effector responses. Defective signal transduction underlies many pathologies, including cancer, diabetes, autoimmunity and about 400 other human diseases. Therefore, there is high impetus to define the composition and architecture of cellular communications networks in humans. The major components of intracellular signaling networks are protein kinases and protein phosphatases, which catalyze the reversible phosphorylation of proteins. Here, we have focused on identification of kinase-substrate interactions through prediction of the phosphorylation site specificity from knowledge of the primary amino acid sequence of the catalytic domain of each kinase. The presented method predicts 488 different kinase catalytic domain substrate specificity matrices in 478 typical and 4 atypical human kinases that rely on both positive and negative determinants for scoring individual phosphosites for their suitability as kinase substrates. This represents a marked advancement over existing methods such as those used in NetPhorest (179 kinases in 76 groups) and NetworKIN (123 kinases), which consider only positive determinants for kinase substrate prediction. Comparison of our predicted matrices with experimentally-derived matrices from about 9,000 known kinase-phosphosite substrate pairs revealed a high degree of concordance with the established preferences of about 150 well studied protein kinases. Furthermore for many of the better known kinases, the predicted optimal phosphosite sequences were more accurate than the consensus phosphosite sequences inferred by simple alignment of the phosphosites of known kinase substrates. Application of this improved kinase substrate prediction algorithm to the primary structures of over 23, 000 proteins encoded by the human genome has permitted the identification of about 650, 000 putative phosphosites, which are posted on the

  3. Creatine kinase in ischemic and inflammatory disorders.

    PubMed

    Kitzenberg, David; Colgan, Sean P; Glover, Louise E

    2016-12-01

    The creatine/phosphocreatine pathway plays a conserved and central role in energy metabolism. Compartmentalization of specific creatine kinase enzymes permits buffering of local high energy phosphates in a thermodynamically favorable manner, enabling both rapid energy storage and energy transfer within the cell. Augmentation of this metabolic pathway by nutritional creatine supplementation has been shown to elicit beneficial effects in a number of diverse pathologies, particularly those that incur tissue ischemia, hypoxia or oxidative stress. In these settings, creatine and phosphocreatine prevent depletion of intracellular ATP and internal acidification, enhance post-ischemic recovery of protein synthesis and promote free radical scavenging and stabilization of cellular membranes. The creatine kinase energy system is itself further regulated by hypoxic signaling, highlighting the existence of endogenous mechanisms in mammals that can enhance creatine metabolism during oxygen deprivation to promote tissue resolution and homeostasis. Here, we review recent insights into the creatine kinase pathway, and provide rationale for dietary creatine supplementation in human ischemic and inflammatory pathologies.

  4. Phosphatidylinositol kinase activities in Trypanosoma cruzi epimastigotes.

    PubMed

    Gimenez, Alba Marina; Gesumaría, María Celeste; Schoijet, Alejandra C; Alonso, Guillermo D; Flawiá, Mirtha M; Racagni, Graciela E; Machado, Estela E

    2015-01-01

    Phosphatidylinositol (PtdIns) metabolism through phosphatidylinositol kinase (PIKs) activities plays a central role in different signaling pathways. In Trypanosoma cruzi, causative agent of Chagas disease, PIKs have been proposed as target for drug design in order to combat this pathogen. In this work, we studied the classes of PI4K, PIPK and PI3K that could participate in signaling pathways in T. cruzi epimastigote forms. For this reason, we analyzed their enzymatic parameters and detailed responses to avowed kinase inhibitors (adenosine, sodium deoxycholate, wortmannin and LY294002) and activators (Ca(2+), phosphatidic acid, spermine and heparin). Our results suggest the presence and activity of a class III PI4K, a class I PIPK, a class III PI3K previously described (TcVps34) and a class I PI3K. Class I PI3K enzyme, here named TcPI3K, was cloned and expressed in a bacterial system, and their product was tested for kinase activity. The possible participation of TcPI3K in central cellular events of the parasite is also discussed.

  5. Hexose-6-kinases in germinating honey locust cotyledons: substrate specificity of D-fructo-6-kinase.

    PubMed

    Myers, D; Matheson, N K

    1994-11-01

    Extracts of the cotyledons of germinated honey locust (Gleditsia triacanthos) seeds, which contain galactomannan as a reserve polysaccharide in the endosperm, were fractionated by chromatography and the fractions examined for the presence of a specific manno-6-kinase which could phosphorylate the D-mannose released by hydrolysis of galactomannan. One particulate hexokinase (the major hexose-6-kinase fraction) and two soluble hexokinase fractions (the minor portion), as well as a soluble fructo-6-kinase fraction, were initially separated. From chromatography, electrophoresis and kinetic studies, no evidence for a specific manno-kinase was obtained. This and the level and kinetic behaviour of the particulate hexokinase implicated it as the enzyme catalysing the phosphorylation of released D-mannose. The fructo-kinase activity was further separated into three fractions. Kinetic studies on one of these with native and synthetic substrates indicated that the structural requirements for the monosaccharide substrate were a beta-D-anomeric 2-OH in the furanose ring, a 4-OH trans to the D-5-CH2OH and a -CH2OH substituent on C2 (trans to the 5-CH2OH) which could be modified. The orientation of the hydroxyl on C-3 had only a limited effect.

  6. Pea DNA Topoisomerase I Is Phosphorylated and Stimulated by Casein Kinase 2 and Protein Kinase C

    PubMed Central

    Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

    2003-01-01

    DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg2+-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants. PMID:12913165

  7. Protein kinase Calpha activation by RET: evidence for a negative feedback mechanism controlling RET tyrosine kinase.

    PubMed

    Andreozzi, Francesco; Melillo, Rosa Marina; Carlomagno, Francesca; Oriente, Francesco; Miele, Claudia; Fiory, Francesca; Santopietro, Stefania; Castellone, Maria Domenica; Beguinot, Francesco; Santoro, Massimo; Formisano, Pietro

    2003-05-15

    We have studied the role of protein kinase C (PKC) in signaling of the RET tyrosine kinase receptor. By using a chimeric receptor (E/R) in which RET kinase can be tightly controlled by the addition of epidermal growth factor (EGF), we have found that RET triggering induces a strong increase of PKCalpha, PKCdelta and PKCzeta activity and that PKCalpha, not PKCdelta and PKCzeta, forms a ligand-dependent protein complex with E/R. We have identified tyrosine 1062 in the RET carboxyl-terminal tail as the docking site for PKCalpha. Block of PKC activity by bisindolylmaleimide or chronic phorbol esters treatment decreased EGF-induced serine/threonine phosphorylation of E/R, while it caused a similarly sized increase of EGF-induced E/R tyrosine kinase activity and mitogenic signaling. Conversely, acute phorbol esters treatment, which promotes PKC activity, increased the levels of E/R serine/threonine phosphorylation and significantly decreased its phosphotyrosine content. A threefold reduction of tyrosine phosphorylation levels of the constitutively active RET/MEN2A oncoprotein was observed upon coexpression with PKCalpha. We conclude that RET binds to and activates PKCalpha. PKCalpha, in turn, causes RET phosphorylation and downregulates RET tyrosine kinase and downstream signaling, thus functioning as a negative feedback loop to modulate RET activity.

  8. A-kinase Anchoring Protein 79/150 Recruits Protein Kinase C to Phosphorylate Roundabout Receptors.

    PubMed

    Samelson, Bret K; Gore, Bryan B; Whiting, Jennifer L; Nygren, Patrick J; Purkey, Alicia M; Colledge, Marcie; Langeberg, Lorene K; Dell'Acqua, Mark L; Zweifel, Larry S; Scott, John D

    2015-05-29

    Anchoring proteins direct protein kinases and phosphoprotein phosphatases toward selected substrates to control the efficacy, context, and duration of neuronal phosphorylation events. The A-kinase anchoring protein AKAP79/150 interacts with protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2B (calcineurin) to modulate second messenger signaling events. In a mass spectrometry-based screen for additional AKAP79/150 binding partners, we have identified the Roundabout axonal guidance receptor Robo2 and its ligands Slit2 and Slit3. Biochemical and cellular approaches confirm that a linear sequence located in the cytoplasmic tail of Robo2 (residues 991-1070) interfaces directly with sites on the anchoring protein. Parallel studies show that AKAP79/150 interacts with the Robo3 receptor in a similar manner. Immunofluorescent staining detects overlapping expression patterns for murine AKAP150, Robo2, and Robo3 in a variety of brain regions, including hippocampal region CA1 and the islands of Calleja. In vitro kinase assays, peptide spot array mapping, and proximity ligation assay staining approaches establish that human AKAP79-anchored PKC selectively phosphorylates the Robo3.1 receptor subtype on serine 1330. These findings imply that anchored PKC locally modulates the phosphorylation status of Robo3.1 in brain regions governing learning and memory and reward. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Cellular trafficking of the IL-1RI-associated kinase-1 requires intact kinase activity

    SciTech Connect

    Boel, Gaby-Fleur . E-mail: boel@mail.dife.de; Jurrmann, Nadine; Brigelius-Flohe, Regina

    2005-06-24

    Upon stimulation of cells with interleukin-1 (IL-1) the IL-1 receptor type I (IL-1RI) associated kinase-1 (IRAK-1) transiently associates to and dissociates from the IL-1RI and thereafter translocates into the nucleus. Here we show that nuclear translocation of IRAK-1 depends on its kinase activity since translocation was not observed in EL-4 cells overexpressing a kinase negative IRAK-1 mutant (EL-4{sup IRAK-1-K239S}). IRAK-1 itself, an endogenous substrate with an apparent molecular weight of 24 kDa (p24), and exogenous substrates like histone and myelin basic protein are phosphorylated by nuclear located IRAK-1. Phosphorylation of p24 cannot be detected in EL-4{sup IRAK-1-K239S} cells. IL-1-dependent recruitment of IRAK-1 to the IL-1RI and subsequent phosphorylation of IRAK-1 is a prerequisite for nuclear translocation of IRAK-1. It is therefore concluded that intracellular localization of IRAK-1 depends on its kinase activity and that IRAK-1 may also function as a kinase in the nucleus as shown by a new putative endogenous substrate.

  10. beta-subunits of Snf1 kinase are required for kinase function and substrate definition.

    PubMed

    Schmidt, M C; McCartney, R R

    2000-09-15

    The Snf1 kinase and its mammalian homolog, the AMP-activated protein kinase, are heterotrimeric enzymes composed of a catalytic alpha-subunit, a regulatory gamma-subunit and a beta-subunit that mediates heterotrimer formation. Saccharomyces cerevisiae encodes three beta-subunit genes, SIP1, SIP2 and GAL83. Earlier studies suggested that these subunits may not be required for Snf1 kinase function. We show here that complete and precise deletion of all three beta-subunit genes inactivates the Snf1 kinase. The sip1Delta sip2Delta gal83Delta strain is unable to derepress invertase, grows poorly on alternative carbon sources and fails to direct the phosphorylation of the Mig1 and Sip4 proteins in vivo. The SIP1 sip2Delta gal83Delta strain manifests a subset of Snf phenotypes (Raf(+), Gly(-)) observed in the snf1Delta 10 strain (Raf(-), Gly(-)), suggesting that individual beta-subunits direct the Snf1 kinase to a subset of its targets in vivo. Indeed, deletion of individual beta-subunit genes causes distinct differences in the induction and phosphorylation of Sip4, strongly suggesting that the beta-subunits play an important role in substrate definition.

  11. G protein-coupled receptor kinases: more than just kinases and not only for GPCRs

    PubMed Central

    Gurevich, Eugenia V.; Tesmer, John J. G.; Mushegian, Arcady; Gurevich, Vsevolod V.

    2011-01-01

    G protein-coupled receptor (GPCR) kinases (GRKs) are best known for their role in homologous desensitization of GPCRs. GRKs phosphorylate activated receptors and promote high affinity binding of arrestins, which precludes G protein coupling. GRKs have a multidomain structure, with the kinase domain inserted into a loop of a regulator of G protein signaling homology domain. Unlike many other kinases, GRKs do not need to be phosphorylated in their activation loop to achieve an activated state. Instead, they are directly activated by docking with active GPCRs. In this manner they are able to selectively phosphorylate Ser/Thr residues on only the activated form of the receptor, unlike related kinases such as protein kinase A. GRKs also phosphorylate a variety of non-GPCR substrates and regulate several signaling pathways via direct interactions with other proteins in a phosphorylation-independent manner. Multiple GRK subtypes are present in virtually every animal cell, with the highest expression levels found in neurons, with their extensive and complex signal regulation. Insufficient or excessive GRK activity was implicated in a variety of human disorders, ranging from heart failure to depression to Parkinson’s disease. As key regulators of GPCR-dependent and -independent signaling pathways, GRKs are emerging drug targets and promising molecular tools for therapy. Targeted modulation of expression and/or of activity of several GRK isoforms for therapeutic purposes was recently validated in cardiac disorders and Parkinson’s disease. PMID:21903131

  12. Conservation and Early Expression of Zebrafish Tyrosine Kinases Support the Utility of Zebrafish as a Model for Tyrosine Kinase Biology

    PubMed Central

    Challa, Anil Kumar

    2013-01-01

    Abstract Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome. PMID:23234507

  13. Conservation and early expression of zebrafish tyrosine kinases support the utility of zebrafish as a model for tyrosine kinase biology.

    PubMed

    Challa, Anil Kumar; Chatti, Kiranam

    2013-09-01

    Tyrosine kinases have significant roles in cell growth, apoptosis, development, and disease. To explore the use of zebrafish as a vertebrate model for tyrosine kinase signaling and to better understand their roles, we have identified all of the tyrosine kinases encoded in the zebrafish genome and quantified RNA expression of selected tyrosine kinases during early development. Using profile hidden Markov model analysis, we identified 122 zebrafish tyrosine kinase genes and proposed unambiguous gene names where needed. We found them to be organized into 39 nonreceptor and 83 receptor type, and 30 families consistent with human tyrosine kinase family assignments. We found five human tyrosine kinase genes (epha1, bmx, fgr, srm, and insrr) with no identifiable zebrafish ortholog, and one zebrafish gene (yrk) with no identifiable human ortholog. We also found that receptor tyrosine kinase genes were duplicated more often than nonreceptor tyrosine kinase genes in zebrafish. We profiled expression levels of 30 tyrosine kinases representing all families using direct digital detection at different stages during the first 24 hours of development. The profiling experiments clearly indicate regulated expression of tyrosine kinases in the zebrafish, suggesting their role during early embryonic development. In summary, our study has resulted in the first comprehensive description of the zebrafish tyrosine kinome.

  14. Characterization of irreversible kinase inhibitors by directly detecting covalent bond formation: a tool for dissecting kinase drug resistance.

    PubMed

    Klüter, Sabine; Simard, Jeffrey R; Rode, Haridas B; Grütter, Christian; Pawar, Vijaykumar; Raaijmakers, Hans C A; Barf, Tjeerd A; Rabiller, Matthias; van Otterlo, Willem A L; Rauh, Daniel

    2010-12-10

    Targeting protein kinases in cancer therapy with irreversible small-molecule inhibitors is moving to the forefront of kinase-inhibitor research and is thought to be an effective means of overcoming mutation-associated drug resistance in epidermal growth factor receptor kinase (EGFR). We generated a detection technique that allows direct measurements of covalent bond formation without relying on kinase activity, thereby allowing the straightforward investigation of the influence of steric clashes on covalent inhibitors in different resistant kinase mutants. The obtained results are discussed together with structural biology and biochemical studies of catalytic activity in both wild-type and gatekeeper mutated kinase variants to draw conclusions about the impact of steric hindrance and increased catalytic activity in drug-resistant kinase variants.

  15. Design of an encodable tyrosine kinase-inducible domain: detection of tyrosine kinase activity by terbium luminescence.

    PubMed

    Zondlo, Susan Carr; Gao, Feng; Zondlo, Neal J

    2010-04-28

    Tyrosine kinases are critical mediators of intracellular signaling and of intracellular responses to extracellular signaling. Changes in tyrosine kinase activity are implicated in numerous human diseases, including cancers, diabetes, and pathogen infectivity. To address questions in tyrosine phosphorylation, we have designed a protein tyrosine kinase-inducible domain, a small, genetically encodable protein motif whose structure is dependent on its tyrosine phosphorylation state. Tyrosine kinase-inducible domain peptides are based on EF-hand loops in which a structurally critical Glu12 residue is replaced by tyrosine at residue 11 or at residue 15 of the protein. Tyrosine kinase-inducible domain peptides bind terbium(III) in a phosphorylation-dependent manner, showing strong terbium luminescence when phosphorylated but weak terbium luminescence when not phosphorylated. Lanthanide binding was confirmed by NMR. A tyrosine kinase-inducible domain peptide, pKID-Abl, was designed to incorporate a recognition sequence of the Abl kinase. Incubation of pKID-Abl with Abl kinase resulted in a large increase in terbium luminescence. This increase in luminescence was abolished when pKID-Abl and Abl kinase were incubated with the Abl kinase inhibitor Gleevec. In addition, incubation of phosphorylated pKID-Abl with the tyrosine phosphatase YOP resulted in a large reduction in terbium luminescence. pKID-Abl was employed as a fluorescent sensor of Abl tyrosine kinase activity in HeLa cell extracts, exhibiting low luminescence with extracts from serum-starved cells and increased luminescence using extracts from EGF-treated cells. These results indicate that tyrosine kinase-inducible domains may be used as sensors of tyrosine kinase and tyrosine phosphatase activity and in the detection of tyrosine kinase inhibitors.

  16. Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis.

    PubMed

    Pillai, Smitha; Nguyen, Jonathan; Johnson, Joseph; Haura, Eric; Coppola, Domenico; Chellappan, Srikumar

    2015-12-10

    TANK Binding Kinase 1 (TBK1) is a non-canonical IκB kinase that contributes to KRAS-driven lung cancer. Here we report that TBK1 plays essential roles in mammalian cell division. Specifically, levels of active phospho-TBK1 increase during mitosis and localize to centrosomes, mitotic spindles and midbody, and selective inhibition or silencing of TBK1 triggers defects in spindle assembly and prevents mitotic progression. TBK1 binds to the centrosomal protein CEP170 and to the mitotic apparatus protein NuMA, and both CEP170 and NuMA are TBK1 substrates. Further, TBK1 is necessary for CEP170 centrosomal localization and binding to the microtubule depolymerase Kif2b, and for NuMA binding to dynein. Finally, selective disruption of the TBK1-CEP170 complex augments microtubule stability and triggers defects in mitosis, suggesting that TBK1 functions as a mitotic kinase necessary for microtubule dynamics and mitosis.

  17. Mitogen-Activated Protein Kinases and Mitogen Kinase Phosphatase 1: A Critical Interplay in Macrophage Biology

    PubMed Central

    Lloberas, Jorge; Valverde-Estrella, Lorena; Tur, Juan; Vico, Tania; Celada, Antonio

    2016-01-01

    Macrophages are necessary in multiple processes during the immune response or inflammation. This review emphasizes the critical role of the mitogen-activated protein kinases (MAPKs) and mitogen kinase phosphatase-1 (MKP-1) in the functional activities of macrophages. While the phosphorylation of MAPKs is required for macrophage activation or proliferation, MKP-1 dephosphorylates these kinases, thus playing a balancing role in the control of macrophage behavior. MKP-1 is a nuclear-localized dual-specificity phosphatase whose expression is regulated at multiple levels, including at the transcriptional and post-transcriptional level. The regulatory role of MKP-1 in the interplay between MAPK phosphorylation/dephosphorylation makes this molecule a critical regulator of macrophage biology and inflammation. PMID:27446931

  18. Targeting Spleen Tyrosine Kinase-Bruton's Tyrosine Kinase Axis for Immunologically Mediated Glomerulonephritis

    PubMed Central

    Chen, Jin-Shuen; Chang, Li-Chien; Huang, Shyh-Jer

    2014-01-01

    The importance of B-cell activation and immune complex-mediated Fc-receptor activation in the pathogenesis of immunologically mediated glomerulonephritis has long been recognized. The two nonreceptor tyrosine kinases, spleen tyrosine kinase (Syk) and Bruton's tyrosine kinase (Btk), are primarily expressed by hematopoietic cells, and participate in B-cell-receptor- and Fc-receptor-mediated activation. Pharmacological inhibitors of Syk or Btk are undergoing preclinical development and clinical trials for several immune diseases; and Syk inhibitors have been shown to reduce disease activity in rheumatoid arthritis patients. However, the clinical therapeutic efficacies of these inhibitors in glomerulonephritis have not been evaluated. Herein, we review recent studies of Syk and Btk inhibitors in several experimental primary and secondary glomerulonephritis models. These inhibitors suppressed development of glomerular injury, and also ameliorated established kidney disease. Thus, targeting Syk and Btk signaling pathways is a potential therapeutic strategy for glomerulonephritis, and further evaluation is recommended. PMID:24795896

  19. The association of phosphoinositide 3-kinase enhancer A with hepatic insulin receptor enhances its kinase activity.

    PubMed

    Chan, Chi Bun; Liu, Xia; He, Kunyan; Qi, Qi; Jung, Dae Y; Kim, Jason K; Ye, Keqiang

    2011-07-01

    Dysfunction of hepatic insulin receptor tyrosine kinase (IRTK) causes the development of type 2 diabetes. However, the molecular mechanism regulating IRTK activity in the liver remains poorly understood. Here, we show that phosphoinositide 3-kinase enhancer A (PIKE-A) is a new insulin-dependent enhancer of hepatic IRTK. Liver-specific Pike-knockout (LPKO) mice display glucose intolerance with impaired hepatic insulin sensitivity. Specifically, insulin-provoked phosphoinositide 3-kinase/Akt signalling is diminished in the liver of LPKO mice, leading to the failure of insulin-suppressed gluconeogenesis and hyperglycaemia. Thus, hepatic PIKE-A has a key role in mediating insulin signal transduction and regulating glucose homeostasis in the liver.

  20. Targeting the phosphoinositide 3-kinase pathway in hematologic malignancies

    PubMed Central

    Jabbour, Elias; Ottmann, Oliver G.; Deininger, Michael; Hochhaus, Andreas

    2014-01-01

    The phosphoinositide 3-kinase pathway represents an important anticancer target because it has been implicated in cancer cell growth, survival, and motility. Recent studies show that PI3K may also play a role in the development of resistance to currently available therapies. In a broad range of cancers, various components of the phosphoinositide 3-kinase signaling axis are genetically modified, and the pathway can be activated through many different mechanisms. The frequency of genetic alterations in the phosphoinositide 3-kinase pathway, coupled with the impact in oncogenesis and disease progression, make this signaling axis an attractive target in anticancer therapy. A better understanding of the critical function of the phosphoinositide 3-kinase pathway in leukemias and lymphomas has led to the clinical evaluation of novel rationally designed inhibitors in this setting. Three main categories of phosphoinositide 3-kinase inhibitors have been developed so far: agents that target phosphoinositide 3-kinase and mammalian target of rapamycin (dual inhibitors), pan-phosphoinositide 3-kinase inhibitors that target all class I isoforms, and isoform-specific inhibitors that selectively target the α, -β, -γ, or -δ isoforms. Emerging data highlight the promise of phosphoinositide 3-kinase inhibitors in combination with other therapies for the treatment of patients with hematologic malignancies. Further evaluation of phosphoinositide 3-kinase inhibitors in first-line or subsequent regimens may improve clinical outcomes. This article reviews the role of phosphoinositide 3-kinase signaling in hematologic malignancies and the potential clinical utility of inhibitors that target this pathway. PMID:24425689

  1. The Structure of Lombricine Kinase: Implications for Phosphagen Conformational Changes

    SciTech Connect

    Bush, D. Jeffrey; Kirillova, Olga; Clark, Shawn A.; Davulcu, Omar; Fabiola, Felcy; Xie, Qing; Somasundaram, Thayumanasamy; Ellington, W. Ross; Chapman, Michael S.

    2012-05-29

    Lombricine kinase is a member of the phosphagen kinase family and a homolog of creatine and arginine kinases, enzymes responsible for buffering cellular ATP levels. Structures of lombricine kinase from the marine worm Urechis caupo were determined by x-ray crystallography. One form was crystallized as a nucleotide complex, and the other was substrate-free. The two structures are similar to each other and more similar to the substrate-free forms of homologs than to the substrate-bound forms of the other phosphagen kinases. Active site specificity loop 309-317, which is disordered in substrate-free structures of homologs and is known from the NMR of arginine kinase to be inherently dynamic, is resolved in both lombricine kinase structures, providing an improved basis for understanding the loop dynamics. Phosphagen kinases undergo a segmented closing on substrate binding, but the lombricine kinase ADP complex is in the open form more typical of substrate-free homologs. Through a comparison with prior complexes of intermediate structure, a correlation was revealed between the overall enzyme conformation and the substrate interactions of His{sup 178}. Comparative modeling provides a rationale for the more relaxed specificity of these kinases, of which the natural substrates are among the largest of the phosphagen substrates.

  2. The three-dimensional structure of shikimate kinase.

    PubMed

    Krell, T; Coggins, J R; Lapthorn, A J

    1998-05-22

    The three-dimensional structure of shikimate kinase from Erwinia chrysanthemi has been determined by multiple isomorphous replacement. Two models are presented: a high resolution 1.9 A model and a 2.6 A model which contains bound Mg-ADP. The enzyme is an alpha/beta protein consisting of a central sheet of five parallel beta-strands flanked by alpha-helices with overall topology similar to adenylate kinase. Evidence is presented that shikimate kinase undergoes major conformational changes on ligand binding. It resembles adenylate kinase in having a P-loop containing core structure and two flexible domains which undergo induced fit movement on substrate binding. The binding of Mg2+ in the active site of shikimate kinase involves direct interaction with two protein side-chains which is different from the situation found in adenylate kinase. Shikimate kinase has a readily identifiable Walker A-motif and a recognisable but modified Walker B-motif. Comparison of shikimate kinase to adenylate kinase has led to the identification of an adenine-binding motif (I/VDAXQ/NXP). Difference Fourier calculations have revealed the shikimate binding site which corresponds to the location of the AMP-binding site in adenylate kinase. A model for shikimate-binding is presented.

  3. A framework for classification of prokaryotic protein kinases.

    PubMed

    Tyagi, Nidhi; Anamika, Krishanpal; Srinivasan, Narayanaswamy

    2010-05-26

    Overwhelming majority of the Serine/Threonine protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Serine/Threonine protein kinases recognized from genomes of prokaryotes have been used to develop a classification framework for prokaryotic Ser/Thr protein kinases. We have used traditional sequence alignment and phylogenetic approaches and clustered the prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence database we recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, we also identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular organization which indicates a degree of complexity and protein-protein interactions in the

  4. A Framework for Classification of Prokaryotic Protein Kinases

    PubMed Central

    Tyagi, Nidhi; Anamika, Krishanpal; Srinivasan, Narayanaswamy

    2010-01-01

    Background Overwhelming majority of the Serine/Threonine protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Serine/Threonine protein kinases recognized from genomes of prokaryotes have been used to develop a classification framework for prokaryotic Ser/Thr protein kinases. Methodology/Principal Findings We have used traditional sequence alignment and phylogenetic approaches and clustered the prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence database we recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, we also identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. Conclusion/Significance Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular organization which indicates a

  5. Unveiling the Novel Dual Specificity Protein Kinases in Bacillus anthracis

    PubMed Central

    Arora, Gunjan; Sajid, Andaleeb; Arulanandh, Mary Diana; Singhal, Anshika; Mattoo, Abid R.; Pomerantsev, Andrei P.; Leppla, Stephen H.; Maiti, Souvik; Singh, Yogendra

    2012-01-01

    Dual specificity protein kinases (DSPKs) are unique enzymes that can execute multiple functions in the cell, which are otherwise performed exclusively by serine/threonine and tyrosine protein kinases. In this study, we have characterized the protein kinases Bas2152 (PrkD) and Bas2037 (PrkG) from Bacillus anthracis. Transcriptional analyses of these kinases showed that they are expressed in all phases of growth. In a serendipitous discovery, both kinases were found to be DSPKs. PrkD was found to be similar to the eukaryotic dual specificity Tyr phosphorylation-regulated kinase class of dual specificity kinases, which autophosphorylates on Ser, Thr, and Tyr residues and phosphorylates Ser and Thr residues on substrates. PrkG was found to be a bona fide dual specificity protein kinase that mediates autophosphorylation and substrate phosphorylation on Ser, Thr, and Tyr residues. The sites of phosphorylation in both of the kinases were identified through mass spectrometry. Phosphorylation on Tyr residues regulates the kinase activity of PrkD and PrkG. PrpC, the only known Ser/Thr protein phosphatase, was also found to possess dual specificity. Genistein, a known Tyr kinase inhibitor, was found to inhibit the activities of PrkD and PrkG and affect the growth of B. anthracis cells, indicating a possible role of these kinases in cell growth and development. In addition, the glycolytic enzyme pyruvate kinase was found to be phosphorylated by PrkD on Ser and Thr residues but not by PrkG. Thus, this study provides the first evidence of DSPKs in B. anthracis that belong to different classes and have different modes of regulation. PMID:22711536

  6. Polyomavirus middle-T antigen associates with the kinase domain of Src-related tyrosine kinases.

    PubMed Central

    Dunant, N M; Senften, M; Ballmer-Hofer, K

    1996-01-01

    Middle-T antigen of mouse polyomavirus, an oncogenic DNA virus, associates with and activates the cellular tyrosine kinases c-Src, c-Yes, and Fyn. This interaction is essential for polyomavirus-mediated transformation of cells in culture and tumor formation in animals. To determine the domain of c-Src directing association with middle-T, mutant c-Src proteins lacking the amino-terminal unique domain and the myristylation signal, the SH2 domain, the SH3 domain, or all three of these domains were coexpressed with middle-T in NIH 3T3 cells. All mutants were found to associate with middle-T, demonstrating that the kinase domain of c-Src, including the carboxy-terminal regulatory tail, is sufficient for association with middle-T. Moreover, we found that Hck, another member of the Src kinase family, does not bind middle-T, while chimeric kinases consisting of the amino-terminal domains of c-Src fused to the kinase domain of Hck or the amino-terminal domains of Hck fused to the kinase domain of c-Src associated with middle-T. Hck mutated at its carboxy-terminal regulatory residue, tyrosine 501, was also found to associate with middle-T. These results suggest that in Hck, the postulated intramolecular interaction between the carboxy-terminal regulatory tyrosine and the SH2 domain prevents association with middle-T. This intramolecular interaction apparently also limits the ability of c-Src to associate with middle-T, since removal of the SH2 or SH3 domain increases the efficiency with which middle-T binds c-Src. PMID:8627648

  7. Evaluation of a tyrosine kinase peptide microarray for tyrosine kinase inhibitor therapy selection in cancer

    PubMed Central

    Labots, Mariette; Gotink, Kristy J; Dekker, Henk; Azijli, Kaamar; van der Mijn, Johannes C; Huijts, Charlotte M; Piersma, Sander R; Jiménez, Connie R; Verheul, Henk M W

    2016-01-01

    Personalized cancer medicine aims to accurately predict the response of individual patients to targeted therapies, including tyrosine kinase inhibitors (TKIs). Clinical implementation of this concept requires a robust selection tool. Here, using both cancer cell lines and tumor tissue from patients, we evaluated a high-throughput tyrosine kinase peptide substrate array to determine its readiness as a selection tool for TKI therapy. We found linearly increasing phosphorylation signal intensities of peptides representing kinase activity along the kinetic curve of the assay with 7.5–10 μg of lysate protein and up to 400 μM adenosine triphosphate (ATP). Basal kinase activity profiles were reproducible with intra- and inter-experiment coefficients of variation of <15% and <20%, respectively. Evaluation of 14 tumor cell lines and tissues showed similar consistently high phosphorylated peptides in their basal profiles. Incubation of four patient-derived tumor lysates with the TKIs dasatinib, sunitinib, sorafenib and erlotinib primarily caused inhibition of substrates that were highly phosphorylated in the basal profile analyses. Using recombinant Src and Axl kinase, relative substrate specificity was demonstrated for a subset of peptides, as their phosphorylation was reverted by co-incubation with a specific inhibitor. In conclusion, we demonstrated robust technical specifications of this high-throughput tyrosine kinase peptide microarray. These features required as little as 5–7 μg of protein per sample, facilitating clinical implementation as a TKI selection tool. However, currently available peptide substrates can benefit from an enhancement of the differential potential for complex samples such as tumor lysates. We propose that mass spectrometry-based phosphoproteomics may provide such an enhancement by identifying more discriminative peptides. PMID:27980342

  8. A Fluorescence-Based Thermal Shift Assay Identifies Inhibitors of Mitogen Activated Protein Kinase Kinase 4

    PubMed Central

    Krishna, Sankar N.; Luan, Chi-Hao; Mishra, Rama K.; Xu, Li; Scheidt, Karl A.; Anderson, Wayne F.; Bergan, Raymond C.

