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Sample records for receptors protein kinase

  1. Identification of intracellular receptor proteins for activated protein kinase C.

    PubMed Central

    Mochly-Rosen, D; Khaner, H; Lopez, J

    1991-01-01

    Protein kinase C (PKC) translocates from the cytosol to the particulate fraction on activation. This activation-induced translocation of PKC is thought to reflect PKC binding to the membrane lipids. However, immunological and biochemical data suggest that PKC may bind to proteins in the cytoskeletal elements in the particulate fraction and in the nuclei. Here we describe evidence for the presence of intracellular receptor proteins that bind activated PKC. Several proteins from the detergent-insoluble material of the particulate fraction bound PKC in the presence of phosphatidylserine and calcium; binding was further increased with the addition of diacylglycerol. Binding of PKC to two of these proteins was concentration-dependent, saturable, and specific, suggesting that these binding proteins are receptors for activated C-kinase, termed here "RACKs." PKC binds to RACKs via a site on PKC distinct from the substrate binding site. We suggest that binding to RACKs may play a role in activation-induced translocation of PKC. Images PMID:1850844

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

    PubMed

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

    2015-05-29

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

  3. [Regulation of G protein-coupled receptor kinase activity].

    PubMed

    Haga, T; Haga, K; Kameyama, K; Nakata, H

    1994-09-01

    Recent progress on the activation of G protein-coupled receptor kinases is reviewed. beta-Adrenergic receptor kinase (beta ARK) is activated by G protein beta gamma -subunits, which interact with the carboxyl terminal portion of beta ARK. Muscarinic receptor m2-subtypes are phosphorylated by beta ARK1 in the central part of the third intracellular loop (I3). Phosphorylation of I3-GST fusion protein by beta ARK1 is synergistically stimulated by the beta gamma -subunits and mastoparan or a peptide corresponding to portions adjacent to the transmembrane segments of m2-receptors or by beta gamma -subunits and the agonist-bound I3-deleted m2 variant. These results indicate that agonist-bound receptors serve as both substrates and activators of beta ARK.

  4. The Two Faces of Receptor Interacting Protein Kinase-1

    PubMed Central

    Weinlich, Ricardo; Green, Douglas R.

    2014-01-01

    Receptor Interacting Protein Kinase-1 (RIPK1), a key player in inflammation and cell death, assumes opposite functions depending on the cellular context and its posttranslational modifications. Genetic evidence supported by biochemical and cellular biology approaches shed light on the circumstances in which RIPK1 promotes or inhibits these processes. PMID:25459879

  5. Isolation of Drosophila genes encoding G protein-coupled receptor kinases.

    PubMed Central

    Cassill, J A; Whitney, M; Joazeiro, C A; Becker, A; Zuker, C S

    1991-01-01

    G protein-coupled receptors are regulated via phosphorylation by a variety of protein kinases. Recently, termination of the active state of two such receptors, the beta-adrenergic receptor and rhodopsin, has been shown to be mediated by agonist- or light-dependent phosphorylation of the receptor by members of a family of protein-serine/threonine kinases (here referred to as G protein-coupled receptor kinases). We now report the isolation of a family of genes encoding a set of Drosophila protein kinases that appear to code for G protein-coupled receptor kinases. These proteins share a high degree of sequence homology with the bovine beta-adrenergic receptor kinase. The presence of a conserved family of G protein-coupled receptor kinases in vertebrates and invertebrates points to the central role of these kinases in signal transduction cascades. Images PMID:1662381

  6. GRK mythology: G-protein receptor kinases in cardiovascular disease.

    PubMed

    Dorn, Gerald W

    2009-05-01

    G-protein receptor kinases (GRKs) are indispensable for terminating signaling of G-protein coupled receptors (GPCR) through receptor desensitization and downregulation. Increased neurohormone levels in heart failure and the adverse consequences of constant neurohormonal stimulation suggest an important protective role for mechanisms that desensitize neurohormone receptor responses. For that reason, GRK2, the first GRK identified in the heart, has been extensively studied in heart failure, cardiac hypertrophy, and myocardial infarction. However, our understanding of the roles of GRKs in general, and the differential effects of cardiac receptor phosphorylation by individual cardiac-expressed GRKs, have evolved considerably in the last few years. Here, recent developments are reviewed, with an emphasis on novel GRK functions and signaling pathways.

  7. Protein engineering of bacterial histidine kinase receptor systems.

    PubMed

    Xie, Wei; Blain, Katherine Y; Kuo, Mario Meng-Chiang; Choe, Senyon

    2010-07-01

    Two-component systems (TCS) involving the His-Asp phosphotransfer are commonly utilized for signal transduction in prokaryotes in which the two essential components are a sensor histidine kinase (HK) receptor and a response regulator (RR). Despite great efforts in structural and functional characterization of signal perception mechanisms, the exact signaling mechanisms remain elusive for many TCSs. Mimicking the natural TCS signaling pathways, chimeric receptor kinases and response regulators have been constructed through the process of swapping modular domains of related TCSs. To design chimeras with new signaling pathways, domains from different proteins that have little relationship at the primary structural level but carrying desirable functional properties can be conjoined to engineer novel TCSs. These chimeras maintain the ability to respond to environmental stimulants by regulating protein phosphorylation to produce downstream output signals. Depending on the nature of external signals, chimeric TCSs can serve as a novel tool not only to examine the natural signaling mechanisms in TCSs, but also for industrial and clinical applications.

  8. Solubilized placental membrane protein inhibits insulin receptor tyrosine kinase activity

    SciTech Connect

    Strout, H.V. Jr.; Slater, E.E.

    1987-05-01

    Regulation of insulin receptor (IR) tyrosine kinase (TK) activity may be important in modulating insulin action. Utilizing an assay which measures IR phosphorylation of angiotensin II (AII), the authors investigated whether fractions of TX-100 solubilized human placental membranes inhibited IR dependent AII phosphorylation. Autophosphorylated IR was incubated with membrane fractions before the addition of AII, and kinase inhibition measured by the loss of TSP incorporated in AII. An inhibitory activity was detected which was dose, time, and temperature dependent. The inhibitor was purified 200-fold by sequential chromatography on wheat germ agglutinin, DEAE, and hydroxyapatite. This inhibitory activity was found to correlate with an 80 KD protein which was electroeluted from preparative slab gels and rabbit antiserum raised. Incubation of membrane fractions with antiserum before the IRTK assay immunoprecipitated the inhibitor. Protein immunoblots of crude or purified fractions revealed only the 80 KD protein. Since IR autophosphorylation is crucial to IRTK activity, the authors investigated the state of IR autophosphorylation after treatment with inhibitor; no change was detected by phosphoamino acid analysis.

  9. Control of EGF receptor function by protein kinase C

    SciTech Connect

    Whiteley, B.J.

    1986-01-01

    Treatment of human epidermoid carcinoma A431 cells with nanomolar concentrations of the potent tumor promotor, phorbol 12-myristate 13-acetate (PMA), is shown to attentuate the ability of epidermal growth factor (EGF) or serum to activate Na/sup +//H/sup +/ exchange, which is measured as an amiloride-inhibitable pH/sub i/ increase or /sup 22/Na/sup +/ uptake. The ability of PMA to directly activate Na/sup +//H/sup +/ exchange is also reported, but PMA-induced pH/sub i/ increases are modest with respect to those of EGF or serum and require relatively high concentrations of PMA. The effects of PMA on mitogen receptor-stimulated Na/sup +//H/sup +/ exchange were examined in the mouse fibroblast NR6 cell line using platelet-derived growth factor (PDGF). The results were similar to those in A431 cells, except that PMA in NR6 cells causes pH/sub i/ increases at lower concentrations. Phorbol diester action is mediated by the activity of the enzyme protein kinase C. The results summarized above support the hypothesis that PMA-induced protein kinase C activity opposes mitogenic stimulation. The presumed endogenous PMA analog is diacylglycerol, which is generated by phosphoinositide hydrolysis and has been reported to be produced in response to the mitogens, EGF and PDGF.

  10. Flunitrazepam rapidly reduces GABAA receptor subunit protein expression via a protein kinase C-dependent mechanism

    PubMed Central

    Johnston, Jonathan D; Price, Sally A; Bristow, David R

    1998-01-01

    Acute flunitrazepam (1 μM) exposure for 1 h reduced GABAA receptor α1 (22±4%, mean±s.e.mean) and β2/3 (21±4%) subunit protein levels in cultured rat cerebellar granule cells. This rapid decrease in subunit proteins was completely prevented by bisindolymaleimide 1 (1 μM), an inhibitor of protein kinase C, but not by N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide (H-89, 4.8 μM), an inhibitor of protein kinases A and G. These results suggest the existence of a benzodiazepine-induced mechanism to rapidly alter GABAA receptor protein expression, that appears to be dependent on protein kinase C activity. PMID:9723942

  11. Glucagon receptor activates extracellular signal-regulated protein kinase 1/2 via cAMP-dependent protein kinase

    PubMed Central

    Jiang, Youwei; Cypess, Aaron M.; Muse, Evan D.; Wu, Cui-Rong; Unson, Cecilia G.; Merrifield, R. B.; Sakmar, Thomas P.

    2001-01-01

    We prepared a stable cell line expressing the glucagon receptor to characterize the effect of Gs-coupled receptor stimulation on extracellular signal-regulated protein kinase 1/2 (ERK1/2) activity. Glucagon treatment of the cell line caused a dose-dependent increase in cAMP concentration, activation of cAMP-dependent protein kinase (PKA), and transient release of intracellular calcium. Glucagon treatment also caused rapid dose-dependent phosphorylation and activation of mitogen-activated protein kinase kinase/ERK kinase (MEK1/2) and ERK1/2. Inhibition of either PKA or MEK1/2 blocked ERK1/2 activation by glucagon. However, no significant activation of several upstream activators of MEK, including Ras, Rap1, and Raf, was observed in response to glucagon treatment. In addition, chelation of intracellular calcium reduced glucagon-mediated ERK1/2 activation. In transient transfection experiments, glucagon receptor mutants that bound glucagon but failed to increase intracellular cAMP and calcium concentrations showed no glucagon-stimulated ERK1/2 phosphorylation. We conclude that glucagon-induced MEK1/2 and ERK1/2 activation is mediated by PKA and that an increase in intracellular calcium concentration is required for maximal ERK activation. PMID:11517300

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

    PubMed Central

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

    1993-01-01

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

  13. Monocyte chemoattractant protein-1-induced CCR2B receptor desensitization mediated by the G protein-coupled receptor kinase 2

    PubMed Central

    Aragay, A. M.; Mellado, M.; Frade, J. M. R.; Martin, A. M.; Jimenez-Sainz, M. C.; Martinez-A, C.; Mayor, F.

    1998-01-01

    Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine cytokine family, whose physiological function is mediated by binding to the CCR2 and CCR4 receptors, which are members of the G protein-coupled receptor family. MCP-1 plays a critical role in both activation and migration of leukocytes. Rapid chemokine receptor desensitization is very likely essential for accurate chemotaxis. In this report, we show that MCP-1 binding to the CCR2 receptor in Mono Mac 1 cells promotes the rapid desensitization of MCP-1-induced calcium flux responses. This desensitization correlates with the Ser/Thr phosphorylation of the receptor and with the transient translocation of the G protein-coupled receptor kinase 2 (GRK2, also called β-adrenergic kinase 1 or βARK1) to the membrane. We also demonstrate that GRK2 and the uncoupling protein β-arrestin associate with the receptor, forming a macromolecular complex shortly after MCP-1 binding. Calcium flux responses to MCP-1 in HEK293 cells expressing the CCR2B receptor were also markedly reduced upon cotransfection with GRK2 or the homologous kinase GRK3. Nevertheless, expression of the GRK2 dominant-negative mutant βARK-K220R did not affect the initial calcium response, but favored receptor response to a subsequent challenge by agonists. The modulation of the CCR2B receptor by GRK2 suggests an important role for this kinase in the regulation of monocyte and lymphocyte response to chemokines. PMID:9501202

  14. Tyrosine kinase receptor transactivation associated to G protein-coupled receptors.

    PubMed

    Almendro, Vanessa; García-Recio, Susana; Gascón, Pedro

    2010-09-01

    G protein-coupled receptors (GPCRs) comprise a large family of membrane receptors involved in signal transduction. These receptors are linked to a variety of physiological and biological processes such as regulation of neurotransmission, growth, cell differentiation and oncogenesis among others. Some of the effects of GPCRs are known to be mediated by the activation of MAPK pathways. Several GPCRs are also able to transactivate receptors with tyrosine kinase activity (TKR) such as EGFR and HER2 and thus to control DNA synthesis and cell proliferation. The interaction between these receptors not only plays an important physiological role but its disregulation can induce pathological states such as cancer. For this reason, the crosstalk between these two types of receptors can be considered a possible mechanism for cell transformation, tumor progression, reactivation of the metastatic disease, and the acquisition of resistance to therapies targeting TKR receptors. The transactivation of some TKRs by GPCRs is related to the lost of response of TKRs to inhibitors of TK activity, mainly by the activation of the c-Src protein which can directly phosphorylate and activate the cytoplasmic domain of a TKR. For these reason, the dual inhibition of GPCRs and TKRs in some types of cancer has been proposed as a better strategy to kill tumor cells. Increased understanding of the mechanisms that interconnect the two pathways regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies.

  15. The protein kinase LKB1 negatively regulates bone morphogenetic protein receptor signaling

    PubMed Central

    Raja, Erna; Edlund, Karolina; Kahata, Kaoru; Zieba, Agata; Morén, Anita; Watanabe, Yukihide; Voytyuk, Iryna; Botling, Johan; Söderberg, Ola; Micke, Patrick; Pyrowolakis, George; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-01-01

    The protein kinase LKB1 regulates cell metabolism and growth and is implicated in intestinal and lung cancer. Bone morphogenetic protein (BMP) signaling regulates cell differentiation during development and tissue homeostasis. We demonstrate that LKB1 physically interacts with BMP type I receptors and requires Smad7 to promote downregulation of the receptor. Accordingly, LKB1 suppresses BMP-induced osteoblast differentiation and affects BMP signaling in Drosophila wing longitudinal vein morphogenesis. LKB1 protein expression and Smad1 phosphorylation analysis in a cohort of non-small cell lung cancer patients demonstrated a negative correlation predominantly in a subset enriched in adenocarcinomas. Lung cancer patient data analysis indicated strong correlation between LKB1 loss-of-function mutations and high BMP2 expression, and these two events further correlated with expression of a gene subset functionally linked to apoptosis and migration. This new mechanism of BMP receptor regulation by LKB1 has ramifications in physiological organogenesis and disease. PMID:26701726

  16. Structure of Human G Protein-Coupled Receptor Kinase 2 in Complex with the Kinase Inhibitor Balanol

    SciTech Connect

    Tesmer, John J.G.; Tesmer, Valerie M.; Lodowski, David T.; Steinhagen, Henning; Huber, Jochen

    2010-07-19

    G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with G{beta}{gamma} in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.

  17. Biochemical characterization of the protein tyrosine kinase homology domain of the ErbB3 (HER3) receptor protein.

    PubMed

    Sierke, S L; Cheng, K; Kim, H H; Koland, J G

    1997-03-15

    The putative protein tyrosine kinase domain (TKD) of the ErbB3 (HER3) receptor protein was generated as a histidine-tagged recombinant protein (hisTKD-B3) and characterized enzymologically. CD spectroscopy indicated that the hisTKD-B3 protein assumed a native conformation with a secondary structure similar to that of the epidermal growth factor (EGF) receptor TKD. However, when compared with the EGF receptor-derived protein, hisTKD-B3 exhibited negligible intrinsic protein tyrosine kinase activity. Immune complex kinase assays of full-length ErbB3 proteins also yielded no evidence of catalytic activity. A fluorescence assay previously used to characterize the nucleotide-binding properties of the EGF receptor indicated that the ErbB3 protein was unable to bind nucleotide. The hisTKD-B3 protein was subsequently found to be an excellent substrate for the EGF receptor protein tyrosine kinase, which suggested that in vivo phosphorylation of ErbB3 in response to EGF could be attributed to a direct cross-phosphorylation by the EGF receptor protein tyrosine kinase.

  18. Biochemical characterization of the protein tyrosine kinase homology domain of the ErbB3 (HER3) receptor protein.

    PubMed Central

    Sierke, S L; Cheng, K; Kim, H H; Koland, J G

    1997-01-01

    The putative protein tyrosine kinase domain (TKD) of the ErbB3 (HER3) receptor protein was generated as a histidine-tagged recombinant protein (hisTKD-B3) and characterized enzymologically. CD spectroscopy indicated that the hisTKD-B3 protein assumed a native conformation with a secondary structure similar to that of the epidermal growth factor (EGF) receptor TKD. However, when compared with the EGF receptor-derived protein, hisTKD-B3 exhibited negligible intrinsic protein tyrosine kinase activity. Immune complex kinase assays of full-length ErbB3 proteins also yielded no evidence of catalytic activity. A fluorescence assay previously used to characterize the nucleotide-binding properties of the EGF receptor indicated that the ErbB3 protein was unable to bind nucleotide. The hisTKD-B3 protein was subsequently found to be an excellent substrate for the EGF receptor protein tyrosine kinase, which suggested that in vivo phosphorylation of ErbB3 in response to EGF could be attributed to a direct cross-phosphorylation by the EGF receptor protein tyrosine kinase. PMID:9148746

  19. Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs

    PubMed Central

    Hines, John; Gough, Jonathan D.; Corson, Timothy W.; Crews, Craig M.

    2013-01-01

    Posttranslational knockdown of a specific protein is an attractive approach for examining its function within a system. Here we introduce phospho-dependent proteolysis targeting chimeras (phosphoPROTACs), a method to couple the conditional degradation of targeted proteins to the activation state of particular kinase-signaling pathways. We generated two phosphoPROTACs that couple the tyrosine phosphorylation sequences of either the nerve growth factor receptor, TrkA (tropomyosin receptor kinase A), or the neuregulin receptor, ErbB3 (erythroblastosis oncogene B3), with a peptide ligand for the E3 ubiquitin ligase von Hippel Lindau protein. These phosphoPROTACs recruit either the neurotrophic signaling effector fibroblast growth factor receptor substrate 2α or the survival-promoting phosphatidylinositol-3-kinase, respectively, to be ubiquitinated and degraded upon activation of specific receptor tyrosine kinases and phosphorylation of the phosphoPROTACs. We demonstrate the ability of these phosphoPROTACs to suppress the short- and long-term effects of their respective activating receptor tyrosine kinase pathways both in vitro and in vivo. In addition, we show that activation of phosphoPROTACs is entirely dependent on their kinase-mediated phosphorylation, as phenylalanine-containing null variants are inactive. Furthermore, stimulation of unrelated growth factor receptors does not induce target protein knockdown. Although comparable in efficiency to RNAi, this approach has the added advantage of providing a degree of temporal and dosing control as well as cell-type selectivity unavailable using nucleic acid-based strategies. By varying the autophosphorylation sequence of a phosphoPROTAC, it is conceivable that other receptor tyrosine kinase/effector pairings could be similarly exploited to achieve other biological effects. PMID:23674677

  20. Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs.

    PubMed

    Hines, John; Gough, Jonathan D; Corson, Timothy W; Crews, Craig M

    2013-05-28

    Posttranslational knockdown of a specific protein is an attractive approach for examining its function within a system. Here we introduce phospho-dependent proteolysis targeting chimeras (phosphoPROTACs), a method to couple the conditional degradation of targeted proteins to the activation state of particular kinase-signaling pathways. We generated two phosphoPROTACs that couple the tyrosine phosphorylation sequences of either the nerve growth factor receptor, TrkA (tropomyosin receptor kinase A), or the neuregulin receptor, ErbB3 (erythroblastosis oncogene B3), with a peptide ligand for the E3 ubiquitin ligase von Hippel Lindau protein. These phosphoPROTACs recruit either the neurotrophic signaling effector fibroblast growth factor receptor substrate 2α or the survival-promoting phosphatidylinositol-3-kinase, respectively, to be ubiquitinated and degraded upon activation of specific receptor tyrosine kinases and phosphorylation of the phosphoPROTACs. We demonstrate the ability of these phosphoPROTACs to suppress the short- and long-term effects of their respective activating receptor tyrosine kinase pathways both in vitro and in vivo. In addition, we show that activation of phosphoPROTACs is entirely dependent on their kinase-mediated phosphorylation, as phenylalanine-containing null variants are inactive. Furthermore, stimulation of unrelated growth factor receptors does not induce target protein knockdown. Although comparable in efficiency to RNAi, this approach has the added advantage of providing a degree of temporal and dosing control as well as cell-type selectivity unavailable using nucleic acid-based strategies. By varying the autophosphorylation sequence of a phosphoPROTAC, it is conceivable that other receptor tyrosine kinase/effector pairings could be similarly exploited to achieve other biological effects.

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

    PubMed

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

    2016-09-16

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

  2. Conformational transitions and interactions underlying the function of membrane embedded receptor protein kinases.

    PubMed

    Bocharov, Eduard V; Sharonov, Georgy V; Bocharova, Olga V; Pavlov, Konstantin V

    2017-01-25

    Among membrane receptors, the single-span receptor protein kinases occupy a broad but specific functional niche determined by distinctive features of the underlying transmembrane signaling mechanisms that are briefly overviewed on the basis of some of the most representative examples, followed by a more detailed discussion of several hierarchical levels of organization and interactions involved. All these levels, including single-molecule interactions (e.g., dimerization, liganding, chemical modifications), local processes (e.g. lipid membrane perturbations, cytoskeletal interactions), and larger scale phenomena (e.g., effects of membrane surface shape or electrochemical potential gradients) appear to be closely integrated to achieve the observed diversity of the receptor functioning. Different species of receptor protein kinases meet their specific functional demands through different structural features defining their responses to stimulation, but certain common patterns exist. Signaling by receptor protein kinases is typically associated with the receptor dimerization and clustering, ligand-induced rearrangements of receptor domains through allosteric conformational transitions with involvement of lipids, release of the sequestered lipids, restriction of receptor diffusion, cytoskeleton and membrane shape remodeling. Understanding of complexity and continuity of the signaling processes can help identifying currently neglected opportunities for influencing the receptor signaling with potential therapeutic implications. This article is part of a Special Issue entitled: Interactions between membrane receptors in cellular membranes edited by Kalina Hristova.

  3. Insights into the regulation of 5-HT2A serotonin receptors by scaffolding proteins and kinases.

    PubMed

    Allen, John A; Yadav, Prem N; Roth, Bryan L

    2008-11-01

    5-HT(2A) serotonin receptors are essential molecular targets for the actions of LSD-like hallucinogens and atypical antipsychotic drugs. 5-HT(2A) serotonin receptors also mediate a variety of physiological processes in peripheral and central nervous systems including platelet aggregation, smooth muscle contraction, and the modulation of mood and perception. Scaffolding proteins have emerged as important regulators of 5-HT(2A) receptors and our recent studies suggest multiple scaffolds exist for 5-HT(2A) receptors including PSD95, arrestin, and caveolin. In addition, a novel interaction has emerged between p90 ribosomal S6 kinase and 5-HT(2A) receptors which attenuates receptor signaling. This article reviews our recent studies and emphasizes the role of scaffolding proteins and kinases in the regulation of 5-HT(2A) trafficking, targeting and signaling.

  4. 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

  5. The PR5K receptor protein kinase from Arabidopsis thaliana is structurally related to a family of plant defense proteins.

    PubMed Central

    Wang, X; Zafian, P; Choudhary, M; Lawton, M

    1996-01-01

    We have isolated an Arabidopsis thaliana gene that codes for a receptor related to antifungal pathogenesis-related (PR) proteins. The PR5K gene codes for a predicted 665-amino acid polypeptide that comprises an extracellular domain related to the PR5 proteins, a central transmembrane-spanning domain, and an intracellular protein-serine/threonine kinase. The extracellular domain of PR5K (PR5-like receptor kinase) is most highly related to acidic PR5 proteins that accumulate in the extracellular spaces of plants challenged with pathogenic microorganisms. The kinase domain of PR5K is related to a family of protein-serine/threonine kinases that are involved in the expression of self-incompatibility and disease resistance. PR5K transcripts accumulate at low levels in all tissues examined, although particularly high levels are present in roots and inflorescence stems. Treatments that induce authentic PR5 proteins had no effect on the level of PR5K transcripts, suggesting that the receptor forms part of a preexisting surveillance system. When the kinase domain of PR5K was expressed in Escherichia coli, the resulting polypeptide underwent autophosphorylation, consistent with its predicted enzyme activity. These results are consistent with PR5K encoding a functional receptor kinase. Moreover, the structural similarity between the extracellular domain of PR5K and the antimicrobial PR5- proteins suggests a possible interaction with common or related microbial targets. Images Fig. 2 Fig. 3 Fig. 4 PMID:8637920

  6. Activation of a GTP-binding protein and a GTP-binding-protein-coupled receptor kinase (beta-adrenergic-receptor kinase-1) by a muscarinic receptor m2 mutant lacking phosphorylation sites.

    PubMed

    Kameyama, K; Haga, K; Haga, T; Moro, O; Sadée, W

    1994-12-01

    A mutant of the human muscarinic acetylcholine receptor m2 subtype (m2 receptor), lacking a large part of the third intracellular loop, was expressed and purified using the baculovirus/insect cell culture system. The mutant was not phosphorylated by beta-adrenergic-receptor kinase, as expected from the previous assignment of phosphorylation sites to the central part of the third intracellular loop. However, the m2 receptor mutant was capable of stimulating beta-adrenergic-receptor-kinase-1-mediated phosphorylation of a glutathione S-transferase fusion protein containing the m2 phosphorylation sites in an agonist-dependent manner. Both mutant and wild-type m2 receptors reconstituted with the guanine-nucleotide-binding regulatory proteins (G protein), G(o) and G(i)2, displayed guanine-nucleotide-sensitive high-affinity agonist binding, as assessed by displacement of [3H]quinuclidinyl-benzilate binding with carbamoylcholine, and both stimulated guanosine 5'-3-O-[35S]thiotriphosphate ([35S]GTP[S]) binding in the presence of carbamoylcholine and GDP. The Ki values of carbamoylcholine effects on [3H]quinuclidinyl-benzilate binding were indistinguishable for the mutant and wild-type m2 receptors. Moreover, the phosphorylation of the wild-type m2 receptor by beta-adrenergic-receptor kinase-1 did not affect m2 interaction with G proteins as assessed by the binding of [3H]quinuclidinyl benzilate or [35S]GTP[S]. These results indicate that (a) the m2 receptor serves both as an activator and as a substrate of beta-adrenergic-receptor kinase, and (b) a large part of the third intracellular loop of the m2 receptor does not contribute to interaction with G proteins and its phosphorylation by beta-adrenergic-receptor kinase does not uncouple the receptor and G proteins in reconstituted lipid vesicles.

