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Sample records for modulating protein kinase

  1. Leishmania amazonensis: PKC-like protein kinase modulates the (Na++K+)ATPase activity.

    PubMed

    Almeida-Amaral, Elmo Eduardo de; Caruso-Neves, Celso; Lara, Lucienne Silva; Pinheiro, Carla Mônica; Meyer-Fernandes, José Roberto

    2007-08-01

    The present study aimed to identify the presence of protein kinase C-like (PKC-like) in Leishmania amazonensis and to elucidate its possible role in the modulation of the (Na(+)+K(+))ATPase activity. Immunoblotting experiments using antibody against a consensus sequence (Ac 543-549) of rabbit protein kinase C (PKC) revealed the presence of a protein kinase of 80 kDa in L. amazonensis. Measurements of protein kinase activity showed the presence of both (Ca(2+)-dependent) and (Ca(2+)-independent) protein kinase activity in plasma membrane and cytosol. Phorbol ester (PMA) activation of the Ca(2+)-dependent protein kinase stimulated the (Na(+)+K(+))ATPase activity, while activation of the Ca(2+)-independent protein kinase was inhibitory. Both effects of protein kinase on the (Na(+)+K(+))ATPase of the plasma membrane were lower than that observed in intact cells. PMA induced the translocation of protein kinase from cytosol to plasma membrane, indicating that the maximal effect of protein kinase on the (Na(+)+K(+))ATPase activity depends on the synergistic action of protein kinases from both plasma membrane and cytosol. This is the first demonstration of a protein kinase activated by PMA in L. amazonensis and the first evidence for a possible role in the regulation of the (Na(+)+K(+))ATPase activity in this trypanosomatid. Modulation of the (Na(+)+K(+))ATPase by protein kinase in a trypanosomatid opens up new possibilities to understand the regulation of ion homeostasis in this parasite. PMID:17475255

  2. DIRECT MODULATION OF THE PROTEIN KINASE A CATALYTIC SUBUNIT α BY GROWTH FACTOR RECEPTOR TYROSINE KINASES

    PubMed Central

    Caldwell, George B.; Howe, Alan K.; Nickl, Christian K.; Dostmann, Wolfgang R.; Ballif, Bryan A.; Deming, Paula B.

    2011-01-01

    The cyclic-AMP-dependent protein kinase A (PKA) regulates processes such as cell proliferation and migration following activation of growth factor receptor tyrosine kinases (RTKs), yet the signaling mechanisms that link PKA with growth factor receptors remain largely undefined. Here we report that RTKs can directly modulate the function of the catalytic subunit of PKA (PKA-C) through post-translational modification. In vitro kinase assays revealed that both the epidermal growth factor and platelet derived growth factor receptors (EGFR and PDGFR, respectively) tyrosine phosphorylate PKA-C. Mass spectrometry identified tyrosine 330 (Y330) as a receptor-mediated phosphorylation site and mutation of Y330 to phenylalanine (Y330F) all but abolished the RTK-mediated phosphorylation of PKA-C in vitro. Y330 resides within a conserved region at the C-terminal tail of PKA-C that allosterically regulates enzymatic activity. Therefore, the effect of phosphorylation at Y330 on the activity of PKA-C was investigated. The Km for a peptide substrate was markedly decreased when PKA-C subunits were tyrosine phosphorylated by the receptors as compared to un-phosphorylated controls. Importantly, tyrosine-phosphorylated PKA-C subunits were detected in cells stimulated with EGF, PDGF and FGF2 and in fibroblasts undergoing PDGF-mediated chemotaxis. These results demonstrate a direct, functional interaction between RTKs and PKA-C and identify tyrosine phosphorylation as a novel mechansim for regulating PKA activity. PMID:21866565

  3. Modulation of the protein kinase activity of mTOR.

    PubMed

    Lawrence, J C; Lin, T A; McMahon, L P; Choi, K M

    2004-01-01

    mTOR is a founding member of a family of protein kinases having catalytic domains homologous to those in phosphatidylinositol 3-OH kinase. mTOR participates in the control by insulin of the phosphorylation of lipin, which is required for adipocyte differentiation, and the two translational regulators, p70S6K and PHAS-I. The phosphorylation of mTOR, itself, is stimulated by insulin in Ser2448, a site that is also phosphorylated by protein kinase B (PKB) in vitro and in response to activation of PKB activity in vivo. Ser2448 is located in a short stretch of amino acids not found in the two TOR proteins in yeast. A mutant mTOR lacking this stretch exhibited increased activity, and binding of the antibody, mTAb-1, to this region markedly increased mTOR activity. In contrast, rapamycin-FKBP12 inhibited mTOR activity towards both PHAS-I and p70S6K, although this complex inhibited the phosphorylation of some sites more than that of others. Mutating Ser2035 to Ile in the FKBP12-rapamycin binding domain rendered mTOR resistant to inhibition by rapamycin. Unexpectedly, this mutation markedly decreased the ability of mTOR to phosphorylate certain sites in both PHAS-I and p70S6K. The results support the hypotheses that rapamycin disrupts substrate recognition instead of directly inhibiting phosphotransferase activity and that mTOR activity in cells is controlled by the phosphorylation of an inhibitory regulatory domain containing the mTAb-1 epitope. PMID:14560959

  4. Rho-associated protein kinase modulates neurite extension by regulating microtubule remodeling and vinculin distribution

    PubMed Central

    Chen, Ke’en; Zhang, Wenbin; Chen, Jing; Li, Sumei; Guo, Guoqing

    2013-01-01

    Rho-associated protein kinase is an essential regulator of cytoskeletal dynamics during the process of neurite extension. However, whether Rho kinase regulates microtubule remodeling or the distribution of adhesive proteins to mediate neurite outgrowth remains unclear. By specifically modulating Rho kinase activity with pharmacological agents, we studied the morpho-dynamics of neurite outgrowth. We found that lysophosphatidic acid, an activator of Rho kinase, inhibited neurite outgrowth, which could be reversed by Y-27632, an inhibitor of Rho kinase. Meanwhile, reorganization of microtubules was noticed during these processes, as indicated by their significant changes in the soma and growth cone. In addition, exposure to lysophosphatidic acid led to a decreased membrane distribution of vinculin, a focal adhesion protein in neurons, whereas Y-27632 recruited vinculin to the membrane. Taken together, our data suggest that Rho kinase regulates rat hippocampal neurite growth and microtubule formation via a mechanism associated with the redistribution of vinculin. PMID:25206623

  5. Modulation of skeletal muscle sodium channels by human myotonin protein kinase.

    PubMed Central

    Mounsey, J P; Xu, P; John, J E; Horne, L T; Gilbert, J; Roses, A D; Moorman, J R

    1995-01-01

    In myotonic muscular dystrophy, abnormal muscle Na currents underlie myotonic discharges. Since the myotonic muscular dystrophy gene encodes a product, human myotonin protein kinase, with structural similarity to protein kinases, we tested the idea that human myotonin protein kinase modulates skeletal muscle Na channels. Coexpression of human myotonin protein kinase with rat skeletal muscle Na channels in Xenopus oocytes reduced the amplitude of Na currents and accelerated current decay. The effect required the presence of a potential phosphorylation site in the inactivation mechanism of the channel. The mutation responsible for human disease, trinucleotide repeats in the 3' untranslated region, did not prevent the effect. The consequence of an abnormal amount of the kinase would be altered muscle cell excitability, consistent with the clinical finding of myotonia in myotonic dystrophy. Images PMID:7738201

  6. Interaction between protein kinase C and protein kinase A can modulate transmitter release at the rat neuromuscular synapse.

    PubMed

    Santafé, M M; Garcia, N; Lanuza, M A; Tomàs, M; Tomàs, J

    2009-02-15

    We used intracellular recording to investigate the functional interaction between protein kinase C (PKC) and protein kinase A (PKA) signal transduction cascades in the control of transmitter release in the neuromuscular synapses from adult rats. Our results indicate that: 1) PKA and PKC are independently involved in asynchronous release. 2) Evoked acetylcholine (ACh) release is enhanced with the PKA agonist Sp-8-BrcAMP and the PKC agonist phorbol ester (PMA). 3) PKA has a constitutive role in promoting a component of normal evoked transmitter release because, when the kinase is inhibited with H-89, the release diminishes. However, the PKC inhibitor calphostin C (CaC) does not affect ACh release. 4) PKA regulates neurotransmission without PKC involvement because, after PMA or CaC modulation of the PKC activity, coupling to the ACh release of PKA can normally be stimulated with Sp-8-BrcAMP or inhibited with H-89. 5) After PKA inhibition with H-89, PKC stimulation with PMA (or inhibition with CaC) does not lead to any change in evoked ACh release. However, in PKA-stimulated preparations with Sp-8-BrcAMP, PKC becomes tonically active, thus potentiating a component of release that can now be blocked with CaC. In normal conditions, therefore, PKA was able to modulate ACh release independently of PKC activity, whereas PKA stimulation caused the PKC coupling to evoked release. In contrast, PKA inhibition prevent PKC stimulation (with the phorbol ester) and coupling to ACh output. There was therefore some dependence of PKC on PKA activity in the fine control of the neuromuscular synaptic functionalism and ACh release. PMID:18816790

  7. Bovine prion protein as a modulator of protein kinase CK2.

    PubMed

    Meggio, F; Negro, A; Sarno, S; Ruzzene, M; Bertoli, A; Sorgato, M C; Pinna, L A

    2000-11-15

    On the basis of far-Western blot and plasmon resonance (BIAcore) experiments, we show here that recombinant bovine prion protein (bPrP) (25-242) strongly interacts with the catalytic alpha/alpha' subunits of protein kinase CK2 (also termed 'casein kinase 2'). This association leads to increased phosphotransferase activity of CK2alpha, tested on calmodulin or specific peptides as substrate. We also show that bPrP counteracts the inhibition of calmodulin phosphorylation promoted by the regulatory beta subunits of CK2. A truncated form of bPrP encompassing the C-terminal domain (residues 105-242) interacts with CK2 but does not affect its catalytic activity. The opposite is found with the N-terminal fragment of bPrP (residues 25-116), although the stimulation of catalysis is less efficient than with full-size bPrP. These results disclose the potential of the PrP to modulate the activity of CK2, a pleiotropic protein kinase that is particularly abundant in the brain. PMID:11062072

  8. Protein kinase A and protein kinase C modulators have reciprocal effects on mesenchymal condensation during skin appendage morphogenesis.

    PubMed

    Noveen, A; Jiang, T X; Chuong, C M

    1995-10-01

    The molecular signaling of secondary induction is a fundamental process in organogenesis during embryonic development. To study the signal transduction pathways involved, we used developing chicken skin as a model and focused on the roles of intracellular signaling during feather morphogenesis. Protein kinase C (PKC) immunoreactivity increases in the whole layer of forming dermis around H and H stage 30. This is followed by a gradual and highly localized decrease of PKC expression immediately beneath each forming feather germ. In contrast, cAMP response element binding protein (CREB) is ubiquitously expressed in both epithelium and mesenchyme. From stage 29 on, phosphorylated CREB (P-CREB), reflecting the activity of protein kinase A (PKA), begins to be seen in placode but not in interplacode epithelia. P-CREB is also expressed in bud mesenchyme transiently between stages 33 and 36, but not in the interbud mesenchyme. The presence and activity of PKC, PKA, and P-CREB in developing chicken skin are further characterized by immunoblot, kinase activity, and gel shift assays. To explore their physiological significance, embryonic chicken dorsal skin explants were treated with different modulators in medium or in beads for localized effects. The results showed that PKA activators and PKC inhibitors can expand a feather bud domain by enhancing dermal condensation, while PKC activators and PKA inhibitors can expand interbud domains. Neural cell adhesion molecule (N-CAM) is involved in dermal condensation. We observed that activation of PKA causes diffused expression of N-CAM in mesenchyme while activation of PKC causes the disappearance of N-CAM in precondensed mesenchymal regions. A model of how the well-concerted PKA and PKC signaling may be involved in the formation and size regulation of dermal condensation is presented. PMID:7556946

  9. Direct Modulation of Heterotrimeric G Protein-coupled Signaling by a Receptor Kinase Complex.

    PubMed

    Tunc-Ozdemir, Meral; Urano, Daisuke; Jaiswal, Dinesh Kumar; Clouse, Steven D; Jones, Alan M

    2016-07-01

    Plants and some protists have heterotrimeric G protein complexes that activate spontaneously without canonical G protein-coupled receptors (GPCRs). In Arabidopsis, the sole 7-transmembrane regulator of G protein signaling 1 (AtRGS1) modulates the G protein complex by keeping it in the resting state (GDP-bound). However, it remains unknown how a myriad of biological responses is achieved with a single G protein modulator. We propose that in complete contrast to G protein activation in animals, plant leucine-rich repeat receptor-like kinases (LRR RLKs), not GPCRs, provide this discrimination through phosphorylation of AtRGS1 in a ligand-dependent manner. G protein signaling is directly activated by the pathogen-associated molecular pattern flagellin peptide 22 through its LRR RLK, FLS2, and co-receptor BAK1. PMID:27235398

  10. Modulation of neurosteroid potentiation by protein kinases at synaptic- and extrasynaptic-type GABAA receptors

    PubMed Central

    Adams, Joanna M.; Thomas, Philip; Smart, Trevor G.

    2015-01-01

    GABAA receptors are important for inhibition in the CNS where neurosteroids and protein kinases are potent endogenous modulators. Acting individually, these can either enhance or depress receptor function, dependent upon the type of neurosteroid or kinase and the receptor subunit combination. However, in vivo, these modulators probably act in concert to fine-tune GABAA receptor activity and thus inhibition, although how this is achieved remains unclear. Therefore, we investigated the relationship between these modulators at synaptic-type α1β3γ2L and extrasynaptic-type α4β3δ GABAA receptors using electrophysiology. For α1β3γ2L, potentiation of GABA responses by tetrahydro-deoxycorticosterone was reduced after inhibiting protein kinase C, and enhanced following its activation, suggesting this kinase regulates neurosteroid modulation. In comparison, neurosteroid potentiation was reduced at α1β3S408A,S409Aγ2L receptors, and unaltered by PKC inhibitors or activators, indicating that phosphorylation of β3 subunits is important for regulating neurosteroid activity. To determine whether extrasynaptic-type GABAA receptors were similarly modulated, α4β3δ and α4β3S408A,S409Aδ receptors were investigated. Neurosteroid potentiation was reduced at both receptors by the kinase inhibitor staurosporine. By contrast, neurosteroid-mediated potentiation at α4S443Aβ3S408A,S409Aδ receptors was unaffected by protein kinase inhibition, strongly suggesting that phosphorylation of α4 and β3 subunits is required for regulating neurosteroid activity at extrasynaptic receptors. Western blot analyses revealed that neurosteroids increased phosphorylation of β3S408,S409 implying that a reciprocal pathway exists for neurosteroids to modulate phosphorylation of GABAA receptors. Overall, these findings provide important insight into the regulation of GABAA receptors in vivo, and into the mechanisms by which GABAergic inhibitory transmission may be simultaneously tuned by

  11. PRO40 Is a Scaffold Protein of the Cell Wall Integrity Pathway, Linking the MAP Kinase Module to the Upstream Activator Protein Kinase C

    PubMed Central

    Teichert, Ines; Steffens, Eva Katharina; Schnaß, Nicole; Fränzel, Benjamin; Krisp, Christoph; Wolters, Dirk A.; Kück, Ulrich

    2014-01-01

    Mitogen-activated protein kinase (MAPK) pathways are crucial signaling instruments in eukaryotes. Most ascomycetes possess three MAPK modules that are involved in key developmental processes like sexual propagation or pathogenesis. However, the regulation of these modules by adapters or scaffolds is largely unknown. Here, we studied the function of the cell wall integrity (CWI) MAPK module in the model fungus Sordaria macrospora. Using a forward genetic approach, we found that sterile mutant pro30 has a mutated mik1 gene that encodes the MAPK kinase kinase (MAPKKK) of the proposed CWI pathway. We generated single deletion mutants lacking MAPKKK MIK1, MAPK kinase (MAPKK) MEK1, or MAPK MAK1 and found them all to be sterile, cell fusion-deficient and highly impaired in vegetative growth and cell wall stress response. By searching for MEK1 interaction partners via tandem affinity purification and mass spectrometry, we identified previously characterized developmental protein PRO40 as a MEK1 interaction partner. Although fungal PRO40 homologs have been implicated in diverse developmental processes, their molecular function is currently unknown. Extensive affinity purification, mass spectrometry, and yeast two-hybrid experiments showed that PRO40 is able to bind MIK1, MEK1, and the upstream activator protein kinase C (PKC1). We further found that the PRO40 N-terminal disordered region and the central region encompassing a WW interaction domain are sufficient to govern interaction with MEK1. Most importantly, time- and stress-dependent phosphorylation studies showed that PRO40 is required for MAK1 activity. The sum of our results implies that PRO40 is a scaffold protein for the CWI pathway, linking the MAPK module to the upstream activator PKC1. Our data provide important insights into the mechanistic role of a protein that has been implicated in sexual and asexual development, cell fusion, symbiosis, and pathogenicity in different fungal systems. PMID:25188365

  12. A role for cyclin-dependent kinase(s) in the modulation of fast anterograde axonal transport: effects defined by olomoucine and the APC tumor suppressor protein

    NASA Technical Reports Server (NTRS)

    Ratner, N.; Bloom, G. S.; Brady, S. T.

    1998-01-01

    Proteins that interact with both cytoskeletal and membrane components are candidates to modulate membrane trafficking. The tumor suppressor proteins neurofibromin (NF1) and adenomatous polyposis coli (APC) both bind to microtubules and interact with membrane-associated proteins. The effects of recombinant NF1 and APC fragments on vesicle motility were evaluated by measuring fast axonal transport along microtubules in axoplasm from squid giant axons. APC4 (amino acids 1034-2844) reduced only anterograde movements, whereas APC2 (aa 1034-2130) or APC3 (aa 2130-2844) reduced both anterograde and retrograde transport. NF1 had no effect on organelle movement in either direction. Because APC contains multiple cyclin-dependent kinase (CDK) consensus phosphorylation motifs, the kinase inhibitor olomoucine was examined. At concentrations in which olomoucine is specific for cyclin-dependent kinases (5 microM), it reduced only anterograde transport, whereas anterograde and retrograde movement were both affected at concentrations at which other kinases are inhibited as well (50 microM). Both anterograde and retrograde transport also were inhibited by histone H1 and KSPXK peptides, substrates for proline-directed kinases, including CDKs. Our data suggest that CDK-like axonal kinases modulate fast anterograde transport and that other axonal kinases may be involved in modulating retrograde transport. The specific effect of APC4 on anterograde transport suggests a model in which the binding of APC to microtubules may limit the activity of axonal CDK kinase or kinases in restricted domains, thereby affecting organelle transport.

  13. Protein kinase C modulates transcriptional activation by the juvenile hormone receptor methoprene-tolerant.

    PubMed

    Ojani, Reyhaneh; Liu, Pengcheng; Fu, Xiaonan; Zhu, Jinsong

    2016-03-01

    Juvenile hormone (JH) controls many biological events in insects by triggering dramatic changes in gene expression in target cells. The Methoprene-tolerant (MET) protein, an intracellular JH receptor, acts as a transcriptional regulator and binds to the promoters of tissue- and stage-specific JH target genes when JH is present. Our recent study has demonstrated that the transcriptional activation by MET is modulated by a membrane-initiated JH signaling pathway, involving phospholipase C (PLC) and calcium/calmodulin-dependent protein kinase II (CaMKII). Here we report that protein kinase C (PKC) is another essential intermediate of this pathway. PKC was activated by JH and this action was PLC-dependent. Inhibition of the PKC activity substantially weakened the JH-induced gene expression in mosquito cells. RNAi experiments indicated that several PKC isoforms were involved in the JH action during the post-emergence development of adult female mosquitoes. JH treatment considerably increased the binding of MET to the promoters of JH response genes in cultured mosquito abdomens that were collected from newly emerged female adults. The JH-induced DNA binding of MET was hindered when the abdomens were treated with a PKC inhibitor and JH. Therefore, the results suggest that PKC modulates the transactivation activity of MET by enhancing the binding of MET to JH response elements in the JH target genes. This mechanism may allow for variable and stage- and tissue-specific genomic responses to JH. PMID:26689644

  14. Lithium blocks ethanol-induced modulation of protein kinases in the developing brain

    SciTech Connect

    Chakraborty, Goutam; Saito, Mitsuo; Mao, Rui-Fen; Wang, Ray; Vadasz, Csaba; Saito, Mariko

    2008-03-14

    Lithium has been shown to be neuroprotective against various insults including ethanol exposure. We previously reported that ethanol-induced apoptotic neurodegeneration in the postnatal day 7 (P7) mice is associated with decreases in phosphorylation levels of Akt, glycogen synthase kinase-3{beta} (GSK-3{beta}), and AMP-activated protein kinase (AMPK), and alteration in lipid profiles in the brain. Here, P7 mice were injected with ethanol and lithium, and the effects of lithium on ethanol-induced alterations in phosphorylation levels of protein kinases and lipid profiles in the brain were examined. Immunoblot and immunohistochemical analyses showed that lithium significantly blocked ethanol-induced caspase-3 activation and reduction in phosphorylation levels of Akt, GSK-3{beta}, and AMPK. Further, lithium inhibited accumulation of cholesterol ester (ChE) and N-acylphosphatidylethanolamine (NAPE) triggered by ethanol in the brain. These results suggest that Akt, GSK-3{beta}, and AMPK are involved in ethanol-induced neurodegeneration and the neuroprotective effects of lithium by modulating both apoptotic and survival pathways.

  15. Modulation of Leishmania major aquaglyceroporin activity by a mitogen-activated protein kinase

    PubMed Central

    Mandal, Goutam; Sharma, Mansi; Kruse, Martin; Sander-Juelch, Claudia; Munro, Laura Anne; Wang, Yong; Vilg, Jenny Veide; Tamás, Markus J; Bhattacharjee, Hiranmoy; Wiese, Martin; Mukhopadhyay, Rita

    2012-01-01

    Summary Leishmania major aquaglyceroporin (LmjAQP1) adventitiously facilitates the uptake of antimonite [Sb(III)], an active form of Pentostam® or Glucantime®, which are the first line of defense against all forms of leishmaniasis. The present paper shows that LmjAQP1 activity is modulated by the mitogen-activated protein kinase, LmjMPK2. Leishmania parasites co-expressing LmjAQP1 and LmjMPK2 show increased Sb(III) uptake and increased Sb(III) sensitivity. When subjected to a hypo-osmotic stress, these cells show faster volume recovery than cells expressing LmjAQP1 alone. LmjAQP1 is phosphorylated in vivo at Thr197 and this phosphorylation requires LmjMPK2 activity. Lys42 of LmjMPK2 is critical for its kinase activity. Cells expressing altered T197A LmjAQP1 or K42A LmjMPK2 showed decreased Sb(III) influx and a slower volume recovery than cells expressing wild type proteins. Phosphorylation of LmjAQP1 led to a decrease in its turnover rate affecting LmjAQP1 activity. Although LmjAQP1 is localized to the flagellum of promastigotes, upon phosphorylation, it is relocalized to the entire surface of the parasite. L. mexicana promastigotes with an MPK2 deletion showed reduced Sb(III) uptake and slower volume recovery than wild type cells. This is the first report where a parasite aquaglyceroporin activity is post-translationally modulated by a MAP kinase. PMID:22779703

  16. Pharmacological modulation of protein kinases as a new approach to treat addiction to cocaine and opiates.

    PubMed

    García-Pardo, María Pilar; Roger-Sanchez, Concepción; Rodríguez-Arias, Marta; Miñarro, Jose; Aguilar, María Asunción

    2016-06-15

    Drug addiction shares brain mechanisms and molecular substrates with learning and memory processes, such as the stimulation of glutamate receptors and their downstream signalling pathways. In the present work we provide an up-to-date review of studies that have demonstrated the implication of the main memory-related calcium-dependent protein kinases in opiate and cocaine addiction. The effects of these drugs of abuse in different animal models of drug reward, dependence and addiction are altered by manipulation of the mitogen-activated protein kinase (MAPK) family, particularly extracellular signal regulated kinase (ERK), calcium/calmodulin-dependent kinase II (CaMKII), the protein kinase C (PKC) family (including PKMζ), cAMP-dependent protein kinase A (PKA), cGMP-dependent protein kinase G (PKG), the phosphatidylinositol 3-kinase (PI3K) pathway and its downstream target mammalian target of Rapamycin (mTOR), cyclin-dependent kinase 5 (Cdk5), heat-shock proteins (Hsp) and other enzymes and proteins. Research suggests that drugs of abuse induce dependence and addiction by modifying the signalling pathways that involve these memory-related protein kinases, and supports the idea that drug addiction is an excessive aberrant learning disorder in which the maladaptive memory of drug-associated cues maintains compulsive drug use and contributes to relapse. Moreover, the studies we review offer new pharmacological strategies to treat opiate and cocaine dependence based on the manipulation of these protein kinases. In particular, disruption of reconsolidation of drug-related memories may have a high therapeutic value in the treatment of drug addiction. PMID:27056740

  17. The Parkinson's Disease-Associated Protein Kinase LRRK2 Modulates Notch Signaling through the Endosomal Pathway.

    PubMed

    Imai, Yuzuru; Kobayashi, Yoshito; Inoshita, Tsuyoshi; Meng, Hongrui; Arano, Taku; Uemura, Kengo; Asano, Takeshi; Yoshimi, Kenji; Zhang, Chang-Liang; Matsumoto, Gen; Ohtsuka, Toshiyuki; Kageyama, Ryoichiro; Kiyonari, Hiroshi; Shioi, Go; Nukina, Nobuyuki; Hattori, Nobutaka; Takahashi, Ryosuke

    2015-09-01

    Leucine-rich repeat kinase 2 (LRRK2) is a key molecule in the pathogenesis of familial and idiopathic Parkinson's disease (PD). We have identified two novel LRRK2-associated proteins, a HECT-type ubiquitin ligase, HERC2, and an adaptor-like protein with six repeated Neuralized domains, NEURL4. LRRK2 binds to NEURL4 and HERC2 via the LRRK2 Ras of complex proteins (ROC) domain and NEURL4, respectively. HERC2 and NEURL4 link LRRK2 to the cellular vesicle transport pathway and Notch signaling, through which the LRRK2 complex promotes the recycling of the Notch ligand Delta-like 1 (Dll1)/Delta (Dl) through the modulation of endosomal trafficking. This process negatively regulates Notch signaling through cis-inhibition by stabilizing Dll1/Dl, which accelerates neural stem cell differentiation and modulates the function and survival of differentiated dopaminergic neurons. These effects are strengthened by the R1441G ROC domain-mutant of LRRK2. These findings suggest that the alteration of Notch signaling in mature neurons is a component of PD etiology linked to LRRK2. PMID:26355680

  18. Past Strategies and Future Directions for Identifying AMP-Activated Protein Kinase (AMPK) Modulators

    PubMed Central

    Sinnett, Sarah E.; Brenman, Jay E.

    2014-01-01

    AMP-activated protein kinase (AMPK) is a promising therapeutic target for cancer, type II diabetes, and other illnesses characterized by abnormal energy utilization. During the last decade, numerous labs have published a range of methods for identifying novel AMPK modulators. The current understanding of AMPK structure and regulation, however, has propelled a paradigm shift in which many researchers now consider ADP to be an additional regulatory nucleotide of AMPK. How can the AMPK community apply this new understanding of AMPK signaling to translational research? Recent insights into AMPK structure, regulation, and holoenzyme-sensitive signaling may provide the hindsight needed to clearly evaluate the strengths and weaknesses of past AMPK drug discovery efforts. Improving future strategies for AMPK drug discovery will require pairing the current understanding of AMPK signaling with improved experimental designs. PMID:24583089

  19. Protein kinase A-dependent phosphorylation modulates DNA-binding activity of hepatocyte nuclear factor 4.

    PubMed

    Viollet, B; Kahn, A; Raymondjean, M

    1997-08-01

    Hepatocyte nuclear factor 4 (HNF4), a liver-enriched transcription factor of the nuclear receptor superfamily, is critical for development and liver-specific gene expression. Here, we demonstrate that its DNA-binding activity is modulated posttranslationally by phosphorylation in vivo, ex vivo, and in vitro. In vivo, HNF4 DNA-binding activity is reduced by fasting and by inducers of intracellular cyclic AMP (cAMP) accumulation. A consensus protein kinase A (PKA) phosphorylation site located within the A box of its DNA-binding domain has been identified, and its role in phosphorylation-dependent inhibition of HNF4 DNA-binding activity has been investigated. Mutants of HNF4 in which two potentially phosphorylatable serines have been replaced by either neutral or charged amino acids were able to bind DNA in vitro with affinity similar to that of the wild-type protein. However, phosphorylation by PKA strongly repressed the binding affinity of the wild-type factor but not that of HNF4 mutants. Accordingly, in transfection assays, expression vectors for the mutated HNF4 proteins activated transcription more efficiently than that for the wild-type protein-when cotransfected with the PKA catalytic subunit expression vector. Therefore, HNF4 is a direct target of PKA which might be involved in the transcriptional inhibition of liver genes by cAMP inducers. PMID:9234678

  20. Protein kinase A-dependent phosphorylation modulates DNA-binding activity of hepatocyte nuclear factor 4.

    PubMed Central

    Viollet, B; Kahn, A; Raymondjean, M

    1997-01-01

    Hepatocyte nuclear factor 4 (HNF4), a liver-enriched transcription factor of the nuclear receptor superfamily, is critical for development and liver-specific gene expression. Here, we demonstrate that its DNA-binding activity is modulated posttranslationally by phosphorylation in vivo, ex vivo, and in vitro. In vivo, HNF4 DNA-binding activity is reduced by fasting and by inducers of intracellular cyclic AMP (cAMP) accumulation. A consensus protein kinase A (PKA) phosphorylation site located within the A box of its DNA-binding domain has been identified, and its role in phosphorylation-dependent inhibition of HNF4 DNA-binding activity has been investigated. Mutants of HNF4 in which two potentially phosphorylatable serines have been replaced by either neutral or charged amino acids were able to bind DNA in vitro with affinity similar to that of the wild-type protein. However, phosphorylation by PKA strongly repressed the binding affinity of the wild-type factor but not that of HNF4 mutants. Accordingly, in transfection assays, expression vectors for the mutated HNF4 proteins activated transcription more efficiently than that for the wild-type protein-when cotransfected with the PKA catalytic subunit expression vector. Therefore, HNF4 is a direct target of PKA which might be involved in the transcriptional inhibition of liver genes by cAMP inducers. PMID:9234678

  1. Coronin 1 Regulates Cognition and Behavior through Modulation of cAMP/Protein Kinase A Signaling

    PubMed Central

    Zhang, Chun-Lei; Moshous, Despina; Studer, Vera; Schneider, Jacques; Genoud, Christel; Fossoud, Catherine; Gambino, Frédéric; Khelfaoui, Malik; Müller, Christian; Bartholdi, Deborah; Rossez, Helene; Stiess, Michael; Houbaert, Xander; Jaussi, Rolf; Frey, Daniel; Kammerer, Richard A.; Deupi, Xavier; de Villartay, Jean-Pierre; Lüthi, Andreas; Humeau, Yann; Pieters, Jean

    2014-01-01

    Cognitive and behavioral disorders are thought to be a result of neuronal dysfunction, but the underlying molecular defects remain largely unknown. An important signaling pathway involved in the regulation of neuronal function is the cyclic AMP/Protein kinase A pathway. We here show an essential role for coronin 1, which is encoded in a genomic region associated with neurobehavioral dysfunction, in the modulation of cyclic AMP/PKA signaling. We found that coronin 1 is specifically expressed in excitatory but not inhibitory neurons and that coronin 1 deficiency results in loss of excitatory synapses and severe neurobehavioral disabilities, including reduced anxiety, social deficits, increased aggression, and learning defects. Electrophysiological analysis of excitatory synaptic transmission in amygdala revealed that coronin 1 was essential for cyclic–AMP–protein kinase A–dependent presynaptic plasticity. We further show that upon cell surface stimulation, coronin 1 interacted with the G protein subtype Gαs to stimulate the cAMP/PKA pathway. The absence of coronin 1 or expression of coronin 1 mutants unable to interact with Gαs resulted in a marked reduction in cAMP signaling. Strikingly, synaptic plasticity and behavioral defects of coronin 1–deficient mice were restored by in vivo infusion of a membrane-permeable cAMP analogue. Together these results identify coronin 1 as being important for cognition and behavior through its activity in promoting cAMP/PKA-dependent synaptic plasticity and may open novel avenues for the dissection of signal transduction pathways involved in neurobehavioral processes. PMID:24667537

  2. Melatonin decreases breast cancer metastasis by modulating Rho-associated kinase protein-1 expression

    PubMed Central

    Borin, Thaiz Ferraz; Arbab, Ali Syed; Gelaleti, Gabriela Bottaro; Ferreira, Lívia Carvalho; Moschetta, Marina Gobbe; Jardim-Perassi, Bruna Victorasso; Iskander, ASM; Varma, Nadimpalli Ravi S.; Shankar, Adarsh; Coimbra, Verena Benedick; Fabri, Vanessa Alves; de Oliveira, Juliana Garcia; de Campos Zuccari, Debora Aparecida Pires

    2016-01-01

    The occurrence of metastasis, an important breast cancer prognostic factor, depends on cell migration/invasion mechanisms, which can be controlled by regulatory and effector molecules such as Rho-associated kinase protein (ROCK-1). Increased expression of this protein promotes tumor growth and metastasis, which can be restricted by ROCK-1 inhibitors. Melatonin has shown oncostatic, antimetastatic, and anti-angiogenic effects and can modulate ROCK-1 expression. Metastatic and nonmetastatic breast cancer cell lines were treated with melatonin as well as with specific ROCK-1 inhibitor (Y27632). Cell viability, cell migration/invasion, and ROCK-1 gene expression and protein expression were determined in vitro. In vivo lung metastasis study was performed using female athymic nude mice treated with either melatonin or Y27832 for 2 and 5 wk. The metastases were evaluated by X-ray computed tomography and single photon emission computed tomography (SPECT) and by immunohistochemistry for ROCK-1 and cytokeratin proteins. Melatonin and Y27632 treatments reduced cell viability and invasion/migration of both cell lines and decreased ROCK-1 gene expression in metastatic cells and protein expression in nonmetastatic cell line. The numbers of ‘hot’ spots (lung metastasis) identified by SPECT images were significantly lower in treated groups. ROCK-1 protein expression also was decreased in metastatic foci of treated groups. Melatonin has shown to be effective in controlling metastatic breast cancer in vitro and in vivo, not only via inhibition of the proliferation of tumor cells but also through direct antagonism of metastatic mechanism of cells rendered by ROCK-1 inhibition. When Y27632 was used, the effects were similar to those found with melatonin treatment. PMID:26292662

  3. Zonula occludens toxin modulates tight junctions through protein kinase C-dependent actin reorganization, in vitro.

    PubMed Central

    Fasano, A; Fiorentini, C; Donelli, G; Uzzau, S; Kaper, J B; Margaretten, K; Ding, X; Guandalini, S; Comstock, L; Goldblum, S E

    1995-01-01

    The intracellular signaling involved in the mechanism of action of zonula occludens toxin (ZOT) was studied using several in vitro and ex vivo models. ZOT showed a selective effect among various cell lines tested, suggesting that it may interact with a specific receptor, whose surface expression on various cells differs. When tested in IEC6 cell monolayers, ZOT-containing supernatants induced a redistribution of the F-actin cytoskeleton. Similar results were obtained with rabbit ileal mucosa, where the reorganization of F-actin paralleled the increase in tissue permeability. In endothelial cells, the cytoskeletal rearrangement involved a decrease of the soluble G-actin pool (-27%) and a reciprocal increase in the filamentous F-actin pool (+22%). This actin polymerization was time- and dose-dependent, and was reversible. Pretreatment with a specific protein kinase C inhibitor, CGP41251, completely abolished the ZOT effects on both tissue permeability and actin polymerization. In IEC6 cells ZOT induced a peak increment of the PKC-alpha isoform after 3 min incubation. Taken together, these results suggest that ZOT activates a complex intracellular cascade of events that regulate tight junction permeability, probably mimicking the effect of physiologic modulator(s) of epithelial barrier function. Images PMID:7635964

  4. Protein kinase A modulation of CaV1.4 calcium channels.

    PubMed

    Sang, Lingjie; Dick, Ivy E; Yue, David T

    2016-01-01

    The regulation of L-type Ca(2+) channels by protein kinase A (PKA) represents a crucial element within cardiac, skeletal muscle and neurological systems. Although much work has been done to understand this regulation in cardiac CaV1.2 Ca(2+) channels, relatively little is known about the closely related CaV1.4 L-type Ca(2+) channels, which feature prominently in the visual system. Here we find that CaV1.4 channels are indeed modulated by PKA phosphorylation within the inhibitor of Ca(2+)-dependent inactivation (ICDI) motif. Phosphorylation of this region promotes the occupancy of calmodulin on the channel, thus increasing channel open probability (PO) and Ca(2+)-dependent inactivation. Although this interaction seems specific to CaV1.4 channels, introduction of ICDI1.4 to CaV1.3 or CaV1.2 channels endows these channels with a form of PKA modulation, previously unobserved in heterologous systems. Thus, this mechanism may not only play an important role in the visual system but may be generalizable across the L-type channel family. PMID:27456671

  5. Protein kinase A modulation of CaV1.4 calcium channels

    NASA Astrophysics Data System (ADS)

    Sang, Lingjie; Dick, Ivy E.; Yue, David T.

    2016-07-01

    The regulation of L-type Ca2+ channels by protein kinase A (PKA) represents a crucial element within cardiac, skeletal muscle and neurological systems. Although much work has been done to understand this regulation in cardiac CaV1.2 Ca2+ channels, relatively little is known about the closely related CaV1.4 L-type Ca2+ channels, which feature prominently in the visual system. Here we find that CaV1.4 channels are indeed modulated by PKA phosphorylation within the inhibitor of Ca2+-dependent inactivation (ICDI) motif. Phosphorylation of this region promotes the occupancy of calmodulin on the channel, thus increasing channel open probability (PO) and Ca2+-dependent inactivation. Although this interaction seems specific to CaV1.4 channels, introduction of ICDI1.4 to CaV1.3 or CaV1.2 channels endows these channels with a form of PKA modulation, previously unobserved in heterologous systems. Thus, this mechanism may not only play an important role in the visual system but may be generalizable across the L-type channel family.

  6. Protein kinase A modulation of CaV1.4 calcium channels

    PubMed Central

    Sang, Lingjie; Dick, Ivy E.; Yue, David T.