    2013-01-01

    Prostate cancer (PCa) is the second highest cause of cancer death in United States males. If the metastatic movement of PCa cells could be inhibited, then mortality from PCa could be greatly reduced. Mitogen-activated protein kinase kinase 4 (MAP2K4) has previously been shown to activate pro-invasion signaling pathways in human PCa. Recognizing that MAP2K4 represents a novel and validated therapeutic target, we sought to develop and characterize an efficient process for the identification of small molecules that target MAP2K4. Using a fluorescence-based thermal shift assay (FTS) assay, we first evaluated an 80 compound library of known kinase inhibitors, thereby identifying 8 hits that thermally stabilized MAP2K4 in a concentration dependent manner. We then developed an in vitro MAP2K4 kinase assay employing the biologically relevant downstream substrates, JNK1 and p38 MAPK, to evaluate kinase inhibitory function. In this manner, we validated the performance of our initial FTS screen. We next applied this approach to a 2000 compound chemically diverse library, identified 7 hits, and confirmed them in the in vitro kinase assay. Finally, by coupling our structure-activity relationship data to MAP2K4's crystal structure, we constructed a model for ligand binding. It predicts binding of our identified inhibitory compounds to the ATP binding pocket. Herein we report the creation of a robust inhibitor-screening platform with the ability to inform the discovery and design of new and potent MAP2K4 inhibitors. PMID:24339940

  9. Protein kinase C-associated kinase regulates NF-κB activation through inducing IKK activation.

    PubMed

    Kim, Sang-Woo; Schifano, Matthew; Oleksyn, David; Jordan, Craig T; Ryan, Daniel; Insel, Richard; Zhao, Jiyong; Chen, Luojing

    2014-10-01

    Activation of the transcription factor NF-κB induced by extracellular stimuli requires IKKα and IKKβ kinase activity. How IKKα and IKKβ are activated by various upstream signaling molecules is not fully understood. We previously showed that protein kinase C-associated kinase (PKK, also known as DIK/RIP4), which belongs to the receptor-interacting protein (RIP) kinase family, mediates the B cell activating factor of the TNF family (BAFF)-induced NF-κB activation in diffuse large B cell lymphoma (DLBCL) cell lines. Here we have investigated the mechanism underlying NF-κB activation regulated by PKK. Our results suggest that PKK can activate both the classical and the alternative NF-κB activation pathways. PKK associates with IKKα and IKKβ in mammalian cells and induces activation of both IKKα and IKKβ via phosphorylation of their serine residues 176/180 and 177/181, respectively. Unlike other members of the RIP family that activate NF-κB through a kinase-independent pathway, PKK appears to activate IKK and NF-κB mainly in a kinase-dependent manner. Suppression of PKK expression by RNA interference inhibits phosphorylation of IKKα and IKKβ as well as activation of NF-κB in human cancer cell lines. Thus, PKK regulates NF-κB activation by modulating activation of IKKα and IKKβ in mammalian cells. We propose that PKK may provide a critical link between IKK activation and various upstream signaling cascades, and may represent a potential target for inhibiting abnormal NF-κB activation in human cancers.

  10. Evaluation of the enzyme activity of protozoan protein kinases by using an in vitro kinase assay.

    PubMed

    Kato, Kentaro

    2016-10-01

    The life cycles of parasites are more complicated than those of other biological species. Protein kinases (PKs) encoded by parasites are the main triggers of life stage conversions. Phosphorylation by cellular PKs regulates important cellular processes, and the protozoan genome contains many PKs. Some PK inhibitors inhibit specific parasite life cycle event. In this report, I present a practical approach to expressing and purifying protozoan PKs by using a wheat germ cell-free protein synthesis system and I assess the phosphorylation activities of protozoan PKs by using an in vitro kinase assay. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  11. The elusive activity of the Yersinia protein kinase A kinase domain is revealed.

    PubMed

    Laskowski-Arce, Michelle A; Orth, Kim

    2007-10-01

    Yersinia spp. pathogens use their type III secretion system to translocate effectors that manipulate host signaling pathways during infection. Although molecular targets for five of the six known Yersinia effectors are known, the target for the serine/threonine kinase domain of Yersinia protein kinase A (YpkA) has remained elusive. Recently, Navarro et al. (2007) demonstrated that YpkA phosphorylates Galphaq, and inhibits Galphaq-mediated signaling. Inhibition by YpkA could contribute to one of the most documented symptoms of Yersinia pestis infection, extensive bleeding.

  12. Macro creatine kinase type 1: a cause of spuriously elevated serum creatine kinase associated with leukoencephalopathy in a child.

    PubMed

    Bodensteiner, John B

    2014-07-01

    Macro creatine kinase type 1 is a complex formed by the creatine kinase isoenzyme BB and monoclonal IgG and occurs in about 1% of patients studied. First identified as a cause of spurious elevation of the total serum creatine kinase in patients suspected of myocardial infarction, the test has been largely replaced by the measurement of troponin levels. We present a child with delayed milestones and persistently elevated total serum creatine kinase measurements (∼ 1000-4000 IU) normal electromyogram and brisk myotatic reflexes. Creatine kinase isoenzymes and brain imaging showed the presence of macro creatine kinase type 1 and extensive signal abnormality of the cerebral white matter. Macro creatine kinase type 1 has been associated with several conditions though it has not been described in association with leukoencephalopathy or in patients this young. Macro creatine kinase type 1 can be a cause of elevated total creatine kinase in patients without primary muscle disease. The significance of the relationship of the macro creatine kinase to the leukoencephalopathy in this patient is unknown.

  13. Stimulation of receptor-associated kinase, tyrosine kinase, and MAP kinase is required for prolactin-mediated macromolecular biosynthesis and mitogenesis in Nb2 lymphoma.

    PubMed

    Carey, G B; Liberti, J P

    1995-01-10

    Lactogens [prolactin (Prl) and growth hormone] stimulate phosphorylation of the 40S ribosomal protein, S6, in Nb2 cells by mechanisms that do not involve participation of cAMP or protein kinase A, protein kinase C, or cGMP-dependent protein kinase. However, inhibition of tyrosine kinase (TK) abrogates Prl-mediated macromolecular biosynthesis. Inasmuch as lactogen signaling may involve sequential activation of protein kinases, the effect of Prl on the well-characterized mitogen-activated protein kinase (MAPK) and S6 kinase (S6K), the enzyme responsible for S6 phosphorylation in vivo, and their relationship to Nb2 macromolecular biosynthesis and mitogenesis were investigated. The results show that MAPK stimulation is transient (peak activity, 30 min) and precedes that of S6K, which reaches a maximum at 1.5-2 h, and slowly returns towards control levels at 6 h. Both staurosporine which inhibits GH receptor-associated kinase (JAK2) and genistein (GEN), an inhibitor of membrane-associated and cytoplasmic TKs, abrogate Prl-stimulated TK, MAPK, and S6K. Rapamycin (RAP), a specific inhibitor of p70S6K, completely blocks S6K but does not affect TK and MAPK. TK and MAPK activity correlates with Prl-stimulated anabolism, i.e., protein and DNA synthesis and mitogenesis. Thus, concentrations of STR and GEN which abrogate TK and MAPK inhibit anabolism virtually 100%. However, RAP, which inhibits S6K (ca. 100%) but not TK or MAPK, only delays Prl-mediated anabolism. These results indicate that Prl signaling in Nb2 cells involves a protein kinase cascade and that regulation of receptor-associated kinase, TK, and MAPK correlates with anabolism. The role of S6K (and S6 phosphorylation) appears to be ancillary.

  14. AKAP-Lbc nucleates a protein kinase D activation scaffold.

    PubMed

    Carnegie, Graeme K; Smith, F Donelson; McConnachie, George; Langeberg, Lorene K; Scott, John D

    2004-09-24

    The transmission of cellular signals often proceeds through multiprotein complexes where enzymes are positioned in proximity to their upstream activators and downstream substrates. In this report we demonstrate that the A-kinase anchoring protein AKAP-Lbc assembles an activation complex for the lipid-dependent enzyme protein kinase D (PKD). Using a combination of biochemical, enzymatic, and immunofluorescence techniques, we show that the anchoring protein contributes to PKD activation in two ways: it recruits an upstream kinase PKCeta and coordinates PKA phosphorylation events that release activated protein kinase D. Thus, AKAP-Lbc synchronizes PKA and PKC activities in a manner that leads to the activation of a third kinase. This configuration illustrates the utility of kinase anchoring as a mechanism to constrain the action of broad-spectrum enzymes.

  15. The secret life of kinases: functions beyond catalysis

    PubMed Central

    2011-01-01

    Protein phosphorylation participates in the regulation of all fundamental biological processes, and protein kinases have been intensively studied. However, while the focus was on catalytic activities, accumulating evidence suggests that non-catalytic properties of protein kinases are essential, and in some cases even sufficient for their functions. These non-catalytic functions include the scaffolding of protein complexes, the competition for protein interactions, allosteric effects on other enzymes, subcellular targeting, and DNA binding. This rich repertoire often is used to coordinate phosphorylation events and enhance the specificity of substrate phosphorylation, but also can adopt functions that do not rely on kinase activity. Here, we discuss such kinase independent functions of protein and lipid kinases focussing on kinases that play a role in the regulation of cell proliferation, differentiation, apoptosis, and motility. PMID:22035226

  16. High quality, small molecule-activity datasets for kinase research

    PubMed Central

    Sharma, Rajan; Schürer, Stephan C.; Muskal, Steven M.

    2016-01-01

    Kinases regulate cell growth, movement, and death. Deregulated kinase activity is a frequent cause of disease. The therapeutic potential of kinase inhibitors has led to large amounts of published structure activity relationship (SAR) data. Bioactivity databases such as the Kinase Knowledgebase (KKB), WOMBAT, GOSTAR, and ChEMBL provide researchers with quantitative data characterizing the activity of compounds across many biological assays. The KKB, for example, contains over 1.8M kinase structure-activity data points reported in peer-reviewed journals and patents. In the spirit of fostering methods development and validation worldwide, we have extracted and have made available from the KKB 258K structure activity data points and 76K associated unique chemical structures across eight kinase targets. These data are freely available for download within this data note. PMID:27429748

  17. Exploring Missense Mutations in Tyrosine Kinases Implicated with Neurodegeneration.

    PubMed

    Sami, Neha; Kumar, Vijay; Islam, Asimul; Ali, Sher; Ahmad, Faizan; Hassan, Imtaiyaz

    2017-09-01

    Protein kinases are one of the largest families of evolutionarily related proteins and the third most common protein class of human genome. All the protein kinases share the same structural organization. They are made up of an extracellular domain, transmembrane domain and an intra cellular kinase domain. Missense mutations in these kinases have been studied extensively and correlated with various neurological disorders. Individual mutations in the kinase domain affect the functions of protein. The enhanced or reduced expression of protein leads to hyperactivation or inactivation of the signalling pathways, resulting in neurodegeneration. Here, we present extensive analyses of missense mutations in the tyrosine kinase focussing on the neurodegenerative diseases encompassing structure function relationship. This is envisaged to enhance our understanding about the neurodegeneration and possible therapeutic measures.

  18. Rational Redesign of a Functional Protein Kinase-Substrate Interaction

    PubMed Central

    2017-01-01

    Eukaryotic protein kinases typically phosphorylate substrates in the context of specific sequence motifs, contributing to specificity essential for accurate signal transmission. Protein kinases recognize their target sequences through complementary interactions within the active site cleft. As a step toward the construction of orthogonal kinase signaling systems, we have re-engineered the protein kinase Pim1 to alter its phosphorylation consensus sequence. Residues in the Pim1 catalytic domain interacting directly with a critical arginine residue in the substrate were substituted to produce a kinase mutant that instead accommodates a hydrophobic residue. We then introduced a compensating mutation into a Pim1 substrate, the pro-apoptotic protein BAD, to reconstitute phosphorylation both in vitro and in living cells. Coexpression of the redesigned kinase with its substrate in cells protected them from apoptosis. Such orthogonal kinase–substrate pairs provide tools to probe the functional consequences of specific phosphorylation events in living cells and to design synthetic signaling pathways. PMID:28314095

  19. Drosophila melanogaster deoxyribonucleoside kinase activates gemcitabine

    SciTech Connect

    Knecht, Wolfgang; Mikkelsen, Nils Egil; Clausen, Anders Ranegaard; Willer, Mette; Gojkovic, Zoran

    2009-05-01

    Drosophila melanogaster multisubstrate deoxyribonucleoside kinase (Dm-dNK) can additionally sensitize human cancer cell lines towards the anti-cancer drug gemcitabine. We show that this property is based on the Dm-dNK ability to efficiently phosphorylate gemcitabine. The 2.2 A resolution structure of Dm-dNK in complex with gemcitabine shows that the residues Tyr70 and Arg105 play a crucial role in the firm positioning of gemcitabine by extra interactions made by the fluoride atoms. This explains why gemcitabine is a good substrate for Dm-dNK.

  20. Asymmetric Synthesis of Akt Kinase Inhibitor Ipatasertib.

    PubMed

    Han, Chong; Savage, Scott; Al-Sayah, Mohammad; Yajima, Herbert; Remarchuk, Travis; Reents, Reinhard; Wirz, Beat; Iding, Hans; Bachmann, Stephan; Fantasia, Serena M; Scalone, Michelangelo; Hell, André; Hidber, Pirmin; Gosselin, Francis

    2017-09-15

    A highly efficient asymmetric synthesis of the Akt kinase inhibitor ipatasertib (1) is reported. The bicyclic pyrimidine 2 starting material was prepared via a nitrilase biocatalytic resolution, halogen-metal exchange/anionic cyclization, and a highly diastereoselective biocatalytic ketone reduction as key steps. The route also features a halide activated, Ru-catalyzed asymmetric hydrogenation of a vinylogous carbamic acid to produce α-aryl-β-amino acid 3 in high yield and enantioselectivity. The API was assembled in a convergent manner through a late-stage amidation/deprotection/monohydrochloride salt formation sequence.

  1. Receptor-like kinases shape the plant.

    PubMed

    De Smet, Ive; Voss, Ute; Jürgens, Gerd; Beeckman, Tom

    2009-10-01

    To generate the various tissues and organs that build up the adult body, plants and animals require organized formative cell divisions and correct cell specification. In plants, these processes are controlled mainly by phytohormones and transcriptional networks. Recently, ligand-receptor-like kinase signalling pathways have been revealed as additional potentially crucial regulators of cell specification in plants. We review here the importance of such signalling cascades for plant growth and development, and we discuss, where possible, similarities to well-investigated cascades in animals.

  2. Leishmania MAP kinases--familiar proteins in an unusual context.

    PubMed

    Wiese, Martin

    2007-08-01

    Mitogen-activated protein kinases are well-known mediators of signal transduction of higher eukaryotes regulating important processes like proliferation, differentiation, stress response and apoptosis. In Leishmania, the typical three-tiered module of MAP kinase signal transduction pathways is present. However, typical activators like cell surface receptors and substrates such as RNA polymerase II transcription factors are missing. Here, I describe the set of 15 putative mitogen-activated protein kinases encoded in the Leishmania genome and discuss their potential function.

  3. 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.

  4. Kinase inhibitor recognition by use of a multivariable QSAR model.

    PubMed

    Sprous, D G; Zhang, John; Zhang, Lei; Wang, Zhaolin; Tepper, M A

    2006-01-01

    We have applied a retrosynthetic program to determine the scaffold and R-group chemical space seen within a library of known kinase inhibitors and non-kinase drug-like molecules. Comparison of the differences quickly revealed that kinase inhibitors are distinct in several chemical fragment and physical properties. We then applied these descriptors in a multivariable quantitative structure-activity relationship (QSAR) model with the goal to distinguish kinase inhibitors from non-kinase drug-like molecules. This model is heuristic in that it was trained over a dataset of 258 known kinase inhibitors and 230 non-kinase drug molecules. The final model recognized 98% of the training set as being kinase inhibitors and had a false positive rate of 15%. This trait for false positives was accepted out of a desire to maintain diversity and not miss possible good kinase inhibitors for screening. The model was validated by reserving a portion of the datasets as test sets, which were not included in the QSAR model building stage. This was done repetitively for different percentiles of the total dataset population. It was seen that model recognition and false positive were only slightly damaged well down to a 70% reserve (30% dataset used for QSAR model training while 70% used for reserve test set). Beyond 70%, the QSAR models were inconsistent, signifying that the training sets were inadequately diverse to represent the greater reserve test sets. We applied this model to evaluate the commercial kinase libraries available from Asinex, BioFocus, ChemDiv and LifeChemicals to facilitate purchase decisions for compounds for HTS for lead compounds. We observed that there are significant differences in populations of recognizable kinase inhibitors across the vendors analyzed, with BioFocus showing the greatest population of kinase like molecules.

  5. Phosphatidylinositol 3-kinase is required for integrin-stimulated AKT and Raf-1/mitogen-activated protein kinase pathway activation.

    PubMed Central

    King, W G; Mattaliano, M D; Chan, T O; Tsichlis, P N; Brugge, J S

    1997-01-01

    Cell attachment to fibronectin stimulates the integrin-dependent interaction of p85-associated phosphatidylinositol (PI) 3-kinase with integrin-dependent focal adhesion kinase (FAK) as well as activation of the Ras/mitogen-activated protein (MAP) kinase pathway. However, it is not known if this PI 3-kinase-FAK interaction increases the synthesis of the 3-phosphorylated phosphoinositides (3-PPIs) or what role, if any, is played by activated PI 3-kinase in integrin signaling. We demonstrate here the integrin-dependent accumulation of the PI 3-kinase products, PI 3,4-bisphosphate [PI(3,4)P2] and PI(3,4,5)P3, as well as activation of AKT kinase, a serine/threonine kinase that can be stimulated by binding of PI(3,4)P2. The PI 3-kinase inhibitors wortmannin and LY294002 significantly decreased the integrin-induced accumulation of the 3-PPIs and activation of AKT kinase, without having significant effects on the levels of PI(4,5)P2 or tyrosine phosphorylation of paxillin. These inhibitors also reduced cell adhesion/spreading onto fibronectin but had no effect on attachment to polylysine. Interestingly, integrin-mediated Erk-2, Mek-1, and Raf-1 activation, but not Ras-GTP loading, was inhibited at least 80% by wortmannin and LY294002. In support of the pharmacologic results, fibronectin activation of Erk-2 and AKT kinases was completely inhibited by overexpression of a dominant interfering p85 subunit of PI 3-kinase. We conclude that integrin-mediated adhesion to fibronectin results in the accumulation of the PI 3-kinase products PI(3,4)P2 and PI(3,4,5)P3 as well as the PI 3-kinase-dependent activation of the kinases Raf-1, Mek-1, Erk-2, and AKT and that PI 3-kinase may function upstream of Raf-1 but downstream of Ras in integrin activation of Erk-2 MAP and AKT kinases. PMID:9234699

  6. Nonmuscle Myosin IIA Regulates Platelet Contractile Forces Through Rho Kinase and Myosin Light-Chain Kinase.

    PubMed

    Feghhi, Shirin; Tooley, Wes W; Sniadecki, Nathan J

    2016-10-01

    Platelet contractile forces play a major role in clot retraction and help to hold hemostatic clots against the vessel wall. Platelet forces are produced by its cytoskeleton, which is composed of actin and nonmuscle myosin filaments. In this work, we studied the role of Rho kinase, myosin light-chain kinase, and myosin in the generation of contractile forces by using pharmacological inhibitors and arrays of flexible microposts to measure platelet forces. When platelets were seeded onto microposts, they formed aggregates on the tips of the microposts. Forces produced by the platelets in the aggregates were measured by quantifying the deflection of the microposts, which bent in proportion to the force of the platelets. Platelets were treated with small molecule inhibitors of myosin activity: Y-27632 to inhibit the Rho kinase (ROCK), ML-7 to inhibit myosin light-chain kinase (MLCK), and blebbistatin to inhibit myosin ATPase activity. ROCK inhibition reduced platelet forces, demonstrating the importance of the assembly of actin and myosin phosphorylation in generating contractile forces. Similarly, MLCK inhibition caused weaker platelet forces, which verifies that myosin phosphorylation is needed for force generation in platelets. Platelets treated with blebbistatin also had weaker forces, which indicates that myosin's ATPase activity is necessary for platelet forces. Our studies demonstrate that myosin ATPase activity and the regulation of actin-myosin assembly by ROCK and MLCK are needed for the generation of platelet forces. Our findings illustrate and explain the importance of myosin for clot compaction in hemostasis and thrombosis.

  7. Characterization of cyclin-dependent kinases and Cdc2/Cdc28 kinase subunits in Trichomonas vaginalis.

    PubMed

    Amador, Erick; López-Pacheco, Karla; Morales, Nataly; Coria, Roberto; López-Villaseñor, Imelda

    2017-04-01

    Cyclin-dependent kinases (CDKs) have important roles in regulating key checkpoints between stages of the cell cycle. Their activity is tightly regulated through a variety of mechanisms, including through binding with cyclin proteins and the Cdc2/Cdc28 kinase subunit (CKS), and their phosphorylation at specific amino acids. Studies of the components involved in cell cycle control in parasitic protozoa are limited. Trichomonas vaginalis is the causative agent of trichomoniasis in humans and is therefore important in public health; however, some of the basic biological processes used by this organism have not been defined. Here, we characterized proteins potentially involved in cell cycle regulation in T. vaginalis. Three genes encoding protein kinases were identified in the T. vaginalis genome, and the corresponding recombinant proteins (TvCRK1, TvCRK2, TvCRK5) were studied. These proteins displayed similar sequence features to CDKs. Two genes encoding CKSs were also identified, and the corresponding recombinant proteins were found to interact with TvCRK1 and TvCRK2 by a yeast two-hybrid system. One putative cyclin B protein from T. vaginalis was found to bind to and activate the kinase activities of TvCRK1 and TvCRK5, but not TvCRK2. This work is the first characterization of proteins involved in cell cycle control in T. vaginalis.

  8. DHEA improves glucose uptake via activations of protein kinase C and phosphatidylinositol 3-kinase.

    PubMed

    Ishizuka, T; Kajita, K; Miura, A; Ishizawa, M; Kanoh, Y; Itaya, S; Kimura, M; Muto, N; Mune, T; Morita, H; Yasuda, K

    1999-01-01

    We have examined the effect of adrenal androgen, dehydroepiandrosterone (DHEA), on glucose uptake, phosphatidylinositol (PI) 3-kinase, and protein kinase C (PKC) activity in rat adipocytes. DHEA (1 microM) provoked a twofold increase in 2-[3H]deoxyglucose (DG) uptake for 30 min. Pretreatment with DHEA increased insulin-induced 2-[3H]DG uptake without alterations of insulin specific binding and autophosphorylation of insulin receptor. DHEA also stimulated PI 3-kinase activity. [3H]DHEA bound to purified PKC containing PKC-alpha, -beta, and -gamma. DHEA provoked the translocation of PKC-beta and -zeta from the cytosol to the membrane in rat adipocytes. These results suggest that DHEA stimulates both PI 3-kinase and PKCs and subsequently stimulates glucose uptake. Moreover, to clarify the in vivo effect of DHEA on Goto-Kakizaki (GK) and Otsuka Long-Evans fatty (OLETF) rats, animal models of non-insulin-dependent diabetes mellitus (NIDDM) were treated with 0.4% DHEA for 2 wk. Insulin- and 12-O-tetradecanoyl phorbol-13-acetate-induced 2-[3H]DG uptakes of adipocytes were significantly increased, but there was no significant increase in the soleus muscles in DHEA-treated GK/Wistar or OLETF/Long-Evans Tokushima (LETO) rats when compared with untreated GK/Wistar or OLETF/LETO rats. These results indicate that in vivo DHEA treatment can result in increased insulin-induced glucose uptake in two different NIDDM rat models.

  9. A Novel Calcium-Dependent Kinase Inhibitor, Bumped Kinase Inhibitor 1517, Cures Cryptosporidiosis in Immunosuppressed Mice.

    PubMed

    Castellanos-Gonzalez, Alejandro; Sparks, Hayley; Nava, Samantha; Huang, Wenlin; Zhang, Zhongsheng; Rivas, Kasey; Hulverson, Matthew A; Barrett, Lynn K; Ojo, Kayode K; Fan, Erkang; Van Voorhis, Wesley C; White, Arthur Clinton

    2016-12-15

    Cryptosporidium is recognized as one of the main causes of childhood diarrhea worldwide. However, the current treatment for cryptosporidiosis is suboptimal. Calcium flux is essential for entry in apicomplexan parasites. Calcium-dependent protein kinases (CDPKs) are distinct from protein kinases of mammals, and the CDPK1 of the apicomplexan Cryptosporidium lack side chains that typically block a hydrophobic pocket in protein kinases. We exploited this to develop bumped kinase inhibitors (BKIs) that selectively target CDPK1. We have shown that several BKIs of Cryptosporidium CDPK1 potently reduce enzymatic activity and decrease parasite numbers when tested in vitro. In the present work, we studied the anticryptosporidial activity of BKI-1517, a novel BKI. The half maximal effective concentration for Cryptosporidium parvum in HCT-8 cells was determined to be approximately 50 nM. Silencing experiments of CDPK1 suggest that BKI-1517 acts on CDPK1 as its primary target. In a mouse model of chronic infection, 5 of 6 SCID/beige mice (83.3%) were cured after treatment with a single daily dose of 120 mg/kg BKI-1517. No side effects were observed. These data support advancing BKI-1517 as a lead compound for drug development for cryptosporidiosis.

  10. Targeting mitogen-activated protein kinase kinase (MEK) in solid tumors.

    PubMed

    Duffy, Austin; Kummar, Shivaani

    2009-12-01

    The Raf-mitogen activated protein kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) protein kinase signaling cascade is an important intracellular pathway whose activation influences many fundamental cellular processes and whose aberrancy is associated with cancer cell growth. In addition to activation from within by, for example, Raf mutations, this pathway is frequently activated from above by mutated Ras or epidermal growth factor receptor (EGFR). Given the near ubiquity of derangements affecting at least part of this network in cancer, there is a strong and clear rationale for interrupting it. In recent times, in colorectal and lung cancer, Ras and EGFR mutant status have been shown to be critically important and mutually exclusive predictors of response to anti-EGFR therapies. These developments underline the importance of targeting downstream effectors, and MEK inhibition has been the subject of intense scientific and clinical research for some time now. This article reviews the current status of MEK inhibitors with regard to their clinical development.