  7. The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucine-rich repeats.

    PubMed Central

    Torii, K U; Mitsukawa, N; Oosumi, T; Matsuura, Y; Yokoyama, R; Whittier, R F; Komeda, Y

    1996-01-01

    Arabidopsis Landsberg erecta is one of the most popular ecotypes and is used widely for both molecular and genetic studies. It harbors the erecta (er) mutation, which confers a compact inflorescence, blunt fruits, and short petioles. We have identified five er mutant alleles from ecotypes Columbia and Wassilewskija. Phenotypic characterization of the mutant alleles suggests a role for the ER gene in regulating the shape of organs originating from the shoot apical meristem. We cloned the ER gene, and here, we report that it encodes a putative receptor protein kinases. The deduced ER protein contains a cytoplasmic protein kinase catalytic domain, a transmembrane region, and an extracellular domain consisting of leucine-rich repeats, which are thought to interact with other macromolecules. Our results suggest that cell-cell communication mediated by a receptor kinase has an important role in plant morphogenesis. PMID:8624444

  8. Immunoprecipitation of Plasma Membrane Receptor-Like Kinases for Identification of Phosphorylation Sites and Associated Proteins.

    PubMed

    Kadota, Yasuhiro; Macho, Alberto P; Zipfel, Cyril

    2016-01-01

    Membrane proteins are difficult to study for numerous reasons. The surface of membrane proteins is relatively hydrophobic and sometimes very unstable, additionally requiring detergents for their extraction from the membrane. This leads to challenges at all levels, including expression, solubilization, purification, identification of associated proteins, and the identification of post-translational modifications. However, recent advances in immunoprecipitation technology allow to isolate membrane proteins efficiently, facilitating the study of protein-protein interactions, the identification of novel associated proteins, and to identify post-translational modifications, such as phosphorylation. Here, we describe an optimized immunoprecipitation protocol for plant plasma membrane receptor-like kinases.

  9. Sch proteins are localized on endoplasmic reticulum membranes and are redistributed after tyrosine kinase receptor activation.

    PubMed Central

    Lotti, L V; Lanfrancone, L; Migliaccio, E; Zompetta, C; Pelicci, G; Salcini, A E; Falini, B; Pelicci, P G; Torrisi, M R

    1996-01-01

    The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein. PMID:8628261

  10. Phosphorylation of the purified cardiac ryanodine receptor by exogenous and endogenous protein kinases.

    PubMed Central

    Hohenegger, M; Suko, J

    1993-01-01

    The ryanodine receptor is the main Ca(2+)-release structure in skeletal and cardiac sarcoplasmic reticulum. In both tissues, phosphorylation of the ryanodine receptor has been proposed to be involved in the regulation of Ca2+ release. In the present study, we have examined the ability of the purified cardiac ryanodine receptor to serve as a substrate for phosphorylation by exogenously added catalytic subunit of the cyclic AMP (cAMP)-dependent protein kinase (PK-A), cyclic GMP (cGMP)-dependent protein kinase (PK-G), or calmodulin-dependent protein kinase (PK-CaM). A large amount of phosphate incorporation was observed for PK-CaM (938 +/- 48 pmol of Pi/mg of purified channel protein), whereas the level of phosphorylation was considerably lower with PK-A or PK-G (345 +/- 139 and 96 +/- 6 pmol/mg respectively). In addition, endogenous PK-CaM activity co-migrates with the ryanodine receptor through several steps of purification, suggesting a strong association of the two proteins. This endogenous PK-CaM activity is abolished by a PK-CaM-specific synthetic peptide inhibitor. Endogenous cAMP- and cGMP-dependent phosphorylation was not observed in the purified ryanodine-receptor preparation. Taken together, these observations imply that PK-CaM is the physiologically relevant protein kinase, capable of phosphorylating the channel protein to a minimum stoichiometry of 2 mol of Pi per mol of tetramer. Images Figure 2 Figure 3 Figure 4 PMID:8257417

  11. Insulin receptor binding and protein kinase activity in muscles of trained rats

    SciTech Connect

    Dohm, G.L.; Sinha, M.K.; Caro, J.F.

    1987-02-01

    Exercise has been shown to increase insulin sensitivity, and muscle is quantitatively the most important tissue of insulin action. Since the first step in insulin action is the binding to a membrane receptor, the authors postulated that exercise training would change insulin receptors in muscle and in this study they have investigated this hypothesis. Female rats initially weighing approx. 100 g were trained by treadmill running for 2 h/day, 6 days/wk for 4 wk at 25 m/min (0 grade). Insulin receptors from vastus intermedius muscles were solubilized by homogenizing in a buffer containing 1% Triton X-100 and then partially purified by passing the soluble extract over a wheat germ agglutinin column. The 4 wk training regimen resulted in a 65% increase in citrate synthase activity in red vastus lateralis muscle, indicating an adaptation to exercise ( SVI). Insulin binding by the partially purified receptor preparations was approximately doubled in muscle of trained rats at all insulin concentrations, suggesting an increase in the number of receptors. Training did not alter insulin receptor structure as evidenced by electrophoretic mobility under reducing and nonreducing conditions. Basal insulin receptor protein kinase activity was higher in trained than untrained animals and this was likely due to the greater number of receptors. However, insulin stimulation of the protein kinase activity was depressed by training. These results demonstrate that endurance training does alter receptor number and function in muscle and these changes may be important in increasing insulin sensitivity after exercise training.

  12. Cellular progesterone receptor phosphorylation in response to ligands activating protein kinases

    SciTech Connect

    Rao, K.V.; Peralta, W.D.; Greene, G.L.; Fox, C.F.

    1987-08-14

    Progesterone receptors were immunoprecipitated with monoclonal antibodies KD68 from lysates of human breast carcinoma T47D cells labelled to steady state specific activity with /sup 32/Pi. The 120 kDa /sup 32/P-labelled progesterone receptor band was resolved by polyacrylamide gel electrophoresis and identified by autoradiography. Phosphoamino acid analysis revealed serine phosphorylation, but no threonine or tyrosine phosphorylation. Treatment of the /sup 32/Pi-labelled cells with EGF, TPA or dibutyryl cAMP had no significant quantitative effect on progesterone receptor phosphorylation, though the EGF receptor and the cAMP-dependent protein kinases have been reported to catalyze phosphorylation of purified avian progesterone receptor preparations in cell free systems. Progesterone receptor phosphorylation on serine residues was increased by 2-fold in cells treated with 10 nM progesterone; EGF had no effect on progesterone-mediated progesterone receptor phosphorylation.

  13. SH2 domain proteins as high-affinity receptor tyrosine kinase substrates.

    PubMed

    Sierke, S L; Koland, J G

    1993-09-28

    Activation of a growth factor receptor tyrosine kinase (RTK) is accompanied by a rapid autophosphorylation of the receptor on tyrosine residues. Receptor activation has been shown to promote the association of signal-transducing proteins containing SH2 domains (second domain of src homology). These receptor-associated proteins can, in turn, be phosphorylated by the RTK, an event which presumably regulates their activities. It has been suggested that SH2 domains in signal-transducing proteins target these proteins as substrates of the activated RTK. To test this hypothesis, recombinant proteins were generated that contained tyrosine phosphorylation sites of the erbB3 receptor and/or the SH2 domain of c-src. Incorporation of the SH2 domain led to a decrease in KM and an increase in Vmax for the substrate. The KM determined for one chimeric SH2/erbB3 substrate was among the lowest reported for epidermal growth factor RTK substrates. Experiments with a truncated kinase lacking C-terminal autophosphorylation sites indicated that the reduction in KM for these substrates was mediated by interactions between the substrate SH2 domain and phosphotyrosine residues of the RTK. These interactions could also inhibit RTK activity. These results demonstrate that the SH2 domain can effectively target substrates to a RTK and that SH2 domain proteins can regulate RTK activity.

  14. A receptor-like kinase from Arabidopsis thaliana is a calmodulin-binding protein.

    PubMed Central

    Charpenteau, Martine; Jaworski, Krzysztof; Ramirez, Bertha C; Tretyn, Andrzej; Ranjeva, Raoul; Ranty, Benoît

    2004-01-01

    Screening a cDNA expression library with a radiolabelled calmodulin (CaM) probe led to the isolation of AtCaMRLK, a receptor-like kinase (RLK) of Arabidopsis thaliana. AtCaMRLK polypeptide sequence shows a modular organization consisting of the four distinctive domains characteristic of receptor kinases: an amino terminal signal sequence, a domain containing seven leucine-rich repeats, a single putative membrane-spanning segment and a protein kinase domain. Using truncated versions of the protein and a synthetic peptide, we demonstrated that a region of 23 amino acids, located near the kinase domain of AtCaMRLK, binds CaM in a calcium-dependent manner. Real-time binding experiments showed that AtCaMRLK interacted in vitro with AtCaM1, a canonical CaM, but not with AtCaM8, a divergent isoform of the Ca2+ sensor. The bacterially expressed kinase domain of the protein was able to autophosphorylate and to phosphorylate the myelin basic protein, using Mn2+ preferentially to Mg2+ as an ion activator. Site-directed mutagenesis of the conserved lysine residue (Lys423) to alanine, in the kinase subdomain II, resulted in a complete loss of kinase activity. CaM had no influence on the autophosphorylation activity of AtCaMRLK. AtCaMRLK was expressed in reproductive and vegetative tissues of A. thaliana, except in leaves. Disruption in the AtCaMRLK coding sequence by insertion of a DsG transposable element in an Arabidopsis mutant did not generate a discernible phenotype. The CaM-binding motif of AtCaMRLK was found to be conserved in several other members of the plant RLK family, suggesting a role for Ca2+/CaM in the regulation of RLK-mediated pathways. PMID:14720124

  15. The prostaglandin EP1 receptor potentiates kainate receptor activation via a protein kinase C pathway and exacerbates status epilepticus

    PubMed Central

    Rojas, Asheebo; Gueorguieva, Paoula; Lelutiu, Nadia; Quan, Yi; Shaw, Renee; Dingledine, Raymond

    2014-01-01

    Prostaglandin E2 (PGE2) regulates membrane excitability, synaptic transmission, plasticity, and neuronal survival. The consequences of PGE2 release following seizures has been the subject of much study. Here we demonstrate that the prostaglandin E2 receptor 1 (EP1, or Ptger1) modulates native kainate receptors, a family of ionotropic glutamate receptors widely expressed throughout the central nervous system. Global ablation of the EP1 gene in mice (EP1-KO) had no effect on seizure threshold after kainate injection but reduced the likelihood to enter status epilepticus. EP1-KO mice that did experience typical status epilepticus had reduced hippocampal neurodegeneration and a blunted inflammatory response. Further studies with native prostanoid and kainate receptors in cultured cortical neurons, as well as with recombinant prostanoid and kainate receptors expressed in Xenopus oocytes, demonstrated that EP1 receptor activation potentiates heteromeric but not homomeric kainate receptors via a second messenger cascade involving phospholipase C, calcium and protein kinase C. Three critical GluK5 C-terminal serines underlie the potentiation of the GluK2/GluK5 receptor by EP1 activation. Taken together, these results indicate that EP1 receptor activation during seizures, through a protein kinase C pathway, increases the probability of kainic acid induced status epilepticus, and independently promotes hippocampal neurodegeneration and a broad inflammatory response. PMID:24952362

  16. G-protein-coupled receptor kinase 2 terminates G-protein-coupled receptor function in steroid hormone 20-hydroxyecdysone signaling

    PubMed Central

    Zhao, Wen-Li; Wang, Di; Liu, Chun-Yan; Zhao, Xiao-Fan

    2016-01-01

    G-protein-coupled receptors (GPCRs) transmit extracellular signals across the cell membrane. GPCR kinases (GRKs) desensitize GPCR signals in the cell membrane. However, the role and mechanism of GRKs in the desensitization of steroid hormone signaling are unclear. In this study, we propose that GRK2 is phosphorylated by protein kinase C (PKC) in response to induction by the steroid hormone 20-hydroxyecdysone (20E), which determines its translocation to the cell membrane of the lepidopteran Helicoverpa armigera. GRK2 protein expression is increased during the metamorphic stage because of induction by 20E. Knockdown of GRK2 in larvae causes accelerated pupation, an increase in 20E-response gene expression, and advanced apoptosis and metamorphosis. 20E induces translocation of GRK2 from the cytoplasm to the cell membrane via steroid hormone ecdysone-responsive GPCR (ErGPCR-2). GRK2 is phosphorylated by PKC on serine 680 after induction by 20E, which leads to the translocation of GRK2 to the cell membrane. GRK2 interacts with ErGPCR-2. These data indicate that GRK2 terminates the ErGPCR-2 function in 20E signaling in the cell membrane by a negative feedback mechanism. PMID:27412951

  17. Cyclic AMP-dependent protein kinase phosphorylation facilitates GABA(B) receptor-effector coupling.

    PubMed

    Couve, A; Thomas, P; Calver, A R; Hirst, W D; Pangalos, M N; Walsh, F S; Smart, T G; Moss, S J

    2002-05-01

    GABA (gamma-aminobutyric acid)(B) receptors are heterodimeric G protein-coupled receptors that mediate slow synaptic inhibition in the central nervous system. Here we show that the functional coupling of GABA(B)R1/GABA(B)R2 receptors to inwardly rectifying K(+) channels rapidly desensitizes. This effect is alleviated after direct phosphorylation of a single serine residue (Ser892) in the cytoplasmic tail of GABA(B)R2 by cyclic AMP (cAMP)-dependent protein kinase (PKA). Basal phosphorylation of this residue is evident in rat brain membranes and in cultured neurons. Phosphorylation of Ser892 is modulated positively by pathways that elevate cAMP concentration, such as those involving forskolin and beta-adrenergic receptors. GABA(B) receptor agonists reduce receptor phosphorylation, which is consistent with PKA functioning in the control of GABA(B)-activated currents. Mechanistically, phosphorylation of Ser892 specifically enhances the membrane stability of GABA(B) receptors. We conclude that signaling pathways that activate PKA may have profound effects on GABA(B) receptor-mediated synaptic inhibition. These results also challenge the accepted view that phosphorylation is a universal negative modulator of G protein-coupled receptors.

  18. Insights into protein interaction networks reveal non-receptor kinases as significant druggable targets for psoriasis.

    PubMed

    Sundarrajan, Sudharsana; Lulu, Sajitha; Arumugam, Mohanapriya

    2015-07-25

    Psoriasis is a chronic disease of the skin characterized by hyper proliferation and inflammation of the epidermis and dermal components of the skin. T-cell-dependent inflammatory process in skin governs the pathogenesis of psoriasis. An in-silico search strategy was utilized to identify psoriatic therapeutic drug targets. The gene expression profiling of psoriatic skin identified a total of 427 differentially expressed genes (DEGs). Gene ontology investigation of DEGs identified genes involved in calcium binding, apoptosis, keratinisation, lipid transportation and homeostasis apart from immune mediated processes. The protein interaction networks identified proteins involved in various signaling mechanisms with high degree of interconnections. The gene modules derived from the main network were enriched with rich kinome. These sub-networks were dominated by the presence of non-receptor kinase family members which are major signal transmitters in immune response. The computational approach has aided in the identification of non-receptor kinases as potential targets for psoriasis drug development.

  19. A role for protein kinase A and protein kinase M zeta in muscarinic acetylcholine receptor-initiated persistent synaptic enhancement in rat hippocampus in vivo.

    PubMed

    Hayes, J; Li, S; Anwyl, R; Rowan, M J

    2008-01-24

    Antagonists at presynaptic muscarinic autoreceptors increase endogenous acetylcholine (ACh) release and enhance cognition but little is known regarding their actions on plasticity at glutamatergic synapses. Here the mechanisms of the persistent enhancement of hippocampal excitatory transmission induced by the M2/M4 muscarinic ACh receptor antagonist methoctramine were investigated in vivo. The persistent facilitatory effect of i.c.v. methoctramine in the CA1 region of urethane-anesthetized rats was mimicked by gallamine, an M2 receptor antagonist, supporting a role for this receptor subtype. Neither the N-methyl-D-aspartate (NMDA) receptor antagonists D-(-)-2-amino phosphonopentanoic acid (d-AP5) and memantine, nor the metabotropic glutamate receptor subtype 1a antagonist (S)-(+)-alpha-amino-4-carboxy-2-methylbenzeneacetic acid (LY367385) significantly affected the methoctramine-induced persistent synaptic enhancement, indicating a lack of requirement for these glutamate receptors. The selective kinase inhibitors Rp-adenosine-3', 5'-cyclic monophosphorothioate (Rp-cAMPS) and the myrostylated pseudosubstrate peptide, Myr-Ser-Ile-Tyr-Arg-Arg-Gly-Ala-Arg-Arg-Trp-Arg-Lys-Leu-OH (ZIP), were used to investigate the roles of protein kinase A (PKA) and the atypical protein kinase C, protein kinase Mzeta (PKM zeta), respectively. Remarkably, pretreatment with either agent prevented the induction of the persistent synaptic enhancement by methoctramine and post-methoctramine treatment with Rp-cAMPS transiently reversed the enhancement. These findings are strong evidence that antagonism of M2 muscarinic ACh receptors in vivo induces an NMDA receptor-independent persistent synaptic enhancement that requires activation of both PKA and PKM zeta.

  20. Ca2+-dependent inhibition of G protein-coupled receptor kinase 2 by calmodulin.

    PubMed

    Haga, K; Tsuga, H; Haga, T

    1997-02-11

    Agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptor m2 subtypes (m2 receptors) or rhodopsin by G protein-coupled receptor kinase 2 (GRK2) was found to be inhibited by calmodulin in a Ca2+-dependent manner. The phosphorylation was fully inhibited in the absence of G protein betagamma subunits and partially inhibited in the presence of betagamma subunits. The dose-response curve for stimulation by betagamma subunits of the m2 and rhodopsin phosphorylation was shifted to the higher concentration of betagamma subunits by addition of Ca2+-calmodulin. The phosphorylation by GRK2 of a glutathione S-transferase fusion protein containing a peptide corresponding to the central part of the third intracellular loop of m2 receptors (I3-GST) was not affected by Ca2+-calmodulin in the presence or absence of betagamma subunits, but the agonist-dependent stimulation of I3-GST phosphorylation by an I3-deleted m2 receptor mutant in the presence of betagamma subunits was suppressed by Ca2+-calmodulin. These results indicate that Ca2+-calmodulin does not directly interact with the catalytic site of GRK2 but inhibits the kinase activity of GRK2 by interfering with the activation of GRK2 by agonist-bound m2 receptors and G protein betagamma subunits. In agreement with the assumption that GRK2 activity is suppressed by the increase in intracellular Ca2+, the sequestration of m2 receptors expressed in Chinese hamster ovary cells was found to be attenuated by the treatment with a Ca2+ ionophore, A23187.

  1. Activation of G Protein-Coupled Receptor Kinase 1 Involves Interactions between Its N-Terminal Region and Its Kinase Domain

    SciTech Connect

    Huang, Chih-chin; Orban, Tivadar; Jastrzebska, Beata; Palczewski, Krzysztof; Tesmer, John J.G.

    2012-03-16

    G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors (GPCRs) to initiate receptor desensitization. In addition to the canonical phosphoacceptor site of the kinase domain, activated receptors bind to a distinct docking site that confers higher affinity and activates GRKs allosterically. Recent mutagenesis and structural studies support a model in which receptor docking activates a GRK by stabilizing the interaction of its 20-amino acid N-terminal region with the kinase domain. This interaction in turn stabilizes a closed, more active conformation of the enzyme. To investigate the importance of this interaction for the process of GRK activation, we first validated the functionality of the N-terminal region in rhodopsin kinase (GRK1) by site-directed mutagenesis and then introduced a disulfide bond to cross-link the N-terminal region of GRK1 with its specific binding site on the kinase domain. Characterization of the kinetic and biophysical properties of the cross-linked protein showed that disulfide bond formation greatly enhances the catalytic efficiency of the peptide phosphorylation, but receptor-dependent phosphorylation, Meta II stabilization, and inhibition of transducin activation were unaffected. These data indicate that the interaction of the N-terminal region with the kinase domain is important for GRK activation but does not dictate the affinity of GRKs for activated receptors.

  2. G protein-coupled receptor kinase GRK5 phosphorylates moesin and regulates metastasis in prostate cancer.

    PubMed

    Chakraborty, Prabir Kumar; Zhang, Yushan; Coomes, Alexandra S; Kim, Wan-Ju; Stupay, Rachel; Lynch, Lauren D; Atkinson, Tamieka; Kim, Jae I; Nie, Zhongzhen; Daaka, Yehia

    2014-07-01

    G protein-coupled receptor kinases (GRK) regulate diverse cellular functions ranging from metabolism to growth and locomotion. Here, we report an important contributory role for GRK5 in human prostate cancer. Inhibition of GRK5 kinase activity attenuated the migration and invasion of prostate cancer cells and, concordantly, increased cell attachment and focal adhesion formation. Mass spectrometric analysis of the phosphoproteome revealed the cytoskeletal-membrane attachment protein moesin as a putative GRK5 substrate. GRK5 regulated the subcellular distribution of moesin and colocalized with moesin at the cell periphery. We identified amino acid T66 of moesin as a principal GRK5 phosphorylation site and showed that enforcing the expression of a T66-mutated moesin reduced cell spreading. In a xenograft model of human prostate cancer, GRK5 silencing reduced tumor growth, invasion, and metastasis. Taken together, our results established GRK5 as a key contributor to the growth and metastasis of prostate cancer.

  3. Substance P suppresses GABAA receptor function via protein kinase C in primary sensory neurones of bullfrogs.

    PubMed Central

    Yamada, K; Akasu, T

    1996-01-01

    1. The effects of substance P (SP) and related tachykinins on the function of gamma-aminobutyric acid-A (GABAA) receptors were examined in acutely dissociated neurones of bullfrog dorsal root ganglia (DRG) by using whole-cell voltage-clamp techniques. 2. Application of SP (10 nM to 1 microM) depressed inward currents produced by GABAA receptor activation (IGABA). Neurokinin A (NKA) and neurokinin B (NKB) also depressed IGABA; the rank order of agonist potency was SP > NKA > NKB. Spantide ([D-Arg1, D-Trp7,9,Leu11]SP) and L-703,606, NK1 receptor antagonists, blocked the SP-induced depression of IGABA. 3. SP irreversibly depressed IGABA, when neurones were intracellularly dialysed with GTP gamma S. Intracellular application of GDP beta S prevented the SP-induced depression of IGABA. Pertussis toxin (PTX) did not block the inhibitory effect of SP on IGABA. 4. The depression of IGABA produced by SP was inhibited by H-7 and PKC(19-36), protein kinase C (PKC) inhibitors, but not by H-9 and HA-1004, protein kinase A inhibitors. IGABA was suppressed by application of sn-1,2-dioctanoyl glycerol (DOG), a PKC activator. 5. It is concluded that activation of neurokinin-1 (NK1) receptors downregulates the function of the GABAA receptor of primary sensory neurones through a PTX-insensitive G-protein. PKC may be involved in the transduction pathway of the tachykinin-induced inhibition of the GABAA receptor. PMID:8910228

  4. Molecular Mechanism of Selectivity among G Protein-Coupled Receptor Kinase 2 Inhibitors

    SciTech Connect

    Thal, David M.; Yeow, Raymond Y.; Schoenau, Christian; Huber, Jochen; Tesmer, John J.G.

    2012-07-11

    G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-G{beta}{gamma} complex in the presence of two of these inhibitors. Comparison with the apoGRK2-G{beta}{gamma} structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.

  5. Crosstalk between G protein-coupled receptors (GPCRs) and tyrosine kinase receptor (TXR) in the heart after morphine withdrawal.

    PubMed

    Almela, Pilar; García-Carmona, Juan-Antonio; Martínez-Laorden, Elena; Milanés, María-Victoria; Laorden, María-Luisa

    2013-01-01

    G protein-coupled receptors (GPCRs) comprise a large family of membrane receptors involved in signal transduction. These receptors are linked to a variety of physiological and biological processes such as regulation of neurotransmission, growth, and cell differentiation among others. Some of the effects of GPCRs are known to be mediated by the activation of mitogen-activated extracellular kinase (MAPK) pathways. Cross-talk among various signal pathways plays an important role in activation of intracellular and intranuclear signal transduction cascades. Naloxone-induced morphine withdrawal leads to an up-regulation of adenyl cyclase-mediated signaling, resulting in high expression of protein kinase (PK) A. In addition, there is also an increased expression of extracellular signal regulated kinase (ERK), one member of MAPK. For this reason, the crosstalk between these GPCRs and receptors with tyrosine kinase activity (TKR) can be considered a possible mechanism for adaptive changes that occurs after morphine withdrawal. Morphine withdrawal activates ERK1/2 and phosphorylated tyrosine hydroxylase (TH) at Ser31 in the right and left ventricle. When N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), a PKA inhibitor was infused, the ability of morphine withdrawal to activate ERK, which phosphorylates TH at Ser31, was reduced. The present finding demonstrated that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation of TH. Increasing understanding of the mechanisms that interconnect the two pathway regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies.