    2016-01-01

    The regulation of L-type Ca2+ channels by protein kinase A (PKA) represents a crucial element within cardiac, skeletal muscle and neurological systems. Although much work has been done to understand this regulation in cardiac CaV1.2 Ca2+ channels, relatively little is known about the closely related CaV1.4 L-type Ca2+ channels, which feature prominently in the visual system. Here we find that CaV1.4 channels are indeed modulated by PKA phosphorylation within the inhibitor of Ca2+-dependent inactivation (ICDI) motif. Phosphorylation of this region promotes the occupancy of calmodulin on the channel, thus increasing channel open probability (PO) and Ca2+-dependent inactivation. Although this interaction seems specific to CaV1.4 channels, introduction of ICDI1.4 to CaV1.3 or CaV1.2 channels endows these channels with a form of PKA modulation, previously unobserved in heterologous systems. Thus, this mechanism may not only play an important role in the visual system but may be generalizable across the L-type channel family. PMID:27456671

  7. N-Terminal Mutations Modulate Yeast Snf1 Protein Kinase Function

    PubMed Central

    Estruch, F.; Treitel, M. A.; Yang, X.; Carlson, M.

    1992-01-01

    The SNF1 protein kinase is required for expression of glucose-repressed genes in response to glucose deprivation. The SNF4 protein is physically associated with SNF1 and positively affects the kinase activity. We report here the characterization of a dominant mutation, SNF1-G53R, that was isolated as a suppressor of the requirement for SNF4. The mutant SNF1-G53R protein is still responsive to SNF4 but has greatly elevated kinase activity in immune complex assays; in contrast, the activity is wild type in a protein blot assay. Deletion of the region N-terminal to the kinase domain (codons 5-52) reduces kinase activity in vitro, but the mutant SNF1-ΔN kinase is still dependent on SNF4. The N terminus is not required for the regulatory response to glucose. In gel filtration chromatography, the SNF1, SNF1-G53R and SNF1-ΔN proteins showed different elution profiles, consistent with differential formation of high molecular weight complexes. Taken together, the results suggest that the N terminus positively affects the function of the SNF1 kinase and may be involved in interaction with a positive effector other than SNF4. We also showed that the conserved threonine residue 210 in subdomain VIII, which is a phosphorylation site in other kinases, is essential for SNF1 activity. Finally, we present evidence that when the C terminus is deleted, overexpression of the SNF1 kinase domain is deleterious to the cell. PMID:1468623

  8. Protein kinase CK2 phosphorylates Hsp105 alpha at Ser509 and modulates its function.

    PubMed Central

    Ishihara, Keiichi; Yamagishi, Nobuyuki; Hatayama, Takumi

    2003-01-01

    The 105 kDa heat-shock protein (Hsp) Hsp105 alpha is a mammalian stress protein that belongs to the HSP105/HSP110 family. We have shown previously that Hsp105 alpha exists as non-phosphorylated and phosphorylated forms in vivo, and is phosphorylated by protein kinase CK2 (CK2) in vitro. In this study, to elucidate the role of phosphorylation of Hsp105 alpha, we first analysed the site of phosphorylation of Hsp105 alpha by CK2. Peptide mapping analysis of Hsp105 alpha phosphorylated by CK2 and in vitro phosphorylation experiments using various deletion and substitution mutants of Hsp105 alpha revealed that Hsp105 alpha is phosphorylated at Ser(509) in the beta-sheet domain. Furthermore, Ser(509) in Hsp105 alpha was also phosphorylated in mammalian COS-7 cells, although other sites were phosphorylated as well. Next, we examined the effects of phosphorylation of Hsp105 alpha on its functions using CK2-phosphorylated Hsp105 alpha. Interestingly, Hsp105 alpha suppressed 70 kDa heat-shock cognate protein (Hsc70)-mediated protein folding, whereas the phosphorylation of Hsp105 alpha at Ser(509) abolished the inhibitory activity of Hsp105 alpha in vitro. In accordance with these findings, wild-type Hsp105 alpha, which was thought to be phosphorylated in vivo, had no effect on Hsp70-mediated refolding of heat-denatured luciferase, whereas a non-phosphorylatable mutant of Hsp105 alpha suppressed the Hsp70-mediated refolding of heat-denatured luciferase in mammalian cells. Thus it was suggested that CK2 phosphorylates Hsp105 alpha at Ser(509) and modulates the function of Hsp105 alpha. The regulation of Hsp105 alpha function by phosphorylation may play an important role in a variety of cellular events. PMID:12558502

  9. Bile acids modulate the Golgi membrane fission process via a protein kinase Ceta and protein kinase D-dependent pathway in colonic epithelial cells.

    PubMed

    Byrne, Anne-Marie; Foran, Eilis; Sharma, Ruchika; Davies, Anthony; Mahon, Ciara; O'Sullivan, Jacintha; O'Donoghue, Diarmuid; Kelleher, Dermot; Long, Aideen

    2010-04-01

    Deoxycholic acid (DCA) is a secondary bile acid that modulates signalling pathways in epithelial cells. DCA has been implicated in pathogenesis of colon carcinoma, particularly by activation of the protein kinase C (PKC) pathway. Ursodeoxycholic acid (UDCA), a tertiary bile acid, has been observed to have chemopreventive effects. The aim of this study was to investigate the effect of DCA and UDCA on the subcellular localization and activity of PKCeta and its downstream effects on Golgi structure in a colon cancer cell model. PKCeta expression was localized to the Golgi in HCT116 colon cancer cells. DCA induced fragmentation of the Golgi in these cells following activation of PKCeta and its downstream effector protein kinase D (PKD). Pretreatment of cells with UDCA or a glucocorticoid, dexamethasone, inhibited DCA-induced PKCeta/PKD activation and Golgi fragmentation. Knockdown of glucocorticoid receptor (GR) expression using small interfering RNA or inhibition using the GR antagonist mifepristone attenuated the inhibitory effect of UDCA on Golgi fragmentation. Elevated serum and faecal levels of DCA have been previously reported in patients with ulcerative colitis (UC) and colon cancer. Analysis of Golgi architecture in vivo using tissue microarrays revealed Golgi fragmentation in UC and colorectal cancer tissue. We have demonstrated that DCA can disrupt the structure of the Golgi, an organelle critical for normal cell function. Inhibition of this DCA-induced Golgi fragmentation by UDCA was mediated via the GR. This represents a potential mechanism of observed chemopreventive effects of UDCA in benign and malignant disease of the colon. PMID:20093383

  10. A cluster of protein kinases and phosphatases modulated in fetal Down syndrome (trisomy 21) brain.

    PubMed

    Weitzdoerfer, Rachel; Toran, Nuria; Subramaniyan, Saraswathi; Pollak, Arnold; Dierssen, Mara; Lubec, Gert

    2015-06-01

    Down syndrome (DS; trisomy 21) is the most frequent cause of mental retardation with major cognitive and behavioral deficits. Although a series of aberrant biochemical pathways has been reported, work on signaling proteins is limited. It was, therefore, the aim of the study to test a selection of protein kinases and phosphatases known to be essential for memory and learning mechanisms in fetal DS brain. 12 frontal cortices from DS brain were compared to 12 frontal cortices from controls obtained at legal abortions. Proteins were extracted from brains and western blotting with specific antibodies was carried out. Primary results were used for networking (IntAct Molecular Interaction Database) and individual predicted pathway components were subsequently quantified by western blotting. Levels of calcium-calmodulin kinase II alpha, transforming growth factor beta-activated kinase 1 as well as phosphatase and tensin homolog (PTEN) were reduced in cortex of DS subjects and network generation pointed to interaction between PTEN and the dendritic spine protein drebrin that was subsequently determined and reduced levels were observed. The findings of reduced levels of cognitive-function-related protein kinases and the phosphatase may be relevant for interpretation of previous work and may be useful for the design of future studies on signaling in DS brain. Moreover, decreased drebrin levels may point to dendritic spine abnormalities. PMID:25740605

  11. 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

  12. Protein kinase C modulates aryl hydrocarbon receptor nuclear translocator protein-mediated transactivation potential in a dimer context.

    PubMed

    Long, W P; Chen, X; Perdew, G H

    1999-04-30

    Protein kinase C (PKC)- and protein kinase A (PKA)-mediated modulation of the transactivation potential of human aryl hydrocarbon receptor nuclear translocator (hARNT), a basic helix-loop-helix (bHLH)-PAS transcription factor, and the bHLH-ZIP transcription factors USF-1 (for upstream regulatory factor 1) and c-Myc were examined. An 81 nM dose of the PKC activator phorbol-12-myristate-13-acetate (PMA), shown here to specifically activate PKC in COS-1 cells, or a 1 nM dose of the PKA activator 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP) results in 2. 6- and 1.9-fold enhancements, respectively, in hARNT-mediated transactivation of the class B, E-box-driven reporter pMyc3E1bLuc relative to identically transfected, carrier solvent-treated COS-1 cells. In contrast, 81 nM PMA and 1 nM 8-Br-cAMP did not enhance transactivation of pMyc3E1bLuc-driven by USF-1 and c-Myc expression relative to identically transfected, carrier-treated COS-1 cells. Co-transfection of pcDNA3/ARNT-474-Flag, expressing a hARNT carboxyl-terminal transactivation domain deletion, and pMyc3E1bLuc does not result in induction of reporter activity, suggesting PMA's effects do not involve formation of unknown hARNT-protein heterodimers. Additionally, PMA had no effect on hARNT expression relative to Me2SO-treated cells. Metabolic 32P labeling of hARNT in cells treated with carrier solvent or 81 nM PMA demonstrates that PMA does not increase the overall phosphorylation level of hARNT. These results demonstrate, for the first time, that the transactivation potential of ARNT in a dimer context can be specifically modulated by PKC or PKA stimulation and that the bHLH-PAS and bHLH-ZIP transcription factors are differentially regulated by these pathways in COS-1 cells. PMID:10212212

  13. 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

  14. Microbiota Modulates Behavior and Protein Kinase C mediated cAMP response element-binding protein Signaling

    PubMed Central

    Zeng, Li; Zeng, Benhua; Wang, Haiyang; Li, Bo; Huo, Ran; Zheng, Peng; Zhang, Xiaotong; Du, Xiangyu; Liu, Meiling; Fang, Zheng; Xu, Xuejiao; Zhou, Chanjuan; Chen, Jianjun; Li, Wenxia; Guo, Jing; Wei, Hong; Xie, Peng

    2016-01-01

    Evolutionary pressure drives gut microbiota–host coevolution and results in complex interactions between gut microbiota and neural development; however, the molecular mechanisms by which the microbiota governs host behavior remain obscure. Here, we report that colonization early in life is crucial for the microbiota to modulate brain development and behavior; later colonization or deletion of microbiota cannot completely reverse the behaviors. Microarray analysis revealed an association between absence of gut microbiota and expression in cAMP responding element-binding protein (CREB) regulated genes in the hippocampus. The absence of gut microbiota from birth was shown to be associated with decreased CREB expression, followed by decreases of protein kinase C beta (PRKCB) and AMPA receptors expression, and an increase of phosphorylation CREB (pCREB) expression. Microbiota colonization in adolescence restored CREB and pCREB expression, but did not alter PRKCB and AMPARs expression. The removal of the gut microbiota from SPF mice using antibiotics only reduced pCREB expression. These findings suggest that (i) colonization of the gut microbiota early in life might facilitate neurodevelopment via PKC–CREB signaling and (ii) although GF mice and ABX mice display reduced anxiety-related behaviors, the molecular mechanisms behind this might differ. PMID:27444685

  15. Microbiota Modulates Behavior and Protein Kinase C mediated cAMP response element-binding protein Signaling.

    PubMed

    Zeng, Li; Zeng, Benhua; Wang, Haiyang; Li, Bo; Huo, Ran; Zheng, Peng; Zhang, Xiaotong; Du, Xiangyu; Liu, Meiling; Fang, Zheng; Xu, Xuejiao; Zhou, Chanjuan; Chen, Jianjun; Li, Wenxia; Guo, Jing; Wei, Hong; Xie, Peng

    2016-01-01

    Evolutionary pressure drives gut microbiota-host coevolution and results in complex interactions between gut microbiota and neural development; however, the molecular mechanisms by which the microbiota governs host behavior remain obscure. Here, we report that colonization early in life is crucial for the microbiota to modulate brain development and behavior; later colonization or deletion of microbiota cannot completely reverse the behaviors. Microarray analysis revealed an association between absence of gut microbiota and expression in cAMP responding element-binding protein (CREB) regulated genes in the hippocampus. The absence of gut microbiota from birth was shown to be associated with decreased CREB expression, followed by decreases of protein kinase C beta (PRKCB) and AMPA receptors expression, and an increase of phosphorylation CREB (pCREB) expression. Microbiota colonization in adolescence restored CREB and pCREB expression, but did not alter PRKCB and AMPARs expression. The removal of the gut microbiota from SPF mice using antibiotics only reduced pCREB expression. These findings suggest that (i) colonization of the gut microbiota early in life might facilitate neurodevelopment via PKC-CREB signaling and (ii) although GF mice and ABX mice display reduced anxiety-related behaviors, the molecular mechanisms behind this might differ. PMID:27444685

  16. Acceleration of crossbridge kinetics by protein kinase A phosphorylation of cardiac myosin binding protein C modulates cardiac function.

    PubMed

    Tong, Carl W; Stelzer, Julian E; Greaser, Marion L; Powers, Patricia A; Moss, Richard L

    2008-10-24

    Normal cardiac function requires dynamic modulation of contraction. beta1-adrenergic-induced protein kinase (PK)A phosphorylation of cardiac myosin binding protein (cMyBP)-C may regulate crossbridge kinetics to modulate contraction. We tested this idea with mechanical measurements and echocardiography in a mouse model lacking 3 PKA sites on cMyBP-C, ie, cMyBP-C(t3SA). We developed the model by transgenic expression of mutant cMyBP-C with Ser-to-Ala mutations on the cMyBP-C knockout background. Western blots, immunofluorescence, and in vitro phosphorylation combined to show that non-PKA-phosphorylatable cMyBP-C expressed at 74% compared to normal wild-type (WT) and was correctly positioned in the sarcomeres. Similar expression of WT cMyBP-C at 72% served as control, ie, cMyBP-C(tWT). Skinned myocardium responded to stretch with an immediate increase in force, followed by a transient relaxation of force and finally a delayed development of force, ie, stretch activation. The rate constants of relaxation, k(rel) (s-1), and delayed force development, k(df) (s-1), in the stretch activation response are indicators of crossbridge cycling kinetics. cMyBP-C(t3SA) myocardium had baseline k(rel) and k(df) similar to WT myocardium, but, unlike WT, k(rel) and k(df) were not accelerated by PKA treatment. Reduced dobutamine augmentation of systolic function in cMyBP-C(t3SA) hearts during echocardiography corroborated the stretch activation findings. Furthermore, cMyBP-C(t3SA) hearts exhibited basal echocardiographic findings of systolic dysfunction, diastolic dysfunction, and hypertrophy. Conversely, cMyBP-C(tWT) hearts performed similar to WT. Thus, PKA phosphorylation of cMyBP-C accelerates crossbridge kinetics and loss of this regulation leads to cardiac dysfunction. PMID:18802026

  17. The Atlantic salmon protein tyrosine kinase Tyk2: molecular cloning, modulation of expression and function.

    PubMed

    Sobhkhez, Mehrdad; Hansen, Tom; Iliev, Dimitar B; Skjesol, Astrid; Jørgensen, Jorunn B

    2013-12-01

    Tyk2, a member of the Janus Kinase (JAK) family of protein tyrosine kinases, is required for interferon-α/β binding and signaling in higher vertebrates. Currently, little is known about the role of the different JAKs in signaling responses to interferon (IFN) in lower vertebrates including fish. In this paper we report the identification and characterization of Atlantic salmon (Salmo salar) Tyk2. Four cDNA sequences, two containing an open reading frame encoding full-length Tyk protein and two with an up-stream in frame stop codon, were identified. The deduced amino acid sequences of the salmon full-length Tyk2 proteins showed highest identity with Tyk2 from other species and their transcripts were ubiquitously expressed. Like in mammals the presented data suggests that salmon Tyk2 is auto-phosporylated when ectopically expressed in cells. In our experiments, full-length salmon Tyk2 overexpressed in CHSE-cells phosphorylated itself, while both a kinase-deficient mutant and the truncated Tyk2 (Tyk-short) were inactive. Interestingly, the overexpression of full length Tyk2 was shown to up-regulate the transcript levels of the IFN induced gene Mx, thus indicating the involvement of salmon Tyk2 in the salmon IFN I pathway. PMID:23872231

  18. AMP-activated Protein Kinase Signaling Activation by Resveratrol Modulates Amyloid-β Peptide Metabolism*

    PubMed Central

    Vingtdeux, Valérie; Giliberto, Luca; Zhao, Haitian; Chandakkar, Pallavi; Wu, Qingli; Simon, James E.; Janle, Elsa M.; Lobo, Jessica; Ferruzzi, Mario G.; Davies, Peter; Marambaud, Philippe

    2010-01-01

    Alzheimer disease is an age-related neurodegenerative disorder characterized by amyloid-β (Aβ) peptide deposition into cerebral amyloid plaques. The natural polyphenol resveratrol promotes anti-aging pathways via the activation of several metabolic sensors, including the AMP-activated protein kinase (AMPK). Resveratrol also lowers Aβ levels in cell lines; however, the underlying mechanism responsible for this effect is largely unknown. Moreover, the bioavailability of resveratrol in the brain remains uncertain. Here we show that AMPK signaling controls Aβ metabolism and mediates the anti-amyloidogenic effect of resveratrol in non-neuronal and neuronal cells, including in mouse primary neurons. Resveratrol increased cytosolic calcium levels and promoted AMPK activation by the calcium/calmodulin-dependent protein kinase kinase-β. Direct pharmacological and genetic activation of AMPK lowered extracellular Aβ accumulation, whereas AMPK inhibition reduced the effect of resveratrol on Aβ levels. Furthermore, resveratrol inhibited the AMPK target mTOR (mammalian target of rapamycin) to trigger autophagy and lysosomal degradation of Aβ. Finally, orally administered resveratrol in mice was detected in the brain where it activated AMPK and reduced cerebral Aβ levels and deposition in the cortex. These data suggest that resveratrol and pharmacological activation of AMPK have therapeutic potential against Alzheimer disease. PMID:20080969

  19. Selective Modulation of Protein Kinase C α over Protein Kinase C ε by Curcumin and Its Derivatives in CHO-K1 Cells.

    PubMed

    Pany, Satyabrata; Majhi, Anjoy; Das, Joydip

    2016-04-12

    Members of the protein kinase C (PKC) family of serine/threonine kinases regulate various cellular functions, including cell growth, differentiation, metabolism, and apoptosis. Modulation of isoform-selective activity of PKC by curcumin (1), the active constituent of Curcuma L., is poorly understood, and the literature data are inconsistent and obscure. The effect of curcumin (1) and its analogues, 4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl oleate (2), (9Z,12Z)-4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl octadeca-9,12-dienoate (3), (9Z,12Z,15Z)-4-[(2Z,6E)-3-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-5-oxohepta-2,6-dien-1-yl]-2-methoxyphenyl octadeca-9,12,15-trienoate (4), and (1E,6E)-1-[4-(hexadecyloxy)-3-methoxyphenyl]-7-(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione (5), and didemethylcurcumin (6) on the membrane translocation of PKCα, a conventional PKC, and PKCε, a novel PKC, has been studied in CHO-K1 cells, in which these PKC isoforms are endogenously expressed. Translocation of PKC from the cytosol to the membrane was measured using immunoblotting and confocal microscopy. 1 and 6 inhibited the TPA-induced membrane translocation of PKCα but not of PKCε. Modification of the hydroxyl group of curcumin with a long aliphatic chain containing unsaturated double bonds in 2-4 completely abolished this inhibition property. Instead, 2-4 showed significant translocation of PKCα but not of PKCε to the membrane. No membrane translocation was observed with 1, 6, or the analogue 5 having a saturated long chain for either PKCα or PKCε. 1 and 6 inhibited TPA-induced activation of ERK1/2, and 2-4 activated it. ERK1/2 is the downstream readout of PKC. These results show that the hydroxyl group of curcumin is important for PKC activity and the curcumin template can be useful in developing isoform specific PKC modulators for regulating a particular disease state. PMID

  20. AMP-Activated Protein Kinase and Glycogen Synthase KinaseModulate the Severity of Sepsis-Induced Lung Injury

    PubMed Central

    Liu, Zhongyu; Bone, Nathaniel; Jiang, Shaoning; Park, Dae Won; Tadie, Jean-Marc; Deshane, Jessy; Rodriguez, Cilina Ann; Pittet, Jean-Francois; Abraham, Edward; Zmijewski, Jaroslaw W

    2015-01-01

    Alterations in metabolic and bioenergetic homeostasis contribute to sepsis-mediated organ injury. However, how AMP-activated protein kinase (AMPK), a major sensor and regulator of energy expenditure and production, affects development of organ injury and loss of innate capacity during polymicrobial sepsis remains unclear. In the present experiments, we found that cross-talk between the AMPK and GSK3β signaling pathways controls chemotaxis and the ability of neutrophils and macrophages to kill bacteria ex vivo. In mice with polymicrobial abdominal sepsis or more severe sepsis induced by the combination of hemorrhage and intraabdominal infection, administration of the AMPK activator metformin or the GSK3β inhibitor SB216763 reduced the severity of acute lung injury (ALI). Improved survival in metformin-treated septic mice was correlated with preservation of mitochondrial complex V (ATP synthase) function and increased amounts of ETC complex III and IV. Although immunosuppression is a consequence of sepsis, metformin effectively increased innate immune capacity to eradicate P. aeruginosa in the lungs of septic mice. We also found that AMPK activation diminished accumulation of the immunosuppressive transcriptional factor HIF-1α as well as the development of endotoxin tolerance in LPS-treated macrophages. Furthermore, AMPK-dependent preservation of mitochondrial membrane potential also prevented LPS-mediated dysfunction of neutrophil chemotaxis. These results indicate that AMPK activation reduces the severity of polymicrobial sepsis-induced lung injury and prevents the development of sepsis-associated immunosuppression. PMID:26650187

  1. Protein interaction module-assisted function X (PIMAX) approach to producing challenging proteins including hyperphosphorylated tau and active CDK5/p25 kinase complex.

    PubMed

    Sui, Dexin; Xu, Xinjing; Ye, Xuemei; Liu, Mengyu; Mianecki, Maxwell; Rattanasinchai, Chotirat; Buehl, Christopher; Deng, Xiexiong; Kuo, Min-Hao

    2015-01-01

    Many biomedically critical proteins are underrepresented in proteomics and biochemical studies because of the difficulty of their production in Escherichia coli. These proteins might possess posttranslational modifications vital to their functions, tend to misfold and be partitioned into bacterial inclusion bodies, or act only in a stoichiometric dimeric complex. Successful production of these proteins requires efficient interaction between these proteins and a specific "facilitator," such as a protein-modifying enzyme, a molecular chaperone, or a natural physical partner within the dimeric complex. Here we report the design and application of a protein interaction module-assisted function X (PIMAX) system that effectively overcomes these hurdles. By fusing two proteins of interest to a pair of well-studied protein-protein interaction modules, we were able to potentiate the association of these two proteins, resulting in successful production of an enzymatically active cyclin-dependent kinase complex and hyperphosphorylated tau protein, which is intimately linked to Alzheimer disease. Furthermore, using tau isoforms quantitatively phosphorylated by GSK-3β and CDK5 kinases via PIMAX, we demonstrated the hyperphosphorylation-stimulated tau oligomerization in vitro, paving the way for new Alzheimer disease drug discoveries. Vectors for PIMAX can be easily modified to meet the needs of different applications. This approach thus provides a convenient and modular suite with broad implications for proteomics and biomedical research. PMID:25385071

  2. AMP-activated protein kinase modulates tau phosphorylation and tau pathology in vivo.

    PubMed

    Domise, Manon; Didier, Sébastien; Marinangeli, Claudia; Zhao, Haitian; Chandakkar, Pallavi; Buée, Luc; Viollet, Benoit; Davies, Peter; Marambaud, Philippe; Vingtdeux, Valérie

    2016-01-01

    Neurofibrillary tangles (NFTs) are the pathological hallmark of neurodegenerative diseases commonly known as tauopathies. NFTs result from the intracellular aggregation of abnormally and hyperphosphorylated tau proteins. Tau functions, which include the regulation of microtubules dynamics, are dependent on its phosphorylation status. As a consequence, any changes in tau phosphorylation can have major impacts on synaptic plasticity and memory. Recently, it has been demonstrated that AMP-activated protein kinase (AMPK) was deregulated in the brain of Alzheimer's disease (AD) patients where it co-localized with phosphorylated tau in pre-tangle and tangle-bearing neurons. Besides, it was found that AMPK was a tau kinase in vitro. Here, we find that endogenous AMPK activation in mouse primary neurons induced an increase of tau phosphorylation at multiple sites, whereas AMPK inhibition led to a rapid decrease of tau phosphorylation. We further show that AMPK mice deficient for one of the catalytic alpha subunits displayed reduced endogenous tau phosphorylation. Finally, we found that AMPK deficiency reduced tau pathology in the PS19 mouse model of tauopathy. These results show that AMPK regulates tau phosphorylation in mouse primary neurons as well as in vivo, and thus suggest that AMPK could be a key player in the development of AD pathology. PMID:27230293

  3. AMP-activated protein kinase modulates tau phosphorylation and tau pathology in vivo

    PubMed Central

    Domise, Manon; Didier, Sébastien; Marinangeli, Claudia; Zhao, Haitian; Chandakkar, Pallavi; Buée, Luc; Viollet, Benoit; Davies, Peter; Marambaud, Philippe; Vingtdeux, Valérie

    2016-01-01

    Neurofibrillary tangles (NFTs) are the pathological hallmark of neurodegenerative diseases commonly known as tauopathies. NFTs result from the intracellular aggregation of abnormally and hyperphosphorylated tau proteins. Tau functions, which include the regulation of microtubules dynamics, are dependent on its phosphorylation status. As a consequence, any changes in tau phosphorylation can have major impacts on synaptic plasticity and memory. Recently, it has been demonstrated that AMP-activated protein kinase (AMPK) was deregulated in the brain of Alzheimer’s disease (AD) patients where it co-localized with phosphorylated tau in pre-tangle and tangle-bearing neurons. Besides, it was found that AMPK was a tau kinase in vitro. Here, we find that endogenous AMPK activation in mouse primary neurons induced an increase of tau phosphorylation at multiple sites, whereas AMPK inhibition led to a rapid decrease of tau phosphorylation. We further show that AMPK mice deficient for one of the catalytic alpha subunits displayed reduced endogenous tau phosphorylation. Finally, we found that AMPK deficiency reduced tau pathology in the PS19 mouse model of tauopathy. These results show that AMPK regulates tau phosphorylation in mouse primary neurons as well as in vivo, and thus suggest that AMPK could be a key player in the development of AD pathology. PMID:27230293

  4. A calcium sensor - protein kinase signaling module diversified in plants and is retained in all lineages of Bikonta species.

    PubMed

    Beckmann, Linda; Edel, Kai H; Batistič, Oliver; Kudla, Jörg

    2016-01-01

    Calcium (Ca(2+)) signaling is a universal mechanism of signal transduction and involves Ca(2+) signal formation and decoding of information by Ca(2+) binding proteins. Calcineurin B-like proteins (CBLs), which upon Ca(2+) binding activate CBL-interacting protein kinases (CIPKs) regulate a multitude of physiological processes in plants. Here, we combine phylogenomics and functional analyses to investigate the occurrence and structural conservation of CBL and CIPK proteins in 26 species representing all major clades of eukaryotes. We demonstrate the presence of at least singular CBL-CIPK pairs in representatives of Archaeplastida, Chromalveolates and Excavates and their general absence in Opisthokonta and Amoebozoa. This denotes CBL-CIPK complexes as evolutionary ancient Ca(2+) signaling modules that likely evolved in the ancestor of all Bikonta. Furthermore, we functionally characterize the CBLs and CIPK from the parabasalid human pathogen Trichomonas vaginalis. Our results reveal strict evolutionary conservation of functionally important structural features, preservation of biochemical properties and a remarkable cross-kingdom protein-protein interaction potential between CBLs and CIPKs from Arabidopsis thaliana and T. vaginalis. Together our findings suggest an ancient evolutionary origin of a functional CBL-CIPK signaling module close to the root of eukaryotic evolution and provide insights into the initial evolution of signaling networks and Ca(2+) signaling specificity. PMID:27538881

  5. A calcium sensor – protein kinase signaling module diversified in plants and is retained in all lineages of Bikonta species

    PubMed Central

    Beckmann, Linda; Edel, Kai H.; Batistič, Oliver; Kudla, Jörg

    2016-01-01

    Calcium (Ca2+) signaling is a universal mechanism of signal transduction and involves Ca2+ signal formation and decoding of information by Ca2+ binding proteins. Calcineurin B-like proteins (CBLs), which upon Ca2+ binding activate CBL-interacting protein kinases (CIPKs) regulate a multitude of physiological processes in plants. Here, we combine phylogenomics and functional analyses to investigate the occurrence and structural conservation of CBL and CIPK proteins in 26 species representing all major clades of eukaryotes. We demonstrate the presence of at least singular CBL-CIPK pairs in representatives of Archaeplastida, Chromalveolates and Excavates and their general absence in Opisthokonta and Amoebozoa. This denotes CBL-CIPK complexes as evolutionary ancient Ca2+ signaling modules that likely evolved in the ancestor of all Bikonta. Furthermore, we functionally characterize the CBLs and CIPK from the parabasalid human pathogen Trichomonas vaginalis. Our results reveal strict evolutionary conservation of functionally important structural features, preservation of biochemical properties and a remarkable cross-kingdom protein-protein interaction potential between CBLs and CIPKs from Arabidopsis thaliana and T. vaginalis. Together our findings suggest an ancient evolutionary origin of a functional CBL-CIPK signaling module close to the root of eukaryotic evolution and provide insights into the initial evolution of signaling networks and Ca2+ signaling specificity. PMID:27538881

  6. Cellular Casein Kinase 2 and Protein Phosphatase 2A Modulate Replication Site Assembly of Bluetongue Virus*

    PubMed Central

    Mohl, Bjorn-Patrick; Roy, Polly

    2016-01-01

    A number of cytoplasmic replicating viruses produce cytoplasmic inclusion bodies or protein aggregates; however, a hallmark of viruses of the Reoviridae family is that they utilize these sites for purposes of replication and capsid assembly, functioning as viral assembly factories. Here we have used bluetongue virus (BTV) as a model system for this broad family of important viruses to understand the mechanisms regulating inclusion body assembly. Newly synthesized viral proteins interact with sequestered viral RNA molecules prior to capsid assembly and double-stranded RNA synthesis within viral inclusion bodies (VIBs). VIBs are predominantly comprised of a BTV-encoded non-structural protein 2 (NS2). Previous in vitro studies indicated that casein kinase 2 (CK2) mediated the phosphorylation of NS2, which regulated the propensity of NS2 to form larger aggregates. Using targeted pharmacological reagents, specific mutation in the viral genome by reverse genetics and confocal microscopy, here we demonstrate that CK2 activity is important for BTV replication. Furthermore, we show that a novel host cell factor, protein phosphatase 2A, is involved in NS2 dephosphorylation and that, together with CK2, it regulates VIB morphology and virus replication. Thus, these two host enzymes influence the dynamic nature of VIB assembly/disassembly, and these concerted activities may be relevant to the assembly and the release of these cores from VIBs. PMID:27226558

  7. Involvement of protein kinase C and IP3-mediated Ca2+ release in activity modulation by paraoxon in snail neurons.

    PubMed

    Vatanparast, Jafar; Janahmadi, Mahyar; Asgari, Ali Reza

    2007-10-01

    We have previously reported that paraoxon, an organophosphate compound, at submicromolar concentrations effectively suppresses Ca2+ action potentials and modulates the activity of snail neurons. This effect was unrelated to acetylcholinesterase inhibition but was found to involve the direct or indirect modulation of ion channels [Vatanparast, J., Janahmadi, M., Asgari, A.R., Sepehri, H., Haeri-Rohani, A., 2006a. Paraoxon suppresses Ca2+ action potential and afterhyperpolarization in snail neurons: Relevance to the hyperexcitability induction. Brain Res. 1083 (1), 110-117]. In the present work, the interaction of paraoxon with protein kinase C (PKC) and inositol 1,4,5-trisphosphate (IP3)-mediated Ca2+ release, on the modulation of Ca2+ action potentials and neuronal activity was investigated. Phorbol 12, 13 dibutyrate (PdBu), the activator of PKC, suppressed afterhyperpolarization and increased the activity of snail neurons without any significant effect on the Ca2+ action potential duration. Pretreatment with PKC activator attenuated the suppressing effect of paraoxon on the duration of Ca2+ action potentials. Staurosporine, a selective blocker of PKC, did not block the effect of paraoxon on Ca2+ action potential suppression and hyperexcitability induction. Our findings did not support the involvement PKC in the paraoxon induced Ca2+ action potential suppression and neuronal activity modulation, although activation of this protein kinase could attenuate some effects of paraoxon. Pretreatment with 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an antagonist of IP3-mediated Ca2+ release, abolished the secondary silencing effect of paraoxon, which is observed after primary paraoxon-induced hyperexcitability. It was concluded that slow activation of intracellular cascades by paraoxon could induce an IP3 mediated Ca2+ release from intracellular stores and participate to its secondary silencing effect by mechanisms dependent on intracellular

  8. Functional homology of protein kinases required for sexual differentiation in Schizosaccharomyces pombe and Saccharomyces cerevisiae suggests a conserved signal transduction module in eukaryotic organisms.

    PubMed Central

    Neiman, A M; Stevenson, B J; Xu, H P; Sprague, G F; Herskowitz, I; Wigler, M; Marcus, S

    1993-01-01

    We present genetic evidence that three presumptive protein kinases of Schizosaccharomyces pombe, byr2, byr1, and spk1 that are structurally related to protein kinases of Saccharomyces cerevisiae, STE11, STE7, and FUS3, respectively, are also functionally related. In some cases, introduction of the heterologous protein kinase into a mutant was sufficient for complementation. In other cases (as in a ste11- mutant of S. cerevisiae), expression of two S. pombe protein kinases (byr2 and byr1) was required to observe complementation, suggesting that byr2 and byr1 act cooperatively. Complementation in S. pombe mutants is observed as restoration of sporulation and conjugation and in S. cerevisiae as restoration of conjugation, pheromone-induced cell cycle arrest, and pheromone-induced transcription of the FUS1 gene. We also show that the S. pombe kinases bear a similar relationship to the mating pheromone receptor apparatus as do their S. cerevisiae counterparts. Our results indicate that pheromone-induced signal transduction employs a conserved set of kinases in these two evolutionarily distant yeasts despite an apparently significant difference in function of the heterotrimeric G proteins. We suggest that the STE11/byr2, STE7/byr1, and FUS3/spk1 kinases comprise a signal transduction module that may be conserved in higher eukaryotes. Consistent with this hypothesis, we show that a mammalian mitogen-activated protein (MAP) kinase, ERK2, can partially replace spk1 function in S. pombe. Images PMID:8443406

  9. Manganese modulation of MAPK pathways: effects on upstream mitogen activated protein kinase kinases (MKKs) and mitogen activated kinase phosphatase-1 (MKP-1) in microglial cells

    PubMed Central

    Crittenden, Patrick L.; Filipov, Nikolay M.