  11. Glycogen Synthase Kinase 3β Interaction Protein Functions as an A-kinase Anchoring Protein*

    PubMed Central

    Hundsrucker, Christian; Skroblin, Philipp; Christian, Frank; Zenn, Hans-Michael; Popara, Viola; Joshi, Mangesh; Eichhorst, Jenny; Wiesner, Burkhard; Herberg, Friedrich W.; Reif, Bernd; Rosenthal, Walter; Klussmann, Enno

    2010-01-01

    A-kinase anchoring proteins (AKAPs) include a family of scaffolding proteins that target protein kinase A (PKA) and other signaling proteins to cellular compartments and thereby confine the activities of the associated proteins to distinct regions within cells. AKAPs bind PKA directly. The interaction is mediated by the dimerization and docking domain of regulatory subunits of PKA and the PKA-binding domain of AKAPs. Analysis of the interactions between the dimerization and docking domain and various PKA-binding domains yielded a generalized motif allowing the identification of AKAPs. Our bioinformatics and peptide array screening approaches based on this signature motif identified GSKIP (glycogen synthase kinase 3β interaction protein) as an AKAP. GSKIP directly interacts with PKA and GSK3β (glycogen synthase kinase 3β). It is widely expressed and facilitates phosphorylation and thus inactivation of GSK3β by PKA. GSKIP contains the evolutionarily conserved domain of unknown function 727. We show here that this domain of GSKIP and its vertebrate orthologues binds both PKA and GSK3β and thereby provides a mechanism for the integration of PKA and GSK3β signaling pathways. PMID:20007971

  12. Kinase Pathway Database: An Integrated Protein-Kinase and NLP-Based Protein-Interaction Resource

    PubMed Central

    Koike, Asako; Kobayashi, Yoshiyuki; Takagi, Toshihisa

    2003-01-01

    Protein kinases play a crucial role in the regulation of cellular functions. Various kinds of information about these molecules are important for understanding signaling pathways and organism characteristics. We have developed the Kinase Pathway Database, an integrated database involving major completely sequenced eukaryotes. It contains the classification of protein kinases and their functional conservation, ortholog tables among species, protein–protein, protein–gene, and protein–compound interaction data, domain information, and structural information. It also provides an automatic pathway graphic image interface. The protein, gene, and compound interactions are automatically extracted from abstracts for all genes and proteins by natural-language processing (NLP).The method of automatic extraction uses phrase patterns and the GENA protein, gene, and compound name dictionary, which was developed by our group. With this database, pathways are easily compared among species using data with more than 47,000 protein interactions and protein kinase ortholog tables. The database is available for querying and browsing at http://kinasedb.ontology.ims.u-tokyo.ac.jp/. PMID:12799355

  13. Quick evaluation of kinase inhibitors by surface plasmon resonance using single-site specifically biotinylated kinases.

    PubMed

    Kitagawa, Daisuke; Gouda, Masaki; Kirii, Yasuyuki

    2014-03-01

    In evaluating kinase inhibitors, kinetic parameters such as association/dissociation rate constants are valuable information, as are equilibrium parameters KD and IC50 values. Surface plasmon resonance (SPR) is a powerful technique to investigate these parameters. However, results are often complicated because of impaired conformations by inappropriate conditions required for protein immobilization and/or heterogeneity of the orientation of immobilization. In addition, conventional SPR experiments are generally time-consuming. Here we introduce the use of single-site specifically biotinylated kinases combined with a multichannel SPR device to improve such problems. Kinetic parameters of four compounds-staurosporine, dasatinib, sunitinib, and lapatinib-against six kinases were determined by the ProteOn XPR36 system. The very slow off-rate of lapatinib from the epidermal growth factor receptor and dasatinib from Bruton's tyrosine kinase and colony stimulating factor 1 receptor (CSF1R) were confirmed. Furthermore, IC50 values were determined by an activity-based assay. Evaluating both physicochemical and biochemical properties would help to understand the detailed character of the compound.

  14. Protein kinase Cδ regulates vaccinia-related kinase 1 in DNA damage–induced apoptosis

    PubMed Central

    Park, Choon-Ho; Choi, Bo-Hwa; Jeong, Min-Woo; Kim, Sangjune; Kim, Wanil; Song, Yun Seon; Kim, Kyong-Tai

    2011-01-01

    Vaccinia-related kinase 1 (VRK1) is a novel serine/threonine kinase that plays an important role in cell proliferation. However, little is known about the upstream regulators of VRK1 activity. Here we provide evidence for a role of protein kinase Cδ (PKCδ) in the regulation of murine VRK1. We show that PKCδ interacts with VRK1, phosphorylates the Ser-355 residue in the putative regulatory region, and negatively regulates its kinase activity in vitro. Intriguingly, PKCδ-induced cell death was facilitated by phosphorylation of VRK1 when cells were exposed to a DNA-damaging agent. In addition, p53 played a critical role in the regulation of DNA damage–induced cell death accompanied by PKCδ-mediated modulation of VRK1. In p53-deficient cells, PKCδ-mediated phosphorylation of VRK1 had no effect on cell viability. However, cells overexpressing p53 exhibited significant reduction of cell viability when cotransfected with both VRK1 and PKCδ. Taken together, these results indicate that PKCδ regulates phosphorylation and down-regulation of VRK1, thereby contributing to cell cycle arrest and apoptotic cell death in a p53-dependent manner. PMID:21346188

  15. Nuclear localization of Lyn tyrosine kinase mediated by inhibition of its kinase activity

    SciTech Connect

    Ikeda, Kikuko; Nakayama, Yuji; Togashi, Yuuki; Obata, Yuuki; Kuga, Takahisa; Kasahara, Kousuke; Fukumoto, Yasunori; Yamaguchi, Naoto

    2008-11-01

    Src-family kinases, cytoplasmic enzymes that participate in various signaling events, are found at not only the plasma membrane but also subcellular compartments, such as the nucleus, the Golgi apparatus and late endosomes/lysosomes. Lyn, a member of the Src-family kinases, is known to play a role in DNA damage response and cell cycle control in the nucleus. However, it is still unclear how the localization of Lyn to the nucleus is regulated. Here, we investigated the mechanism of the distribution of Lyn between the cytoplasm and the nucleus in epitheloid HeLa cells and hematopoietic THP-1 cells. Lyn was definitely detected in purified nuclei by immunofluorescence and immunoblotting analyses. Nuclear accumulation of Lyn was enhanced upon treatment of cells with leptomycin B (LMB), an inhibitor of Crm1-mediated nuclear export. Moreover, Lyn mutants lacking the sites for lipid modification were highly accumulated in the nucleus upon LMB treatment. Intriguingly, inhibition of the kinase activity of Lyn by SU6656, Csk overexpression, or point mutation in the ATP-binding site induced an increase in nuclear Lyn levels. These results suggest that Lyn being imported into and rapidly exported from the nucleus preferentially accumulates in the nucleus by inhibition of the kinase activity and lipid modification.

  16. Gene looping facilitates TFIIH kinase-mediated termination of transcription

    PubMed Central

    Medler, Scott; Ansari, Athar

    2015-01-01

    TFIIH is a general transcription factor with kinase and helicase activities. The kinase activity resides in the Kin28 subunit of TFIIH. The role of Kin28 kinase in the early steps of transcription is well established. Here we report a novel role of Kin28 in the termination of transcription. We show that RNAPII reads through a termination signal upon kinase inhibition. Furthermore, the recruitment of termination factors towards the 3′ end of a gene was compromised in the kinase mutant, thus confirming the termination defect. A concomitant decrease in crosslinking of termination factors near the 5′ end of genes was also observed in the kinase-defective mutant. Simultaneous presence of termination factors towards both the ends of a gene is indicative of gene looping; while the loss of termination factor occupancy from the distal ends suggest the abolition of a looped gene conformation. Accordingly, CCC analysis revealed that the looped architecture of genes was severely compromised in the Kin28 kinase mutant. In a looping defective sua7-1 mutant, even the enzymatically active Kin28 kinase could not rescue the termination defect. These results strongly suggest a crucial role of Kin28 kinase-dependent gene looping in the termination of transcription in budding yeast. PMID:26286112

  17. Structural and mechanistic insights into Mps1 kinase activation

    SciTech Connect

    Wang, Wei; Yang, Yuting; Gao, Yuefeng; Xu, Quanbin; Wang, Feng; Zhu, Songcheng; Old, William; Resing, Katheryn; Ahn, Natalie; Lei, Ming; Liu, Xuedong

    2010-11-05

    Mps1 is one of the several essential kinases whose activation is required for robust mitotic spindle checkpoint signalling. The activity of Mps1 is tightly regulated and increases dramatically during mitosis or in response to spindle damage. To understand the molecular mechanism underlying Mps1 regulation, we determined the crystal structure of the kinase domain of Mps1. The 2.7-{angstrom}-resolution crystal structure shows that the Mps1 kinase domain adopts a unique inactive conformation. Intramolecular interactions between the key Glu residue in the {alpha}C helix of the N-terminal lobe and the backbone amides in the catalytic loop lock the kinase in the inactive conformation. Autophosphorylation appears to be a priming event for kinase activation. We identified Mps1 autophosphorylation sites in the activation and the P+1 loops. Whereas activation loop autophosphorylation enhances kinase activity, autophosphorylation at the P+1 loop (T686) is associated with the active kinase. Mutation of T686 autophosphorylation site impairs both autophosphorylation and transphosphorylation. Furthermore, we demonstrated that phosphorylation of T676 may be a priming event for phosphorylation at T686. Finally, we identified two critical lysine residues in the loop between helices {alpha}EF and {alpha}F that are essential for substrate recruitment and maintaining high levels of kinase activity. Our studies reveal critical biochemical mechanisms for Mps1 kinase regulation.

  18. Structure and dynamic regulation of Src-family kinases.

    PubMed

    Engen, J R; Wales, T E; Hochrein, J M; Meyn, M A; Banu Ozkan, S; Bahar, I; Smithgall, T E

    2008-10-01

    Src-family kinases are modular signaling proteins involved in a diverse array of cellular processes. All members of the Src family share the same domain organization, with modular SH3, SH2 and kinase domains followed by a C-terminal negative regulatory tail. X-ray crystallographic analyses of several Src family members have revealed critical roles for the SH3 and SH2 domains in the down-regulation of the kinase domain. This review focuses on biological, biophysical, and computational studies that reveal conformationally distinct active states within this unique kinase family.

  19. LIM kinases: function, regulation and association with human disease.

    PubMed

    Scott, Rebecca W; Olson, Michael F

    2007-06-01

    The LIM kinase family consists of just two members: LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2). With uniquely organised signalling domains, LIM kinases are regulated by several upstream signalling pathways, principally acting downstream of Rho GTPases to influence the architecture of the actin cytoskeleton by regulating the activity of the cofilin family proteins cofilin1, cofilin2 and destrin. Although the LIM kinases are very homologous, particularly when comparing kinase domains, there is emerging evidence that each may be subject to different regulatory pathways and may contribute to both distinct and overlapping cellular and developmental functions. Normal central nervous system development is reliant upon the presence of LIMK1, and its deletion has been implicated in the development of the human genetic disorder Williams syndrome. Normal testis development, on the other hand, is disrupted by the deletion of LIMK2. In addition, the possible involvement of each kinase in cardiovascular disorders as well as cancer has recently emerged. The LIM kinases have been proposed to play an important role in tumour-cell invasion and metastasis; fine-tuning the balance between phosphorylated and non-phosphorylated cofilin may be a significant determinant of tumour-cell metastatic potential. In this review, we outline the structure, regulation and function of LIM kinases and their functions at cellular and organismal levels, as well as their possible contributions to human disease.

  20. Diversity, classification and function of the plant protein kinase superfamily

    PubMed Central

    Lehti-Shiu, Melissa D.; Shiu, Shin-Han

    2012-01-01

    Eukaryotic protein kinases belong to a large superfamily with hundreds to thousands of copies and are components of essentially all cellular functions. The goals of this study are to classify protein kinases from 25 plant species and to assess their evolutionary history in conjunction with consideration of their molecular functions. The protein kinase superfamily has expanded in the flowering plant lineage, in part through recent duplications. As a result, the flowering plant protein kinase repertoire, or kinome, is in general significantly larger than other eukaryotes, ranging in size from 600 to 2500 members. This large variation in kinome size is mainly due to the expansion and contraction of a few families, particularly the receptor-like kinase/Pelle family. A number of protein kinases reside in highly conserved, low copy number families and often play broadly conserved regulatory roles in metabolism and cell division, although functions of plant homologues have often diverged from their metazoan counterparts. Members of expanded plant kinase families often have roles in plant-specific processes and some may have contributed to adaptive evolution. Nonetheless, non-adaptive explanations, such as kinase duplicate subfunctionalization and insufficient time for pseudogenization, may also contribute to the large number of seemingly functional protein kinases in plants. PMID:22889912

  1. How protein kinases co-ordinate mitosis in animal cells.

    PubMed

    Ma, Hoi Tang; Poon, Randy Y C

    2011-04-01

    Mitosis is associated with profound changes in cell physiology and a spectacular surge in protein phosphorylation. To accomplish these, a remarkably large portion of the kinome is involved in the process. In the present review, we will focus on classic mitotic kinases, such as cyclin-dependent kinases, Polo-like kinases and Aurora kinases, as well as more recently characterized players such as NIMA (never in mitosis in Aspergillus nidulans)-related kinases, Greatwall and Haspin. Together, these kinases co-ordinate the proper timing and fidelity of processes including centrosomal functions, spindle assembly and microtubule-kinetochore attachment, as well as sister chromatid separation and cytokinesis. A recurrent theme of the mitotic kinase network is the prevalence of elaborated feedback loops that ensure bistable conditions. Sequential phosphorylation and priming phosphorylation on substrates are also frequently employed. Another important concept is the role of scaffolds, such as centrosomes for protein kinases during mitosis. Elucidating the entire repertoire of mitotic kinases, their functions, regulation and interactions is critical for our understanding of normal cell growth and in diseases such as cancers.

  2. In Vitro Characterization of Derrone as an Aurora Kinase Inhibitor.

    PubMed

    Hoang, Nhung Thi My; Phuong, Thuong Thien; Nguyen, Trang Thi Nhu; Tran, Yen Thi Hai; Nguyen, Anh Thi Ngoc; Nguyen, Thanh Lai; Bui, Khanh Thi Van

    2016-06-01

    Among mitotic kinases, Aurora kinases are the most widely studied, since their expression is restricted to mitosis. They play a key role in chromosome segregation and cell polyploidy. Aurora kinases are important therapeutic targets, and several research groups have directed their efforts toward the identification of kinase inhibitors. The aim of this study is to screen and characterize Aurora kinase inhibitors from natural substances extracted from plants that are used in the Vietnamese pharmacopoeia. We have characterized in vitro Derrone, extracted from Erythrina orientalis L. MURR, as a novel Aurora kinase inhibitor. This compound exhibited an ability to inhibit the phosphorylation of histone H3 at ser10 both in kinase assay and at the cellular level. The compound was more effective against Aurora kinase B, with a lower IC50 value as compared to Aurora A. Moreover, it impaired the mitotic spindle checkpoint and led to endoreduplication in cancer cells, a phenomenon caused by an Aurora B inhibitor. Interestingly, using the xCelligence system and real-time cell analysis (RTCA) software, we set up a comparison of cell proliferation profiles between cancer cells treated with Derrone and VX680-a well-known Aurora kinase inhibitor-and we found that these profiles exhibited considerable similarity in cell morphology, growth, and death. Additionally, Derrone significantly inhibited the formation and growth of MCF7 tumor spheroids.

  3. Role of Jak kinases and STATs in cytokine signal transduction.

    PubMed

    Leonard, W J

    2001-04-01

    The Janus family tyrosine kinase-signal transducer and activator of transcription (Jak-STAT) signaling pathway is broadly used by interferons and type I cytokines. These cytokines and interferons activate Janus family tyrosine kinases (Jak kinases), which in turn phosphorylate and thereby activate STAT proteins. Before activation, STAT proteins are cytosolic proteins; after activation, however, they are translocated to the nucleus where they function as transcription factors. This review summarizes salient features of the Jak-STAT pathway and focuses on the functional role of the different Jak kinases and STATs in vivo.

  4. Diversity, classification and function of the plant protein kinase superfamily.

    PubMed

    Lehti-Shiu, Melissa D; Shiu, Shin-Han

    2012-09-19

    Eukaryotic protein kinases belong to a large superfamily with hundreds to thousands of copies and are components of essentially all cellular functions. The goals of this study are to classify protein kinases from 25 plant species and to assess their evolutionary history in conjunction with consideration of their molecular functions. The protein kinase superfamily has expanded in the flowering plant lineage, in part through recent duplications. As a result, the flowering plant protein kinase repertoire, or kinome, is in general significantly larger than other eukaryotes, ranging in size from 600 to 2500 members. This large variation in kinome size is mainly due to the expansion and contraction of a few families, particularly the receptor-like kinase/Pelle family. A number of protein kinases reside in highly conserved, low copy number families and often play broadly conserved regulatory roles in metabolism and cell division, although functions of plant homologues have often diverged from their metazoan counterparts. Members of expanded plant kinase families often have roles in plant-specific processes and some may have contributed to adaptive evolution. Nonetheless, non-adaptive explanations, such as kinase duplicate subfunctionalization and insufficient time for pseudogenization, may also contribute to the large number of seemingly functional protein kinases in plants.

  5. Developing irreversible inhibitors of the protein kinase cysteinome

    PubMed Central

    Liu, Qingsong; Sabnis, Yogesh; Zhao, Zheng; Zhang, Tinghu; Buhrlage, Sara J.; Jones, Lyn H.; Gray, Nathanael S.

    2013-01-01

    Protein kinases are a large family of approximately 530 highly conserved enzymes that transfer a γ-phosphate group from ATP to a variety of amino acid residues such as tyrosine, serine and threonine which serves as a ubiquitous mechanism for cellular signal transduction. The clinical success of a number of kinase-directed drugs and the frequent observation of disease causing mutations in protein kinases suggest that a large number of kinases may represent therapeutically relevant targets. To-date the majority of clinical and preclinical kinase inhibitors are ATP-competitive, non-covalent inhibitors that achieve selectivity through recognition of unique features of particular protein kinases. Recently there has been renewed interest in the development of irreversible inhibitors that form covalent bonds with cysteine or other nucleophilic residues in the ATP-binding pocket. Irreversible kinase inhibitors have a number of potential advantages including prolonged pharmacodynamics, suitability for rational design, high potency and ability to validate pharmacological specificity through mutation of the reactive cysteine residue. Here we review recent efforts to develop cysteine-targeted irreversible protein kinase inhibitors and discuss their modes of recognizing the ATP-binding pocket and their biological activity profiles. In addition, we provided an informatics assessment of the potential ‘kinase-cysteinome’ and discuss strategies for the efficient development of new covalent inhibitors. PMID:23438744

  6. The acetate kinase of Clostridum acetobutylicum strain P262.

    PubMed

    Diez-Gonzalez, F; Russell, J B; Hunter, J B

    1996-12-01

    Clostridum acetobutylicum strain P262 fermented glucose, pyruvate, or lactate, and the butyrate production was substrate-dependent. Differences in butyrate yield could not be explained by changes in butyrate kinase activities, but the butyrate production was inversely related to acetate kinase activity. The acetate kinase had a pH optimum of 8.0, a Km for acetate of 160 mM, and a kcat of 16, 800 min-1. The enyzme had a native molecular mass of 78 kDa; the size of 42 kDa on SDS-PAGE indicated that the acetate kinase of strain P262 was a homodimer.

  7. Constitutive Activity in an Ancestral Form of Abl Tyrosine Kinase.

    PubMed

    Aleem, Saadat U; Craddock, Barbara P; Miller, W Todd

    2015-01-01

    The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase that is found in all metazoans, and is ubiquitously expressed in mammalian tissues. The Abl tyrosine kinase plays important roles in the regulation of mammalian cell physiology. Abl-like kinases have been identified in the genomes of unicellular choanoflagellates, the closest relatives to the Metazoa, and in related unicellular organisms. Here, we have carried out the first characterization of a premetazoan Abl kinase, MbAbl2, from the choanoflagellate Monosiga brevicollis. The enzyme possesses SH3, SH2, and kinase domains in a similar arrangement to its mammalian counterparts, and is an active tyrosine kinase. MbAbl2 lacks the N-terminal myristoylation and cap sequences that are critical regulators of mammalian Abl kinase activity, and we show that MbAbl2 is constitutively active. When expressed in mammalian cells, MbAbl2 strongly phosphorylates cellular proteins on tyrosine, and transforms cells much more potently than mammalian Abl kinase. Thus, MbAbl2 appears to lack the autoinhibitory mechanism that tightly constrains the activity of mammalian Abl kinases, suggesting that this regulatory apparatus arose more recently in metazoan evolution.

  8. Constitutive Activity in an Ancestral Form of Abl Tyrosine Kinase

    PubMed Central

    Miller, W. Todd

    2015-01-01

    The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase that is found in all metazoans, and is ubiquitously expressed in mammalian tissues. The Abl tyrosine kinase plays important roles in the regulation of mammalian cell physiology. Abl-like kinases have been identified in the genomes of unicellular choanoflagellates, the closest relatives to the Metazoa, and in related unicellular organisms. Here, we have carried out the first characterization of a premetazoan Abl kinase, MbAbl2, from the choanoflagellate Monosiga brevicollis. The enzyme possesses SH3, SH2, and kinase domains in a similar arrangement to its mammalian counterparts, and is an active tyrosine kinase. MbAbl2 lacks the N-terminal myristoylation and cap sequences that are critical regulators of mammalian Abl kinase activity, and we show that MbAbl2 is constitutively active. When expressed in mammalian cells, MbAbl2 strongly phosphorylates cellular proteins on tyrosine, and transforms cells much more potently than mammalian Abl kinase. Thus, MbAbl2 appears to lack the autoinhibitory mechanism that tightly constrains the activity of mammalian Abl kinases, suggesting that this regulatory apparatus arose more recently in metazoan evolution. PMID:26090675

  9. 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.

  10. Kinase drug discovery--what's next in the field?

    PubMed

    Cohen, Philip; Alessi, Dario R

    2013-01-18

    Over the past 15 years protein kinases have become the pharmaceutical industry's most important class of drug target in the field of cancer. Some 20 drugs that target kinases have been approved for clinical use over the past decade, and hundreds more are undergoing clinical trials. However, the recent approval of the first protein kinase inhibitors for the treatment of inflammatory diseases, coupled with an enhanced understanding of the signaling networks that control the immune system, suggests that there will be a surge of interest in this area over the next 10 years. In this connection, we discuss opportunities for targeting protein kinases in the MyD88 signaling network for the development of drugs to treat chronic inflammatory and autoimmune diseases. Activating mutations in protein kinases underlie many other diseases and conditions, and we also discuss why the protein kinases SPAK/OSR1 and LRRK2 have recently become interesting targets for the treatment of hypertension and Parkinson's disease, respectively, and the progress that has been made in developing LRRK2 inhibitors. Finally we suggest that more focus on the identification of inhibitors of kinase activation, rather than kinase activity, may pay dividends in identifying exquisitely specific inhibitors of signal transduction cascades, and we also highlight "pseudo-kinases" as an attractive and unexplored area for drug development that merits much more attention in the years to come.

  11. Protein kinase A contributes to the negative control of Snf1 protein kinase in Saccharomyces cerevisiae.

    PubMed

    Barrett, LaKisha; Orlova, Marianna; Maziarz, Marcin; Kuchin, Sergei

    2012-02-01

    Snf1 protein kinase regulates responses to glucose limitation and other stresses. Snf1 activation requires phosphorylation of its T-loop threonine by partially redundant upstream kinases (Sak1, Tos3, and Elm1). Under favorable conditions, Snf1 is turned off by Reg1-Glc7 protein phosphatase. The reg1 mutation causes increased Snf1 activation and slow growth. To identify new components of the Snf1 pathway, we searched for mutations that, like snf1, suppress reg1 for the slow-growth phenotype. In addition to mutations in genes encoding known pathway components (SNF1, SNF4, and SAK1), we recovered "fast" mutations, designated fst1 and fst2. Unusual morphology of the mutants in the Σ1278b strains employed here helped us identify fst1 and fst2 as mutations in the RasGAP genes IRA1 and IRA2. Cells lacking Ira1, Ira2, or Bcy1, the negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA), exhibited reduced Snf1 pathway activation. Conversely, Snf1 activation was elevated in cells lacking the Gpr1 sugar receptor, which contributes to PKA signaling. We show that the Snf1-activating kinase Sak1 is phosphorylated in vivo on a conserved serine (Ser1074) within an ideal PKA motif. However, this phosphorylation alone appears to play only a modest role in regulation, and Sak1 is not the only relevant target of the PKA pathway. Collectively, our results suggest that PKA, which integrates multiple regulatory inputs, could contribute to Snf1 regulation under various conditions via a complex mechanism. Our results also support the view that, like its mammalian counterpart, AMP-activated protein kinase (AMPK), yeast Snf1 participates in metabolic checkpoint control that coordinates growth with nutrient availability.

  12. Identification of a fungi-specific lineage of protein kinases closely related to tyrosine kinases.

    PubMed

    Zhao, Zhongtao; Jin, Qiaojun; Xu, Jin-Rong; Liu, Huiquan

    2014-01-01

    Tyrosine kinases (TKs) specifically catalyze the phosphorylation of tyrosine residues in proteins and play essential roles in many cellular processes. Although TKs mainly exist in animals, recent studies revealed that some organisms outside the Opisthokont clade also contain TKs. The fungi, as the sister group to animals, are thought to lack TKs. To better understand the origin and evolution of TKs, it is important to investigate if fungi have TK or TK-related genes. We therefore systematically identified possible TKs across the fungal kingdom by using the profile hidden Markov Models searches and phylogenetic analyses. Our results confirmed that fungi lack the orthologs of animal TKs. We identified a fungi-specific lineage of protein kinases (FslK) that appears to be a sister group closely related to TKs. Sequence analysis revealed that members of the FslK clade contain all the conserved protein kinase sub-domains and thus are likely enzymatically active. However, they lack key amino acid residues that determine TK-specific activities, indicating that they are not true TKs. Phylogenetic analysis indicated that the last common ancestor of fungi may have possessed numerous members of FslK. The ancestral FslK genes were lost in Ascomycota and Ustilaginomycotina and Pucciniomycotina of Basidiomycota during evolution. Most of these ancestral genes, however, were retained and expanded in Agaricomycetes. The discovery of the fungi-specific lineage of protein kinases closely related to TKs helps shed light on the origin and evolution of TKs and also has potential implications for the importance of these kinases in mushroom fungi.

  13. Genomic survey of premetazoans shows deep conservation of cytoplasmic tyrosine kinases and multiple radiations of receptor tyrosine kinases.

    PubMed

    Suga, Hiroshi; Dacre, Michael; de Mendoza, Alex; Shalchian-Tabrizi, Kamran; Manning, Gerard; Ruiz-Trillo, Iñaki

    2012-05-01

    The evolution of multicellular metazoans from a unicellular ancestor is one of the most important advances in the history of life. Protein tyrosine kinases play important roles in cell-to-cell communication, cell adhesion, and differentiation in metazoans; thus, elucidating their origins and early evolution is crucial for understanding the origin of metazoans. Although tyrosine kinases exist in choanoflagellates, few data are available about their existence in other premetazoan lineages. To unravel the origin of tyrosine kinases, we performed a genomic and polymerase chain reaction (PCR)-based survey of the genes that encode tyrosine kinases in the two described filasterean species, Capsaspora owczarzaki and Ministeria vibrans, the closest relatives to the Metazoa and Choanoflagellata clades. We present 103 tyrosine kinase-encoding genes identified in the whole genome sequence of C. owczarzaki and 15 tyrosine kinase-encoding genes cloned by PCR from M. vibrans. Through detailed phylogenetic analysis, comparison of the organizations of the protein domains, and resequencing and revision of tyrosine kinase sequences previously found in some whole genome sequences, we demonstrate that the basic repertoire of metazoan cytoplasmic tyrosine kinases was established before the divergence of filastereans from the Metazoa and Choanoflagellata clades. In contrast, the receptor tyrosine kinases diversified extensively in each of the filasterean, choanoflagellate, and metazoan clades. This difference in the divergence patterns between cytoplasmic tyrosine kinases and receptor tyrosine kinases suggests that receptor tyrosine kinases that had been used for receiving environmental cues were subsequently recruited as a communication tool between cells at the onset of metazoan multicellularity.