  6. Crosstalk between G protein-coupled receptors (GPCRs) and tyrosine kinase receptor (TXR) in the heart after morphine withdrawal

    PubMed Central

    Almela, Pilar; García-Carmona, Juan-Antonio; Martínez-Laorden, Elena; Milanés, María-Victoria; Laorden, María-Luisa

    2013-01-01

    G protein-coupled receptors (GPCRs) comprise a large family of membrane receptors involved in signal transduction. These receptors are linked to a variety of physiological and biological processes such as regulation of neurotransmission, growth, and cell differentiation among others. Some of the effects of GPCRs are known to be mediated by the activation of mitogen-activated extracellular kinase (MAPK) pathways. Cross-talk among various signal pathways plays an important role in activation of intracellular and intranuclear signal transduction cascades. Naloxone-induced morphine withdrawal leads to an up-regulation of adenyl cyclase-mediated signaling, resulting in high expression of protein kinase (PK) A. In addition, there is also an increased expression of extracellular signal regulated kinase (ERK), one member of MAPK. For this reason, the crosstalk between these GPCRs and receptors with tyrosine kinase activity (TKR) can be considered a possible mechanism for adaptive changes that occurs after morphine withdrawal. Morphine withdrawal activates ERK1/2 and phosphorylated tyrosine hydroxylase (TH) at Ser31 in the right and left ventricle. When N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), a PKA inhibitor was infused, the ability of morphine withdrawal to activate ERK, which phosphorylates TH at Ser31, was reduced. The present finding demonstrated that the enhancement of ERK1/2 expression and the phosphorylation state of TH at Ser31 during morphine withdrawal are dependent on PKA and suggest cross-talk between PKA and ERK1/2 transduction pathway mediating morphine withdrawal-induced activation of TH. Increasing understanding of the mechanisms that interconnect the two pathway regulated by GPCRs and TKRs may facilitate the design of new therapeutic strategies. PMID:24409147

  7. Phenotypic Regulation of the Sphingosine 1-Phosphate Receptor Miles Apart by G Protein-Coupled Receptor Kinase 2

    PubMed Central

    2016-01-01

    The evolutionarily conserved DRY motif at the end of the third helix of rhodopsin-like, class-A G protein-coupled receptors (GPCRs) is a major regulator of receptor stability, signaling activity, and β-arrestin-mediated internalization. Substitution of the DRY arginine with histidine in the human vasopressin receptor results in a loss-of-function phenotype associated with diabetes insipidus. The analogous R150H substitution of the DRY motif in zebrafish sphingosine-1 phosphate receptor 2 (S1p2) produces a mutation, miles apart m93 (milm93), that not only disrupts signaling but also impairs heart field migration. We hypothesized that constitutive S1p2 desensitization is the underlying cause of this strong zebrafish developmental defect. We observed in cell assays that the wild-type S1p2 receptor is at the cell surface whereas in distinct contrast the S1p2 R150H receptor is found in intracellular vesicles, blocking G protein but not arrestin signaling activity. Surface S1p2 R150H expression could be restored by inhibition of G protein-coupled receptor kinase 2 (GRK2). Moreover, we observed that β-arrestin 2 and GRK2 colocalize with S1p2 in developing zebrafish embryos and depletion of GRK2 in the S1p2 R150H miles apart zebrafish partially rescued cardia bifida. The ability of reduced GRK2 activity to reverse a developmental phenotype associated with constitutive desensitization supports efforts to genetically or pharmacologically target this kinase in diseases involving biased GPCR signaling. PMID:25555130

  8. Analysis of Phosphorylation of the Receptor-Like Protein Kinase HAESA during Arabidopsis Floral Abscission

    PubMed Central

    Taylor, Isaiah; Wang, Ying; Seitz, Kati; Baer, John; Bennewitz, Stefan; Mooney, Brian P.; Walker, John C.

    2016-01-01

    Receptor-like protein kinases (RLKs) are the largest family of plant transmembrane signaling proteins. Here we present functional analysis of HAESA, an RLK that regulates floral organ abscission in Arabidopsis. Through in vitro and in vivo analysis of HAE phosphorylation, we provide evidence that a conserved phosphorylation site on a region of the HAE protein kinase domain known as the activation segment positively regulates HAE activity. Additional analysis has identified another putative activation segment phosphorylation site common to multiple RLKs that potentially modulates HAE activity. Comparative analysis suggests that phosphorylation of this second activation segment residue is an RLK specific adaptation that may regulate protein kinase activity and substrate specificity. A growing number of RLKs have been shown to exhibit biologically relevant dual specificity toward serine/threonine and tyrosine residues, but the mechanisms underlying dual specificity of RLKs are not well understood. We show that a phospho-mimetic mutant of both HAE activation segment residues exhibits enhanced tyrosine auto-phosphorylation in vitro, indicating phosphorylation of this residue may contribute to dual specificity of HAE. These results add to an emerging framework for understanding the mechanisms and evolution of regulation of RLK activity and substrate specificity. PMID:26784444

  9. Prostaglandin E2 restrains macrophage maturation via E prostanoid receptor 2/protein kinase A signaling

    PubMed Central

    Zasłona, Zbigniew; Serezani, Carlos H.; Okunishi, Katsuhide; Aronoff, David M.

    2012-01-01

    Prostaglandin E2 (PGE2) is a lipid mediator that acts by ligating 4 distinct G protein–coupled receptors, E prostanoid (EP) 1 to 4. Previous studies identified the importance of PGE2 in regulating macrophage functions, but little is known about its effect on macrophage maturation. Macrophage maturation was studied in vitro in bone marrow cell cultures, and in vivo in a model of peritonitis. EP2 was the most abundant PGE2 receptor expressed by bone marrow cells, and its expression further increased during macrophage maturation. EP2-deficient (EP2−/−) macrophages exhibited enhanced in vitro maturation compared with wild-type cells, as evidenced by higher F4/80 expression. An EP2 antagonist also increased maturation. In the peritonitis model, EP2−/− mice exhibited a higher percentage of F4/80high/CD11bhigh cells and greater expression of macrophage colony-stimulating factor receptor (M-CSFR) in both the blood and the peritoneal cavity. Subcutaneous injection of the PGE2 analog misoprostol decreased M-CSFR expression in bone marrow cells and reduced the number of peritoneal macrophages in wild-type mice but not EP2−/− mice. The suppressive effect of EP2 ligation on in vitro macrophage maturation was mimicked by a selective protein kinase A agonist. Our findings reveal a novel role for PGE2/EP2/protein kinase A signaling in the suppression of macrophage maturation. PMID:22234697

  10. Protein receptor for activated C kinase 1 is involved in morphine reward in mice.

    PubMed

    Wan, L; Su, L; Xie, Y; Liu, Y; Wang, Y; Wang, Z

    2009-07-07

    Opiate addiction is associated with upregulation of cAMP signaling in the brain. cAMP-responsive element binding protein (CREB), a nuclear transcription factor, is a downstream component of the extracellular signal-regulated protein kinase (ERK) pathway, which has been shown to regulate different physiological and psychological responses of drug addiction. RACK1, the protein receptor for activated C kinase 1, is a multifunctional scaffolding protein known to be a key regulator of various signaling cascades in the CNS. RACK1 functions specifically in integrin mediated activation of ERK cascade and targets active ERK. We examined if RACK1 is involved in the mechanism of drug addiction by regulating CREB in mouse hippocampus and prefrontal cortex. Several expressions were observed. Chronic administration of morphine made the expression of RACK1 and CREB mRNA increase in hippocampus and prefrontal cortex. The expression of RACK1 and CREB protein was strongly positive in CA1, CA3 and dentate gyrus (DG) of the hippocampus of morphine-treated mice brain, especially the pyramidal neurons in the DG of the hippocampus. Using the small interfering RNA technology, we determined that the expression of CREB mRNA was decreased in hippocampus and prefrontal cortex of morphine-treated mice. The expression of RACK1 and CREB protein was negative in CA1, CA3 and DG of hippocampus. These findings suggest that morphine reward can influence the expression of RACK1 in mouse hippocampus and prefrontal cortex through regulating CREB transcription.

  11. Expansion of the Receptor-Like Kinase/Pelle Gene Family and Receptor-Like Proteins in Arabidopsis1[w

    PubMed Central

    Shin-Han, Shiu; Bleecker, Anthony B.

    2003-01-01

    Receptor-like kinases (RLKs) are a family of transmembrane proteins with versatile N-terminal extracellular domains and C-terminal intracellular kinases. They control a wide range of physiological responses in plants and belong to one of the largest gene families in the Arabidopsis genome with more than 600 members. Interestingly, this gene family constitutes 60% of all kinases in Arabidopsis and accounts for nearly all transmembrane kinases in Arabidopsis. Analysis of four fungal, six metazoan, and two Plasmodium sp. genomes indicates that the family was represented in all but fungal genomes, indicating an ancient origin for the family with a more recent expansion only in the plant lineages. The RLK/Pelle family can be divided into several subfamilies based on three independent criteria: the phylogeny based on kinase domain sequences, the extracellular domain identities, and intron locations and phases. A large number of receptor-like proteins (RLPs) resembling the extracellular domains of RLKs are also found in the Arabidopsis genome. However, not all RLK subfamilies have corresponding RLPs. Several RLK/Pelle subfamilies have undergone differential expansions. More than 33% of the RLK/Pelle members are found in tandem clusters, substantially higher than the genome average. In addition, 470 of the RLK/Pelle family members are located within the segmentally duplicated regions in the Arabidopsis genome and 268 of them have a close relative in the corresponding regions. Therefore, tandem duplications and segmental/whole-genome duplications represent two of the major mechanisms for the expansion of the RLK/Pelle family in Arabidopsis. PMID:12805585

  12. Regulation of beta-adrenergic receptor signaling by S-nitrosylation of G-protein-coupled receptor kinase 2.

    PubMed

    Whalen, Erin J; Foster, Matthew W; Matsumoto, Akio; Ozawa, Kentaro; Violin, Jonathan D; Que, Loretta G; Nelson, Chris D; Benhar, Moran; Keys, Janelle R; Rockman, Howard A; Koch, Walter J; Daaka, Yehia; Lefkowitz, Robert J; Stamler, Jonathan S

    2007-05-04

    beta-adrenergic receptors (beta-ARs), prototypic G-protein-coupled receptors (GPCRs), play a critical role in regulating numerous physiological processes. The GPCR kinases (GRKs) curtail G-protein signaling and target receptors for internalization. Nitric oxide (NO) and/or S-nitrosothiols (SNOs) can prevent the loss of beta-AR signaling in vivo, but the molecular details are unknown. Here we show in mice that SNOs increase beta-AR expression and prevent agonist-stimulated receptor downregulation; and in cells, SNOs decrease GRK2-mediated beta-AR phosphorylation and subsequent recruitment of beta-arrestin to the receptor, resulting in the attenuation of receptor desensitization and internalization. In both cells and tissues, GRK2 is S-nitrosylated by SNOs as well as by NO synthases, and GRK2 S-nitrosylation increases following stimulation of multiple GPCRs with agonists. Cys340 of GRK2 is identified as a principal locus of inhibition by S-nitrosylation. Our studies thus reveal a central molecular mechanism through which GPCR signaling is regulated.

  13. Cross-interactions of two p38 mitogen-activated protein (MAP) kinase inhibitors and two cholecystokinin (CCK) receptor antagonists with the CCK1 receptor and p38 MAP kinase.

    PubMed

    Morel, Caroline; Ibarz, Géraldine; Oiry, Catherine; Carnazzi, Eric; Bergé, Gilbert; Gagne, Didier; Galleyrand, Jean-Claude; Martinez, Jean

    2005-06-03

    Although SB202190 and SB203580 are described as specific p38 MAP kinase inhibitors, several reports have indicated that other enzymes are also sensitive to SB203580. Using a pharmacological approach, we report for the first time that compounds SB202190 and SB203580 were able to directly and selectively interact with a G-protein-coupled receptor, namely the cholecystokinin receptor subtype CCK1, but not with the CCK2 receptor. We demonstrated that these compounds were non-competitive antagonists of the CCK1 receptor at concentrations typically used to inhibit protein kinases. By chimeric construction of the CCK2 receptor, we determined the involvement of two CCK1 receptor intracellular loops in the binding of SB202190 and SB203580. We also showed that two CCK antagonists, L364,718 and L365,260, were able to regulate p38 mitogen-activated protein (MAP) kinase activity. Using a reporter gene strategy and immunoblotting experiments, we demonstrated that both CCK antagonists inhibited selectively the enzymatic activity of p38 MAP kinase. Kinase assays suggested that this inhibition resulted from a direct interaction with both CCK antagonists. Molecular modeling simulations suggested that this interaction occurs in the ATP binding pocket of p38 MAP kinase. These results suggest that SB202190 and SB203580 bind to the CCK1 receptor and, as such, these compounds should be used with caution in models that express this receptor. We also found that L364,718 and L365,260, two CCK receptor antagonists, directly interacted with p38 MAP kinase and inhibited its activity. These findings suggest that the CCK1 receptor shares structural analogies with the p38 MAP kinase ATP binding site. They open the way to potential design of either a new family of MAP kinase inhibitors from CCK1 receptor ligand structures or new CCK1 receptor ligands based on p38 MAP kinase inhibitor structures.

  14. Identification and Structure-Function Analysis of Subfamily Selective G Protein-Coupled Receptor Kinase Inhibitors

    SciTech Connect

    Homan, Kristoff T.; Larimore, Kelly M.; Elkins, Jonathan M.; Szklarz, Marta; Knapp, Stefan; Tesmer, John J.G.

    2015-02-13

    Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.

  15. APS, an adapter protein with a PH and SH2 domain, is a substrate for the insulin receptor kinase.

    PubMed Central

    Ahmed, Z; Smith, B J; Kotani, K; Wilden, P; Pillay, T S

    1999-01-01

    APS (adapter protein with a PH and SH2 domain) is the newest member of a family of tyrosine kinase adapter proteins including SH2-B and Lnk. We previously identified SH2-B as an insulin-receptor-binding protein and substrate [Kotani, Wilden and Pillay (1998) Biochem J. 335, 103-109]. Here we show that APS interacts with the insulin receptor kinase activation loop through its SH2 domain and insulin stimulates the tyrosine-phosphorylation of APS. Furthermore, the phosphorylation of activation-loop tyrosine residues 1158 and 1162 are required for this interaction. PMID:10417330

  16. syk protein tyrosine kinase regulates Fc receptor gamma-chain-mediated transport to lysosomes.

    PubMed Central

    Bonnerot, C; Briken, V; Brachet, V; Lankar, D; Cassard, S; Jabri, B; Amigorena, S

    1998-01-01

    B- and T-cell receptors, as well as most Fc receptors (FcR), are part of a large family of membrane proteins named immunoreceptors and are expressed on all cells of the immune system. Immunoreceptors' biological functions rely on two of their fundamental attributes: signal transduction and internalization. The signals required for these two functions are present in the chains associated with immunoreceptors, within conserved amino acid motifs called immunoreceptor tyrosine-based activation motifs (ITAMs). We have examined the role of the protein tyrosine kinase (PTK) syk, a critical effector of immunoreceptor-mediated cell signalling through ITAMs, in FcR-associated gamma-chain internalization and lysosomal targeting. A point mutation in the immunoreceptor-associated gamma-chain ITAM affecting syk activation, as well as overexpression of a syk dominant negative mutant, inhibited signal transduction without affecting receptor coated-pit localization or internalization. In contrast, blocking of gamma-chain-mediated syk activation impaired FcR transport from endosomes to lysosomes and selectively inhibited the presentation of certain T-cell epitopes. Therefore, activation of the PTK syk is dispensable for receptor internalization, but necessary for cell signalling and for gamma-chain-mediated FcR delivery to lysosomes. PMID:9707420

  17. Identification of tyrosine phosphorylation sites in human Gab-1 protein by EGF receptor kinase in vitro.

    PubMed

    Lehr, S; Kotzka, J; Herkner, A; Klein, E; Siethoff, C; Knebel, B; Noelle, V; Brüning, J C; Klein, H W; Meyer, H E; Krone, W; Müller-Wieland, D

    1999-01-05

    Grb2-associated binder-1 (Gab-1) has been identified recently in a cDNA library of glioblastoma tumors and appears to play a central role in cellular growth response, transformation, and apoptosis. Structural and functional features indicate that Gab-1 is a multisubstrate docking protein downstream in the signaling pathways of different receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR). Therefore, the aim of the study was to characterize the phosphorylation of recombinant human Gab-1 (hGab-1) protein by EGFR in vitro. Using the pGEX system to express the entire protein and different domains of hGab-1 as glutathione S-transferase proteins, kinetic data for phosphorylation of these proteins by wheat germ agglutinine-purified EGFR and the recombinant EGFR (rEGFR) receptor kinase domain were determined. Our data revealed similar affinities of hGab-1-C for both receptor preparations (KM = 2.7 microM for rEGFR vs 3.2 microM for WGA EGFR) as well as for the different recombinant hGab-1 domains. To identify the specific EGFR phosphorylation sites, hGab-1-C was sequenced by Edman degradation and mass spectrometry. The entire protein was phosphorylated by rEGFR at eight tyrosine residues (Y285, Y373, Y406, Y447, Y472, Y619, Y657, and Y689). Fifty percent of the identified radioactivity was incorporated in tyrosine Y657 as the predominant peak in HPLC analysis, a site exhibiting features of a potential Syp (PTP1D) binding site. Accordingly, GST-pull down assays with A431 and HepG2 cell lysates showed that phosphorylated intact hGab-1 was able to bind Syp. This binding appears to be specific, because it was abolished by changing the Y657 of hGab-1 to F657. These results demonstrate that hGab-1 is a high-affinity substrate for the EGFR and the major tyrosine phosphorylation site Y657 in the C terminus is a specific binding site for the tyrosine phosphatase Syp.

  18. Cloning and Characterization of a Receptor-Like Protein Kinase Gene Associated with Senescence

    PubMed Central

    Hajouj, Taleb; Michelis, Regina; Gepstein, Shimon

    2000-01-01

    Senescence-associated genes are up-regulated during plant senescence and many have been implicated in encoding enzymes involved in the metabolism of senescing tissues. Using the differential display technique, we identified a SAG in bean (Phaseolus vulgaris) leaf that was exclusively expressed during senescence and was designated senescence-associated receptor-like kinase (SARK). The deduced SARK polypeptide consists of a signal peptide, a leucine-rich repeat in the extracellular region, a single membrane-spanning domain, and the characteristic serine/threonine protein kinase domain. The mRNA level for SARK increased prior to the loss of chlorophyll and the decrease of chlorophyll a/b-binding protein mRNA. Detached mature bean leaves, which senesce at an accelerated rate compared with leaves on intact plants, showed a similar temporal pattern of SARK message accumulation. Light and cytokinin, which delayed the initiation of leaf senescence, also delayed SARK gene expression; in contrast, darkness and ethylene, which accelerated senescence, advanced the initial appearance of the SARK transcript. SARK protein accumulation exhibited a temporal pattern similar to that of its mRNA. A possible role for SARK in the regulation of leaf senescence was considered. PMID:11080306

  19. [Characterization of a putative S locus encoded receptor protein kinase and its role in self-incompatibility

    SciTech Connect

    Not Available

    1993-01-01

    The serine/threonine protein kinase (SRK) protein was predicted to be similar to the growth factor receptor tyrosine kinases in animals but its amino acid sequence of the catalytic domain is more similar to that of the catalytic domains of protein serine/threonine kinases than to protein tyrosine kinases. We have shown that the SRK protein has intrinsic scrine/threonine kinase activity. We subcloned the protein kinase-homologous domain of the SRK[sub 6] cDNA into the bacterial expression vector pGEX-3X and we have constructed a second plasmid identical to the first except that it carried a conservative mutation that substituted Arg for the Lys[sup 524] codon of SRK6 This lysine corresponds to the ATP-binding site, is essential in protein kinases, and is a common target for site-directed mutagenesis as a means to obtain kinase-defective proteins. Cultures bearing the wild-type and mutant SRK catalytic domains each produced an approximately 64 kD protein that reacted with anti-SRK6 antibodies. Following pulse-labeling with [sup 32]P we found that the wild-type SRK6 protein but not the mutant form was detectably phosphorylated. Phosphoamino acid analysis of the affinity purified [sup 32]p-labeled GST-SRK6 fusion protein demonstrated that SRK was phosphorylated predominantly on semine and to a lesser extent on threonine, but not on tyrosine. Thus, SRK6 is a functional serine/threonine protein kinase.

  20. Receptor protein kinase gene encoded at the self-incompatibility locus

    DOEpatents

    Nasrallah, June B.; Nasrallah, Mikhail E.; Stein, Joshua

    1996-01-01

    Described herein is a S receptor kinase gene (SRK), derived from the S locus in Brassica oleracea, having a extracellular domain highly similar to the secreted product of the S-locus glycoprotein gene.

  1. Regulation of glycine receptor diffusion properties and gephyrin interactions by protein kinase C

    PubMed Central

    Specht, Christian G; Grünewald, Nora; Pascual, Olivier; Rostgaard, Nina; Schwarz, Günter; Triller, Antoine

    2011-01-01

    Glycine receptors (GlyRs) can dynamically exchange between synaptic and extrasynaptic locations through lateral diffusion within the plasma membrane. Their accumulation at inhibitory synapses depends on the interaction of the β-subunit of the GlyR with the synaptic scaffold protein gephyrin. An alteration of receptor–gephyrin binding could thus shift the equilibrium between synaptic and extrasynaptic GlyRs and modulate the strength of inhibitory neurotransmission. Using a combination of dynamic imaging and biochemical approaches, we have characterised the molecular mechanism that links the GlyR–gephyrin interaction with GlyR diffusion and synaptic localisation. We have identified a protein kinase C (PKC) phosphorylation site within the cytoplasmic domain of the β-subunit of the GlyR (residue S403) that causes a reduction of the binding affinity between the receptor and gephyrin. In consequence, the receptor's diffusion in the plasma membrane is accelerated and GlyRs accumulate less strongly at synapses. We propose that the regulation of GlyR dynamics by PKC thus contributes to the plasticity of inhibitory synapses and may be involved in maladaptive forms of synaptic plasticity. PMID:21829170

  2. Role of macrophage-stimulating protein and its receptor, RON tyrosine kinase, in ciliary motility.

    PubMed Central

    Sakamoto, O; Iwama, A; Amitani, R; Takehara, T; Yamaguchi, N; Yamamoto, T; Masuyama, K; Yamanaka, T; Ando, M; Suda, T

    1997-01-01

    Macrophage-stimulating protein (MSP) is an 80-kD serum protein with homology to hepatocyte growth factor (HGF). Its receptor, RON tyrosine kinase, is a new member of the HGF receptor family. The MSP-RON signaling pathway has been implicated in the functional regulation of mononuclear phagocytes. However, the function of this pathway in other types of cells has not been elucidated. Here we show that in contrast to the HGF receptor, which was expressed at the basolateral surface, RON was localized at the apical surface of ciliated epithelia in the airways and oviduct. In addition, MSP was found in the bronchoalveolar space at biologically significant concentrations. MSP bound to RON on normal human bronchial epithelial cells with a high affinity (Kd = 0.5 nM) and induced autophosphorylation of RON. Activation of RON by MSP led to a significant increase in ciliary beat frequency of human nasal cilia. These findings indicate that the ciliated epithelium of the mucociliary transport apparatus is a novel target of MSP. Ciliary motility is critical for mucociliary transport. Our findings suggest that the MSP-RON signaling pathway is a novel regulatory system of mucociliary function and might be involved in the host defense and fertilization. PMID:9045873

  3. Molecular Mechanism for Inhibition of G Protein-Coupled Receptor Kinase 2 by a Selective RNA Aptamer

    SciTech Connect

    Tesmer, Valerie M.; Lennarz, Sabine; Mayer, Günter; Tesmer, John J.G.

    2012-08-31

    Cardiovascular homeostasis is maintained in part by the rapid desensitization of activated heptahelical receptors that have been phosphorylated by G protein-coupled receptor kinase 2 (GRK2). However, during chronic heart failure GRK2 is upregulated and believed to contribute to disease progression. We have determined crystallographic structures of GRK2 bound to an RNA aptamer that potently and selectively inhibits kinase activity. Key to the mechanism of inhibition is the positioning of an adenine nucleotide into the ATP-binding pocket and interactions with the basic {alpha}F-{alpha}G loop region of the GRK2 kinase domain. Constraints imposed on the RNA by the terminal stem of the aptamer also play a role. These results highlight how a high-affinity aptamer can be used to selectively trap a novel conformational state of a protein kinase.

  4. Differential role of protein kinase C in desensitization of muscarinic receptor induced by phorbol esters and receptor agonists

    SciTech Connect

    Lai, Wi Sheung.

    1989-01-01

    PKC, a phorbol ester receptor, copurified with specific binding sites of ({sup 3}H)phorbol-12,13,-dibutyrate (({sup 3}H)PDBu). The specific binding of ({sup 3}H)PDBu to intact cells was saturable to a single class of binding sites. The PKC and phorbol ester receptors in N1E-115 cells can be down regulated by prolonged phorbol ester incubation. Phorbol 12-myristate 13-acetate (PMA) suppressed muscarinic receptor-mediated cyclic GMP response in a time-dependent and a concentration-dependent fashion and the suppressive effect of PMA could be attenuated by a protein kinase inhibitor, H-7, as well as by down-regulation of the PKC through long-term incubation with PDBu. Exposure of the cells to the muscarinic agonist carbamylcholine also desensitized subsequent CBC-mediated cyclic GMP response. However, pretreatment with carbamylcholine did not desensitize histamine-induced cyclic GMP formation while treatment with PMA suppressed this histamine-mediated response. Preincubation of the cells with CBC, but not with phorbol ester, resulted in down-regulation of muscarinic receptors. The loss of muscarinic receptors induced by agonist even occurred when the phosphoinositide hydrolysis response was suppressed.

  5. Lectin Receptor Kinases Participate in Protein-Protein Interactions to Mediate Plasma Membrane-Cell Wall Adhesions in Arabidopsis1

    PubMed Central

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces. PMID:16361528

  6. Lectin receptor kinases participate in protein-protein interactions to mediate plasma membrane-cell wall adhesions in Arabidopsis.

    PubMed

    Gouget, Anne; Senchou, Virginie; Govers, Francine; Sanson, Arnaud; Barre, Annick; Rougé, Pierre; Pont-Lezica, Rafael; Canut, Hervé

    2006-01-01

    Interactions between plant cell walls and plasma membranes are essential for cells to function properly, but the molecules that mediate the structural continuity between wall and membrane are unknown. Some of these interactions, which are visualized upon tissue plasmolysis in Arabidopsis (Arabidopsis thaliana), are disrupted by the RGD (arginine-glycine-aspartic acid) tripeptide sequence, a characteristic cell adhesion motif in mammals. In planta induced-O (IPI-O) is an RGD-containing protein from the plant pathogen Phytophthora infestans that can disrupt cell wall-plasma membrane adhesions through its RGD motif. To identify peptide sequences that specifically bind the RGD motif of the IPI-O protein and potentially play a role in receptor recognition, we screened a heptamer peptide library displayed in a filamentous phage and selected two peptides acting as inhibitors of the plasma membrane RGD-binding activity of Arabidopsis. Moreover, the two peptides also disrupted cell wall-plasma membrane adhesions. Sequence comparison of the RGD-binding peptides with the Arabidopsis proteome revealed 12 proteins containing amino acid sequences in their extracellular domains common with the two RGD-binding peptides. Eight belong to the receptor-like kinase family, four of which have a lectin-like extracellular domain. The lectin domain of one of these, At5g60300, recognized the RGD motif both in peptides and proteins. These results imply that lectin receptor kinases are involved in protein-protein interactions with RGD-containing proteins as potential ligands, and play a structural and signaling role at the plant cell surfaces.