    2010-01-01

    Multiple studies demonstrate that manganese (Mn) exposure potentiates inflammatory mediator output from activated glia; this increased output is associated with enhanced mitogen activated protein kinase (MAPK: p38, ERK, and JNK) activity. We hypothesized that Mn activates MAPK by activating the kinases upstream of MAPK, i.e., MKK-3/6, MKK-1/2, and MKK-4 (responsible for activation of p38, ERK, and JNK, respectively), and/or by inhibiting a major phosphatase responsible for MAPK inactivation, MKP-1. Exposure of N9 microglia to Mn (250μM), LPS (100 ng/ml), or Mn+LPS increased MKK-3/6 and MKK-4 activity at 1 h; the effect of Mn+LPS on MKK-4 activation was greater than the rest. At 4 h, Mn, LPS, and Mn+LPS increased MKK-3/6 and MKK-1/2 phosphorylation, whereas MKK-4 was activated only by Mn and Mn+LPS. Besides activating MKK-4 via Ser257/Thr261 phosphorylation, Mn (4 h) prevented MKK-4’s phosphorylation on Ser80, which negatively regulates MKK-4 activity. Exposure to Mn or Mn+LPS (1 h) decreased both mRNA and protein expression of MKP-1, the negative MAPK regulator. In addition, we observed that at 4 h, but not at 1 h, a time point coinciding with increased MAPK activity, Mn+LPS markedly increased TNF-α , IL-6, and Cox-2 mRNA, suggesting a delayed effect. The fact that all three major groups of MKKs, MKK-1/2, MKK-3/6, and MKK-4 are activated by Mn suggests that Mn-induced activation of MAPK occurs via traditional mechanisms, which perhaps involve the MAPKs farthest upstream, MKKKs (MAP3Ks). In addition, for all MKKs, Mn-induced activation was persistent at least for 4 h, indicating a long-term effect. PMID:20589745

  10. Modulation by protein kinase C of nitric oxide and cyclic GMP poffation in cultured cerebellar granule cells.

    PubMed

    Riccio, A; Esposito, E; Eboli, M L

    1996-04-29

    The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated in primary cultures of rat cerebellar neurons. Incubation of the cells with L-arginine and nicotinamide-adenine dinucleotide phosphate (NADPH) produced detectable levels of NO, as quantified by photometric assay [0.14 +/- 0.03 nmol/h/dish (2.5 x 10(6) cells)]. The NO producing activity was paralleled by concomitant accumulation of cyclic GMP (cGMP) (0.12 +/- 0.02 pmol/dish). Downregulation of PKC by prolonged treatment with phorbol esters or inhibition of the kinase by treatment with 4taurosporine raised the basal levels of NO and cGMP five fold. When granule cells were incubated in the absence of extracellular Mg2+, N-methyl-D-aspartate and to a lesser extent, glutamate became effective in enhancing NO formation and cGMP accumulation with respect to the control. The NO and cGMP increases induced by the two agonists were almost doubled by treatment of the cells with staurosporine or depletion of PKC. Calphostin C. an inhibitor of the regulatory domain of PKC, was as effective as staurosporine in increasing the formation of NO in both resting and excited cells. These results indicate that downregulation or inhibition of PKC increase NOS activity in cerebellar neurons, and suggest that phosphorylation of NOS by PKC negatively modulates the catalytic activity of the enzyme in these cells. PMID:8773779

  11. Src family protein tyrosine kinase (PTK) modulates the effect of SGK1 and WNK4 on ROMK channels.

    PubMed

    Yue, Peng; Lin, Dao-Hong; Pan, Chun-Yang; Leng, Qiang; Giebisch, Gerhard; Lifton, Richard P; Wang, Wen-Hui

    2009-09-01

    WNK4 (with no lysine kinase 4) inhibits ROMK channel activity in the distal nephron by stimulating clathrin-dependent endocytosis, an effect attenuated by SGK1 (serum-glucocorticoids-induced kinase)-mediated phosphorylation. It has been suggested that increased ROMK activity because of SGK1-mediated inhibition of WNK4 plays a role in promoting renal K secretion in response to elevated serum K or high K (HK) intake. In contrast, intravascular volume depletion also increases SGK1 activity but fails to stimulate ROMK channels and K secretion. Because HK intake decreases Src family protein tyrosine kinase (PTK) activity an inhibitor of ROMK channels, it is possible that Src family PTK may modulate the effects of SGK1 on WNK4. Here, we show that c-Src prevents SGK1 from attenuating WNK4's inhibition of ROMK activity. This effect of c-Src was WNK4-dependent because c-Src had no effect on ROMK harboring mutation at the site of c-Src phosphorylation (R1Y337A) in the absence of WNK4. Moreover, expression c-Src diminished the SGK1-mediated increase in serine phosphorylation of WNK4, suggesting that c-Src enhances WNK4-mediated inhibition of ROMK channels by suppressing the SGK1-induced phosphorylation. This notion is also supported by the observation that c-Src was not able to modulate the interaction between SGK1 and WNK4 mutants (WNK4(S1169A) or WNK4(S1169D)) in which an SGK1-phosphorylation site (serine 1169) was mutated by alanine or aspartate. We conclude that c-Src inhibits SGK1-mediated phosphorylation hereby restoring the WNK4-mediated inhibition of ROMK channels thus suppressing K secretion. PMID:19706464

  12. The Parkinson’s Disease-Associated Protein Kinase LRRK2 Modulates Notch Signaling through the Endosomal Pathway

    PubMed Central

    Imai, Yuzuru; Kobayashi, Yoshito; Inoshita, Tsuyoshi; Meng, Hongrui; Arano, Taku; Uemura, Kengo; Asano, Takeshi; Yoshimi, Kenji; Zhang, Chang-Liang; Matsumoto, Gen; Ohtsuka, Toshiyuki; Kageyama, Ryoichiro; Kiyonari, Hiroshi; Shioi, Go; Nukina, Nobuyuki; Hattori, Nobutaka; Takahashi, Ryosuke

    2015-01-01

    Leucine-rich repeat kinase 2 (LRRK2) is a key molecule in the pathogenesis of familial and idiopathic Parkinson’s disease (PD). We have identified two novel LRRK2-associated proteins, a HECT-type ubiquitin ligase, HERC2, and an adaptor-like protein with six repeated Neuralized domains, NEURL4. LRRK2 binds to NEURL4 and HERC2 via the LRRK2 Ras of complex proteins (ROC) domain and NEURL4, respectively. HERC2 and NEURL4 link LRRK2 to the cellular vesicle transport pathway and Notch signaling, through which the LRRK2 complex promotes the recycling of the Notch ligand Delta-like 1 (Dll1)/Delta (Dl) through the modulation of endosomal trafficking. This process negatively regulates Notch signaling through cis-inhibition by stabilizing Dll1/Dl, which accelerates neural stem cell differentiation and modulates the function and survival of differentiated dopaminergic neurons. These effects are strengthened by the R1441G ROC domain-mutant of LRRK2. These findings suggest that the alteration of Notch signaling in mature neurons is a component of PD etiology linked to LRRK2. PMID:26355680

  13. The Protein Kinase A Pathway Regulates Zearalenone Production by Modulating Alternative ZEB2 Transcription.

    PubMed

    Park, Ae Ran; Fu, Minmin; Shin, Ji Young; Son, Hokyoung; Lee, Yin-Won

    2016-05-28

    Zearalenone (ZEA) is an estrogenic mycotoxin that is produced by several Fusarium species, including Fusarium graminearum. One of the ZEA biosynthetic genes, ZEB2, encodes two isoforms of Zeb2 by alternative transcription, forming an activator (Zeb2L-Zeb2L homooligomer) and an inhibitor (Zeb2L-Zeb2S heterodimer) that directly regulate the ZEA biosynthetic genes in F. graminearum. Cyclic AMP-dependent protein kinase A (PKA) signaling regulates secondary metabolic processes in several filamentous fungi. In this study, we investigated the effects of the PKA signaling pathway on ZEA biosynthesis. Through functional analyses of PKA catalytic and regulatory subunits (CPKs and PKR), we found that the PKA pathway negatively regulates ZEA production. Genetic and biochemical evidence further demonstrated that the PKA pathway specifically represses ZEB2L transcription and also takes part in posttranscriptional regulation of ZEB2L during ZEA production. Our findings reveal the intriguing mechanism that the PKA pathway regulates secondary metabolite production by reprograming alternative transcription. PMID:26907763

  14. Alpha-latrotoxin modulates the secretory machinery via receptor-mediated activation of protein kinase C.

    PubMed

    Liu, Jie; Wan, Qunfang; Lin, Xianguang; Zhu, Hongliang; Volynski, Kirill; Ushkaryov, Yuri; Xu, Tao

    2005-09-01

    The hypothesis whether alpha-latrotoxin (LTX) could directly regulate the secretory machinery was tested in pancreatic beta cells using combined techniques of membrane capacitance (Cm) measurement and Ca2+ uncaging. Employing ramp increase in [Ca2+]i to stimulate exocytosis, we found that LTX lowers the Ca2+ threshold required for exocytosis without affecting the size of the readily releasable pool (RRP). The burst component of exocytosis in response to step-like [Ca2+]i increase generated by flash photolysis of caged Ca2+ was also speeded up by LTX treatment. LTX increased the maximum rate of exocytosis compared with control responses with similar postflash [Ca2+]i and shifted the Ca2+ dependence of the exocytotic machinery toward lower Ca2+ concentrations. LTXN4C, a LTX mutant which cannot form membrane pores or penetrate through the plasma membrane but has similar affinity for the receptors as the wild-type LTX, mimicked the effect of LTX. Moreover, the effects of both LTX and LTXN4C) were independent of intracellular or extracellular Ca2+ but required extracellular Mg2+. Our data propose that LTX, by binding to the membrane receptors, sensitizes the fusion machinery to Ca2+ and, hence, may permit release at low [Ca2+]i level. This sensitization is mediated by activation of protein kinase C. PMID:16101679

  15. Protein Kinase C Epsilon Promotes Cerebral Ischemic Tolerance Via Modulation of Mitochondrial Sirt5

    PubMed Central

    Morris-Blanco, Kahlilia C.; Dave, Kunjan R.; Saul, Isabel; Koronowski, Kevin B.; Stradecki, Holly M.; Perez-Pinzon, Miguel A.

    2016-01-01

    Sirtuin 5 (SIRT5) is a mitochondrial-localized NAD+-dependent lysine desuccinylase and a major regulator of the mitochondrial succinylome. We wanted to determine whether SIRT5 is activated by protein kinase C epsilon (PKCε)-mediated increases in mitochondrial Nampt and whether SIRT5 regulates mitochondrial bioenergetics and neuroprotection against cerebral ischemia. In isolated mitochondria from rat cortical cultures, PKCε activation increased SIRT5 levels and desuccinylation activity in a Nampt-dependent manner. PKCε activation did not lead to significant modifications in SIRT3 activity, the major mitochondrial lysine deacetylase. Assessments of mitochondrial bioenergetics in the cortex of wild type (WT) and SIRT5−/− mice revealed that SIRT5 regulates oxygen consumption in the presence of complex I, complex II, and complex IV substrates. To explore the potential role of SIRT5 in PKCε-mediated protection, we compared WT and SIRT5−/− mice by employing both in vitro and in vivo ischemia paradigms. PKCε-mediated decreases in cell death following oxygen-glucose deprivation were abolished in cortical cultures harvested from SIRT5−/− mice. Furthermore, PKCε failed to prevent cortical degeneration following MCAO in SIRT5−/− mice. Collectively this demonstrates that SIRT5 is an important mitochondrial enzyme for protection against metabolic and ischemic stress following PKCε activation in the brain. PMID:27435822

  16. Src-family protein tyrosine kinase phosphorylates WNK4 and modulates its inhibitory effect on KCNJ1 (ROMK)

    PubMed Central

    Lin, Dao-Hong; Yue, Peng; Yarborough, Orlando; Scholl, Ute I.; Giebisch, Gerhard; Lifton, Richard P.; Rinehart, Jesse; Wang, Wen-Hui

    2015-01-01

    With-no-lysine kinase 4 (WNK4) inhibits the activity of the potassium channel KCNJ1 (ROMK) in the distal nephron, thereby contributing to the maintenance of potassium homeostasis. This effect is inhibited via phosphorylation at Ser1196 by serum/glucocorticoid-induced kinase 1 (SGK1), and this inhibition is attenuated by the Src-family protein tyrosine kinase (SFK). Using Western blot and mass spectrometry, we now identify three sites in WNK4 that are phosphorylated by c-Src: Tyr1092, Tyr1094, and Tyr1143, and show that both c-Src and protein tyrosine phosphatase type 1D (PTP-1D) coimmunoprecipitate with WNK4. Mutation of Tyr1092 or Tyr1143 to phenylalanine decreased the association of c-Src or PTP-1D with WNK4, respectively. Moreover, the Tyr1092Phe mutation markedly reduced ROMK inhibition by WNK4; this inhibition was completely absent in the double mutant WNK4Y1092/1094F. Similarly, c-Src prevented SGK1-induced phosphorylation of WNK4 at Ser1196, an effect that was abrogated in the double mutant. WNK4Y1143F inhibited ROMK activity as potently as wild-type (WT) WNK4, but unlike WT, the inhibitory effect of WNK4Y1143F could not be reversed by SGK1. The failure to reverse WNK4Y1143F-induced inhibition of ROMK by SGK1 was possibly due to enhancing endogenous SFK effect on WNK4 by decreasing the WNK4–PTP-1D association because inhibition of SFK enabled SGK1 to reverse WNK4Y1143F-induced inhibition of ROMK. We conclude that WNK4 is a substrate of SFKs and that the association of c-Src and PTP-1D with WNK4 at Tyr1092 and Tyr1143 plays an important role in modulating the inhibitory effect of WNK4 on ROMK. PMID:25805816

  17. Src-family protein tyrosine kinase phosphorylates WNK4 and modulates its inhibitory effect on KCNJ1 (ROMK).

    PubMed

    Lin, Dao-Hong; Yue, Peng; Yarborough, Orlando; Scholl, Ute I; Giebisch, Gerhard; Lifton, Richard P; Rinehart, Jesse; Wang, Wen-Hui

    2015-04-01

    With-no-lysine kinase 4 (WNK4) inhibits the activity of the potassium channel KCNJ1 (ROMK) in the distal nephron, thereby contributing to the maintenance of potassium homeostasis. This effect is inhibited via phosphorylation at Ser1196 by serum/glucocorticoid-induced kinase 1 (SGK1), and this inhibition is attenuated by the Src-family protein tyrosine kinase (SFK). Using Western blot and mass spectrometry, we now identify three sites in WNK4 that are phosphorylated by c-Src: Tyr(1092), Tyr(1094), and Tyr(1143), and show that both c-Src and protein tyrosine phosphatase type 1D (PTP-1D) coimmunoprecipitate with WNK4. Mutation of Tyr(1092) or Tyr(1143) to phenylalanine decreased the association of c-Src or PTP-1D with WNK4, respectively. Moreover, the Tyr1092Phe mutation markedly reduced ROMK inhibition by WNK4; this inhibition was completely absent in the double mutant WNK4(Y1092/1094F). Similarly, c-Src prevented SGK1-induced phosphorylation of WNK4 at Ser(1196), an effect that was abrogated in the double mutant. WNK4(Y1143F) inhibited ROMK activity as potently as wild-type (WT) WNK4, but unlike WT, the inhibitory effect of WNK4(Y1143F) could not be reversed by SGK1. The failure to reverse WNK4(Y1143F)-induced inhibition of ROMK by SGK1 was possibly due to enhancing endogenous SFK effect on WNK4 by decreasing the WNK4-PTP-1D association because inhibition of SFK enabled SGK1 to reverse WNK4(Y1143F)-induced inhibition of ROMK. We conclude that WNK4 is a substrate of SFKs and that the association of c-Src and PTP-1D with WNK4 at Tyr(1092) and Tyr(1143) plays an important role in modulating the inhibitory effect of WNK4 on ROMK. PMID:25805816

  18. N,N-Dimethylsphingosine is a potent competitive inhibitor of sphingosine kinase but not of protein kinase C: modulation of cellular levels of sphingosine 1-phosphate and ceramide.

    PubMed

    Edsall, L C; Van Brocklyn, J R; Cuvillier, O; Kleuser, B; Spiegel, S

    1998-09-15

    Sphingosine 1-phosphate (SPP), a lipid second messenger formed by the action of sphingosine kinase, has been implicated in regulating diverse biological processes, including growth, survival, and differentiation. N,N-Dimethylsphingosine (DMS) inhibits sphingosine kinase and has been used to investigate the biological roles of SPP; however, little is known of the mechanism of inhibition of sphingosine kinase by DMS. In addition, DMS has been shown to inhibit protein kinase C in vitro. Here we report that DMS is a competitive inhibitor of sphingosine kinase from U937 monoblastic leukemia cells, Swiss 3T3 fibroblasts, and PC12 pheochromocytoma cells. DMS decreases basal levels of SPP and prevents increases in SPP in response to physiological stimuli known to activate sphingosine kinase. DMS also effectively increases cellular levels of ceramide in a variety of cell types, and resetting of the ceramide/SPP rheostat may account for the pro-apoptotic effects of DMS. Moreover, DMS, at concentrations which effectively inhibit sphingosine kinase, has no effect on protein kinase C activity or its membrane translocation. Thus, DMS acts as a specific competitive inhibitor of sphingosine kinase in diverse cell types and is a useful tool to elucidate the role of SPP as an intracellular second messenger. PMID:9737868

  19. Dietary modulation of rat colonic cAMP-dependent protein kinase activity.

    PubMed

    Aukema, H M; Davidson, L A; Chang, W C; Lupton, J R; Derr, J N; Chapkin, R S

    1994-10-20

    Malignant transformation of cells is associated with enhanced proliferation and alterations in cAMP-dependent protein kinase (PKA) activity. To investigate the role of PKA in normal colonic cell proliferation, PKA was characterized in rat colonic mucosa. In addition, rats were fed diets containing different fats (corn oil, fish oil) and fibers (pectin, cellulose, fiber free) to elicit varying levels of colonic cell proliferation in order to study this signaling system under normal physiologic conditions. Overall, PKA activities were higher in cytosolic compared to membrane fractions. PKA type II (PKA II) isozyme contributed 89 +/- 1% and 96 +/- 1% of total PKA activity in cytosolic and membrane fractions, respectively. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the presence of mRNA for both the alpha and beta isoforms of the regulatory subunits of PKA II. PKA activities were 21-33% higher in distal membrane and total distal fractions in rats fed a cellulose/corn oil diet compared to animals consuming the other fiber/fat diets. These effects were seen only in the distal colon, where the number of cells per crypt column was elevated only in animals fed the cellulose/corn oil diet relative to other diets. Diet-induced mitogenic responses did not involve significant changes in the relative activity of PKA I and II isozymes. These data demonstrate that dietary effects on PKA activity in the distal colon may be related to changes in cell differentiation as indicated by the number of cells per crypt column. PMID:7948042

  20. The activated glucocorticoid receptor modulates presumptive autoregulation of ribosomal protein S6 protein kinase, p70 S6K.

    PubMed

    Shah, O Jameel; Iniguez-Lluhi, Jorge A; Romanelli, Angela; Kimball, Scot R; Jefferson, Leonard S

    2002-01-25

    Protein metabolism in eukaryotic organisms is defined by a synthesis-degradation equilibrium that is subject to regulation by hormonal and nutritional signals. In mammalian tissues such as skeletal muscle, glucocorticoid hormones specify a catabolic response that influences both protein synthetic and protein degradative pathways. With regard to the former, glucocorticoids attenuate mRNA translation at two levels: translational efficiency, i.e. translation initiation, and translational capacity, i.e. ribosome biogenesis. Glucocorticoids may impair translational capacity through the ribosomal S6 protein kinase (p70 S6K), a recognized glucocorticoid target and an effector of ribosomal protein synthesis. We demonstrate here that the reduction in growth factor-activated p70 S6K activity by glucocorticoids depends upon a functional glucocorticoid receptor (GR) and that the GR is both necessary and sufficient to render p70 S6K subject to glucocorticoid regulation. Furthermore, the DNA binding and transcriptional activation but not repression properties of the GR are indispensable for p70 S6K regulation. Finally, a mutational analysis of the p70 S6K carboxyl terminus indicates that this region confers glucocorticoid sensitivity, and thus glucocorticoids may facilitate autoinhibition of the enzyme ultimately reducing the efficiency with which T389 is phosphorylated. PMID:11705993

  1. E6 variants of human papillomavirus 18 differentially modulate the protein kinase B/phosphatidylinositol 3-kinase (akt/PI3K) signaling pathway

    SciTech Connect

    Contreras-Paredes, Adriana

    2009-01-05

    Intra-type genome variations of high risk Human papillomavirus (HPV) have been associated with a differential threat for cervical cancer development. In this work, the effect of HPV18 E6 isolates in Akt/PKB and Mitogen-associated protein kinase (MAPKs) signaling pathways and its implication in cell proliferation were analyzed. E6 from HPV types 16 and 18 are able to bind and promote degradation of Human disc large (hDlg). Our results show that E6 variants differentially modulate hDlg degradation, rebounding in levels of activated PTEN and PKB. HPV18 E6 variants are also able to upregulate phospho-PI3K protein, strongly correlating with activated MAPKs and cell proliferation. Data was supported by the effect of E6 silencing in HPV18-containing HeLa cells, as well as hDlg silencing in the tested cells. Results suggest that HPV18 intra-type variations may derive in differential abilities to activate cell-signaling pathways such as Akt/PKB and MAPKs, directly involved in cell survival and proliferation.

  2. Modulation of beta2- and beta3-adrenoceptor-mediated relaxation of rat oesophagus smooth muscle by protein kinase C.

    PubMed

    Oostendorp, Jaap; Obels, Peter Ph; Terpstra, A Rene; Nelemans, S Adriaan; Zaagsma, Johan

    2004-07-01

    Although a prominent role for protein kinase C (PKC) in the cross-talk between the phosphoinositide pathway and beta2-adrenoceptor signalling has been indicated, modulation of beta3-adrenoceptor function by PKC has not been studied thus far. In the present study, we have compared the relative capacity of PKC in modulating beta2- and beta3-adrenoceptor-mediated relaxation of methacholine-contracted rat oesophagus smooth muscle. To this purpose the effects of the PKC-inhibitor GF 109203X (2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide) on relaxation induced by fenoterol, formoterol, (-)-noradrenaline, BRL 35135 (4-[2-[(2-hydroxy-2-(chlorophenyl)ethyl)amino]-propyl]-phenoxyacetic-acidmethylester) and IBMX (3-isobutyl-1-methyl-xanthine) were studied, in the absence and presence of the selective beta2-adrenoceptor antagonist ICI 118,551 (erythro-1(7-methylindan-4-yloxy)-3-(isopropylamin)-butan-2-ol). Our results show that inhibition of PKC resulted in differential augmentation of both beta2- and beta3-adrenoceptor-mediated relaxation. In contrast, relaxation induced by IBMX was not influenced at all by GF 109203X. The beta2-adrenoceptor bears phosphorylation sites for several kinases, including PKC. Since the beta3-adrenoceptor lacks these consensus sites, the results may also indicate that PKC-mediated Galphas phosphorylation is involved in the cross-talk between the muscarinic receptor-mediated phosphoinositide pathway and beta2- and, particularly, beta3-adrenoceptor signalling. PMID:15219823

  3. DNA Unwinding Functions of Minute Virus of Mice NS1 Protein Are Modulated Specifically by the Lambda Isoform of Protein Kinase C

    PubMed Central

    Dettwiler, Sabine; Rommelaere, Jean; Nüesch, Jürg P. F.

    1999-01-01

    The parvovirus minute virus of mice NS1 protein is a multifunctional protein involved in a variety of processes during virus propagation, ranging from viral DNA replication to promoter regulation and cytotoxic action to the host cell. Since NS1 becomes phosphorylated during infection, it was proposed that the different tasks of this protein might be regulated in a coordinated manner by phosphorylation. Indeed, comparing biochemical functions of native NS1 with its dephosphorylated counterpart showed that site-specific nicking of the origin and the helicase and ATPase activities are remarkably reduced upon NS1 dephosphorylation while site-specific affinity of the protein to the origin became enhanced. As a consequence, the dephosphorylated polypeptide is deficient for initiation of DNA replication. By adding fractionated cell extracts to a kinase-free in vitro replication system, the combination of two protein components containing members of the protein kinase C (PKC) family was found to rescue the replication activity of the dephosphorylated NS1 protein upon addition of PKC cofactors. One of these components, termed HA-1, also stimulated NS1 helicase function in response to acidic lipids but not phorbol esters, indicating the involvement of atypical PKC isoforms in the modulation of this NS1 function (J. P. F. Nüesch, S. Dettwiler, R. Corbau, and J. Rommelaere, J. Virol. 72:9966–9977, 1998). The present study led to the identification of atypical PKCλ/ι as the active component of HA-1 responsible for the regulation of NS1 DNA unwinding and replicative functions. Moreover, a target PKCλ phosphorylation site was localized at S473 of NS1. By site-directed mutagenesis, we showed that this residue is essential for NS1 helicase activity but not promoter regulation, suggesting a possible modulation of NS1 functions by PKCλ phosphorylation at residue S473. PMID:10438831

  4. Protein kinase A modulates transforming growth factor-β signaling through a direct interaction with Smad4 protein.

    PubMed

    Yang, Huibin; Li, Gangyong; Wu, Jing-Jiang; Wang, Lidong; Uhler, Michael; Simeone, Diane M

    2013-03-22

    Transforming growth factor β (TGFβ) signaling normally functions to regulate embryonic development and cellular homeostasis. It is increasingly recognized that TGFβ signaling is regulated by cross-talk with other signaling pathways. We previously reported that TGFβ activates protein kinase A (PKA) independent of cAMP through an interaction of an activated Smad3-Smad4 complex and the regulatory subunit of the PKA holoenzyme (PKA-R). Here we define the interaction domains of Smad4 and PKA-R and the functional consequences of this interaction. Using a series of Smad4 and PKA-R truncation mutants, we identified amino acids 290-300 of the Smad4 linker region as critical for the specific interaction of Smad4 and PKA-R. Co-immunoprecipitation assays showed that the B cAMP binding domain of PKA-R was sufficient for interaction with Smad4. Targeting of B domain regions conserved among all PKA-R isoforms and exposed on the molecular surface demonstrated that amino acids 281-285 and 320-329 were required for complex formation with Smad4. Interactions of these specific regions of Smad4 and PKA-R were necessary for TGFβ-mediated increases in PKA activity, CREB (cAMP-response element-binding protein) phosphorylation, induction of p21, and growth inhibition. Moreover, this Smad4-PKA interaction was required for TGFβ-induced epithelial mesenchymal transition, invasion of pancreatic tumor cells, and regulation of tumor growth in vivo. PMID:23362281

  5. Intracellular sodium modulates the state of protein kinase C phosphorylation of rat proximal tubule Na+,K+-ATPase.

    PubMed

    Ibarra, F R; Cheng, S X Jun; Agrén, M; Svensson, L-B; Aizman, O; Aperia, A

    2002-06-01

    The natriuretic hormone dopamine and the antinatriuretic hormone noradrenaline, acting on alpha-adrenergic receptors, have been shown to bidirectionally modulate the activity of renal tubular Na+,K+-adenosine triphosphate (ATPase). Here we have examined whether intracellular sodium concentration influences the effects of these bidirectional forces on the state of phosphorylation of Na+,K+-ATPase. Proximal tubules dissected from rat kidney were incubated with dopamine or the alpha-adrenergic agonist, oxymetazoline, and transiently permeabilized in a medium where sodium concentration ranged between 5 and 70 mM. The variations of sodium concentration in the medium had a proportional effect on intracellular sodium. Dopamine and protein kinase C (PKC) phosphorylate the catalytic subunit of rat Na+,K+-ATPase on the Ser23 residue. The level of PKC induced Na+,K+-ATPase phosphorylation was determined using an antibody that only recognizes Na+,K+-ATPase, which is not phosphorylated on its PKC site. Under basal conditions Na+,K+-ATPase was predominantly in its phosphorylated state. When intracellular sodium was increased, Na+,K+-ATPase was predominantly in its dephosphorylated state. Phosphorylation of Na+,K+-ATPase by dopamine was most pronounced when intracellular sodium was high, and dephosphorylation by oxymetazoline was most pronounced when intracellular sodium was low. The oxymetazoline effect was mimicked by the calcium ionophore A23187. An inhibitor of the calcium-dependent protein phosphatase, calcineurin, increased the state of Na+,K+-ATPase phosphorylation. The results imply that phosphorylation of renal Na+,K+-ATPase activity is modulated by the level of intracellular sodium and that this effect involves PKC and calcium signalling pathways. The findings may have implication for the regulation of salt excretion and sodium homeostasis. PMID:12028137

  6. E sub 1 BF is an essential RNA polymerase I transcription factor with an intrinsic protein kinase activity that can modulate rRNA gene transcription

    SciTech Connect

    Ji Zhang; Huifeng Niu; Jacob, S.T. )

    1991-10-01

    The authors previously described the purification and characterization of E{sub 1}BF, a rat rRNA gene core promoter-binding factor that consists of two polypeptides of 89 and 79 kDa. When this factor was incubated in the absence of any exogenous protein kinase under conditions optimal for protein phosphorylation, the 79-kDa polypeptide of E{sub 1}BF was selectively phosphorylated. The labeled phosphate could be removed from the E{sub 1}BF polypeptide by treatment with calf intestinal alkaline phosphatase or potato acid phosphatase. Elution of the protein from the E{sub 1}BF-promoter complex formed in an electrophoretic mobility-shift assay followed by incubation of the concentrated eluent with ({gamma}-{sup 32}P)ATP resulted in the selective labeling o the 79-kDa band. The E{sub 1}BF-associated protein kinase did not phosphorylate casein or histone H1. These data demonstrate that (1) polymerase I promoter-binding factor E{sub 1}BF contains an intrinsic substrate-specific protein kinase and (2) E{sub 1}BF is an essential polymerase I transcription factor that can modulate rRNA gene transcription by protein phosphorylation. Further, these studies have provided a direct means to identify a protein kinase or any other enzyme that can interact with a specific DNA sequence.

  7. Protein phosphatase 1 modulates the inhibitory effect of With-no-Lysine kinase 4 on ROMK channels.

    PubMed

    Lin, Dao-Hong; Yue, Peng; Rinehart, Jesse; Sun, Peng; Wang, Zhijian; Lifton, Richard; Wang, Wen-Hui

    2012-07-01

    With-no-Lysine kinase 4 (WNK4) inhibited ROMK (Kir1.1) channels and the inhibitory effect of WNK4 was abolished by serum-glucocorticoid-induced kinase 1 (SGK1) but restored by c-Src. The aim of the present study is to explore the mechanism by which Src-family tyrosine kinase (SFK) modulates the effect of SGK1 on WNK4 and to test the role of SFK-WNK4-SGK1 interaction in regulating ROMK channels in the kidney. Immunoprecipitation demonstrated that protein phosphatase 1 (PP1) binds to WNK4 at amino acid (aa) residues 695-699 (PP1(#1)) and at aa 1211-1215 (PP1(#2)). WNK4(-PP1#1) and WNK4(-PP1#2), in which the PP1(#1) or PP1(#2) binding site was deleted or mutated, inhibited ROMK channels as potently as WNK4. However, c-Src restored the inhibitory effect of WNK4 but not WNK4(-PP1#1) on ROMK channels in the presence of SGK1. Moreover, expression of c-Src inhibited SGK1-induced phosphorylation of WNK4 but not WNK4(-PP1#1) at serine residue 1196 (Ser(1196)). In contrast, coexpression of c-Src restored the inhibitory effect of WNK4(-PP1#2) on ROMK in the presence of SGK1 and diminished SGK1-induced WNK4 phosphorylation at Ser(1196) in cells transfected with WNK4(-PP1#2). This suggests the possibility that c-Src regulates the interaction between WNK4 and SGK1 through activating PP1 binding to aa 695-9 thereby decreasing WNK4 phosphorylation and restoring the inhibitory effect of WNK4. This mechanism plays a role in suppressing ROMK channel activity during the volume depletion because inhibition of SFK or serine/threonine phosphatases increases ROMK channel activity in the cortical collecting duct of rats on a low-Na diet. We conclude that regulation of phosphatase activity by SFK plays a role in determining the effect of aldosterone on ROMK channels and on renal K secretion. PMID:22513846

  8. A computational model on the modulation of mitogen-activated protein kinase (MAPK) and Akt pathways in heregulin-induced ErbB signalling.

    PubMed Central

    Hatakeyama, Mariko; Kimura, Shuhei; Naka, Takashi; Kawasaki, Takuji; Yumoto, Noriko; Ichikawa, Mio; Kim, Jae-Hoon; Saito, Kazuki; Saeki, Mihoro; Shirouzu, Mikako; Yokoyama, Shigeyuki; Konagaya, Akihiko

    2003-01-01

    ErbB tyrosine kinase receptors mediate mitogenic signal cascade by binding a variety of ligands and recruiting the different cassettes of adaptor proteins. In the present study, we examined heregulin (HRG)-induced signal transduction of ErbB4 receptor and found that the phosphatidylinositol 3'-kinase (PI3K)-Akt pathway negatively regulated the extracellular signal-regulated kinase (ERK) cascade by phosphorylating Raf-1 on Ser(259). As the time-course kinetics of Akt and ERK activities seemed to be transient and complex, we constructed a mathematical simulation model for HRG-induced ErbB4 receptor signalling to explain the dynamics of the regulation mechanism in this signal transduction cascade. The model reflected well the experimental results observed in HRG-induced ErbB4 cells and in other modes of growth hormone-induced cell signalling that involve Raf-Akt cross-talk. The model suggested that HRG signalling is regulated by protein phosphatase 2A as well as Raf-Akt cross-talk, and protein phosphatase 2A modulates the kinase activity in both the PI3K-Akt and MAPK (mitogen-activated protein kinase) pathways. PMID:12691603

  9. Protein kinase CK2 modulates IL-6 expression in inflammatory breast cancer.

    PubMed

    Drygin, Denis; Ho, Caroline B; Omori, Mayuko; Bliesath, Joshua; Proffitt, Chris; Rice, Rachel; Siddiqui-Jain, Adam; O'Brien, Sean; Padgett, Claire; Lim, John K C; Anderes, Kenna; Rice, William G; Ryckman, David

    2011-11-11

    Inflammatory breast cancer is driven by pro-angiogenic and pro-inflammatory cytokines. One of them Interleukin-6 (IL-6) is implicated in cancer cell proliferation and survival, and promotes angiogenesis, inflammation and metastasis. While IL-6 has been shown to be upregulated by several oncogenes, the mechanism behind this phenomenon is not well characterized. Here we demonstrate that the pleotropic Serine/Threonine kinase CK2 is implicated in the regulation of IL-6 expression in a model of inflammatory breast cancer. We used siRNAs targeted toward CK2 and a selective small molecule inhibitor of CK2, CX-4945, to inhibit the expression and thus suppress the secretion of IL-6 in in vitro as well as in vivo models. Moreover, we report that in a clinical trial, CX-4945 was able to dramatically reduce IL-6 levels in plasma of an inflammatory breast cancer patient. Our data shed a new light on the regulation of IL-6 expression and position CX-4945 and potentially other inhibitors of CK2, for the treatment of IL-6-driven cancers and possibly other diseases where IL-6 is instrumental, including rheumatoid arthritis. PMID:22027148

  10. Mitogen-activated Protein Kinase Phosphatase-1 Modulates Regional Effects of Injurious Mechanical Ventilation in Rodent Lungs

    PubMed Central

    Park, Moo Suk; Edwards, Michael G.; Sergew, Amen; Riches, David W. H.; Albert, Richard K.

    2012-01-01

    Rationale: Mechanical ventilation induces heterogeneous lung injury by mitogen-activated protein kinase (MAPK) and nuclear factor-κB. Mechanisms regulating regional injury and protective effects of prone positioning are unclear. Objectives: To determine the key regulators of the lung regional protective effects of prone positioning in rodent lungs exposed to injurious ventilation. Methods: Adult rats were ventilated with high (18 ml/kg, positive end-expiratory pressure [PEEP] 0) or low Vt (6 ml/kg; PEEP 3 cm H2O; 3 h) in supine or prone position. Dorsal–caudal lung mRNA was analyzed by microarray and MAPK phosphatases (MKP)-1 quantitative polymerase chain reaction. MKP-1−/− or wild-type mice were ventilated with very high (24 ml/kg; PEEP 0) or low Vt (6–7 ml/kg; PEEP 3 cm H2O). The MKP-1 regulator PG490-88 (MRx-108; 0.75 mg/kg) or phosphate-buffered saline was administered preventilation. Injury was assessed by lung mechanics, bronchioalveolar lavage cell counts, protein content, and lung injury scoring. Immunoblotting for MKP-1, and IκBα and cytokine ELISAs were performed on lung lysates. Measurements and Main Results: Prone positioning was protective against injurious ventilation in rats. Expression profiling demonstrated MKP-1 20-fold higher in rats ventilated prone rather than supine and regional reduction in p38 and c-jun N-terminal kinase activation. MKP-1−/− mice experienced amplified injury. PG490-88 improved static lung compliance and injury scores, reduced bronchioalveolar lavage cell counts and cytokine levels, and induced MKP-1 and IκBα. Conclusions: Injurious ventilation induces MAPK in an MKP-1–dependent fashion. Prone positioning is protective and induces MKP-1. PG490-88 induced MKP-1 and was protective against high Vt in a nuclear factor-κB–dependent manner. MKP-1 is a potential target for modulating regional effects of injurious ventilation. PMID:22582160

  11. Protein kinase CK2 modulates IL-6 expression in inflammatory breast cancer

    SciTech Connect

    Drygin, Denis Ho, Caroline B.; Omori, Mayuko; Bliesath, Joshua; Proffitt, Chris; Rice, Rachel; Siddiqui-Jain, Adam; O'Brien, Sean; Padgett, Claire; Lim, John K.C.; Anderes, Kenna; Rice, William G.; Ryckman, David

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer We examine the potential cross-talk between CK2 and IL-6. Black-Right-Pointing-Pointer Inhibition of CK2 by siRNA or CX-4945 inhibits expression of IL-6 in models of IBC. Black-Right-Pointing-Pointer Treatment of IBC patient in the clinic with CX-4945 reduces her IL-6 plasma levels. Black-Right-Pointing-Pointer We demonstrate that CK2 is a potential therapeutic target for IL-6 driven diseases. -- Abstract: Inflammatory breast cancer is driven by pro-angiogenic and pro-inflammatory cytokines. One of them Interleukin-6 (IL-6) is implicated in cancer cell proliferation and survival, and promotes angiogenesis, inflammation and metastasis. While IL-6 has been shown to be upregulated by several oncogenes, the mechanism behind this phenomenon is not well characterized. Here we demonstrate that the pleotropic Serine/Threonine kinase CK2 is implicated in the regulation of IL-6 expression in a model of inflammatory breast cancer. We used siRNAs targeted toward CK2 and a selective small molecule inhibitor of CK2, CX-4945, to inhibit the expression and thus suppress the secretion of IL-6 in in vitro as well as in vivo models. Moreover, we report that in a clinical trial, CX-4945 was able to dramatically reduce IL-6 levels in plasma of an inflammatory breast cancer patient. Our data shed a new light on the regulation of IL-6 expression and position CX-4945 and potentially other inhibitors of CK2, for the treatment of IL-6-driven cancers and possibly other diseases where IL-6 is instrumental, including rheumatoid arthritis.

  12. Inhibition of growth of established human glioma cell lines by modulators of the protein kinase-C system

    SciTech Connect

    Couldwell, W.T.; Antel, J.P.; Apuzzo, M.L.; Yong, V.W. )

    1990-10-01

    The protein kinase-C (PKC) second messenger system contributes to regulation of cell growth and differentiation. This study was undertaken to examine the effects of modulators of the PKC enzyme system on the state of differentiation and proliferation rates of human gliomas in vitro. The administration of the PKC-activating phorbol esters 4-beta-phorbol-12,13-dibutyrate (PDB) and phorbol-12-myristate-13-acetate (PMA) resulted in a dose-related inhibition of growth of human glioma cell lines in vitro as measured by 3H-thymidine uptake. The synthetic nonphorbol PKC activator (SC-9) produced an even more pronounced decrease of 3H-thymidine uptake. Diacylglycerol, an endogenous activator of the system, applied externally, transiently decreased the proliferation, in concordance with its short-lived existence in vivo. Conversely, the administration of 4-alpha-phorbol-12,13-didecanoate (alpha-PDD), a phorbol ester that binds but does not activate the enzyme, had no effect on the proliferation rate. At the dosages that maximally decreased proliferation, there was no evidence of direct glioma cell lysis induced by these agents as measured by a chromium-release assay. Immunocytochemical analysis and cytofluorometric measurement of glial fibrillary acidic protein (GFAP) staining in the treated cultures revealed an increase in GFAP staining over control cultures. In contrast to the response of glioma cells, nonmalignant human adult astrocytes treated with the PKC activators responded by increasing their proliferation rate. The authors postulate that the diametrically opposed effects of PKC activators on nonmalignant astrocytes versus glioma growth may be due to a high intrinsic PKC activity in glioma cells, with resultant down-regulation of enzyme activity following the administration of the pharmacological activators.

  13. The Arabidopsis SR45 Splicing Factor, a Negative Regulator of Sugar Signaling, Modulates SNF1-Related Protein Kinase 1 Stability.