  14. Src-homology 3 domain of protein kinase p59fyn mediates binding to phosphatidylinositol 3-kinase in T cells.

    PubMed Central

    Prasad, K V; Janssen, O; Kapeller, R; Raab, M; Cantley, L C; Rudd, C E

    1993-01-01

    The Src-related tyrosine kinase p59fyn(T) plays an important role in the generation of intracellular signals from the T-cell antigen receptor TCR zeta/CD3 complex. A key question concerns the nature and the binding sites of downstream components that interact with this Src-related kinase. p59fyn(T) contains Src-homology 2 and 3 domains (SH2 and SH3) with a capacity to bind to intracellular proteins. One potential downstream target is phosphatidylinositol 3-kinase (PI 3-kinase). In this study, we demonstrate that anti-CD3 and anti-Fyn immunoprecipitates possess PI 3-kinase activity as assessed by TLC and HPLC. Both free and receptor-bound p59fyn(T) were found to bind to the lipid kinase. Further, our results indicate that Src-related kinases have developed a novel mechanism to interact with PI 3-kinase. Precipitation using GST fusion proteins containing Fyn SH2, SH3, and SH2/SH3 domains revealed that PI 3-kinase bound principally to the SH3 domain of Fyn. Fyn SH3 bound directly to the p85 subunit of PI 3-kinase as expressed in a baculoviral system. Anti-CD3 crosslinking induced an increase in the detection of Fyn SH3-associated PI 3-kinase activity. Thus PI 3-kinase is a target of SH3 domains and is likely to play a major role in the signals derived from the TCR zeta/CD3-p59fyn complex. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8394019

  15. Identification and Targeting of Upstream Tyrosine Kinases Mediating PI3 Kinase Activation in PTEN-Deficient Prostate Cancer

    DTIC Science & Technology

    2009-06-01

    by Ras, and is amplified by PTEN loss in the majority of advanced prostate cancers (PCa). We found that RTK inhibitors lapatinib and sorafenib could...PI3 kinase activity, but combined treatment with lapatinib and sorafenib (a multi-kinase inhibitor) was as effective as a direct PI3 kinase... sorafenib , contributed to PI3K pathway activation in PTEN deficient LNCaP cells. Moreover, this inhibition correlated with loss of tyrosine

  16. Thermodynamic study of yeast phosphoglycerate kinase.

    PubMed

    Hu, C Q; Sturtevant, J M

    1987-01-13

    Enthalpies of binding of MgADP, MgATP, and 3-phosphoglycerate to yeast phosphoglycerate kinase have been determined by flow calorimetry at 9.95-32.00 degrees C. Combination of these data with published dissociation constants [Scopes, R.K. (1978) Eur. J. Biochem. 91, 119-129] yielded the following thermodynamic parameters for the binding of 3-phosphoglycerate at 25 degrees C: delta Go = -6.76 +/- 0.11 kcal mol-1, delta H = 3.74 +/- 0.08 kcal mol-1, delta So = 35.2 +/- 0.6 cal K-1 mol-1, and delta Cp = 0.12 +/- 0.32 kcal K-1 mol-1. The thermal unfolding of phosphoglycerate kinase in the absence and presence of the ligands listed above was studied by differential scanning calorimetry. The temperature of half-completion, t 1/2, of the denaturation and the denaturational enthalpy are increased by the binding of the ligands, the increase in t 1/2 being a manifestation of Le Chatelier's principle and that in enthalpy reflecting the enthalpy of dissociation of the ligand. Only one denaturational peak was observed under all conditions, and in contrast with the case of yeast hexokinase [Takahashi, K., Casey, J.L., & Sturtevant, J.M. (1981) Biochemistry 20, 4693-4697], no definitive evidence for the unfolding of more than one domain was obtained.

  17. Coated vesicles contain a phosphatidylinositol kinase.

    PubMed

    Campbell, C R; Fishman, J B; Fine, R E

    1985-09-15

    When coated vesicles (CVs) are incubated with [gamma-32P]ATP, radioactivity is rapidly incorporated into a compound identified by thin layer chromatography as phosphatidylinositol 4-phosphate. This activity has been identified in CVs isolated from bovine brain as well as from rat liver and chick embryo skeletal muscle. Phosphatidylinositol (PI) kinase is not separated from CVs during agarose electrophoresis, which produces CVs of greater than 95% purity, indicating that the activity present does not derive from contamination. The specific activity of these highly purified CVs was demonstrated to be approximately twice that of synaptic plasma membranes, further ruling out contamination from this source. The PI kinase remains associated with the vesicle upon removal of clathrin and its associated proteins and is solubilized by nonionic detergents, suggesting it is an integral membrane protein. We have been unable to demonstrate the formation of significant amounts of phosphatidylinositol 4,5-bisphosphate in any of our CV preparations. In the presence of exogenous PI, activity is stimulated, with maximal phosphorylation occurring at 0.1 mM. The enzyme appears to be maximally stimulated by 200 mM MgCl2 and 1 mM ATP and is most active at pH 7.25. Calculations indicate that, under optimal conditions, approximately 25 molecules of PIP are produced per CV within 60 s, suggesting that these structures may play an important role in cellular PI metabolism.

  18. Ras, Raf, and MAP kinase in melanoma.

    PubMed

    Solus, Jason F; Kraft, Stefan

    2013-07-01

    A growing understanding of the biology and molecular mechanisms of melanoma has led to the identification of a number of driver mutations for this aggressive tumor. The most common mutations affect signaling of the Ras/Raf/MAPK (mitogen-activated protein kinase) pathway. This review will focus on mutations in genes encoding proteins that play a role in the MAPK pathway and that have been implicated in melanoma biology, such as BRAF, NRAS, and MEK (MAPK kinase), and detail the current understanding of their role in melanoma progression from a molecular biology perspective. Furthermore, this review will also consider some additional mutations in genes such as KIT, GNAQ, and GNA11, which can be seen in certain subtypes of melanoma and whose gene products interact with the MAPK pathway. In addition, the association of these molecular changes with clinical and classical histopathologic characteristics of melanoma will be outlined and their role in diagnosis of melanocytic lesions discussed. Finally, a basic overview of the current targeted therapy landscape, as far as relevant to the pathologist, will be provided.

  19. Protein kinase C activity in boar sperm.

    PubMed

    Teijeiro, J M; Marini, P E; Bragado, M J; Garcia-Marin, L J

    2017-03-01

    Male germ cells undergo different processes within the female reproductive tract to successfully fertilize the oocyte. These processes are triggered by different extracellular stimuli leading to activation of protein phosphorylation. Protein kinase C (PKC) is a key regulatory enzyme in signal transduction mechanisms involved in many cellular processes. Studies in boar sperm demonstrated a role for PKC in the intracellular signaling involved in motility and cellular volume regulation. Experiments using phorbol 12-myristate 13-acetate (PMA) showed increases in the Serine/Threonine phosphorylation of substrates downstream of PKC in boar sperm. In order to gain knowledge about those cellular processes regulated by PKC, we evaluate the effects of PMA on boar sperm motility, lipid organization of plasma membrane, integrity of acrosome membrane and sperm agglutination. Also, we investigate the crosstalk between PKA and PKC intracellular pathways in spermatozoa from this species. The results presented here reveal a participation of PKC in sperm motility regulation and membrane fluidity changes, which is probably associated to acrosome reaction and to agglutination. Also, we show the existence of a hierarchy in the kinases pathway. Previous works on boar sperm suggest a pathway in which PKA is positioned upstream to PKC and this new results support such model.

  20. 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.

  1. Evolutionary hypothesis of the Mevalonate Kinase Deficiency.

    PubMed

    Vuch, J; Marcuzzi, A; Bianco, A M; Tommasini, A; Zanin, V; Crovella, S

    2013-01-01

    Mevalonate Kinase Deficiency (MKD) is an autosomal-recessively inherited disorder of cholesterol biosynthesis with higher prevalence in the Netherlands and other North European countries. MKD is due to mutations in the second enzyme of mevalonate pathway (mevalonate kinase, MK/MVK) which results in reduced enzymatic activity and in the consequent shortage of downstream compounds. In most severe cases the deregulation of mevalonate pathway is associated with a decrease in serum cholesterol. More than 100 pathological mutations have been described in the MVK gene so far, and a founder effect has been hypothesized as responsible for the diffusion of the most frequent disease-associated mutations. In the acute phase of disease, patients affected with MKD present low cholesterol levels comparable to their basal physiologic conditions, already characterized by lower cholesterol levels when compared to healthy individuals. Low cholesterol levels are widely known to correlate with the reduction of cardiovascular events. We hypothesize a selective advantage for heterozygote carriers of the most frequent MVK mutations in those countries where the diet is characterized by high consumption of saturated animal fats rich in cholesterol. This could explain the maintenance in North European population of the main mutations leading to MKD and the distribution world-wide of these mutations that followed the migrations of North European populations.

  2. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho.

    PubMed Central

    Matsui, T; Amano, M; Yamamoto, T; Chihara, K; Nakafuku, M; Ito, M; Nakano, T; Okawa, K; Iwamatsu, A; Kaibuchi, K

    1996-01-01

    The small GTP binding protein Rho is implicated in cytoskeletal responses to extracellular signals such as lysophosphatidic acid to form stress fibers and focal contacts. Here we have purified a Rho-interacting protein with a molecular mass of approximately 164 kDa (p164) from bovine brain. This protein bound to GTPgammaS (a non-hydrolyzable GTP analog).RhoA but not to GDP.RhoA or GTPgammaS.RhoA with a mutation in the effector domain (RhoAA37).p164 had a kinase activity which was specifically stimulated by GTPgammaS.RhoA. We obtained the cDNA encoding p164 on the basis of its partial amino acid sequences and named it Rho-associated kinase (Rho-kinase). Rho-kinase has a catalytic domain in the N-terminal portion, a coiled coil domain in the middle portion and a zinc finger-like motif in the C-terminal portion. The catalytic domain shares 72% sequence homology with that of myotonic dystrophy kinase and the coiled coil domain contains a Rho-interacting interface. When COS7 cells were cotransfected with Rho-kinase and activated RhoA, some Rho-kinase was recruited to membranes. Thus it is likely that Rho-kinase is a putative target serine/threonine kinase for Rho and serves as a mediator of the Rho-dependent signaling pathway. Images PMID:8641286

  3. Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases

    SciTech Connect

    Marcotte, Douglas J.; Liu, Yu-Ting; Arduini, Robert M.; Hession, Catherine A.; Miatkowski, Konrad; Wildes, Craig P.; Cullen, Patrick F.; Hong, Victor; Hopkins, Brian T.; Mertsching, Elisabeth; Jenkins, Tracy J.; Romanowski, Michael J.; Baker, Darren P.; Silvian, Laura F.

    2010-11-15

    Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 {angstrom} resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 {angstrom} resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.

  4. Protein-tyrosine phosphorylation interaction network in Bacillus subtilis reveals new substrates, kinase activators and kinase cross-talk

    PubMed Central

    Shi, Lei; Pigeonneau, Nathalie; Ventroux, Magali; Derouiche, Abderahmane; Bidnenko, Vladimir; Mijakovic, Ivan; Noirot-Gros, Marie-Françoise

    2014-01-01

    Signal transduction in eukaryotes is generally transmitted through phosphorylation cascades that involve a complex interplay of transmembrane receptors, protein kinases, phosphatases and their targets. Our previous work indicated that bacterial protein-tyrosine kinases and phosphatases may exhibit similar properties, since they act on many different substrates. To capture the complexity of this phosphorylation-based network, we performed a comprehensive interactome study focused on the protein-tyrosine kinases and phosphatases in the model bacterium Bacillus subtilis. The resulting network identified many potential new substrates of kinases and phosphatases, some of which were experimentally validated. Our study highlighted the role of tyrosine and serine/threonine kinases and phosphatases in DNA metabolism, transcriptional control and cell division. This interaction network reveals significant crosstalk among different classes of kinases. We found that tyrosine kinases can bind to several modulators, transmembrane or cytosolic, consistent with a branching of signaling pathways. Most particularly, we found that the division site regulator MinD can form a complex with the tyrosine kinase PtkA and modulate its activity in vitro. In vivo, it acts as a scaffold protein which anchors the kinase at the cell pole. This network highlighted a role of tyrosine phosphorylation in the spatial regulation of the Z-ring during cytokinesis. PMID:25374563

  5. Identification of the regulatory autophosphorylation site of autophosphorylation-dependent protein kinase (auto-kinase). Evidence that auto-kinase belongs to a member of the p21-activated kinase family.

    PubMed

    Yu, J S; Chen, W J; Ni, M H; Chan, W H; Yang, S D

    1998-08-15

    Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both

  6. Identification and characterization of two wheat Glycogen Synthase Kinase 3/ SHAGGY-like kinases

    PubMed Central

    2013-01-01

    Background Plant Glycogen Synthase Kinase 3/ SHAGGY-like kinases (GSKs) have been implicated in numerous biological processes ranging from embryonic, flower, stomata development to stress and wound responses. They are key regulators of brassinosteroid signaling and are also involved in the cross-talk between auxin and brassinosteroid pathways. In contrast to the human genome that contains two genes, plant GSKs are encoded by a multigene family. Little is known about Liliopsida resp. Poaceae in comparison to Brassicaceae GSKs. Here, we report the identification and structural characterization of two GSK homologs named TaSK1 and TaSK2 in the hexaploid wheat genome as well as a widespread phylogenetic analysis of land plant GSKs. Results Genomic and cDNA sequence alignments as well as chromosome localization using nullisomic-tetrasomic lines provided strong evidence for three expressed gene copies located on homoeolog chromosomes for TaSK1 as well as for TaSK2. Predicted proteins displayed a clear GSK signature. In vitro kinase assays showed that TaSK1 and TaSK2 possessed kinase activity. A phylogenetic analysis of land plant GSKs indicated that TaSK1 and TaSK2 belong to clade II of plant GSKs, the Arabidopsis members of which are all involved in Brassinosteroid signaling. Based on a single ancestral gene in the last common ancestor of all land plants, paralogs were acquired and retained through paleopolyploidization events, resulting in six to eight genes in angiosperms. More recent duplication events have increased the number up to ten in some lineages. Conclusions To account for plant diversity in terms of functionality, morphology and development, attention has to be devoted to Liliopsida resp Poaceae GSKs in addition to Arabidopsis GSKs. In this study, molecular characterization, chromosome localization, kinase activity test and phylogenetic analysis (1) clarified the homologous/paralogous versus homoeologous status of TaSK sequences, (2) pointed out their

  7. Rho kinase acts as a downstream molecule to participate in protein kinase Cε regulation of vascular reactivity after hemorrhagic shock in rats.

    PubMed

    Li, Tao; Zhu, Yu; Zang, Jia-tao; Peng, Xiao-yong; Lan, Dan; Yang, Guang-ming; Xu, Jing; Liu, Liang-ming

    2014-09-01

    Our previous study demonstrated that Rho kinase and protein kinase C (PKC) played important parts in the regulation of vascular reactivity after shock. Using superior mesenteric arteries (SMAs) from hemorrhagic shock rats and hypoxia-treated vascular smooth muscle cells (VSMCs), relationship of PKCε regulation of vascular reactivity to Rho kinase, as well as the signal transduction after shock, was investigated. The results showed that inhibition of Rho kinase with the Rho kinase-specific inhibitor Y-27632 antagonized the PKCε-specific agonist carbachol and highly expressed PKCε-induced increase of vascular reactivity in SMAs and VSMCs, whereas inhibition of PKCε with its specific inhibitory peptide did not antagonize the Rho kinase agonist (U-46619)-induced increase of vascular reactivity in SMAs and VSMCs. Activation of PKCε or highly expressed PKCε upregulated the activity of Rho kinase and the phosphorylation of PKC-dependent phosphatase inhibitor 17 (CPI-17), zipper interacting protein kinase (ZIPK), and integrin-linked kinase (ILK), whereas activation of Rho kinase increased only CPI-17 phosphorylation. The specific neutralization antibodies of ZIPK and ILK antagonized PKCε-induced increases in the activity of Rho kinase, but CPI-17 neutralization antibody did not antagonize this effect. These results suggested that Rho kinase takes part in the regulation of PKCε on vascular reactivity after shock. Rho kinase is downstream of PKCε. Protein kinase Cε activates Rho kinase via ZIPK and ILK; CPI-17 is downstream of Rho kinase.

  8. The specificities of protein kinase inhibitors: an update.

    PubMed Central

    Bain, Jenny; McLauchlan, Hilary; Elliott, Matthew; Cohen, Philip

    2003-01-01

    We have previously examined the specificities of 28 commercially available compounds, reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases [Davies, Reddy, Caivano and Cohen (2000) Biochem. J. 351, 95-105]. In the present study, we have extended this analysis to a further 14 compounds. Of these, indirubin-3'-monoxime, SP 600125, KT 5823 and ML-9 were found to inhibit a number of protein kinases and conclusions drawn from their use in cell-based assays are likely to be erroneous. Kenpaullone, Alsterpaullone, Purvalanol, Roscovitine, pyrazolopyrimidine 1 (PP1), PP2 and ML-7 were more specific, but still inhibited two or more protein kinases with similar potency. Our results suggest that the combined use of Roscovitine and Kenpaullone may be useful for identifying substrates and physiological roles of cyclin-dependent protein kinases, whereas the combined use of Kenpaullone and LiCl may be useful for identifying substrates and physiological roles of glycogen synthase kinase 3. The combined use of SU 6656 and either PP1 or PP2 may be useful for identifying substrates of Src family members. Epigallocatechin 3-gallate, one of the main polyphenolic constituents of tea, inhibited two of the 28 protein kinases in the panel, dual-specificity, tyrosine-phosphorylated and regulated kinase 1A (DYRK1A; IC(50)=0.33 microM) and p38-regulated/activated kinase (PRAK; IC(50)=1.0 microM). PMID:12534346

  9. Antiproliferative effect of Aurora kinase targeting in mesothelioma.

    PubMed

    Crispi, Stefania; Fagliarone, Claudia; Biroccio, Annamaria; D'Angelo, Carmen; Galati, Rossella; Sacchi, Ada; Vincenzi, Bruno; Baldi, Alfonso; Verdina, Alessandra

    2010-12-01

    The Aurora proteins are a small family of serine/threonine kinase that function in various stages of mitosis. Current interest in Aurora kinase relates to its role in tumours, and its potential as a therapeutic target. In this work we studied the expression of Aurora kinases A and B and related genes in human mesothelioma tissues and in five mesothelioma cell lines. Moreover, we analyzed the effects of ZM447439 (ZM), an Aurora kinase inhibitor, on cellular growth. Results evidenced an over-expression of Aurora kinase A and related genes in human mesothelioma tissues and an over-expression of Aurora kinases A and B in all cell lines. Moreover, we demonstrated that ZM447439 was able to inhibit cell growth in all cell lines and that this inhibition was due to a specific effect as demonstrated by the reduction in the level of Histone H3 phosphorylation. Our findings support a role of Aurora kinase in mesothelioma and the possibility of using Aurora kinase inhibitors in therapeutic modalities.

  10. Chromosome dynamics: new light on Aurora B kinase function.

    PubMed

    Shannon, Katie B; Salmon, E D

    2002-07-09

    Aurora B family kinases play an essential role in chromosome segregation and cytokinesis. Recent work suggests that the kinase activity is required for bipolar chromosome orientation, kinetochore assembly, spindle checkpoint and microtubule dynamics. Aurora B also has additional functions in chromosome condensation and cohesion.

  11. Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling.

    PubMed Central

    Shepherd, P R; Withers, D J; Siddle, K

    1998-01-01

    Insulin plays a key role in regulating a wide range of cellular processes. However, until recently little was known about the signalling pathways that are involved in linking the insulin receptor with downstream responses. It is now apparent that the activation of class 1a phosphoinositide 3-kinase (PI 3-kinase) is necessary and in some cases sufficient to elicit many of insulin's effects on glucose and lipid metabolism. The lipid products of PI 3-kinase act as both membrane anchors and allosteric regulators, serving to localize and activate downstream enzymes and their protein substrates. One of the major ways these lipid products of PI 3-kinase act in insulin signalling is by binding to pleckstrin homology (PH) domains of phosphoinositide-dependent protein kinase (PDK) and protein kinase B (PKB) and in the process regulating the phosphorylation of PKB by PDK. Using mechanisms such as this, PI 3-kinase is able to act as a molecular switch to regulate the activity of serine/threonine-specific kinase cascades important in mediating insulin's effects on endpoint responses. PMID:9677303

  12. Allosteric activation of apicomplexan calcium-dependent protein kinases

    SciTech Connect

    Ingram, Jessica R.; Knockenhauer, Kevin E.; Markus, Benedikt M.; Mandelbaum, Joseph; Ramek, Alexander; Shan, Yibing; Shaw, David E.; Schwartz, Thomas U.; Ploegh, Hidde L.; Lourido, Sebastian

    2015-08-24

    Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics, the effects of 1B7–kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes.

  13. Allosteric activation of apicomplexan calcium-dependent protein kinases

    DOE PAGES

    Ingram, Jessica R.; Knockenhauer, Kevin E.; Markus, Benedikt M.; ...

    2015-08-24

    Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics,more » the effects of 1B7–kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes.« less

  14. The Role of HAP Kinases in Breast Cancer.

    DTIC Science & Technology

    1996-09-01

    these MEKs and have obtained the following results. Germinal center kinase ( GCK ) can potently activate the SAPKs and SEK1 in cotransfections. GCK ...interacts with the small GTPase Rab8. Optimal activation of the SAPKs by GCK in situ requires the C-terminal PEST motif. RIP, a kinase associated with the

  15. Genomic analysis of the eukaryotic protein kinase superfamily: a perspective

    PubMed Central

    Hanks, Steven K

    2003-01-01

    Protein kinases with a conserved catalytic domain make up one of the largest 'superfamilies' of eukaryotic proteins and play many key roles in biology and disease. Efforts to identify and classify all the members of the eukaryotic protein kinase superfamily have recently culminated in the mining of essentially complete human genome data. PMID:12734000

  16. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2001-07-03

    The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  17. Ancestral resurrection reveals evolutionary mechanisms of kinase plasticity

    PubMed Central

    Howard, Conor J; Hanson-Smith, Victor; Kennedy, Kristopher J; Miller, Chad J; Lou, Hua Jane; Johnson, Alexander D; Turk, Benjamin E; Holt, Liam J

    2014-01-01

    Protein kinases have evolved diverse specificities to enable cellular information processing. To gain insight into the mechanisms underlying kinase diversification, we studied the CMGC protein kinases using ancestral reconstruction. Within this group, the cyclin dependent kinases (CDKs) and mitogen activated protein kinases (MAPKs) require proline at the +1 position of their substrates, while Ime2 prefers arginine. The resurrected common ancestor of CDKs, MAPKs, and Ime2 could phosphorylate substrates with +1 proline or arginine, with preference for proline. This specificity changed to a strong preference for +1 arginine in the lineage leading to Ime2 via an intermediate with equal specificity for proline and arginine. Mutant analysis revealed that a variable residue within the kinase catalytic cleft, DFGx, modulates +1 specificity. Expansion of Ime2 kinase specificity by mutation of this residue did not cause dominant deleterious effects in vivo. Tolerance of cells to new specificities likely enabled the evolutionary divergence of kinases. DOI: http://dx.doi.org/10.7554/eLife.04126.001 PMID:25310241

  18. Purification of leucoplast pyruvate kinase from developing castor bean endosperm.

    PubMed

    Plaxton, W C; Dennis, D T; Knowles, V L

    1990-12-01

    Leucoplast pyruvate kinase from endosperm of developing castor oil seeds (Ricinus communis L.; cv Baker) has been purified 1370-fold to a specific activity of 41.1 micromoles pyruvate produced per minute per milligram protein. Nondenaturing polyacrylamide gel electrophoresis of the purified enzyme resulted in a single protein staining band that co-migrated with pyruvate kinase activity. However, following sodium dodecyl sulfate polyacrylamide electrophoresis, two major protein staining bands of 57.5 and 44 kilodaltons, which occurred in an approximate 2:1 ratio, respectively, were observed. The native molecular mass was approximately 305 kilodaltons. Rabbit antiserum raised against the final enzyme preparation effectively immunoprecipitated leucoplast pyruvate kinase. The 57.5- and 44-kilodalton polypeptides are immunologically related as both proteins cross-reacted strongly on Western blots probed with the rabbit anti-(developing castor seed endosperm leucoplast pyruvate kinase) immunoglobulin that had been affinity-purified against the 57.5-kilodalton polypeptide. In contrast, pyruvate kinases from the following sources showed no immunological cross-reactivity with the same immunoglobulin: the cytosolic enzyme from developing or germinating castor bean endosperm; chloroplastic pyruvate kinase from expanding leaves of the castor oil plant; chloroplastic or cytosolic pyruvate kinase from the green alga, Selenastrum minutum; and mammalian or bacterial pyruvate kinases.

  19. Application Of Kinase Inhibitors For Anti-Aging Intervention.

    PubMed

    Cano, Mercedes; Ayala, Antonio; Marotta, Francesco; Argüelles, Sandro

    2017-07-14

    Protein phosphorylation, mediated by protein kinases, has important physiological and pathological implications in our lives . Targeting kinase is one of the most interesting of the emerging topics in the pharmaceutical industry, especially since there is a focus on cancer therapy. Given that kinases may be involved in the aging process the focus will be on using the kinase inhibitor for anti-aging intervention to enhance healthspan and increase longevity. In this review , we will summarize: (i) the impact of the phosphoproteomic approach to elucidate molecular mechanisms of diseases; (ii) importance of the drug discovery approach for targeting kinases; (iii) the dysregulation of Janus kinase (JAK) / signal-transducing adapter molecules (STAT) and p70 ribosomal protein S6 kinase (S6Ks) pathway in aging and the age-related process; (iv) the epidemiological studies available in order to see whether a correlation between JAK/STAT and S6Ks mRNA expression levels exist in cancer and in patient outcome; (v) finally, we will show selected inhibitors of these kinases approved by the US Food and Drug Administration (FDA). Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  20. A Flexible Workflow for Automated Bioluminescent Kinase Selectivity Profiling.

    PubMed

    Worzella, Tracy; Butzler, Matt; Hennek, Jacquelyn; Hanson, Seth; Simdon, Laura; Goueli, Said; Cowan, Cris; Zegzouti, Hicham

    2017-04-01

    Kinase profiling during drug discovery is a necessary process to confirm inhibitor selectivity and assess off-target activities. However, cost and logistical limitations prevent profiling activities from being performed in-house. We describe the development of an automated and flexible kinase profiling workflow that combines ready-to-use kinase enzymes and substrates in convenient eight-tube strips, a bench-top liquid handling device, ADP-Glo Kinase Assay (Promega, Madison, WI) technology to quantify enzyme activity, and a multimode detection instrument. Automated methods were developed for kinase reactions and quantification reactions to be assembled on a Gilson (Middleton, WI) PIPETMAX, following standardized plate layouts for single- and multidose compound profiling. Pipetting protocols were customized at runtime based on user-provided information, including compound number, increment for compound titrations, and number of kinase families to use. After the automated liquid handling procedures, a GloMax Discover (Promega) microplate reader preloaded with SMART protocols was used for luminescence detection and automatic data analysis. The functionality of the automated workflow was evaluated with several compound-kinase combinations in single-dose or dose-response profiling formats. Known target-specific inhibitions were confirmed. Novel small molecule-kinase interactions, including off-target inhibitions, were identified and confirmed in secondary studies. By adopting this streamlined profiling process, researchers can quickly and efficiently profile compounds of interest on site.

  1. A Flexible Workflow for Automated Bioluminescent Kinase Selectivity Profiling

    PubMed Central

    Worzella, Tracy; Butzler, Matt; Hennek, Jacquelyn; Hanson, Seth; Simdon, Laura; Goueli, Said; Cowan, Cris; Zegzouti, Hicham

    2016-01-01

    Kinase profiling during drug discovery is a necessary process to confirm inhibitor selectivity and assess off-target activities. However, cost and logistical limitations prevent profiling activities from being performed in-house. We describe the development of an automated and flexible kinase profiling workflow that combines ready-to-use kinase enzymes and substrates in convenient eight-tube strips, a bench-top liquid handling device, ADP-Glo Kinase Assay (Promega, Madison, WI) technology to quantify enzyme activity, and a multimode detection instrument. Automated methods were developed for kinase reactions and quantification reactions to be assembled on a Gilson (Middleton, WI) PIPETMAX, following standardized plate layouts for single- and multidose compound profiling. Pipetting protocols were customized at runtime based on user-provided information, including compound number, increment for compound titrations, and number of kinase families to use. After the automated liquid handling procedures, a GloMax Discover (Promega) microplate reader preloaded with SMART protocols was used for luminescence detection and automatic data analysis. The functionality of the automated workflow was evaluated with several compound-kinase combinations in single-dose or dose-response profiling formats. Known target-specific inhibitions were confirmed. Novel small molecule-kinase interactions, including off-target inhibitions, were identified and confirmed in secondary studies. By adopting this streamlined profiling process, researchers can quickly and efficiently profile compounds of interest on site. PMID:28095176

  2. Regulation of tomato Prf by Pto-like protein kinases.

    PubMed

    Mucyn, Tatiana S; Wu, Ai-Jiuan; Balmuth, Alexi L; Arasteh, Julia Maryam; Rathjen, John P

    2009-04-01

    Tomato Prf encodes a nucleotide-binding domain shared by Apaf-1, certain R proteins, and CED-4 fused to C-terminal leucine-rich repeats (NBARC-LRR) protein that is required for bacterial immunity to Pseudomonas syringae and sensitivity to the organophosphate fenthion. The signaling pathways involve two highly related protein kinases. Pto kinase mediates direct recognition of the bacterial effector proteins AvrPto or AvrPtoB. Fen kinase is required for fenthion sensitivity and recognition of bacterial effectors related to AvrPtoB. The role of Pto and its association with Prf has been characterized but Fen is poorly described. We show that, similar to Pto, Fen requires N-myristoylation and kinase activity for signaling and interacts with the N-terminal domain of Prf. Thus, the mechanisms of activation of Prf by the respective protein kinases are similar. Prf-Fen interaction is underlined by coregulatory mechanisms in which Prf negatively regulates Fen, most likely by controlling kinase activity. We further characterized negative regulation of Prf by Pto, and show that regulation is mediated by the previously described negative regulatory patch. Remarkably, the effectors released negative regulation of Prf in a manner dependent on Pto kinase activity. The data suggest a model in which Prf associates generally with Pto-like kinases in tightly regulated complexes, which are activated by effector-mediated disruption of negative regulation. Release of negative regulation may be a general feature of activation of NBARC-LRR proteins by cognate effectors.