  7. G protein-coupled receptor kinase and beta-arrestin-mediated desensitization of the angiotensin II type 1A receptor elucidated by diacylglycerol dynamics.

    PubMed

    Violin, Jonathan D; Dewire, Scott M; Barnes, William G; Lefkowitz, Robert J

    2006-11-24

    Receptor desensitization progressively limits responsiveness of cells to chronically applied stimuli. Desensitization in the continuous presence of agonist has been difficult to study with available assay methods. Here, we used a fluorescence resonance energy transfer-based live cell assay for the second messenger diacylglycerol to measure desensitization of a model seven-transmembrane receptor, the Gq-coupled angiotensin II type 1(A) receptor, expressed in human embryonic kidney 293 cells. In response to angiotensin II, we observed a transient diacylglycerol response reflecting activation and complete desensitization of the receptor within 2-5 min. By utilizing a variety of approaches including graded tetracycline-inducible receptor expression, mutated receptors, and overexpression or short interfering RNA-mediated silencing of putative components of the cellular desensitization machinery, we conclude that the rate and extent of receptor desensitization are critically determined by the following: receptor concentration in the plasma membrane; the presence of phosphorylation sites on the carboxyl terminus of the receptor; kinase activity of G protein-coupled receptor kinase 2, but not of G protein-coupled receptor kinases 3, 5, or 6; and stoichiometric expression of beta-arrestin. The findings introduce the use of the biosensor diacylglycerol reporter as a powerful means for studying Gq-coupled receptor desensitization and document that, at the levels of receptor overexpression commonly used in such studies, the properties of the desensitization process are markedly perturbed and do not reflect normal cellular physiology.

  8. Receptor protein kinase FERONIA controls leaf starch accumulation by interacting with glyceraldehyde-3-phosphate dehydrogenase.

    PubMed

    Yang, Tao; Wang, Long; Li, Chiyu; Liu, Ying; Zhu, Sirui; Qi, Yinyao; Liu, Xuanming; Lin, Qinglu; Luan, Sheng; Yu, Feng

    2015-09-11

    Cell expansion is coordinated by several cues, but available energy is the major factor determining growth. Receptor protein kinase FERONIA (FER) is a master regulator of cell expansion, but the details of its control mechanisms are not clear. Here we show that FER interacts with cytosolic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GAPC1 and GAPC2), that catalyzes a key reaction in glycolysis, which contributes to energy production. When there is an FER deficiency, there are corresponding decreases in the enzyme activity of GAPDH and increased amounts of starch. More importantly, gapc1/2 mutants mimic fer4 mutants. These data indicate that FER regulated starch content is an evolutionarily conserved function in plants that connects the cell expansion and energy metabolism pathways.

  9. G Protein Coupled Receptor Kinase 3 Regulates Breast Cancer Migration, Invasion, and Metastasis

    PubMed Central

    Billard, Matthew J.; Fitzhugh, David J.; Parker, Joel S.; Brozowski, Jaime M.; McGinnis, Marcus W.; Timoshchenko, Roman G.; Serafin, D. Stephen; Lininger, Ruth; Klauber-Demore, Nancy; Sahagian, Gary; Truong, Young K.; Sassano, Maria F.; Serody, Jonathan S.; Tarrant, Teresa K.

    2016-01-01

    Triple negative breast cancer (TNBC) is a heterogeneous disease that has a poor prognosis and limited treatment options. Chemokine receptor interactions are important modulators of breast cancer metastasis; however, it is now recognized that quantitative surface expression of one important chemokine receptor, CXCR4, may not directly correlate with metastasis and that its functional activity in breast cancer may better inform tumor pathogenicity. G protein coupled receptor kinase 3 (GRK3) is a negative regulator of CXCR4 activity, and we show that GRK expression correlates with tumorigenicity, molecular subtype, and metastatic potential in human tumor microarray analysis. Using established human breast cancer cell lines and an immunocompetent in vivo mouse model, we further demonstrate that alterations in GRK3 expression levels in tumor cells directly affect migration and invasion in vitro and the establishment of distant metastasis in vivo. The effects of GRK3 modulation appear to be specific to chemokine-mediated migration behaviors without influencing tumor cell proliferation or survival. These data demonstrate that GRK3 dysregulation may play an important part in TNBC metastasis. PMID:27049755

  10. Antifibrotic effects of noscapine through activation of prostaglandin E2 receptors and protein kinase A.

    PubMed

    Kach, Jacob; Sandbo, Nathan; La, Jennifer; Denner, Darcy; Reed, Eleanor B; Akimova, Olga; Koltsova, Svetlana; Orlov, Sergei N; Dulin, Nickolai O

    2014-03-14

    Myofibroblast differentiation is a key process in the pathogenesis of fibrotic disease. We have shown previously that differentiation of myofibroblasts is regulated by microtubule polymerization state. In this work, we examined the potential antifibrotic effects of the antitussive drug, noscapine, recently found to bind microtubules and affect microtubule dynamics. Noscapine inhibited TGF-β-induced differentiation of cultured human lung fibroblasts (HLFs). Therapeutic noscapine treatment resulted in a significant attenuation of pulmonary fibrosis in the bleomycin model of the disease. Noscapine did not affect gross microtubule content in HLFs, but inhibited TGF-β-induced stress fiber formation and activation of serum response factor without affecting Smad signaling. Furthermore, noscapine stimulated a rapid and profound activation of protein kinase A (PKA), which mediated the antifibrotic effect of noscapine in HLFs, as assessed with the PKA inhibitor, PKI. In contrast, noscapine did not activate PKA in human bronchial or alveolar epithelial cells. Finally, activation of PKA and the antifibrotic effect of noscapine in HLFs were blocked by the EP2 prostaglandin E2 receptor antagonist, PF-04418948, but not by the antagonists of EP4, prostaglandin D2, or prostacyclin receptors. Together, we demonstrate for the first time the antifibrotic effect of noscapine in vitro and in vivo, and we describe a novel mechanism of noscapine action through EP2 prostaglandin E2 receptor-mediated activation of PKA in pulmonary fibroblasts.

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

    PubMed Central

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

    1991-01-01

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

  12. Interaction between protein kinase Cmu and the vanilloid receptor type 1.

    PubMed

    Wang, Yun; Kedei, Noemi; Wang, Min; Wang, Q Jane; Huppler, Anna R; Toth, Attila; Tran, Richard; Blumberg, Peter M

    2004-12-17

    The capsaicin receptor VR1 is a polymodal nociceptor activated by multiple stimuli. It has been reported that protein kinase C plays a role in the sensitization of VR1. Protein kinase D/PKCmu is a member of the protein kinase D serine/threonine kinase family that exhibits structural, enzymological, and regulatory features distinct from those of the PKCs, with which they are related. As part of our effort to optimize conditions for evaluating VR1 pharmacology, we found that treatment of Chinese hamster ovary (CHO) cells heterologously expressing rat VR1 (CHO/rVR1) with butyrate enhanced rVR1 expression and activity. The expression of PKCmu and PKCbeta1, but not of other PKC isoforms, was also enhanced by butyrate treatment, suggesting the possibility that these two isoforms might contribute to the enhanced activity of rVR1. In support of this hypothesis, we found the following. 1) Overexpression of PKCmu enhanced the response of rVR1 to capsaicin and low pH, and expression of a dominant negative variant of PKCmu reduced the response of rVR1. 2) Reduction of endogenous PKCmu using antisense oligonucleotides decreased the response of exogenous rVR1 expressed in CHO cells as well as of endogenous rVR1 in dorsal root ganglion neurons. 3) PKCmu localized to the plasma membrane when overexpressed in CHO/rVR1 cells. 4) PKCmu directly bound to rVR1 expressed in CHO cells as well as to endogenous rVR1 in dorsal root ganglia or to an N-terminal fragment of rVR1, indicating a direct interaction between PKCmu and rVR1. 5) PKCmu directly phosphorylated rVR1 or a longer N-terminal fragment (amino acids 1-118) of rVR1 but not a shorter one (amino acids 1-99). 6) Mutation of S116A in rVR1 blocked both the phosphorylation of rVR1 by PKCmu and the enhancement by PKCmu of the rVR1 response to capsaicin. We conclude that PKCmu functions as a direct modulator of rVR1.

  13. Regulation of ERBB Receptor Tyrosine Kinase Activities in Breast Cancer by the KEK Proteins

    DTIC Science & Technology

    1999-11-01

    can also inhibit transformation in mouse mammary tumor cells with deregulated expression of receptors and ligands of the ErbB family. 14. SUBJECT...Reportable outcomes Conclusions References Appendices 7 4 5. INTRODUCTION: In 20-30% of breast tumors , ErbB2, a receptor tyrosine kinase (RTK) of the...inhibit transformation in mouse mammary tumor cells with deregulated expression of receptors and ligands of the ErbB family. In the second year of

  14. THE GRK4 SUBFAMILY OF G PROTEIN-COUPLED RECEPTOR KINASES: ALTERNATIVE SPLICING, GENE ORGANIZATION, AND SEQUENCE CONSERVATION

    EPA Science Inventory

    The GRK4 subfamily of G protein-coupled receptor kinases. Alternative splicing, gene organization, and sequence conservation.

    Premont RT, Macrae AD, Aparicio SA, Kendall HE, Welch JE, Lefkowitz RJ.

    Department of Medicine, Howard Hughes Medical Institute, Duke Univer...

  15. 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

  16. Somatocrinin receptor coupled with cAMP-dependent protein kinase on anterior pituitary granules.

    PubMed

    Lewin, M J; Reyl-Desmars, F; Ling, N

    1983-11-01

    The molecular mechanism of growth hormone release by synthetic somatocrinin was investigated on purified hog anterior pituitary secretory granules; the granules were found to contain a cAMP-dependent protein kinase that catalyzed [gamma-32P]-ATP histone phosphorylation with maximal rates ranging from 1 to 5 nmol of Pi incorporated per mg of protein per 20 min. The activity of this enzyme was further stimulated by somatocrinin. Stimulation was observed at concentrations as low as 0.3 pM, and the half-maximal effect was obtained with 35 +/- 8 pM (n = 4). Michaelis-Menten analysis of phosphorylation kinetics suggested that the peptide did not change significantly the reaction's Vmax, but produced a dramatic increase in enzyme affinity for cAMP: the apparent Km for the nucleotide decreased from 400 X 10(-9) M under unstimulated conditions to 15 X 10(-9) M in the presence of 100 pM somatocrinin. Furthermore, a Hill plot of concentration-dependence curve indicated the existence of negative cooperativity. At the concentration of 35 pM, the less potent analogs of somatocrinin [designated hpGRF-44 to indicate source (human pancreas, hp), activity (growth hormone-releasing factor, GRF), and amino acid composition], hpGRF-(1-37) and [Phe1]hpGRF-(1-40) had 20% and 7%, respectively, of the effect of somatocrinin. The biologically inactive analog hpGRF-(2-40) had no evident effect at concentrations up to 0.1 microM. Therefore, we suggest that somatocrinin stimulation of growth hormone release involves activation of exocytosis through a phosphorylation mechanism mediated by a granular receptor coupled with a cAMP-dependent protein kinase.

  17. Regulated exocytosis contributes to protein kinase C potentiation of vanilloid receptor activity.

    PubMed

    Morenilla-Palao, Cruz; Planells-Cases, Rosa; García-Sanz, Nuria; Ferrer-Montiel, Antonio

    2004-06-11

    The vanilloid receptor-1 (TRPV1) plays a key role in the perception of peripheral thermal and inflammatory pain. TRPV1 expression and channel activity are notably up-regulated by proalgesic agents. The transduction pathways involved in TRPV1 sensitization are still elusive. We have used a yeast two-hybrid screen to identify proteins that associate with the N terminus of TRPV1. We report that two vesicular proteins, Snapin and synaptotagmin IX (Syt IX), strongly interact in vitro and in vivo with the TRPV1 N-terminal domain. In primary dorsal root ganglion neurons, TRPV1 co-distributes in vesicles with Syt IX and the vesicular protein synaptobrevin. Neither Snapin nor Syt IX affected channel function, but they notably inhibited protein kinase C (PKC)-induced potentiation of TRPV1 channel activity with a potency that rivaled the blockade evoked by botulinum neurotoxin A, a potent blocker of neuronal exocytosis. Noteworthily, we found that PKC activation induced a rapid delivery of functional TRPV1 channels to the plasma membrane. Botulinum neurotoxin A blocked the TRPV1 membrane translocation induced by PKC that was activated with a phorbol ester or the metabotropic glutamate receptor mGluR5. Therefore, our results indicate that PKC signaling promotes at least in part the SNARE-dependent exocytosis of TRPV1 to the cell surface. Taken together, these findings imply that activity-dependent delivery of channels to the neuronal surface may contribute to the buildup and maintenance of thermal inflammatory hyperalgesia in peripheral nociceptor terminals.

  18. Expression of epidermal growth factor receptor sequences as E. coli fusion proteins: applications in the study of tyrosine kinase function.

    PubMed

    Koland, J G; O'Brien, K M; Cerione, R A

    1990-01-15

    To investigate the functions of key domains of the epidermal growth factor receptor (EGFR), various EGFR-derived peptide sequences were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. The purified fusion proteins (GST-TK0-8) were tested as substrates for the tyrosine kinase activities of the EGFR and c-src. Both the GST-TK4 fusion protein, which contains the major C-terminal tyrosine autophosphorylation sites of the EGFR, and GST-TK7, which contains the connecting sequence between the EGFR kinase domain and the C-terminal autophosphorylation domain, were strongly phosphorylated by the EGFR and c-src. Hence the candidate tyrosine phosphorylation sites present in the connecting sequences of the EGFR, as well as the known autophosphorylation sites of the EGFR, can be phosphorylated by the two tyrosine kinases. The protein GST-TK7 was phosphorylated by c-src with a KM of 5-10 microM, which indicated a potential interaction between the connecting segment of the EGFR and the c-src kinase. The GST fusion proteins were also used to map the sites recognized by two anti-EGFR monoclonal antibodies and a polyclonal serum raised against an EGFR tyrosine kinase domain fragment. The recognition site of one monoclonal antibody was determined to be in a short sequence surrounding tyr1068, a primary site of autophosphorylation in the C-terminal domain of the receptor. The anti-peptide polyclonal serum recognized only sequences in the GST-TK7 fusion protein, and hence binds to the connecting sequence between the kinase core and the C-terminal domain. These antibodies will therefore be useful reagents for studying the function of two key structural elements of the EGFR tyrosine kinase. The GST-TK fusion proteins should have many other applications in the study of EGFR catalysis and mitogenic signalling.

  19. The Interplay of AMP-activated Protein Kinase and Androgen Receptor in Prostate Cancer Cells†

    PubMed Central

    Shen, Min; Zhang, Zhen; Ratnam, Manohar; Dou, Q. Ping

    2013-01-01

    AMP-activated protein kinase (AMPK) has recently emerged as a potential target for cancer therapy due to the observation that activation of AMPK inhibits tumor cell growth. It is well-known that androgen receptor (AR) signaling is a major driver for the development and progression of prostate cancer and that downregulation of AR is a critical step in the induction of apoptosis in prostate cancer cells. However, little is known about the potential interaction between AMPK and AR signaling pathways. In the current study, we showed that activation of AMPK by metformin caused decrease of AR protein level through suppression of AR mRNA expression and promotion of AR protein degradation, demonstrating that AMPK activation is upstream of AR downregulation. We also showed that inhibition of AR function by an anti-androgen or its siRNA enhanced AMPK activation and growth inhibition whereas overexpression of AR delayed AMPK activation and increased prostate cancer cellular resistance to metformin treatment, suggesting that AR suppresses AMPK signaling-mediated growth inhibition in a feedback mechanism. Our findings thus reveal a novel AMPK-AR regulatory loop in prostate cancer cells and should have a potential clinical significance. PMID:24129850

  20. Glycosylphosphatidylinositol-anchored proteins as chaperones and co-receptors for FERONIA receptor kinase signaling in Arabidopsis.

    PubMed

    Li, Chao; Yeh, Fang-Ling; Cheung, Alice Y; Duan, Qiaohong; Kita, Daniel; Liu, Ming-Che; Maman, Jacob; Luu, Emily J; Wu, Brendan W; Gates, Laura; Jalal, Methun; Kwong, Amy; Carpenter, Hunter; Wu, Hen-Ming

    2015-06-08

    The Arabidopsis receptor kinase FERONIA (FER) is a multifunctional regulator for plant growth and reproduction. Here we report that the female gametophyte-expressed glycosylphosphatidylinositol-anchored protein (GPI-AP) LORELEI and the seedling-expressed LRE-like GPI-AP1 (LLG1) bind to the extracellular juxtamembrane region of FER and show that this interaction is pivotal for FER function. LLG1 interacts with FER in the endoplasmic reticulum and on the cell surface, and loss of LLG1 function induces cytoplasmic retention of FER, consistent with transport of FER from the endoplasmic reticulum to the plasma membrane in a complex with LLG1. We further demonstrate that LLG1 is a component of the FER-regulated RHO GTPase signaling complex and that fer and llg1 mutants display indistinguishable growth, developmental and signaling phenotypes, analogous to how lre and fer share similar reproductive defects. Together our results support LLG1/LRE acting as a chaperone and co-receptor for FER and elucidate a mechanism by which GPI-APs enable the signaling capacity of a cell surface receptor.

  1. Sequestration of human muscarinic acetylcholine receptor hm1-hm5 subtypes: effect of G protein-coupled receptor kinases GRK2, GRK4, GRK5 and GRK6.

    PubMed

    Tsuga, H; Okuno, E; Kameyama, K; Haga, T

    1998-03-01

    Sequestration of porcine muscarinic acetylcholine receptor m2 subtypes (m2 receptors) expressed in COS-7 cells is facilitated by coexpression of G protein-coupled receptor kinases 2 (GRK2). We examined the effect of coexpression of GRK2, GRK4 delta, GRK5 and GRK6 on sequestration of human m1-m5 receptors expressed in COS-7 cells, which was assessed as loss of [3H]N-methylscopolamine binding activity from the cell surface. Sequestration of m4 receptors as well as m2 receptors was facilitated by coexpression of GRK2 and attenuated by coexpression of the dominant negative form of GRK2 (DN-GRK2). Sequestration of m3 and m5 receptors also was facilitated by coexpression of GRK2 but not affected by coexpression of DN-GRK2. On the other hand, proportions of sequestered m1 receptors were not significantly different with coexpression of GRK2 and DN-GRK2. GRK4 delta, GRK5 and GRK6 did not facilitate sequestration of m1-m5 receptors in COS-7 cells, except that the sequestration of m2 receptors tended to be facilitated by coexpression of GRK4 delta, GRK5 and GRK6. However, coexpression of GRK4 delta, GRK5, but not GRK6, in BHK-21 cells facilitated sequestration of m2, but not m3, receptors. These results indicate that the effect of GRK2 to facilitate receptor sequestration is not restricted to m2 receptors but is generalized to other muscarinic receptors except m1 receptors and that other kinases, including GRK4 delta, GRK5 and endogenous kinase(s) in COS-7 cells, also contribute to sequestration of m2 and m4 receptors.

  2. The Dtk receptor tyrosine kinase, which binds protein S, is expressed during hematopoiesis.

    PubMed

    Crosier, P S; Freeman, S A; Orlic, D; Bodine, D M; Crosier, K E

    1996-02-01

    Dtk (Tyro 3/Sky/Rse/Brt/Tif) belongs to a recently recognized subfamily of receptor tyrosine kinases that also includes Ufo (Axl/Ark) and Mer (Eyk). Ligands for Dtk and Ufo have been identified as protein S and the related molecule Gas6, respectively. This study examined expression of Dtk during ontogeny of the hematopoietic system and compared the pattern of expression with that of Ufo. Both receptors were abundantly expressed in differentiating embryonic stem cells, yolk sac blood islands, para-aortic splanchnopleural mesoderm, fractionated AA4+ fetal liver cells, and fetal thymus from day 14 until birth. Although Ufo was expressed at moderate levels in adult bone marrow, expression of Dtk in this tissue was barely detectable. In adult bone marrow subpopulations fractionated using counterflow centrifugal elutriation, immunomagnetic bead selection for lineage-depletion and FACS sorting for c-kit expression, very low levels of Dtk and/or Ufo were detected in some cell fractions. These results suggest that Dtk and Ufo are likely to be involved in the regulation of hematopoiesis, particularly during the embryonic stages of blood cell development.

  3. 5-HT2A SEROTONIN RECEPTOR BIOLOGY: Interacting proteins, kinases and paradoxical regulation

    PubMed Central

    Roth, Bryan L

    2011-01-01

    5-hydroxytryptamine2A (5-HT2A) serotonin receptors are important pharmacological targets for a large number of central nervous system and peripheral serotonergic medications. In this review article I summarize work mainly from my lab regarding serotonin receptor anatomy, pharmacology, signaling and regulation. I highlight the role of serotonin receptor interacting proteins and the emerging paradigm of G-protein coupled receptor functional selectivity. PMID:21288474

  4. LIG Family Receptor Tyrosine Kinase-Associated Proteins Modulate Growth Factor Signals During Neural Development

    PubMed Central

    Mandai, Kenji; Guo, Ting; Hillaire, Coryse St.; Meabon, James S.; Kanning, Kevin C.; Bothwell, Mark; Ginty, David D.

    2009-01-01

    SUMMARY Genome-wide screens were performed to identify transmembrane proteins that mediate axonal growth, guidance and target field innervation of somatosensory neurons. One gene, Linx (alias Islr2), encoding a leucine-rich repeat and immunoglobulin (LIG) family protein, is expressed in a subset of developing sensory and motor neurons. Domain and genomic structures of Linx and other LIG family members suggest that they are evolutionarily related to Trk receptor tyrosine kinases (RTKs). Several LIGs, including Linx are expressed in subsets of somatosensory and motor neurons and select members interact with TrkA and Ret RTKs. Moreover, axonal projection defects in mice harboring a null mutation in Linx resemble those in mice lacking Ngf, TrkA and Ret. In addition, Linx modulates NGF–TrkA- and GDNF–GFRα1/Ret-mediated axonal extension in cultured sensory and motor neurons, respectively. These findings show that LIGs physically interact with RTKs and modulate their activities to control axonal extension, guidance and branching. PMID:19755105

  5. G-protein-coupled receptor kinase 2 and endothelial dysfunction: molecular insights and pathophysiological mechanisms

    PubMed Central

    Taguchi, Kumiko; Matsumoto, Takayuki; Kobayashi, Tsuneo

    2015-01-01

    Smooth muscle cells (SMC) and endothelial cells are the major cell types in blood vessels. The principal function of vascular SMC in the body is to regulate blood flow and pressure through contraction and relaxation. The endothelium performs a crucial role in maintaining vascular integrity by achieving whole-organ metabolic homeostasis via the production of factors associated with vasoconstriction or vasorelaxation. In this review, we have focused on the production of nitric oxide (NO), a vasorelaxation factor. The extent of NO production represents a key marker in vascular health. A decrease in NO is capable of inducing pathological conditions associated with endothelial dysfunction, such as obesity, diabetes, cardiovascular disease, and atherosclerosis. Recent studies have strongly implicated the involvement of G-protein-coupled receptor kinase 2 (GRK2) in the progression of cardiovascular disease. Vasculature which is affected by insulin resistance and type 2 diabetes expresses high levels of GRK2, which may induce endothelial dysfunction by reducing intracellular NO. GRK2 activation also induces changes in the subcellular localization of GRK2 itself and also of β-arrestin 2, a downstream protein. In this review, we describe the pathophysiological mechanisms of insulin resistance and diabetes, focusing on the signal transduction for NO production via GRK2 and β-arrestin 2, providing novel insights into the potential field of translational investigation in the treatment of diabetic complications. PMID:26447102

  6. G-Protein-Coupled Receptor Kinase 2 as a Potential Modulator of the Hallmarks of Cancer.

    PubMed

    Nogués, Laura; Reglero, Clara; Rivas, Verónica; Neves, María; Penela, Petronila; Mayor, Federico

    2017-03-01

    Malignant features-such as sustained proliferation, refractoriness to growth suppressors, resistance to cell death or aberrant motility, and metastasis-can be triggered by a variety of mutations and signaling adaptations. Signaling nodes can act as cancer-associated factors by cooperating with oncogene-governed pathways or participating in compensatory transduction networks to strengthen tumor properties. G-protein-coupled receptor kinase 2 (GRK2) is arising as one of such nodes. Via its complex network of connections with other cellular proteins, GRK2 contributes to the modulation of basic cellular functions-such as cell proliferation, survival, or motility-and is involved in metabolic homeostasis, inflammation, or angiogenic processes. Moreover, altered GRK2 levels are starting to be reported in different tumoral contexts and shown to promote breast tumorigenesis or to trigger the tumoral angiogenic switch. The ability to modulate several of the hallmarks of cancer puts forward GRK2 as an oncomodifier, able to modulate carcinogenesis in a cell-type specific way.

  7. G-protein-coupled receptor kinase 2 and endothelial dysfunction: molecular insights and pathophysiological mechanisms.

    PubMed

    Taguchi, Kumiko; Matsumoto, Takayuki; Kobayashi, Tsuneo

    2015-01-01

    Smooth muscle cells (SMC) and endothelial cells are the major cell types in blood vessels. The principal function of vascular SMC in the body is to regulate blood flow and pressure through contraction and relaxation. The endothelium performs a crucial role in maintaining vascular integrity by achieving whole-organ metabolic homeostasis via the production of factors associated with vasoconstriction or vasorelaxation. In this review, we have focused on the production of nitric oxide (NO), a vasorelaxation factor. The extent of NO production represents a key marker in vascular health. A decrease in NO is capable of inducing pathological conditions associated with endothelial dysfunction, such as obesity, diabetes, cardiovascular disease, and atherosclerosis. Recent studies have strongly implicated the involvement of G-protein-coupled receptor kinase 2 (GRK2) in the progression of cardiovascular disease. Vasculature which is affected by insulin resistance and type 2 diabetes expresses high levels of GRK2, which may induce endothelial dysfunction by reducing intracellular NO. GRK2 activation also induces changes in the subcellular localization of GRK2 itself and also of β-arrestin 2, a downstream protein. In this review, we describe the pathophysiological mechanisms of insulin resistance and diabetes, focusing on the signal transduction for NO production via GRK2 and β-arrestin 2, providing novel insights into the potential field of translational investigation in the treatment of diabetic complications.