    PubMed

    Carvalho, Raquel F; Szakonyi, Dóra; Simpson, Craig G; Barbosa, Inês C R; Brown, John W S; Baena-González, Elena; Duque, Paula

    2016-08-01

    The ability to sense and respond to sugar signals allows plants to cope with environmental and metabolic changes by adjusting growth and development accordingly. We previously reported that the SR45 splicing factor negatively regulates glucose signaling during early seedling development in Arabidopsis thaliana Here, we show that under glucose-fed conditions, the Arabidopsis sr45-1 loss-of-function mutant contains higher amounts of the energy-sensing SNF1-Related Protein Kinase 1 (SnRK1) despite unaffected SnRK1 transcript levels. In agreement, marker genes for SnRK1 activity are upregulated in sr45-1 plants, and the glucose hypersensitivity of sr45-1 is attenuated by disruption of the SnRK1 gene. Using a high-resolution RT-PCR panel, we found that the sr45-1 mutation broadly targets alternative splicing in vivo, including that of the SR45 pre-mRNA itself. Importantly, the enhanced SnRK1 levels in sr45-1 are suppressed by a proteasome inhibitor, indicating that SR45 promotes targeting of the SnRK1 protein for proteasomal destruction. Finally, we demonstrate that SR45 regulates alternative splicing of the Arabidopsis 5PTase13 gene, which encodes an inositol polyphosphate 5-phosphatase previously shown to interact with and regulate the stability of SnRK1 in vitro, thus providing a mechanistic link between SR45 function and the modulation of degradation of the SnRK1 energy sensor in response to sugars. PMID:27436712

  14. The C-terminal Kinase and ERK-binding Domains of Drosophila S6KII (RSK) Are Required for Phosphorylation of the Protein and Modulation of Circadian Behavior*

    PubMed Central

    Tangredi, Michelle M.; Ng, Fanny S.; Jackson, F. Rob

    2012-01-01

    A detailed structure/function analysis of Drosophila p90 ribosomal S6 kinase (S6KII) or its mammalian homolog RSK has not been performed in the context of neuronal plasticity or behavior. We previously reported that S6KII is required for normal circadian periodicity. Here we report a site-directed mutagenesis of S6KII and analysis of mutants, in vivo, that identifies functional domains and phosphorylation sites critical for the regulation of circadian period. We demonstrate, for the first time, a role for the S6KII C-terminal kinase that is independent of its known role in activation of the N-terminal kinase. Both S6KII C-terminal kinase activity and its ERK-binding domain are required for wild-type circadian period and normal phosphorylation status of the protein. In contrast, the N-terminal kinase of S6KII is dispensable for modulation of circadian period and normal phosphorylation of the protein. We also show that particular sites of S6KII phosphorylation, Ser-515 and Thr-732, are essential for normal circadian behavior. Surprisingly, the phosphorylation of S6KII residues, in vivo, does not follow a strict sequential pattern, as implied by certain cell-based studies of mammalian RSK protein. PMID:22447936

  15. Modulation of Cyclins, p53 and Mitogen-Activated Protein Kinases Signaling in Breast Cancer Cell Lines by 4-(3,4,5-Trimethoxyphenoxy)benzoic Acid

    PubMed Central

    Lee, Kuan-Han; Ho, Wen-Yueh; Wu, Shu-Jing; Omar, Hany A.; Huang, Po-Jui; Wang, Clay C. C.; Hung, Jui-Hsiang

    2014-01-01

    Despite the advances in cancer therapy and early detection, breast cancer remains a leading cause of cancer-related deaths among females worldwide. The aim of the current study was to investigate the antitumor activity of a novel compound, 4-(3,4,5-trimethoxyphenoxy)benzoic acid (TMPBA) and its mechanism of action, in breast cancer. Results indicated the relatively high sensitivity of human breast cancer cell-7 and MDA-468 cells towards TMPBA with IC50 values of 5.9 and 7.9 μM, respectively compared to hepatocarcinoma cell line Huh-7, hepatocarcinoma cell line HepG2, and cervical cancer cell line Hela cells. Mechanistically, TMPBA induced apoptotic cell death in MCF-7 cells as indicated by 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining, cell cycle analysis and the activation of caspase-3. Western blot analysis revealed the ability of TMPBA to target pathways mediated by mitogen-activated protein (MAP) kinases, 5′ adenosine monophosphate-activated protein kinase (AMPK), and p53, of which the concerted action underlined its antitumor efficacy. In addition, TMPBA induced alteration of cyclin proteins’ expression and consequently modulated the cell cycle. Taken together, the current study underscores evidence that TMPBA induces apoptosis in breast cancer cells via the modulation of cyclins and p53 expression as well as the modulation of AMPK and mitogen-activated protein kinases (MAPK) signaling. These findings support TMPBA’s clinical promise as a potential candidate for breast cancer therapy. PMID:24406729

  16. Constitutive modulation of Raf-1 protein kinase is associated with differential gene expression of several known and unknown genes.

    PubMed Central

    Patel, S.; Wang, F. H.; Whiteside, T. L.; Kasid, U.

    1997-01-01

    BACKGROUND: Raf-1, a cytoplasmic serine/threonine protein kinase, plays an important role in mitogen- and damage-responsive cellular signal transduction pathways. Consistent with this notion is the fact that constitutive modulation of expression and/or activity of Raf-1 protein kinase modifies cell growth, proliferation, and cell survival. Although these effects are controlled at least in part by transcriptional mechanisms, the role of Raf-1 in the regulation of specific gene expression is unclear. MATERIALS AND METHODS: Differential display of mRNA was used to identify the genes differentially expressed in human head and neck squamous carcinoma cells (PCI-06A) transfected with either the antisense c-raf-1 cDNA (PCI-06A-Raf(AS)), or a portion of cDNA coding for the kinase domain of Raf-1 (PCI-06A-Raf(K)). The differentially expressed fragments were cloned and sequenced, and they were used as probes to compare the expression patterns in parent transfectants by Northern blot analysis. In addition, expression patterns of the novel genes were examined in normal tissues and cancer cell lines. RESULTS: Six differentially expressed cDNA fragments were identified and sequenced. Northern blot analysis revealed that four of these fragments representing human alpha 1-antichymotrypsin (alpha 1-ACT), mitochondrial cytochrome c oxidase subunit II (COX-II), and two as-yet unidentified cDNAs (KAS-110 and KAS-111) were relatively overexpressed in PCI-06A-Raf(AS) transfectants compared with PCI-06A-Raf(K) transfectants. The other two cDNA fragments representing human elongation factor-1 alpha (HEF-1 alpha) and ornithine decarboxylase antizyme (OAz) were overexpressed in PCI-06A-Raf(K) transfectants compared with PCI-06A-Raf(AS) transfectants. The KAS-110 (114 bp) and KAS-111 (202 bp) cDNAs did not show significant matches with sequences in the GenEMBL, TIGR, and HGS DNA databases, and these may represent novel genes. The KAS-110 and KAS-111 transcripts, approximately 0.9 kb and

  17. Nuclear association of cyclin D1 in human fibroblasts: tight binding to nuclear structures and modulation by protein kinase inhibitors.

    PubMed

    Scovassi, A I; Stivala, L A; Rossi, L; Bianchi, L; Prosperi, E

    1997-11-25

    The association of cyclin D1 with nuclear structures was investigated in normal human fibroblasts by using hypotonic detergent extraction procedures, immunofluorescence quantitation with flow cytometry, and Western blot analysis. About 20% of the total cellular levels of cyclin D1 was found to be tightly bound to nuclear structures, being the complex formation resistant to DNase I treatment and to high salt extraction. Maximal levels of the insoluble form of the protein were found in the middle to late G1 phase of the cell cycle. Cell fractionation and immunoprecipitation techniques after in vivo 32P-labeling showed that both soluble and nuclear-bound forms of cyclin D1 were phosphorylated. Both fractions were reactive to an anti-phosphotyrosine antibody, while only the latter was detectable with an anti-phosphoserine antibody. Treatment with the protein kinase inhibitor staurosporine, which induces a cell cycle arrest in early G1 phase, strongly reduced cyclin D1 phosphorylation. Concomitantly, the ratio of nuclear-bound/total cyclin D1 levels was reduced by about 60%, compared with the control value. The protein kinase A specific inhibitor isoquinoline-sulfonamide (H-89) induced a similar reduction in the ratio, with no significant modification in the total amount of protein. In contrast, both calphostin C and bisindolylmaleimide, specific inhibitors of protein kinase C, consistently increased by 30-50% the ratio of nuclear-bound/total amount of the cyclin protein. These results suggest that, during the G1 phase, formation of an insoluble complex of cyclin D1 occurs at nuclear matrix structures and that this association is mediated by a protein kinase A-dependent pathway. PMID:9417875

  18. Steady-State Modulation of Voltage-Gated K+ Channels in Rat Arterial Smooth Muscle by Cyclic AMP-Dependent Protein Kinase and Protein Phosphatase 2B

    PubMed Central

    Brignell, Jennifer L.; Perry, Matthew D.; Nelson, Carl P.; Willets, Jonathon M.; Challiss, R. A. John; Davies, Noel W.

    2015-01-01

    Voltage-gated potassium channels (Kv) are important regulators of membrane potential in vascular smooth muscle cells, which is integral to controlling intracellular Ca2+ concentration and regulating vascular tone. Previous work indicates that Kv channels can be modulated by receptor-driven alterations of cyclic AMP-dependent protein kinase (PKA) activity. Here, we demonstrate that Kv channel activity is maintained by tonic activity of PKA. Whole-cell recording was used to assess the effect of manipulating PKA signalling on Kv and ATP-dependent K+ channels of rat mesenteric artery smooth muscle cells. Application of PKA inhibitors, KT5720 or H89, caused a significant inhibition of Kv currents. Tonic PKA-mediated activation of Kv appears maximal as application of isoprenaline (a β-adrenoceptor agonist) or dibutyryl-cAMP failed to enhance Kv currents. We also show that this modulation of Kv by PKA can be reversed by protein phosphatase 2B/calcineurin (PP2B). PKA-dependent inhibition of Kv by KT5720 can be abrogated by pre-treatment with the PP2B inhibitor cyclosporin A, or inclusion of a PP2B auto-inhibitory peptide in the pipette solution. Finally, we demonstrate that tonic PKA-mediated modulation of Kv requires intact caveolae. Pre-treatment of the cells with methyl-β-cyclodextrin to deplete cellular cholesterol, or adding caveolin-scaffolding domain peptide to the pipette solution to disrupt caveolae-dependent signalling each attenuated PKA-mediated modulation of the Kv current. These findings highlight a novel, caveolae-dependent, tonic modulatory role of PKA on Kv channels providing new insight into mechanisms and the potential for pharmacological manipulation of vascular tone. PMID:25793374

  19. Sensitization of human colon cancer cells to sodium butyrate-induced apoptosis by modulation of sphingosine kinase 2 and protein kinase D

    SciTech Connect

    Xiao, Min; Liu, Yungang; Zou, Fei

    2012-01-01

    Sphingosine kinases (SphKs) have been recognized as important proteins regulating cell proliferation and apoptosis. Of the two isoforms of SphK (SphK1 and SphK2), little is known about the functions of SphK2. Sodium butyrate (NaBT) has been established as a promising chemotherapeutic agent, but the precise mechanism for its effects is unknown. In this study, we investigated the role of SphK2 in NaBT-induced apoptosis of HCT116 colon cancer cells. The results indicated that following NaBT treatment SphK2 was translocated from the nucleus to the cytoplasm, leading to its accumulation in the cytoplasm; in the meantime, only mild apoptosis occurred. However, downregulation of SphK2 resulted in sensitized apoptosis, and overexpression of SphK2 led to even lighter apoptosis; these strongly indicate an inhibitory role of SphK2 in cell apoptosis induced by NaBT. After knocking down protein kinase D (PKD), another protein reported to be critical in cell proliferation/apoptosis process, by using siRNA, blockage of cytoplasmic accumulation of SphK2 and sensitized apoptosis following NaBT treatment were observed. The present study suggests that PKD and SphK2 may form a mechanism for the resistance of cancer cells to tumor chemotherapies, such as HCT116 colon cancer cells to NaBT, and these two proteins may become molecular targets for designation of new tumor-therapeutic drugs. -- Highlights: Black-Right-Pointing-Pointer In the present study sodium butyrate (10 mM) induced mild apoptosis of cancer cells. Black-Right-Pointing-Pointer The apoptosis was negatively regulated by cytoplasmic Sphingosine Kinase 2 (SphK2). Black-Right-Pointing-Pointer Translocation of SphK2 from nucleus to cytoplasm was mediated by protein kinase D. Black-Right-Pointing-Pointer Downregulation of SphK2 or protein kinase D leads to sensitized cell apoptosis.

  20. Spa2p functions as a scaffold-like protein to recruit the Mpk1p MAP kinase module to sites of polarized growth.

    PubMed

    van Drogen, Frank; Peter, Matthias

    2002-10-01

    Scaffold proteins play a major role in regulating MAP kinase pathways. In yeast, the Mpk1p-MAP kinase pathway functions to maintain the integrity of the cytoskeleton and the cell wall. In this module, the MEKK Bck1p functions upstream of the MEKs Mkk1p and Mkk2p, which in turn activate the MAP kinase Mpk1p. Mpk1p regulates several nuclear targets, including the transcription factors Rlm1p and SBF, and the two HMG1-like proteins NHP6A and NHP6B. Here we show that Mpk1p constitutively shuttles between the nucleus and the cytoplasm, and both Mpk1p and Mkk1p localize to sites of polarized growth in a Spa2p-dependent manner. Spa2p belongs to a group of proteins that includes Bni1p, Bud6p, and Pea2p, which are involved in the dynamic organization of the actin cytoskeleton during polarized growth. FRAP analysis shows that Spa2p-GFP is stably anchored at bud tips, whereas Mpk1p binds transiently. Spa2p interacts with Mkk1p and Mpk1p, and membrane bound Spa2p is sufficient to recruit Mkk1p and Mpk1p but not other MAP kinases to the cell cortex. Taken together, these results suggest that Spa2p functions as a scaffold-like protein for the cell wall integrity pathway during polarized growth. PMID:12361575

  1. The glial cell modulator ibudilast attenuates neuroinflammation and enhances retinal ganglion cell viability in glaucoma through protein kinase A signaling.

    PubMed

    Cueva Vargas, Jorge L; Belforte, Nicolas; Di Polo, Adriana

    2016-09-01

    Glaucoma is a neurodegenerative disease and the leading cause of irreversible blindness worldwide. Vision deficits in glaucoma result from the selective loss of retinal ganglion cells (RGC). Glial cell-mediated neuroinflammation has been proposed to contribute to disease pathophysiology, but whether this response is harmful or beneficial for RGC survival is not well understood. To test this, we characterized the role of ibudilast, a clinically approved cAMP phosphodiesterase (PDE) inhibitor with preferential affinity for PDE type 4 (PDE4). Here, we demonstrate that intraocular administration of ibudilast dampened macroglia and microglia reactivity in the retina and optic nerve hence decreasing production of proinflammatory cytokines in a rat model of ocular hypertension. Importantly, ibudilast promoted robust RGC soma survival, prevented axonal degeneration, and improved anterograde axonal transport in glaucomatous eyes without altering intraocular pressure. Intriguingly, ocular hypertension triggered upregulation of PDE4 subtype A in Müller glia, and ibudilast stimulated cAMP accumulation in these cells. Co-administration of ibudilast with Rp-cAMPS, a cell-permeable and non-hydrolysable cAMP analog that inhibits protein kinase A (PKA), completely blocked ibudilast-induced neuroprotection. Collectively, these data demonstrate that ibudilast, a safe and well-tolerated glial cell modulator, attenuates gliosis, decreases levels of proinflammatory mediators, and enhances neuronal viability in glaucoma through activation of the cAMP/PKA pathway. This study provides insight into PDE4 signaling as a potential target to counter the harmful effects associated with chronic gliosis and neuroinflammation in glaucoma. PMID:27163643

  2. Proteomic characterization of HIV-modulated membrane receptors, kinases and signaling proteins involved in novel angiogenic pathways

    PubMed Central

    Rasheed, Suraiya; Yan, Jasper S; Hussain, Adil; Lai, Bruce

    2009-01-01

    Background Kaposi's sarcoma (KS), hemangioma, and other angioproliferative diseases are highly prevalent in HIV-infected individuals. While KS is etiologically linked to the human herpesvirus-8 (HHV8) infection, HIV-patients without HHV-8 and those infected with unrelated viruses also develop angiopathies. Further, HIV-Tat can activate protein-tyrosine-kinase (PTK-activity) of the vascular endothelial growth factor receptor involved in stimulating angiogenic processes. However, Tat by itself or HHV8-genes alone cannot induce angiogenesis in vivo unless specific proteins/enzymes are produced synchronously by different cell-types. We therefore tested a hypothesis that chronic HIV-replication in non-endothelial cells may produce novel factors that provoke angiogenic pathways. Methods Genome-wide proteins from HIV-infected and uninfected T-lymphocytes were tested by subtractive proteomics analyses at various stages of virus and cell growth in vitro over a period of two years. Several thousand differentially regulated proteins were identified by mass spectrometry (MS) and >200 proteins were confirmed in multiple gels. Each protein was scrutinized extensively by protein-interaction-pathways, bioinformatics, and statistical analyses. Results By functional categorization, 31 proteins were identified to be associated with various signaling events involved in angiogenesis. 88% proteins were located in the plasma membrane or extracellular matrix and >90% were found to be essential for regeneration, neovascularization and angiogenic processes during embryonic development. Conclusion Chronic HIV-infection of T-cells produces membrane receptor-PTKs, serine-threonine kinases, growth factors, adhesion molecules and many diffusible signaling proteins that have not been previously reported in HIV-infected cells. Each protein has been associated with endothelial cell-growth, morphogenesis, sprouting, microvessel-formation and other biological processes involved in angiogenesis (p

  3. Imposed glutathione-mediated redox switch modulates the tobacco wound-induced protein kinase and salicylic acid-induced protein kinase activation state and impacts on defence against Pseudomonas syringae

    PubMed Central

    Matern, Sanja; Peskan-Berghoefer, Tatjana; Gromes, Roland; Kiesel, Rebecca Vazquez; Rausch, Thomas

    2015-01-01

    The role of the redox-active tripeptide glutathione in plant defence against pathogens has been studied extensively; however, the impact of changes in cellular glutathione redox potential on signalling processes during defence reactions has remained elusive. This study explored the impact of elevated glutathione content on the cytosolic redox potential and on early defence signalling at the level of mitogen-activated protein kinases (MAPKs), as well as on subsequent defence reactions, including changes in salicylic acid (SA) content, pathogenesis-related gene expression, callose depositions, and the hypersensitive response. Wild-type (WT) Nicotiana tabacum L. and transgenic high-glutathione lines (HGL) were transformed with the cytosol-targeted sensor GRX1-roGFP2 to monitor the cytosolic redox state. Surprisingly, HGLs displayed an oxidative shift in their cytosolic redox potential and an activation of the tobacco MAPKs wound-induced protein kinase (WIPK) and SA-induced protein kinase (SIPK). This activation occurred in the absence of any change in free SA content, but was accompanied by constitutively increased expression of several defence genes. Similarly, rapid activation of MAPKs could be induced in WT tobacco by exposure to either reduced or oxidized glutathione. When HGL plants were challenged with adapted or non-adapted Pseudomonas syringae pathovars, the cytosolic redox shift was further amplified and the defence response was markedly increased, showing a priming effect for SA and callose; however, the initial and transient hyperactivation of MAPK signalling was attenuated in HGLs. The results suggest that, in tobacco, MAPK and SA signalling may operate independently, both possibly being modulated by the glutathione redox potential. Possible mechanisms for redox-mediated MAPK activation are discussed. PMID:25628332

  4. Protein kinase C epsilon is localized to the Golgi via its zinc-finger domain and modulates Golgi function.

    PubMed Central

    Lehel, C; Olah, Z; Jakab, G; Anderson, W B

    1995-01-01

    Protein kinase C (PKC) is a multigene family of serine/threonine kinases that are central to many signal transduction pathways. Among the PKC isozymes, only PKC epsilon has been reported to exhibit full oncogenic potential. PKC epsilon also displays unique substrate specificity and intracellular localization. To examine the interrelationship between the biological effects and domain structure of PKC epsilon, NIH 3T3 cells were stably transfected to overexpress different epitope-tagged fragments of PKC epsilon. The overexpressed proteins each contain the epsilon-tag peptide at the C terminus to allow ready detection with an antibody specific for the tag. The holo-PKC epsilon was found to localize with the Golgi network and other compartments, whereas the zinc-finger domain localized exclusively at the Golgi. Golgi-specific glycosaminoglycan sulfation was strongly inhibited in cells overexpressing either holo-PKC epsilon or its zinc-finger domain, while the secretion of sulfated glycosaminoglycans into the medium was impaired in cells expressing the PKC epsilon zinc-finger domain. Thus, these results suggest that PKC epsilon may be involved in specifically regulating Golgi-related processes. Further, the results indicate that PKC epsilon domains other than the kinase domain may also have biological activity and that the zinc-finger domain may function as a subcellular localization signal. Images Fig. 1 Fig. 2 Fig. 3 PMID:7877991

  5. The regulation of GRP78 and messenger RNA levels by hypoxia is modulated by protein kinase C activators and inhibitors

    SciTech Connect

    Koong, A.C.; Auger, E.A.; Chen, E.Y.; Giaccia, A.J.

    1994-04-01

    In this study, we have shown that steady-state levels of glucose-regulated 78 kDa (GRP78) protein and messenger RNA increase during a 5-h exposure to 0.02% oxygen. This increase in GRP78 protein and mRNA induced by hypoxia can be abolished by a 1-h pretreatment of cells before hypoxia with the protein kinase C (PKC) inhibitors staurosporine and H7 at concentrations at which the drugs themselves do not cause cytotoxicity. Although all studies using protein kinase inhibitors must be interpreted with caution, staurosporine and H7 have been shown to be potent inhibitors of PKC activity, suggesting a role for PKC in mediating the transcriptional regulation of GRP78 by hypoxia. Further support for PKC in regulating GRP78 gene expression by hypoxia stems from gel-mobility shift studies in mixtures of nuclear extracts from aerobic or hypoxic cells with a 36 bp region of the GRP78 promoter (-170 to -135). Binding of this factor could be inhibited by pretreating cells with the PKC inhibitor staurosporine before hypoxia or activated by treating cells with the PKC-activating phorbol ester TPA. These data suggest that activation of this hypoxia-responsive factor is sensitive to oxygen levels and seems to be mediated through a PKC signal transduction pathway. 13 refs., 4 figs.

  6. α-Tocopherol at Nanomolar Concentration Protects PC12 Cells from Hydrogen Peroxide-Induced Death and Modulates Protein Kinase Activities

    PubMed Central

    Zakharova, Irina O.; Sokolova, Tatyana V.; Bayunova, Liubov V.; Vlasova, Yulia A.; Rychkova, Maria P.; Avrova, Natalia F.

    2012-01-01

    The aim of this work was to compare protective and anti-apoptotic effects of α-tocopherol at nanomolar and micromolar concentrations against 0.2 mM H2O2-induced toxicity in the PC12 neuronal cell line and to reveal protein kinases that contribute to α-tocopherol protective action. The protection by 100 nM α-tocopherol against H2O2-induced PC12 cell death was pronounced if the time of pre-incubation with α-tocopherol was 3–18 h. For the first time, the protective effect of α-tocopherol was shown to depend on its concentration in the nanomolar range (1 nM < 10 nM < 100 nM), if the pre-incubation time was 18 h. Nanomolar and micromolar α-tocopherol decreased the number of PC12 cells in late apoptosis induced by H2O2 to the same extent if pre-incubation time was 18 h. Immunoblotting data showed that α-tocopherol markedly diminished the time of maximal activation of extracellular signal-regulated kinase 1/2 (ERK 1/2) and protein kinase B (Akt)-induced in PC12 cells by H2O2. Inhibitors of MEK 1/2, PI 3-kinase and protein kinase C (PKC) diminished the protective effect of α-tocopherol against H2O2-initiated toxicity if the pre-incubation time was long. The modulation of ERK 1/2, Akt and PKC activities appears to participate in the protection by α-tocopherol against H2O2-induced death of PC12 cells. The data obtained suggest that inhibition by α-tocopherol in late stage ERK 1/2 and Akt activation induced by H2O2 in PC12 cells makes contribution to its protective effect, while total inhibition of these enzymes is not protective. PMID:23109870

  7. 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

  8. Protein Kinase CK2 Expression Predicts Relapse Survival in ERα Dependent Breast Cancer, and Modulates ERα Expression in Vitro

    PubMed Central

    Williams, Marlon D.; Nguyen, Thu; Carriere, Patrick P.; Tilghman, Syreeta L.; Williams, Christopher

    2015-01-01

    The heterotetrameric protein kinase CK2 has been associated with oncogenic transformation, and our previous studies have shown that it may affect estrogenic signaling. Here, we investigate the role of the protein kinase CK2 in regulating ERα (estrogen receptor α) signaling in breast cancer. We determined the correlation of CK2α expression with relapse free breast cancer patient survival utilizing Kaplan Meier Plotter (kmplot.com/analysis/) to mine breast cancer microarrays repositories. Patients were stratified according to ERα status, histological grade, and hormonal therapy. Luciferase reporter assays and flow cytometry were implemented to determine the impact of CK2 inhibition on ERE-mediated gene expression and expression of ERα protein. CK2α expression is associated with shorter relapse free survival among ERα (+) patients with grade 1 or 2 tumors, as well as among those patients receiving hormonal therapy. Biochemical inhibition of CK2 activity results in increased ER-transactivation as well as increased expression among ERα (+) and ERα (−) breast cancer cell lines. These findings suggest that CK2 may contribute to estrogen-independent cell proliferation and breast tumor progression, and may potentially serve as a biomarker and pharmacological target in breast cancer. PMID:26703694

  9. Osthole regulates inflammatory mediator expression through modulating NF-κB, mitogen-activated protein kinases, protein kinase C, and reactive oxygen species.

    PubMed

    Liao, Pei-Chun; Chien, Shih-Chang; Ho, Chen-Lung; Wang, Eugene I-Chen; Lee, Shu-Ching; Kuo, Yueh-Hsiung; Jeyashoke, Narumon; Chen, Jie; Dong, Wei-Chih; Chao, Louis Kuoping; Hua, Kuo-Feng

    2010-10-13

    Osthole, a coumarin compound, has been reported to exhibit various biological activities; however the cellular mechanism of its immune modulating activity has not yet been fully addressed. In this study we isolated osthole from the seeds of Cnidium monnieri and demonstrated that osthole inhibited TNF-α, NO and COX-2 expression in LPS-stimulated macrophages, without reducing the expression of IL-6. Furthermore, the phosphorylation of p38, JNK1/2, PKC-α and PKC-ε induced by LPS was inhibited by osthole; however, the phosphorylation of ERK1/2 and PKC-δ was not reduced by osthole. Osthole also inhibited NF-κB activation and ROS release in LPS-stimulated macrophages. Our current results indicated that osthole is the major anti-inflammatory ingredient of Cnidium monnieri seed ethanol extract. PMID:20839800

  10. Hexamethylenebisacetamide modulation of thyroglobulin and protein levels in thyroid cells is not mediated by phosphatidylinositol-3-kinase: a study with wortmannin.

    PubMed

    Aouani, A; Samih, N; Amphoux-Fazekas, T; Hovsépian, S; Fayet, G

    1999-04-01

    Hexamethylenebisacetamide (HMBA) induces in murine erythroleukemia cells (MELC) the commitment to terminal differentiation leading to globin gene expression. In the thyroid, HMBA acts as a growth factor and also as a differentiating agent. In the present paper, we studied the effect of HMBA on the very specific thyroid marker thyroglobulin (Tg) in two different thyroid cell systems, i.e., porcine cells in primary culture and ovine cells in long term culture. Using wortmannin, a specific inhibitor of phosphatidylinositol-3-kinase, we investigated whether this enzyme is involved in HMBA mode of action. We found that HMBA is a positive modulator of Tg production in porcine cells, but a negative effector in the OVNIS cell line. As all HMBA effects studied in the present paper, i.e., Tg production and total protein levels, are not inhibited by wortmannin, we suggest the non-involvement of phosphatidylinositol-3-kinase in HMBA mode of action. PMID:10650339

  11. Phosphorylation of coat protein by protein kinase CK2 regulates cell-to-cell movement of Bamboo mosaic virus through modulating RNA binding.

    PubMed

    Hung, Chien-Jen; Huang, Ying-Wen; Liou, Ming-Ru; Lee, Ya-Chien; Lin, Na-Sheng; Meng, Menghsiao; Tsai, Ching-Hsiu; Hu, Chung-Chi; Hsu, Yau-Heiu

    2014-11-01

    In this study, we investigated the fine regulation of cell-to-cell movement of Bamboo mosaic virus (BaMV). We report that the coat protein (CP) of BaMV is phosphorylated in planta at position serine 241 (S241), in a process involving Nicotiana benthamiana casein kinase 2α (NbCK2α). BaMV CP and NbCK2α colocalize at the plasmodesmata, suggesting that phosphorylation of BaMV may be involved in its movement. S241 was mutated to examine the effects of temporal and spatial dysregulation of phosphorylation on i) the interactions between CP and viral RNA and ii) the regulation of cell-to-cell movement. Replacement of S241 with alanine did not affect RNA binding affinity but moderately impaired cell-to-cell movement. A negative charge at position 241 reduced the ability of CP to bind RNA and severely interfered with cell-to-cell movement. Deletion of residues 240 to 242 increased the affinity of CP to viral RNA and dramatically impaired cell-to-cell movement. A threonine at position 241 changed the binding preference of CP toward genomic RNA and inhibited cell-to-cell movement. Together, these results reveal a fine regulatory mechanism for the cell-to-cell movement of BaMV, which involves the modulation of RNA binding affinity through appropriate phosphorylation of CP by NbCK2α. PMID:25025779

  12. Disease Resistance and Abiotic Stress Tolerance in Rice Are Inversely Modulated by an Abscisic Acid–Inducible Mitogen-Activated Protein KinaseW⃞

    PubMed Central

    Xiong, Lizhong; Yang, Yinong

    2003-01-01

    Mitogen-activated protein kinase (MAPK) cascades play an important role in mediating stress responses in eukaryotic organisms. However, little is known about the role of MAPKs in modulating the interaction of defense pathways activated by biotic and abiotic factors. In this study, we have isolated and functionally characterized a stress-responsive MAPK gene (OsMAPK5) from rice. OsMAPK5 is a single-copy gene but can generate at least two differentially spliced transcripts. The OsMAPK5 gene, its protein, and kinase activity were inducible by abscisic acid as well as various biotic (pathogen infection) and abiotic (wounding, drought, salt, and cold) stresses. To determine its biological function, we generated and analyzed transgenic rice plants with overexpression (using the 35S promoter of Cauliflower mosaic virus) or suppression (using double-stranded RNA interference [dsRNAi]) of OsMAPK5. Interestingly, suppression of OsMAPK5 expression and its kinase activity resulted in the constitutive expression of pathogenesis-related (PR) genes such as PR1 and PR10 in the dsRNAi transgenic plants and significantly enhanced resistance to fungal (Magnaporthe grisea) and bacterial (Burkholderia glumae) pathogens. However, these same dsRNAi lines had significant reductions in drought, salt, and cold tolerance. By contrast, overexpression lines exhibited increased OsMAPK5 kinase activity and increased tolerance to drought, salt, and cold stresses. These results strongly suggest that OsMAPK5 can positively regulate drought, salt, and cold tolerance and negatively modulate PR gene expression and broad-spectrum disease resistance. PMID:12615946

  13. 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

  14. Protein Crystals of Raf Kinase

    NASA Technical Reports Server (NTRS)

    1995-01-01

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

  15. Vitexin protects brain against ischemia/reperfusion injury via modulating mitogen-activated protein kinase and apoptosis signaling in mice.

    PubMed

    Wang, Yanan; Zhen, Yilan; Wu, Xian; Jiang, Qin; Li, Xiaoliang; Chen, Zhiwu; Zhang, Gongliang; Dong, Liuyi

    2015-03-15

    Vitexin is a major bioactive flavonoid compound derived from the dried leaf of hawthorn (Crataegus pinnatifida), a widely used conventional folk medicine in China. Recent studies have shown that vitexin presents neuroprotective effects in vitro. Whether this protective effect applies to the cerebral ischemia/reperfusion (I/R) injury remains elusive. In the present study, we examined the potential neuroprotective effect of vitexin against cerebral I/R injury and underlying mechanisms. A focal cerebral I/R model in male Kunming mice was induced by middle cerebral artery occlusion (MCAO) for 2 h followed by reperfusion for 22 h. The neurological function and infarct volume were assessed by using Long's five-point scale system and triphenyl-tetrazolium chloride (TTC) staining technique, respectively. Neuronal damage was evaluated by histological staining. Extracellular signal-regulated kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38 phosphorylation, and apoptosis were measured via Western blot at 24 h after reperfusion. As a result, systemic vitexin treatment significantly reduced neurological deficit, cerebral infarct volume and neuronal damage when compared with the I/R group. Western blot analyses revealed that vitexin markedly upregulated p-ERK1/2 and downregulated p-JNK and p-p38. Meanwhile, vitexin increased Bcl-2 expression and suppressed the overexpression of Bax in the I/R injury mice. In conclusion, the results indicate that vitexin protects brain against cerebral I/R injury, and this effect may be regulated by mitogen-activated protein kinase (MAPK) and apoptosis signaling pathways. PMID:25837275

  16. Protein kinase C delta (PKCδ) splice variant modulates senescence via hTERT in adipose-derived stem cells

    PubMed Central

    Carter, Gay; Patel, Rekha; Apostolatos, André; Murr, Michel; Cooper, Denise R.

    2014-01-01

    Background Adipose-derived stem cells (ADSC) were isolated and characterized from lean and obese subjects. We previously reported that distinct differences were observed in differentiating lean and obese preadipocytes. Protein kinase C delta (PKCδ) is alternatively spliced and has important roles in apoptosis. PKCδI promotes apoptosis and PKCδVIII promotes survival. Our previous data indicated an increase in the survival kinase, PKCδVIII in ADSC derived from an obese donor. We also determined that obese adipocytes were resistant to apoptosis. Here, we determine the relationship between a survival kinase PKCδVIII and hTERT expression in adipose derived stem cells from a lean and obese subject. Methods We evaluated the telomerase activity and human telomerase reverse transcriptase (hTERT) expression in lean and obese ADSC. The lean and obese ADSC were purchased as cryopreserved cells from ZenBio™ (Research Triangle Park, NC, USA). Analyses were performed using PRISM™ software and analyzed using two-tailed Student’s t-test. Results We observed an increase in telomerase in differentiating obese ADSC using western blot analysis. We determined the levels of hTERT splice variants. hTERT α+/β+ splice variant was increased after transfected of PKCδVIII. We next determined whether PKCδVIII over-expression affected the levels of telomerase. The results indicate an increase in telomerase with PKCδVIII over-expression. Conclusions Over-expression of PKCδVIII in lean ADSC substantially increased expression of hTERT and telomerase. The decreased senescence seen in obese ADSC may in part be attributed to PKCδVIII. Obese ADSC undergo lower senescence and may have increased growth potential. These results propose a larger epigenetic modification in obese ADSC compared to lean ADSC.

  17. Protein kinase D1 modulates aldosterone-induced ENaC activity in a renal cortical collecting duct cell line.

    PubMed

    McEneaney, Victoria; Dooley, Ruth; Yusef, Yamil R; Keating, Niamh; Quinn, Ursula; Harvey, Brian J; Thomas, Warren

    2010-08-30

    Aldosterone treatment of M1-CCD cells stimulated an increase in epithelial Na(+) channel (ENaC) alpha-subunit expression that was mainly localized to the apical membrane. PKD1-suppressed cells constitutively expressed ENaCalpha at low abundance, with no increase after aldosterone treatment. In the PKD1-suppressed cells, ENaCalpha was mainly localized proximal to the basolateral surface of the epithelium both before and after aldosterone treatment. Apical membrane insertion of ENaCbeta in response to aldosterone treatment was also sensitive to PKD1 suppression as was the aldosterone-induced rise in the amiloride-sensitive, trans-epithelial current (I(TE)). The interaction of the mineralocorticoid receptor (MR) with specific elements in the promoters of aldosterone responsive genes is stabilized by ligand interaction and phosphorylation. PKD1 suppression inhibited aldosterone-induced SGK-1 expression. The nuclear localization of MR was also blocked by PKD1 suppression and MEK antagonism implicating both these kinases in MR nuclear stabilization. PKD1 thus modulates aldosterone-induced ENaC activity through the modulation of sub-cellular trafficking and the stabilization of MR nuclear localization. PMID:20434520

  18. Ca2+/Calmodulin-Dependent Protein Kinase II Is a Modulator of CARMA1-Mediated NF-κB Activation†

    PubMed Central

    Ishiguro, Kazuhiro; Green, Todd; Rapley, Joseph; Wachtel, Heather; Giallourakis, Cosmas; Landry, Aimee; Cao, Zhifang; Lu, Naifang; Takafumi, Ando; Goto, Hidemi; Daly, Mark J.; Xavier, Ramnik J.

    2006-01-01

    CARMA1 is a central regulator of NF-κB activation in lymphocytes. CARMA1 and Bcl10 functionally interact and control NF-κB signaling downstream of the T-cell receptor (TCR). Computational analysis of expression neighborhoods of CARMA1-Bcl10MALT 1 for enrichment in kinases identified calmodulin-dependent protein kinase II (CaMKII) as an important component of this pathway. Here we report that Ca2+/CaMKII is redistributed to the immune synapse following T-cell activation and that CaMKII is critical for NF-κB activation induced by TCR stimulation. Furthermore, CaMKII enhances CARMA1-induced NF-κB activation. Moreover, we have shown that CaMKII phosphorylates CARMA1 on Ser109 and that the phosphorylation facilitates the interaction between CARMA1 and Bcl10. These results provide a novel function for CaMKII in TCR signaling and CARMA1-induced NF-κB activation. PMID:16809782

  19. Modulation of mitogen-activated protein kinases (MAPK) activity in response to different immune stimuli in haemocytes of the common periwinkle Littorina littorea.

    PubMed

    Iakovleva, Nadya V; Gorbushin, Alexander M; Storey, Kenneth B

    2006-09-01

    The modulation of mitogen-activated protein kinase (MAPK) activity in haemocytes of the common periwinkle (Littorina littorea) in response to immune challenges by lipopolysaccharide from Echerichia coli (LPS), mannan from baker's yeast Saccharomyces cerevisiae and secretory-excretory products (SEP) of trematodes Himasthla elongata (Echinostomatidae) or after the treatment with phorbol ester (PMA) has been studied by Western blotting using affinity purified rabbit polyclonal antibodies. Exposure of the cells in suspension to PMA, LPS and mannan triggered an activation of p38 and ERK2. The JNK-mediated cascade was modulated differently by the elicitors examined. PMA treatment caused a transient activation of the JNK54 isoform, LPS exposure resulted in a decrease in activity of JNK46, and mannan had no effect on JNK phosphorylation status. Incubation of periwinkle haemocytes in culture medium containing trematode SEP did not affect the activity of any MAPK. PMID:16533608

  20. AKT serine/threonine protein kinase modulates baicalin-triggered autophagy in human bladder cancer T24 cells.

    PubMed

    Lin, Chingju; Tsai, Shih-Chang; Tseng, Michael T; Peng, Shu-Fen; Kuo, Sheng-Chu; Lin, Meng-Wei; Hsu, Yuan-Man; Lee, Miau-Rong; Amagaya, Sakae; Huang, Wen-Wen; Wu, Tian-Shung; Yang, Jai-Sing

    2013-03-01

    Baicalin is one of the major compounds in the traditional Chinese medicinal herb from Scutellaria baicalensis Georgi. We investigated the molecular mechanisms of cell autophagy induced by baicalin in human bladder cancer T24 cells. Baicalin inhibited cell survival as shown by MTT assay and increased cell death by trypan blue exclusion assay in a concentration-dependent manner. Baicalin did not induce apoptotic cell death in T24 cells by TUNEL and caspase-3 activity assay. Baicalin induced the acidic vesicular organelle cell autophagy marker, manifested by acridine orange (AO) and monodansylcadaverine (MDC) staining and cleavage of microtubule-associated protein 1 light chain 3 (LC3). The protein expression levels of the Atg 5, Atg 7, Atg 12, Beclin-1 and LC3-II were upregulated in T24 cells after baicalin treatment. Inhibition of autophagy by 3-methyl-adenine (an inhibitor of class III phosphatidylinositol-3 kinase; 3-MA) reduced the cleavage of LC3 in T24 cells after baicalin treatment. Furthermore, protein expression levels of phospho-AKT (Ser473) and enzyme activity of AKT were downregulated in T24 cells after baicalin treatment. In conclusion, baicalin triggered cell autophagy through the AKT signaling pathway in T24 cells. PMID:23354080

  1. 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.