  3. Molecular and structural insight into plasmodium falciparum RIO2 kinase.

    PubMed

    Chouhan, Devendra K; Sharon, Ashoke; Bal, Chandralata

    2013-02-01

    Among approximately 65 kinases of the malarial genome, RIO2 (right open reading frame) kinase belonging to the atypical class of kinase is unique because along with a kinase domain, it has a highly conserved N-terminal winged helix (wHTH) domain. The wHTH domain resembles the wing like domain found in DNA binding proteins and is situated near to the kinase domain. Ligand binding to this domain may reposition the kinase domain leading to inhibition of enzyme function and could be utilized as a novel allosteric site to design inhibitor. In the present study, we have generated a model of RIO2 kinase from Plasmodium falciparum utilizing multiple modeling, simulation approach. A novel putative DNA-binding site is identified for the first time in PfRIO2 kinase to understand the DNA binding events involving wHTH domain and flexible loop. Induced fit DNA docking followed by minimization, molecular dynamics simulation, energetic scoring and binding mode studies are used to reveal the structural basis of PfRIO2-ATP-DNA complex. Ser105 as a potential site of phosphorylation is revealed through the structural studies of ATP binding in PfRIO2. Overall the present study discloses the structural facets of unknown PfRIO2 complex and opens an avenue toward exploration of novel drug target.

  4. Allosteric activation of apicomplexan calcium-dependent protein kinases

    PubMed Central

    Ingram, Jessica R.; Knockenhauer, Kevin E.; Markus, Benedikt M.; Mandelbaum, Joseph; Ramek, Alexander; Shan, Yibing; Shaw, David E.; Schwartz, Thomas U.; Ploegh, Hidde L.; Lourido, Sebastian

    2015-01-01

    Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, through molecular dynamics, the effects of 1B7–kinase interactions. In contrast to other Ca2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes. PMID:26305940

  5. Auto-phosphorylation Represses Protein Kinase R Activity.

    PubMed

    Wang, Die; de Weerd, Nicole A; Willard, Belinda; Polekhina, Galina; Williams, Bryan R G; Sadler, Anthony J

    2017-03-10

    The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity.

  6. Auto-phosphorylation Represses Protein Kinase R Activity

    PubMed Central

    Wang, Die; de Weerd, Nicole A.; Willard, Belinda; Polekhina, Galina; Williams, Bryan R. G.; Sadler, Anthony J.

    2017-01-01

    The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity. PMID:28281686

  7. Resveratrol dimers are novel sphingosine kinase 1 inhibitors and affect sphingosine kinase 1 expression and cancer cell growth and survival

    PubMed Central

    Lim, Keng Gat; Gray, Alexander I; Pyne, Susan; Pyne, Nigel J

    2012-01-01

    BACKGROUND AND PURPOSE Sphingosine kinase 1 catalyses formation of the bioactive lipid, sphingosine 1-phosphate, which protects cancer cells from apoptosis. Therefore, sphingosine kinase 1 is a novel target for intervention with anti-cancer agents. We have assessed the effect of the anti-cancer agent, resveratrol and its dimers (ampelopsin A and balanocarpol) on sphingosine kinase 1 activity and on survival of MCF-7 breast cancer cells. EXPERIMENTAL APPROACH Ampelopsin A and balanocarpol were purified from Hopea dryobalanoides and their effect on sphingosine kinase 1 activity and expression, [3H] thymidine incorporation, ERK-1/2 phosphorylation and PARP activity assessed in MCF-7 cells. KEY RESULTS Resveratrol, ampelopsin A and balanocarpol were novel inhibitors of sphingosine kinase 1 activity. Balanocarpol was a mixed inhibitor (with sphingosine) of sphingosine kinase 1 with a Kic= 90 ± 10 µM and a Kiu of ∼500 µM. Balanocarpol and ampelopsin A also induced down-regulation of sphingosine kinase 1 expression and reduced DNA synthesis, while balanocarpol stimulated PARP cleavage in MCF-7 breast cancer cells. Resveratrol was a competitive inhibitor (with sphingosine) of sphingosine kinase 1 with a Kic= 160 ± 40 µM, reduced sphingosine kinase 1 expression and induced PARP cleavage in MCF-7 cells. CONCLUSIONS AND IMPLICATIONS Each molecule of balanocarpol may bind at least two sphingosine kinase 1 catalytic molecules to reduce the activity of each simultaneously. These findings suggest that resveratrol, ampelopsin A and balanocarpol could perturb sphingosine kinase 1-mediated signalling and this might explain their activity against MCF-7 breast cancer cells. LINKED ARTICLE This article is commented on by Hergst and Yun, pp. 1603–1604 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2012.01898.x PMID:22251058

  8. A high-throughput, multiplexed kinase assay using a benchtop orbitrap mass spectrometer to investigate the effect of kinase inhibitors on kinase signaling pathways.

    PubMed

    Kunz, Ryan C; McAllister, Fiona E; Rush, John; Gygi, Steven P

    2012-07-17

    Protein phosphorylation is an important and ubiquitous post-translational modification in eukaryotic biological systems. The KAYAK (Kinase ActivitY Assay for Kinome profiling) assay measures the phosphorylation rates of dozens of peptide substrates simultaneously, directly from cell lysates. Here, we simplified the assay by removing the phosphopeptide enrichment step, increasing throughput while maintaining similar data quality. We term this new method, direct-KAYAK, because kinase activities were measured directly from reaction mixtures after desalting. In addition, new peptides were included to profile additional kinase pathways and redundant substrate peptides were removed. Finally, the method is now performed in 96-well plate format using a benchtop orbitrap mass spectrometer and the Pinpoint software package for improved data analysis. We applied the new high-throughput method to measure IC(50) values for kinases involved in monocyte-to-macrophage differentiation, a process important for inflammation and the immune response.

  9. VZV ORF47 serine protein kinase and its viral substrates.

    PubMed

    Kenyon, Teri K; Grose, Charles

    2010-01-01

    ORF47, a serine protein kinase of varicella-zoster virus (VZV) and homolog of herpes simplex virus UL13, is an interesting modulator of VZV pathogenesis. This chapter summarizes research showing that ORF47 protein kinase activity, by virtue of phosphorylation of or binding to various viral substrates, regulates VZV proteins during all phases of viral infection and has a pronounced effect on the trafficking of gE, the predominant VZV glycoprotein, which in turn is critical for cell-to-cell spread of the virus. Casein kinase II, an ubiquitous cellular protein kinase, recognizes a similar but less stringent phosphorylation consensus sequence and can partially compensate for lack of ORF47 activity in VZV-infected cells. Differences between the phosphorylation consensus sites of the viral and cellular kinases are outlined in detail.

  10. 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

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

    PubMed

    Vlasova-St Louis, Irina; Bohjanen, Paul R

    2016-01-25

    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.

  12. Rho-associated coiled-coil containing kinases (ROCK)

    PubMed Central

    Julian, Linda; Olson, Michael F

    2014-01-01

    Rho-associated coiled-coil containing kinases (ROCK) were originally identified as effectors of the RhoA small GTPase.1–5 They belong to the AGC family of serine/threonine kinases6 and play vital roles in facilitating actomyosin cytoskeleton contractility downstream of RhoA and RhoC activation. Since their discovery, ROCK kinases have been extensively studied, unveiling their manifold functions in processes including cell contraction, migration, apoptosis, survival, and proliferation. Two mammalian ROCK homologs have been identified, ROCK1 (also called ROCK I, ROKβ, Rho-kinase β, or p160ROCK) and ROCK2 (also known as ROCK II, ROKα, or Rho kinase), hereafter collectively referred to as ROCK. In this review, we will focus on the structure, regulation, and functions of ROCK. PMID:25010901

  13. Tyrosine kinase signaling and the emergence of multicellularity

    PubMed Central

    Miller, W. Todd

    2012-01-01

    Tyrosine phosphorylation is an essential element of signal transduction in multicellular animals. Although tyrosine kinases were originally regarded as specific to the metazoan lineage, it is now clear that they evolved prior to the split between unicellular and multicellular eukaryotes (≈ 600 million years ago). Genome analyses of choanoflagellates and other protists show an abundance of tyrosine kinases that rivals the most complex animals. Some of these kinases are orthologs of metazoan enzymes (e.g., Src), but others display unique domain compositions not seen in any metazoan. Biochemical experiments have highlighted similarities and differences between the unicellular and multicellular tyrosine kinases. In particular, it appears that the complex systems of kinase autoregulation may have evolved later in the metazoan lineage. PMID:22480439

  14. Rho-kinase inhibition in the therapy of cardiovascular disease.

    PubMed

    Lai, Andrew; Frishman, William H

    2005-01-01

    Rho is a GTPase known to be a major mediator in the formation of stress fibers and focal adhesions, cell morphology, and smooth muscle contraction. Its role in smooth muscle contraction has led to exploration into the connection between Rho-mediated kinase activity and cardiovascular disease. The role of Rho-kinase in calcium sensitization for vascular smooth muscle contraction has recently been characterized. Inappropriate coronary artery vasoconstriction resulting from increased Rho-kinase in the vascular system is likely involved in the pathogenesis of exercise-induced myocardial ischemia, spontaneous coronary artery spasm, and hypertension. In clinical trials, Rho-kinase inhibitors such as fasudil and Y-27632 have demonstrated antiischemic, antivasospastic, and antihypertensive effects. These compounds have also exhibited the ability to blunt progression of cardiomyocyte hypertrophy and cardiac remodeling in heart failure. As such, Rho-kinase inhibition represents a potential novel therapeutic approach in cardiovascular disease.

  15. Irreversible Nek2 kinase inhibitors with cellular activity

    PubMed Central

    Henise, Jeffrey C.; Taunton, Jack

    2013-01-01

    A structure-based approach was used to design irreversible, cysteine-targeted inhibitors of the human centrosomal kinase, Nek2. Potent inhibition of Nek2 kinase activity in biochemical and cell-based assays required a noncatalytic cysteine residue (Cys22), located near the glycine-rich loop in a subset of human kinases. Elaboration of an oxindole scaffold led to our most selective compound, oxindole propynamide 16 (JH295). Propynamide 16 irreversibly inhibited cellular Nek2 without affecting the mitotic kinases, Cdk1, Aurora B, or Plk1. Moreover, 16 did not perturb bipolar spindle assembly or the spindle assembly checkpoint. To our knowledge, 16 is the first small molecule shown to inactivate Nek2 kinase activity in cells. PMID:21627121

  16. [The role of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis].

    PubMed

    Nerusheva, O O; Dorogova, N V; Gubanova, N V; Omel'ianchuk, L V

    2008-09-01

    The cellular function of the gilgamesh mutation (89B9-12) of casein kinase gene in Drosophila spermatogenesis was studied. It was demonstrated that the sterility resulting from this mutation is connected with the abnormalities in spermatid individualization. A phylogenetic study of the protein sequences of casein kinases 1 from various organisms was conducted. The Gilgamesh protein was shown to be phylogenetically closer to the cytoplasmic casein kinase family, represented by the YCK3, YCK2, and YCK1 proteins of Saccharomyces cerevisiae and animal gamma-casein kinases. It is known that these yeast casein kinases are involved in vesicular trafficking, which, in turn, is related in its genetic control to the cell membrane remodeling during spermatid individualization. Thus, the data of phylogenetic analysis fit well the results obtained by studying the mutation phenotype.

  17. Structure of the kinase domain of Gilgamesh from Drosophila melanogaster.

    PubMed

    Han, Ni; Chen, CuiCui; Shi, Zhubing; Cheng, Dianlin

    2014-04-01

    The CK1 family kinases regulate multiple cellular aspects and play important roles in Wnt/Wingless and Hedgehog signalling. The kinase domain of Drosophila Gilgamesh isoform I (Gilgamesh-I), a homologue of human CK1-γ, was purified and crystallized. Crystals of methylated Gilgamesh-I kinase domain with a D210A mutation diffracted to 2.85 Å resolution and belonged to space group P43212, with unit-cell parameters a = b = 52.025, c = 291.727 Å. The structure of Gilgamesh-I kinase domain, which was determined by molecular replacement, has conserved catalytic elements and an active conformation. Structural comparison indicates that an extended loop between the α1 helix and the β4 strand exists in the Gilgamesh-I kinase domain. This extended loop may regulate the activity and function of Gilgamesh-I.

  18. Structure of the kinase domain of Gilgamesh from Drosophila melanogaster

    PubMed Central

    Han, Ni; Chen, CuiCui; Shi, Zhubing; Cheng, Dianlin

    2014-01-01

    The CK1 family kinases regulate multiple cellular aspects and play important roles in Wnt/Wingless and Hedgehog signalling. The kinase domain of Drosophila Gilgamesh isoform I (Gilgamesh-I), a homologue of human CK1-γ, was purified and crystallized. Crystals of methylated Gilgamesh-I kinase domain with a D210A mutation diffracted to 2.85 Å resolution and belonged to space group P43212, with unit-cell parameters a = b = 52.025, c = 291.727 Å. The structure of Gilgamesh-I kinase domain, which was determined by molecular replacement, has conserved catalytic elements and an active conformation. Structural comparison indicates that an extended loop between the α1 helix and the β4 strand exists in the Gilgamesh-I kinase domain. This extended loop may regulate the activity and function of Gilgamesh-I. PMID:24699734

  19. Complex kinase requirements for Chlamydia trachomatis Tarp phosphorylation.

    PubMed

    Mehlitz, Adrian; Banhart, Sebastian; Hess, Simone; Selbach, Matthias; Meyer, Thomas F

    2008-12-01

    Chlamydia trachomatis translocates the effector protein Tarp (translocated actin-recruiting phosphoprotein) into the host cell cytoplasm where it is quickly tyrosine phosphorylated. Abl and Src kinases have been implicated in Tarp phosphorylation; however, we observed that the situation is more complex. Chemical inhibition of Src family kinases confirmed a role for these kinases in Tarp phosphorylation. Infection of Src, Yes, Fyn (SYF)-deficient cells showed a dampened, but incompletely blocked, Tarp phosphorylation. Inhibition of Abl in an SYF background still did not completely block Tarp phosphorylation. Consequently, we tested additional kinases and found that Syk, but not Btk or Jak2, is a potent kinase of Tarp in vitro. Inhibition of Syk in an SYF background further blocked Tarp phosphorylation. Under these conditions, inclusion formation still proceeded normally. These data reveal a highly promiscuous substrate property of Tarp and set the stage for further functional characterization of Tarp phosphorylation during host cell infection.

  20. Brassinosteroid signal transduction from receptor kinases to transcription factors.

    PubMed

    Kim, Tae-Wuk; Wang, Zhi-Yong

    2010-01-01

    Brassinosteroids (BRs) are growth-promoting steroid hormones in plants. Genetic studies in Arabidopsis illustrated the essential roles of BRs in a wide range of developmental processes and helped identify many genes involved in BR biosynthesis and signal transduction. Recently, proteomic studies identified missing links. Together, these approaches established the BR signal transduction cascade, which includes BR perception by the BRI1 receptor kinase at the cell surface, activation of BRI1/BAK1 kinase complex by transphosphorylation, subsequent phosphorylation of the BSK kinases, activation of the BSU1 phosphatase, dephosphorylation and inactivation of the BIN2 kinase, and accumulation of unphosphorylated BZR transcription factors in the nucleus. Mass spectrometric analyses are providing detailed information on the phosphorylation events involved in each step of signal relay. Thus, the BR signaling pathway provides a paradigm for understanding receptor kinase-mediated signal transduction as well as tools for the genetic improvement of the productivity of crop plants.

  1. Targeting the HER-kinase axis in cancer.

    PubMed

    Gross, Mitchell E; Shazer, Ronald L; Agus, David B

    2004-02-01

    The human epidermal growth factor receptor (HER) family of receptor tyrosine kinases controls critical pathways involved in the differentiation, growth, division, and motility of normal epithelial cells. Most human solid tumors are of epithelial origin. The process of malignant transformation and progression in many cancers may depend on activation of ligands and receptors that function as part of the HER-kinase pathway. This signaling axis has earned increased attention because of the development of antibodies and small-molecule tyrosine kinase inhibitors that specifically target components of the HER-kinase axis for cancer therapy. This review focuses on the basic biology underlying HER-kinase pathway activation and the current state of development for agents that target this axis. In particular, the importance of pan-HER inhibitors is discussed.

  2. A family of human cdc2-related protein kinases.

    PubMed Central

    Meyerson, M; Enders, G H; Wu, C L; Su, L K; Gorka, C; Nelson, C; Harlow, E; Tsai, L H

    1992-01-01

    The p34cdc2 protein kinase is known to regulate important transitions in the eukaryotic cell cycle. We have identified 10 human protein kinases based on their structural relation to p34cdc2. Seven of these kinases are novel and the products of five share greater than 50% amino acid sequence identity with p34cdc2. The seven novel genes are broadly expressed in human cell lines and tissues with each displaying some cell type or tissue specificity. The cdk3 gene, like cdc2 and cdk2, can complement cdc28 mutants of Saccharomyces cerevisiae, suggesting that all three of these protein kinases can play roles in the regulation of the mammalian cell cycle. The identification of a large family of cdc2-related kinases opens the possibility of combinatorial regulation of the cell cycle together with the emerging large family of cyclins. Images PMID:1639063

  3. Structural investigation of protein kinase C inhibitors.

    PubMed

    Barak, D; Shibata, M; Rein, R

    1991-01-01

    The phospholipid and Ca2+ dependent protein kinase (PKC) plays an essential role in a variety of cellular events. Inhibition of PKC was shown to arrest growth in tumor cell cultures making it a target for possible antitumor therapy. Calphostins are potent inhibitors of PKC with high affinity for the enzyme regulatory site. Structural characteristics of calphostins, which confer the inhibitory activity, are investigated by comparing their optimized structures with the existing models for PKC activation. The resulting model of inhibitory activity assumes interaction with two out of the three electrostatic interaction sites postulated for activators. The model shows two sites of hydrophobic interaction and enables the inhibitory activity of gossypol to be accounted for.

  4. Protein kinase C isozymes and addiction.

    PubMed

    Olive, M Foster; Messing, Robert O

    2004-04-01

    Protein kinase C (PKC) has long been recognized an important family of enzymes that regulate numerous aspects of neuronal signal transduction, neurotransmitter synthesis, release and reuptake, receptor and ion channel function, neuronal excitability, development, and gene expression. Much evidence has implicated PKCs in the effects of several drugs of abuse, and in behavioral responses to these drugs. The present review summarizes the effects of both acute and chronic exposure to various drugs of abuse on individual PKC isozymes in the brain. In addition, we summarize recent studies utilizing mice with targeted deletions of the genes for PKCgamma and PKCepsilon. These studies suggest that individual PKC isozymes play a role in the development of drug dependence and addiction.

  5. The anaplastic lymphoma kinase testing conundrum.

    PubMed

    Conde, Esther; Taniere, Philippe; Lopez-Rios, Fernando

    2015-02-01

    Given the excellent results of the clinical trials with anaplastic lymphoma kinase (ALK) inhibitors, the importance of accurately identifying ALK-positive lung carcinoma patients has never been greater. It brings with it a pressing need for harmonized development of companion diagnostics, for economic, scientific and medical reasons. Therefore, it is crucial that ALK testing assays become more standardized both in performance (analytical phase) and interpretation (post-analytical phase). We find that both methods currently recommended by College of American Pathologists/International Association for the Study of Lung Cancer/Association for Molecular Pathology guidelines (FISH and Immunohistochemistry) are reasonable approaches for primary routine ALK testing, if at least 50 tumor cells are scored and protocols are strictly followed. Moreover, due to the high demand to study multiple predictive biomarkers on different assay platforms, quick and reliable approaches to achieve this are essential to guide treatment decisions.

  6. Serum creatine kinase after intramuscular injections.

    PubMed Central

    Konikoff, F.; Halevy, J.; Theodor, E.

    1985-01-01

    Serum creatine kinase (CK) activity was measured after intramuscular injections in 44 patients hospitalized for non-cardiac reasons. The drugs injected were: diazepam, dipyrone, metoclopramide, meperidine, pentazocine and procaine penicillin. Only 3 out of 44 patients (7%) demonstrated significant elevation of CK levels following the intramuscular injections. In these 3 patients the elevation was mainly due to a rise of the MM-isoenzyme fraction with MB levels increased in one patient. These findings do not justify the common clinical notion of regarding intramuscular injections as a frequent cause of serum CK elevation. It is concluded that high CK serum values in a patient with chest pain should always be considered with utmost suspicion, disregarding the possible effects of a previous intramuscular injection. PMID:4022892

  7. Tyr(less) kinase signaling during mitosis.

    PubMed

    Elowe, Sabine

    2017-04-18

    Tyrosine phosphorylation is rare, representing only about 0.5% of phosphorylations in the cell under basal conditions. While mitogenic tyrosine kinase signaling has been extensively explored, the role of phosphotyrosine signaling across the cell cycle and in particular during mitosis is poorly understood. Two recent, independent studies tackled this question from different angles to reveal exciting new insights into the role of this modification during cell division. Caron et al. (1) exploited mitotic phosphoproteomics data sets to determine the extent of mitotic tyrosine phosphorylation, and St-Denis et al. (2) identified protein tyrosine phosphatases from all subfamilies as regulators of mitotic progression or spindle formation. These studied collectively revealed that tyrosine phosphorylation may play a more prominent and active role in mitotic progression than previously appreciated.

  8. The Immunomodulatory Functions of Diacylglycerol Kinase ζ

    PubMed Central

    Singh, Brenal K.; Kambayashi, Taku

    2016-01-01

    The generation of diacylglycerol (DAG) is critical for promoting immune cell activation, regulation, and function. Diacylglycerol kinase ζ (DGKζ) serves as an important negative regulator of DAG by enzymatically converting DAG into phosphatidic acid (PA) to shut down DAG-mediated signaling. Consequently, the loss of DGKζ increases DAG levels and the duration of DAG-mediated signaling. However, while the enhancement of DAG signaling is thought to augment immune cell function, the loss of DGKζ can result in both immunoactivation and immunomodulation depending on the cell type and function. In this review, we discuss how different immune cell functions can be selectively modulated by DGKζ. Furthermore, we consider how targeting DGKζ can be potentially beneficial for the resolution of human diseases by either promoting immune responses important for protection against infection or cancer or dampening immune responses in immunopathologic conditions such as allergy and septic shock. PMID:27656643

  9. Dealing with osmostress through MAP kinase activation

    PubMed Central

    de Nadal, Eulàlia; Alepuz, Paula M.; Posas, Francesc

    2002-01-01

    In response to changes in the extracellular environment, cells coordinate intracellular activities to maximize their probability of survival and proliferation. Eukaryotic cells, from yeast to mammals, transduce diverse extracellular stimuli through the cell by multiple mitogen-activated protein kinase (MAPK) cascades. Exposure of cells to increases in extracellular osmolarity results in rapid activation of a highly conserved family of MAPKs, known as stress-activated MAPKs (SAPKs). Activation of SAPKs is essential for the induction of adaptive responses required for cell survival upon osmostress. Recent studies have begun to shed light on the broad effects of SAPK activation in the modulation of several aspects of cell physiology, ranging from the control of gene expression to the regulation of cell division. PMID:12151331

  10. Protein Scaffolds in MAP Kinase Signalling

    PubMed Central

    Brown, Matthew D.; Sacks, David B.

    2009-01-01

    The mitogen-activated protein kinase (MAPK) pathway allows cells to interpret external signals and respond in an appropriate way. Diverse cellular functions, ranging from differentiation and proliferation to migration and inflammation, are regulated by MAPK signalling. Therefore, cells have developed mechanisms by which this single pathway modulates numerous cellular responses from a wide range of activating factors. This specificity is achieved by several mechanisms, including temporal and spatial control of MAPK signalling components. Key to this control are protein scaffolds, which are multidomain proteins that interact with components of the MAPK cascade in order to assemble signalling complexes. Studies conducted on different scaffolds, in different biological systems, have shown that scaffolds exert substantial control over MAPK signalling, influencing the signal intensity, time course and, importantly, the cellular responses. Protein scaffolds, therefore, are integral elements in the modulation of the MAPK network in fundamental physiological processes. PMID:19091303

  11. Antibodies directed against receptor tyrosine kinases

    PubMed Central

    FAUVEL, Bénédicte; Yasri, Aziz

    2014-01-01

    Approximately 30 therapeutic monoclonal antibodies have already been approved for cancers and inflammatory diseases, and monoclonal antibodies continue to be one of the fastest growing classes of therapeutic molecules. Because aberrant signaling by receptor tyrosine kinases (RTKs) is a commonly observed factor in cancer, most of the subclasses of RTKs are being extensively studied as potential targets for treating malignancies. The first two RTKs that have been targeted by antibody therapy, with five currently marketed antibodies, are the growth factor receptors EGFR and HER2. However, due to systemic side effects, refractory patients and the development of drug resistance, these treatments are being challenged by emerging therapeutics. This review examines current monoclonal antibody therapies against RTKs. After an analysis of agents that have already been approved, we present an analysis of antibodies in clinical development that target RTKs. Finally, we highlight promising RTKs that are emerging as new oncological targets for antibody-based therapy. PMID:24859229

  12. Structural investigation of protein kinase C inhibitors

    NASA Technical Reports Server (NTRS)

    Barak, D.; Shibata, M.; Rein, R.

    1991-01-01

    The phospholipid and Ca2+ dependent protein kinase (PKC) plays an essential role in a variety of cellular events. Inhibition of PKC was shown to arrest growth in tumor cell cultures making it a target for possible antitumor therapy. Calphostins are potent inhibitors of PKC with high affinity for the enzyme regulatory site. Structural characteristics of calphostins, which confer the inhibitory activity, are investigated by comparing their optimized structures with the existing models for PKC activation. The resulting model of inhibitory activity assumes interaction with two out of the three electrostatic interaction sites postulated for activators. The model shows two sites of hydrophobic interaction and enables the inhibitory activity of gossypol to be accounted for.

  13. Characterization of two kinases involved in thiamine pyrophosphate and pyridoxal phosphate biosynthesis in Bacillus subtilis: 4-amino-5-hydroxymethyl-2methylpyrimidine kinase and pyridoxal kinase.

    PubMed

    Park, Joo-Heon; Burns, Kristin; Kinsland, Cynthia; Begley, Tadhg P

    2004-03-01

    Two Bacillus subtilis genes encoding two proteins (currently annotated ThiD and YjbV) were overexpressed and characterized. YjbV has 4-amino-5-hydroxymethyl-2-methylpyrimidine and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate kinase activity and should be reannotated ThiD, and B. subtilis ThiD has pyridoxine, pyridoxal, and pyridoxamine kinase activity and should be reannotated PdxK.

  14. 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.

  15. Combined Cancer Immunotherapy Against Aurora Kinase A.

    PubMed

    Kaštánková, Iva; Poláková, Ingrid; Dušková, Martina; Šmahel, Michal

    2016-05-01

    Aurora kinase A (AURKA) is a centrosomal protein that is overexpressed in a number of human malignancies and can contribute to tumor progression. As we used this protein as a target of DNA immunization, we increased its immunogenicity by the addition of the PADRE helper epitope and decreased its potential oncogenicity by mutagenesis of the kinase domain. For in vitro analysis of induced immune responses in mice, we identified the Aurka(220-228) nonapeptide representing an H-2Kb epitope. As DNA vaccination against the Aurka self-antigen by a gene gun did not show any antitumor effect, we combined DNA immunization with anti-CD25 treatment that depletes mainly regulatory T cells. Whereas 1 anti-CD25 dose injected before DNA vaccination did not enhance the activation of Aurka-specific splenocytes, 3 doses administered on days of immunizations augmented about 10-fold immunity against Aurka. However, an opposite effect was found for antitumor immunity-only 1 anti-CD25 dose combined with DNA vaccination reduced tumor growth. Moreover, the administration of 3 doses of anti-CD25 antibody alone accelerated tumor growth. Analysis of tumor-infiltrating cells showed that 3 anti-CD25 doses not only efficiently depleted regulatory T cells but also activated helper T cells and CD3(-)CD25(+) cells. Next, we found that blockade of the PD-1 receptor initiated 1 week after the first immunization was necessary for significant inhibition of tumor growth with therapeutic DNA vaccination against Aurka combined with depletion of CD25 cells. Our results suggest that combined cancer immunotherapy should be carefully evaluated to achieve the optimal antitumor effect.

  16. Conformational Dynamics and Allostery in Pyruvate Kinase*

    PubMed Central

    Donovan, Katherine A.; Zhu, Shaolong; Liuni, Peter; Peng, Fen; Kessans, Sarah A.; Wilson, Derek J.

    2016-01-01

    Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the “domain rotation model” suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the “rigid body reorientation model” proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1,6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1,6-bisphosphate destabilizes an α-helix that bridges the allosteric and active site domains within the monomeric unit. This destabilizes the β-strands within the (β/α)8-barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1,6-bisphosphate affects the active site. PMID:26879751

  17. Photoinduced structural changes to protein kinase A

    NASA Astrophysics Data System (ADS)

    Rozinek, Sarah C.; Thomas, Robert J.; Brancaleon, Lorenzo

    2014-03-01

    The importance of porphyrins in organisms is underscored by the ubiquitous biological and biochemical functions that are mediated by these compounds and by their potential biomedical and biotechnological applications. Protoporphyrin IX (PPIX) is the precursor to heme and has biomedical applications such as its use as a photosensitizer in phototherapy and photodetection of cancer. Among other applications, our group has demonstrated that low-irradiance exposure to laser irradiation of PPIX, Fe-PPIX, or meso-tetrakis (4-sulfonatophenyl) porphyrin (TSPP) non-covalently docked to a protein causes conformational changes in the polypeptide. Such approach can have remarkable consequences in the study of protein structure/function relationship and can be used to prompt non-native protein properties. Therefore we have investigated protein kinase A (PKA), a more relevant protein model towards the photo-treatment of cancer. PKA's enzymatic functions are regulated by the presence of cyclic adenosine monophosphate for intracellular signal transduction involved in, among other things, stimulation of transcription, tumorigenesis in Carney complex and migration of breast carcinoma cells. Since phosphorylation is a necessary step in some cancers and inflammatory diseases, inhibiting the protein kinase, and therefore phosphorylation, may serve to treat these diseases. Changes in absorption, steady-state fluorescence, and fluorescence lifetime indicate: 1) both TSPP and PPIX non-covalently bind to PKA where they maintain photoreactivity; 2) absorptive photoproduct formation occurs only when PKA is bound to TSPP and irradiated; and 3) PKA undergoes secondary structural changes after irradiation with either porphyrin bound. These photoinduced changes could affect the protein's enzymatic and signaling capabilities.