  8. LIG family receptor tyrosine kinase-associated proteins modulate growth factor signals during neural development.

    PubMed

    Mandai, Kenji; Guo, Ting; St Hillaire, Coryse; Meabon, James S; Kanning, Kevin C; Bothwell, Mark; Ginty, David D

    2009-09-10

    Genome-wide screens were performed to identify transmembrane proteins that mediate axonal growth, guidance and target field innervation of somatosensory neurons. One gene, Linx (alias Islr2), encoding a leucine-rich repeat and immunoglobulin (LIG) family protein, is expressed in a subset of developing sensory and motor neurons. Domain and genomic structures of Linx and other LIG family members suggest that they are evolutionarily related to Trk receptor tyrosine kinases (RTKs). Several LIGs, including Linx, are expressed in subsets of somatosensory and motor neurons, and select members interact with TrkA and Ret RTKs. Moreover, axonal projection defects in mice harboring a null mutation in Linx resemble those in mice lacking Ngf, TrkA, and Ret. In addition, Linx modulates NGF-TrkA- and GDNF-GFRalpha1/Ret-mediated axonal extension in cultured sensory and motor neurons, respectively. These findings show that LIGs physically interact with RTKs and modulate their activities to control axonal extension, guidance and branching.

  9. Inhibition of T-cell antigen receptor-mediated transmembrane signaling by protein kinase C activation.

    PubMed Central

    Abraham, R T; Ho, S N; Barna, T J; Rusovick, K M; McKean, D J

    1988-01-01

    The murine T-lymphoma cell line LBRM-33 is known to require synergistic signals delivered through the antigen receptor (Ti-CD3) complex, together with interleukin 1 (IL-1), for activation of IL-2 gene expression and IL-2 production. Although 12-O-tetradecanoylphorbol-13-acetate (TPA) was capable of replacing IL-1 as an activating stimulus under certain conditions, biologic studies indicated that TPA failed to synergize with Ti-CD3-dependent stimuli under conditions in which IL-1 was clearly active. Acute exposure to TPA and other active phorbol esters resulted in a concentration-dependent inhibition of the increases in phosphoinositide hydrolysis and intracellular free Ca2+ concentration stimulated by phytohemagglutinin or anti-Ti antibodies. TPA treatment induced no direct alteration of phospholipase C enzymatic activities in LBRM-33 cells. In contrast, both Ti-CD3 cross-linkage and TPA rapidly stimulated the phosphorylation of identical CD3 complex polypeptides, presumably via activation of protein kinase C. Exposure of LBRM-33 cells to TPA resulted in a time-dependent, partial down-regulation of surface Ti-CD3 expression. Thus, TPA treatment inhibited the responsiveness of LBRM-33 cells to Ti-CD3-dependent stimuli by inducing an early desensitization of Ti-CD3 receptors, followed by a decrease in membrane receptor expression. These studies indicate that phorbol esters deliver bidirectional signals that both inhibit Ti-CD3-dependent phosphoinositide hydrolysis and augment IL-2 production in LBRM-33 cells. Images PMID:2977423

  10. p90 ribosomal S6 kinase 2 exerts a tonic brake on G protein-coupled receptor signaling.

    PubMed

    Sheffler, Douglas J; Kroeze, Wesley K; Garcia, Bonnie G; Deutch, Ariel Y; Hufeisen, Sandra J; Leahy, Patrick; Brüning, Jens C; Roth, Bryan L

    2006-03-21

    G protein-coupled receptors (GPCRs) are essential for normal central CNS function and represent the proximal site(s) of action for most neurotransmitters and many therapeutic drugs, including typical and atypical antipsychotic drugs. Similarly, protein kinases mediate many of the downstream actions for both ionotropic and metabotropic receptors. We report here that genetic deletion of p90 ribosomal S6 kinase 2 (RSK2) potentiates GPCR signaling. Initial studies of 5-hydroxytryptamine (5-HT)(2A) receptor signaling in fibroblasts obtained from RSK2 wild-type (+/+) and knockout (-/-) mice showed that 5-HT(2A) receptor-mediated phosphoinositide hydrolysis and both basal and 5-HT-stimulated extracellular signal-regulated kinase 1/2 phosphorylation are augmented in RSK2 knockout fibroblasts. Endogenous signaling by other GPCRs, including P2Y-purinergic, PAR-1-thrombinergic, beta1-adrenergic, and bradykinin-B receptors, was also potentiated in RSK2-deficient fibroblasts. Importantly, reintroduction of RSK2 into RSK2-/- fibroblasts normalized signaling, thus demonstrating that RSK2 apparently modulates GPCR signaling by exerting a "tonic brake" on GPCR signal transduction. Our results imply the existence of a novel pathway regulating GPCR signaling, modulated by downstream members of the extracellular signal-related kinase/mitogen-activated protein kinase cascade. The loss of RSK2 activity in humans leads to Coffin-Lowry syndrome, which is manifested by mental retardation, growth deficits, skeletal deformations, and psychosis. Because RSK2-inactivating mutations in humans lead to Coffin-Lowry syndrome, our results imply that alterations in GPCR signaling may account for some of its clinical manifestations.

  11. Contraction inhibits insulin-stimulated insulin receptor substrate-1/2-associated phosphoinositide 3-kinase activity, but not protein kinase B activation or glucose uptake, in rat muscle.

    PubMed Central

    Whitehead, J P; Soos, M A; Aslesen, R; O'rahilly, S; Jensen, J

    2000-01-01

    The initial stages of insulin-stimulated glucose uptake are thought to involve tyrosine phosphorylation of insulin receptor substrates (IRSs), which recruit and activate phosphoinositide 3-kinase (PI 3-kinase), leading to the activation of protein kinase B (PKB) and other downstream effectors. In contrast, contraction stimulates glucose uptake via a PI 3-kinase-independent mechanism. The combined effects of insulin and contraction on glucose uptake are additive. However, it has been reported that contraction causes a decrease in insulin-stimulated IRS-1-associated PI 3-kinase activity. To investigate this paradox, we have examined the effects of contraction on insulin-stimulated glucose uptake and proximal insulin-signalling events in isolated rat epitrochlearis muscle. Stimulation by insulin or contraction produced a 3-fold increase in glucose uptake, with the effects of simultaneous treatment by insulin and contraction being additive. Wortmannin completely blocked the additive effect of insulin in contracting skeletal muscle, indicating that this is a PI 3-kinase-dependent effect. Insulin-stimulated recruitment of PI 3-kinase to IRS-1 was unaffected by contraction; however, insulin produced no discernible increase in PI 3-kinase activity in IRS-1 or IRS-2 immunocomplexes in contracting skeletal muscle. Consistent with this, contraction inhibited insulin-stimulated p70(S6K) activation. In contrast, insulin-stimulated activation of PKB was unaffected by contraction. Thus, in contracting skeletal muscle, insulin stimulates glucose uptake and activates PKB, but not p70(S6K), by a PI 3-kinase-dependent mechanism that is independent of changes in IRS-1- and IRS-2-associated PI 3-kinase activity. PMID:10903138

  12. Bacterial chemoreceptor arrays are hexagonally packed trimers of receptor dimers networked by rings of kinase and coupling proteins

    PubMed Central

    Briegel, Ariane; Li, Xiaoxiao; Bilwes, Alexandrine M.; Hughes, Kelly T.; Jensen, Grant J.; Crane, Brian R.

    2012-01-01

    Chemoreceptor arrays are supramolecular transmembrane machines of unknown structure that allow bacteria to sense their surroundings and respond by chemotaxis. We have combined X-ray crystallography of purified proteins with electron cryotomography of native arrays inside cells to reveal the arrangement of the component transmembrane receptors, histidine kinases (CheA) and CheW coupling proteins. Trimers of receptor dimers lie at the vertices of a hexagonal lattice in a “two-facing-two” configuration surrounding a ring of alternating CheA regulatory domains (P5) and CheW couplers. Whereas the CheA kinase domains (P4) project downward below the ring, the CheA dimerization domains (P3) link neighboring rings to form an extended, stable array. This highly interconnected protein architecture underlies the remarkable sensitivity and cooperative nature of transmembrane signaling in bacterial chemotaxis. PMID:22355139

  13. Fps/Fes and Fer non-receptor protein-tyrosine kinases regulate collagen- and ADP-induced platelet aggregation.

    PubMed

    Senis, Y A; Sangrar, W; Zirngibl, R A; Craig, A W B; Lee, D H; Greer, P A

    2003-05-01

    Fps/Fes and Fer proto-oncoproteins are structurally related non-receptor protein-tyrosine kinases implicated in signaling downstream from cytokines, growth factors and immune receptors. We show that Fps/Fes and Fer are expressed in human and mouse platelets, and are activated following stimulation with collagen and collagen-related peptide (CRP), suggesting a role in GPVI receptor signaling. Fer was also activated following stimulation with thrombin and a protease-activated receptor4 (PAR4)-activating peptide, suggesting a role in signaling downstream from the G protein-coupled PAR4. There were no detectable perturbations in CRP-induced activation of Syk, PLCgamma2, cortactin, Erk, Jnk, Akt or p38 in platelets from mice lacking Fps/Fes, Fer, or both kinases. Platelets lacking Fps/Fes, from a targeted fps/fes null strain of mice, showed increased rates and amplitudes of collagen-induced aggregation, relative to wild-type platelets. P-Selectin expression was also elevated on the surface of Fps/Fes-null platelets in response to CRP. Fer-deficient platelets, from mice targeted with a kinase-inactivating mutation, disaggregated more rapidly than wild-type platelets in response to ADP. This report provides the first evidence that Fps/Fes and Fer are expressed in platelets and become activated downstream from the GPVI collagen receptor, and that Fer is activated downstream from a G-protein coupled receptor. Furthermore, using targeted mouse models we show that deficiency in Fps/Fes or Fer resulted in disregulated platelet aggregation and disaggregation, demonstrating a role for these kinases in regulating platelet functions.

  14. Role of receptor desensitization, phosphatase induction and intracellular cyclic AMP in the termination of mitogen-activated protein kinase activity in UTP-stimulated EAhy 926 endothelial cells.

    PubMed Central

    Graham, A; McLees, A; Malarkey, K; Gould, G W; Plevin, R

    1996-01-01

    We have investigated the mechanisms that bring about the termination of mitogen-activated protein kinase (MAP kinase) activation in response to UTP in EAhy 926 endothelial cells. UTP-stimulated MAP kinase activity was transient, returning to basal values by 60 min. At this time MAP kinase activation was desensitized; re-application of UTP did not further activate MAP kinase, full re-activation of MAP kinase being only apparent after a 1-2 h wash period. However, activation of MAP kinase by UTP could be sustained beyond 60 min by preincubation of the cells with the protein synthesis inhibitor cycloheximide. UTP also stimulated expression of MAP kinase phosphatase-1 and this was abolished after pretreatment with cycloheximide. Pretreatment of cells with forskolin abolished the initial activation of MAP kinase kinase or c-Raf-1 by UTP, but only affected MAP kinase activity during prolonged stimulation. The effect of forskolin on prolonged MAP kinase activation was also prevented by cycloheximide. These results suggest that the termination of MAP kinase activity in response to UTP involves a number of interacting mechanisms including receptor desensitization and the induction of a phosphatase. However, several pieces of evidence do not support a major role for MAP kinase phosphatase-1 in termination of the MAP kinase signal. Raising intracellular cyclic AMP may also be involved but only after an initial protein-synthesis step and by a mechanism that does not involve the inactivation of c-Raf-1 or MAP kinase kinase. PMID:8615830

  15. G Protein-Coupled Receptor Kinase 2 Promotes Flaviviridae Entry and Replication

    PubMed Central

    Le Sommer, Caroline; Barrows, Nicholas J.; Bradrick, Shelton S.; Pearson, James L.; Garcia-Blanco, Mariano A.

    2012-01-01

    Flaviviruses cause a wide range of severe diseases ranging from encephalitis to hemorrhagic fever. Discovery of host factors that regulate the fate of flaviviruses in infected cells could provide insight into the molecular mechanisms of infection and therefore facilitate the development of anti-flaviviral drugs. We performed genome-scale siRNA screens to discover human host factors required for yellow fever virus (YFV) propagation. Using a 2×2 siRNA pool screening format and a duplicate of the screen, we identified a high confidence list of YFV host factors. To find commonalities between flaviviruses, these candidates were compared to host factors previously identified for West Nile virus (WNV) and dengue virus (DENV). This comparison highlighted a potential requirement for the G protein-coupled receptor kinase family, GRKs, for flaviviral infection. The YFV host candidate GRK2 (also known as ADRBK1) was validated both in siRNA-mediated knockdown HuH-7 cells and in GRK−/− mouse embryonic fibroblasts. Additionally, we showed that GRK2 was required for efficient propagation of DENV and Hepatitis C virus (HCV) indicating that GRK2 requirement is conserved throughout the Flaviviridae. Finally, we found that GRK2 participates in multiple distinct steps of the flavivirus life cycle by promoting both entry and RNA synthesis. Together, our findings identified GRK2 as a novel regulator of flavivirus infection and suggest that inhibition of GRK2 function may constitute a new approach for treatment of flavivirus associated diseases. PMID:23029581

  16. Inhibition of Receptor Interacting Protein Kinases Attenuates Cardiomyocyte Hypertrophy Induced by Palmitic Acid.

    PubMed

    Zhao, Mingyue; Lu, Lihui; Lei, Song; Chai, Hua; Wu, Siyuan; Tang, Xiaoju; Bao, Qinxue; Chen, Li; Wu, Wenchao; Liu, Xiaojing

    2016-01-01

    Palmitic acid (PA) is known to cause cardiomyocyte dysfunction. Cardiac hypertrophy is one of the important pathological features of PA-induced lipotoxicity, but the mechanism by which PA induces cardiomyocyte hypertrophy is still unclear. Therefore, our study was to test whether necroptosis, a receptor interacting protein kinase 1 and 3 (RIPK1 and RIPK3-) dependent programmed necrosis, was involved in the PA-induced cardiomyocyte hypertrophy. We used the PA-treated primary neonatal rat cardiac myocytes (NCMs) or H9c2 cells to study lipotoxicity. Our results demonstrated that cardiomyocyte hypertrophy was induced by PA treatment, determined by upregulation of hypertrophic marker genes and cell surface area enlargement. Upon PA treatment, the expression of RIPK1 and RIPK3 was increased. Pretreatment with the RIPK1 inhibitor necrostatin-1 (Nec-1), the PA-induced cardiomyocyte hypertrophy, was attenuated. Knockdown of RIPK1 or RIPK3 by siRNA suppressed the PA-induced myocardial hypertrophy. Moreover, a crosstalk between necroptosis and endoplasmic reticulum (ER) stress was observed in PA-treated cardiomyocytes. Inhibition of RIPK1 with Nec-1, phosphorylation level of AKT (Ser473), and mTOR (Ser2481) was significantly reduced in PA-treated cardiomyocytes. In conclusion, RIPKs-dependent necroptosis might be crucial in PA-induced myocardial hypertrophy. Activation of mTOR may mediate the effect of necroptosis in cardiomyocyte hypertrophy induced by PA.

  17. Receptor-interacting protein kinase 3 promotes platelet activation and thrombosis.

    PubMed

    Zhang, Yiwen; Zhang, Jian; Yan, Rong; Tian, Jingluan; Zhang, Yang; Zhang, Jie; Chen, Mengxing; Cui, Qingya; Zhao, Lili; Hu, Renping; Jiang, Miao; Li, Zhenyu; Ruan, Changgeng; He, Sudan; Dai, Kesheng

    2017-03-14

    Previous studies have shown that receptor-interacting protein kinase 3 (RIP3) is involved in many important biological processes, including necroptosis, apoptosis, and inflammation. Here we show that RIP3 plays a critical role in regulating platelet functions and in vivo thrombosis and hemostasis. Tail bleeding times were significantly longer in RIP3-knockout (RIP3(-/-)) mice compared with their wild-type (WT) littermates. In an in vivo model of arteriole thrombosis, mice lacking RIP3 exhibited prolonged occlusion times. WT mice repopulated with RIP3(-/-) bone marrow-derived cells had longer occlusion times than RIP3(-/-) mice repopulated with WT bone marrow-derived cells, suggesting a role for RIP3-deficient platelets in arterial thrombosis. Consistent with these findings, we observed that RIP3 was expressed in both human and mice platelets. Deletion of RIP3 in mouse platelets caused a marked defect in aggregation and attenuated dense granule secretion in response to low doses of thrombin or a thromboxane A2 analog, U46619. Phosphorylation of Akt induced by U46619 or thrombin was diminished in RIP3(-/-) platelets. Moreover, RIP3 interacted with Gα13 Platelet spreading on fibrinogen and clot retraction were impaired in the absence of RIP3. RIP3 inhibitor dose-dependently inhibited platelet aggregation in vitro and prevented arterial thrombus formation in vivo. These data demonstrate a role for RIP3 in promoting in vivo thrombosis and hemostasis by amplifying platelet activation. RIP3 may represent a novel promising therapeutic target for thrombotic diseases.

  18. Agonist-Biased Signaling via Proteinase Activated Receptor-2: Differential Activation of Calcium and Mitogen-Activated Protein Kinase Pathways

    PubMed Central

    Ramachandran, Rithwik; Mihara, Koichiro; Mathur, Maneesh; Rochdi, Moulay Driss; Bouvier, Michel; DeFea, Kathryn

    2009-01-01

    We evaluated the ability of different trypsin-revealed tethered ligand (TL) sequences of rat proteinase-activated receptor 2 (rPAR2) and the corresponding soluble TL-derived agonist peptides to trigger agonist-biased signaling. To do so, we mutated the proteolytically revealed TL sequence of rPAR2 and examined the impact on stimulating intracellular calcium transients and mitogen-activated protein (MAP) kinase. The TL receptor mutants, rPAR2-Leu37Ser38, rPAR2-Ala37–38, and rPAR2-Ala39–42 were compared with the trypsin-revealed wild-type rPAR2 TL sequence, S37LIGRL42—. Upon trypsin activation, all constructs stimulated MAP kinase signaling, but only the wt-rPAR2 and rPAR2-Ala39–42 triggered calcium signaling. Furthermore, the TL-derived synthetic peptide SLAAAA-NH2 failed to cause PAR2-mediated calcium signaling but did activate MAP kinase, whereas SLIGRL-NH2 triggered both calcium and MAP kinase signaling by all receptors. The peptides AAIGRL-NH2 and LSIGRL-NH2 triggered neither calcium nor MAP kinase signals. Neither rPAR2-Ala37–38 nor rPAR2-Leu37Ser38 constructs recruited β-arrestins-1 or -2 in response to trypsin stimulation, whereas both β-arrestins were recruited to these mutants by SLIGRL-NH2. The lack of trypsin-triggered β-arrestin interactions correlated with impaired trypsin-activated TL-mutant receptor internalization. Trypsin-stimulated MAP kinase activation by the TL-mutated receptors was not blocked by inhibitors of Gαi (pertussis toxin), Gαq [N-cyclohexyl-1-(2,4-dichlorophenyl)-1,4-dihydro-6-methylindeno[1,2-c]pyrazole-3-carboxamide (GP2A)], Src kinase [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine (PP1)], or the epidermal growth factor (EGF) receptor [4-(3′-chloroanilino)-6,7-dimethoxy-quinazoline (AG1478)], but was inhibited by the Rho-kinase inhibitor (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 2HCl (Y27362). The data indicate that the proteolytically revealed TL sequence(s) and the mode

  19. Protein Kinase Cβ Modulates Ligand-induced Cell Surface Death Receptor Accumulation

    PubMed Central

    Meng, Xue Wei; Heldebrant, Michael P.; Flatten, Karen S.; Loegering, David A.; Dai, Haiming; Schneider, Paula A.; Gomez, Timothy S.; Peterson, Kevin L.; Trushin, Sergey A.; Hess, Allan D.; Smith, B. Douglas; Karp, Judith E.; Billadeau, Daniel D.; Kaufmann, Scott H.

    2010-01-01

    Although treatment with the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) is known to protect a subset of cells from induction of apoptosis by death ligands such as Fas ligand and tumor necrosis factor-α-related apoptosis-inducing ligand, the mechanism of this protection is unknown. This study demonstrated that protection in short term apoptosis assays and long term proliferation assays was maximal when Jurkat or HL-60 human leukemia cells were treated with 2–5 nm PMA. Immunoblotting demonstrated that multiple PKC isoforms, including PKCα, PKCβ, PKCϵ, and PKCθ, translocated from the cytosol to a membrane-bound fraction at these PMA concentrations. When the ability of short hairpin RNA (shRNA) constructs that specifically down-regulated each of these isoforms was examined, PKCβ shRNA uniquely reversed PMA-induced protection against cell death. The PKCβ-selective small molecule inhibitor enzastaurin had a similar effect. Although mass spectrometry suggested that Fas is phosphorylated on a number of serines and threonines, mutation of these sites individually or collectively had no effect on Fas-mediated death signaling or PMA protection. Further experiments demonstrated that PMA diminished ligand-induced cell surface accumulation of Fas and DR5, and PKCβ shRNA or enzastaurin reversed this effect. Moreover, enzastaurin sensitized a variety of human tumor cell lines and clinical acute myelogenous leukemia isolates, which express abundant PKCβ, to tumor necrosis factor-α related apoptosis-inducing ligand-induced death in the absence of PMA. Collectively, these results identify a specific PKC isoform that modulates death receptor-mediated cytotoxicity as well as a small molecule inhibitor that mitigates the inhibitory effects of PKC activation on ligand-induced death receptor trafficking and cell death. PMID:19887445

  20. Role of Non-receptor Protein Tyrosine Kinases During Phospholipase C-γ1 Related Uterine Contractions in the Rat

    PubMed Central

    Phillippe, Mark; Sweet, Leigh M.; Bradley, Diana F.; Engle, Daniel

    2011-01-01

    Activated phospholipase Cγ1 (PLC-γ1), produced in response to tyrosine phosphorylation, appears to play an important role during uterine contractions. These studies sought to determine which non-receptor protein tyrosine kinases (PTKs) are involved in the tyrosine phosphorylation and activation of PLC-γ1 in uterine tissue from the rat. In vitro uterine contraction studies were performed utilizing isoform specific PTK inhibitors. Western blots were performed utilizing antibodies to phosphotyrosine-PLC-γ1, total PLC-γ1, c-Src kinase and Lck kinase. Spontaneous, stretch-stimulated, and bpV(phen) (a tyrosine phosphatase inhibitor) enhanced uterine contractions were significantly suppressed in response to Damnacanthal (a Lck kinase inhibitor) and PP1 (a c-Src kinase inhibitor); whereas, several other PTK isoform inhibitors had no significant effect. Damnacanthal and PP1 also significantly suppressed bpV(phen)-enhanced tyrosine phosphorylation of PLC-γ1 compared to other PTK isoform inhibitors. Western blots confirmed expression of the Lck and c-Src kinases in uterine tissue. In conclusion, the Lck and c-Src kinases appear to play an important role in regulating tyrosine phosphorylation of PLC-γ1 and contractile activity in the rat uterus. PMID:19208792

  1. Molecular cloning of a putative receptor protein kinase gene encoded at the self-incompatibility locus of Brassica oleracea

    SciTech Connect

    Stein, J.C.; Howlett, B.; Boyes, D.C.; Nasrallah, M.E.; Nasrallah, J.B. )

    1991-10-01

    Self-recognition between pollen and stigma during pollination in Brassica oleracea is genetically controlled by the multiallelic self-incompatibility locus (S). The authors describe the S receptor kinase (SRK) gene, a previously uncharacterized gene that residues at the S locus. The nucleotide sequences of genomic DNA and of cDNAs corresponding to SRK predict a putative transmembrane receptor having serine/threonine-specific protein kinase activity. Its extracellular domain exhibits striking homology to the secreted product of the S-locus genotypes are highly polymorphic and have apparently evolved in unison with genetically linked alleles of SLG. SRK directs the synthesis of several alternative transcripts, which potentially encode different protein products, and these transcripts were detected exclusively in reproductive organs. The identification of SRK may provide new perspectives into the signal transduction mechanism underlying pollen recognition.

  2. Adrenal G protein-coupled receptor kinase-2 in regulation of sympathetic nervous system activity in heart failure.

    PubMed

    McCrink, Katie A; Brill, Ava; Lymperopoulos, Anastasios

    2015-09-26

    Heart failure (HF), the number one cause of death in the western world, is caused by the insufficient performance of the heart leading to tissue underperfusion in response to an injury or insult. It comprises complex interactions between important neurohormonal mechanisms that try but ultimately fail to sustain cardiac output. The most prominent such mechanism is the sympathetic (adrenergic) nervous system (SNS), whose activity and outflow are greatly elevated in HF. SNS hyperactivity confers significant toxicity to the failing heart and markedly increases HF morbidity and mortality via excessive activation of adrenergic receptors, which are G protein-coupled receptors. Thus, ligand binding induces their coupling to heterotrimeric G proteins that transduce intracellular signals. G protein signaling is turned-off by the agonist-bound receptor phosphorylation courtesy of G protein-coupled receptor kinases (GRKs), followed by βarrestin binding, which prevents the GRK-phosphorylated receptor from further interaction with the G proteins and simultaneously leads it inside the cell (receptor sequestration). Recent evidence indicates that adrenal GRK2 and βarrestins can regulate adrenal catecholamine secretion, thereby modulating SNS activity in HF. The present review gives an account of all these studies on adrenal GRKs and βarrestins in HF and discusses the exciting new therapeutic possibilities for chronic HF offered by targeting these proteins pharmacologically.