  2. Involvement of Ca2+/calmodulin-dependent protein kinase II in the modulation of indolamines in diabetic and hyperglycemic rats.

    PubMed

    Ramakrishnan, R; Prabhakaran, K; Jayakumar, A R; Gunasekaran, P; Sheeladevi, R; Suthanthirarajan, N

    2005-05-15

    Hyperglycemia and acidosis are the key factors in diabetic complications. It has been shown that acute or chronic diabetes alters serotonin levels in brain. However, the mechanism of hyperglycemia- or acidosis-induced changes in serotonin levels remains poorly understood. Because Ca2+-dependent protein kinases play a major role in the regulation of serotonin synthesis and release, we investigated the effect of diabetes, hyperglycemia, and acidosis on the level of indolamines [5-hydroxytryptamine (5-HT) and/or 5-hydroxyindoleacetic acid (5-HIAA)] and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) enzyme activity or protein expression in different brain regions. Alloxan-induced (45 mg/kg bw) diabetic rats (30 days) showed increased level of 5-HT in striatum (ST; 183%), midbrain (MB; 199%), pons medulla (PM; 151%), cerebellum (CB; 214%), and cerebral cortex (CCX; 162%) compared with control (P < 0.05), and these changes were reversed after insulin administration. Rats treated with glucose (500 mg/kg bw) for 30 days showed a 146%, 183%, 208%, and 177% (P < 0.05) increase in 5-HT levels in ST, PM, CB, and CCX, respectively. 5-HIAA level increased in hippocampus (HC; 172%) and in MB (145%; P < 0.05). In addition, rats treated with sodium acetoacetate (NaAcAc) for 30 days (60 mg/kg bw) showed significant increases (P < 0.05) of 5-HT level in ST (152%) and MB (174%). However, the levels of 5-HIAA increased only in MB (151%, P < 0.05). Rats treated with NH4Cl, which induced acidosis (150 mg/kg bw), showed an increased level of 5-HT only in HC (165%, P < 0.05). The increased activity and protein expression of CaMKII in ST, MB, PM, CB, and CCX under diabetic conditions were correlated with the levels of indolamines changes during diabetic, hyperglycemic, or acidotic conditions. These results suggest that CaMKII may be involved in the regulation of indolamines in diabetic animals. PMID:15846780

  3. All-trans retinoic acid modulates mitogen-activated protein kinase pathway activation in human scleral fibroblasts through retinoic acid receptor beta

    PubMed Central

    Huo, Lijun; Cui, Dongmei; Yang, Xiao; Gao, Zhenya; Trier, Klaus

    2013-01-01

    Purpose All-trans retinoic acid (ATRA) is known to inhibit the proliferation of human scleral fibroblasts (HSFs) and to modulate the scleral intercellular matrix composition, and may therefore serve as a mediator for controlling eye growth. Cell proliferation is regulated by the mitogen-activated protein kinase (MAPK) pathway. The aim of the current study was to investigate whether changed activation of the MAPK pathway could be involved in the response of HSFs exposed to ATRA. Methods HSFs were cultured in Dulbecco Modified Eagle's Medium/F12 (DMEM/F12) and exposed to 1 μmol/l ATRA for 10 min, 30 min, 1 h, 8 h, or 24 h. The activation of extracellular signal-regulated kinase (ERK 1/2), p38, and c-Jun N-terminal kinase (JNK) in HSFs was assessed with western blot analysis and immunocytofluorescence. Results After exposure to ATRA for 24 h, the HSFs appeared shrunken and thinner than the control cells. The intercellular spaces were wider, and the HSFs appeared less numerous than in the control culture. Western blot showed decreased activation of ERK 1/2 in the HSFs from 30 min (p=0.01) to 24 h (p<0.01) after the start of exposure to ATRA, and increased activation of the JNK protein from 10 to 30 min (p<0.01) after the start of exposure to ATRA. Indirect immunofluorescence confirmed changes in activation of ERK 1/2 and JNK in HSFs exposed to ATRA. No change in activation of p38 in HSFs was observed after exposure to ATRA. Pretreatment of the HSFs with LE135, an antagonist of retinoic acid receptor beta (RARβ), abolished the ATRA-induced changes inactivation of ERK 1/2 and JNK. Conclusions ATRA inhibits HSF proliferation by a mechanism associated with modulation of ERK 1/2 and JNK activation and depends on stimulation of retinoic acid receptor beta. PMID:23946634

  4. LDL-cholesterol reduction in patients with hypercholesterolemia by modulation of adenosine triphosphate-citrate lyase and adenosine monophosphate-activated protein kinase

    PubMed Central

    Filippov, Sergey; Pinkosky, Stephen L.; Newton, Roger S.

    2014-01-01

    Purpose of review To review the profile of ETC-1002, as shown in preclinical and clinical studies, including LDL-cholesterol (LDL-C)-lowering activity and beneficial effects on other cardiometabolic risk markers as they relate to the inhibition of adenosine triphosphate-citrate lyase and the activation of adenosine monophosphate-activated protein kinase. Recent findings ETC-1002 is an adenosine triphosphate-citrate lyase inhibitor/adenosine monophosphate-activated protein kinase activator currently in Phase 2b clinical development. In seven Phase 1 and Phase 2a clinical studies, ETC-1002 dosed once daily for 2–12 weeks has lowered LDL-C and reduced high-sensitivity C-reactive protein by up to 40%, with neutral to positive effects on glucose levels, blood pressure, and body weight. Importantly, use of ETC-1002 in statin-intolerant patients has shown statin-like lowering of LDL-C without the muscle pain and weakness responsible for discontinuation of statin use by many patients. ETC-1002 has also been shown to produce an incremental benefit, lowering LDL-C as an add-on therapy to a low-dose statin. In over 300 individuals in studies of up to 12 weeks, ETC-1002 has been well tolerated with no serious adverse effects. Summary Because adenosine triphosphate-citrate lyase and adenosine monophosphate-activated protein kinase play central roles in regulating lipid and glucose metabolism, pharmacological modulation of these two enzymes could provide an important therapeutic alternative for statin-intolerant patients with hypercholesterolemia. PMID:24978142

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. Adult cardiac fibroblast proliferation is modulated by calcium/calmodulin-dependent protein kinase II in normal and hypertrophied hearts.

    PubMed

    Martin, Tamara P; Lawan, Ahmed; Robinson, Emma; Grieve, David J; Plevin, Robin; Paul, Andrew; Currie, Susan

    2014-02-01

    Increased adult cardiac fibroblast proliferation results in an increased collagen deposition responsible for the fibrosis accompanying pathological remodelling of the heart. The mechanisms regulating cardiac fibroblast proliferation remain poorly understood. Using a minimally invasive transverse aortic banding (MTAB) mouse model of cardiac hypertrophy, we have assessed fibrosis and cardiac fibroblast proliferation. We have investigated whether calcium/calmodulin-dependent protein kinase IIδ (CaMKIIδ) regulates proliferation in fibroblasts isolated from normal and hypertrophied hearts. It is known that CaMKIIδ plays a central role in cardiac myocyte contractility, but nothing is known of its role in adult cardiac fibroblast function. The MTAB model used here produces extensive hypertrophy and fibrosis. CaMKIIδ protein expression and activity is upregulated in MTAB hearts and, specifically, in cardiac fibroblasts isolated from hypertrophied hearts. In response to angiotensin II, cardiac fibroblasts isolated from MTAB hearts show increased proliferation rates. Inhibition of CaMKII with autocamtide inhibitory peptide inhibits proliferation in cells isolated from both sham and MTAB hearts, with a significantly greater effect evident in MTAB cells. These results are the first to show selective upregulation of CaMKIIδ in adult cardiac fibroblasts following cardiac hypertrophy and to assign a previously unrecognised role to CaMKII in regulating adult cardiac fibroblast function in normal and diseased hearts. PMID:23881186

  16. Mitogen and stress-activated protein kinase 1 (MSK1) modulates photic entrainment of the suprachiasmatic circadian clock

    PubMed Central

    Cao, Ruifeng; Butcher, Greg Q.; Karelina, Kate; Arthur, J. Simon C.; Obrietan, Karl

    2013-01-01

    The master circadian clock in mammals, the suprachiasmatic nucleus (SCN), is under the entraining influence of the external light cycle. At a mechanistic level, intracellular signaling via the p42/44 mitogen-activated protein kinase (MAPK) pathway appears to play a central role in light-evoked clock entrainment; however, the precise downstream mechanisms by which this pathway influences clock timing are not known. Within this context, we have previously reported that light stimulates activation of the MAPK effector mitogen stress activated kinase 1 (MSK1) in the SCN. In this study we utilized MSK1-/- mice to further investigate the potential role of MSK1 in circadian clock timing and entrainment. Locomotor activity analysis revealed that MSK1 null mice entrained to a 12h light/dark cycle and exhibited circadian free-running rhythms in constant darkness. Interestingly, the free running period in MSK1 null mice was significantly longer than WT control animals, and MSK1 null mice exhibited a significantly greater variance in activity onset. Further, MSK1 null mice exhibited a significant reduction in the phase delaying response to an early night light pulse (100 lux, 15 min), and, using an 8-hr phase-advancing “jet-lag” experimental paradigm MSK1 knockout animals exhibited a significantly delayed rate of re-entrainment. At the molecular level, early night light-evoked CREB phosphorylation, histone phosphorylation and Period1 gene expression were markedly attenuated in MSK1-/- animals relative to WT mice. Together, these data provide key new insights into the molecular mechanisms by which MSK1 affects the SCN clock. PMID:23127194

  17. 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

  18. Modulation of human basophil histamine release by protein kinase C inhibitors differs with secretagogue and with inhibitor.

    PubMed

    Bergstrand, H; Lundquist, B; Karabelas, K; Michelsen, P

    1992-03-01

    To assess possible involvement of protein kinase C (PKC) in human basophil degranulation, the present work compared effects of various purported PKC inhibitors on leukocyte histamine release triggered by different stimuli. The effects recorded varied with the inhibitor and the secretagogue used; moreover, with a given secretagogue, different inhibitors often displayed different activities. Thus, histamine release triggered by the PKC activator 4 beta-phorbol 12-myristate 13-acetate was blocked by K252a, staurosporine and the purported specific PKC inhibitor Ro 31-7549, and reduced by calphostin C, H-7, TMB-8 and W-7 but not affected by polymyxin B; it was augmented by 2.1 microM palmitoyl carnitine. The leukocyte response induced by another putative activator of PKC, 1,2-isopropylidene-3-decanoyl-sn-glycerol, was also enhanced by 2.1 microM palmitoyl carnitine, slightly increased by staurosporine, TMB-8 and W-7 but not affected by calphostin C, H-7, K252a or Ro 31-7549, whereas the hyperosmolar mannitol-induced response was reduced by H-7, calphostin C, TMB-8 and W-7 and slightly augmented by staurosporine. Anti-IgE-induced histamine release was blocked by staurosporine and K252a and reduced by calphostin C, sphingosine, TMB-8 and W-7 but not affected by H-7, polymyxin B or retinal. It was enhanced by Ro 31-7549. In contrast, leukocyte histamine release induced by calcium ionophore A23187 or by ionomycin was blocked by retinal, TMB-8 and W-7 and reduced by calphostin C and palmitoyl carnitine but enhanced by H-7, staurosporine and polymyxin B; K252a and Ro 31-7549 did not affect such responses. Formyl-methionyl-leucyl-phenylalanine-triggered histamine release was barely affected by any agent used. Thus, the specific PKC inhibitor Ro 31-7549 selectively blocked 4 beta-phorbol 12-myristate 13-acetate-triggered leukocyte histamine release. These results imply that examined secretagogues trigger human leukocyte histamine release through partly separate pathways

  19. A calcium and free fatty acid-modulated protein kinase as putative effector of the fusicoccin 14-3-3 receptor.

    PubMed Central

    van der Hoeven, P C; Siderius, M; Korthout, H A; Drabkin, A V; de Boer, A H

    1996-01-01

    A protein kinase that is activated by calcium and cis-unsaturated fatty acids has been characterized from oat (Avena sativa L.) root plasma membranes. The kinase phosphorylates a synthetic peptide with a motif (-R-T-L-S-) that can be phosphorylated by both protein kinase C (PKC) and calcium-dependent protein kinase (CDPK)-type kinases. Calphostin C and chelerythrine, two PKC inhibitors, completely inhibited the kinase activity with values of inhibitor concentration for 50% inhibition of 0.7 and 30 microns, respectively. At low Ca2+ concentrations cis-unsaturated fatty acids (linolenic acid, linoleic acid, arachidonic acid, and oleic acid) stimulated the kinase activity almost 10-fold. The two inhibitors of the kinase, calphostin C and chelerythrin, strongly reduced the fusicoccin (FC)-induced H+ extrusion, and the activators of the kinase, the cis-unsaturated fatty acids, prevented [3H]FC binding to the FC 14-3-3 receptor. CDPK antibodies cross-reacted with a 43-kD band in the plasma membrane and in a purified FC receptor fraction. A polypeptide with the same apparent molecular mass was recognized by a synthetic peptide that has a sequence homologous to the annexin-like domain from barely 14-3-3. The possibility of the involvement of a kinase, with properties from both CDPK and PKC, and a phospholipase A2 in the FC Signal transduction pathway is discussed. PMID:8754686

  20. Leishmania Infection Engages Non-Receptor Protein Kinases Differentially to Persist in Infected Hosts

    PubMed Central

    Zhang, Naixin; Kima, Peter E.

    2016-01-01

    Protein kinases play important roles in the regulation of cellular activities. In cells infected by pathogens, there is an increasing appreciation that dysregulated expression of protein kinases promotes the success of intracellular infections. In Leishmania-infected cells, expression and activation of protein kinases, such as the mitogen-activated protein kinases, kinases in the PI3-kinase signaling pathway, and kinases in the NF-κB-signaling pathway, are modulated in some manner. Several recent reviews have discussed our current understanding of the roles of these kinases in Leishmania infections. Apart from the kinases in the pathways enumerated above, there are other host cell protein kinases that are activated during the Leishmania infection of mammalian cells whose roles also appear to be significant. This review discusses recent observations on the Abl family of protein kinases and the protein kinase regulated by RNA in Leishmania infections. PMID:27148265

  1. Homeodomain-interacting protein kinase 2, a novel autoimmune regulator interaction partner, modulates promiscuous gene expression in medullary thymic epithelial cells.

    PubMed

    Rattay, Kristin; Claude, Janine; Rezavandy, Esmail; Matt, Sonja; Hofmann, Thomas G; Kyewski, Bruno; Derbinski, Jens

    2015-02-01

    Promiscuous expression of a plethora of tissue-restricted Ags (TRAs) by medullary thymic epithelial cells (mTECs) plays an essential role in T cell tolerance. Although the cellular mechanisms by which promiscuous gene expression (pGE) imposes T cell tolerance have been well characterized, the underlying molecular mechanisms remain poorly understood. The autoimmune regulator (AIRE) is to date the only validated molecule known to regulate pGE. AIRE is part of higher-order multiprotein complexes, which promote transcription, elongation, and splicing of a wide range of target genes. How AIRE and its partners mediate these various effects at the molecular level is still largely unclear. Using a yeast two-hybrid screen, we searched for novel AIRE-interacting proteins and identified the homeodomain-interacting protein kinase 2 (HIPK2) as a novel partner. HIPK2 partially colocalized with AIRE in nuclear bodies upon cotransfection and in human mTECs in situ. Moreover, HIPK2 phosphorylated AIRE in vitro and suppressed the coactivator activity of AIRE in a kinase-dependent manner. To evaluate the role of Hipk2 in modulating the function of AIRE in vivo, we compared whole-genome gene signatures of purified mTEC subsets from TEC-specific Hipk2 knockout mice with control mice and identified a small set of differentially expressed genes. Unexpectedly, most differentially expressed genes were confined to the CD80(lo) mTEC subset and preferentially included AIRE-independent TRAs. Thus, although it modulates gene expression in mTECs and in addition affects the size of the medullary compartment, TEC-specific HIPK2 deletion only mildly affects AIRE-directed pGE in vivo. PMID:25552543

  2. BGLF4 Kinase Modulates the Structure and Transport Preference of the Nuclear Pore Complex To Facilitate Nuclear Import of Epstein-Barr Virus Lytic Proteins

    PubMed Central

    Chang, Chou-Wei; Lee, Chung-Pei; Su, Mei-Tzu; Tsai, Ching-Hwa

    2014-01-01

    nucleocapsids. Because nuclear pore complexes are effective gateways that control nucleocytoplasmic traffic, most EBV proteins without canonical NLSs are retained in the cytoplasm until they form complexes with their NLS-containing partners for nuclear targeting. In this study, we found that EBV BGLF4 protein kinase interacts with the Nup62 and Nup153 and induces the redistribution of FG-Nups. BGLF4 modulates the function of the NPC to inhibit the nuclear import of host NLS-containing proteins. Simultaneously, the nuclear import of non-NLS-containing EBV lytic proteins was enhanced, possibly through phosphorylation of Nup62 and Nup153, nuclear pore dilation, or microtubule reorganization. Overall, our data suggest that BGLF4-induced modification of nuclear pore transport may block nuclear targeting of cellular proteins and increase the import of viral proteins to promote viral lytic replication. PMID:25410863

  3. 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

  4. Energy-dependent modulation of glucagon-like signaling in Drosophila via the AMP-activated protein kinase.

    PubMed

    Braco, Jason T; Gillespie, Emily L; Alberto, Gregory E; Brenman, Jay E; Johnson, Erik C

    2012-10-01

    Adipokinetic hormone (AKH) is the equivalent of mammalian glucagon, as it is the primary insect hormone that causes energy mobilization. In Drosophila, current knowledge of the mechanisms regulating AKH signaling is limited. Here, we report that AMP-activated protein kinase (AMPK) is critical for normal AKH secretion during periods of metabolic challenges. Reduction of AMPK in AKH cells causes a suite of behavioral and physiological phenotypes resembling AKH cell ablations. Specifically, reduced AMPK function increases life span during starvation and delays starvation-induced hyperactivity. Neither AKH cell survival nor gene expression is significantly impacted by reduced AMPK function. AKH immunolabeling was significantly higher in animals with reduced AMPK function; this result is paralleled by genetic inhibition of synaptic release, suggesting that AMPK promotes AKH secretion. We observed reduced secretion in AKH cells bearing AMPK mutations employing a specific secretion reporter, confirming that AMPK functions in AKH secretion. Live-cell imaging of wild-type AKH neuroendocrine cells shows heightened excitability under reduced sugar levels, and this response was delayed and reduced in AMPK-deficient backgrounds. Furthermore, AMPK activation in AKH cells increases intracellular calcium levels in constant high sugar levels, suggesting that the underlying mechanism of AMPK action is modification of ionic currents. These results demonstrate that AMPK signaling is a critical feature that regulates AKH secretion, and, ultimately, metabolic homeostasis. The significance of these findings is that AMPK is important in the regulation of glucagon signaling, suggesting that the organization of metabolic networks is highly conserved and that AMPK plays a prominent role in these networks. PMID:22798489

  5. Energy-Dependent Modulation of Glucagon-Like Signaling in Drosophila via the AMP-Activated Protein Kinase

    PubMed Central

    Braco, Jason T.; Gillespie, Emily L.; Alberto, Gregory E.; Brenman, Jay E.; Johnson, Erik C.

    2012-01-01

    Adipokinetic hormone (AKH) is the equivalent of mammalian glucagon, as it is the primary insect hormone that causes energy mobilization. In Drosophila, current knowledge of the mechanisms regulating AKH signaling is limited. Here, we report that AMP-activated protein kinase (AMPK) is critical for normal AKH secretion during periods of metabolic challenges. Reduction of AMPK in AKH cells causes a suite of behavioral and physiological phenotypes resembling AKH cell ablations. Specifically, reduced AMPK function increases life span during starvation and delays starvation-induced hyperactivity. Neither AKH cell survival nor gene expression is significantly impacted by reduced AMPK function. AKH immunolabeling was significantly higher in animals with reduced AMPK function; this result is paralleled by genetic inhibition of synaptic release, suggesting that AMPK promotes AKH secretion. We observed reduced secretion in AKH cells bearing AMPK mutations employing a specific secretion reporter, confirming that AMPK functions in AKH secretion. Live-cell imaging of wild-type AKH neuroendocrine cells shows heightened excitability under reduced sugar levels, and this response was delayed and reduced in AMPK-deficient backgrounds. Furthermore, AMPK activation in AKH cells increases intracellular calcium levels in constant high sugar levels, suggesting that the underlying mechanism of AMPK action is modification of ionic currents. These results demonstrate that AMPK signaling is a critical feature that regulates AKH secretion, and, ultimately, metabolic homeostasis. The significance of these findings is that AMPK is important in the regulation of glucagon signaling, suggesting that the organization of metabolic networks is highly conserved and that AMPK plays a prominent role in these networks. PMID:22798489

  6. Diets enriched with cereal brans or inulin modulate protein kinase C activity and isozyme expression in rat colonic mucosa.

    PubMed

    Pajari, A M; Oikarinen, S; Gråsten, S; Mutanen, M

    2000-11-01

    The role of dietary fibres in colon carcinogenesis is controversial. To elucidate the mechanisms by which different dietary fibre sources may affect colonic tumour development, we studied the effects of diets enriched with cereal brans or inulin on protein kinase C (PKC) activity and isozyme expression in rat colon. Male Wistar rats (twelve per group) were fed one of the following AIN-93G-based diets (Reeves et al. 1993) for 4 weeks: a non-fibre high-fat diet or one of the four high-fat diets supplemented with either rye, oat or wheat bran or inulin at 100 g/kg diet. The fat concentration (20 g/100 g) and fatty acid composition of the non-fibre high-fat diet was designed to approximate that in a typical Western-type diet. In the proximal colon, rats fed the inulin diet had a significantly higher membrane PKC activity and a higher membrane PKC delta level than rats fed the non-fibre diet In the distal colon, rats fed the inulin and oat bran diets had a higher total PKC activity and a higher membrane PKC beta 2 level than rats fed the wheat-bran diet. Rats in the non-fibre and wheat-bran groups had the lowest concentrations of luminal diacylglycerol. In conclusion, feeding of wheat bran resulted in low distal PKC activity and expression of PKC beta 2, a PKC isozyme related to colonic cell proliferation and increased susceptibility for colon carcinogenesis, which may explain in part the protective effect of wheat bran against tumour development in a number of experimental colon cancer studies. The increase in PKC activity and PKC beta 2 expression by feeding inulin may be a drawback of inulin as a functional food. PMID:11177176

  7. Modulation of Spc1 stress-activated protein kinase activity by methylglyoxal through inhibition of protein phosphatase in the fission yeast Schizosaccharomyces pombe

    SciTech Connect

    Takatsume, Yoshifumi; Izawa, Shingo; Inoue, Yoshiharu

    2007-11-30

    Methylglyoxal, a ubiquitous metabolite derived from glycolysis has diverse physiological functions in yeast cells. Previously, we have reported that extracellularly added methylglyoxal activates Spc1, a stress-activated protein kinase (SAPK), in the fission yeast Schizosaccharomyces pombe [Y. Takatsume, S. Izawa, Y. Inoue, J. Biol. Chem. 281 (2006) 9086-9092]. Phosphorylation of Spc1 by treatment with methylglyoxal in S. pombe cells defective in glyoxalase I, an enzyme crucial for the metabolism of methylglyoxal, continues for a longer period than in wild-type cells. Here we show that methylglyoxal inhibits the activity of the protein phosphatase responsible for the dephosphorylation of Spc1 in vitro. In addition, we found that methylglyoxal inhibits human protein tyrosine phosphatase 1B (PTP1B) also. We propose a model for the regulation of the activity of the Spc1-SAPK signaling pathway by methylglyoxal in S. pombe.

  8. Molecular basis for the modulation of native T-type Ca2+ channels in vivo by Ca2+ /calmodulin-dependent protein kinase II

    PubMed Central

    Yao, Junlan; Davies, Lucinda A.; Howard, Jason D.; Adney, Scott K.; Welsby, Philip J.; Howell, Nancy; Carey, Robert M.; Colbran, Roger J.; Barrett, Paula Q.

    2006-01-01

    Ang II receptor activation increases cytosolic Ca2+ levels to enhance the synthesis and secretion of aldosterone, a recently identified early pathogenic stimulus that adversely influences cardiovascular homeostasis. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a downstream effector of the Ang II–elicited signaling cascade that serves as a key intracellular Ca2+ sensor to feedback-regulate Ca2+ entry through voltage-gated Ca2+ channels. However, the molecular mechanism(s) by which CaMKII regulates these important physiological targets to increase Ca2+ entry remain unresolved. We show here that CaMKII forms a signaling complex with α1H T-type Ca2+ channels, directly interacting with the intracellular loop connecting domains II and III of the channel pore (II-III loop). Activation of the kinase mediated the phosphorylation of Ser1198 in the II-III loop and the positive feedback regulation of channel gating both in intact cells in situ and in cells of the native adrenal zona glomerulosa stimulated by Ang II in vivo. These data define the molecular basis for the in vivo modulation of native T-type Ca2+ channels by CaMKII and suggest that the disruption of this signaling complex in the zona glomerulosa may provide a new therapeutic approach to limit aldosterone production and cardiovascular disease progression. PMID:16917542

  9. BAM 1 and RECEPTOR-LIKE PROTEIN KINASE 2 constitute a signaling pathway and modulate CLE peptide-triggered growth inhibition in Arabidopsis root.

    PubMed

    Shimizu, Noriko; Ishida, Takashi; Yamada, Masashi; Shigenobu, Shuji; Tabata, Ryo; Kinoshita, Atsuko; Yamaguchi, Katsushi; Hasebe, Mitsuyasu; Mitsumasu, Kanako; Sawa, Shinichiro

    2015-12-01

    Ligand receptor-based signaling is a means of cell-to-cell communication for coordinating developmental and physiological processes in multicellular organisms. In plants, cell-producing meristems utilize this signaling to regulate their activities and ensure for proper development. Shoot and root systems share common requirements for carrying out this process; however, its molecular basis is largely unclear. It has been suggested that synthetic CLV3/EMBRYO SURROUNDING REGION (CLE) peptide shrinks the root meristem through the actions of CLAVATA2 (CLV2) and the RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) pathway in Arabidopsis thaliana. Our genetic screening for mutations that resist CLE peptide signaling in roots determined that BAM1, which is a member of the leucine-rich repeat receptor-like kinase (LRR-RLK) family, is also involved in this pathway. BAM1 is preferentially expressed in the root tip, including the quiescent center and its surrounding stem cells. Our genetic analysis revealed that BAM1 functions together with RPK2. Using coimmunoprecipitation assay, we showed that BAM1 is capable of forming heteromeric complexes with RPK2. These findings suggest that the BAM1 and RPK2 receptors constitute a signaling pathway that modulates cell proliferation in the root meristem and that related molecules are employed in root and shoot meristems. PMID:26083273

  10. Rapeseed calcineurin B-like protein CBL4, interacting with CBL-interacting protein kinase CIPK24, modulates salt tolerance in plants.

    PubMed

    Liu, Wu-Zhen; Deng, Min; Li, Liang; Yang, Bo; Li, Hongwei; Deng, Hanqing; Jiang, Yuan-Qing

    2015-11-20

    Calcium is a ubiquitous intracellular secondary messenger in eukaryotes. Upon stress challenge, cytosolic Ca(2+) fluctuation could be sensed and bound by calcineurin B-like proteins (CBLs), which further regulate a group of Ser/Thr protein kinases called CBL-interacting protein kinases (CIPKs) to relay the signal and induce cellular responses. Although the CBL-CIPK network has been demonstrated to play crucial roles in plant development and responses to various environmental stresses in Arabidopsis, little is known about their function in rapeseed. In the present study, we characterized CBL4 gene from rapeseed. We found that CBL4 is localized at the plasma membrane and it interacted with CIPK24 in both yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays. Unlike the orthologs in Arabidopsis, rapeseed CIPK24 did not interact with CBL10. Furthermore, expression of rapeseed CBL4 rescued the salt-sensitive phenotype of sos3-1 mutant and overexpression of rapeseed CBL4 in Arabidopsis showed enhanced tolerance of salt stress than wild-type. Overall, the results clarified the function of CBL4 in rapeseed. PMID:26462466

  11. Regulation of mitogen-activated protein kinase by protein kinase C and mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle.

    PubMed

    Trappanese, Danielle M; Sivilich, Sarah; Ets, Hillevi K; Kako, Farah; Autieri, Michael V; Moreland, Robert S

    2016-06-01

    Vascular smooth muscle contraction is primarily regulated by phosphorylation of myosin light chain. There are also modulatory pathways that control the final level of force development. We tested the hypothesis that protein kinase C (PKC) and mitogen-activated protein (MAP) kinase modulate vascular smooth muscle activity via effects on MAP kinase phosphatase-1 (MKP-1). Swine carotid arteries were mounted for isometric force recording and subjected to histamine stimulation in the presence and absence of inhibitors of PKC [bisindolylmaleimide-1 (Bis)], MAP kinase kinase (MEK) (U0126), and MKP-1 (sanguinarine) and flash frozen for measurement of MAP kinase, PKC-potentiated myosin phosphatase inhibitor 17 (CPI-17), and caldesmon phosphorylation levels. CPI-17 was phosphorylated in response to histamine and was inhibited in the presence of Bis. Caldesmon phosphorylation levels increased in response to histamine stimulation and were decreased in response to MEK inhibition but were not affected by the addition of Bis. Inhibition of PKC significantly increased p42 MAP kinase, but not p44 MAP kinase. Inhibition of MEK with U0126 inhibited both p42 and p44 MAP kinase activity. Inhibition of MKP-1 with sanguinarine blocked the Bis-dependent increase of MAP kinase activity. Sanguinarine alone increased MAP kinase activity due to its effects on MKP-1. Sanguinarine increased MKP-1 phosphorylation, which was inhibited by inhibition of MAP kinase. This suggests that MAP kinase has a negative feedback role in inhibiting MKP-1 activity. Therefore, PKC catalyzes MKP-1 phosphorylation, which is reversed by MAP kinase. Thus the fine tuning of vascular contraction is due to the concerted effort of PKC, MAP kinase, and MKP-1. PMID:27053523

  12. In vivo phosphorylation of the Na,K-ATPase alpha subunit in sciatic nerves of control and diabetic rats: effects of protein kinase modulators.

    PubMed Central

    Borghini, I; Geering, K; Gjinovci, A; Wollheim, C B; Pralong, W F

    1994-01-01

    The phosphorylation state of the Na,K-ATPase alpha subunit has been examined in 32P-labeled sciatic nerves of control and streptozotocin-treated diabetic rats. Intact nerves were challenged with protein kinase (PK) modulators and alpha-subunit 32P labeling was analyzed after immunoprecipitation. In control nerves, the PKC activator phorbol 12-myristate 13-acetate (PMA) had little effect on alpha-subunit 32P labeling. In contrast, staurosporine, a PKC inhibitor, and extracellular calcium omission decreased it. In Ca(2+)-free conditions, PMA restored the labeling to basal levels. The cAMP-raising agent forskolin reduced the 32P labeling of the alpha subunit. The results suggest that nerve Na,K-ATPase is tonically phosphorylated by PKC in a Ca(2+)-dependent manner and that PKA modulates the phosphorylation process. In nerves of diabetic rats, PMA increased 32P labeling of the alpha subunit. In contrast to staurosporine or extracellular calcium omission, the decreased state of phosphorylation seen with forskolin was no longer significant in diabetic nerves. No change in the level of alpha-subunit isoforms (alpha 1 or alpha 2) was detected by Western blot analysis in such nerves. In conclusion, the altered effect of PK activators on Na,K-ATPase phosphorylation state is consistent with the view that a defect in PKC activation exists in diabetic nerves. Images PMID:8016140

  13. IMPACT Is a Developmentally Regulated Protein in Neurons That Opposes the Eukaryotic Initiation Factor 2α Kinase GCN2 in the modulation of Neurite Outgrowth*

    PubMed Central

    Roffé, Martín; Hajj, Glaucia N. M.; Azevedo, Hátylas F.; Alves, Viviane S.; Castilho, Beatriz A.

    2013-01-01

    The product of the mouse Imprinted and Ancient gene, IMPACT, is preferentially expressed in neurons. We have previously shown that IMPACT overexpression inhibits the activation of the protein kinase GCN2, which signals amino acid starvation. GCN2 phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in inhibition of general protein synthesis but increased translation of specific messages, such as ATF4. GCN2 is also involved in the regulation of neuronal functions, controlling synaptic plasticity, memory, and feeding behavior. We show here that IMPACT abundance increases during differentiation of neurons and neuron-like N2a cells, whereas GCN2 displays lowered activation levels. Upon differentiation, IMPACT associates with translating ribosomes, enhances translation initiation, and down-regulates the expression of ATF4. We further show that endogenous IMPACT promotes neurite outgrowth whereas GCN2 is a strong inhibitor of spontaneous neuritogenesis. Together, these results uncover the participation of the GCN2-IMPACT module of translational regulation in a highly controlled step in the development of the nervous system. PMID:23447528

  14. IMPACT is a developmentally regulated protein in neurons that opposes the eukaryotic initiation factor 2α kinase GCN2 in the modulation of neurite outgrowth.

    PubMed

    Roffé, Martín; Hajj, Glaucia N M; Azevedo, Hátylas F; Alves, Viviane S; Castilho, Beatriz A

    2013-04-12

    The product of the mouse Imprinted and Ancient gene, IMPACT, is preferentially expressed in neurons. We have previously shown that IMPACT overexpression inhibits the activation of the protein kinase GCN2, which signals amino acid starvation. GCN2 phosphorylates the α-subunit of eukaryotic translation initiation factor 2 (eIF2α), resulting in inhibition of general protein synthesis but increased translation of specific messages, such as ATF4. GCN2 is also involved in the regulation of neuronal functions, controlling synaptic plasticity, memory, and feeding behavior. We show here that IMPACT abundance increases during differentiation of neurons and neuron-like N2a cells, whereas GCN2 displays lowered activation levels. Upon differentiation, IMPACT associates with translating ribosomes, enhances translation initiation, and down-regulates the expression of ATF4. We further show that endogenous IMPACT promotes neurite outgrowth whereas GCN2 is a strong inhibitor of spontaneous neuritogenesis. Together, these results uncover the participation of the GCN2-IMPACT module of translational regulation in a highly controlled step in the development of the nervous system. PMID:23447528

  15. Inverse modulation of the energy sensor Snf1-related protein kinase 1 on hypoxia adaptation and salt stress tolerance in Arabidopsis thaliana.

    PubMed

    Im, Jong Hee; Cho, Young-Hee; Kim, Geun-Don; Kang, Geun-Ho; Hong, Jung-Woo; Yoo, Sang-Dong

    2014-10-01

    Terrestrial plants are exposed to complex stresses of high salt-induced abscisic acid (ABA) and submergence-induced hypoxia when seawater floods fields. Many studies have investigated plant responses to individual stress conditions, but not so much for coupled or sequentially imposed stresses. We examined molecular regulatory mechanisms of gene expression underlying the cellular responses involved in crosstalk between salt and hypoxia stresses. Salt/ABA- and AtMYC2-dependent induction of a synthetic ABA-responsive element and the native RD22 promoters were utilized in our cell-based functional assays. Such promoter-based reporter induction was largely inhibited by hypoxia and hypoxia-inducible AKIN10 activity. Biochemical analyses showed that AKIN10 negatively modulates AtMYC2 protein accumulation via proteasome activity upon AKIN10 kinase activity-dependent protein modification. Further genetic analysis using transgenic plants expressing AKIN10 provided evidence that AKIN10 activity undermined AtMYC2-dependent salt tolerance. Our findings unravel a novel molecular interaction between the key signalling constituents leading crosstalk between salt and hypoxia stresses in Arabidopsis thaliana under the detrimental condition of submergence in saltwater. PMID:24890857

  16. Allosteric modulation of AURKA kinase activity by a small-molecule inhibitor of its protein-protein interaction with TPX2.

    PubMed

    Janeček, Matej; Rossmann, Maxim; Sharma, Pooja; Emery, Amy; Huggins, David J; Stockwell, Simon R; Stokes, Jamie E; Tan, Yaw S; Almeida, Estrella Guarino; Hardwick, Bryn; Narvaez, Ana J; Hyvönen, Marko; Spring, David R; McKenzie, Grahame J; Venkitaraman, Ashok R

    2016-01-01

    The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the 'Y pocket') that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases. PMID:27339427

  17. Allosteric modulation of AURKA kinase activity by a small-molecule inhibitor of its protein-protein interaction with TPX2

    PubMed Central

    Janeček, Matej; Rossmann, Maxim; Sharma, Pooja; Emery, Amy; Huggins, David J.; Stockwell, Simon R.; Stokes, Jamie E.; Tan, Yaw S.; Almeida, Estrella Guarino; Hardwick, Bryn; Narvaez, Ana J.; Hyvönen, Marko; Spring, David R.; McKenzie, Grahame J.; Venkitaraman, Ashok R.

    2016-01-01

    The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the ‘Y pocket’) that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases. PMID:27339427

  18. Cytoskeletal protein kinases: titin and its relations in mechanosensing.

    PubMed

    Gautel, Mathias

    2011-07-01

    Titin, the giant elastic ruler protein of striated muscle sarcomeres, contains a catalytic kinase domain related to a family of intrasterically regulated protein kinases. The most extensively studied member of this branch of the human kinome is the Ca(2+)-calmodulin (CaM)-regulated myosin light-chain kinases (MLCK). However, not all kinases of the MLCK branch are functional MLCKs, and about half lack a CaM binding site in their C-terminal autoinhibitory tail (AI). A unifying feature is their association with the cytoskeleton, mostly via actin and myosin filaments. Titin kinase, similar to its invertebrate analogue twitchin kinase and likely other "MLCKs", is not Ca(2+)-calmodulin-activated. Recently, local protein unfolding of the C-terminal AI has emerged as a common mechanism in the activation of CaM kinases. Single-molecule data suggested that opening of the TK active site could also be achieved by mechanical unfolding of the AI. Mechanical modulation of catalytic activity might thus allow cytoskeletal signalling proteins to act as mechanosensors, creating feedback mechanisms between cytoskeletal tension and tension generation or cellular remodelling. Similar to other MLCK-like kinases like DRAK2 and DAPK1, TK is linked to protein turnover regulation via the autophagy/lysosomal system, suggesting the MLCK-like kinases have common functions beyond contraction regulation. PMID:21416260

  19. Protein Kinase C Pharmacology: Refining the Toolbox

    PubMed Central

    Wu-Zhang, Alyssa X.; Newton, Alexandra C.