  18. Anaplastic lymphoma kinase status in rhabdomyosarcomas.

    PubMed

    Yoshida, Akihiko; Shibata, Tatsuhiro; Wakai, Susumu; Ushiku, Tetsuo; Tsuta, Koji; Fukayama, Masashi; Makimoto, Atsushi; Furuta, Koh; Tsuda, Hitoshi

    2013-06-01

    Rhabdomyosarcoma is a rare soft tissue sarcoma that typically affects children, adolescents, and young adults. Despite treatment via a multidisciplinary approach, the prognosis of advance-stage rhabdomyosarcomas remains poor, and a new treatment strategy is needed. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is a potential target for specific inhibitors. In this study, we investigated 116 rhabdomyosarcomas using a polymer-based ALK immunostaining method and correlated the results with clinicopathological parameters. In addition, we examined ALK status using dual-color fluorescence in situ hybridization, PCR, and sequencing. In immunohistochemical analysis, ALK was detected in 2 (6%) of 33 embryonal rhabdomyosarcomas, 42 (69%) of 61 alveolar rhabdomyosarcomas, and 0 (0%) of 22 other subtypes, including pleomorphic, adult-spindle-cell/sclerosing, and epithelioid variants. Compared with ALK-negative alveolar rhabdomyosarcomas, ALK-positive ones are presented with metastatic spread more frequently and showed a greater extent of myogenin reactivity. Overall survival was not associated with ALK expression. FOXO1 rearrangement was significantly associated with ALK immunoreactivity. The median ALK copy number was greater in ALK-positive tumors than in ALK-negative tumors. Most (93%) cases tested showed no selective increase in the ALK gene dosage. ALK selective amplification and low-level selective gain were noted in one and three cases, respectively. Further, a high-polysomy pattern (≥4 ALK copies in ≥40% of cells) was observed in seven cases. A significant increase in the ALK copy number was exclusive to the ALK-immunopositive cohort, but it was uncommon, accounting for only 30% of the 37 ALK-positive rhabdomyosarcomas. ALK gene rearrangement was not observed in either cohort, while an ALK somatic mutation (I1277T) was found in one ALK-negative embryonal case. Although it remains controversial whether ALK expression without gene rearrangement

  19. Assembly and activation of a kinase ribozyme

    PubMed Central

    Burke, Donald H.; Rhee, Steven S.

    2010-01-01

    RNA activities can be regulated by modulating the relative energies of all conformations in a folding landscape; however, it is often unknown precisely how peripheral elements perturb the overall landscape in the absence of discrete alternative folds (inactive ensemble). This work explores the effects of sequence and secondary structure in governing kinase ribozyme activity. Kin.46 catalyzes thiophosphoryl transfer from ATPγS onto the 5′ hydroxyl of polynucleotide substrates, and is regulated 10,000-fold by annealing an effector oligonucleotide to form activator helix P4. Transfer kinetics for an extensive series of ribozyme variants identified several dispensable internal single-stranded segments, in addition to a potential pseudoknot at the active site between segments J1/4 and J3/2 that is partially supported by compensatory rescue. Standard allosteric mechanisms were ruled out, such as formation of discrete repressive structures or docking P4 into the rest of the ribozyme via backbone 2′ hydroxyls. Instead, P4 serves both to complete an important structural element (100-fold contribution to the reaction relative to a P4-deleted variant) and to mitigate nonspecific, inhibitory effects of the single-stranded tail (an additional 100-fold contribution to the apparent rate constant, kobs). Thermodynamic activation parameters ΔH‡ and ΔS‡, calculated from the temperature dependence of kobs, varied with tail length and sequence. Inhibitory effects of the unpaired tail are largely enthalpic for short tails and are both enthalpic and entropic for longer tails. These results refine the structural view of this kinase ribozyme and highlight the importance of nonspecific ensemble effects in conformational regulation by peripheral elements. PMID:20935068

  20. Tyrosine Kinase Receptor Expression in Canine Liposarcoma.

    PubMed

    Avallone, G; Pellegrino, V; Roccabianca, P; Lepri, E; Crippa, L; Beha, G; De Tolla, L; Sarli, G

    2017-03-01

    The expression of tyrosine kinase receptors is attracting major interest in human and veterinary oncological pathology because of their role as targets for adjuvant therapies. Little is known about tyrosine kinase receptor (TKR) expression in canine liposarcoma (LP), a soft tissue sarcoma. The aim of this study was to evaluate the immunohistochemical expression of the TKRs fibroblast growth factor receptor 1 (FGFR1) and platelet-derived growth factor receptor-β (PDGFRβ); their ligands, fibroblast growth factor 2 (FGF2) and platelet-derived growth factor B (PDGFB); and c-kit in canine LP. Immunohistochemical labeling was categorized as high or low expression and compared with the mitotic count and MIB-1-based proliferation index. Fifty canine LPs were examined, classified, and graded. Fourteen cases were classified as well differentiated, 7 as myxoid, 25 as pleomorphic, and 4 as dedifferentiated. Seventeen cases were grade 1, 26 were grade 2, and 7 were grade 3. A high expression of FGF2, FGFR1, PDGFB, and PDGFRβ was identified in 62% (31/50), 68% (34/50), 81.6% (40/49), and 70.8% (34/48) of the cases, respectively. c-kit was expressed in 12.5% (6/48) of the cases. Mitotic count negatively correlated with FGF2 ( R = -0.41; P < .01), being lower in cases with high FGF2 expression, and positively correlated with PDGFRβ ( R = 0.33; P < .01), being higher in cases with high PDGFRβ expression. No other statistically significant correlations were identified. These results suggest that the PDGFRβ-mediated pathway may have a role in the progression of canine LP and may thus represent a promising target for adjuvant cancer therapies.

  1. Creatine kinase activity is associated with blood pressure.

    PubMed

    Brewster, Lizzy M; Mairuhu, Gideon; Bindraban, Navin R; Koopmans, Richard P; Clark, Joseph F; van Montfrans, Gert A

    2006-11-07

    We previously hypothesized that high activity of creatine kinase, the central regulatory enzyme of energy metabolism, facilitates the development of high blood pressure. Creatine kinase rapidly provides adenosine triphosphate to highly energy-demanding processes, including cardiovascular contraction, and antagonizes nitric oxide-mediated functions. Relatively high activity of the enzyme, particularly in resistance arteries, is thought to enhance pressor responses and increase blood pressure. Tissue creatine kinase activity is reported to be high in black people, a population subgroup with greater hypertension risk; the proposed effects of high creatine kinase activity, however, are not "race dependent." We therefore assessed whether creatine kinase is associated with blood pressure in a multiethnic population. We analyzed a stratified random sample of the population of Amsterdam, The Netherlands, consisting of 1444 citizens (503 white European, 292 South Asian, 580 black, and 69 of other ethnicity) aged 34 to 60 years. We used linear regression analysis to investigate the association between blood pressure and normal serum creatine kinase after rest, as a substitute measure of tissue activity. Creatine kinase was independently associated with blood pressure, with an increase in systolic and diastolic pressure, respectively, of 8.0 (95% CI, 3.3 to 12.7) and 4.7 (95% CI, 1.9 to 7.5) mm Hg per log creatine kinase increase after adjustment for age, sex, body mass index, and ethnicity. Creatine kinase is associated with blood pressure. Further studies are needed to explore the nature of this association, including how variation in cardiovascular creatine kinase activity may affect pressor responses.

  2. 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. Copyright © 2013 Elsevier Ltd. All rights reserved.

  3. Multiple host kinases contribute to Akt activation during Salmonella infection.

    PubMed

    Roppenser, Bernhard; Kwon, Hyunwoo; Canadien, Veronica; Xu, Risheng; Devreotes, Peter N; Grinstein, Sergio; Brumell, John H

    2013-01-01

    SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4) P2/PI(3-5) P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4) P2/PI(3-5) P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

  4. Structural and evolutionary divergence of eukaryotic protein kinases in Apicomplexa.

    PubMed

    Talevich, Eric; Mirza, Amar; Kannan, Natarajan

    2011-11-02

    The Apicomplexa constitute an evolutionarily divergent phylum of protozoan pathogens responsible for widespread parasitic diseases such as malaria and toxoplasmosis. Many cellular functions in these medically important organisms are controlled by protein kinases, which have emerged as promising drug targets for parasitic diseases. However, an incomplete understanding of how apicomplexan kinases structurally and mechanistically differ from their host counterparts has hindered drug development efforts to target parasite kinases. We used the wealth of sequence data recently made available for 15 apicomplexan species to identify the kinome of each species and quantify the evolutionary constraints imposed on each family of apicomplexan kinases. Our analysis revealed lineage-specific adaptations in selected families, namely cyclin-dependent kinase (CDK), calcium-dependent protein kinase (CDPK) and CLK/LAMMER, which have been identified as important in the pathogenesis of these organisms. Bayesian analysis of selective constraints imposed on these families identified the sequence and structural features that most distinguish apicomplexan protein kinases from their homologs in model organisms and other eukaryotes. In particular, in a subfamily of CDKs orthologous to Plasmodium falciparum crk-5, the activation loop contains a novel PTxC motif which is absent from all CDKs outside Apicomplexa. Our analysis also suggests a convergent mode of regulation in a subset of apicomplexan CDPKs and mammalian MAPKs involving a commonly conserved arginine in the αC helix. In all recognized apicomplexan CLKs, we find a set of co-conserved residues involved in substrate recognition and docking that are distinct from metazoan CLKs. We pinpoint key conserved residues that can be predicted to mediate functional differences from eukaryotic homologs in three identified kinase families. We discuss the structural, functional and evolutionary implications of these lineage-specific variations and

  5. Structural and evolutionary divergence of eukaryotic protein kinases in Apicomplexa

    PubMed Central

    2011-01-01

    Background The Apicomplexa constitute an evolutionarily divergent phylum of protozoan pathogens responsible for widespread parasitic diseases such as malaria and toxoplasmosis. Many cellular functions in these medically important organisms are controlled by protein kinases, which have emerged as promising drug targets for parasitic diseases. However, an incomplete understanding of how apicomplexan kinases structurally and mechanistically differ from their host counterparts has hindered drug development efforts to target parasite kinases. Results We used the wealth of sequence data recently made available for 15 apicomplexan species to identify the kinome of each species and quantify the evolutionary constraints imposed on each family of apicomplexan kinases. Our analysis revealed lineage-specific adaptations in selected families, namely cyclin-dependent kinase (CDK), calcium-dependent protein kinase (CDPK) and CLK/LAMMER, which have been identified as important in the pathogenesis of these organisms. Bayesian analysis of selective constraints imposed on these families identified the sequence and structural features that most distinguish apicomplexan protein kinases from their homologs in model organisms and other eukaryotes. In particular, in a subfamily of CDKs orthologous to Plasmodium falciparum crk-5, the activation loop contains a novel PTxC motif which is absent from all CDKs outside Apicomplexa. Our analysis also suggests a convergent mode of regulation in a subset of apicomplexan CDPKs and mammalian MAPKs involving a commonly conserved arginine in the αC helix. In all recognized apicomplexan CLKs, we find a set of co-conserved residues involved in substrate recognition and docking that are distinct from metazoan CLKs. Conclusions We pinpoint key conserved residues that can be predicted to mediate functional differences from eukaryotic homologs in three identified kinase families. We discuss the structural, functional and evolutionary implications of these

  6. Arginine kinase shows nucleoside diphosphate kinase-like activity toward deoxythymidine diphosphate.

    PubMed

    Lopez-Zavala, Alonso A; Sotelo-Mundo, Rogerio R; Hernandez-Flores, Jose M; Lugo-Sanchez, Maria E; Sugich-Miranda, Rocio; Garcia-Orozco, Karina D

    2016-06-01

    Arginine kinase (AK) (ATP: L-arginine phosphotransferase, E.C. 2.7.3.3) catalyzes the reversible transfer of ATP γ-phosphate group to L-arginine to synthetize phospho-arginine as a high-energy storage. Previous studies suggest additional roles for AK in cellular processes. Since AK is found only in invertebrates and it is homologous to creatine kinase from vertebrates, the objective of this work was to demonstrate nucleoside diphosphate kinase-like activity for shrimp AK. For this, AK from marine shrimp Litopenaeus vannamei (LvAK) was purified and its activity was assayed for phosphorylation of TDP using ATP as phosphate donor. Moreover, by using high-pressure liquid chromatography (HPLC) the phosphate transfer reaction was followed. Also, LvAK tryptophan fluorescence emission changes were detected by dTDP titration, suggesting that the hydrophobic environment of Trp 221, which is located in the top of the active site, is perturbed upon dTDP binding. The kinetic constants for both substrates Arg and dTDP were calculated by isothermal titration calorimetry (ITC). Besides, docking calculations suggested that dTDP could bind LvAK in the same cavity where ATP bind, and LvAK basic residues (Arg124, 126 and 309) stabilize the dTDP phosphate groups and the pyrimidine base interact with His284 and Ser122. These results suggest that LvAK bind and phosphorylate dTDP being ATP the phosphate donor, thus describing a novel alternate nucleoside diphosphate kinase-like activity for this enzyme.

  7. Cbk1 kinase and Bck2 control MAP kinase activation and inactivation during heat shock

    PubMed Central

    Kuravi, Venkata K.; Kurischko, Cornelia; Puri, Manasi; Luca, Francis C.

    2011-01-01

    Saccharomyces cerevisiae Cbk1 kinase is a LATS/NDR tumor suppressor orthologue and component of the Regulation of Ace2 and Morphogenesis signaling network. Cbk1 was previously implicated in regulating polarized morphogenesis, gene expression, and cell integrity. Here we establish that Cbk1 is critical for heat shock and cell wall stress signaling via Bck2, a protein associated with the Pkc1-Mpk1 cell integrity pathway. We demonstrate that cbk1 and bck2 loss-of-function mutations prevent Mpk1 kinase activation and Mpk1-dependent gene expression but do not disrupt Mpk1 Thr-190/Tyr-192 phosphorylation. Bck2 overexpression partially restores Mpk1-dependent Rlm1 transcription factor activity in cbk1 mutants, suggesting that Bck2 functions downstream of Cbk1. We demonstrate that Bck2 precisely colocalizes with the mitogen-activated protein kinase (MAPK) phosphatase Sdp1. During heat shock, Bck2 and Sdp1 transiently redistribute from nuclei and the cytosol to mitochondria and other cytoplasmic puncta before returning to their pre-stressed localization patterns. Significantly, Cbk1 inhibition delays the return of Bck2 and Sdp1 to their pre-stressed localization patterns and delays Mpk1 Thr-190/Tyr-192 dephosphorylation upon heat shock adaptation. We conclude that Cbk1 and Bck2 are required for Mpk1 activation during heat shock and cell wall stress and for Mpk1 dephosphorylation during heat shock adaptation. These data provide the first evidence that Cbk1 kinase regulates MAPK-dependent stress signaling and provide mechanistic insight into Sdp1 phosphatase regulation. PMID:22031291

  8. Venus Kinase Receptors: Prospects in Signaling and Biological Functions of These Invertebrate Kinases

    PubMed Central

    Dissous, Colette; Morel, Marion; Vanderstraete, Mathieu

    2014-01-01

    Venus kinase receptors (VKRs) form a family of invertebrate receptor tyrosine kinases (RTKs) initially discovered in the parasitic platyhelminth Schistosoma mansoni. VKRs are single transmembrane receptors that contain an extracellular venus fly trap structure similar to the ligand-binding domain of G protein-coupled receptors of class C, and an intracellular tyrosine kinase domain close to that of insulin receptors. VKRs are found in a large variety of invertebrates from cnidarians to echinoderms and are highly expressed in larval stages and in gonads, suggesting a role of these proteins in embryonic and larval development as well as in reproduction. VKR gene silencing could demonstrate the function of these receptors in oogenesis as well as in spermatogenesis in S. mansoni. VKRs are activated by amino acids and are highly responsive to arginine. As many other RTKs, they form dimers when activated by ligands and induce intracellular pathways involved in protein synthesis and cellular growth, such as MAPK and PI3K/Akt/S6K pathways. VKRs are not present in vertebrates or in some invertebrate species. Questions remain open about the origin of this little-known RTK family in evolution and its role in emergence and specialization of Metazoa. What is the meaning of maintenance or loss of VKR in some phyla or species in terms of development and physiological functions? The presence of VKRs in invertebrates of economical and medical importance, such as pests, vectors of pathogens, and platyhelminth parasites, and the implication of these RTKs in gametogenesis and reproduction processes are valuable reasons to consider VKRs as interesting targets in new programs for eradication/control of pests and infectious diseases, with the main advantage in the case of parasite targeting that VKR counterparts are absent from the vertebrate host kinase panel. PMID:24860549

  9. The role of MAP2 kinases and p38 kinase in acute murine liver injury models.

    PubMed

    Zhang, Jun; Min, Robert W M; Le, Khanh; Zhou, Sheng; Aghajan, Mariam; Than, Tin A; Win, Sanda; Kaplowitz, Neil

    2017-06-29

    c-Jun N-terminal kinase (JNK) mediates hepatotoxicity through interaction of its phospho-activated form with a mitochondrial outer membrane protein, Sh3bp5 or Sab, leading to dephosphorylation of intermembrane Src and consequent impaired mitochondrial respiration and enhanced ROS release. ROS production from mitochondria activates MAP3 kinases, such as MLK3 and ASK1, which continue to activate a pathway to sustain JNK activation, and amplifies the toxic effect of acetaminophen (APAP) and TNF/galactosamine (TNF/GalN). Downstream of MAP3K, in various contexts MKK4 activates both JNK and p38 kinases and MKK7 activates only JNK. The relative role of MKK4 versus 7 in liver injury is largely unexplored, as is the potential role of p38 kinase, which might be a key mediator of toxicity in addition to JNK. Antisense oligonucleotides (ASO) to MKK4, MKK7 and p38 (versus scrambled control) were used for in vivo knockdown, and in some experiments PMH were used after in vivo knockdown. Mice were treated with APAP or TNF/GalN and injury assessed. MKK4 and MKK7 were expressed in liver and each was efficiently knocked down with two different ASOs. Massive liver injury and ALT elevation were abrogated by MKK4 but not MKK7 ASO pretreatment in both injury models. The protection was confirmed in PMH. Knockdown of MKK4 completely inhibited basal P-p38 in both cytoplasm and mitochondria. However, ALT levels and histologic injury in APAP-treated mice were not altered with p38 knockdown versus scrambled control. p38 knockdown significantly increased P-JNK levels in cytoplasm but not mitochondria after APAP treatment. In conclusion, MKK4 is the major MAP2K, which activates JNK in acute liver injury. p38, the other downstream target of MKK4, does not contribute to liver injury from APAP or TNF/galactosamine.

  10. Protein-tyrosine Phosphatase and Kinase Specificity in Regulation of SRC and Breast Tumor Kinase* ♦

    PubMed Central

    Fan, Gaofeng; Aleem, Saadat; Yang, Ming; Miller, W. Todd; Tonks, Nicholas K.

    2015-01-01

    Despite significant evidence to the contrary, the view that phosphatases are “nonspecific” still pervades the field. Systems biology approaches to defining how signal transduction pathways are integrated at the level of whole organisms also often downplay the contribution of phosphatases, defining them as “erasers” that serve merely to restore the system to its basal state. Here, we present a study that counteracts the idea of “nonspecific phosphatases.” We have characterized two structurally similar and functionally related kinases, BRK and SRC, which are regulated by combinations of activating autophosphorylation and inhibitory C-terminal sites of tyrosine phosphorylation. We demonstrated specificity at the level of the kinases in that SRMS phosphorylated the C terminus of BRK, but not SRC; in contrast, CSK is the kinase responsible for C-terminal phosphorylation of SRC, but not BRK. For the phosphatases, we observed that RNAi-mediated suppression of PTP1B resulted in opposing effects on the activity of BRK and SRC and have defined the mechanisms underlying this specificity. PTP1B inhibited BRK by directly dephosphorylating the Tyr-342 autophosphorylation site. In contrast, PTP1B potentiated SRC activity, but not by dephosphorylating SRC itself directly; instead, PTP1B regulated the interaction between CBP/PAG and CSK. SRC associated with, and phosphorylated, the transmembrane protein CBP/PAG at Tyr-317, resulting in CSK recruitment. We identified PAG as a substrate of PTP1B, and dephosphorylation abolished recruitment of the inhibitory kinase CSK. Overall, these findings illustrate how the combinatorial effects of PTKs and PTPs may be integrated to regulate signaling, with both classes of enzymes displaying exquisite specificity. PMID:25897081

  11. Protein kinase C phosphorylates AMP-activated protein kinase α1 Ser487

    PubMed Central

    Heathcote, Helen R.; Mancini, Sarah J.; Strembitska, Anastasiya; Jamal, Kunzah; Reihill, James A.; Palmer, Timothy M.; Gould, Gwyn W.; Salt, Ian P.

    2016-01-01

    The key metabolic regulator, AMP-activated protein kinase (AMPK), is reported to be down-regulated in metabolic disorders, but the mechanisms are poorly characterised. Recent studies have identified phosphorylation of the AMPKα1/α2 catalytic subunit isoforms at Ser487/491, respectively, as an inhibitory regulation mechanism. Vascular endothelial growth factor (VEGF) stimulates AMPK and protein kinase B (Akt) in cultured human endothelial cells. As Akt has been demonstrated to be an AMPKα1 Ser487 kinase, the effect of VEGF on inhibitory AMPK phosphorylation in cultured primary human endothelial cells was examined. Stimulation of endothelial cells with VEGF rapidly increased AMPKα1 Ser487 phosphorylation in an Akt-independent manner, without altering AMPKα2 Ser491 phosphorylation. In contrast, VEGF-stimulated AMPKα1 Ser487 phosphorylation was sensitive to inhibitors of protein kinase C (PKC) and PKC activation using phorbol esters or overexpression of PKC-stimulated AMPKα1 Ser487 phosphorylation. Purified PKC and Akt both phosphorylated AMPKα1 Ser487 in vitro with similar efficiency. PKC activation was associated with reduced AMPK activity, as inhibition of PKC increased AMPK activity and phorbol esters inhibited AMPK, an effect lost in cells expressing mutant AMPKα1 Ser487Ala. Consistent with a pathophysiological role for this modification, AMPKα1 Ser487 phosphorylation was inversely correlated with insulin sensitivity in human muscle. These data indicate a novel regulatory role of PKC to inhibit AMPKα1 in human cells. As PKC activation is associated with insulin resistance and obesity, PKC may underlie the reduced AMPK activity reported in response to overnutrition in insulin-resistant metabolic and vascular tissues. PMID:27784766

  12. Venus kinase receptors: prospects in signaling and biological functions of these invertebrate kinases.

    PubMed

    Dissous, Colette; Morel, Marion; Vanderstraete, Mathieu

    2014-01-01

    Venus kinase receptors (VKRs) form a family of invertebrate receptor tyrosine kinases (RTKs) initially discovered in the parasitic platyhelminth Schistosoma mansoni. VKRs are single transmembrane receptors that contain an extracellular venus fly trap structure similar to the ligand-binding domain of G protein-coupled receptors of class C, and an intracellular tyrosine kinase domain close to that of insulin receptors. VKRs are found in a large variety of invertebrates from cnidarians to echinoderms and are highly expressed in larval stages and in gonads, suggesting a role of these proteins in embryonic and larval development as well as in reproduction. VKR gene silencing could demonstrate the function of these receptors in oogenesis as well as in spermatogenesis in S. mansoni. VKRs are activated by amino acids and are highly responsive to arginine. As many other RTKs, they form dimers when activated by ligands and induce intracellular pathways involved in protein synthesis and cellular growth, such as MAPK and PI3K/Akt/S6K pathways. VKRs are not present in vertebrates or in some invertebrate species. Questions remain open about the origin of this little-known RTK family in evolution and its role in emergence and specialization of Metazoa. What is the meaning of maintenance or loss of VKR in some phyla or species in terms of development and physiological functions? The presence of VKRs in invertebrates of economical and medical importance, such as pests, vectors of pathogens, and platyhelminth parasites, and the implication of these RTKs in gametogenesis and reproduction processes are valuable reasons to consider VKRs as interesting targets in new programs for eradication/control of pests and infectious diseases, with the main advantage in the case of parasite targeting that VKR counterparts are absent from the vertebrate host kinase panel.

  13. Protein-tyrosine Phosphatase and Kinase Specificity in Regulation of SRC and Breast Tumor Kinase.

    PubMed

    Fan, Gaofeng; Aleem, Saadat; Yang, Ming; Miller, W Todd; Tonks, Nicholas K

    2015-06-26

    Despite significant evidence to the contrary, the view that phosphatases are "nonspecific" still pervades the field. Systems biology approaches to defining how signal transduction pathways are integrated at the level of whole organisms also often downplay the contribution of phosphatases, defining them as "erasers" that serve merely to restore the system to its basal state. Here, we present a study that counteracts the idea of "nonspecific phosphatases." We have characterized two structurally similar and functionally related kinases, BRK and SRC, which are regulated by combinations of activating autophosphorylation and inhibitory C-terminal sites of tyrosine phosphorylation. We demonstrated specificity at the level of the kinases in that SRMS phosphorylated the C terminus of BRK, but not SRC; in contrast, CSK is the kinase responsible for C-terminal phosphorylation of SRC, but not BRK. For the phosphatases, we observed that RNAi-mediated suppression of PTP1B resulted in opposing effects on the activity of BRK and SRC and have defined the mechanisms underlying this specificity. PTP1B inhibited BRK by directly dephosphorylating the Tyr-342 autophosphorylation site. In contrast, PTP1B potentiated SRC activity, but not by dephosphorylating SRC itself directly; instead, PTP1B regulated the interaction between CBP/PAG and CSK. SRC associated with, and phosphorylated, the transmembrane protein CBP/PAG at Tyr-317, resulting in CSK recruitment. We identified PAG as a substrate of PTP1B, and dephosphorylation abolished recruitment of the inhibitory kinase CSK. Overall, these findings illustrate how the combinatorial effects of PTKs and PTPs may be integrated to regulate signaling, with both classes of enzymes displaying exquisite specificity. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition

    PubMed Central

    Chang, Ching Ching; Few, Ling Ling; Konrad, Manfred; See Too, Wei Cun

    2016-01-01

    Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme’s activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis. PMID:27149373

  15. Identification and characterization of a novel sucrose-non-fermenting protein kinase/AMP-activated protein kinase-related protein kinase, SNARK.

    PubMed Central

    Lefebvre, D L; Bai, Y; Shahmolky, N; Sharma, M; Poon, R; Drucker, D J; Rosen, C F

    2001-01-01

    Subtraction hybridization after the exposure of keratinocytes to ultraviolet radiation identified a differentially expressed cDNA that encodes a protein of 630 amino acid residues possessing significant similarity to the catalytic domain of the sucrose-non-fermenting protein kinase (SNF1)/AMP-activated protein kinase (AMPK) family of serine/threonine protein kinases. Northern blotting and reverse-transcriptase-mediated PCR demonstrated that mRNA transcripts for the SNF1/AMPK-related kinase (SNARK) were widely expressed in rodent tissues. The SNARK gene was localized to human chromosome 1q32 by fluorescent in situ hybridization. SNARK was translated in vitro to yield a single protein band of approx. 76 kDa; Western analysis of transfected baby hamster kidney (BHK) cells detected two SNARK-immunoreactive bands of approx. 76-80 kDa. SNARK was capable of autophosphorylation in vitro; immunoprecipitated SNARK exhibited phosphotransferase activity with the synthetic peptide substrate HMRSAMSGLHLVKRR (SAMS) as a kinase substrate. SNARK activity was significantly increased by AMP and 5-amino-4-imidazolecarboxamide riboside (AICAriboside) in rat keratinocyte cells, implying that SNARK might be activated by an AMPK kinase-dependent pathway. Furthermore, glucose deprivation increased SNARK activity 3-fold in BHK fibroblasts. These findings identify SNARK as a glucose- and AICAriboside-regulated member of the AMPK-related gene family that represents a new candidate mediator of the cellular response to metabolic stress. PMID:11284715

  16. A Quantitative Mass Spectrometry-based Approach for Identifying Protein Kinase-Clients and Quantifying Kinase Activity

    USDA-ARS?s Scientific Manuscript database

    The Homo sapiens and Arabidopsis thaliana genomes are believed to encode >500 and >1,000 protein kinases, respectively. Despite this abundance, few bona fide kinase-client relationships have been described in detail. Mass spectrometry (MS)-based approaches have been integral to the large-scale mapp...

  17. Novel detection method for chemiluminescence derived from the Kinase-Glo luminescent kinase assay platform: Advantages over traditional microplate luminometers

    PubMed Central

    Bell, Ryan A.V.; Storey, Kenneth B.