  3. High‐fat diet induces protein kinase A and G‐protein receptor kinase phosphorylation of β2‐adrenergic receptor and impairs cardiac adrenergic reserve in animal hearts

    PubMed Central

    Hu, Yuting; Wang, Qingtong; Liu, Yongming; Li, Ning; Xu, Bing; Kim, Sungjin; Chiamvimonvat, Nipavan

    2017-01-01

    Key points Patients with diabetes show a blunted cardiac inotropic response to β‐adrenergic stimulation despite normal cardiac contractile reserve.Acute insulin stimulation impairs β‐adrenergically induced contractile function in isolated cardiomyocytes and Langendorff‐perfused hearts.In this study, we aimed to examine the potential effects of hyperinsulinaemia associated with high‐fat diet (HFD) feeding on the cardiac β2‐adrenergic receptor signalling and the impacts on cardiac contractile function.We showed that 8 weeks of HFD feeding leads to reductions in cardiac functional reserve in response to β‐adrenergic stimulation without significant alteration of cardiac structure and function, which is associated with significant changes in β2‐adrenergic receptor phosphorylation at protein kinase A and G‐protein receptor kinase sites in the myocardium.The results suggest that clinical intervention might be applied to subjects in early diabetes without cardiac symptoms to prevent further cardiac complications. Abstract Patients with diabetes display reduced exercise capability and impaired cardiac contractile reserve in response to adrenergic stimulation. We have recently uncovered an insulin receptor and adrenergic receptor signal network in the heart. The aim of this study was to understand the impacts of high‐fat diet (HFD) on the insulin–adrenergic receptor signal network in hearts. After 8 weeks of HFD feeding, mice exhibited diabetes, with elevated insulin and glucose concentrations associated with body weight gain. Mice fed an HFD had normal cardiac structure and function. However, the HFD‐fed mice displayed a significant elevation of phosphorylation of the β2‐adrenergic receptor (β2AR) at both the protein kinase A site serine 261/262 and the G‐protein‐coupled receptor kinase site serine 355/356 and impaired adrenergic reserve when compared with mice fed on normal chow. Isolated myocytes from HFD‐fed mice also displayed a

  4. Regulation of oxytocin receptor responsiveness by G protein-coupled receptor kinase 6 in human myometrial smooth muscle.

    PubMed

    Willets, Jonathon M; Brighton, Paul J; Mistry, Rajendra; Morris, Gavin E; Konje, Justin C; Challiss, R A John

    2009-08-01

    Oxytocin plays an important role in the progression, timing, and modulation of uterine contraction during labor and is widely used as an uterotonic agent. We investigated the mechanisms regulating oxytocin receptor (OTR) signaling in human primary myometrial smooth muscle cells and the ULTR cell-line. Oxytocin produced concentration-dependent increases in both total [(3)H]inositol phosphate accumulation and intracellular Ca(2+) concentration ([Ca(2+)](i)); however, responses were greater and more reproducible in the ULTR cell line. Assessment of phospholipase C activity in single cells revealed that the OTR desensitizes rapidly (within 5 min) in the presence of oxytocin (100 nm). To characterize OTR desensitization further, cells were stimulated with a maximally effective concentration of oxytocin (100 nm, 30 sec) followed by a variable washout period and a second identical application of oxytocin. This brief exposure to oxytocin caused a marked decrease (>70%) in OTR responsiveness to rechallenge and was fully reversed by increasing the time period between agonist challenges. To assess involvement of G protein-coupled receptor kinases (GRKs) in OTR desensitization, cells were transfected with small interfering RNAs to cause specific > or =75% knockdown of GRKs 2, 3, 5, or 6. In both primary myometrial and ULTR cells, knockdown of GRK6 largely prevented oxytocin-induced OTR desensitization; in contrast, selective depletion of GRKs 2, 3, or 5 was without effect. These data indicate that GRK6 recruitment is a cardinal effector of OTR responsiveness and provide mechanistic insight into the likely in vivo regulation of OTR signaling in uterine smooth muscle.

  5. Insulin receptors mediate growth effects in cultured fetal neurons. II. Activation of a protein kinase that phosphorylates ribosomal protein S6

    SciTech Connect

    Heidenreich, K.A.; Toledo, S.P. )

    1989-09-01

    As an initial attempt to identify early steps in insulin action that may be involved in the growth responses of neurons to insulin, we investigated whether insulin receptor activation increases the phosphorylation of ribosomal protein S6 in cultured fetal neurons and whether activation of a protein kinase is involved in this process. When neurons were incubated for 2 h with 32Pi, the addition of insulin (100 ng/ml) for the final 30 min increased the incorporation of 32Pi into a 32K microsomal protein. The incorporation of 32Pi into the majority of other neuronal proteins was unaltered by the 30-min exposure to insulin. Cytosolic extracts from insulin-treated neurons incubated in the presence of exogenous rat liver 40S ribosomes and (gamma-32P)ATP displayed a 3- to 8-fold increase in the phosphorylation of ribosomal protein S6 compared to extracts from untreated cells. Inclusion of cycloheximide during exposure of the neurons to insulin did not inhibit the increased cytosolic kinase activity. Activation of S6 kinase activity by insulin was dose dependent (seen at insulin concentration as low as 0.1 ng/ml) and reached a maximum after 20 min of incubation. Addition of phosphatidylserine, diolein, and Ca2+ to the in vitro kinase reaction had no effect on the phosphorylation of ribosomal protein S6. Likewise, treatment of neurons with (Bu)2cAMP did not alter the phosphorylation of ribosomal protein S6 by neuronal cytosolic extracts. We conclude that insulin activates a cytosolic protein kinase that phosphorylates ribosomal S6 in neurons and is distinct from protein kinase-C and cAMP-dependent protein kinase. Stimulation of this kinase may play a role in insulin signal transduction in neurons.

  6. Nicotine enhances the cyclic AMP-dependent protein kinase-mediated phosphorylation of alpha4 subunits of neuronal nicotinic receptors.

    PubMed

    Hsu, Y N; Edwards, S C; Wecker, L

    1997-12-01

    Studies determined whether alpha4beta2 or alpha3beta2 neuronal nicotinic receptors expressed in Xenopus oocytes are substrates for cyclic AMP-dependent protein kinase (PKA) and whether nicotine affects receptor phosphorylation. The cRNAs for the subunits were coinjected into oocytes, and cells were incubated for 24 h in the absence or presence of nicotine (50 nM for alpha4beta2 and 500 nM for alpha3beta2 receptors). Nicotine did not interfere with the isolation of the receptors. When receptors isolated from oocytes expressing alpha4beta2 receptors were incubated with [gamma-32P]ATP and the catalytic subunit of PKA, separated by electrophoresis, and visualized by autoradiography, a labeled phosphoprotein with the predicted molecular size of the alpha4 subunit was present. Phosphorylation of alpha4 subunits of alpha4beta2 receptors increased within the first 5 min of incubation with nicotine and persisted for 24 h. In contrast, receptors isolated from oocytes expressing alpha3beta2 receptors did not exhibit a labeled phosphoprotein corresponding to the size of the alpha3 subunit. Results suggest that the PKA-mediated phosphorylation of alpha4 and not alpha3 subunits may explain the differential inactivation by nicotine of these receptor subtypes expressed in oocytes.

  7. 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

  8. Internalization and recycling of 5-HT2A receptors activated by serotonin and protein kinase C-mediated mechanisms

    PubMed Central

    Bhattacharyya, Samarjit; Puri, Sapna; Miledi, Ricardo; Panicker, Mitradas M.

    2002-01-01

    Serotonin (5-HT), a major neurotransmitter, has a large number of G protein-coupled receptors in mammals. On activation by exposure to their ligand, 5-HT2 receptor subtypes increase IP3 levels and undergo desensitization and internalization. To visualize the receptor in cells during these processes, we have constructed a 5-HT2A-enhanced GFP (SR2-GFP) fusion receptor. We show that this fusion receptor undergoes internalization on exposure to its natural ligand, 5-HT. Because 5-HT2A receptors activate the phospholipase C pathway, we studied the effect of protein kinase C (PKC) on the internalization process and found that activation of PKC by its specific activator phorbol 12-myristate 13-acetate, in the absence of 5-HT, leads to internalization of the receptor. Moreover, inhibition of PKC by its inhibitor sphingosine in the presence of 5-HT prevents the internalization process, suggesting that activation of PKC is sufficient and necessary for the internalization of 5-HT2A receptors. We also show that SR2-GFP recycles back to the plasma membrane after 5-HT-dependent internalization, suggesting a mechanism for resensitization. In addition, receptors that have been internalized on addition of phorbol 12-myristate 13-acetate in the absence of 5-HT also recycle to the surface, with a time course similar to that seen after activation of the receptors by 5-HT. Our study suggests that 5-HT2A receptors internalize and return to the surface after both serotonin- and PKC-mediated processes. This study reveals a role for PKC in receptor internalization and also shows that 5-HT2A receptors are recycled. PMID:12388782

  9. Differential Role of G Protein-Coupled Receptor Kinase 5 in Physiological Versus Pathological Cardiac Hypertrophy

    PubMed Central

    Traynham, Christopher J.; Cannavo, Alessandro; Zhou, Yan; Vouga, Alexandre G.; Woodall, Benjamin P.; Hullmann, Jonathan; Ibetti, Jessica; Gold, Jessica I.; Chuprun, J. Kurt; Gao, Erhe; Koch, Walter J.

    2015-01-01

    Rationale G protein-coupled receptor (GPCR) kinases (GRKs) are dynamic regulators of cellular signaling. GRK5 is highly expressed within myocardium and is up-regulated in heart failure (HF). Although GRK5 is a critical regulator of cardiac GPCR signaling, recent data has uncovered non-canonical activity of GRK5 within nuclei that plays a key role in pathological hypertrophy. Targeted cardiac elevation of GRK5 in mice leads to exaggerated hypertrophy and early HF after transverse aortic constriction (TAC) due to GRK5 nuclear accumulation. Objective In this study we investigated the role of GRK5 in physiological, swimming induced hypertrophy (SIH). Methods and Results Cardiac-specific GRK5 transgenic mice (TgGRK5) and non-transgenic littermate control (NLC) mice were subjected to a 21-day high intensity swim protocol (or no swim sham controls). SIH and specific molecular and genetic indices of physiological hypertrophy were assessed including nuclear localization of GRK5 and compared to TAC. Unlike after TAC, swim-trained TgGRK5 and NLC mice exhibited similar increases in cardiac growth. Mechanistically, SIH did not lead to GRK5 nuclear accumulation, which was confirmed in vitro as insulin-like growth factor-1, a known mediator of physiological hypertrophy, was unable to induce GRK5 nuclear translocation in myocytes. We found specific patterns of altered gene expression between TAC and SIH with GRK5 overexpression. Further, SIH in post-TAC TgGRK5 mice was able to preserve cardiac function. Conclusions These data suggest that while nuclear-localized GRK5 is a pathological mediator after stress, this non-canonical nuclear activity of GRK5 is not induced during physiological hypertrophy. PMID:26515328

  10. Development of natural product-derived receptor tyrosine kinase inhibitors based on conservation of protein domain fold.

    PubMed

    Kissau, Lars; Stahl, Petra; Mazitschek, Ralph; Giannis, Athannasios; Waldmann, Herbert

    2003-07-03

    Receptor tyrosine kinases (RTKs) such as Tie-2, IGF1R, Her-2/Neu, EGFR, and VEGFR1-3 play crucial roles in the control of cell growth and differentiation. Inhibition of such RTKs has become a major focus of current anticancer drug development, and therefore the discovery of new classes of inhibitors for these signal-transducing proteins is of prime importance. We have recently proposed a novel concept for improving the hit-finding process by employing natural products as biologically validated starting points in structural space for compound library development. In this concept, natural products are regarded as evolutionary chosen ligands for protein domains which are structurally conserved yet genetically mobile. Here we report on the discovery of novel and highly selective VEGFR-2 and -3, Tie-2, and IGF1R inhibitors derived from the naturally occurring Her-2/Neu kinase inhibitor nakijiquinone C and developed on the basis of this concept. Based on the structure of the natural product, a small library (74 members) was synthesized and investigated for inhibition of kinases with highly similar ATP-binding domains. The library yielded inhibitors with IC(50)s in the low micromolar range with high frequency (7 out of 74). In particular, four inhibitors of Tie-2 were found, a kinase critically involved in the formation of new blood vessels from preexisting ones (angiogenesis) and believed to be a new promising target in antitumor therapy. These results support the "domain concept". To advance the development of improved inhibitors, extensive molecular modeling studies were undertaken, including the construction of new homology models for VEGFR-2 and Tie-2. These studies revealed residues in the kinase structure which are crucial to the development of tailor-made receptor tyrosine kinase inhibitors.

  11. E6-Associated Protein Dependent Estrogen Receptor Regulation of Protein Kinase A Regulatory Subunit R2A Expression in Neuroblastoma.

    PubMed

    Obeid, Jean-Pierre; Zeidan, Youssef H; Zafar, Nawal; El Hokayem, Jimmy

    2017-02-18

    E6ap is a known transcriptional coregulator for estrogen receptor alpha (Er, Erα) in the presence of estrogen. Protein kinase A (PKA) contains two regulatory subunits derived from four genes. Recent evidence demonstrates that PKA regulates E6ap activity. Data generated in our lab indicated estrogen dependent regulation of Pkar2a levels. Our project sets to investigate a possible feedback mechanism constituting of Erα and E6ap transcriptional regulation of Pkar2a expression. Western blot evaluated protein regulation correlations with E2 in mouse neuroblastoma lines. Bioinformatics detected estrogen response element (ERE) sequences. quantitative polymerase chain reaction (qPCR) validated the western blot results. ERE oligonucleotides were synthesized. Reporter gene transcriptional activity was evaluated via Luciferase assay output. Electromobility shift assay (EMSA) assessed direct binding between Erα relevant sequences. Chromatin immunoprecipitation (ChIP) and Re-ChIP were conducted in quantifying protein complex recruitment levels. Pkar2a protein expression directly correlated with E2, and four putative ERE sequences were identified. Pkar2a mRNA expression reverted to baseline with either E2 or E6ap absent. In the presence of E2, ERE-1 and ERE-4 possessed Luciferase reporter gene transcriptional capabilities. ERE-1 portrayed band shifts, representing direct binding to Erα with E2 supplementation. With E2, ERE-1 significantly enhanced Erα and E6ap recruitment levels to the Pkar2a promoter. Pkar2a is directly regulated by Erα and E6ap in the presence of estrogen stimulus. This work indicates a feedback mechanism in the interplay between PKA and E6ap, which may prove crucial for the role of both proteins in cancers and neurogenetic diseases like Angelman syndrome.

  12. Teaching resources. Protein kinases.

    PubMed

    Caplan, Avrom

    2005-02-22

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

  13. SmShb, the SH2-Containing Adaptor Protein B of Schistosoma mansoni Regulates Venus Kinase Receptor Signaling Pathways.

    PubMed

    Morel, Marion; Vanderstraete, Mathieu; Cailliau, Katia; Hahnel, Steffen; Grevelding, Christoph G; Dissous, Colette

    2016-01-01

    Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (RTKs) formed by an extracellular Venus Fly Trap (VFT) ligand binding domain associated via a transmembrane domain with an intracellular tyrosine kinase (TK) domain. Schistosoma mansoni VKRs, SmVKR1 and SmVKR2, are both implicated in reproductive activities of the parasite. In this work, we show that the SH2 domain-containing protein SmShb is a partner of the phosphorylated form of SmVKR1. Expression of these proteins in Xenopus oocytes allowed us to demonstrate that the SH2 domain of SmShb interacts with the phosphotyrosine residue (pY979) located in the juxtamembrane region of SmVKR1. This interaction leads to phosphorylation of SmShb on tyrosines and promotes SmVKR1 signaling towards the JNK pathway. SmShb transcripts are expressed in all parasite stages and they were found in ovary and testes of adult worms, suggesting a possible colocalization of SmShb and SmVKR1 proteins. Silencing of SmShb in adult S. mansoni resulted in an accumulation of mature sperm in testes, indicating a possible role of SmShb in gametogenesis.

  14. SmShb, the SH2-Containing Adaptor Protein B of Schistosoma mansoni Regulates Venus Kinase Receptor Signaling Pathways

    PubMed Central

    Morel, Marion; Vanderstraete, Mathieu; Cailliau, Katia; Hahnel, Steffen; Grevelding, Christoph G.; Dissous, Colette

    2016-01-01

    Venus kinase receptors (VKRs) are invertebrate receptor tyrosine kinases (RTKs) formed by an extracellular Venus Fly Trap (VFT) ligand binding domain associated via a transmembrane domain with an intracellular tyrosine kinase (TK) domain. Schistosoma mansoni VKRs, SmVKR1 and SmVKR2, are both implicated in reproductive activities of the parasite. In this work, we show that the SH2 domain-containing protein SmShb is a partner of the phosphorylated form of SmVKR1. Expression of these proteins in Xenopus oocytes allowed us to demonstrate that the SH2 domain of SmShb interacts with the phosphotyrosine residue (pY979) located in the juxtamembrane region of SmVKR1. This interaction leads to phosphorylation of SmShb on tyrosines and promotes SmVKR1 signaling towards the JNK pathway. SmShb transcripts are expressed in all parasite stages and they were found in ovary and testes of adult worms, suggesting a possible colocalization of SmShb and SmVKR1 proteins. Silencing of SmShb in adult S. mansoni resulted in an accumulation of mature sperm in testes, indicating a possible role of SmShb in gametogenesis. PMID:27636711

  15. Ror receptor tyrosine kinases: orphans no more

    PubMed Central

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

    2015-01-01

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

  16. G Protein-coupled Receptor Kinase-mediated Phosphorylation Regulates Post-endocytic Trafficking of the D2 Dopamine Receptor*S⃞

    PubMed Central

    Namkung, Yoon; Dipace, Concetta; Javitch, Jonathan A.; Sibley, David R.

    2009-01-01

    We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D2 dopamine receptor (DAR). Agonist activation of D2 DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D2 DAR phosphorylation. Mutational analyses resulted in the identification of eight serine/threonine residues within the third cytoplasmic loop of the receptor that are phosphorylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(-), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(-) receptors underwent a similar degree of agonist-induced desensitization as assessed using [35S]GTPγS binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using bioluminescence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(-) receptors. Thus, phosphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the D2 DARs to the cell surface, subsequent to agonist-induced endocytosis, the GRK(-) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(-) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphorylated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor. PMID:19332542

  17. Phosphorylation of the Drosophila transient receptor potential ion channel is regulated by the phototransduction cascade and involves several protein kinases and phosphatases.

    PubMed

    Voolstra, Olaf; Bartels, Jonas-Peter; Oberegelsbacher, Claudia; Pfannstiel, Jens; Huber, Armin

    2013-01-01

    Protein phosphorylation plays a cardinal role in regulating cellular processes in eukaryotes. Phosphorylation of proteins is controlled by protein kinases and phosphatases. We previously reported the light-dependent phosphorylation of the Drosophila transient receptor potential (TRP) ion channel at multiple sites. TRP generates the receptor potential upon stimulation of the photoreceptor cell by light. An eye-enriched protein kinase C (eye-PKC) has been implicated in the phosphorylation of TRP by in vitro studies. Other kinases and phosphatases of TRP are elusive. Using phosphospecific antibodies and mass spectrometry, we here show that phosphorylation of most TRP sites depends on the phototransduction cascade and the activity of the TRP ion channel. A candidate screen to identify kinases and phosphatases provided in vivo evidence for an involvement of eye-PKC as well as other kinases and phosphatases in TRP phosphorylation.

  18. Alpha7-nicotinic acetylcholine receptors affect growth regulation of human mesothelioma cells: role of mitogen-activated protein kinase pathway.

    PubMed

    Trombino, Sonya; Cesario, Alfredo; Margaritora, Stefano; Granone, PierLuigi; Motta, Giovanni; Falugi, Carla; Russo, Patrizia

    2004-01-01

    This study presents data suggesting that both human mesothelioma (cell lines and human mesothelioma biopsies) and human normal mesothelial cells express receptors for acetylcholine and that stimulation of these receptors by nicotine prompted cell growth via activation of nicotinic cholinergic receptors. Thus, these data demonstrate that: (a) human mesothelioma cells and human biopsies of mesothelioma as well as of normal pleural mesothelial cells express functionally alpha-7 nicotinic acethlycholine receptors, evaluated by alpha-bungarotoxin-FITC binding, receptor binding assay, Western blot, and reverse transcription-PCR; (b) choline acetyltransferase immunostaining is present in mesothelioma cells; (c) mesothelioma cell growth is modulated by the cholinergic system in which agonists (i.e., nicotine) has a proliferative effect, and antagonists (i.e., curare) has an inhibitory effect, evaluated by cell cloning, DNA synthesis and cell cycle; (d) nicotine induces Ca(+2) influx, evaluated by [(45)Ca(2+)] uptake, and consequently activation of mitogen-activated protein kinase pathway (extracellular signal-regulated kinase and p90(RSK) phosphorylation), evaluated by Western blot; and (e) apoptosis mechanisms in mesothelioma cells are under the control of the cholinergic system (nicotine antiapoptotic via induction of nuclear factor-kappaB complexes and phosphorylation of Bad at Ser(112); curare proapoptotic via G(0)-G(1) arrest p21(waf-1) dependent but p53 independent). The involvement of the nonneuronal cholinergic system in mesothelioma appears reasonable and open up new therapeutic strategies.

  19. 5-HT1A receptor-mediated phosphorylation of extracellular signal-regulated kinases (ERK1/2) is modulated by regulator of G protein signaling protein 19.

    PubMed

    Wang, Qin; Terauchi, Akiko; Yee, Christopher H; Umemori, Hisashi; Traynor, John R

    2014-09-01

    The 5-HT1A receptor is a G protein coupled receptor (GPCR) that activates G proteins of the Gαi/o family. 5-HT1A receptors expressed in the raphe, hippocampus and prefrontal cortex are implicated in the control of mood and are targets for anti-depressant drugs. Regulators of G protein signaling (RGS) proteins are members of a large family that play important roles in signal transduction downstream of G protein coupled receptors (GPCRs). The main role of RGS proteins is to act as GTPase accelerating proteins (GAPs) to dampen or negatively regulate GPCR-mediated signaling. We have shown that a mouse expressing Gαi2 that is insensitive to all RGS protein GAP activity has an anti-depressant-like phenotype due to increased signaling of postsynaptic 5-HT1A receptors, thus implicating the 5-HT1A receptor-Gαi2 complex as an important target. Here we confirm that RGS proteins act as GAPs to regulate signaling to adenylate cyclase and the mitogen-activated protein kinase (MAPK) pathway downstream of the 5-HT1A receptor, using RGS-insensitive Gαi2 protein expressed in C6 cells. We go on to use short hairpin RNA (shRNA) to show that RGS19 is responsible for the GAP activity in C6 cells and also that RGS19 acts as a GAP for 5-HT1A receptor signaling in human neuroblastoma SH-SY5Y cells and primary hippocampal neurons. In addition, in both cell types the synergy between 5-HT1A receptor and the fibroblast growth factor receptor 1 in stimulating the MAPK pathway is enhanced following shRNA reduction of RGS19 expression. Thus RGS19 may be a viable new target for anti-depressant medications.

  20. Activation of the protein-tyrosine kinase associated with the bombesin receptor complex in small cell lung carcinomas.

    PubMed Central

    Gaudino, G; Cirillo, D; Naldini, L; Rossino, P; Comoglio, P M

    1988-01-01

    It has been hypothesized that bombesin-like peptides produced by small cell lung carcinomas may sustain deregulated proliferation through an autocrine mechanism. We have shown that the neuropeptide bombesin leads to the activation of a protein-tyrosine kinase that phosphorylates a 115-kDa protein (p115) associated with the bombesin receptor complex in mouse Swiss 3T3 fibroblasts. We now report that phosphotyrosine antibodies recognize a 115-kDa protein, phosphorylated on tyrosine, in four human small cell lung carcinoma cell lines producing bombesin but not in a nonproducer "variant" line. p115 from detergent-treated small cell lung carcinoma cells binds to bombesin-Sepharose and can be phosphorylated on tyrosine in the presence of radiolabeled ATP and Mn2+. As for the p115 immunoprecipitated from mouse fibroblast, the small cell lung carcinoma p115 can be phosphorylated in an immunocomplex kinase assay. However, the latter does not require the presence of exogenous bombesin for activity. Binding data, obtained by using radiolabeled ligand, suggest receptor occupancy in the cell lines producing bombesin. These observations are consistent with the hypothesis that proliferation in some human small cell lung carcinoma lines is under autocrine control, regulated through activation of bombesin receptors. Images PMID:2451242

  1. Characterization of a putative S-locus encoded receptor protein kinase and its role in self-incompatibility. Progress report

    SciTech Connect

    Nasrallah, J.B.

    1994-05-01

    The major results of our research effort include the determination of the S-Receptor Kinase (SRK) gene structure, the demonstration of S-haplotype-associated SRK polymorphisms and possible co-evolution of SRK and SLG, the characterization of the temporal and spatial expression patterns of SRK, and the demonstration that SRK has intrinsic serine/threonine kinase activity. Our results have indicated that SLG originated from an SRK-like gene by a gene duplication event and suggested a possible molecular basis for leaky S haplotypes. The data have allowed us to develop a model of self-incompatibility based on the interaction of SRK and SLG and the activation of SRK in response to self-pollination. More generally, the information that we have obtained is potentially relevant to understanding mechanisms of signalling inplants. Thus, the interaction of membrane-based receptor protein kinases with secreted forms of their extracellular domains may represent a generalized mechanism by which receptors signal across the plant cell wall.

  2. Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes

    PubMed Central

    Tamás, Peter; Hawley, Simon A.; Clarke, Rosemary G.; Mustard, Kirsty J.; Green, Kevin; Hardie, D. Grahame; Cantrell, Doreen A.

    2006-01-01

    The adenosine monophosphate (AMP)–activated protein kinase (AMPK) has a crucial role in maintaining cellular energy homeostasis. This study shows that human and mouse T lymphocytes express AMPKα1 and that this is rapidly activated in response to triggering of the T cell antigen receptor (TCR). TCR stimulation of AMPK was dependent on the adaptors LAT and SLP76 and could be mimicked by the elevation of intracellular Ca2+ with Ca2+ ionophores or thapsigargin. AMPK activation was also induced by energy stress and depletion of cellular adenosine triphosphate (ATP). However, TCR and Ca2+ stimulation of AMPK required the activity of Ca2+–calmodulin-dependent protein kinase kinases (CaMKKs), whereas AMPK activation induced by increased AMP/ATP ratios did not. These experiments reveal two distinct pathways for the regulation of AMPK in T lymphocytes. The role of AMPK is to promote ATP conservation and production. The rapid activation of AMPK in response to Ca2+ signaling in T lymphocytes thus reveals that TCR triggering is linked to an evolutionally conserved serine kinase that regulates energy metabolism. Moreover, AMPK does not just react to cellular energy depletion but also anticipates it. PMID:16818670

  3. Concerted transcriptional activation of the low density lipoprotein receptor gene by insulin and luteinizing hormone in cultured porcine granulosa-luteal cells: possible convergence of protein kinase a, phosphatidylinositol 3-kinase, and mitogen-activated protein kinase signaling pathways.