    2014-01-01

    SYNOPSIS Protein kinase C (PKC) has been in the limelight since the discovery three decades ago that it acts as a major receptor for the tumor-promoting phorbol esters. Phorbol esters, with their potent ability to activate two of the three classes of PKC isozymes, have remained the best pharmacological tool for directly modulating PKC activity. However, with the discovery of other phorbol ester-responsive proteins, the advent of various small-molecule and peptide modulators, and the need to distinguish isozyme-specific activity, the pharmacology of PKC has become increasingly complex. Not surprisingly, many of the compounds originally touted as direct modulators of PKC have subsequently been shown to hit many other cellular targets and, in some cases, not even directly modulate PKC. The complexities and reversals in PKC pharmacology have led to widespread confusion about the current status of the pharmacological tools available to control PKC activity. Here, we aim to clarify the cacophony in the literature regarding the current state of bona fide and discredited cellular PKC modulators, including activators, small-molecule inhibitors, and peptides, and also address the use of genetically-encoded reporters and of PKC mutants to measure the effects of these drugs on the spatiotemporal dynamics of signaling by specific isozymes. PMID:23662807

  20. Epigenetic modulation of the protein kinase A RIIα (PRKAR2A) gene by histone deacetylases 1 and 2 in human smooth muscle cells

    PubMed Central

    Karolczak-Bayatti, Magdalena; Loughney, Andrew D; Robson, Stephen C; Europe-Finner, G Nicholas

    2011-01-01

    Abstract Recently we reported that the expression of the protein kinase A (PKA) regulatory subunit RIIα is dynamically regulated in human smooth muscle cells of the uterus. We showed that expression levels of mRNA/protein were substantially increased during pregnancy and decreased upon labour, changes that were mirrored by particulate type II PKA activity. This implied an important role for RIIα in maintaining uterine quiescence during pregnancy. Consequently the purpose of the present study was to identify potential mechanisms by which expression of the RIIα gene was regulated in this tissue. We indicate here that the three SpI-III (GC) binding domains within the proximal promoter region of the human RIIα gene may play important roles in modulating expression of the gene in human myometrial cells. We show that all three GC binding domains are involved in binding Sp1, Sp3, histone deacetylase (HDACs) 1/2 and RbAp48 transcriptional complexes. The functional significance of these binding domains was further analysed employing in vitro luciferase reporter assays with full-length/truncated RIIα promoter constructs. Importantly we show that treatment of primary human myometrial cell cultures with the general class I/II HDAC inhibitor trichostatin A results in an increase in mRNA/protein levels. Moreover the increase in mRNA levels appeared to be preceded by an increase in aH3, PolIIa, Sp3 and HDAC 2 binding to the three SpI-III (GC) binding sites within the RIIα promoter. These results enable us to provide a model whereby RIIα expression is epigenetically regulated in human myometrial smooth muscle cells by histone deacetylase(s) activity within the GC-rich proximal promoter region of the gene. PMID:19818097

  1. Modulation of the human cardiac sodium channel alpha-subunit by cAMP-dependent protein kinase and the responsible sequence domain.

    PubMed Central

    Frohnwieser, B; Chen, L Q; Schreibmayer, W; Kallen, R G

    1997-01-01

    1. In order to investigate the modulation of human hH1 sodium channel alpha-subunits by cAMP-dependent protein kinase (PKA), the channel was expressed in oocytes of Xenopus laevis. 2. Cytosolic injection of cAMP, as well as of SP-cyclic 3',5'-hydrogen phosphorothioate adenosine triethylammonium salt (SP-cAMPS, the S-diastereoisomeric configuration of the compound with respect to the phosphorus atom), resulted in a marked and significant increase in peak sodium current (INa,p). Cytosolic injections of RP-cyclic 3',5'-hydrogen phosphorothioate adenosine triethylammonium salt (RP-cAMPS; a compound inhibitory to PKA) had no effect on peak current. 3. Kinetic parameters of steady-state activation, inactivation and recovery from inactivation were unchanged following stimulation of PKA activity, but a 42 +/- 5% (mean +/- S.E.M.) increase in maximal sodium conductance (delta gmax) could account for the observed increase in INa,p. 4. A set of chimerical sodium channels made from portions of the human cardiac hH1 alpha-subunit and the rat skeletal muscle SkM1 alpha-subunit (which is not affected by PKA stimulation) was generated. These were used to localize the structural determinant in the hH1 sequence responsible for PKA modulation of hH1. From our data we conclude that the effects of PKA on hH1 are conferred by the large cytosolic loop interconnecting transmembrane domains I and II, which is not conserved among sodium channel subtypes. Images Figure 1 Figure 5 Figure 6 PMID:9032680

  2. 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. PMID:27226575

  3. Long Wavelength Monitoring of Protein Kinase Activity

    PubMed Central

    Oien, Nathan P.; Nguyen, Luong T.; Jernigan, Finith E.; Priestman, Melanie A.

    2014-01-01

    A family of long wavelength protein kinase fluorescent reporters is described in which the probing wavelength is pre-programmed using readily available fluorophores. These agents can assess protein kinase activity within the optical window of tissue, as exemplified by monitoring endogenous cAMP-dependent protein kinase activity (1) in erythrocyte lysates and (2) in intact erythrocytes using a light-activatable reporter. PMID:24604833

  4. Signaling of the human calcium-sensing receptor expressed in HEK293-cells is modulated by protein kinases A and C.

    PubMed

    Bösel, J; John, M; Freichel, M; Blind, E

    2003-02-01

    In this study, the human calcium-sensing receptor (CaR) stably expressed in HEK293 cells was investigated with regard to the phosphorylation-induced desensitization of its signaling pathway. The receptor is known to activate the phospholipase C/inositol-1,4,5-trisphosphate (IP 3 ) signaling cascade, thus stimulating protein kinase C (PKC). In contrast, the adenylylcyclase/cAMP signaling pathway that activates protein kinase A (PKA) is believed to be coupled to the receptor via an inhibitory G-protein. We elucidated the roles of PKC and PKA by measuring Ca 2+o -stimulated accumulation of total inositol phosphates and by individually and simultaneously inhibiting the two kinases pharmacologically in HEK293 cells, which stably expressed the human CaR. Pharmacological inhibition of PKC resulted in a 5-fold enhancement of IP 3 signaling, whereas blocking PKA had almost no effect. IP 3 signaling activity increased even more (10-fold) however, when the two kinases were inhibited simultaneously. Apart from validating the role of PKC as a potent down-regulator of signaling of the human CaR in this cell system, this study suggests that both kinases synergize in inhibiting Ca 2+o -stimulated IP 3 signaling in CaR-transfected HEK293 cells. PMID:12605346

  5. Mitogen activated protein kinase at the nuclear pore complex

    PubMed Central

    Faustino, Randolph S; Maddaford, Thane G; Pierce, Grant N

    2011-01-01

    Abstract Mitogen activated protein (MAP) kinases control eukaryotic proliferation, and import of kinases into the nucleus through the nuclear pore complex (NPC) can influence gene expression to affect cellular growth, cell viability and homeostatic function. The NPC is a critical regulatory checkpoint for nucleocytoplasmic traffic that regulates gene expression and cell growth, and MAP kinases may be physically associated with the NPC to modulate transport. In the present study, highly enriched NPC fractions were isolated and investigated for associated kinases and/or activity. Endogenous kinase activity was identified within the NPC fraction, which phosphorylated a 30 kD nuclear pore protein. Phosphomodification of this nucleoporin, here termed Nup30, was inhibited by apigenin and PD-98059, two MAP kinase antagonists as well as with SB-202190, a pharmacological blocker of p38. Furthermore, high throughput profiling of enriched NPCs revealed constitutive presence of all members of the MAP kinase family, extracellular regulated kinases (ERK), p38 and Jun N-terminal kinase. The NPC thus contains a spectrum of associated MAP kinases that suggests an intimate role for ERK and p38 in regulation of nuclear pore function. PMID:20497490

  6. Structural Basis of Ribosomal S6 Kinase 1 (RSK1) Inhibition by S100B Protein: MODULATION OF THE EXTRACELLULAR SIGNAL-REGULATED KINASE (ERK) SIGNALING CASCADE IN A CALCIUM-DEPENDENT WAY.

    PubMed

    Gógl, Gergő; Alexa, Anita; Kiss, Bence; Katona, Gergely; Kovács, Mihály; Bodor, Andrea; Reményi, Attila; Nyitray, László

    2016-01-01

    Mitogen-activated protein kinases (MAPK) promote MAPK-activated protein kinase activation. In the MAPK pathway responsible for cell growth, ERK2 initiates the first phosphorylation event on RSK1, which is inhibited by Ca(2+)-binding S100 proteins in malignant melanomas. Here, we present a detailed in vitro biochemical and structural characterization of the S100B-RSK1 interaction. The Ca(2+)-dependent binding of S100B to the calcium/calmodulin-dependent protein kinase (CaMK)-type domain of RSK1 is reminiscent of the better known binding of calmodulin to CaMKII. Although S100B-RSK1 and the calmodulin-CAMKII system are clearly distinct functionally, they demonstrate how unrelated intracellular Ca(2+)-binding proteins could influence the activity of the CaMK domain-containing protein kinases. Our crystallographic, small angle x-ray scattering, and NMR analysis revealed that S100B forms a "fuzzy" complex with RSK1 peptide ligands. Based on fast-kinetics experiments, we conclude that the binding involves both conformation selection and induced fit steps. Knowledge of the structural basis of this interaction could facilitate therapeutic targeting of melanomas. PMID:26527685

  7. Plant protein kinase substrates identification using protein microarrays.

    PubMed

    Ma, Shisong; Dinesh-Kumar, Savithramma P

    2015-01-01

    Protein kinases regulate signaling pathways by phosphorylating their targets. They play critical roles in plant signaling networks. Although many important protein kinases have been identified in plants, their substrates are largely unknown. We have developed and produced plant protein microarrays with more than 15,000 purified plant proteins. Here, we describe a detailed protocol to use these microarrays to identify plant protein kinase substrates via in vitro phosphorylation assays on these arrays. PMID:25930701

  8. Phosphorylation of Krüppel-like factor 3 (KLF3/BKLF) and C-terminal binding protein 2 (CtBP2) by homeodomain-interacting protein kinase 2 (HIPK2) modulates KLF3 DNA binding and activity.

    PubMed

    Dewi, Vitri; Kwok, Alister; Lee, Stella; Lee, Ming Min; Tan, Yee Mun; Nicholas, Hannah R; Isono, Kyo-ichi; Wienert, Beeke; Mak, Ka Sin; Knights, Alexander J; Quinlan, Kate G R; Cordwell, Stuart J; Funnell, Alister P W; Pearson, Richard C M; Crossley, Merlin

    2015-03-27

    Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3. PMID:25659434

  9. Modulation of nicotinic ACh-, GABAA- and 5-HT3-receptor functions by external H-7, a protein kinase inhibitor, in rat sensory neurones

    PubMed Central

    Hu, Hong-Zhen; Li, Zhi-Wang

    1997-01-01

    The effects of external H-7, a potent protein kinase inhibitor, on the responses mediated by γ-aminobutyric acid A type (GABAA)-, nicotinic acetylcholine (nicotinic ACh)-, ionotropic 5-hydroxytryptamine (5-HT3)-, adenosine 5′-triphosphate (ATP)-, N-methyl-D-aspartate (NMDA)- and kainate (KA)-receptors were studied in freshly dissociated rat dorsal root ganglion neurone by use of whole cell patch-clamp technique. External H-7 (1–1000 μM) produced a reversible, dose-dependent inhibition of whole cell currents activated by GABA, ACh and 5-HT. Whole-cell currents evoked by ATP, 2-methylthio-ATP, NMDA and KA were insensitive to external H-7. External H-7 shifted the dose-response curve of GABA-activated currents downward without changing the EC50 significantly (from 15.0±4.0 μM to 18.0±5.0 μM). The maximum response to GABA was depressed by 34.0±5.3%. This inhibitory action of H-7 was voltage-independent. Intracellular application of H-7 (20 μM), cyclic AMP (1 mM) and BAPTA (10 mM) could not reverse the H-7 inhibition of GABA-activated currents. The results suggest that external H-7 selectively and allosterically modulates the functions of GABAA-, nicotine ACh- and 5-HT3 receptors via a common conserved site in the external domain of these receptors. PMID:9401786

  10. Giant protein kinases: domain interactions and structural basis of autoregulation.

    PubMed Central

    Kobe, B; Heierhorst, J; Feil, S C; Parker, M W; Benian, G M; Weiss, K R; Kemp, B E

    1996-01-01

    The myosin-associated giant protein kinases twitchin and titin are composed predominantly of fibronectin- and immunoglobulin-like modules. We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C-terminal immunoglobulin-like domain. The structure of the longer fragment shows that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues. Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin-dependent kinase I. Images PMID:9003756

  11. The Predikin webserver: improved prediction of protein kinase peptide specificity using structural information

    PubMed Central

    Saunders, Neil F. W.

    2008-01-01

    The Predikin webserver allows users to predict substrates of protein kinases. The Predikin system is built from three components: a database of protein kinase substrates that links phosphorylation sites with specific protein kinase sequences; a perl module to analyse query protein kinases and a web interface through which users can submit protein kinases for analysis. The Predikin perl module provides methods to (i) locate protein kinase catalytic domains in a sequence, (ii) classify them by type or family, (iii) identify substrate-determining residues, (iv) generate weighted scoring matrices using three different methods, (v) extract putative phosphorylation sites in query substrate sequences and (vi) score phosphorylation sites for a given kinase, using optional filters. The web interface provides user-friendly access to each of these functions and allows users to obtain rapidly a set of predictions that they can export for further analysis. The server is available at http://predikin.biosci.uq.edu.au. PMID:18477637

  12. Sphingosine kinases and their metabolites modulate endolysosomal trafficking in photoreceptors

    PubMed Central

    Yonamine, Ikuko; Bamba, Takeshi; Nirala, Niraj K.; Jesmin, Nahid; Kosakowska-Cholody, Teresa; Nagashima, Kunio; Fukusaki, Eiichiro

    2011-01-01

    Internalized membrane proteins are either transported to late endosomes and lysosomes for degradation or recycled to the plasma membrane. Although proteins involved in trafficking and sorting have been well studied, far less is known about the lipid molecules that regulate the intracellular trafficking of membrane proteins. We studied the function of sphingosine kinases and their metabolites in endosomal trafficking using Drosophila melanogaster photoreceptors as a model system. Gain- and loss-of-function analyses show that sphingosine kinases affect trafficking of the G protein–coupled receptor Rhodopsin and the light-sensitive transient receptor potential (TRP) channel by modulating the levels of dihydrosphingosine 1 phosphate (DHS1P) and sphingosine 1 phosphate (S1P). An increase in DHS1P levels relative to S1P leads to the enhanced lysosomal degradation of Rhodopsin and TRP and retinal degeneration in wild-type photoreceptors. Our results suggest that sphingosine kinases and their metabolites modulate photoreceptor homeostasis by influencing endolysosomal trafficking of Rhodopsin and TRP. PMID:21321100

  13. Fibronectin phosphorylation by ecto-protein kinase

    SciTech Connect

    Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru )

    1988-12-01

    The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with ({gamma}-{sup 32})ATP for 10 min at 37{degree}C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with ({gamma}-{sup 32}P)ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation.

  14. 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.

  15. 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

  16. Dynamic architecture of a protein kinase

    PubMed Central

    McClendon, Christopher L.; Kornev, Alexandr P.; Gilson, Michael K.; Taylor, Susan S.

    2014-01-01

    Protein kinases are dynamically regulated signaling proteins that act as switches in the cell by phosphorylating target proteins. To establish a framework for analyzing linkages between structure, function, dynamics, and allostery in protein kinases, we carried out multiple microsecond-scale molecular-dynamics simulations of protein kinase A (PKA), an exemplar active kinase. We identified residue–residue correlated motions based on the concept of mutual information and used the Girvan–Newman method to partition PKA into structurally contiguous “communities.” Most of these communities included 40–60 residues and were associated with a particular protein kinase function or a regulatory mechanism, and well-known motifs based on sequence and secondary structure were often split into different communities. The observed community maps were sensitive to the presence of different ligands and provide a new framework for interpreting long-distance allosteric coupling. Communication between different communities was also in agreement with the previously defined architecture of the protein kinase core based on the “hydrophobic spine” network. This finding gives us confidence in suggesting that community analyses can be used for other protein kinases and will provide an efficient tool for structural biologists. The communities also allow us to think about allosteric consequences of mutations that are linked to disease. PMID:25319261

  17. Protein kinase C, an elusive therapeutic target?

    PubMed Central

    Mochly-Rosen, Daria; Das, Kanad; Grimes, Kevin V

    2013-01-01

    Preface Protein kinase C (PKC) has been a tantalizing target for drug discovery ever since it was first identified as the receptor for the tumor promoter phorbol ester in 19821. Although initial therapeutic efforts focused on cancer, additional diseases, including diabetic complications, heart failure, myocardial infarction, pain and bipolar disease were targeted as researchers developed a better understanding of the roles that PKC’s eight conventional and novel isozymes play in health and disease. Unfortunately, both academic and pharmaceutical efforts have yet to result in the approval of a single new drug that specifically targets PKC. Why does PKC remain an elusive drug target? This review will provide a short account of some of the efforts, challenges and opportunities in developing PKC modulators to address unmet clinical needs. PMID:23197040

  18. 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

  19. Expression and phosphorylation of a MARCKS-like protein in gastric chief cells: further evidence for modulation of pepsinogen secretion by interaction of Ca2+/calmodulin with protein kinase C.

    PubMed

    Raufman, J P; Malhotra, R; Xie, Q; Raffaniello, R D

    1997-03-01

    In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72-kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine-Rich C Kinase Substrate) protein. We examined expression and phosphorylation of the MARCKS-like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody resulted in staining of a myristoylated 72-kDA protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells, we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 microM), on pepsinogen secretion and phosphorylation of the 72-KDa MARCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 microM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 microM CGP 41 251) abolished PMA-induced secretion, but did not alter calcium-induced secretion. This indicates that calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with 32P-orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2-dimensional polyacrylamide gels. In the presence of basal calcium, PMA (100 nM) caused a > two-fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 microM calcium caused an approx. 50% attenuation of PMA-induced phosphorylation of pp72. Experiments with a MARCKS "phosphorylation/calmodulin binding domain peptide" indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells

  20. Homeodomain-interacting protein kinase-2 (HIPK2) phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77 and modulates its DNA binding affinity.

    PubMed

    Zhang, Qingchun; Wang, Yinsheng

    2007-12-01

    The chromosomal high-mobility group A (HMGA) proteins, composed of HMGA1a, HMGA1b and HMGA2, play important roles in the regulation of numerous processes in eukaryotic cells, such as transcriptional regulation, DNA repair, RNA processing, and chromatin remodeling. The biological activities of HMGA1 proteins are highly regulated by their post-translational modifications (PTMs), including acetylation, methylation, and phosphorylation. Recently, it was found that the homeodomain-interacting protein kinase-2 (HIPK2), a newly identified serine/threonine kinase, co-immunoprecipitated with, and phosphorylated, HMGA1 proteins. However, the sites and the biological significance of the phosphorylation have not been elucidated. Here, we found that HIPK2 phosphorylates HMGA1a at Ser-35, Thr-52, and Thr-77, and HMGA1b at Thr-41 and Thr-66. In addition, we demonstrated that cdc2, which is known to phosphorylate HMGA1 proteins, could induce the phosphorylation of HMGA1 proteins at the same Ser/Thr sites. The two kinases, however, exhibited different site preferences for the phosphorylation: The preference for HIPK2 phosphorylation followed the order of Thr-77 > Thr-52 > Ser-35, whereas the order for cdc2 phosphorylation was Thr-52 > Thr-77 > Ser-35. Moreover, we found that the HIPK2-phosphorylated HMGA1a reduced the binding affinity of HMGA1a to human germ line promoter, and the drop in binding affinity induced by HIPK2 phosphorylation was lower than that introduced by cdc2 phosphorylation, which is consistent with the notion that the second AT-hook in HMGA1a is more important for DNA binding than the third AT-hook. PMID:17960875

  1. THI1, a Thiamine Thiazole Synthase, Interacts with Ca2+-Dependent Protein Kinase CPK33 and Modulates the S-Type Anion Channels and Stomatal Closure in Arabidopsis.

    PubMed

    Li, Chun-Long; Wang, Mei; Wu, Xiao-Meng; Chen, Dong-Hua; Lv, Hong-Jun; Shen, Jian-Lin; Qiao, Zhu; Zhang, Wei

    2016-02-01

    Thiamine is required for both plant growth and development. Here, the involvement of a thiamine thiazole synthase, THI1, has been demonstrated in both guard cell abscisic acid (ABA) signaling and the drought response in Arabidopsis (Arabidopsis thaliana). THI1 overexpressors proved to be more sensitive to ABA than the wild type with respect to both the activation of guard cell slow type anion channels and stomatal closure; this effectively reduced the rate of water loss from the plant and thereby enhanced its level of drought tolerance. A yeast two-hybrid strategy was used to screen a cDNA library from epidermal strips of leaves for THI1 regulatory factors, and identified CPK33, a Ca(2+)-dependent protein kinase, as interactor with THI1 in a plasma membrane-delimited manner. Loss-of-function cpk33 mutants were hypersensitive to ABA activation of slow type anion channels and ABA-induced stomatal closure, while the CPK33 overexpression lines showed opposite phenotypes. CPK33 kinase activity was essential for ABA-induced stomatal closure. Consistent with their contrasting regulatory role over stomatal closure, THI1 suppressed CPK33 kinase activity in vitro. Together, our data reveal a novel regulatory role of thiamine thiazole synthase to kinase activity in guard cell signaling. PMID:26662273

  2. Protective effect of paeoniflorin on irradiation-induced cell damage involved in modulation of reactive oxygen species and the mitogen-activated protein kinases.

    PubMed

    Li, Chun Rong; Zhou, Zhe; Zhu, Dan; Sun, Yu Ning; Dai, Jin Ming; Wang, Sheng Qi

    2007-01-01

    Ionizing radiation can induce DNA damage and cell death by generating reactive oxygen species (ROS). The objective of this study was to investigate the radioprotective effect of paeoniflorin (PF, a main bioactive component in the traditional Chinese herb peony) on irradiated thymocytes and discover the possible mechanisms of protection. We found 60Co gamma-ray irradiation increased cell death and DNA fragmentation in a dose-dependent manner while increasing intracellular ROS. Pretreatment of thymocytes with PF (50-200 microg/ml) reversed this tendency and attenuated irradiation-induced ROS generation. Hydroxyl-scavenging action of PF in vitro was detected through electron spin resonance assay. Several anti-apoptotic characteristics of PF, including the ability to diminish cytosolic Ca2+ concentration, inhibit caspase-3 activation, and upregulate Bcl-2 and downregulate Bax in 4Gy-irradiated thymocytes were determined. Extracellular regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 kinase were activated by 4Gy irradiation, whereas its activations were partly blocked by pretreatment of cells with PF. The presence of ERK inhibitor PD98059, JNK inhibitor SP600125 and p38 inhibitor SB203580 decreased cell death in 4Gy-irradiated thymocytes. These results suggest PF protects thymocytes against irradiation-induced cell damage by scavenging ROS and attenuating the activation of the mitogen-activated protein kinases. PMID:17097910

  3. A protein kinase associated with paired helical filaments in Alzheimer disease.

    PubMed Central

    Vincent, I J; Davies, P

    1992-01-01

    We have identified a protein kinase in immunoaffinity-purified preparations of paired helical filaments from brain tissue of individuals with Alzheimer disease. The kinase phosphorylates the filament proteins in vitro in a manner independent of second messenger regulation or of modulation by heparin and polyamines. Physiological concentrations of hemin, an oxidized heme porphyrin, inhibit the kinase and abolish Alz-50 immunoreactivity of the proteins. Since paired helical filaments are composed of hyperphosphorylated proteins, association of a protein kinase with the filaments provides a mechanism for abnormal processing of the proteins in disease. Images PMID:1557394

  4. Signals fly when kinases meet Rho-of-plants (ROP) small G-proteins.

    PubMed

    Fehér, Attila; Lajkó, Dézi Bianka

    2015-08-01

    Rho-type small GTP-binding plant proteins function as two-state molecular switches in cellular signalling. There is accumulating evidence that Rho-of-plants (ROP) signalling is positively controlled by plant receptor kinases, through the ROP guanine nucleotide exchange factor proteins. These signalling modules regulate cell polarity, cell shape, hormone responses, and pathogen defence, among other things. Other ROP-regulatory proteins might also be subjected to protein phosphorylation by cellular kinases (e.g., mitogen-activated protein kinases or calcium-dependent protein kinases), in order to integrate various cellular signalling pathways with ROP GTPase-dependent processes. In contrast to the role of kinases in upstream ROP regulation, much less is known about the potential link between ROP GTPases and downstream kinase signalling. In other eukaryotes, Rho-type G-protein-activated kinases are widespread and have a key role in many cellular processes. Recent data indicate the existence of structurally different ROP-activated kinases in plants, but their ROP-dependent biological functions still need to be validated. In addition to these direct interactions, ROPs may also indirectly control the activity of mitogen-activated protein kinases or calcium-dependent protein kinases. These kinases may therefore function as upstream as well as downstream kinases in ROP-mediated signalling pathways, such as the phosphatidylinositol monophosphate kinases involved in cell polarity establishment. PMID:26089155

  5. AKAP79 Selectively Enhances Protein Kinase C Regulation of GluR1 at a Ca2+-Calmodulin-dependent Protein Kinase II/Protein Kinase C Site*

    PubMed Central

    Tavalin, Steven J.

    2008-01-01

    Enhancement of AMPA receptor activity in response to synaptic plasticity inducing stimuli may arise, in part, through phosphorylation of the GluR1 AMPA receptor subunit at Ser-831. This site is a substrate for both Ca2+-calmodulin-dependent protein kinase II (CaMKII) and protein kinase C (PKC). However, neuronal protein levels of CaMKII may exceed those of PKC by an order of magnitude. Thus, it is unclear how PKC could effectively regulate this common target site. The multivalent neuronal scaffold A-kinase-anchoring protein 79 (AKAP79) is known to bind PKC and is linked to GluR1 by synapse-associated protein 97 (SAP97). Here, biochemical studies demonstrate that AKAP79 localizes PKC activity near the receptor, thus accelerating Ser-831 phosphorylation. Complementary electrophysiological studies indicate that AKAP79 selectively shifts the dose-dependence for PKC modulation of GluR1 receptor currents ∼20-fold, such that low concentrations of PKC are as effective as much higher CaMKII concentrations. By boosting PKC activity near a target substrate, AKAP79 provides a mechanism to overcome limitations in kinase abundance thereby ensuring faithful signal propagation and efficient modification of AMPA receptor-mediated responses. PMID:18305116

  6. Revisiting protein kinase-substrate interactions: Toward therapeutic development.

    PubMed

    de Oliveira, Paulo Sérgio L; Ferraz, Felipe Augusto N; Pena, Darlene A; Pramio, Dimitrius T; Morais, Felipe A; Schechtman, Deborah

    2016-01-01

    Despite the efforts of pharmaceutical companies to develop specific kinase modulators, few drugs targeting kinases have been completely successful in the clinic. This is primarily due to the conserved nature of kinases, especially in the catalytic domains. Consequently, many currently available inhibitors lack sufficient selectivity for effective clinical application. Kinases phosphorylate their substrates to modulate their activity. One of the important steps in the catalytic reaction of protein phosphorylation is the correct positioning of the target residue within the catalytic site. This positioning is mediated by several regions in the substrate binding site, which is typically a shallow crevice that has critical subpockets that anchor and orient the substrate. The structural characterization of this protein-protein interaction can aid in the elucidation of the roles of distinct kinases in different cellular processes, the identification of substrates, and the development of specific inhibitors. Because the region of the substrate that is recognized by the kinase can be part of a linear consensus motif or a nonlinear motif, advances in technology beyond simple linear sequence scanning for consensus motifs were needed. Cost-effective bioinformatics tools are already frequently used to predict kinase-substrate interactions for linear consensus motifs, and new tools based on the structural data of these interactions improve the accuracy of these predictions and enable the identification of phosphorylation sites within nonlinear motifs. In this Review, we revisit kinase-substrate interactions and discuss the various approaches that can be used to identify them and analyze their binding structures for targeted drug development. PMID:27016527

  7. G-protein coupled receptor kinases in inflammation and disease

    PubMed Central

    Packiriswamy, Nandakumar; Parameswaran, Narayanan

    2015-01-01

    G-protein coupled receptor kinases (GRKs) are serine/threonine protein kinases originally discovered for their role in G-protein coupled receptor (GPCR) phosphorylation. Recent studies have demonstrated a much broader function for this kinase family including phosphorylation of cytosolic substrates involved in cell signaling pathways stimulated by GPCRs as well as non-GPCRs. In addition, GRKs modulate signaling via phosphorylation-independent functions. Because of these various biochemical functions, GRKs have been shown to affect critical physiological and pathophysiological processes and thus are considered as drug targets in diseases such as heart failure. Role of GRKs in inflammation and inflammatory diseases is an evolving area of research and several studies including work from our lab in the recent years have demonstrated critical role of GRKs in the immune system. In this review we discuss the classical and the newly emerging functions of GRKs in the immune system and their role in inflammation and disease processes. PMID:26226012

  8. 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

  9. Involvement of protein kinase C in the modulation of morphine-induced analgesia and the inhibitory effects of exposure to 60-hz magnetic fields in the land snail, Cepaea nemoralis

    SciTech Connect

    Kavaliers, M.; Ossenkopp, K.P. )

    1990-02-26

    One of the more consistent and dramatic effects of exposure to magnetic fields is the attenuation of morphine-induced analgesia. Results of previous studies have implicated alterations in calcium channel functioning and Ca{sup ++} flux in the mediation of these effects. It is generally accepted that Ca{sup ++}-activated-phospholipid-dependent protein kinase (Protein kinase C; PKC) plays an important role in relaying trans-membrane signaling in diverse Ca{sup ++} dependent cellular processes. In experiment 1 we observed that morphine-induced analgesia in the land snail, Cepaea nemoralis, as measured by the latency of an avoidance behavior to a warmed surface, was reduced by the PKC activator, SC-9, and was enhanced by the PKC inhibitors, H-7 and H-9. In contrast, HA-10004, a potent inhibitor of other protein kinases, but only a very weak inhibitor of PKC, had no effect on morphine-induced analgesia. In experiment 2 exposure of snails for 30 minutes to a 1.0 gauss (rms) 60-Hz magnetic field reduced morphine-induced analgesia. This inhibitory effect of the magnetic field was reduced by the PKC inhibitors, H-7 and H-9, and was augmented by the PKC activator SC-9. These results suggest that: (i) PKC is involved in the modulation of morphine-induced analgesia and, (ii) the inhibitory effects of magnetic fields involve PKC.

  10. Regulation of death induction and chemosensitizing action of 3-bromopyruvate in myeloid leukemia cells: energy depletion, oxidative stress, and protein kinase activity modulation.

    PubMed

    Calviño, Eva; Estañ, María Cristina; Sánchez-Martín, Carlos; Brea, Rocío; de Blas, Elena; Boyano-Adánez, María del Carmen; Rial, Eduardo; Aller, Patricio

    2014-02-01

    3-Bromopyruvate (3-BrP) is an alkylating, energy-depleting drug that is of interest in antitumor therapies, although the mechanisms underlying its cytotoxicity are ill-defined. We show here that 3-BrP causes concentration-dependent cell death of HL60 and other human myeloid leukemia cells, inducing both apoptosis and necrosis at 20-30 μM and a pure necrotic response at 60 μM. Low concentrations of 3-BrP (10-20 μM) brought about a rapid inhibition of glycolysis, which at higher concentrations was followed by the inhibition of mitochondrial respiration. The combination of these effects causes concentration-dependent ATP depletion, although this cannot explain the lethality at intermediate 3-BrP concentrations (20-30 μM). The oxidative stress caused by exposure to 3-BrP was evident as a moderate overproduction of reactive oxygen species and a concentration-dependent depletion of glutathione, which was an important determinant of 3-BrP toxicity. In addition, 3-BrP caused glutathione-dependent stimulation of p38 mitogen-activated protein kinase (MAPK), mitogen-induced extracellular kinase (MEK)/extracellular signal-regulated kinase (ERK), and protein kinase B (Akt)/mammalian target of rapamycin/p70S6K phosphorylation or activation, as well as rapid LKB-1/AMP kinase (AMPK) activation, which was later followed by Akt-mediated inactivation. Experiments with pharmacological inhibitors revealed that p38 MAPK activation enhances 3-BrP toxicity, which is conversely restrained by ERK and Akt activity. Finally, 3-BrP was seen to cooperate with antitumor agents like arsenic trioxide and curcumin in causing cell death, a response apparently mediated by both the generation of oxidative stress induced by 3-BrP and the attenuation of Akt and ERK activation by curcumin. In summary, 3-BrP cytotoxicity is the result of several combined regulatory mechanisms that might represent important targets to improve therapeutic efficacy. PMID:24307199

  11. A Mitogen-Activated Protein Kinase Cascade Module, MKK3-MPK6 and MYC2, Is Involved in Blue Light-Mediated Seedling Development in Arabidopsis[C][W

    PubMed Central

    Sethi, Vishmita; Raghuram, Badmi; Sinha, Alok Krishna; Chattopadhyay, Sudip

    2014-01-01

    Mitogen-activated protein kinase (MAPK) pathways are involved in several signal transduction processes in eukaryotes. Light signal transduction pathways have been extensively studied in plants; however, the connection between MAPK and light signaling pathways is currently unknown. Here, we show that MKK3-MPK6 is activated by blue light in a MYC2-dependent manner. MPK6 physically interacts with and phosphorylates a basic helix-loop-helix transcription factor, MYC2, and is phosphorylated by a MAPK kinase, MKK3. Furthermore, MYC2 binds to the MPK6 promoter and regulates its expression in a feedback regulatory mechanism in blue light signaling. We present mutational and physiological studies that illustrate the function of the MKK3-MPK6-MYC2 module in Arabidopsis thaliana seedling development and provide a revised mechanistic view of photomorphogenesis. PMID:25139007

  12. Regulation of Axonal Transport by Protein Kinases.

    PubMed

    Gibbs, Katherine L; Greensmith, Linda; Schiavo, Giampietro

    2015-10-01

    The intracellular transport of organelles, proteins, lipids, and RNA along the axon is essential for neuronal function and survival. This process, called axonal transport, is mediated by two classes of ATP-dependent motors, kinesins, and cytoplasmic dynein, which carry their cargoes along microtubule tracks. Protein kinases regulate axonal transport through direct phosphorylation of motors, adapter proteins, and cargoes, and indirectly through modification of the microtubule network. The misregulation of axonal transport by protein kinases has been implicated in the pathogenesis of several nervous system disorders. Here, we review the role of protein kinases acting directly on axonal transport and discuss how their deregulation affects neuronal function, paving the way for the exploitation of these enzymes as novel drug targets. PMID:26410600

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

    DOEpatents

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

    2004-10-12

    The present invention relates to 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such 2-N-substituted 6-(4-methoxybenzylamino)-9-isopropylpurines to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  14. Protein kinase domain of twitchin has protein kinase activity and an autoinhibitory region.

    PubMed

    Lei, J; Tang, X; Chambers, T C; Pohl, J; Benian, G M

    1994-08-19

    Twitchin is a 753-kDa polypeptide located in the muscle A-bands of the nematode, Caenorhabditis elegans. It consists of multiple copies of both fibronectin III and immunoglobulin C2 domains and, near the C terminus, a protein kinase domain with greatest homology to the catalytic domains of myosin light chain kinases. We have expressed and purified from Escherichia coli twitchin's protein kinase catalytic core and flanking sequences that do not include fibronectin III and immunoglobulin C2 domains. The protein was shown to phosphorylate a model substrate and to undergo autophosphorylation. The autophosphorylation occurs at a slow rate, attaining a maximum at 3 h with a stoichiometry of about 1.0 mol of phosphate/mol of protein, probably through an intramolecular mechanism. Sequence analysis of proteolytically derived phosphopeptides revealed that autophosphorylation occurred N-terminal to the catalytic core, predominantly at Thr-5910, with possible minor sites at Ser5912 and/or Ser-5913. This portion of twitchin (residues 5890-6268) was also phosphorylated in vitro by protein kinase C in the absence of calcium and phosphotidylserine, but not by cAMP-dependent protein kinase. By comparing the activities of three twitchin segments, the enzyme appears to be inhibited by the 60-amino acid residues lying just C-terminal to the kinase catalytic core. Thus, like a number of other protein kinases including myosin light chain kinases, the twitchin kinase appears to be autoregulated. PMID:8063727

  15. Ca/sup 2 +/-stimulated catecholamine release from. cap alpha. -toxin-permeabilized PC12 cells: biochemical evidence for exocytosis and its modulation by protein kinase C and G protein

    SciTech Connect

    Ahnert-Hilger, G.; Braeutigam, M.; Gratzl, M.

    1987-12-01

    Two possible cellular pathways of catecholamines from the chromaffin vesicles of PC12 cells to the surrounding medium are explored in this study. The direct one circumventing the cytoplasm can be activated in ..cap alpha..-toxin-permeabilized cells with micromolar levels of free Ca/sup 2 +/. Catecholamine metabolites formed in the cytoplasm (i.e., 3,4-dihydroxyphenylacetic acid and 3,4-dihydroxyphenylethanol) are neither formed nor released from the cells under these conditions. However, when vesicular catecholamines were discharged into the cytoplasm by addition of the ionophore nigericin, such metabolites are formed and released into the medium independent of Ca/sup 2 +/. Both types of experiments provide direct evidence for the operation of Ca/sup 2 +/-induced exocytosis of dopamine and noradrenaline in permeabilized PC12 cells. The Ca/sup 2 +/ dependence of dopamine or noradrenaline release, as measured by the determination of the endogenous catecholamines using the high-performance liquid chromatography technique, exhibits two different phases. One is already activated below ..mu..M free Ca/sup 2 +/ and plateaus at 1-5 ..mu..M free Ca/sup 2 +/, while a second occurs in the presence of larger amounts of free Ca/sup 2 +/ (10-100 ..mu..M). Ca/sup 2 +/-induced catecholamine release from the permeabilized cells can be modulated in different ways: It is enhanced by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate and the diacylglycerol 1-oleyl-2-acetylglycerol provided Mg/sup 2 +//ATP is present, and it is inhibited by guanosine 5'-O-(3-thiotriphosphate). The latter effect is abolished by pretreatment of the cells with pertussis toxin but not by cholera toxin. Thus, it appears that Ca/sup 2 +/-induced exocytosis can be modulated via the protein kinase C system, as well as via GTP binding proteins.

  16. A protein kinase screen of Neurospora crassa mutant strains reveals that the SNF1 protein kinase promotes glycogen synthase phosphorylation.