    2014-01-01

    The efficacy of cellular signal transduction is of paramount importance for the proper functioning of a cell and an organism as a whole. Protein kinases are responsible for much of this transmission and thus have been the focal point of extensive research. While there are numerous commercially available protein kinase assays, the Kinase-Glo luminescent kinase assay (Promega) provides an easy-to-use and high throughput platform for determining protein kinase activity. This assay is said to require the use of a microplate spectrophotometer capable of detecting a luminescent signal. This study shows that:•The ChemiGenius Bioimaging system (Syngene), typically used for visualizing chemiluminescence from Western blots, provides an alternative detection system for Kinase-Glo luminescence.•The novel detection system confers an advantage over traditional luminometers, in that it allows visualization of the luminescent wells, which allows for the real-time analysis and correction of experimental errors (i.e. bubble formation).•Determining kinase kinetics using this detection system produced comparable results to previous studies on the same enzyme (i.e. glycogen synthase kinase 3). PMID:26150941

  18. Novel detection method for chemiluminescence derived from the Kinase-Glo luminescent kinase assay platform: Advantages over traditional microplate luminometers.

    PubMed

    Bell, Ryan A V; Storey, Kenneth B

    2014-01-01

    The efficacy of cellular signal transduction is of paramount importance for the proper functioning of a cell and an organism as a whole. Protein kinases are responsible for much of this transmission and thus have been the focal point of extensive research. While there are numerous commercially available protein kinase assays, the Kinase-Glo luminescent kinase assay (Promega) provides an easy-to-use and high throughput platform for determining protein kinase activity. This assay is said to require the use of a microplate spectrophotometer capable of detecting a luminescent signal. This study shows that:•The ChemiGenius Bioimaging system (Syngene), typically used for visualizing chemiluminescence from Western blots, provides an alternative detection system for Kinase-Glo luminescence.•The novel detection system confers an advantage over traditional luminometers, in that it allows visualization of the luminescent wells, which allows for the real-time analysis and correction of experimental errors (i.e. bubble formation).•Determining kinase kinetics using this detection system produced comparable results to previous studies on the same enzyme (i.e. glycogen synthase kinase 3).

  19. Updated rice kinase database RKD 2.0: enabling transcriptome and functional analysis of rice kinase genes

    USDA-ARS?s Scientific Manuscript database

    Protein kinases catalyze the transfer of a phosphate moiety from a phosphate donor to the substrate molecule, thus, playing critical roles in cell signaling and metabolism. Although plant genomes contain more than 1,000 genes that encode kinases, knowledge is limited about the precise roles for the...

  20. Transphosphorylation of E. coli proteins during production of recombinant protein kinases provides a robust system to characterize kinase specificity

    USDA-ARS?s Scientific Manuscript database

    Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in E.coli. We apply this to the study of the cytoplasmic domain of the plant rec...

  1. Diacylglycerol kinase regulation of protein kinase D during oxidative stress-induced intestinal cell injury

    SciTech Connect

    Song Jun; Li Jing; Mourot, Joshua M.; Mark Evers, B.; Chung, Dai H.

    2008-10-17

    We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury; however, the exact role of DAG kinase (DGK){zeta}, an isoform expressed in intestine, during this process is unknown. We sought to determine the role of DGK during oxidative stress-induced intestinal cell injury and whether DGK acts as an upstream regulator of PKD. Inhibition of DGK with R59022 compound or DGK{zeta} siRNA transfection decreased H{sub 2}O{sub 2}-induced RIE-1 cell apoptosis as measured by DNA fragmentation and increased PKD phosphorylation. Overexpression of kinase-dead DGK{zeta} also significantly increased PKD phosphorylation. Additionally, endogenous nuclear DGK{zeta} rapidly translocated to the cytoplasm following H{sub 2}O{sub 2} treatment. Our findings demonstrate that DGK is involved in the regulation of oxidative stress-induced intestinal cell injury. PKD activation is induced by DGK{zeta}, suggesting DGK is an upstream regulator of oxidative stress-induced activation of the PKD signaling pathway in intestinal epithelial cells.

  2. The New Role for an Old Kinase: Protein Kinase CK2 Regulates Metal Ion Transport

    PubMed Central

    Johnson, Adam J.; Wu, Ming J.

    2016-01-01

    The pleiotropic serine/threonine protein kinase CK2 was the first kinase discovered. It is renowned for its role in cell proliferation and anti-apoptosis. The complexity of this kinase is well reflected by the findings of past decades in terms of its heterotetrameric structure, subcellular location, constitutive activity and the extensive catalogue of substrates. With the advent of non-biased high-throughput functional genomics such as genome-wide deletion mutant screening, novel aspects of CK2 functionality have been revealed. Our recent discoveries using the model organism Saccharomyces cerevisiae and mammalian cells demonstrate that CK2 regulates metal toxicity. Extensive literature search reveals that there are few but elegant works on the role of CK2 in regulating the sodium and zinc channels. As both CK2 and metal ions are key players in cell biology and oncogenesis, understanding the details of CK2’s regulation of metal ion homeostasis has a direct bearing on cancer research. In this review, we aim to garner the recent data and gain insights into the role of CK2 in metal ion transport. PMID:28009816

  3. Glycogen synthase kinase 3β suppresses polyglutamine aggregation by inhibiting Vaccinia-related kinase 2 activity

    PubMed Central

    Lee, Eunju; Ryu, Hye Guk; Kim, Sangjune; Lee, Dohyun; Jeong, Young-Hun; Kim, Kyong-Tai

    2016-01-01

    Huntington’s disease (HD) is a neurodegenerative disorder caused by an abnormal expansion of polyglutamine repeats in the N-terminal of huntingtin. The amount of aggregate-prone protein is controlled by various mechanisms, including molecular chaperones. Vaccinia-related kinase 2 (VRK2) is known to negatively regulate chaperonin TRiC, and VRK2-facilitated degradation of TRiC increases polyQ protein aggregation, which is involved in HD. We found that VRK2 activity was negatively controlled by glycogen synthase kinase 3β (GSK3β). GSK3β directly bound to VRK2 and inhibited the catalytic activity of VRK2 in a kinase activity-independent manner. Furthermore, GSK3β increased the stability of TRiC and decreased the formation of HttQ103-GFP aggregates by inhibiting VRK2. These results indicate that GSK3β signaling may be a regulatory mechanism of HD progression and suggest targets for further therapeutic trials for HD. PMID:27377031

  4. Tank binding kinase 1 is a centrosome-associated kinase necessary for microtubule dynamics and mitosis

    PubMed Central

    Pillai, Smitha; Nguyen, Jonathan; Johnson, Joseph; Haura, Eric; Coppola, Domenico; Chellappan, Srikumar

    2015-01-01

    TANK Binding Kinase 1 (TBK1) is a non-canonical IκB kinase that contributes to KRAS-driven lung cancer. Here we report that TBK1 plays essential roles in mammalian cell division. Specifically, levels of active phospho-TBK1 increase during mitosis and localize to centrosomes, mitotic spindles and midbody, and selective inhibition or silencing of TBK1 triggers defects in spindle assembly and prevents mitotic progression. TBK1 binds to the centrosomal protein CEP170 and to the mitotic apparatus protein NuMA, and both CEP170 and NuMA are TBK1 substrates. Further, TBK1 is necessary for CEP170 centrosomal localization and binding to the microtubule depolymerase Kif2b, and for NuMA binding to dynein. Finally, selective disruption of the TBK1–CEP170 complex augments microtubule stability and triggers defects in mitosis, suggesting that TBK1 functions as a mitotic kinase necessary for microtubule dynamics and mitosis. PMID:26656453

  5. Casein Kinase 1 Functions as both Penultimate and Ultimate Kinase in Regulating Cdc25A Destruction

    PubMed Central

    Honaker, Yuchi; Piwnica-Worms, Helen

    2010-01-01

    The Cdc25A protein phosphatase drives cell cycle transitions by activating cyclin-dependent protein kinases. Failure to regulate Cdc25A leads to deregulated cell cycle progression, bypass of cell cycle checkpoints and genome instability. Ubiquitin-mediated proteolysis plays an important role in balancing Cdc25A levels. Cdc25A contains a DS82G motif whose phosphorylation is targeted by β-TrCP E3 ligase during interphase. Targeting of β-TrCP to Cdc25A requires phosphorylation of serines 79 (S79) and 82 (S82). Here, we report that casein kinase 1 alpha (CK1α) phosphorylates Cdc25A on both S79 and S82 in a hierarchical manner requiring prior phosphorylation of serine 76 by Chk1 or GSK-3β. This facilitates β-TrCP binding and ubiquitin-mediated proteolysis of Cdc25A throughout interphase and following exposure to genotoxic stress. The priming of Cdc25A by at least three kinases (Chk1, GSK-3β, CK1α), some of which also require priming, ensures diverse extra- and intra-cellular signals interface with Cdc25A to precisely control cell division. PMID:20348946

  6. Prostaglandin E2 negatively regulates AMP-activated protein kinase via protein kinase A signaling pathway.

    PubMed

    Funahashi, Koji; Cao, Xia; Yamauchi, Masako; Kozaki, Yasuko; Ishiguro, Naoki; Kambe, Fukushi

    2009-01-01

    We investigated possible involvement of prostaglandin (PG) E2 in regulation of AMP-activated protein kinase (AMPK). When osteoblastic MG63 cells were cultured in serum-deprived media, Thr-172 phosphorylation of AMPK alpha-subunit was markedly increased. Treatment of the cells with PGE2 significantly reduced the phosphorylation. Ser-79 phosphorylation of acetyl-CoA carboxylase, a direct target for AMPK, was also reduced by PGE2. On the other hand, PGE2 reciprocally increased Ser-485 phosphorylation of the alpha-subunit that could be associated with inhibition of AMPK activity. These effects of PGE2 were mimicked by PGE2 receptor EP2 and EP4 agonists and forskolin, but not by EP1 and EP3 agonists, and the effects were suppressed by an adenylate cyclase inhibitor SQ22536 and a protein kinase A inhibitor H89. Additionally, the PGE2 effects were duplicated in primary calvarial osteoblasts. Together, the present study demonstrates that PGE2 negatively regulates AMPK activity via activation of protein kinase A signaling pathway.

  7. Activation of cGMP-dependent protein kinase by protein kinase C.

    PubMed

    Hou, Yali; Lascola, Judith; Dulin, Nickolai O; Ye, Richard D; Browning, Darren D

    2003-05-09

    The cGMP-dependent protein kinases (PKG) are emerging as important components of mainstream signal transduction pathways. Nitric oxide-induced cGMP formation by stimulation of soluble guanylate cyclase is generally accepted as being the most widespread mechanism underlying PKG activation. In the present study, PKG was found to be a target for phorbol 12-myristate 13-acetate (PMA)-responsive protein kinase C (PKC). PKG1alpha became phosphorylated in HEK-293 cells stimulated with PMA and also in vitro using purified components. PKC-dependent phosphorylation was found to activate PKG as measured by phosphorylation of vasodilator-stimulated phosphoprotein, and by in vitro kinase assays. Although there are 11 potential PKC substrate recognition sites in PKG1alpha, threonine 58 was examined due to its proximity to the pseudosubstrate domain. Antibodies generated against the phosphorylated form of this region were used to demonstrate phosphorylation in response to PMA treatment of the cells with kinetics similar to vasodilator-stimulated phosphoprotein phosphorylation. A phospho-mimetic mutation at this site (T58E) generated a partially activated PKG that was more sensitive to cGMP levels. A phospho-null mutation (T58A) revealed that this residue is important but not sufficient for PKG activation by PKC. Taken together, these findings outline a novel signal transduction pathway that links PKC stimulation with cyclic nucleotide-independent activation of PKG.

  8. Aurora Kinases and Potential Medical Applications of Aurora Kinase Inhibitors: A Review

    PubMed Central

    Gavriilidis, Paschalis; Giakoustidis, Alexandros; Giakoustidis, Dimitrios

    2015-01-01

    Aurora kinases (AKs) represent a novel group of serine/threonine kinases. They were originally described in 1995 by David Glover in the course of studies of mutant alleles characterized with unusual spindle pole configuration in Drosophila melanogaster. Thus far, three AKs A, B, and C have been discovered in human healthy and neoplastic cells. Each one locates in different subcellular locations and they are all nuclear proteins. AKs are playing an essential role in mitotic events such as monitoring of the mitotic checkpoint, creation of bipolar mitotic spindle and alignment of centrosomes on it, also regulating centrosome separation, bio-orientation of chromosomes and cytokinesis. Any inactivation of them can have catastrophic consequences on mitotic events of spindle formation, alignment of centrosomes and cytokinesis, resulting in apoptosis. Overexpression of AKs has been detected in diverse solid and hematological cancers and has been linked with a dismal prognosis. After discovery and identification of the first aurora kinase inhibitor (AKI) ZM447439 as a potential drug for targeted therapy in cancer treatment, approximately 30 AKIs have been introduced in cancer treatment. PMID:26345296

  9. Parkinson's disease-associated mutations in leucine-rich repeat kinase 2 augment kinase activity

    PubMed Central

    West, Andrew B.; Moore, Darren J.; Biskup, Saskia; Bugayenko, Artem; Smith, Wanli W.; Ross, Christopher A.; Dawson, Valina L.; Dawson, Ted M.

    2005-01-01

    Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) cause late-onset Parkinson's disease (PD) with a clinical appearance indistinguishable from idiopathic PD. Initial studies suggest that LRRK2 mutations are the most common yet identified determinant of PD susceptibility, transmitted in an autosomal-dominant mode of inheritance. Herein, we characterize the LRRK2 gene and transcript in human brain and subclone the predominant ORF. Exogenously expressed LRRK2 protein migrates at ≈280 kDa and is present largely in the cytoplasm but also associates with the mitochondrial outer membrane. Familial-linked mutations G2019S or R1441C do not have an obvious effect on protein steady-state levels, turnover, or localization. However, in vitro kinase assays using full-length recombinant LRRK2 reveal an increase in activity caused by familial-linked mutations in both autophosphorylation and the phosphorylation of a generic substrate. These results suggest a gain-of-function mechanism for LRRK2-linked disease with a central role for kinase activity in the development of PD. PMID:16269541

  10. Testing the promiscuity of commercial kinase inhibitors against the AGC kinase group using a split-luciferase screen.

    PubMed

    Jester, Benjamin W; Gaj, Alicia; Shomin, Carolyn D; Cox, Kurt J; Ghosh, Indraneel

    2012-02-23

    Using a newly developed competitive binding assay dependent upon the reassembly of a split reporter protein, we have tested the promiscuity of a panel of reported kinase inhibitors against the AGC group. Many non-AGC targeted kinase inhibitors target multiple members of the AGC group. In general, structurally similar inhibitors consistently exhibited activity toward the same target as well as toward closely related kinases. The inhibition data was analyzed to test the predictive value of either using identity scores derived from residues within 6 Å of the active site or identity scores derived from the entire kinase domain. The results suggest that the active site identity in certain cases may be a stronger predictor of inhibitor promiscuity. The overall results provide general guidelines for establishing inhibitor selectivity as well as for the future design of inhibitors that either target or avoid AGC kinases.

  11. RhoA/Rho-Kinase in the Cardiovascular System.

    PubMed

    Shimokawa, Hiroaki; Sunamura, Shinichiro; Satoh, Kimio

    2016-01-22

    Twenty years ago, Rho-kinase was identified as an important downstream effector of the small GTP-binding protein, RhoA. Thereafter, a series of studies demonstrated the important roles of Rho-kinase in the cardiovascular system. The RhoA/Rho-kinase pathway is now widely known to play important roles in many cellular functions, including contraction, motility, proliferation, and apoptosis, and its excessive activity induces oxidative stress and promotes the development of cardiovascular diseases. Furthermore, the important role of Rho-kinase has been demonstrated in the pathogenesis of vasospasm, arteriosclerosis, ischemia/reperfusion injury, hypertension, pulmonary hypertension, and heart failure. Cyclophilin A is secreted by vascular smooth muscle cells and inflammatory cells and activated platelets in a Rho-kinase-dependent manner, playing important roles in a wide range of cardiovascular diseases. Thus, the RhoA/Rho-kinase pathway plays crucial roles under both physiological and pathological conditions and is an important therapeutic target in cardiovascular medicine. Recently, functional differences between ROCK1 and ROCK2 have been reported in vitro. ROCK1 is specifically cleaved by caspase-3, whereas granzyme B cleaves ROCK2. However, limited information is available on the functional differences and interactions between ROCK1 and ROCK2 in the cardiovascular system in vivo. Herein, we will review the recent advances about the importance of RhoA/Rho-kinase in the cardiovascular system. © 2016 American Heart Association, Inc.

  12. Activation of the orphan receptor tyrosine kinase ALK by zinc.

    PubMed

    Bennasroune, Aline; Mazot, Pierre; Boutterin, Marie-Claude; Vigny, Marc

    2010-08-06

    Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase essentially and transiently expressed during development of the central and peripheral nervous system. The nature of the cognate ligand of this receptor in Vertebrates is still a matter of debate. During synaptic transmission the release of ionic zinc found in vesicles of certain glutamatergic and gabaergic terminals may act as a neuromodulator by binding to pre- or post-synaptic receptors. Recently, zinc has been shown to activate the receptor tyrosine kinase, TrkB, independently of neurotrophins. This activation occurs via increasing the Src family kinase activity. In the present study, we investigated whether the ALK activity could be modulated by extracellular zinc. We first showed that zinc alone rapidly activates ALK. This activation is dependent of ALK tyrosine kinase activity and dimerization of the receptor but is independent of Src family kinase activity. In contrast, addition of sodium pyrithione, a zinc ionophore, led to a further activation of ALK. This stronger activation is dependent of Src family kinase but independent of ALK activity and dimerization. In conclusion, zinc could constitute an endogenous ligand of ALK in vertebrates.

  13. Unconventional Functions of Mitotic Kinases in Kidney Tumorigenesis

    PubMed Central

    Hascoet, Pauline; Chesnel, Franck; Le Goff, Cathy; Le Goff, Xavier; Arlot-Bonnemains, Yannick

    2015-01-01

    Human tumors exhibit a variety of genetic alterations, including point mutations, translocations, gene amplifications and deletions, as well as aneuploid chromosome numbers. For carcinomas, aneuploidy is associated with poor patient outcome for a large variety of tumor types, including breast, colon, and renal cell carcinoma. The Renal cell carcinoma (RCC) is a heterogeneous carcinoma consisting of different histologic types. The clear renal cell carcinoma (ccRCC) is the most common subtype and represents 85% of the RCC. Central to the biology of the ccRCC is the loss of function of the Von Hippel–Lindau gene, but is also associated with genetic instability that could be caused by abrogation of the cell cycle mitotic spindle checkpoint and may involve the Aurora kinases, which regulate centrosome maturation. Aneuploidy can also result from the loss of cell–cell adhesion and apical–basal cell polarity that also may be regulated by the mitotic kinases (polo-like kinase 1, casein kinase 2, doublecortin-like kinase 1, and Aurora kinases). In this review, we describe the “non-mitotic” unconventional functions of these kinases in renal tumorigenesis. PMID:26579493

  14. Thrombin activates MAPKAP2 kinase in vascular smooth muscle.

    PubMed

    Brophy, C M; Woodrum, D; Dickinson, M; Beall, A

    1998-05-01

    Thrombin mediates hemostasis by promoting thrombus development and vasospasm, which reduces the size of the arterial injury. Thrombin stimulation of vascular smooth muscle is associated with activation of mitogen-associated protein kinase. The purpose of this investigation was to determine the subsequent cellular signaling events in thrombin-stimulated vascular smooth muscle contraction. Contractile responses of bovine carotid artery smooth muscle were determined in a muscle bath and compared with phosphorylation events with two-dimensional gel electrophoresis. The activity of a novel kinase, mitogen-activated protein kinase-activated protein-2 kinase (MAPKAP2 kinase), was determined by immunoprecipitation and a phosphotransferase assay. A small heat shock protein, HSP27, was identified with immunoblotting. Thrombin induces contraction of vascular smooth muscle and is associated with increased activity of MAPKAP2 kinase and increased phosphorylation of HSP27. Multiple isoforms of HSP27 are the predominant phosphoproteins in vascular smooth muscle, and peptide mapping suggests that the isoforms of HSP27 are structurally related and phosphorylated within similar peptide sequences. Activation of the MAPKAP2 kinase pathway and phosphorylation of HSP27 are associated with thrombin-induced contraction of vascular smooth muscle.

  15. Binding of exogenous brain protein kinase C to liver nuclei

    SciTech Connect

    Misra, U.K.; Wolf, M.; Besterman, J.; Cuatrecasas, P.; Sahyoun, N.

    1986-05-01

    Protein kinase C is found both in the cytosol and bound to membranes. Binding of the enzyme to plasma membranes is controlled by calcium whereas enzyme activators regulate both its membrane binding and enzyme catalysis. Activation of protein kinase C has been implicated in several regulatory processes including gene expression. Accordingly, the possibility of direct interaction of protein kinase C with the nucleus was examined utilizing /sup 3/H-PDBu binding to detect the enzyme. Purified protein kinase C from rat brain could bind to purified rat liver nuclei at 4/sup 0/C or at 21/sup 0/C, and the reaction was completed by 20 min. The binding was linearly dependent on protein kinase C concentration and required free Ca/sup 2 +/ with a K/sub m/sub app// of 0.5 ..mu..M. Chelation of Ca/sup 2 +/ with EGTA resulted in a rapid dissociation of protein kinase C from the nuclei. Differential extraction experiments suggested that about 50% of the enzyme was bound to chromatin and 25% was associated with the nuclear matrix. Moreover, protein kinase C bound to nuclei was able to phosphorylate several endogenous nuclear substrates, including chromatin proteins, in a Ca/sup 2 +/ phosphatidyl serine dependent reaction.

  16. Quantitative Structure-Activity Relationship Modeling of Kinase Selectivity Profiles.

    PubMed

    Kothiwale, Sandeepkumar; Borza, Corina; Pozzi, Ambra; Meiler, Jens

    2017-09-19

    The discovery of selective inhibitors of biological target proteins is the primary goal of many drug discovery campaigns. However, this goal has proven elusive, especially for inhibitors targeting the well-conserved orthosteric adenosine triphosphate (ATP) binding pocket of kinase enzymes. The human kinome is large and it is rather difficult to profile early lead compounds against around 500 targets to gain an upfront knowledge on selectivity. Further, selectivity can change drastically during derivatization of an initial lead compound. Here, we have introduced a computational model to support the profiling of compounds early in the drug discovery pipeline. On the basis of the extensive profiled activity of 70 kinase inhibitors against 379 kinases, including 81 tyrosine kinases, we developed a quantitative structure-activity relation (QSAR) model using artificial neural networks, to predict the activity of these kinase inhibitors against the panel of 379 kinases. The model's performance in predicting activity ranges from 0.6 to 0.8 depending on the kinase, from the area under the curve (AUC) of the receiver operating characteristics (ROC). The profiler is available online at http://www.meilerlab.org/index.php/servers/show?s_id=23.

  17. Creatine kinase inhibits ADP-induced platelet aggregation

    PubMed Central

    Horjus, D. L.; Nieuwland, R.; Boateng, K. B.; Schaap, M. C. L.; van Montfrans, G. A.; Clark, J. F.; Sturk, A.; Brewster, L. M.

    2014-01-01

    Bleeding risk with antiplatelet therapy is an increasing clinical challenge. However, the inter-individual variation in this risk is poorly understood. We assessed whether the level of plasma creatine kinase, the enzyme that utilizes ADP and phosphocreatine to rapidly regenerate ATP, may modulate bleeding risk through a dose-dependent inhibition of ADP-induced platelet activation. Exogenous creatine kinase (500 to 4000 IU/L, phosphocreatine 5 mM) added to human plasma induced a dose-dependent reduction to complete inhibition of ADP-induced platelet aggregation. Accordingly, endogenous plasma creatine kinase, studied in 9 healthy men (mean age 27.9 y, SE 3.3; creatine kinase 115 to 859 IU/L, median 358), was associated with reduced ADP-induced platelet aggregation (Spearman's rank correlation coefficient, −0.6; p < 0.05). After exercise, at an endogenous creatine kinase level of 4664, ADP-induced platelet aggregation was undetectable, normalizing after rest, with a concomitant reduction of creatine kinase to normal values. Thus, creatine kinase reduces ADP-induced platelet activation. This may promote bleeding, in particular when patients use platelet P2Y12 ADP receptor inhibitors. PMID:25298190

  18. Protein kinase C activators inhibit capillary endothelial cell growth

    SciTech Connect

    Doctrow, S.R.

    1986-05-01

    Phorbol 12,13-dibutyrate (PDBu) binds specifically to bovine capillary endothelial (BCE) cells (K/sub d/ = 8nM) and inhibits the proliferation (K/sub 50/ = 6 +/- 4 nM). Under similar conditions, PDBu does not inhibit the growth of bovine aortic endothelial or smooth muscle cells. PDBu markedly attenuates the response of BCE cells to purified human hepatoma-derived growth factor which, in the absence of PDBu, stimulates BCE cell growth by about 3-fold. Several observations suggest that the inhibition of BCE cell growth by PDBu is mediated by protein kinase C: (1) different phorbol compounds inhibit BCE cell growth according to the relative potencies as protein kinase C activators (12-tetradecanoylphorbol 13-acetate > PDBu >> phorbol 12,13-diacetate >>>..beta..-phorbol; ..cap alpha..-phorbol 12,13-didecanoate). (2) Specific binding of PDBu to BCE cells is displaced by sn-1,2-dioctanoylglycerol (diC/sub 8/), a protein kinase C activator and an analog of the putative second messenger activating this kinase in vivo. The weak protein kinase C activator, sn-1,2-dibutyrylglycerol, does not affect PDBu binding. (3) A cytosolic extract from BCE cells contains a Ca/sup 2 +//phosphatidylserine-dependent kinase that is activated by diC/sub 8/ and PDBu, but not by ..beta..-phorbol. These results support a role for protein kinase C in suppressing capillary endothelial cell growth and may therefore have implications in the intracellular regulation of angiogenesis.

  19. Adenosine phosphonoacetic acid is slowly metabolized by NDP kinase.

    PubMed

    Chen, Y; Morera, S; Pasti, C; Angusti, A; Solaroli, N; Véron, M; Janin, J; Manfredini, S; Deville-Bonne, D

    2005-11-01

    NDP kinase catalyzes the last step in the phosphorylation of nucleotides. It is also involved in the activation by cellular kinases of nucleoside analogs used in antiviral therapies. Adenosine phosphonoacetic acid, a close analog of ADP already proposed as an inhibitor of ribonucleotide reductase, was found to be a poor substrate for human NDP kinase, as well as a weak inhibitor with an equilibrium dissociation constant of 0.6 mM to be compared to 0.025 mM for ADP. The X-ray structure of a complex of adenosine phosphonoacetic acid and the NDP kinase from Dictyostelium was determined to 2.0 A resolution showing that the analog adopts a binding mode similar to ADP, but that no magnesium ion is present at the active site. As ACP may also interfere with other cellular kinases, its potential as a drug targeting NDP kinase or ribonucleotide reductase is likely to be limited due to strong side effects. The design of new molecules with a narrower specificity and a stronger affinity will benefit from the detailed knowledge of the complex ACP-NDP kinase.

  20. Structure of the intact ATM/Tel1 kinase.

    PubMed

    Wang, Xuejuan; Chu, Huanyu; Lv, Mengjuan; Zhang, Zhihui; Qiu, Shuwan; Liu, Haiyan; Shen, Xuetong; Wang, Weiwu; Cai, Gang

    2016-05-27

    The ataxia-telangiectasia mutated (ATM) protein is an apical kinase that orchestrates the multifaceted DNA-damage response. Normally, ATM kinase is in an inactive, homodimer form and is transformed into monomers upon activation. Besides a conserved kinase domain at the C terminus, ATM contains three other structural modules, referred to as FAT, FATC and N-terminal helical solenoid. Here we report the first cryo-EM structure of ATM kinase, which is an intact homodimeric ATM/Tel1 from Schizosaccharomyces pombe. We show that two monomers directly contact head-to-head through the FAT and kinase domains. The tandem N-terminal helical solenoid tightly packs against the FAT and kinase domains. The structure suggests that ATM/Tel1 dimer interface and the consecutive HEAT repeats inhibit the binding of kinase substrates and regulators by steric hindrance. Our study provides a structural framework for understanding the mechanisms of ATM/Tel1 regulation as well as the development of new therapeutic agents.

  1. Structure of the intact ATM/Tel1 kinase

    NASA Astrophysics Data System (ADS)

    Wang, Xuejuan; Chu, Huanyu; Lv, Mengjuan; Zhang, Zhihui; Qiu, Shuwan; Liu, Haiyan; Shen, Xuetong; Wang, Weiwu; Cai, Gang

    2016-05-01

    The ataxia-telangiectasia mutated (ATM) protein is an apical kinase that orchestrates the multifaceted DNA-damage response. Normally, ATM kinase is in an inactive, homodimer form and is transformed into monomers upon activation. Besides a conserved kinase domain at the C terminus, ATM contains three other structural modules, referred to as FAT, FATC and N-terminal helical solenoid. Here we report the first cryo-EM structure of ATM kinase, which is an intact homodimeric ATM/Tel1 from Schizosaccharomyces pombe. We show that two monomers directly contact head-to-head through the FAT and kinase domains. The tandem N-terminal helical solenoid tightly packs against the FAT and kinase domains. The structure suggests that ATM/Tel1 dimer interface and the consecutive HEAT repeats inhibit the binding of kinase substrates and regulators by steric hindrance. Our study provides a structural framework for understanding the mechanisms of ATM/Tel1 regulation as well as the development of new therapeutic agents.