    PubMed

    Sekar, N; Veldhuis, J D

    2001-07-01

    -repressive region in this gene. Non-LH receptor-dependent agonists of protein kinase A (PKA), 8-bromo-cAMP (1 mM), and forskolin (10 microM) with or without insulin/IGF-I costimulation likewise augmented LDL receptor promoter expression with similar strong dependency on the -255 to -139 bp 5'-upstream region. To assess more specific PKA-dependent mediation of LH's contribution to combined hormonal drive, the LDL receptor (-1076 to +11 bp) reporter plasmid was cotransfected with a full-sequence rabbit muscle protein kinase inhibitor (PKI) minigene driven constitutively by a Rous sarcoma virus promoter. Expression of the latter PKA antagonist blocked transcriptional stimulation by LH alone as well as that by LH combined with insulin (or IGF-I) by 70-85% without reducing basal transcriptional activity. Transfection of a mutant inactive (Arg to Gly) Rous sarcoma virus/PKI gene confirmed the specificity of the PKI effect. To investigate the convergent role of the insulin/IGF-I effector pathway mediating bihormonal stimulation of LDL receptor promoter expression, transfected granulosa-luteal cells were pretreated for 30 min with two specific inhibitors of phophatidylinositol 3-kinase, wortmannin (100 nM) and LY 294002 (10 microM), or of mitogen-activated protein kinase kinase, PD 98059 (50 microM), U0126 (10 microM), or the latter's inactive derivative, U0124 (10 microM). Both classes of antagonists impeded the ability of insulin or IGF-I to enhance LH-stimulated LDL receptor promoter expression by 60-80%. In conclusion, the present analyses indicate that LH and insulin (or IGF-I) can up-regulate LDL receptor transcriptional activity supraadditively in porcine granulosa-luteal cells 1) via one or more agonistic cis-acting DNA regions located between -255 and -139 bp 5'- upstream of the transcriptional start site, 2) without abrogating sterol-sensitive repressive of this promoter, and 3) by way of intracellular mechanisms that include the PKA, phophatidylinositol 3-kinase, and mitogen

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

    PubMed Central

    Shiu, Shin-Han; Bleecker, Anthony B.

    2001-01-01

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

  5. Activation of lysophosphatidic acid receptor by gintonin inhibits Kv1.2 channel activity: involvement of tyrosine kinase and receptor protein tyrosine phosphatase α.

    PubMed

    Lee, Jun-Ho; Choi, Sun-Hye; Lee, Byung-Hwan; Hwang, Sung-Hee; Kim, Hyeon-Joong; Rhee, Jeehae; Chung, Chihye; Nah, Seung-Yeol

    2013-08-26

    Gintonin is a novel ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand. The primary action of gintonin is to elicit a transient increase in [Ca(2+)]i via activation of LPA receptor subtypes. Voltage-gated potassium (Kv) channels play important roles in synaptic transmission in nervous systems. The previous reports have shown that Kv channels can be regulated by Gαq/11 protein-coupled receptor ligands. In the present study, we examined the effects of gintonin on Kv1.2 channel activity expressed in Xenopus oocytes after injection of RNA encoding the human Kv1.2 α subunit. Gintonin treatment inhibited Kv1.2 channel activity in reversible and concentration-dependent manners. The inhibitory effect of gintonin on Kv1.2 channel activity was blocked by active phospholipase C inhibitor, inositol 1,4,5-triphosphate receptor antagonist, and intracellular Ca(2+) chelator. The co-expression of active receptor protein tyrosine phosphatase α (RPTPα) with Kv1.2 channel greatly attenuated gintonin-mediated inhibition of Kv1.2 channel activity, but attenuation was not observed with catalytically inactive RPTPα. Furthermore, neither genistein, a tyrosine kinase inhibitor, nor site-directed mutation of a tyrosine residue (Y132 to Y132F), which is phosphorylated by tyrosine kinase of the N-terminal of the Kv1.2 channel α subunit, significantly attenuated gintonin-mediated inhibition of Kv1.2 channel activity. These results indicate that the gintonin-mediated Kv1.2 channel regulation involves the dual coordination of both tyrosine kinase and RPTPα coupled to this receptor. Finally, gintonin-mediated regulation of Kv1.2 channel activity might explain one of the modulations of gintonin-mediated neuronal activities in nervous systems.

  6. Nucleotide binding by the epidermal growth factor receptor protein-tyrosine kinase. Trinitrophenyl-ATP as a spectroscopic probe.

    PubMed

    Cheng, K; Koland, J G

    1996-01-05

    The nucleotide binding properties of the epidermal growth factor (EGF) receptor protein-tyrosine kinase were investigated with the fluorescent nucleotide analog 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate (TNP-ATP). TNP-ATP was found to be an active substrate for the autophosphorylation reaction of the recombinant EGF receptor protein-tyrosine kinase domain (TKD). Whereas the Vmax for the TNP-ATP-dependent autophosphorylation reaction was approximately 200-fold lower than that of ATP, the Km for this reaction was similar to that observed with ATP. The nucleotide analog was also shown to be an inhibitor of the ATP-dependent autophosphorylation and substrate phosphorylation reactions of the TKD. Spectroscopic studies demonstrated both a high affinity binding of TNP-ATP to the recombinant TKD and a markedly enhanced fluorescence of the bound nucleotide analog. The fluorescence of enzyme-bound TNP-ATP was attenuated in the presence of ATP, which enabled determination of the dissociation constants for both ATP and the Mn2+ complex of ATP. A truncated form of the EGF receptor TKD lacking the C-terminal autophosphorylation domain exhibited an enhanced affinity for TNP-ATP, which indicated that the autophosphorylation domain occupied the peptide substrate binding site of the TKD and modulated the binding of the nucleotide substrates.

  7. Sodium depletion enhances renal expression of (pro)renin receptor via cyclic GMP-protein kinase G signaling pathway.

    PubMed

    Huang, Jiqian; Siragy, Helmy M

    2012-02-01

    (Pro)renin receptor (PRR) is expressed in renal vasculature, glomeruli, and tubules. The physiological regulation of this receptor is not well established. We hypothesized that sodium depletion increases PRR expression through cGMP- protein kinase G (PKG) signaling pathway. Renal PRR expressions were evaluated in Sprague-Dawley rats on normal sodium or low-sodium diet (LS) and in cultured rat proximal tubular cells and mouse renal inner medullary collecting duct cells exposed to LS concentration. LS augmented PRR expression in renal glomeruli, proximal tubules, distal tubules, and collecting ducts. LS also increased cGMP production and PKG activity. In cells exposed to normal sodium, cGMP analog increased PKG activity and upregulated PRR expression. In cells exposed to LS, blockade of guanylyl cyclase with 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one decreased PKG activity and downregulated PRR expression. PKG inhibition decreased phosphatase protein phosphatase 2A activity; suppressed LS-mediated phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, c-Jun, and nuclear factor-κB p65; and attenuated LS-mediated PRR upregulation. LS also enhanced DNA binding of cAMP response element binding protein 1 to cAMP response elements, nuclear factor-κB p65 to nuclear factor-κB elements, and c-Jun to activator protein 1 elements in PRR promoter in proximal tubular cells. We conclude that sodium depletion upregulates renal PRR expression via the cGMP-PKG signaling pathway by enhancing binding of cAMP response element binding protein 1, nuclear factor-κB p65, and c-Jun to PRR promotor.

  8. A cell-free method for expressing and reconstituting membrane proteins enables functional characterization of the plant receptor-like protein kinase FERONIA.

    PubMed

    Minkoff, Benjamin B; Makino, Shin-Ichi; Haruta, Miyoshi; Beebe, Emily T; Wrobel, Russell L; Fox, Brian G; Sussman, Michael R

    2017-04-07

    There are more than 600 receptor-like kinases (RLKs) in Arabidopsis, but due to challenges associated with the characterization of membrane proteins, only a few have known biological functions. The plant RLK FERONIA is a peptide receptor and has been implicated in plant growth regulation, but little is known about its molecular mechanism of action. To investigate the properties of this enzyme, we used a cell-free wheat germ-based expression system in which mRNA encoding FERONIA was co-expressed with mRNA encoding the membrane scaffold protein variant MSP1D1. With the addition of the lipid cardiolipin, assembly of these proteins into nanodiscs was initiated. FERONIA protein kinase activity in nanodiscs was higher than that of soluble protein and comparable with other heterologously expressed protein kinases. Truncation experiments revealed that the cytoplasmic juxtamembrane domain is necessary for maximal FERONIA activity, whereas the transmembrane domain is inhibitory. An ATP analogue that reacts with lysine residues inhibited catalytic activity and labeled four lysines; mutagenesis demonstrated that two of these, Lys-565 and Lys-663, coordinate ATP in the active site. Mass spectrometric phosphoproteomic measurements further identified phosphorylation sites that were examined using phosphomimetic mutagenesis. The results of these experiments are consistent with a model in which kinase-mediated phosphorylation within the C-terminal region is inhibitory and regulates catalytic activity. These data represent a step further toward understanding the molecular basis for the protein kinase catalytic activity of FERONIA and show promise for future characterization of eukaryotic membrane proteins.

  9. The octopamine receptor OAMB mediates ovulation via Ca2+/calmodulin-dependent protein kinase II in the Drosophila oviduct epithelium.

    PubMed

    Lee, Hyun-Gwan; Rohila, Suman; Han, Kyung-An

    2009-01-01

    Ovulation is an essential physiological process in sexual reproduction; however, the underlying cellular mechanisms are poorly understood. We have previously shown that OAMB, a Drosophila G-protein-coupled receptor for octopamine (the insect counterpart of mammalian norepinephrine), is required for ovulation induced upon mating. OAMB is expressed in the nervous and reproductive systems and has two isoforms (OAMB-AS and OAMB-K3) with distinct capacities to increase intracellular Ca2+ or intracellular Ca2+ and cAMP in vitro. Here, we investigated tissue specificity and intracellular signals required for OAMB's function in ovulation. Restricted OAMB expression in the adult oviduct epithelium, but not the nervous system, reinstated ovulation in oamb mutant females, in which either OAMB isoform was sufficient for the rescue. Consistently, strong immunoreactivities for both isoforms were observed in the wild-type oviduct epithelium. To delineate the cellular mechanism by which OAMB regulates ovulation, we explored protein kinases functionally interacting with OAMB by employing a new GAL4 driver with restricted expression in the oviduct epithelium. Conditional inhibition of Ca2+/Calmodulin-dependent protein kinase II (CaMKII), but not protein kinase A or C, in the oviduct epithelium inhibited ovulation. Moreover, constitutively active CaMKII, but not protein kinase A, expressed only in the adult oviduct epithelium fully rescued the oamb female's phenotype, demonstrating CaMKII as a major downstream molecule conveying the OAMB's ovulation signal. This is consistent with the ability of both OAMB isoforms, whose common intracellular signal in vitro is Ca2+, to reinstate ovulation in oamb females. These observations reveal the critical roles of the oviduct epithelium and its cellular components OAMB and CaMKII in ovulation. It is conceivable that the OAMB-mediated cellular activities stimulated upon mating are crucial for secretory activities suitable for egg transfer from

  10. The Octopamine Receptor OAMB Mediates Ovulation via Ca2+/Calmodulin-Dependent Protein Kinase II in the Drosophila Oviduct Epithelium

    PubMed Central

    Lee, Hyun-Gwan; Rohila, Suman; Han, Kyung-An

    2009-01-01

    Ovulation is an essential physiological process in sexual reproduction; however, the underlying cellular mechanisms are poorly understood. We have previously shown that OAMB, a Drosophila G-protein-coupled receptor for octopamine (the insect counterpart of mammalian norepinephrine), is required for ovulation induced upon mating. OAMB is expressed in the nervous and reproductive systems and has two isoforms (OAMB-AS and OAMB-K3) with distinct capacities to increase intracellular Ca2+ or intracellular Ca2+ and cAMP in vitro. Here, we investigated tissue specificity and intracellular signals required for OAMB's function in ovulation. Restricted OAMB expression in the adult oviduct epithelium, but not the nervous system, reinstated ovulation in oamb mutant females, in which either OAMB isoform was sufficient for the rescue. Consistently, strong immunoreactivities for both isoforms were observed in the wild-type oviduct epithelium. To delineate the cellular mechanism by which OAMB regulates ovulation, we explored protein kinases functionally interacting with OAMB by employing a new GAL4 driver with restricted expression in the oviduct epithelium. Conditional inhibition of Ca2+/Calmodulin-dependent protein kinase II (CaMKII), but not protein kinase A or C, in the oviduct epithelium inhibited ovulation. Moreover, constitutively active CaMKII, but not protein kinase A, expressed only in the adult oviduct epithelium fully rescued the oamb female's phenotype, demonstrating CaMKII as a major downstream molecule conveying the OAMB's ovulation signal. This is consistent with the ability of both OAMB isoforms, whose common intracellular signal in vitro is Ca2+, to reinstate ovulation in oamb females. These observations reveal the critical roles of the oviduct epithelium and its cellular components OAMB and CaMKII in ovulation. It is conceivable that the OAMB-mediated cellular activities stimulated upon mating are crucial for secretory activities suitable for egg transfer from

  11. Tyrosine phosphorylation of two cytosolic proteins of 50 kDa and 35 kDa in rat liver by insulin-receptor kinase in vitro.

    PubMed Central

    Kwok, Y C; Yip, C C

    1987-01-01

    Insulin-receptor tyrosine kinase can phosphorylate a variety of artificial substrates in vitro. Its physiological substrate(s), however, remains unknown. In the present study, we show that immobilized insulin receptors phosphorylate tyrosine residues of two cytosolic proteins of 50 kDa and 35 kDa in rat liver. Phosphorylation of these two proteins required Mn2+- or Mg2+-ATP as the phosphate donor. Phosphorylation was time- and temperature-dependent. Furthermore, the rate of phosphorylation of the two proteins was related to the autophosphorylated state of the insulin receptor. The pI of the phosphorylated 50 kDa and 35 kDa proteins was 5.4 and 5.6 respectively. These proteins were present in low abundance. They were not related to each other, nor to the insulin receptor, as demonstrated by in-gel proteolytic digestion and by immunoprecipitation using antibodies produced against them. They were specific substrates for the insulin receptor kinase, since they were not phosphorylated by epidermal-growth-factor-receptor kinase. These observations suggest that the 50 kDa and 35 kDa cytosolic proteins may be endogenous substrates for the insulin-receptor kinase. Images Fig. 1. Fig. 2. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. PMID:2829823

  12. Puerarin activates endothelial nitric oxide synthase through estrogen receptor-dependent PI3-kinase and calcium-dependent AMP-activated protein kinase

    SciTech Connect

    Hwang, Yong Pil; Kim, Hyung Gyun; Hien, Tran Thi; Jeong, Myung Ho; Jeong, Tae Cheon; Jeong, Hye Gwang

    2011-11-15

    The cardioprotective properties of puerarin, a natural product, have been attributed to the endothelial nitric oxide synthase (eNOS)-mediated production of nitric oxide (NO) in EA.hy926 endothelial cells. However, the mechanism by which puerarin activates eNOS remains unclear. In this study, we sought to identify the intracellular pathways underlying eNOS activation by puerarin. Puerarin induced the activating phosphorylation of eNOS on Ser1177 and the production of NO in EA.hy926 cells. Puerarin-induced eNOS phosphorylation required estrogen receptor (ER)-mediated phosphatidylinositol 3-kinase (PI3K)/Akt signaling and was reversed by AMP-activated protein kinase (AMPK) and calcium/calmodulin-dependent kinase II (CaMKII) inhibition. Importantly, puerarin inhibited the adhesion of tumor necrosis factor (TNF)-{alpha}-stimulated monocytes to endothelial cells and suppressed the TNF-{alpha} induced expression of intercellular cell adhesion molecule-1. Puerarin also inhibited the TNF-{alpha}-induced nuclear factor-{kappa}B activation, which was attenuated by pretreatment with N{sup G}-nitro-L-arginine methyl ester, a NOS inhibitor. These results indicate that puerarin stimulates eNOS phosphorylation and NO production via activation of an estrogen receptor-mediated PI3K/Akt- and CaMKII/AMPK-dependent pathway. Puerarin may be useful for the treatment or prevention of endothelial dysfunction associated with diabetes and cardiovascular disease. -- Highlights: Black-Right-Pointing-Pointer Puerarin induced the phosphorylation of eNOS and the production of NO. Black-Right-Pointing-Pointer Puerarin activated eNOS through ER-dependent PI3-kinase and Ca{sup 2+}-dependent AMPK. Black-Right-Pointing-Pointer Puerarin-induced NO was involved in the inhibition of NF-kB activation. Black-Right-Pointing-Pointer Puerarin may help for prevention of vascular dysfunction and diabetes.

  13. Phospho-dependent functional modulation of GABA(B) receptors by the metabolic sensor AMP-dependent protein kinase.

    PubMed

    Kuramoto, Nobuyuki; Wilkins, Megan E; Fairfax, Benjamin P; Revilla-Sanchez, Raquel; Terunuma, Miho; Tamaki, Keisuke; Iemata, Mika; Warren, Noel; Couve, Andrés; Calver, Andrew; Horvath, Zsolt; Freeman, Katie; Carling, David; Huang, Lan; Gonzales, Cathleen; Cooper, Edward; Smart, Trevor G; Pangalos, Menelas N; Moss, Stephen J

    2007-01-18

    GABA(B) receptors are heterodimeric G protein-coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly rectifying K(+) channels (GIRKs) and inhibiting Ca(2+) channels. We demonstrate here that GABA(B) receptors are intimately associated with 5'AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5'AMP concentration that are caused by enhanced metabolic activity, anoxia, or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABA(B) receptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally, we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a neuroprotective mechanism, which, under conditions of metabolic stress or after ischemia, increases GABA(B) receptor function to reduce excitotoxicity and thereby promotes neuronal survival.

  14. Phospho-dependent functional modulation of GABAB receptors by the metabolic sensor AMP-dependent protein kinase

    PubMed Central

    Kuramoto, Nobuyuki; Wilkins, Megan E; Fairfax, Benjamin P; Revilla-Sanchez, Raquel; Terunuma, Miho; Warren, Noel; Tamaki, Keisuke; Iemata, Mika; Couve, Andrés; Calver, Andrew; Horvath, Zsolt; Freeman, Katie; Carling, David; Huang, Lan; Gonzales, Cathleen; Cooper, Edward; Smart, Trevor G.; Pangalos, Menelas N.; Moss., Stephen J.

    2007-01-01

    GABAB receptors are heterodimeric G-protein coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly-rectifying K+ channels (GIRKs) and inhibiting Ca2+ channels. We demonstrate here that GABAB receptors are intimately associated with 5’AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5’AMP concentration caused by enhanced metabolic activity, anoxia or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABAB receptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a novel neuroprotective mechanism, which under conditions of metabolic stress or after ischemia increases GABAB receptor function to reduce excitotoxicity and thereby promoting neuronal survival. PMID:17224405

  15. Diacylglycerol kinase delta and protein kinase C(alpha) modulate epidermal growth factor receptor abundance and degradation through ubiquitin-specific protease 8.

    PubMed

    Cai, Jinjin; Crotty, Tracy M; Reichert, Ethan; Carraway, Kermit L; Stafforini, Diana M; Topham, Matthew K

    2010-03-05

    Many human epithelial cancers are characterized by abnormal activation of the epidermal growth factor receptor (EGFR), which is often caused by its excessive expression in tumor cells. The abundance of EGFR is modulated, in part, by its ubiquitination, which targets it for degradation. The components responsible for adding ubiquitin to EGFR are well characterized, but this is a reversible process, and the mechanisms that modulate the removal of ubiquitin from the EGFR are not well known. We found that de-ubiquitination of EGFR was regulated by diacylglycerol kinase delta (DGKdelta), a lipid kinase that terminates diacylglycerol signaling. In DGKdelta-deficient cells, ubiquitination of EGFR was enhanced, which attenuated the steady-state levels of EGFR and promoted its ligand-induced degradation. These effects were not caused by changes in the ubiquitinating apparatus, but instead were due to reduced expression of the de-ubiquitinase, ubiquitin-specific protease 8 (USP8). Depletion of protein kinase Calpha (PKCalpha), a target of diacylglycerol, rescued the levels of USP8 and normalized EGFR degradation in DGKdelta-deficient cells. Moreover, the effects of PKCalpha were caused by its inhibition of Akt, which stabilizes USP8. Our data indicate a novel mechanism where DGKdelta and PKCalpha modulate the levels of ubiquitinated EGFR through Akt and USP8.

  16. Sigma-1 receptor stimulation by dehydroepiandrosterone ameliorates cognitive impairment through activation of CaM kinase II, protein kinase C and extracellular signal-regulated kinase in olfactory bulbectomized mice.

    PubMed

    Moriguchi, Shigeki; Yamamoto, Yui; Ikuno, Tatsuya; Fukunaga, Kohji

    2011-06-01

    Dehydroepiandrosterone (DHEA) is one of the most abundant neurosteroids synthesized de novo in the CNS. We here found that sigma-1 receptor stimulation by DHEA improves cognitive function through phosphorylation of synaptic proteins in olfactory bulbectomized (OBX) mouse hippocampus. We have previously reported that calcium/calmodulin-dependent protein kinase II (CaMKII), protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) were impaired in OBX mouse hippocampus. OBX mice were administered once a day for 7-8 days with DHEA (30 or 60 mg/kg p.o.) 10 days after operation. The spatial, cognitive and conditioned fear memories in OBX mice were significantly improved as assessed by Y-maze, novel object recognition and passive avoidance task, respectively. DHEA also improved impaired hippocampal long-term potentiation in OBX mice. Notably, DHEA treatment restored PKCα (Ser-657) autophosphorylation and NR1 (Ser-896) and myristoylated alanine-rich protein kinase C substrate (Ser-152/156) phosphorylation to the control levels in the hippocampal CA1 region. Likewise, DHEA treatment improved CaMKIIα (Thr-286) autophosphorylation and GluR1 (Ser-831) phosphorylation to the control levels in the CA1 region. Furthermore, DHEA treatment improved ERK and cAMP-responsive element-binding protein (Ser-133) phosphorylation to the control levels. Finally, NE-100, sigma-1 receptor antagonist, significantly inhibited the DHEA-induced improvement of memory-related behaviors and CaMKII, PKC and ERK phosphorylation in CA1 region. Taken together, sigma-1 receptor stimulation by DHEA ameliorates OBX-induced impairment in memory-related behaviors and long-term potentiation in the hippocampal CA1 region through activation of CaMKII, PKC and ERK.

  17. G-protein-coupled receptor kinase specificity for beta-arrestin recruitment to the beta2-adrenergic receptor revealed by fluorescence resonance energy transfer.

    PubMed

    Violin, Jonathan D; Ren, Xiu-Rong; Lefkowitz, Robert J

    2006-07-21

    The small family of G-protein-coupled receptor kinases (GRKs) regulate cell signaling by phosphorylating heptahelical receptors, thereby promoting receptor interaction with beta-arrestins. This switches a receptor from G-protein activation to G-protein desensitization, receptor internalization, and beta-arrestin-dependent signal activation. However, the specificity of GRKs for recruiting beta-arrestins to specific receptors has not been elucidated. Here we use the beta(2)-adrenergic receptor (beta(2)AR), the archetypal nonvisual heptahelical receptor, as a model to test functional GRK specificity. We monitor endogenous GRK activity with a fluorescence resonance energy transfer assay in live cells by measuring kinetics of the interaction between the beta(2)AR and beta-arrestins. We show that beta(2)AR phosphorylation is required for high affinity beta-arrestin binding, and we use small interfering RNA silencing to show that HEK-293 and U2-OS cells use different subsets of their expressed GRKs to promote beta-arrestin recruitment, with significant GRK redundancy evident in both cell types. Surprisingly, the GRK specificity for beta-arrestin recruitment does not correlate with that for bulk receptor phosphorylation, indicating that beta-arrestin recruitment is specific for a subset of receptor phosphorylations on specific sites. Moreover, multiple members of the GRK family are able to phosphorylate the beta(2)AR and induce beta-arrestin recruitment, with their relative contributions largely determined by their relative expression levels. Because GRK isoforms vary in their regulation, this partially redundant system ensures beta-arrestin recruitment while providing the opportunity for tissue-specific regulation of the rate of beta-arrestin recruitment.

  18. Spinal 5-HT7 receptors and protein kinase A constrain intermittent hypoxia-induced phrenic long-term facilitation.

    PubMed

    Hoffman, M S; Mitchell, G S

    2013-10-10

    Phrenic long-term facilitation (pLTF) is a form of serotonin-dependent respiratory plasticity induced by acute intermittent hypoxia (AIH). pLTF requires spinal Gq protein-coupled serotonin-2 receptor (5-HT2) activation, new synthesis of brain-derived neurotrophic factor (BDNF) and activation of its high-affinity receptor, TrkB. Intrathecal injections of selective agonists for Gs protein-coupled receptors (adenosine 2A and serotonin-7; 5-HT7) also induce long-lasting phrenic motor facilitation via TrkB "trans-activation." Since serotonin released near phrenic motor neurons may activate multiple serotonin receptor subtypes, we tested the hypothesis that 5-HT7 receptor activation contributes to AIH-induced pLTF. A selective 5-HT7 receptor antagonist (SB-269970, 5mM, 12 μl) was administered intrathecally at C4 to anesthetized, vagotomized and ventilated rats prior to AIH (3, 5-min episodes, 11% O2). Contrary to predictions, pLTF was greater in SB-269970 treated versus control rats (80 ± 11% versus 45 ± 6% 60 min post-AIH; p<0.05). Hypoglossal LTF was unaffected by spinal 5-HT7 receptor inhibition, suggesting that drug effects were localized to the spinal cord. Since 5-HT7 receptors are coupled to protein kinase A (PKA), we tested the hypothesis that PKA inhibits AIH-induced pLTF. Similar to 5-HT7 receptor inhibition, spinal PKA inhibition (KT-5720, 100 μM, 15 μl) enhanced pLTF (99 ± 15% 60 min post-AIH; p<0.05). Conversely, PKA activation (8-br-cAMP, 100 μM, 15 μl) blunted pLTF versus control rats (16 ± 5% versus 45 ± 6% 60 min post-AIH; p<0.05). These findings suggest a novel mechanism whereby spinal Gs protein-coupled 5-HT7 receptors constrain AIH-induced pLTF via PKA activity.

  19. A-Kinase Anchoring Protein 79/150 Coordinates Metabotropic Glutamate Receptor Sensitization of Peripheral Sensory Neurons

    PubMed Central

    Szteyn, Kalina; Rowan, Matthew P.; Gomez, Ruben; Du, Junhui; Carlton, Susan M.; Jeske, Nathaniel A.