    PubMed

    Candido, Thiago De Souza; Gonçalves, Rodrigo Duarte; Felício, Ana Paula; Freitas, Fernanda Zanolli; Cupertino, Fernanda Barbosa; De Carvalho, Ana Carolina Gomes Vieira; Bertolini, Maria Célia

    2014-12-15

    Glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. The activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. To identify the protein kinases affecting glycogen metabolism in Neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. We identified multiple kinases that have already been described as controlling glycogen metabolism in different organisms, such as NcSNF1, NcPHO85, NcGSK3, NcPKA, PSK2 homologue and NcATG1. In addition, many hypothetical kinases have been implicated in the control of glycogen metabolism. Two kinases, NcIME-2 and NcNIMA, already functionally characterized but with no functions related to glycogen metabolism regulation, were also identified. Among the kinases identified, it is important to mention the role of NcSNF1. We showed in the present study that this kinase was implicated in glycogen synthase phosphorylation, as demonstrated by the higher levels of glycogen accumulated during growth, along with a higher glycogen synthase (GSN) ±glucose 6-phosphate activity ratio and a lesser set of phosphorylated GSN isoforms in strain Ncsnf1KO, when compared with the wild-type strain. The results led us to conclude that, in N. crassa, this kinase promotes phosphorylation of glycogen synthase either directly or indirectly, which is the opposite of what is described for Saccharomyces cerevisiae. The kinases also play a role in gene expression regulation, in that gdn, the gene encoding the debranching enzyme, was down-regulated by the proteins identified in the screen. Some kinases affected growth and development, suggesting a connection linking glycogen metabolism with cell growth and development. PMID:25253091

  17. Protein kinase activators alter glial cholesterol esterification

    SciTech Connect

    Jeng, I.; Dills, C.; Klemm, N.; Wu, C.

    1986-05-01

    Similar to nonneural tissues, the activity of glial acyl-CoA cholesterol acyltransferase is controlled by a phosphorylation and dephosphorylation mechanism. Manipulation of cyclic AMP content did not alter the cellular cholesterol esterification, suggesting that cyclic AMP is not a bioregulator in this case. Therefore, the authors tested the effect of phorbol-12-myristate 13-acetate (PMA) on cellular cholesterol esterification to determine the involvement of protein kinase C. PMA has a potent effect on cellular cholesterol esterification. PMA depresses cholesterol esterification initially, but cells recover from inhibition and the result was higher cholesterol esterification, suggesting dual effects of protein kinase C. Studies of other phorbol analogues and other protein kinase C activators such as merezein indicate the involvement of protein kinase C. Oleoyl-acetyl glycerol duplicates the effect of PMA. This observation is consistent with a diacyl-glycerol-protein kinase-dependent reaction. Calcium ionophore A23187 was ineffective in promoting the effect of PMA. They concluded that a calcium-independent and protein C-dependent pathway regulated glial cholesterol esterification.

  18. Purification and characterization of a thylakoid protein kinase

    SciTech Connect

    Coughlan, S.J.; Hind, G.

    1986-01-01

    Control of state transitions in the thylakoid by reversible phosphorylation of the light-harvesting chlorophyll a/b protein complex of photosystem II (LHC-II) is modulated by a kinase. The kinase catalyzing this phosphorylation is associated with the thylakoid membrane, and is regulated by the redox state of the plastoquinone pool. The isolation and partial purification from spinach thylakoids of two protein kinases (CPK1, CPK2) of apparent molecular masses 25 kDa and 38 kDa has been reported. Neither enzyme utilizes isolated LHC-II as a substrate. The partial purification of a third protein kinase (LHCK) which can utilize both lysine-rich histones (IIIs and Vs) and isolated LHC-II as substrate has now been purified to homogeneity and characterized by SDS-polyacrylamide gel electrophoresis as a 64 kDa peptide. From a comparison of the two isolation procedures we have concluded that CPK1 is indeed a protein kinase, but has a lower specific activity than that of LHCK. 8 refs., 4 figs.

  19. [Mitogen-activated protein kinases in atherosclerosis].

    PubMed

    Bryk, Dorota; Olejarz, Wioletta; Zapolska-Downar, Danuta

    2014-01-01

    Intracellular signalling cascades, in which MAPK (mitogen-activated protein kinases) intermediate, are responsible for a biological response of a cell to an external stimulus. MAP kinases, which include ERK1/2 (extracellular signalling-regulated kinase), JNK (c-Jun N-terminal kinase) and p 38 MAPK, regulate the activity of many proteins, enzymes and transcription factors and thus have a wide spectrum of biological effects. Many basic scientific studies have defined numerous details of their pathway organization and activation. There are also more and more studies suggesting that individual MAP kinases probably play an important role in the pathogenesis of atherosclerosis. They may mediate inflammatory processes, endothelial cell activation, monocyte/macrophage recruitment and activation, smooth muscle cell proliferation and T-lymphocyte differentiation, all of which represent crucial mechanisms involved in pathogenesis of atherosclerosis. The specific inhibition of an activity of the respective MAP kinases may prove a new therapeutic approach to attenuate atherosclerotic plaque formation in the future. In this paper, we review the current state of knowledge concerning MAP kinase-dependent cellular and molecular mechanisms underlying atherosclerosis. PMID:24491891

  20. Differential Protein Kinase C-dependent Modulation of Kv7.4 and Kv7.5 Subunits of Vascular Kv7 Channels*

    PubMed Central

    Brueggemann, Lioubov I.; Mackie, Alexander R.; Cribbs, Leanne L.; Freda, Jessica; Tripathi, Abhishek; Majetschak, Matthias; Byron, Kenneth L.

    2014-01-01

    The Kv7 family (Kv7.1–7.5) of voltage-activated potassium channels contributes to the maintenance of resting membrane potential in excitable cells. Previously, we provided pharmacological and electrophysiological evidence that Kv7.4 and Kv7.5 form predominantly heteromeric channels and that Kv7 activity is regulated by protein kinase C (PKC) in response to vasoconstrictors in vascular smooth muscle cells. Direct evidence for Kv7.4/7.5 heteromer formation, however, is lacking. Furthermore, it remains to be determined whether both subunits are regulated by PKC. Utilizing proximity ligation assays to visualize single molecule interactions, we now show that Kv7.4/Kv.7.5 heteromers are endogenously expressed in vascular smooth muscle cells. Introduction of dominant-negative Kv7.4 and Kv7.5 subunits in mesenteric artery myocytes reduced endogenous Kv7 currents by 84 and 76%, respectively. Expression of an inducible protein kinase Cα (PKCα) translocation system revealed that PKCα activation is sufficient to suppress endogenous Kv7 currents in A7r5 rat aortic and mesenteric artery smooth muscle cells. Arginine vasopressin (100 and 500 pm) and the PKC activator phorbol 12-myristate 13-acetate (1 nm) each inhibited human (h) Kv7.5 and hKv7.4/7.5, but not hKv7.4 channels expressed in A7r5 cells. A decrease in hKv7.5 and hKv7.4/7.5 current densities was associated with an increase in PKC-dependent phosphorylation of the channel proteins. These findings provide further evidence for a differential regulation of Kv7.4 and Kv7.5 channel subunits by PKC-dependent phosphorylation and new mechanistic insights into the role of heteromeric subunit assembly for regulation of vascular Kv7 channels. PMID:24297175

  1. 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.

  2. 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

  3. Leptin modulates the expression of catabolic genes in rat nucleus pulposus cells through the mitogen-activated protein kinase and Janus kinase 2/signal transducer and activator of transcription 3 pathways.

    PubMed

    Miao, Daoyi; Zhang, Lingzhou

    2015-08-01

    Obesity has been demonstrated to be involved in the progress of intervertebral disc degeneration (IDD). However, the associated mechanisms remain to be elucidated. The purpose the present study was to examine the effect of leptin on the expression of degeneration-associated genes in rat nucleus pulposus (NP) cells, and determine the possible mechanism. Normal NP cells, obtained from Sprague Dawley rats, were identified using immunocytochemistry for the expression of collagen II and CA125, and treated with leptin and/or interleukin (IL)-β. Subsequently, the mRNA expression levels of matrix metalloproteinase (MMP)-1, MMP-3, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, ADAMTS-5, aggrecan and COL2A1 were detected by reverse transcription-quantitative polymerase chain reaction (RT-q-PCR). Alcian staining and immunocytochemistry were used to examine the expression levels of proteoglycan and collagen II. The pathway activation was investigated using western blotting, and inhibitors of the pathways were used to reveal the effect of these pathways on the NP cells. The results of the RT-qPCR demonstrated that leptin alone upregulated the mRNA expression levels of MMP-1, MMP-13, ADAMTS-4, ADAMTS-5 and COL2A1. Synergy of leptin and IL-β was found in the increased expression levels of MMP-1, MMP-3 and ADAMTS-5. The leptin-treated NP cells exhibited decreased expression of collagen II. The mitrogen-activated protein kinase (MAPK) pathway (c-Jun-N-terminal kinase, phosphorylated extracellular signal-regulated kinase and p38), phosphatidylinositol 3-kinase (PI3K)/Akt pathway and Janus kinase (JAK)2/signal transducer and activator of transcription 3 pathway were all activated by leptin, however, inhibitors of all the pathways, with the exception of the PI3K/Akt pathway, reversed the expression levels of MMP-1 and MMP-13. These results suggested that leptin promoted catabolic metabolism in the rat NP cells via the MAPK and JAK2/STAT3

  4. Leptin modulates the expression of catabolic genes in rat nucleus pulposus cells through the mitogen-activated protein kinase and Janus kinase 2/signal transducer and activator of transcription 3 pathways

    PubMed Central

    MIAO, DAOYI; ZHANG, LINGZHOU

    2015-01-01

    Obesity has been demonstrated to be involved in the progress of intervertebral disc degeneration (IDD). However, the associated mechanisms remain to be elucidated. The purpose the present study was to examine the effect of leptin on the expression of degeneration-associated genes in rat nucleus pulposus (NP) cells, and determine the possible mechanism. Normal NP cells, obtained from Sprague Dawley rats, were identified using immunocytochemistry for the expression of collagen II and CA125, and treated with leptin and/or interleukin (IL)-β. Subsequently, the mRNA expression levels of matrix metalloproteinase (MMP)-1, MMP-3, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4, ADAMTS-5, aggrecan and COL2A1 were detected by reverse transcription-quantitative polymerase chain reaction (RT-q-PCR). Alcian staining and immunocytochemistry were used to examine the expression levels of proteoglycan and collagen II. The pathway activation was investigated using western blotting, and inhibitors of the pathways were used to reveal the effect of these pathways on the NP cells. The results of the RT-qPCR demonstrated that leptin alone upregulated the mRNA expression levels of MMP-1, MMP-13, ADAMTS-4, ADAMTS-5 and COL2A1. Synergy of leptin and IL-β was found in the increased expression levels of MMP-1, MMP-3 and ADAMTS-5. The leptin-treated NP cells exhibited decreased expression of collagen II. The mitrogen-activated protein kinase (MAPK) pathway (c-Jun-N-terminal kinase, phosphorylated extracellular signal-regulated kinase and p38), phosphatidylinositol 3-kinase (PI3K)/Akt pathway and Janus kinase (JAK)2/signal transducer and activator of transcription 3 pathway were all activated by leptin, however, inhibitors of all the pathways, with the exception of the PI3K/Akt pathway, reversed the expression levels of MMP-1 and MMP-13. These results suggested that leptin promoted catabolic metabolism in the rat NP cells via the MAPK and JAK2/STAT3

  5. Modulation of hormone-sensitive lipase and protein kinase A-mediated lipolysis by perilipin A in an adenoviral reconstituted system.

    PubMed

    Souza, Sandra C; Muliro, Kizito V; Liscum, Laura; Lien, Ping; Yamamoto, Mia T; Schaffer, Jean E; Dallal, Gerard E; Wang, Xinzhong; Kraemer, Fredric B; Obin, Martin; Greenberg, Andrew S

    2002-03-01

    Perilipin (Peri) A is a phosphoprotein located at the surface of intracellular lipid droplets in adipocytes. Activation of cyclic AMP-dependent protein kinase (PKA) results in the phosphorylation of Peri A and hormone-sensitive lipase (HSL), the predominant lipase in adipocytes, with concurrent stimulation of adipocyte lipolysis. To investigate the relative contributions of Peri A and HSL in basal and PKA-mediated lipolysis, we utilized NIH 3T3 fibroblasts lacking Peri A and HSL but stably overexpressing acyl-CoA synthetase 1 (ACS1) and fatty acid transport protein 1 (FATP1). When incubated with exogenous fatty acids, ACS1/FATP1 cells accumulated 5 times more triacylglycerol (TG) as compared with NIH 3T3 fibroblasts. Adenoviral-mediated expression of Peri A in ACS1/FATP1 cells enhanced TG accumulation and inhibited lipolysis, whereas expression of HSL fused to green fluorescent protein (GFPHSL) reduced TG accumulation and enhanced lipolysis. Forskolin treatment induced Peri A hyperphosphorylation and abrogated the inhibitory effect of Peri A on lipolysis. Expression of a mutated Peri A Delta 3 (Ser to Ala substitutions at PKA consensus sites Ser-81, Ser-222, and Ser-276) reduced Peri A hyperphosphorylation and blocked constitutive and forskolin-stimulated lipolysis. Thus, perilipin expression and phosphorylation state are critical regulators of lipid storage and hydrolysis in ACS1/FATP1 cells. PMID:11751901

  6. Phosphorylation of FE65 Ser610 by serum- and glucocorticoid-induced kinase 1 modulates Alzheimer's disease amyloid precursor protein processing

    PubMed Central

    Chow, Wan Ning Vanessa; Ngo, Jacky Chi Ki; Li, Wen; Chen, Yu Wai; Tam, Ka Ming Vincent; Chan, Ho Yin Edwin; Miller, Christopher C.J.; Lau, Kwok-Fai

    2015-01-01

    Alzheimer's disease (AD) is a fatal neurodegenerative disease affecting 36 million people worldwide. Genetic and biochemical research indicate that the excessive generation of amyloid-β peptide (Aβ) from amyloid precursor protein (APP), is a major part of AD pathogenesis. FE65 is a brain-enriched adaptor protein that binds to APP. However, the role of FE65 in APP processing and the mechanisms that regulate binding of FE65 to APP are not fully understood. In the present study, we show that serum- and glucocorticoid-induced kinase 1 (SGK1) phosphorylates FE65 on Ser610 and that this phosphorylation attenuates FE65 binding to APP. We also show that FE65 promotes amyloidogenic processing of APP and that FE65 Ser610 phosphorylation inhibits this effect. Furthermore, we found that the effect of FE65 Ser610 phosphorylation on APP processing is linked to a role of FE65 in metabolic turnover of APP via the proteasome. Thus FE65 influences APP degradation via the proteasome and phosphorylation of FE65 Ser610 by SGK1 regulates binding of FE65 to APP, APP turnover and processing. PMID:26188042

  7. Modulation of kinase-inhibitor interactions by auxiliary protein binding: Crystallography studies on Aurora A interactions with VX-680 and with TPX2

    SciTech Connect

    Zhao, Baoguang; Smallwood, Angela; Yang, Jingsong; Koretke, Kristin; Nurse, Kelvin; Calamari, Amy; Kirkpatrick, Robert B.; Lai, Zhihong

    2008-10-24

    VX-680, also known as MK-0457, is an ATP-competitive small molecule inhibitor of the Aurora kinases that has entered phase II clinical trials for the treatment of cancer. We have solved the cocrystal structure of AurA/TPX2/VX-680 at 2.3 {angstrom} resolution. In the crystal structure, VX-680 binds to the active conformation of AurA. The glycine-rich loop in AurA adopts a unique bent conformation, forming a {pi}-{pi} interaction with the phenyl group of VX-680. In contrast, in the published AurA/VX-680 structure, VX-680 binds to AurA in the inactive conformation, interacting with a hydrophobic pocket only present in the inactive conformation. These data suggest that TPX2, a protein cofactor, can alter the binding mode of VX-680 with AurA. More generally, the presence of physiologically relevant cofactor proteins can alter the kinetics, binding interactions, and inhibition of enzymes, and studies with these multiprotein complexes may be beneficial to the discovery and optimization of enzyme inhibitors as therapeutic agents.

  8. 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"…

  9. The protein interaction landscape of the human CMGC kinase group.

    PubMed

    Varjosalo, Markku; Keskitalo, Salla; Van Drogen, Audrey; Nurkkala, Helka; Vichalkovski, Anton; Aebersold, Ruedi; Gstaiger, Matthias

    2013-04-25

    Cellular information processing via reversible protein phosphorylation requires tight control of the localization, activity, and substrate specificity of protein kinases, which to a large extent is accomplished by complex formation with other proteins. Despite their critical role in cellular regulation and pathogenesis, protein interaction information is available for only a subset of the 518 human protein kinases. Here we present a global proteomic analysis of complexes of the human CMGC kinase group. In addition to subgroup-specific functional enrichment and modularity, the identified 652 high-confidence kinase-protein interactions provide a specific biochemical context for many poorly studied CMGC kinases. Furthermore, the analysis revealed a kinase-kinase subnetwork and candidate substrates for CMGC kinases. Finally, the presented interaction proteome uncovered a large set of interactions with proteins genetically linked to a range of human diseases, including cancer, suggesting additional routes for analyzing the role of CMGC kinases in controlling human disease pathways. PMID:23602568

  10. eIF4A RNA Helicase Associates with Cyclin-Dependent Protein Kinase A in Proliferating Cells and Is Modulated by Phosphorylation.

    PubMed

    Bush, Maxwell S; Pierrat, Olivier; Nibau, Candida; Mikitova, Veronika; Zheng, Tao; Corke, Fiona M K; Vlachonasios, Konstantinos; Mayberry, Laura K; Browning, Karen S; Doonan, John H

    2016-09-01

    Eukaryotic initiation factor 4A (eIF4A) is a highly conserved RNA-stimulated ATPase and helicase involved in the initiation of messenger RNA translation. Previously, we found that eIF4A interacts with cyclin-dependent kinase A (CDKA), the plant ortholog of mammalian CDK1. Here, we show that this interaction occurs only in proliferating cells where the two proteins coassociate with 5'-cap-binding protein complexes, eIF4F or the plant-specific eIFiso4F. CDKA phosphorylates eIF4A on a conserved threonine residue (threonine-164) within the RNA-binding motif 1b TPGR. In vivo, a phospho-null (APGR) variant of the Arabidopsis (Arabidopsis thaliana) eIF4A1 protein retains the ability to functionally complement a mutant (eif4a1) plant line lacking eIF4A1, whereas a phosphomimetic (EPGR) variant fails to complement. The phospho-null variant (APGR) rescues the slow growth rate of roots and rosettes, together with the ovule-abortion and late-flowering phenotypes. In vitro, wild-type recombinant eIF4A1 and its phospho-null variant both support translation in cell-free wheat germ extracts dependent upon eIF4A, but the phosphomimetic variant does not support translation and also was deficient in ATP hydrolysis and helicase activity. These observations suggest a mechanism whereby CDK phosphorylation has the potential to down-regulate eIF4A activity and thereby affect translation. PMID:27388680

  11. Eukaryotic Translation Initiation Factor 5A (EIF5A) Regulates Pancreatic Cancer Metastasis by Modulating RhoA and Rho-associated Kinase (ROCK) Protein Expression Levels.

    PubMed

    Fujimura, Ken; Choi, Sunkyu; Wyse, Meghan; Strnadel, Jan; Wright, Tracy; Klemke, Richard

    2015-12-11

    Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with an overall survival rate of less than 5%. The poor patient outcome in PDAC is largely due to the high prevalence of systemic metastasis at the time of diagnosis and lack of effective therapeutics that target disseminated cells. The fact that the underlying mechanisms driving PDAC cell migration and dissemination are poorly understood have hindered drug development and compounded the lack of clinical success in this disease. Recent evidence indicates that mutational activation of K-Ras up-regulates eIF5A, a component of the cellular translational machinery that is critical for PDAC progression. However, the role of eIF5A in PDAC cell migration and metastasis has not been investigated. We report here that pharmacological inhibition or genetic knockdown of eIF5A reduces PDAC cell migration, invasion, and metastasis in vitro and in vivo. Proteomic profiling and bioinformatic analyses revealed that eIF5A controls an integrated network of cytoskeleton-regulatory proteins involved in cell migration. Functional interrogation of this network uncovered a critical RhoA/ROCK signaling node that operates downstream of eIF5A in invasive PDAC cells. Importantly, eIF5A mediates PDAC cell migration and invasion by modulating RhoA/ROCK protein expression levels. Together our findings implicate eIF5A as a cytoskeletal rheostat controlling RhoA/ROCK protein expression during PDAC cell migration and metastasis. Our findings also implicate the eIF5A/RhoA/ROCK module as a potential new therapeutic target to treat metastatic PDAC cells. PMID:26483550

  12. p38 mitogen-activated protein kinase inhibition modulates nucleus pulposus cell apoptosis in spontaneous resorption of herniated intervertebral discs: An experimental study in rats.

    PubMed

    Zhu, Yu; Liu, Jin-Tao; Yang, Li-Yan; Du, Wen-Pei; Li, Xiao-Chun; Qian, Xiang; Yu, Peng-Fei; Liu, Jian-Wen; Jiang, Hong

    2016-05-01

    The present study was performed to investigate the role of p38 mitogen‑activated protein kinase (MAPK) in the resorption of herniated intervertebral discs in 30 rats. In the non‑contained and p38 MAPK inhibition (p38i) groups, two coccygeal intervertebral discs (IVDs) were removed and wounded prior to relocation into the subcutaneous space of the skin of the back. In the contained group, the cartilage endplates maintained their integrity. Furthermore, SB203580 was injected intraperitoneally into the p38i group, whereas saline was injected into the other two groups. In the non‑contained group, the weight of the relocated IVDs decreased to a greater extent over time when compared with the contained and p38i groups. Phosphorylated p38, tumor necrosis factor‑α, and interleukin‑1β were observed to exhibit higher expression levels in the non‑contained group compared with the contained and p38i groups, at weeks 1 and 4 post‑surgery. The expression level of caspase‑3 and the densities of apoptotic disc cells were significantly higher in the non‑contained group compared with the contained and p38i groups at 4 weeks post‑surgery. In conclusion, p38 MAPK induces apoptosis in IVDs, while also accelerating the resorption of the relocated IVDs. Thus, p38 MAPK may be important in spontaneous resorption of IVDs. PMID:27035219

  13. Protein Interacting C-Kinase 1 Modulates Surface Expression of P2Y6 Purinoreceptor, Actin Polymerization and Phagocytosis in Microglia.

    PubMed

    Zhu, Jia; Wang, Zhen; Zhang, Nan; Ma, Jiao; Xu, Shui-Lin; Wang, Yin; Shen, Ying; Li, Yun-Hong

    2016-04-01

    Microglia clean up dead cells and debris through phagocytosis in the central nervous system. UDP-activated P2Y6 receptors (P2Y6Rs) induce the formation of phagocytic cup-like structure and P2Y6R expression is increased during the phagocytosis. However, it remains unclear how surface expression of P2Y6R is increased. PICK1 (protein interacting with C-kinase-1) interacts with various neurotransmitter receptors, transporters, and enzymes. We here report that PICK1 might interact with P2Y6R. Surface P2Y6R was reduced in microglia from PICK1-knockout mice and PICK1-knockdown BV2 cells, which was also confirmed by electrophysiological recordings, showing that P2Y6R-mediated current was increased by PICK1 overexpression but was reduced by PICK1-knockdown in BV2 microglia. Finally, PICK1 was sufficient to affect cytoskeletal aggregation and phagocytosis both in primary microglia and BV2 cells. These results indicate that PICK1 is an important regulator of P2Y6R expression and microglial phagocytosis. PMID:26566795

  14. Cell signaling through protein kinase C oxidation and activation.

    PubMed

    Cosentino-Gomes, Daniela; Rocco-Machado, Nathália; Meyer-Fernandes, José Roberto

    2012-01-01

    Due to the growing importance of cellular signaling mediated by reactive oxygen species (ROS), proteins that are reversibly modulated by these reactant molecules are of high interest. In this context, protein kinases and phosphatases, which act coordinately in the regulation of signal transduction through the phosphorylation and dephosphorylation of target proteins, have been described to be key elements in ROS-mediated signaling events. The major mechanism by which these proteins may be modified by oxidation involves the presence of key redox-sensitive cysteine residues. Protein kinase C (PKC) is involved in a variety of cellular signaling pathways. These proteins have been shown to contain a unique structural feature that is susceptible to oxidative modification. A large number of scientific studies have highlighted the importance of ROS as a second messenger in numerous cellular processes, including cell proliferation, gene expression, adhesion, differentiation, senescence, and apoptosis. In this context, the goal of this review is to discuss the mechanisms by which PKCs are modulated by ROS and how these processes are involved in the cellular response. PMID:23109817

  15. Mycobacterium tuberculosis Serine/Threonine Protein Kinases

    PubMed Central

    PRISIC, SLADJANA; HUSSON, ROBERT N.

    2014-01-01

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

  16. Caveolin-3 Overexpression Attenuates Cardiac Hypertrophy via Inhibition of T-type Ca2+ Current Modulated by Protein Kinase Cα in Cardiomyocytes*

    PubMed Central

    Markandeya, Yogananda S.; Phelan, Laura J.; Woon, Marites T.; Keefe, Alexis M.; Reynolds, Courtney R.; August, Benjamin K.; Hacker, Timothy A.; Roth, David M.; Patel, Hemal H.; Balijepalli, Ravi C.

    2015-01-01

    Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca2+ cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca2+ signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKCα and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca2+ current (ICa, T) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of ICa, T and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of ICa, T mediated by PKCα, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKCα-mediated increase in ICa, T and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy. PMID:26170457

  17. Caveolin-3 Overexpression Attenuates Cardiac Hypertrophy via Inhibition of T-type Ca2+ Current Modulated by Protein Kinase Cα in Cardiomyocytes.

    PubMed

    Markandeya, Yogananda S; Phelan, Laura J; Woon, Marites T; Keefe, Alexis M; Reynolds, Courtney R; August, Benjamin K; Hacker, Timothy A; Roth, David M; Patel, Hemal H; Balijepalli, Ravi C

    2015-09-01

    Pathological cardiac hypertrophy is characterized by subcellular remodeling of the ventricular myocyte with a reduction in the scaffolding protein caveolin-3 (Cav-3), altered Ca(2+) cycling, increased protein kinase C expression, and hyperactivation of calcineurin/nuclear factor of activated T cell (NFAT) signaling. However, the precise role of Cav-3 in the regulation of local Ca(2+) signaling in pathological cardiac hypertrophy is unclear. We used cardiac-specific Cav-3-overexpressing mice and in vivo and in vitro cardiac hypertrophy models to determine the essential requirement for Cav-3 expression in protection against pharmacologically and pressure overload-induced cardiac hypertrophy. Transverse aortic constriction and angiotensin-II (Ang-II) infusion in wild type (WT) mice resulted in cardiac hypertrophy characterized by significant reduction in fractional shortening, ejection fraction, and a reduced expression of Cav-3. In addition, association of PKCα and angiotensin-II receptor, type 1, with Cav-3 was disrupted in the hypertrophic ventricular myocytes. Whole cell patch clamp analysis demonstrated increased expression of T-type Ca(2+) current (ICa, T) in hypertrophic ventricular myocytes. In contrast, the Cav-3-overexpressing mice demonstrated protection from transverse aortic constriction or Ang-II-induced pathological hypertrophy with inhibition of ICa, T and intact Cav-3-associated macromolecular signaling complexes. siRNA-mediated knockdown of Cav-3 in the neonatal cardiomyocytes resulted in enhanced Ang-II stimulation of ICa, T mediated by PKCα, which caused nuclear translocation of NFAT. Overexpression of Cav-3 in neonatal myocytes prevented a PKCα-mediated increase in ICa, T and nuclear translocation of NFAT. In conclusion, we show that stable Cav-3 expression is essential for protecting the signaling mechanisms in pharmacologically and pressure overload-induced cardiac hypertrophy. PMID:26170457

  18. SUMOylation regulates the SNF1 protein kinase

    PubMed Central

    Simpson-Lavy, Kobi J.; Johnston, Mark

    2013-01-01

    The AMP-activated protein kinase (AMPK) is a major stress sensor of mammalian cells. AMPK’s homolog in the yeast Saccharomyces cerevisiae, the SNF1 protein kinase, is a central regulator of carbon metabolism that inhibits the Snf3/Rgt2-Rgt1 glucose sensing pathway and activates genes involved in respiration. We present evidence that glucose induces modification of the Snf1 catalytic subunt of SNF1 with the small ubiquitin-like modifier protein SUMO, catalyzed by the SUMO (E3) ligase Mms21. Our results suggest that SUMOylation of Snf1 inhibits its function in two ways: by interaction of SUMO attached to lysine 549 with a SUMO-interacting sequence motif located near the active site of Snf1, and by targeting Snf1 for destruction via the Slx5-Slx8 (SUMO-directed) ubiquitin ligase. These findings reveal another way SNF1 function is regulated in response to carbon source. PMID:24108357

  19. Protein kinases as drug targets in cancer.

    PubMed

    Arslan, Mehmet Alper; Kutuk, Ozgur; Basaga, Huveyda

    2006-11-01

    Identification of the key roles of protein kinases in signaling pathways leading to development of cancer has caused pharmacological interest to concentrate extensively on targeted therapies as a more specific and effective way for blockade of cancer progression. This review will mainly focus on inhibitors targeting these key components of cellular signaling by employing a technology-based point of view with respect to ATP- and non-ATP-competitive small molecule inhibitors and monoclonal antibodies of selected protein kinases, particularly, mammalian target of rapamycin (mTOR), BCR-ABL, MEK, p38 MAPK, EGFR PDGFR, VEGFR, HER2 and Raf. Inhibitors of the heat shock protein Hsp90 are also included in a separate section, as this protein plays an essential role for the maturation/proper activation of cancer-related protein kinases. In the following review, the molecular details of the mode of action of these inhibitors as well as the emergence of drug resistance encountered in several cases are discussed in light of the structural, molecular and clinical studies conducted so far. PMID:17100568

  20. Mitogen Activated Protein kinase signal transduction pathways in the prostate

    PubMed Central

    Maroni, Paul D; Koul, Sweaty; Meacham, Randall B; Koul, Hari K

    2004-01-01

    The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy. PMID:15219238

  1. cGMP/cGMP-dependent protein kinase pathway modulates nicotine-induced currents through the activation of α-bungarotoxin-insensitive nicotinic acetylcholine receptors from insect neurosecretory cells.

    PubMed

    Mannai, Safa; Bitri, Lofti; Thany, Steeve H

    2016-06-01

    Insect neurosecretory cells, called dorsal unpaired median neurons, are known to express two α-bungarotoxin-insensitive nicotinic acetylcholine receptor (nAChR) subtypes, nAChR1 and nAChR2. It was demonstrated that nAChR1 was sensitive to cAMP/cAMP-dependent protein kinase (PKA) regulation, resulting in a modulation of nicotine currents. In this study, we show that cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase (PKG) pathway modulates nicotine-induced currents, as increased cGMP affects the second compound of the biphasic current-voltage curve, corresponding to the nAChR2 receptors. Indeed, maintaining the guanosine triphosphate level with 100 μM guanosine triphosphate-γ-S increased nicotine currents through nAChR2. We also demonstrated that inhibition of PKG activity with 0.2 μM (8R,9S,11S)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c,d,e)-trinden-1-one (KT5823), a PKG specific inhibitor, reduced nicotine-induced current amplitudes. KT5823 effect on nicotine currents is associated with calcium (Ca(2+) ) activity because inhibition of Ca(2+) concentration with cadmium chloride (CdCl2 ) abolished KT5823-induced inhibition mediated by nAChR2. However, specific inhibition of nitric oxide-guanylyl cyclase (GC) complex by 10 μM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) significantly increased nicotine-induced current amplitudes on both nAChR1 and nAChR2. These results suggest that nicotine-induced currents mediated by both α-bungarotoxin-insensitive nAChR1 and nAChR2 are coupled to the cGMP/PKG pathway. We propose that nicotinic acetylcholine receptor activation induces an increase in intracellular calcium (Ca(2+) ) concentration. Elevation of intracellular Ca(2+) results in the formation of Ca(2+) -calmodulin (CaM) complex, which activates guanylyl cyclase (GC) and/or adenylyl cyclase (AC). Ca(2+) -CaM complex could activate Ca(2+) calmodulin kinase II which

  2. 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.

  3. Identification of four plastid-localized protein kinases.

    PubMed

    Richter, Andreas S; Gartmann, Hans; Fechler, Mona; Rödiger, Anja; Baginsky, Sacha; Grimm, Bernhard

    2016-06-01

    In chloroplasts, protein phosphorylation regulates important processes, including metabolism, photosynthesis, gene expression, and signaling. Because the hitherto known plastid protein kinases represent only a fraction of existing kinases, we aimed at the identification of novel plastid-localized protein kinases that potentially phosphorylate enzymes of the tetrapyrrole biosynthesis (TBS) pathway. We screened publicly available databases for proteins annotated as putative protein kinase family proteins with predicted chloroplast localization. Additionally, we analyzed chloroplast fractions which were separated by sucrose density gradient centrifugation by mass spectrometry. We identified four new candidates for protein kinases, which were confirmed to be plastid localized by expression of GFP-fusion proteins in tobacco leaves. A phosphorylation assay with the purified kinases confirmed the protein kinase activity for two of them. PMID:27214872

  4. Radial Spoke Protein 3 Is a Mammalian Protein Kinase A-anchoring Protein That Binds ERK1/2*

    PubMed Central

    Jivan, Arif; Earnest, Svetlana; Juang, Yu-Chi; Cobb, Melanie H.

    2009-01-01

    Initially identified in Chlamydomonas, RSP3 (radial spoke protein 3) is 1 of more than 20 identified radial spoke structural components of motile cilia and is required for axonemal sliding and flagellar motility. The mammalian orthologs for this and other radial spoke proteins, however, remain to be characterized. We found mammalian RSP3 to bind to the MAPK ERK2 through a yeast two-hybrid screen designed to identify interacting proteins that have a higher affinity for the phosphorylated, active form of the protein kinase. Consistent with the screening result, the human homolog, RSPH3, interacts with and is a substrate for ERK1/2. Moreover, RSPH3 is a protein kinase A-anchoring protein (AKAP) that scaffolds the cAMP-dependent protein kinase holoenzyme. The binding of RSPH3 to the regulatory subunits of cAMP-dependent protein kinase, RIIα and RIIβ, is regulated by ERK1/2 activity and phosphorylation. Here we describe an ERK1/2-interacting AKAP and suggest a mechanism by which cAMP-dependent protein kinase-AKAP binding can be modulated by the activity of other enzymes. PMID:19684019

  5. Regulation of glutamate metabolism by protein kinases in mycobacteria.

    PubMed

    O'Hare, Helen M; Durán, Rosario; Cerveñansky, Carlos; Bellinzoni, Marco; Wehenkel, Anne Marie; Pritsch, Otto; Obal, Gonzalo; Baumgartner, Jens; Vialaret, Jérome; Johnsson, Kai; Alzari, Pedro M

    2008-12-01

    Protein kinase G of Mycobacterium tuberculosis has been implicated in virulence and in regulation of glutamate metabolism. Here we show that this kinase undergoes a pattern of autophosphorylation that is distinct from that of other M. tuberculosis protein kinases characterized to date and we identify GarA as a substrate for phosphorylation by PknG. Autophosphorylation of PknG has little effect on kinase activity but promotes binding to GarA, an interaction that is also detected in living mycobacteria. PknG phosphorylates GarA at threonine 21, adjacent to the residue phosphorylated by PknB (T22), and these two phosphorylation events are mutually exclusive. Like the homologue OdhI from Corynebacterium glutamicum, the unphosphorylated form of GarA is shown to inhibit alpha-ketoglutarate decarboxylase in the TCA cycle. Additionally GarA is found to bind and modulate the activity of a large NAD(+)-specific glutamate dehydrogenase with an unusually low affinity for glutamate. Previous reports of a defect in glutamate metabolism caused by pknG deletion may thus be explained by the effect of unphosphorylated GarA on these two enzyme activities, which may also contribute to the attenuation of virulence. PMID:19019160

  6. The Chromone Alkaloid, Rohitukine, Affords Anti-Cancer Activity via Modulating Apoptosis Pathways in A549 Cell Line and Yeast Mitogen Activated Protein Kinase (MAPK) Pathway

    PubMed Central

    Safia; Kamil, Mohd; Jadiya, Pooja; Sheikh, Saba; Haque, Ejazul; Nazir, Aamir; Lakshmi, Vijai; Mir, Snober S.

    2015-01-01

    The field of cancer research and treatment has made significant progress, yet we are far from having completely safe, efficient and specific therapies that target cancer cells and spare the healthy tissues. Natural compounds may reduce the problems related to cancer treatment. Currently, many plant products are being used to treat cancer. In this study, Rohitukine, a natural occurring chromone alkaloid extracted from Dysoxylum binectariferum, was investigated for cytotoxic properties against budding yeast as well as against lung cancer (A549) cells. We endeavored to specifically study Rohitukine in S. cerevisiae in the context of MAPK pathways as yeast probably represents the experimental model where the organization and regulation of MAPK pathways are best understood. MAPK are evolutionarily conserved protein kinases that transfer extracellular signals to the machinery controlling essential cellular processes like growth, migration, differentiation, cell division and apoptosis. We aimed at carrying out hypothesis driven studies towards targeting the important network of cellular communication, a critical process that gets awry in cancer. Employing mutant strains of genetic model system Saccharomyces cerevisiae. S. cerevisiae encodes five MAPKs involved in control of distinct cellular responses such as growth, differentiation, migration and apoptosis. Our study involves gene knockouts of Slt2 and Hog1 which are functional homologs of human ERK5 and mammalian p38 MAPK, respectively. We performed cytotoxicity assay to evaluate the effect of Rohitukine on cell viability and also determined the effects of drug on generation of reactive oxygen species, induction of apoptosis and expression of Slt2 and Hog1 gene at mRNA level in the presence of drug. The results of this study show a differential effect in the activity of drug between the WT, Slt2 and Hog1 gene deletion strain indicating involvement of MAPK pathway. Further, we investigated Rohitukine induced cytotoxic

  7. 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. PMID:27151216

  8. 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

  9. Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function

    PubMed Central

    Grover, Prerna; Shi, Haibin; Baumgartner, Matthew; Camacho, Carlos J.; Smithgall, Thomas E.