  2. Arginine kinase in Phytomonas, a trypanosomatid parasite of plants.

    PubMed

    Canepa, Gaspar E; Carrillo, Carolina; Miranda, Mariana R; Sayé, Melisa; Pereira, Claudio A

    2011-09-01

    Phytomonas are trypanosomatid plant parasites closely related to parasites that cause several human diseases. Little is known about the biology of these organisms including aspects of their metabolism. Arginine kinase (E.C. 2.7.3.3) is a phosphotransferase which catalyzes the interconversion between the phosphagen phosphoarginine and ATP. This enzyme is present in some invertebrates and is a homolog of another widely distributed phosphosphagen kinase, creatine kinase. In this work, a single canonical arginine kinase isoform was detected in Phytomonas Jma by enzymatic activity assays, PCR, and Western Blot. This arginine kinase is very similar to the canonical isoforms found in T. cruzi and T. brucei, presenting about 70% of amino acid sequence identity and a very similar molecular weight (40kDa). The Phytomonas phosphagen system seems to be very similar to T. cruzi, which has only one isoform, or T. brucei (three isoforms); establishing a difference with other trypanosomatids, such as Leishmania, which completely lacks phosphagen kinases, probably by the presence of the arginine-consuming enzyme, arginase. Finally, phylogenetic analysis suggests that Kinetoplastids' arginine kinase was acquired, during evolution, from the arthropod vectors by horizontal gene transfer. Copyright © 2011 Elsevier Inc. All rights reserved.

  3. Assessing and managing toxicities induced by kinase inhibitors.

    PubMed

    Castoldi, Raffaella E; Pennella, Giulia; Saturno, Grazia S; Grossi, Pietro; Brughera, Marco; Venturi, Miro

    2007-01-01

    Currently, several protein kinase-modulating compounds have received market approval across a range of diverse therapeutic indications. Furthermore, a large number of chemical and biological protein kinase-modulating compounds are undergoing testing at the preclinical and clinical level. Protein kinases are both major pharmacological targets and diagnostically useful. Progression of kinase modulators toward clinically viable therapies is aided by a reversible mechanism of action, short treatment durations and patient-compliant administration routes. However, the physiological role and essential functional activity of protein kinases in many organs and tissues complicates, to different extents, the development of useful, highly potent protein kinase modulators. In this review, we will highlight common problems in the development of these compounds and lessons learned from the extensive preclinical and clinical characterization of some key protein kinase modulators, some of which have either entered and successfully completed clinical trials or have been abandoned as a consequence of unacceptable toxicity issues. We will ultimately explore how molecular profiling tools combined with histopathological endpoints can be adopted to address and further understand these toxicities in humans and understand their relevance and characterization when identified during early animal experiments.

  4. Structure of the intact ATM/Tel1 kinase

    PubMed Central

    Wang, Xuejuan; Chu, Huanyu; Lv, Mengjuan; Zhang, Zhihui; Qiu, Shuwan; Liu, Haiyan; Shen, Xuetong; Wang, Weiwu; Cai, Gang

    2016-01-01

    The ataxia-telangiectasia mutated (ATM) protein is an apical kinase that orchestrates the multifaceted DNA-damage response. Normally, ATM kinase is in an inactive, homodimer form and is transformed into monomers upon activation. Besides a conserved kinase domain at the C terminus, ATM contains three other structural modules, referred to as FAT, FATC and N-terminal helical solenoid. Here we report the first cryo-EM structure of ATM kinase, which is an intact homodimeric ATM/Tel1 from Schizosaccharomyces pombe. We show that two monomers directly contact head-to-head through the FAT and kinase domains. The tandem N-terminal helical solenoid tightly packs against the FAT and kinase domains. The structure suggests that ATM/Tel1 dimer interface and the consecutive HEAT repeats inhibit the binding of kinase substrates and regulators by steric hindrance. Our study provides a structural framework for understanding the mechanisms of ATM/Tel1 regulation as well as the development of new therapeutic agents. PMID:27229179

  5. Comprehensive characterization of the Published Kinase Inhibitor Set.

    PubMed

    Elkins, Jonathan M; Fedele, Vita; Szklarz, Marta; Abdul Azeez, Kamal R; Salah, Eidarus; Mikolajczyk, Jowita; Romanov, Sergei; Sepetov, Nikolai; Huang, Xi-Ping; Roth, Bryan L; Al Haj Zen, Ayman; Fourches, Denis; Muratov, Eugene; Tropsha, Alex; Morris, Joel; Teicher, Beverly A; Kunkel, Mark; Polley, Eric; Lackey, Karen E; Atkinson, Francis L; Overington, John P; Bamborough, Paul; Müller, Susanne; Price, Daniel J; Willson, Timothy M; Drewry, David H; Knapp, Stefan; Zuercher, William J

    2016-01-01

    Despite the success of protein kinase inhibitors as approved therapeutics, drug discovery has focused on a small subset of kinase targets. Here we provide a thorough characterization of the Published Kinase Inhibitor Set (PKIS), a set of 367 small-molecule ATP-competitive kinase inhibitors that was recently made freely available with the aim of expanding research in this field and as an experiment in open-source target validation. We screen the set in activity assays with 224 recombinant kinases and 24 G protein-coupled receptors and in cellular assays of cancer cell proliferation and angiogenesis. We identify chemical starting points for designing new chemical probes of orphan kinases and illustrate the utility of these leads by developing a selective inhibitor for the previously untargeted kinases LOK and SLK. Our cellular screens reveal compounds that modulate cancer cell growth and angiogenesis in vitro. These reagents and associated data illustrate an efficient way forward to increasing understanding of the historically untargeted kinome.

  6. The chromosomal passenger complex activates Polo kinase at centromeres.

    PubMed

    Carmena, Mar; Pinson, Xavier; Platani, Melpi; Salloum, Zeina; Xu, Zhenjie; Clark, Anthony; Macisaac, Fiona; Ogawa, Hiromi; Eggert, Ulrike; Glover, David M; Archambault, Vincent; Earnshaw, William C

    2012-01-01

    The coordinated activities at centromeres of two key cell cycle kinases, Polo and Aurora B, are critical for ensuring that the two sister kinetochores of each chromosome are attached to microtubules from opposite spindle poles prior to chromosome segregation at anaphase. Initial attachments of chromosomes to the spindle involve random interactions between kinetochores and dynamic microtubules, and errors occur frequently during early stages of the process. The balance between microtubule binding and error correction (e.g., release of bound microtubules) requires the activities of Polo and Aurora B kinases, with Polo promoting stable attachments and Aurora B promoting detachment. Our study concerns the coordination of the activities of these two kinases in vivo. We show that INCENP, a key scaffolding subunit of the chromosomal passenger complex (CPC), which consists of Aurora B kinase, INCENP, Survivin, and Borealin/Dasra B, also interacts with Polo kinase in Drosophila cells. It was known that Aurora A/Bora activates Polo at centrosomes during late G2. However, the kinase that activates Polo on chromosomes for its critical functions at kinetochores was not known. We show here that Aurora B kinase phosphorylates Polo on its activation loop at the centromere in early mitosis. This phosphorylation requires both INCENP and Aurora B activity (but not Aurora A activity) and is critical for Polo function at kinetochores. Our results demonstrate clearly that Polo kinase is regulated differently at centrosomes and centromeres and suggest that INCENP acts as a platform for kinase crosstalk at the centromere. This crosstalk may enable Polo and Aurora B to achieve a balance wherein microtubule mis-attachments are corrected, but proper attachments are stabilized allowing proper chromosome segregation.

  7. Low dose exposure of diethylnitrosamine affects mice liver thymidine kinase.

    PubMed

    Pariat, T; Sharan, R N

    1995-11-17

    Swiss albino mice exposed to 5 and 10 mg diethylnitrosamine kg-1 body weight by intravenous route up to four weeks showed cyto- and genotoxic effects. Distortion of cell and nucleus shapes and extensive necrosis were observed. Thymidine kinase activity in the liver declined in diethylnitrosamine dose and duration dependent manners. The adult-form of thymidine kinase isozyme declined continuously during this period. Simultaneously, two isozymic forms of thymidine kinase, with small anodic migrations in an electrophoretic field, were gradually induced. Significance of theses changes in diethylnitrosamine induced precarcinogenic toxicity has been discussed.

  8. Potential of casein kinase I in digestive cancer screening

    PubMed Central

    Modak, Cristina; Chai, Jianyuan

    2009-01-01

    Casein kinase I is a group of ubiquitous Serine/Threonine kinases that have been implicated in both normal cellular functions and several pathological conditions including Alzheimer’s disease and cancer. Recent findings in colon and pancreatic cancer have brought tremendous attention to these molecules as potential therapeutic targets in treatment of digestive cancers. In this review, we summarize up to date what is known about this family of kinases and their involvement in carcinogenesis and other pathological conditions. Our emphasis is on their implications in digestive cancers and their potential for cancer screening and therapy. PMID:21160770

  9. Second-generation inhibitors of Bruton tyrosine kinase.

    PubMed

    Wu, Jingjing; Liu, Christina; Tsui, Stella T; Liu, Delong

    2016-09-02

    Bruton tyrosine kinase (BTK) is a critical effector molecule for B cell development and plays a major role in lymphoma genesis. Ibrutinib is the first-generation BTK inhibitor. Ibrutinib has off-target effects on EGFR, ITK, and Tec family kinases, which explains the untoward effects of ibrutinib. Resistance to ibrutinib was also reported. The C481S mutation in the BTK kinase domain was reported to be a major mechanism of resistance to ibrutinib. This review summarizes the clinical development of novel BTK inhibitors, ACP-196 (acalabrutinib), ONO/GS-4059, and BGB-3111.

  10. Regulated protein kinases and phosphatases in cell cycle decisions.

    PubMed

    Novak, Bela; Kapuy, Orsolya; Domingo-Sananes, Maria Rosa; Tyson, John J

    2010-12-01

    Many aspects of cell physiology are controlled by protein kinases and phosphatases, which together determine the phosphorylation state of targeted substrates. Some of these target proteins are themselves kinases or phosphatases or other components of a regulatory network characterized by feedback and feed-forward loops. In this review we describe some common regulatory motifs involving kinases, phosphatases, and their substrates, focusing particularly on bistable switches involved in cellular decision processes. These general principles are applied to cell cycle transitions, with special emphasis on the roles of regulated phosphatases in orchestrating progression from one phase to the next of the DNA replication-division cycle.

  11. Regulated protein kinases and phosphatases in cell cycle decisions

    PubMed Central

    Novak, Bela; Kapuy, Orsolya; Domingo-Sananes, Maria Rosa; Tyson, John J

    2013-01-01

    Many aspects of cell physiology are controlled by protein kinases and phosphatases, which together determine the phosphorylation state of targeted substrates. Some of these target proteins are themselves kinases or phosphatases or other components of a regulatory network characterized by feedback and feed-forward loops. In this review we describe some common regulatory motifs involving kinases, phosphatases, and their substrates, focusing particularly on bistable switches involved in cellular decision processes. These general principles are applied to cell cycle transitions, with special emphasis on the roles of regulated phosphatases in orchestrating progression from one phase to the next of the DNA replication-division cycle. PMID:20678910

  12. Rho kinase as a target for cerebral vascular disorders

    PubMed Central

    Bond, Lisa M; Sellers, James R; McKerracher, Lisa

    2015-01-01

    The development of novel pharmaceutical treatments for disorders of the cerebral vasculature is a serious unmet medical need. These vascular disorders are typified by a disruption in the delicate Rho signaling equilibrium within the blood vessel wall. In particular, Rho kinase overactivation in the smooth muscle and endothelial layers of the vessel wall results in cytoskeletal modifications that lead to reduced vascular integrity and abnormal vascular growth. Rho kinase is thus a promising target for the treatment of cerebral vascular disorders. Indeed, preclinical studies indicate that Rho kinase inhibition may reduce the formation/growth/rupture of both intracranial aneurysms and cerebral cavernous malformations. PMID:26062400

  13. The phosphatase activity of mammalian polynucleotide kinase takes precedence over its kinase activity in repair of single strand breaks.

    PubMed

    Dobson, Caroline J; Allinson, Sarah L

    2006-01-01

    The dual function mammalian DNA repair enzyme, polynucleotide kinase (PNK), facilitates strand break repair through catalysis of 5'-hydroxyl phosphorylation and 3'-phosphate dephosphorylation. We have examined the relative activities of the kinase and phosphatase functions of PNK using a novel assay, which allows the simultaneous characterization of both activities in processing nicks and gaps containing both 3'-phosphate and 5'-hydroxyl. Under multiple turnover conditions the phosphatase activity of the purified enzyme is significantly more active than its kinase activity. Consistent with this result, phosphorylation of the 5'-hydroxyl is rate limiting in cell extract mediated-repair of a nicked substrate. On characterizing the effects of individually mutating the two active sites of PNK we find that while site-directed mutagenesis of the kinase domain of PNK does not affect its phosphatase activity, disruption of the phosphatase domain also abrogates kinase function. This loss of kinase function requires the presence of a 3'-phosphate, but it need not be present in the same strand break as the 5'-hydroxyl. PNK preferentially binds 3'-phosphorylated substrates and DNA binding to the phosphatase domain blocks further DNA binding by the kinase domain.

  14. Crystal Structures of Human Pyridoxal Kinase in Complex with the Neurotoxins, Ginkgotoxin and Theophylline: Insights into Pyridoxal Kinase Inhibition

    PubMed Central

    Ghatge, Mohini S.; di Salvo, Martino L.; Di Biase, Stefano; Danso-Danquah, Richmond; Musayev, Faik N.; Contestabile, Roberto; Schirch, Verne; Safo, Martin K.

    2012-01-01

    Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase), resulting in deficiency of the active cofactor form of vitamin B6, pyridoxal 5′-phosphate (PLP). Pyridoxal (PL), an inactive form of vitamin B6 is converted to PLP by PL kinase. PLP is the B6 vitamer required as a cofactor for over 160 enzymatic activities essential in primary and secondary metabolism. We have performed structural and kinetic studies on hPL kinase with several potential inhibitors, including ginkgotoxin and theophylline. The structural studies show ginkgotoxin and theophylline bound at the substrate site, and are involved in similar protein interactions as the natural substrate, PL. Interestingly, the phosphorylated product of ginkgotoxin is also observed bound at the active site. This work provides insights into the molecular basis of hPL kinase inhibition and may provide a working hypothesis to quickly screen or identify neurotoxic drugs as potential hPL kinase inhibitors. Such adverse effects may be prevented by administration of an appropriate form of vitamin B6, or provide clues of how to modify these drugs to help reduce their hPL kinase inhibitory effects. PMID:22879864

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

    PubMed

    Amata, Emanuele; Xi, Hualin; Colmenarejo, Gonzalo; Gonzalez-Diaz, Rosario; Cordon-Obras, Carlos; Berlanga, Manuela; Manzano, Pilar; Erath, Jessey; Roncal, Norma E; Lee, Patricia J; Leed, Susan E; Rodriguez, Ana; Sciotti, Richard J; Navarro, Miguel; Pollastri, Michael P

    2016-03-11

    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".

  16. Schizosaccharomyces pombe AGC family kinase Gad8p forms a conserved signaling module with TOR and PDK1-like kinases

    PubMed Central

    Matsuo, Tomohiko; Kubo, Yoshiya; Watanabe, Yoshinori; Yamamoto, Masayuki

    2003-01-01

    The TOR protein is a phosphoinositide kinase-related kinase widely conserved among eukaryotes. Fission yeast tor1 encodes an ortholog of TOR, which is required for sexual development and growth under stressed conditions. We isolated gad8, which encodes a Ser/Thr kinase of the AGC family, as a high-copy suppressor of the sterility of a tor1 mutant. Disruption of gad8 caused phenotypes similar to those of tor1 disruption. Gad8p was less phosphorylated and its kinase activity was undetectable in tor1Δ cells. Three amino acid residues corresponding to conserved phosphorylation sites in the AGC family kinases, namely Thr387 in the activation loop, Ser527 in the turn motif and Ser546 in the hydrophobic motif, were important for the kinase activity of Gad8p. Tor1p was responsible for the phosphorylation of Ser527 and Ser546, whereas Ksg1p, a PDK1-like kinase, appeared to phosphorylate Thr387 directly. Altogether, Tor1p, Ksg1p and Gad8p appear to constitute a signaling module for sexual development and growth under stressed conditions in fission yeast, which resembles the mTOR–PDK1–S6K1 system in mammals and may represent a basic signaling module ubiquitous in eukaryotes. PMID:12805221

  17. Kinase Inhibitor Screening Identifies Cyclin-Dependent Kinases and Glycogen Synthase Kinase 3 as Potential Modulators of TDP-43 Cytosolic Accumulation during Cell Stress.

    PubMed

    Moujalled, Diane; James, Janine L; Parker, Sarah J; Lidgerwood, Grace E; Duncan, Clare; Meyerowitz, Jodi; Nonaka, Takashi; Hasegawa, Masato; Kanninen, Katja M; Grubman, Alexandra; Liddell, Jeffrey R; Crouch, Peter J; White, Anthony R

    2013-01-01

    Abnormal processing of TAR DNA binding protein 43 (TDP-43) has been identified as a major factor in neuronal degeneration during amyotrophic lateral sclerosis (ALS) or frontotemporal lobar degeneration (FTLD). It is unclear how changes to TDP-43, including nuclear to cytosolic translocation and subsequent accumulation, are controlled in these diseases. TDP-43 is a member of the heterogeneous ribonucleoprotein (hnRNP) RNA binding protein family and is known to associate with cytosolic RNA stress granule proteins in ALS and FTLD. hnRNP trafficking and accumulation is controlled by the action of specific kinases including members of the mitogen-activated protein kinase (MAPK) pathway. However, little is known about how kinase pathways control TDP-43 movement and accumulation. In this study, we used an in vitro model of TDP-43-positve stress granule formation to screen for the effect of kinase inhibitors on TDP-43 accumulation. We found that while a number of kinase inhibitors, particularly of the MAPK pathways modulated both TDP-43 and the global stress granule marker, human antigen R (HuR), multiple inhibitors were more specific to TDP-43 accumulation, including inhibitors of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3 (GSK3). Close correlation was observed between effects of these inhibitors on TDP-43, hnRNP K and TIAR, but often with different effects on HuR accumulation. This may indicate a potential interaction between TDP-43, hnRNP K and TIAR. CDK inhibitors were also found to reverse pre-formed TDP-43-positive stress granules and both CDK and GSK3 inhibitors abrogated the accumulation of C-terminal TDP-43 (219-414) in transfected cells. Further studies are required to confirm the specific kinases involved and whether their action is through phosphorylation of the TDP-43 binding partner hnRNP K. This knowledge provides a valuable insight into the mechanisms controlling abnormal cytoplasmic TDP-43 accumulation and may herald new opportunities

  18. Nck-Interacting Ste20 Kinase Couples Eph Receptors to c-Jun N-Terminal Kinase and Integrin Activation

    PubMed Central

    Becker, Elena; Huynh-Do, Uyen; Holland, Sacha; Pawson, Tony; Daniel, Tom O.; Skolnik, Edward Y.

    2000-01-01

    The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62dok, RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62dok most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs. PMID:10669731

  19. Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930.

    PubMed

    Petti, Filippo; Thelemann, April; Kahler, Jen; McCormack, Siobhan; Castaldo, Linda; Hunt, Tony; Nuwaysir, Lydia; Zeiske, Lynn; Haack, Herbert; Sullivan, Laura; Garton, Andrew; Haley, John D

    2005-08-01

    OSI-930, a potent thiophene inhibitor of the Kit, KDR, and platelet-derived growth factor receptor tyrosine kinases, was used to selectively inhibit tyrosine phosphorylation downstream of juxtamembrane mutant Kit in the mast cell leukemia line HMC-1. Inhibition of Kit kinase activity resulted in a rapid dephosphorylation of Kit and inhibition of the downstream signaling pathways. Attenuation of Ras-Raf-Erk (phospho-Erk, phospho-p38), phosphatidyl inositol-3' kinase (phospho-p85, phospho-Akt, phospho-S6), and signal transducers and activators of transcription signaling pathways (phospho-STAT3/5/6) were measured by affinity liquid chromatography tandem mass spectrometry, by immunoblot, and by tissue microarrays of fixed cell pellets. To more globally define additional components of Kit signaling temporally altered by kinase inhibition, a novel multiplex quantitative isobaric peptide labeling approach was used. This approach allowed clustering of proteins by temporal expression patterns. Kit kinase, which dephosphorylates rapidly upon kinase inhibition, was shown to regulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2. Interactions with SH2 domain adapters [growth factor receptor binding protein 2 (Grb2), Cbl, Slp-76] and SH3 domain adapters (HS1, cortactin, CD2BP3) were attenuated by inhibition of Kit kinase activity. Functional crosstalk between Kit and the non-receptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk was observed. Inhibition of Kit modulated phosphorylation-dependent interactions with pathways controlling focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, Wasp, Fhl-3, G25K, Ack-1, Nap1, SH3P12/ponsin) and septin-actin complexes (NEDD5, cdc11, actin). The combined use of isobaric protein quantitation and expression clustering, immunoblot, and tissue microarray strategies allowed temporal measurement signaling pathways modulated by mutant Kit inhibition in a model of mast cell

  20. Ischemia induced activation of heat shock protein 27 kinases and casein kinase 2 in the preconditioned rabbit heart.

    PubMed

    Kim, S O; Baines, C P; Critz, S D; Pelech, S L; Katz, S; Downey, J M; Cohen, M V

    1999-01-01

    Protein kinase C (PKC), p38 MAP kinase, and mitogen-activated protein kinase-activated kinases 2 and 3 (MAPKAPK2 and MAPKAPK3) have been implicated in ischemic preconditioning (PC) of the heart to reduce damage following a myocardial infarct. This study examined whether extracellular signal-regulated kinase (Erk) 1, p70 ribosomal S6 kinase (p70 S6K), casein kinase 2 (CK2), and other hsp27 kinases are also activated by PC, and if they are required for protection in rabbit hearts. CK2 and hsp27 kinase activities declined during global ischemia in control hearts, whereas PC with 5 min ischemia and 10 min reperfusion increased their activities during global ischemia. Resource Q chromatography resolved two distinct peaks of hsp27 phosphotransferase activities; the first peak (at 0.36 M NaCl) appeared to correspond to the 55-kDa MAPKAPK2. Erk1 activity was elevated in both control and PC hearts after post-ischemic reperfusion, but no change was observed in p70 S6K activity. Infarct size (measured by triphenyltetrazolium staining) in isolated rabbit hearts subjected to 30 min regional ischemia and 2 h reperfusion was 31.0+/-2.6% of the risk zone in controls and was 10.3+/-2.2% in PC hearts (p<0.001). Neither the CK2 inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) nor the Mek1/2 inhibitor PD98059 infused during ischemia blocked protection by PC. The activation of CK2 and Erk1 in ischemic preconditioned hearts appear to be epiphenomena and not required for the reduction of infarction from myocardial ischemia.

  1. The adenosine kinase hypothesis of epileptogenesis

    PubMed Central

    Boison, Detlev

    2008-01-01

    Current therapies for epilepsy are largely symptomatic and do not affect the underlying mechanisms of disease progression, i.e. epileptogenesis. Given the large percentage of pharmacoresistant chronic epilepsies, novel approaches are needed to understand and modify the underlying pathogenetic mechanisms. Although different types of brain injury (e.g. status epilepticus, traumatic brain injury, stroke) can trigger epileptogenesis, astrogliosis appears to be a homotypic response and hallmark of epilepsy. Indeed, recent findings indicate that epilepsy might be a disease of astrocyte dysfunction. This review focuses on the inhibitory neuromodulator and endogenous anticonvulsant adenosine, which is largely regulated by astrocytes and its key metabolic enzyme adenosine kinase (ADK). Recent findings support the “ADK hypothesis of epileptogenesis”: (i) Mouse models of epileptogenesis suggest a sequence of events leading from initial downregulation of ADK and elevation of ambient adenosine as an acute protective response, to changes in astrocytic adenosine receptor expression, to astrocyte proliferation and hypertrophy (i.e. astrogliosis), to consequential overexpression of ADK, reduced adenosine and – finally – to spontaneous focal seizure activity restricted to regions of astrogliotic overexpression of ADK. (ii) Transgenic mice overexpressing ADK display increased sensitivity to brain injury and seizures. (iii) Inhibition of ADK prevents seizures in a mouse model of pharmacoresistant epilepsy. (iv) Intrahippocampal implants of stem cells engineered to lack ADK prevent epileptogenesis. Thus, ADK emerges both as a diagnostic marker to predict, as well as a prime therapeutic target to prevent, epileptogenesis. PMID:18249058

  2. Glycolysis without pyruvate kinase in Clostridium thermocellum

    SciTech Connect

    Olson, Daniel G.; Horl, Manuel; Fuhrer, Tobias; Cui, Jingxuan; Zhou, Jilai; Maloney, Marybeth I.; Amador-Noguez, Daniel; Tian, Liang; Sauer, Uwe; Lynd, Lee R.

    2016-12-01

    The metabolism of Clostridium thermocellum is notable in that it assimilates sugar via the EMP pathway but does not possess a pyruvate kinase enzyme. In the wild type organism, there are three proposed pathways for conversion of phosphoenolpyruvate (PEP) to pyruvate, which differ in their cofactor usage. One path uses pyruvate phosphate dikinase (PPDK), another pathway uses the combined activities of PEP carboxykinase (PEPCK) and oxaloacetate decarboxylase (ODC). Yet another pathway, the malate shunt, uses the combined activities of PEPCK, malate dehydrogenase and malic enzyme. First we showed that there is no flux through the ODC pathway by enzyme assay. Flux through the remaining two pathways (PPDK and malate shunt) was determined by dynamic 13C labeling. In the wild-type strain, the malate shunt accounts for about 33 ± 2% of the flux to pyruvate, with the remainder via the PPDK pathway. Deletion of the ppdk gene resulted in a redirection of all pyruvate flux through the malate shunt. Lastly, this provides the first direct evidence of the in-vivo function of the malate shunt.

  3. Focal Adhesion Kinase: The Reversible Molecular Mechanosensor

    NASA Astrophysics Data System (ADS)

    Bell, Samuel; Terentjev, Eugene M.

    2017-06-01

    Sensors are the first element of the pathways that control the response of cells to their environment. After chemical, the next most important cue is mechanical, and protein complexes that produce or enable a chemical signal in response to a mechanical stimulus are called mechanosensors. There is a sharp distinction between sensing an external force or pressure/tension applied to the cell, and sensing the mechanical stiffness of the environment. We call the first mechanosensitivity of the 1st kind, and the latter mechanosensitivity of the 2nd kind. There are two variants of protein complexes that act as mechanosensors of the 2nd kind: producing either a one-off or a reversible action. The latent complex of TGF-$\\beta$ is an example of the one-off action: on the release of active TGF-$\\beta$ signal, the complex is discarded and needs to be replaced. In contrast, focal adhesion kinase (FAK) in a complex with integrin is a reversible mechanosensor, which initiates the chemical signal in its active phosphorylated conformation, but can spontaneously return to its closed folded conformation. Here we study the physical mechanism of the reversible mechanosensor of the 2nd kind, using FAK as a practical example. We find how the rates of conformation changes depend on the substrate stiffness and the pulling force applied from the cell cytoskeleton. The results compare well with the phenotype observations of cells on different substrates.

  4. Glycolysis without pyruvate kinase in Clostridium thermocellum

    DOE PAGES

    Olson, Daniel G.; Horl, Manuel; Fuhrer, Tobias; ...

    2016-12-01

    The metabolism of Clostridium thermocellum is notable in that it assimilates sugar via the EMP pathway but does not possess a pyruvate kinase enzyme. In the wild type organism, there are three proposed pathways for conversion of phosphoenolpyruvate (PEP) to pyruvate, which differ in their cofactor usage. One path uses pyruvate phosphate dikinase (PPDK), another pathway uses the combined activities of PEP carboxykinase (PEPCK) and oxaloacetate decarboxylase (ODC). Yet another pathway, the malate shunt, uses the combined activities of PEPCK, malate dehydrogenase and malic enzyme. First we showed that there is no flux through the ODC pathway by enzyme assay.more » Flux through the remaining two pathways (PPDK and malate shunt) was determined by dynamic 13C labeling. In the wild-type strain, the malate shunt accounts for about 33 ± 2% of the flux to pyruvate, with the remainder via the PPDK pathway. Deletion of the ppdk gene resulted in a redirection of all pyruvate flux through the malate shunt. Lastly, this provides the first direct evidence of the in-vivo function of the malate shunt.« less

  5. Rho kinase inhibition in diabetic kidney disease.

    PubMed

    Komers, Radko

    2013-10-01

    Small GTPases of the Rho family and their down-stream effectors Rho associated kinases (ROCKs) are the molecules that converge a spectrum of pathophysiological signals triggered by the diabetic milieu and represent promising molecular targets for nephroprotective treatment in diabetes. The review discusses recent studies exploring the consequences of diabetes-induced Rho-ROCK activation in the kidney and the effects of ROCK inhibition (ROCKi) in experimental diabetic kidney disease (DKD). Studies in models of type 1 and type 2 diabetes have indicated blood pressure-independent nephroprotective actions of ROCKi in DKD. The underlying mechanisms include attenuation of diabetes-induced increases in renal expression of prosclerotic cytokines and extracellular matrix, anti-oxidant effects and protection of mitochondrial function, resulting in slower development of glomerulosclerosis and interstitial fibrosis. The studies have also shown antiproteinuric effects of ROCKi that could be related to reductions in permeability of the glomerular barrier and beneficial effects on podocytes. Glomerular haemodynamic mechanisms might also be involved. Despite remaining questions in this field, such as the effects in podocytes later in the course of DKD, specificity of currently available ROCKi, or the roles of individual ROCK isoforms, recent evidence in experimental diabetes suggests that ROCKi might in future broaden the spectrum of treatments available for patients with DKD. This is supported by the evidence generated in models of non-diabetic kidney disease and in clinical studies in patients with various cardiovascular disorders.

  6. Structure, biochemistry, and biology of PAK kinases.