    2015-01-01

    The ABL protein-tyrosine kinase regulates intracellular signaling pathways controlling diverse cellular processes and contributes to several forms of cancer. The kinase activity of ABL is repressed by intramolecular interactions involving its regulatory Ncap, SH3 and SH2 domains. Small molecules that allosterically regulate ABL kinase activity through its non-catalytic domains may represent selective probes of ABL function. Here we report a screening assay for chemical modulators of ABL kinase activity that target the regulatory interaction of the SH3 domain with the SH2-kinase linker. This fluorescence polarization (FP) assay is based on a purified recombinant ABL protein consisting of the N-cap, SH3 and SH2 domains plus the SH2-kinase linker (N32L protein) and a short fluorescein-labeled probe peptide that binds to the SH3 domain. In assay development experiments, we found that the probe peptide binds to the recombinant ABL N32L protein in vitro, producing a robust FP signal that can be competed with an excess of unlabeled peptide. The FP signal is not observed with control N32L proteins bearing either an inactivating mutation in the SH3 domain or enhanced SH3:linker interaction. A pilot screen of 1200 FDA-approved drugs identified four compounds that specifically reduced the FP signal by at least three standard deviations from the untreated controls. Secondary assays showed that one of these hit compounds, the antithrombotic drug dipyridamole, enhances ABL kinase activity in vitro to a greater extent than the previously described ABL agonist, DPH. Docking studies predicted that this compound binds to a pocket formed at the interface of the SH3 domain and the linker, suggesting that it activates ABL by disrupting this regulatory interaction. These results show that screening assays based on the non-catalytic domains of ABL can identify allosteric small molecule regulators of kinase function, providing a new approach to selective drug discovery for this important

  10. Cellular reprogramming through mitogen-activated protein kinases

    PubMed Central

    Lee, Justin; Eschen-Lippold, Lennart; Lassowskat, Ines; Böttcher, Christoph; Scheel, Dierk

    2015-01-01

    Mitogen-activated protein kinase (MAPK) cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554) in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins) as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression—including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding, and degradation) steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes. PMID:26579181

  11. Protein kinase CK2 and protein kinase D are associated with the COP9 signalosome

    PubMed Central

    Uhle, Stefan; Medalia, Ohad; Waldron, Richard; Dumdey, Renate; Henklein, Peter; Bech-Otschir, Dawadschargal; Huang, Xiaohua; Berse, Matthias; Sperling, Joseph; Schade, Rüdiger; Dubiel, Wolfgang

    2003-01-01

    The COP9 signalosome (CSN) purified from human erythrocytes possesses kinase activity that phosphoryl ates proteins such as c-Jun and p53 with consequence for their ubiquitin (Ub)-dependent degradation. Here we show that protein kinase CK2 (CK2) and protein kinase D (PKD) co-purify with CSN. Immunoprecipi tation and far-western blots reveal that CK2 and PKD are in fact associated with CSN. As indicated by electron microscopy with gold-labeled ATP, at least 10% of CSN particles are associated with kinases. Kinase activity, most likely due to CK2 and PKD, co-immuno precipitates with CSN from HeLa cells. CK2 binds to ΔCSN3(111–403) and CSN7, whereas PKD interacts with full-length CSN3. CK2 phosphorylates CSN2 and CSN7, and PKD modifies CSN7. Both CK2 and PKD phosphorylate c-Jun as well as p53. CK2 phosphoryl ates Thr155, which targets p53 to degradation by the Ub system. Curcumin, emodin, DRB and resveratrol block CSN-associated kinases and induce degradation of c-Jun in HeLa cells. Curcumin treatment results in elevated amounts of c-Jun–Ub conjugates. We conclude that CK2 and PKD are recruited by CSN in order to regulate Ub conjugate formation. PMID:12628923

  12. Protein kinase C is involved in regulation of Ca2+ channels in plasmalemma of Nitella syncarpa.

    PubMed

    Zherelova, O M

    1989-01-01

    Ca2+ current recordings have been made on Nitella syncarpa cells using the intracellular perfusion and the voltage-clamp technique. TPA (12-O-tetradecanoylphorbol-13-acetate), a substance capable of activating protein kinase C from plasmalemma of Nitella cells, modulates voltage-dependent Ca2+ channels. Polymixin B, inhibitor of protein kinase C, blocks the Nitella plasmalemma Ca2+ channels; the rate of channel blockage depends on the concentration and exposure time of the substance. PMID:2536617

  13. Mixed - Lineage Protein kinases (MLKs) in inflammation, metabolism, and other disease states.

    PubMed

    Craige, Siobhan M; Reif, Michaella M; Kant, Shashi

    2016-09-01

    Mixed lineage kinases, or MLKs, are members of the MAP kinase kinase kinase (MAP3K) family, which were originally identified among the activators of the major stress-dependent mitogen activated protein kinases (MAPKs), JNK and p38. During stress, the activation of JNK and p38 kinases targets several essential downstream substrates that react in a specific manner to the unique stressor and thus determine the fate of the cell in response to a particular challenge. Recently, the MLK family was identified as a specific modulator of JNK and p38 signaling in metabolic syndrome. Moreover, the MLK family of kinases appears to be involved in a very wide spectrum of disorders. This review discusses the newly identified functions of MLKs in multiple diseases including metabolic disorders, inflammation, cancer, and neurological diseases. PMID:27259981

  14. Osmotic stress signaling via protein kinases.

    PubMed

    Fujii, Hiroaki; Zhu, Jian-Kang

    2012-10-01

    Plants face various kinds of environmental stresses, including drought, salinity, and low temperature, which cause osmotic stress. An understanding of the plant signaling pathways that respond to osmotic stress is important for both basic biology and agriculture. In this review, we summarize recent investigations concerning the SNF1-related protein kinase (SnRK) 2 kinase family, which play central roles in osmotic stress responses. SnRK2s are activated by osmotic stress, and a mutant lacking SnRK2s is hypersensitive to osmotic stress. Many questions remain about the signaling pathway upstream and downstream of SnRK2s. Because some SnRK2s also functions in the abscisic acid (ABA) signaling pathway, which has recently been well clarified, study of SnRK2s in ABA signaling can provide clues regarding their roles in osmotic stress signaling. PMID:22828864

  15. Probing the enzyme kinetics, allosteric modulation and activation of α1- and α2-subunit-containing AMP-activated protein kinase (AMPK) heterotrimeric complexes by pharmacological and physiological activators

    PubMed Central

    Rajamohan, Francis; Reyes, Allan R.; Frisbie, Richard K.; Hoth, Lise R.; Sahasrabudhe, Parag; Magyar, Rachelle; Landro, James A.; Withka, Jane M.; Caspers, Nicole L.; Calabrese, Matthew F.; Ward, Jessica; Kurumbail, Ravi G.

    2015-01-01

    AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that serves as a pleotropic regulator of whole body energy homoeostasis. AMPK exists as a heterotrimeric complex, composed of a catalytic subunit (α) and two regulatory subunits (β and γ), each present as multiple isoforms. In the present study, we compared the enzyme kinetics and allosteric modulation of six recombinant AMPK isoforms, α1β1γ1, α1β2γ1, α1β2γ3, α2β1γ1, α2β2γ1 and α2β2γ3 using known activators, A769662 and AMP. The α1-containing complexes exhibited higher specific activities and lower Km values for a widely used peptide substrate (SAMS) compared with α2-complexes. Surface plasmon resonance (SPR)-based direct binding measurements revealed biphasic binding modes with two distinct equilibrium binding constants for AMP, ADP and ATP across all isoforms tested. The α2-complexes were ∼25-fold more sensitive than α1-complexes to dephosphorylation of a critical threonine on their activation loop (pThr172/174). However, α2-complexes were more readily activated by AMP than α1-complexes. Compared with β1-containing heterotrimers, β2-containing AMPK isoforms are less sensitive to activation by A769662, a synthetic activator. These data demonstrate that ligand induced activation of AMPK isoforms may vary significantly based on their AMPK subunit composition. Our studies provide insights for the design of isoform-selective AMPK activators for the treatment of metabolic diseases. PMID:26635351

  16. Protein kinase C theta (PKCθ) modulates the ClC-1 chloride channel activity and skeletal muscle phenotype: a biophysical and gene expression study in mouse models lacking the PKCθ.

    PubMed

    Camerino, Giulia Maria; Bouchè, Marina; De Bellis, Michela; Cannone, Maria; Liantonio, Antonella; Musaraj, Kejla; Romano, Rossella; Smeriglio, Piera; Madaro, Luca; Giustino, Arcangela; De Luca, Annamaria; Desaphy, Jean-François; Camerino, Diana Conte; Pierno, Sabata

    2014-12-01

    In skeletal muscle, the resting chloride conductance (gCl), due to the ClC-1 chloride channel, controls the sarcolemma electrical stability. Indeed, loss-of-function mutations in ClC-1 gene are responsible of myotonia congenita. The ClC-1 channel can be phosphorylated and inactivated by protein kinases C (PKC), but the relative contribution of each PKC isoforms is unknown. Here, we investigated on the role of PKCθ in the regulation of ClC-1 channel expression and activity in fast- and slow-twitch muscles of mouse models lacking PKCθ. Electrophysiological studies showed an increase of gCl in the PKCθ-null mice with respect to wild type. Muscle excitability was reduced accordingly. However, the expression of the ClC-1 channel, evaluated by qRT-PCR, was not modified in PKCθ-null muscles suggesting that PKCθ affects the ClC-1 activity. Pharmacological studies demonstrated that although PKCθ appreciably modulates gCl, other isoforms are still active and concur to this role. The modification of gCl in PKCθ-null muscles has caused adaptation of the expression of phenotype-specific genes, such as calcineurin and myocyte enhancer factor-2, supporting the role of PKCθ also in the settings of muscle phenotype. Importantly, the lack of PKCθ has prevented the aging-related reduction of gCl, suggesting that its modulation may represent a new strategy to contrast the aging process. PMID:24643479

  17. 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

  18. Protein kinase A signalling in Schistosoma mansoni cercariae and schistosomules.

    PubMed

    Hirst, Natasha L; Lawton, Scott P; Walker, Anthony J

    2016-06-01

    Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A regulates multiple processes in eukaryotes by phosphorylating diverse cellular substrates, including metabolic and signalling enzymes, ion channels and transcription factors. Here we provide insight into protein kinase A signalling in cercariae and 24h in vitro cultured somules of the blood parasite, Schistosoma mansoni, which causes human intestinal schistosomiasis. Functional mapping of activated protein kinase A using anti-phospho protein kinase A antibodies and confocal laser scanning microscopy revealed activated protein kinase A in the central and peripheral nervous system, oral-tip sensory papillae, oesophagus and excretory system of intact cercariae. Cultured 24h somules, which biologically represent the skin-resident stage of the parasite, exhibited similar activation patterns in oesophageal and nerve tissues but also displayed striking activation at the tegument and activation in a region resembling the germinal 'stem' cell cluster. The adenylyl cyclase activator, forskolin, stimulated somule protein kinase A activation and produced a hyperkinesia phenotype. The biogenic amines, serotonin and dopamine known to be present in skin also induced protein kinase A activation in somules, whereas neuropeptide Y or [Leu(31),Pro(34)]-neuropeptide Y attenuated protein kinase A activation. However, neuropeptide Y did not block the forskolin-induced somule hyperkinesia. Bioinformatic investigation of potential protein associations revealed 193 medium confidence and 59 high confidence protein kinase A interacting partners in S. mansoni, many of which possess putative protein kinase A phosphorylation sites. These data provide valuable insight into the intricacies of protein kinase A signalling in S. mansoni and a framework for further physiological investigations into the roles of protein kinase A in schistosomes, particularly in the context of interactions between the parasite and the host. PMID:26777870

  19. 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.

  20. Photoswitchable diacylglycerols enable optical control of protein kinase C.

    PubMed

    Frank, James Allen; Yushchenko, Dmytro A; Hodson, David J; Lipstein, Noa; Nagpal, Jatin; Rutter, Guy A; Rhee, Jeong-Seop; Gottschalk, Alexander; Brose, Nils; Schultz, Carsten; Trauner, Dirk

    2016-09-01

    Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling. PMID:27454932

  1. Increased dietary protein attenuates C-reactive protein and creatine kinase responses to exercise-induced energy deficit

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We determined if dietary protein (P) modulates responses of C-reactive protein (CRP) and creatine kinase (CK), biomarkers of inflammation and muscle damage, during exercise-induced energy deficit (DEF). Thirteen healthy men (22 +/- 1 y, VO2peak 60 +/- 2 ml.kg-1.min-1) balanced energy expenditure (EE...

  2. Phosphorylation of ornithine decarboxylase by a polyamine-dependent protein kinase.

    PubMed Central

    Atmar, V J; Kuehn, G D

    1981-01-01

    This paper presents evidence that a polyamine-dependent protein kinase (EC 2.7.1.37) purified from nuclei of the slime mold Physarum polycephalum catalyzes phosphorylation of ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17). The protein kinase had properties similar to OrnDCase antizyme. Phosphocellulose chromatography of nuclear preparations from P. polycephalum yielded the polyamine-dependent protein kinase of subunit Mr 26,000 that was resolved from a second fraction in which the protein kinase copurified with a phosphate-acceptor protein of subunit Mr 70,000. At Na+ concentrations less than approximately 150 mM, a complex formed between the protein kinase and the phosphate-acceptor protein. The complex did not demonstrate protein kinase or OrnDCase activity. The complex was dissociated by greater than 150 mM Na+ into its constituent proteins. The dissociated complex catalyzed phosphorylation of the Mr 70,000 component in the presence of spermidine and spermine, and it also demonstrated OrnDCase activity. The purified Mr 70,000 component from the complex and authentic OrnDCase, purified by procedures previously reported, were virtually identical with respect to OrnDCase activity, capacity to be phosphorylated by the polyamine-dependent protein kinase, amino acid composition, and immunological crossreactivity. Phosphorylation of OrnDCase by the polyamine-dependent protein kinase sharply inhibited OrnDCase activity. Thus, this is an example of posttranslational covalent modification of OrnDCase with concurrent alteration of its catalytic function. It is also an unusual example of control of the first enzyme in a biosynthetic pathway by a protein kinase that is, in turn, modulated by the immediate end products of the pathway. Images PMID:6946489

  3. Cl- Channels in CF: Lack of Activation by Protein Kinase C and cAMP-Dependent Protein Kinase

    NASA Astrophysics Data System (ADS)

    Hwang, Tzyh-Chang; Lu, Luo; Zeitlin, Pamela L.; Gruenert, Dieter C.; Huganir, Richard; Guggino, William B.

    1989-06-01

    Secretory chloride channels can be activated by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase in normal airway epithelial cells but not in cells from individuals with cystic fibrosis (CF). In excised, inside-out patches of apical membrane of normal human airway cells and airway cells from three patients with CF, the chloride channels exhibited a characteristic outwardly rectifying current-voltage relation and depolarization-induced activation. Channels from normal tissues were activated by both cAMP-dependent protein kinase and protein kinase C. However, chloride channels from CF patients could not be activated by either kinase. Thus, gating of normal epithelial chloride channels is regulated by both cAMP-dependent protein kinase and protein kinase C, and regulation by both kinases is defective in CF.

  4. Myogenic signaling of phosphatidylinositol 3-kinase requires the serine-threonine kinase Akt/protein kinase B

    PubMed Central

    Jiang, Bing-Hua; Aoki, Masahiro; Zheng, Jenny Z.; Li, Jian; Vogt, Peter K.

    1999-01-01

    The oncogene p3k, coding for a constitutively active form of phosphatidylinositol 3-kinase (PI 3-kinase), strongly activates myogenic differentiation. Inhibition of endogenous PI 3-kinase activity with the specific inhibitor LY294002, or with dominant-negative mutants of PI 3-kinase, interferes with myotube formation and with the expression of muscle-specific proteins. Here we demonstrate that a downstream target of PI 3-kinase, serine-threonine kinase Akt, plays an important role in myogenic differentiation. Expression of constitutively active forms of Akt dramatically enhances myotube formation and expression of the muscle-specific proteins MyoD, creatine kinase, myosin heavy chain, and desmin. Transdominant negative forms of Akt inhibit myotube formation and the expression of muscle-specific proteins. The inhibition of myotube formation and the reduced expression of muscle-specific proteins caused by the PI 3-kinase inhibitor LY294002 are completely reversed by constitutively active forms of Akt. Wild-type cellular Akt effects a partial reversal of LY294002-induced inhibition of myogenic differentiation. This result suggests that Akt can substitute for PI 3-kinase in the stimulation of myogenesis; Akt may be an essential downstream component of PI 3-kinase-induced muscle differentiation. PMID:10051597

  5. Targeting of calcium/calmodulin-dependent protein kinase II.

    PubMed Central

    Colbran, Roger J

    2004-01-01

    Calcium/calmodulin-dependent protein kinase II (CaMKII) has diverse roles in virtually all cell types and it is regulated by a plethora of mechanisms. Local changes in Ca2+ concentration drive calmodulin binding and CaMKII activation. Activity is controlled further by autophosphorylation at multiple sites, which can generate an autonomously active form of the kinase (Thr286) or can block Ca2+/calmodulin binding (Thr305/306). The regulated actions of protein phosphatases at these sites also modulate downstream signalling from CaMKII. In addition, CaMKII targeting to specific subcellular microdomains appears to be necessary to account for the known signalling specificity, and targeting is regulated by Ca2+/calmodulin and autophosphorylation. The present review focuses on recent studies revealing the diversity of CaMKII interactions with proteins localized to neuronal dendrites. Interactions with various subunits of the NMDA (N-methyl-D-aspartate) subtype of glutamate receptor have attracted the most attention, but binding of CaMKII to cytoskeletal and several other regulatory proteins has also been reported. Recent reports describing the molecular basis of each interaction and their potential role in the normal regulation of synaptic transmission and in pathological situations are discussed. These studies have revealed fundamental regulatory mechanisms that are probably important for controlling CaMKII functions in many cell types. PMID:14653781

  6. Mitogen-activated protein kinase kinase kinase 1 (MAP3K1) integrates developmental signals for eyelid closure

    PubMed Central

    Geh, Esmond; Meng, Qinghang; Mongan, Maureen; Wang, Jingcai; Takatori, Atsushi; Zheng, Yi; Puga, Alvaro; Lang, Richard A.; Xia, Ying

    2011-01-01

    Developmental eyelid closure is an evolutionarily conserved morphogenetic event requiring proliferation, differentiation, cytoskeleton reorganization, and migration of epithelial cells at the tip of the developing eyelid. Many signaling events take place during eyelid closure, but how the signals converge to regulate the morphogenetic process remains an open and intriguing question. Here we show that mitogen-activated protein kinase kinase kinase 1 (MAP3K1) highly expressed in the developing eyelid epithelium, forms with c-Jun, a regulatory axis that orchestrates morphogenesis by integrating two different networks of eyelid closure signals. A TGF-α/EGFR-RhoA module initiates one of these networks by inducing c-Jun expression which, in a phosphorylation-independent manner, binds to the Map3k1 promoter and causes an increase in MAP3K1 expression. RhoA knockout in the ocular surface epithelium disturbs this network by decreasing MAP3K1 expression, and causes delayed eyelid closure in Map3k1 hemizygotes. The second network is initiated by the enzymatic activity of MAP3K1, which phosphorylates and activates a JNK-c-Jun module, leading to AP-1 transactivation and induction of its downstream genes, such as Pai-1. MAP3K1 inactivation reduces AP-1 activity and PAI-1 expression both in cells and developing eyelids. MAP3K1 is therefore the nexus of an intracrine regulatory loop connecting the TGF-α/EGFR/RhoA-c-Jun and JNK-c-Jun-AP-1 pathways in developmental eyelid closure. PMID:21969564

  7. Identification of Protein Kinase Substrates by the Kinase-Interacting Substrate Screening (KISS) Approach.

    PubMed

    Amano, Mutsuki; Nishioka, Tomoki; Yura, Yoshimitsu; Kaibuchi, Kozo

    2016-01-01

    Identifying the substrates of protein kinases to understand their modes of action has been undertaken by various approaches and remains an ongoing challenge. Phosphoproteomic technologies have accelerated the accumulation of data concerning protein phosphorylation and have uncovered vast numbers of phosphorylation sites in vivo. In this unit, a novel in vitro screening approach for protein kinase substrates is presented, based on protein-protein interaction and mass spectrometry-based phosphoproteomic technology. © 2016 by John Wiley & Sons, Inc. PMID:27580705

  8. Protein kinases are potential targets to treat inflammatory bowel disease

    PubMed Central

    Yang, Lei; Yan, Yutao

    2014-01-01

    Protein kinases play a crucial role in the pathogenesis of inflammatory bowel disease (IBD), the two main forms of which are ulcerative colitis and Crohn’s disease. In this article, we will review the mechanisms of involvement of protein kinases in the pathogenesis of and intervention against IBD, in terms of their effects on genetics, microbiota, mucous layer and tight junction, and the potential of protein kinases as therapeutic targets against IBD. PMID:25374761

  9. Phosphatidylinositol 3 kinase modulation of trophoblast cell differentiation

    PubMed Central

    2010-01-01

    Background The trophoblast lineage arises as the first differentiation event during embryogenesis. Trophoblast giant cells are one of several end-stage products of trophoblast cell differentiation in rodents. These cells are located at the maternal-fetal interface and are capable of invasive and endocrine functions, which are necessary for successful pregnancy. Rcho-1 trophoblast stem cells can be effectively used as a model for investigating trophoblast cell differentiation. In this report, we evaluated the role of the phosphatidylinositol 3-kinase (PI3K) signaling pathway in the regulation of trophoblast cell differentiation. Transcript profiles from trophoblast stem cells, differentiated trophoblast cells, and differentiated trophoblast cells following disruption of PI3K signaling were generated and characterized. Results Prominent changes in gene expression accompanied the differentiation of trophoblast stem cells. PI3K modulated the expression of a subset of trophoblast cell differentiation-dependent genes. Among the PI3K-responsive genes were those encoding proteins contributing to the invasive and endocrine phenotypes of trophoblast giant cells. Conclusions Genes have been identified with differential expression patterns associated with trophoblast stem cells and trophoblast cell differentiation; a subset of these genes are regulated by PI3K signaling, including those impacting the differentiated trophoblast giant cell phenotype. PMID:20840781

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

    PubMed Central

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

    2011-01-01

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

  11. 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…

  12. The selectivity of protein kinase inhibitors: a further update

    PubMed Central

    Bain, Jenny; Plater, Lorna; Elliott, Matt; Shpiro, Natalia; Hastie, C. James; Mclauchlan, Hilary; Klevernic, Iva; Arthur, J. Simon C.; Alessi, Dario R.; Cohen, Philip

    2007-01-01

    The specificities of 65 compounds reported to be relatively specific inhibitors of protein kinases have been profiled against a panel of 70–80 protein kinases. On the basis of this information, the effects of compounds that we have studied in cells and other data in the literature, we recommend the use of the following small-molecule inhibitors: SB 203580/SB202190 and BIRB 0796 to be used in parallel to assess the physiological roles of p38 MAPK (mitogen-activated protein kinase) isoforms, PI-103 and wortmannin to be used in parallel to inhibit phosphatidylinositol (phosphoinositide) 3-kinases, PP1 or PP2 to be used in parallel with Src-I1 (Src inhibitor-1) to inhibit Src family members; PD 184352 or PD 0325901 to inhibit MKK1 (MAPK kinase-1) or MKK1 plus MKK5, Akt-I-1/2 to inhibit the activation of PKB (protein kinase B/Akt), rapamycin to inhibit TORC1 [mTOR (mammalian target of rapamycin)–raptor (regulatory associated protein of mTOR) complex], CT 99021 to inhibit GSK3 (glycogen synthase kinase 3), BI-D1870 and SL0101 or FMK (fluoromethylketone) to be used in parallel to inhibit RSK (ribosomal S6 kinase), D4476 to inhibit CK1 (casein kinase 1), VX680 to inhibit Aurora kinases, and roscovitine as a pan-CDK (cyclin-dependent kinase) inhibitor. We have also identified harmine as a potent and specific inhibitor of DYRK1A (dual-specificity tyrosine-phosphorylated and -regulated kinase 1A) in vitro. The results have further emphasized the need for considerable caution in using small-molecule inhibitors of protein kinases to assess the physiological roles of these enzymes. Despite being used widely, many of the compounds that we analysed were too non-specific for useful conclusions to be made, other than to exclude the involvement of particular protein kinases in cellular processes. PMID:17850214

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Calcium-dependent protein kinases (CDPKs or CPKs) are classified as serine/threonine protein kinases but we made the surprising observation that soybean CDPK' and several Arabidopsis isoforms (AtCPK4 and AtCPK34) could also autophosphorylate on tyrosine residues. In studies with His6-GmCDPK', we ide...

  14. Protein Kinases: Emerging Therapeutic Targets in Chronic Lymphocytic Leukemia

    PubMed Central

    Balakrishnan, Kumudha; Gandhi, Varsha

    2014-01-01

    Introduction Although protein kinases are primary targets for inhibition in hematological malignancies, until recently their contribution to chronic lymphocytic leukemia (CLL) was poorly understood. Insights into B cell receptor (BCR) signaling and its role in regulating key cellular functions have shed light on candidate protein kinases that are aberrantly activated in CLL. In this regard, protein kinases are now considered as potential drug targets in CLL. Area covered This review has covered signaling pathways and associated protein kinases in CLL and the kinase inhibitors currently available in preclinical and clinical investigations. Individual protein kinases that are abnormally active in CLL and the functional consequences of their inhibition are discussed. Expert opinion A growing body of evidence suggests that protein kinases are druggable targets for patients with CLL. The emergence of novel and bio-available kinase inhibitors and their promising clinical activity in CLL underscore the oncogenic role of kinases in leukemogenesis. Further investigations directed towards their role as single agents or in combinations may provide insight into understanding the substantial role of kinase mediated signal transduction pathways and their inhibition in B- CLL. PMID:22409342

  15. Artemisia asiatica Nakai Attenuates the Expression of Proinflammatory Mediators in Stimulated Macrophages Through Modulation of Nuclear Factor-κB and Mitogen-Activated Protein Kinase Pathways

    PubMed Central

    Kim, Eun-Kyung; Tang, Yujiao; Cha, Kwang-Suk; Choi, Heeri; Lee, Chun Bok; Yoon, Jin-Hwan; Kim, Sang Bae; Kim, Jong-Shik; Kim, Jong Moon; Han, Weon Cheol; Choi, Suck-Jun; Lee, Sangmin; Choi, Eun-Ju; Kim, Sang-Hyun

    2015-01-01

    Abstract The present study aimed to examine the anti-inflammatory effects and potential mechanism of action of Artemisia asiatica Nakai (A. asiatica Nakai) extract in activated murine macrophages. A. asiatica Nakai extract showed dose-dependent suppression of lipopolysaccharide (LPS)-induced nitric oxide, inducible nitric oxide synthase, and cyclooxygenase-2 activity. It also showed dose-dependent inhibition of nuclear factor-κB (NF-κB) translocation from the cytosol to the nucleus and as an inhibitor of NF-κB-alpha phosphorylation. The extract's inhibitory effects were found to be mediated through NF-κB inhibition and phosphorylation of extracellular signal-regulated kinase 1/2 and p38 in LPS-stimulated J774A.1 murine macrophages, suggesting a potential mechanism for the anti-inflammatory activity of A. asiatica Nakai. To our knowledge, this is the first report of the anti-inflammatory effects of A. asiatica Nakai on J774A.1 murine macrophages; these results may help develop functional foods possessing an anti-inflammatory activity. PMID:26061361

  16. Phosphate Concentration and the Putative Sensor Kinase Protein CckA Modulate Cell Lysis and Release of the Rhodobacter capsulatus Gene Transfer Agent

    PubMed Central

    Westbye, A. B.; Leung, M. M.; Florizone, S. M.; Taylor, T. A.; Johnson, J. A.; Fogg, P. C.

    2013-01-01

    The gene transfer agent of Rhodobacter capsulatus (RcGTA) is a bacteriophage-like genetic element with the sole known function of horizontal gene transfer. Homologues of RcGTA genes are present in many members of the alphaproteobacteria and may serve an important role in microbial evolution. Transcription of RcGTA genes is induced as cultures enter the stationary phase; however, little is known about cis-active sequences. In this work, we identify the promoter of the first gene in the RcGTA structural gene cluster. Additionally, gene transduction frequency depends on the growth medium, and the reason for this is not known. We report that millimolar concentrations of phosphate posttranslationally inhibit the lysis-dependent release of RcGTA from cells in both a complex medium and a defined medium. Furthermore, we found that cell lysis requires the genes rcc00555 and rcc00556, which were expressed and studied in Escherichia coli to determine their predicted functions as an endolysin and holin, respectively. Production of RcGTA is regulated by host systems, including a putative histidine kinase, CckA, and we found that CckA is required for maximal expression of rcc00555 and for maturation of RcGTA to yield gene transduction-functional particles. PMID:23995641

  17. Artemisia asiatica Nakai Attenuates the Expression of Proinflammatory Mediators in Stimulated Macrophages Through Modulation of Nuclear Factor-κB and Mitogen-Activated Protein Kinase Pathways.

    PubMed

    Kim, Eun-Kyung; Tang, Yujiao; Cha, Kwang-Suk; Choi, Heeri; Lee, Chun Bok; Yoon, Jin-Hwan; Kim, Sang Bae; Kim, Jong-Shik; Kim, Jong Moon; Han, Weon Cheol; Choi, Suck-Jun; Lee, Sangmin; Choi, Eun-Ju; Kim, Sang-Hyun

    2015-08-01

    The present study aimed to examine the anti-inflammatory effects and potential mechanism of action of Artemisia asiatica Nakai (A. asiatica Nakai) extract in activated murine macrophages. A. asiatica Nakai extract showed dose-dependent suppression of lipopolysaccharide (LPS)-induced nitric oxide, inducible nitric oxide synthase, and cyclooxygenase-2 activity. It also showed dose-dependent inhibition of nuclear factor-κB (NF-κB) translocation from the cytosol to the nucleus and as an inhibitor of NF-κB-alpha phosphorylation. The extract's inhibitory effects were found to be mediated through NF-κB inhibition and phosphorylation of extracellular signal-regulated kinase 1/2 and p38 in LPS-stimulated J774A.1 murine macrophages, suggesting a potential mechanism for the anti-inflammatory activity of A. asiatica Nakai. To our knowledge, this is the first report of the anti-inflammatory effects of A. asiatica Nakai on J774A.1 murine macrophages; these results may help develop functional foods possessing an anti-inflammatory activity. PMID:26061361

  18. 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.

  19. The extended protein kinase C superfamily.

    PubMed Central

    Mellor, H; Parker, P J

    1998-01-01

    Members of the mammalian protein kinase C (PKC) superfamily play key regulatory roles in a multitude of cellular processes, ranging from control of fundamental cell autonomous activities, such as proliferation, to more organismal functions, such as memory. However, understanding of mammalian PKC signalling systems is complicated by the large number of family members. Significant progress has been made through studies based on comparative analysis, which have defined a number of regulatory elements in PKCs which confer specific location and activation signals to each isotype. Further studies on simple organisms have shown that PKC signalling paradigms are conserved through evolution from yeast to humans, underscoring the importance of this family in cellular signalling and giving novel insights into PKC function in complex mammalian systems. PMID:9601053

  20. Human protein kinase CK2 genes.

    PubMed

    Wirkner, U; Voss, H; Lichter, P; Pyerin, W

    1994-01-01

    We have analyzed the genomic structure of human protein kinase CK2. Of the presumably four genes, the gene encoding the regulatory subunit beta and a processed (pseudo)gene of the catalytic subunit alpha have been characterized completely. In addition, a 18.9 kb-long central part of the gene encoding the catalytic subunit alpha has been characterized. The subunit beta gene spans 4.2 kb and is composed of seven exons. Its promoter region shows several features of a "housekeeping gene" and shares common features with the promoter of the regulatory subunit of cAMP-dependent protein kinase. Conforming to the genomic structure, the beta gene transcripts form a band around 1.1 kb. The central part of the subunit alpha gene contains eight exons comprising bases 102 to 824 of the translated region. Within the introns, 16 Alu repeats were identified, some of which arranged in tandems. The structure of both human CK2 coding genes, alpha and beta, is highly conserved. Several introns are located at corresponding positions in the respective genes of the nematode Caenorhabditis elegans. The processed alpha (pseudo)gene has a complete open reading frame and is 99% homologous to the coding region of the CK2 alpha cDNA. Although the gene has a promoter-like upstream region, no transcript could be identified so far. The genomic clones were used for localization in the human genome. The beta gene was mapped to locus 6p21, the alpha gene to locus 20p13 and the alpha (pseudo)gene to locus 11p15. There is no evidence for additional alpha or beta loci in the human genome. PMID:7735323

  1. 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

  2. Perivascular fat, AMP-activated protein kinase and vascular diseases

    PubMed Central

    Almabrouk, T A M; Ewart, M A; Salt, I P; Kennedy, S

    2014-01-01

    Perivascular adipose tissue (PVAT) is an active endocrine and paracrine organ that modulates vascular function, with implications for the pathophysiology of cardiovascular disease (CVD). Adipocytes and stromal cells contained within PVAT produce mediators (adipokines, cytokines, reactive oxygen species and gaseous compounds) with a range of paracrine effects modulating vascular smooth muscle cell contraction, proliferation and migration. However, the modulatory effect of PVAT on the vascular system in diseases, such as obesity, hypertension and atherosclerosis, remains poorly characterized. AMP-activated protein kinase (AMPK) regulates adipocyte metabolism, adipose biology and vascular function, and hence may be a potential therapeutic target for metabolic disorders such as type 2 diabetes mellitus (T2DM) and the vascular complications associated with obesity and T2DM. The role of AMPK in PVAT or the actions of PVAT have yet to be established, however. Activation of AMPK by pharmacological agents, such as metformin and thiazolidinediones, may modulate the activity of PVAT surrounding blood vessels and thereby contribute to their beneficial effect in cardiometabolic diseases. This review will provide a current perspective on how PVAT may influence vascular function via AMPK. We will also attempt to demonstrate how modulating AMPK activity using pharmacological agents could be exploited therapeutically to treat cardiometabolic diseases. PMID:24490856

  3. 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.

  4. Protein kinase A activity and Hedgehog signaling pathway.

    PubMed

    Kotani, Tomoya

    2012-01-01

    Protein kinase A (PKA) is a well-known kinase that plays fundamental roles in a variety of biological processes. In Hedgehog-responsive cells, PKA plays key roles in proliferation and fate specification by modulating the transduction of Hedgehog signaling. In the absence of Hedgehog, a basal level of PKA activity represses the transcription of Hedgehog target genes. The main substrates of PKA in this process are the Ci/Gli family of bipotential transcription factors, which activate and repress Hedgehog target gene expression. PKA phosphorylates Ci/Gli, promoting the production of the repressor forms of Ci/Gli and thus repressing Hedgehog target gene expression. In contrast, the activation of Hedgehog signaling in response to Hedgehog increases the active forms of Ci/Gli, resulting in Hedgehog target gene expression. Because both decreased and increased levels of PKA activity cause abnormal cell proliferation and alter cell fate specification, the basal level of PKA activity in Hedgehog-responsive cells should be precisely regulated. However, the mechanism by which PKA activity is regulated remains obscure and appears to vary between cell types, tissues, and organisms. To date, two mechanisms have been proposed. One is a classical mechanism in which PKA activity is regulated by a small second messenger, cAMP; the other is a novel mechanism in which PKA activity is regulated by a protein, Misty somites. PMID:22391308

  5. A- Kinase Anchoring Protein 150 Controls Protein Kinase C-mediated Phosphorylation and Sensitization of TRPV1

    PubMed Central

    Jeske, Nathaniel A.; Patwardhan, Amol M.; Ruparel, Nikita B.; Akopian, Armen N; Shapiro, Mark S.; Henry, Michael A.

    2009-01-01

    Post-translational modifications on various receptor proteins have significant effects on receptor activation. For the Transient Receptor Potential family V type 1 (TRPV1) receptor, phosphorylation of certain serine/threonine amino acid residues sensitizes the receptor to activation by capsaicin and heat. Although Protein Kinase C (PKC) phosphorylates TRPV1 on certain serine/threonine residues, it is not completely understood how PKC functionally associates with TRPV1. Recent studies have reported that the A-kinase Anchoring Protein 150 (AKAP150) mediates PKA phosphorylation of TRPV1 in several nociceptive models. Here, we demonstrate that AKAP150 also mediates PKC-directed phosphorylation and sensitization of TRPV1. In cultured rat trigeminal ganglia, immunocytochemical analyses demonstrate co-localization of AKAP150 and PKC isoforms α, δ, ε, and γ in TRPV1-positive neurons. Additional biochemical evidence supports immunocytochemical results, indicating that AKAP150 preferentially associates with certain PKC isoforms in rat trigeminal ganglia neurons. Employing siRNA-mediated knock-down of AKAP150 expression, we demonstrate that PKC-mediated phosphorylation of TRPV1 and sensitization to a capsaicin response is dependent upon functional expression of the AKAP150 scaffolding protein. Furthermore, PKC-induced sensitization to a thermal stimulus is abrogated in AKAP150 knock-out animals relative to wild-type. Collectively, results from these studies indicate that the AKAP150 scaffolding protein functionally modulates PKC-mediated phosphorylation and sensitization of the TRPV1 receptor in rat sensory neurons, suggesting the scaffolding protein to be an integral regulator of peripheral inflammatory hyperalgesia. PMID:19767149

  6. Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

    SciTech Connect

    Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy; Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A.; Rhee, Kirsten; Tisdale, Sarah; Danial, Nika; Benarafa, Charaf; Orduna, Anonio; Anderson, Paul

    2010-10-22

    Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

  7. Regulation of mitochondrial protein import by cytosolic kinases.

    PubMed

    Schmidt, Oliver; Harbauer, Angelika B; Rao, Sanjana; Eyrich, Beate; Zahedi, René P; Stojanovski, Diana; Schönfisch, Birgit; Guiard, Bernard; Sickmann, Albert; Pfanner, Nikolaus; Meisinger, Chris

    2011-01-21

    Mitochondria import a large number of nuclear-encoded proteins via membrane-bound transport machineries; however, little is known about regulation of the preprotein translocases. We report that the main protein entry gate of mitochondria, the translocase of the outer membrane (TOM complex), is phosphorylated by cytosolic kinases-in particular, casein kinase 2 (CK2) and protein kinase A (PKA). CK2 promotes biogenesis of the TOM complex by phosphorylation of two key components, the receptor Tom22 and the import protein Mim1, which in turn are required for import of further Tom proteins. Inactivation of CK2 decreases the levels of the TOM complex and thus mitochondrial protein import. PKA phosphorylates Tom70 under nonrespiring conditions, thereby inhibiting its receptor activity and the import of mitochondrial metabolite carriers. We conclude that cytosolic kinases exert stimulatory and inhibitory effects on biogenesis and function of the TOM complex and thus regulate protein import into mitochondria. PMID:21215441

  8. Transphosphorylation of E. coli proteins during production of recombinant protein kinases provides a robust system to characterize kinase specificity

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed Central

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

    2003-01-01

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

  10. Protein kinase inhibitors against malignant lymphoma

    PubMed Central

    D’Cruz, Osmond J; Uckun, Fatih M

    2013-01-01

    Introduction Tyrosine kinases (TKs) are intimately involved in multiple signal transduction pathways regulating survival, activation, proliferation and differentiation of lymphoid cells. Deregulation or overexpression of specific oncogenic TKs is implicated in maintaining the malignant phenotype in B-lineage lymphoid malignancies. Several novel targeted TK inhibitors (TKIs) have recently emerged as active in the treatment of relapsed or refractory B-cell lymphomas that inhibit critical signaling pathways, promote apoptotic mechanisms or modulate the tumor microenvironment. Areas covered In this review, the authors summarize the clinical outcomes of newer TKIs in various B-cell lymphomas from published and ongoing clinical studies and abstracts from major cancer and hematology conferences. Expert opinion Multiple clinical trials have demonstrated that robust antitumor activity can be obtained with TKIs directed toward specific oncogenic TKs that are genetically deregulated in various subtypes of B-cell lymphomas. Clinical success of targeting TKIs is dependent upon on identifying reliable molecular and clinical markers associated with select cohorts of patients. Further understanding of the signaling pathways should stimulate the identification of novel molecular targets and expand the development of new therapeutic options and individualized therapies. PMID:23496343