    2016-01-01

    Glutamate serves as the primary excitatory neurotransmitter in the nervous system. Previous studies have identified a role for glutamate and group I metabotropic receptors as targets for study in peripheral inflammatory pain. However, the coordination of signaling events that transpire from receptor activation to afferent neuronal sensitization has not been explored. Herein, we identify that scaffolding protein A-Kinase Anchoring Protein 79/150 (AKAP150) coordinates increased peripheral thermal sensitivity following group I metabotropic receptor (mGluR5) activation. In both acute and persistent models of thermal somatosensory behavior, we report that mGluR5 sensitization requires AKAP150 expression. Furthermore, electrophysiological approaches designed to record afferent neuronal activity reveal that mGluR5 sensitization also requires functional AKAP150 expression. In dissociated primary afferent neurons, mGluR5 activation increases TRPV1 responses in an AKAP dependent manner through a mechanism that induces AKAP association with TRPV1. Experimental results presented herein identify a mechanism of receptor-driven scaffolding association with ion channel targets. Importantly, this mechanism could prove significant in the search for therapeutic targets that repress episodes of acute pain from becoming chronic in nature. PMID:26172554

  20. Arabidopsis receptor-like protein30 and receptor-like kinase suppressor of BIR1-1/EVERSHED mediate innate immunity to necrotrophic fungi.

    PubMed

    Zhang, Weiguo; Fraiture, Malou; Kolb, Dagmar; Löffelhardt, Birgit; Desaki, Yoshitake; Boutrot, Freddy F G; Tör, Mahmut; Zipfel, Cyril; Gust, Andrea A; Brunner, Frédéric

    2013-10-01

    Effective plant defense strategies rely in part on the perception of non-self determinants, so-called microbe-associated molecular patterns (MAMPs), by transmembrane pattern recognition receptors leading to MAMP-triggered immunity. Plant resistance against necrotrophic pathogens with a broad host range is complex and yet not well understood. Particularly, it is unclear if resistance to necrotrophs involves pattern recognition receptors. Here, we partially purified a novel proteinaceous elicitor called sclerotinia culture filtrate elicitor1 (SCFE1) from the necrotrophic fungal pathogen Sclerotinia sclerotiorum that induces typical MAMP-triggered immune responses in Arabidopsis thaliana. Analysis of natural genetic variation revealed five Arabidopsis accessions (Mt-0, Lov-1, Lov-5, Br-0, and Sq-1) that are fully insensitive to the SCFE1-containing fraction. We used a forward genetics approach and mapped the locus determining SCFE1 sensitivity to receptor-like protein30 (RLP30). We also show that SCFE1-triggered immune responses engage a signaling pathway dependent on the regulatory receptor-like kinases brassinosteroid insensitive1-associated receptor kinase1 (BAK1) and Suppressor of BIR1-1/evershed (SOBIR1/EVR). Mutants of RLP30, BAK1, and SOBIR1 are more susceptible to S. sclerotiorum and the related fungus Botrytis cinerea. The presence of an elicitor in S. sclerotiorum evoking MAMP-triggered immune responses and sensed by RLP30/SOBIR1/BAK1 demonstrates the relevance of MAMP-triggered immunity in resistance to necrotrophic fungi.

  1. A crucial role for cAMP and protein kinase A in D1 dopamine receptor regulated intracellular calcium transients.

    PubMed

    Dai, Rujuan; Ali, Mohammad K; Lezcano, Nelson; Bergson, Clare

    2008-01-01

    D1-like dopamine receptors stimulate Ca(2+) transients in neurons but the effector coupling and signaling mechanisms underlying these responses have not been elucidated. Here we investigated potential mechanisms using both HEK 293 cells that stably express D1 receptors (D1HEK293) and hippocampal neurons in culture. In D1HEK293 cells, the full D1 receptor agonist SKF 81297 evoked a robust dose-dependent increase in Ca(2+)(i) following 'priming' of endogenous G(q/11)-coupled muscarinic or purinergic receptors. The effect of SKF81297 could be mimicked by forskolin or 8-Br-cAMP. Further, cholera toxin and the cAMP-dependent protein kinase (PKA) inhibitors, KT5720 and H89, as well as thapsigargin abrogated the D1 receptor evoked Ca(2+) transients. Removal of the priming agonist and treatment with the phospholipase C inhibitor U73122 also blocked the SKF81297-evoked responses. D1R agonist did not stimulate IP(3) production, but pretreatment of cells with the D1R agonist potentiated G(q)-linked receptor agonist mobilization of intracellular Ca(2+) stores. In neurons, SKF81297 and SKF83959, a partial D1 receptor agonist, promoted Ca(2+) oscillations in response to G(q/11)-coupled metabotropic glutamate receptor (mGluR) stimulation. The effects of both D1R agonists on the mGluR-evoked Ca(2+) responses were PKA dependent. Altogether the data suggest that dopamine D1R activation and ensuing cAMP production dynamically regulates the efficiency and timing of IP(3)-mediated intracellular Ca(2+) store mobilization.

  2. Separate Intramolecular Targets for Protein Kinase A Control N-Methyl-d-aspartate Receptor Gating and Ca2+ Permeability*

    PubMed Central

    Aman, Teresa K.; Maki, Bruce A.; Ruffino, Thomas J.; Kasperek, Eileen M.; Popescu, Gabriela K.

    2014-01-01

    Protein kinase A (PKA) enhances synaptic plasticity in the central nervous system by increasing NMDA receptor current amplitude and Ca2+ flux in an isoform-dependent yet poorly understood manner. PKA phosphorylates multiple residues on GluN1, GluN2A, and GluN2B subunits in vivo, but the functional significance of this multiplicity is unknown. We examined gating and permeation properties of recombinant NMDA receptor isoforms and of receptors with altered C-terminal domain (CTDs) prior to and after pharmacological inhibition of PKA. We found that PKA inhibition decreased GluN1/GluN2B but not GluN1/GluN2A gating; this effect was due to slower rates for receptor activation and resensitization and was mediated exclusively by the GluN2B CTD. In contrast, PKA inhibition reduced NMDA receptor-relative Ca2+ permeability (PCa/PNa) regardless of the GluN2 isoform and required the GluN1 CTD; this effect was due primarily to decreased unitary Ca2+ conductance, because neither Na+ conductance nor Ca2+-dependent block was altered substantially. Finally, we show that both the gating and permeation effects can be reproduced by changing the phosphorylation state of a single residue: GluN2B Ser-1166 and GluN1 Ser-897, respectively. We conclude that PKA effects on NMDA receptor gating and Ca2+ permeability rely on distinct phosphorylation sites located on the CTD of GluN2B and GluN1 subunits. This separate control of NMDA receptor properties by PKA may account for the specific effects of PKA on plasticity during synaptic development and may lead to drugs targeted to alter NMDA receptor gating or Ca2+ permeability. PMID:24847051

  3. Distinct cellular and subcellular distributions of G protein-coupled receptor kinase and arrestin isoforms in the striatum.

    PubMed

    Bychkov, Evgeny; Zurkovsky, Lilia; Garret, Mika B; Ahmed, Mohamed R; Gurevich, Eugenia V

    2012-01-01

    G protein-coupled receptor kinases (GRKs) and arrestins mediate desensitization of G protein-coupled receptors (GPCR). Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling.

  4. Distinct Cellular and Subcellular Distributions of G Protein-Coupled Receptor Kinase and Arrestin Isoforms in the Striatum

    PubMed Central

    Bychkov, Evgeny; Zurkovsky, Lilia; Garret, Mika B.; Ahmed, Mohamed R.; Gurevich, Eugenia V.

    2012-01-01

    G protein-coupled receptor kinases (GRKs) and arrestins mediate desensitization of G protein-coupled receptors (GPCR). Arrestins also mediate G protein-independent signaling via GPCRs. Since GRK and arrestins demonstrate no strict receptor specificity, their functions in the brain may depend on their cellular complement, expression level, and subcellular targeting. However, cellular expression and subcellular distribution of GRKs and arrestins in the brain is largely unknown. We show that GRK isoforms GRK2 and GRK5 are similarly expressed in direct and indirect pathway neurons in the rat striatum. Arrestin-2 and arrestin-3 are also expressed in neurons of both pathways. Cholinergic interneurons are enriched in GRK2, arrestin-3, and GRK5. Parvalbumin-positive interneurons express more of GRK2 and less of arrestin-2 than medium spiny neurons. The GRK5 subcellular distribution in the human striatal neurons is altered by its phosphorylation: unphosphorylated enzyme preferentially localizes to synaptic membranes, whereas phosphorylated GRK5 is found in plasma membrane and cytosolic fractions. Both GRK isoforms are abundant in the nucleus of human striatal neurons, whereas the proportion of both arrestins in the nucleus was equally low. However, overall higher expression of arrestin-2 yields high enough concentration in the nucleus to mediate nuclear functions. These data suggest cell type- and subcellular compartment-dependent differences in GRK/arrestin-mediated desensitization and signaling. PMID:23139825

  5. Involvement of the mannose receptor and p38 mitogen-activated protein kinase signaling pathway of the microdomain of the integral membrane protein after enteropathogenic Escherichia coli infection.

    PubMed

    Liu, Zhihua; Ma, Yanlei; Moyer, Mary Pat; Zhang, Peng; Shi, Chenzhang; Qin, Huanlong

    2012-04-01

    The microdomain of the integral membrane protein (MIMP) has been shown to adhere to mucin and to antagonize the adhesion of enteropathogenic Escherichia coli (EPEC) to epithelial cells; however, the mechanism has not been fully elucidated. In this study, we further identified the receptor of MIMP on NCM460 cells and investigated the mechanism (the p38 mitogen-activated protein kinase [MAPK] pathway) following the interaction of MIMP and its corresponding receptor, mannose receptor. We first identified the target receptor of MIMP on the surfaces of NCM460 cells using immunoprecipitation-mass spectrometry technology. We also verified the mannose receptor and examined the degradation and activation of the p38 MAPK signaling pathway. The results indicated that MIMP adhered to NCM460 cells by binding to the mannose receptor and inhibited the phosphorylation of p38 MAPK stimulated after EPEC infection via inhibition of the Toll-like receptor 5 pathway. These findings indicated that MIMPs relieve the injury of NCM460 cells after enteropathogenic E. coli infection through the mannose receptor and inhibition of the p38 MAPK signaling pathway, both of which may therefore be potential therapeutic targets for intestinal diseases, such as inflammatory bowel disease.

  6. Regulation of endothelial protein C receptor shedding by cytokines is mediated through differential activation of MAP kinase signaling pathways

    SciTech Connect

    Menschikowski, Mario; Hagelgans, Albert; Eisenhofer, Graeme; Siegert, Gabriele

    2009-09-10

    The endothelial protein C receptor (EPCR) plays a pivotal role in coagulation, inflammation, cell proliferation, and cancer, but its activity is markedly changed by ectodomain cleavage and release as the soluble protein (sEPCR). In this study we examined the mechanisms involved in the regulation of EPCR shedding in human umbilical endothelial cells (HUVEC). Interleukin-1{beta} (IL-1{beta}) and tumor necrosis factor-{alpha} (TNF-{alpha}), but not interferon-{gamma} and interleukin-6, suppressed EPCR mRNA transcription and cell-associated EPCR expression in HUVEC. The release of sEPCR induced by IL-1{beta} and TNF-{alpha} correlated with activation of p38 MAPK and c-Jun N-terminal kinase (JNK). EPCR shedding was also induced by phorbol 12-myristate 13-acetate, ionomycin, anisomycin, thiol oxidants or alkylators, thrombin, and disruptors of lipid rafts. Both basal and induced shedding of EPCR was blocked by the metalloproteinase inhibitors, TAPI-0 and GM6001, and by the reduced non-protein thiols, glutathione, dihydrolipoic acid, dithiothreitol, and N-acetyl-L-cysteine. Because other antioxidants and scavengers of reactive oxygen species failed to block the cleavage of EPCR, a direct suppression of metalloproteinase activity seems responsible for the observed effects of reduced thiols. In summary, the shedding of EPCR in HUVEC is effectively regulated by IL-1{beta} and TNF-{alpha}, and downstream by MAP kinase signaling pathways and metalloproteinases.

  7. [Study of possible involvement of MEK mitogen-activated protein kinase and TGF-β receptor in planarian regeneration processes using pharmacological inhibition analysis].

    PubMed

    Ermakov, A M; Ermakova, O N; Ermolaeva, S A

    2014-01-01

    Possible involvement of MEK mitogen-activated protein kinase and TGF-β receptor in the processes of regeneration and morphogenesis in freshwater planarian flatworms Schmidtea mediterranea was studied using a pharmacological inhibitor analysis. It was found that pharmacological inhibitors of these kinases significantly inhibit the regeneration of the head end of the animals and that this effect is realized due to inhibition of proliferative activity of neoblasts, planarian stem cells. It is shown that that the inhibition of the studied protein kinases in regenerating planarians markedly disturbs stem cell differentiation and morphogenesis.

  8. [Characterization of a putative S locus encoded receptor protein kinase and its role in self-incompatibility]. Progress report, January 1993

    SciTech Connect

    Not Available

    1993-06-01

    The serine/threonine protein kinase (SRK) protein was predicted to be similar to the growth factor receptor tyrosine kinases in animals but its amino acid sequence of the catalytic domain is more similar to that of the catalytic domains of protein serine/threonine kinases than to protein tyrosine kinases. We have shown that the SRK protein has intrinsic scrine/threonine kinase activity. We subcloned the protein kinase-homologous domain of the SRK{sub 6} cDNA into the bacterial expression vector pGEX-3X and we have constructed a second plasmid identical to the first except that it carried a conservative mutation that substituted Arg for the Lys{sup 524} codon of SRK6 This lysine corresponds to the ATP-binding site, is essential in protein kinases, and is a common target for site-directed mutagenesis as a means to obtain kinase-defective proteins. Cultures bearing the wild-type and mutant SRK catalytic domains each produced an approximately 64 kD protein that reacted with anti-SRK6 antibodies. Following pulse-labeling with {sup 32}P we found that the wild-type SRK6 protein but not the mutant form was detectably phosphorylated. Phosphoamino acid analysis of the affinity purified {sup 32}p-labeled GST-SRK6 fusion protein demonstrated that SRK was phosphorylated predominantly on semine and to a lesser extent on threonine, but not on tyrosine. Thus, SRK6 is a functional serine/threonine protein kinase.

  9. Integrin beta and receptor for activated protein kinase C are involved in the cell entry of Bombyx mori cypovirus.

    PubMed

    Zhang, Yiling; Cao, Guangli; Zhu, Liyuan; Chen, Fei; Zar, Mian Sahib; Wang, Simei; Hu, Xiaolong; Wei, Yuhong; Xue, Renyu; Gong, Chengliang

    2017-02-07

    Receptor-mediated endocytosis using a β1 integrin-dependent internalization was considered as the primary mechanism for the initiation of mammalian reovirus infection. Bombyx mori cypovirus (BmCPV) is a member of Reoviridae family which mainly infects the midgut epithelium of silkworm; the cell entry of BmCPV is poorly explored. In this study, co-immunoprecipitation (Co-IP), virus overlay protein binding assay (VOPBA), and BmCPV-protein interaction on the polyvinylidene difluoride membrane (BmCPV-PI-PVDF) methods were employed to screen the interacting proteins of BmCPV, and several proteins including integrin beta and receptor for activated protein kinase C (RACK1) were identified as the candidate interacting proteins for establishing the infection of BmCPV. The infectivity of BmCPV was investigated in vivo and in vitro by RNA interference (RNAi) and antibody blocking methods, and the results showed that the infectivity of BmCPV was significantly reduced by either small interfering RNA-mediated silencing of integrin beta and RACK1 or antibody blocking of integrin beta and RACK1. The expression level of integrin beta or RACK1 is not the highest in the silkworm midgut which is a principal target tissue of BmCPV, suggesting that the molecules other than integrin beta or RACK1 might play a key role in determining the tissue tropism of BmCPV infection. The establishment of BmCPV infection depends on other factors, and these factors interacted with integrin beta and RACK1 to form receptor complex for the cell entry of BmCPV.

  10. Expression and activation of platelet-derived growth factor β receptor, mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) in canine mammary tumours.

    PubMed

    Altamura, Gennaro; Uberti, Barbara Degli; Galiero, Giorgio; Martano, Manuela; Pirro, Antonella; Russo, Marco; Borzacchiello, Giuseppe

    2017-02-01

    Canine mammary tumours are frequent neoplasms mostly affecting intact female dogs, for which no 100% efficient therapy is available. Platelet derived growth factor β receptor (PDGFβR) is a tyrosine kinase receptor (TKR) with a potential role in human breast cancer and a series of canine tumours. In this study we demonstrated, for the first time, expression of PDGFβR and its downstream transduction molecules, mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) and extracellular signal-regulated kinase (ERK), as well as their activated forms in canine mammary tumours by both biochemical analysis and immunohistochemistry. PDGFβR was expressed and hyperphosphorylated in the majority of tumour samples and tumour derived cell lines. Additionally, both MEK and ERK were expressed and activated in cell lines as well as biopsies. TKR inhibitors (TKRi) are currently under investigation as possible therapy in human breast and several canine tumours, thus our in vivo and in vitro findings pave the way for future studies aimed at establishing a potential therapeutic employment of TKRi for the treatment of canine mammary cancer.

  11. Receptor tyrosine kinases in carcinogenesis.

    PubMed

    Zhang, Xiao-Ying; Zhang, Pei-Ying

    2016-11-01

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

  12. The vanilloid receptor (VR1)-mediated effects of anandamide are potently enhanced by the cAMP-dependent protein kinase.

    PubMed

    De Petrocellis, L; Harrison, S; Bisogno, T; Tognetto, M; Brandi, I; Smith, G D; Creminon, C; Davis, J B; Geppetti, P; Di Marzo, V

    2001-06-01

    The endogenous cannabinoid receptor ligand, anandamide (AEA), is a full agonist of the vanilloid receptor type 1 (VR1) for capsaicin. Here, we demonstrate that the potency and efficacy of AEA at VR1 receptors can be significantly increased by the concomitant activation of protein kinase A (PKA). In human embryonic kidney (HEK) cells over-expressing human VR1, AEA induces a rise in cytosolic Ca(2+) concentration that is mediated by this receptor. The EC(50) for this effect was decreased five-fold in the presence of forskolin (FRSK, 1-5 microM) or the cAMP analogue, 8-Br-cAMP (10-100 microM). The effects of 8-Br-cAMP and FRSK were blocked by a selective PKA inhibitor. The FRSK (10 nM) also potently enhanced the sensory neurone- and VR1-mediated constriction by AEA of isolated guinea-pig bronchi, and this effect was abolished by a PKA inhibitor. In rat dorsal root ganglia slices, AEA-induced release of substance P, an effect mediated by VR1 activation, was enhanced three-fold by FRSK (10 nM). Thus, the ability of AEA to stimulate sensory VR1, with subsequent neuropeptide release, appears to be regulated by the state of activation of PKA. This observation supports the hypothesis that endogenous AEA might stimulate VR1 under certain pathophysiological conditions.

  13. Ligand-induced dynamics of heterotrimeric G protein-coupled receptor-like kinase complexes

    PubMed Central

    Tunc-Ozdemir, Meral; Jones, Alan M.

    2017-01-01

    Background Arabidopsis, 7-transmembrane Regulator of G signaling protein 1 (AtRGS1) modulates canonical G protein signaling by promoting the inactive state of heterotrimeric G protein complex on the plasma membrane. It is known that plant leucine-rich repeat receptor–like kinases (LRR RLKs) phosphorylate AtRGS1 in vitro but little is known about the in vivo interaction, molecular dynamics, or the cellular consequences of this interaction. Methods Therefore, a subset of the known RLKs that phosphorylate AtRGS1 were selected for elucidation, namely, BAK1, BIR1, FLS2. Several microscopies for both static and dynamic protein-protein interactions were used to follow in vivo interactions between the RLKs and AtRGS1 after the presentation of the Pathogen-associated Molecular Pattern, Flagellin 22 (Flg22). These microscopies included Förster Resonance Energy Transfer, Bimolecular Fluoresence Complementation, and Cross Number and Brightness Fluorescence Correlation Spectroscopy. In addition, reactive oxygen species and calcium changes in living cells were quantitated using luminometry and R-GECO1 microscopy. Results The LRR RLKs BAK1 and BIR1, interact with AtRGS1 at the plasma membrane. The RLK ligand flg22 sets BAK1 in motion toward AtRGS1 and BIR1 away, both returning to the baseline orientations by 10 minutes. The C-terminal tail of AtRGS1 is important for the interaction with BAK1 and for the tempo of the AtRGS1/BIR1 dynamics. This window of time corresponds to the flg22-induced transient production of reactive oxygen species and calcium release which are both attenuated in the rgs1 and the bak1 null mutants. Conclusions A temporal model of these interactions is proposed. flg22 binding induces nearly instantaneous dimerization between FLS2 and BAK1. Phosphorylated BAK1 interacts with and enables AtRGS1 to move away from BIR1 and AtRGS1 becomes phosphorylated leading to its endocytosis thus leading to de-repression by permitting AtGPA1 to exchange GDP for GTP

  14. RTKdb: database of receptor tyrosine kinase

    PubMed Central

    Grassot, Julien; Mouchiroud, Guy; Perrière, Guy

    2003-01-01

    Receptor Tyrosine Kinases (RTK) are transmembrane receptors specifically found in metazoans. They represent an excellent model for studying evolution of cellular processes in metazoans because they encompass large families of modular proteins and belong to a major family of contingency generating molecules in eukaryotic cells: the protein kinases. Because tyrosine kinases have been under close scrutiny for many years in various species, they are associated with a wealth of information, mainly in mammals. Presently, most categories of RTK were identified in mammals, but in a near future other model species will be sequenced, and will bring us RTKs from other metazoan clades. Thus, collecting RTK sequences would provide a good starting point as a new model for comparative and evolutionary studies applying to multigene families. In this context, we are developing the Receptor Tyrosine Kinase database (RTKdb), which is the only database on tyrosine kinase receptors presently available. In this database, protein sequences from eight model metazoan species are organized under the format previously used for the HOVERGEN, HOBACGEN and NUREBASE systems. RTKdb can be accessed through the PBIL (Pôle Bioinformatique Lyonnais) World Wide Web server at http://pbil.univ-lyon1.fr/RTKdb/, or through the FamFetch graphical user interface available at the same address. PMID:12520021

  15. Coarse-grained molecular simulation of epidermal growth factor receptor protein tyrosine kinase multi-site self-phosphorylation.

    PubMed

    Koland, John G

    2014-01-01

    Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR), the intrinsic protein tyrosine kinase (PTK) activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites) in either of the two C-terminal (CT) domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in molecules such as EGFR

  16. Coarse-Grained Molecular Simulation of Epidermal Growth Factor Receptor Protein Tyrosine Kinase Multi-Site Self-Phosphorylation

    PubMed Central

    Koland, John G.

    2014-01-01

    Upon the ligand-dependent dimerization of the epidermal growth factor receptor (EGFR), the intrinsic protein tyrosine kinase (PTK) activity of one receptor monomer is activated, and the dimeric receptor undergoes self-phosphorylation at any of eight candidate phosphorylation sites (P-sites) in either of the two C-terminal (CT) domains. While the structures of the extracellular ligand binding and intracellular PTK domains are known, that of the ∼225-amino acid CT domain is not, presumably because it is disordered. Receptor phosphorylation on CT domain P-sites is critical in signaling because of the binding of specific signaling effector molecules to individual phosphorylated P-sites. To investigate how the combination of conventional substrate recognition and the unique topological factors involved in the CT domain self-phosphorylation reaction lead to selectivity in P-site phosphorylation, we performed coarse-grained molecular simulations of the P-site/catalytic site binding reactions that precede EGFR self-phosphorylation events. Our results indicate that self-phosphorylation of the dimeric EGFR, although generally believed to occur in trans, may well occur with a similar efficiency in cis, with the P-sites of both receptor monomers being phosphorylated to a similar extent. An exception was the case of the most kinase-proximal P-site-992, the catalytic site binding of which occurred exclusively in cis via an intramolecular reaction. We discovered that the in cis interaction of P-site-992 with the catalytic site was facilitated by a cleft between the N-terminal and C-terminal lobes of the PTK domain that allows the short CT domain sequence tethering P-site-992 to the PTK core to reach the catalytic site. Our work provides several new mechanistic insights into the EGFR self-phosphorylation reaction, and demonstrates the potential of coarse-grained molecular simulation approaches for investigating the complexities of self-phosphorylation in molecules such as EGFR

  17. Conserved herpesvirus protein kinases

    PubMed Central

    Gershburg, Edward; Pagano, Joseph S.

    2008-01-01

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

  18. Receptor-interacting protein kinase 3-mediated programmed cell necrosis in rats subjected to focal cerebral ischemia-reperfusion injury

    PubMed Central

    DONG, YANRU; BAO, CUIFEN; YU, JINGWEI; LIU, XIA

    2016-01-01

    In the current study, the activation of tumor necrosis factor-α receptor 1 (TNFR1) and receptor-interacting protein kinase 3 (RIP3) were investigated following cerebral ischemia-reperfusion injury (CIRI). Healthy SD rats were randomly divided into 3 groups: Sham operation group, model group and inhibitor group. The model group and inhibitor group were further divided into 4 subgroups of 6, 12, 24 and 72 h following CIRI. Using right middle cerebral artery embolization, the CIRI model was generated. To confirm that the CIRI model was established, neurological scores, TTC staining and brain water content measurements were conducted. Immunohistochemistry and western blotting were conducted to investigate the expression of TNFR1 and RIP3 in the cerebral cortex. It was observed that nerve cell necrosis occurred following 6 h of CIRI. The appearance of necrotic cells was gradually increased with increasing CIRI duration. TNFR1 and RIP3 were positively expressed following 6 h of CIRI. With increasing durations of CIRI, the protein expression levels of TNFR1 and RIP3 were significantly increased. Pre-administration with Z-VAD-FMK (zVAD) significantly increased the protein level of RIP3, however, had no effect on the levels of TNFR1, and was accompanied by a reduction in necrosis. In conclusion, RIP3-mediated cell necrosis was enhanced by caspase blockade zVAD and the function of zVAD was independent of TNFR1 signaling following IR. PMID:27220678

  19. Berberine reduces insulin resistance through protein kinase C-dependent up-regulation of insulin receptor expression.

    PubMed

    Kong, Wei-Jia; Zhang, Hao; Song, Dan-Qing; Xue, Rong; Zhao, Wei; Wei, Jing; Wang, Yue-Ming; Shan, Ning; Zhou, Zhen-Xian; Yang, Peng; You, Xue-Fu; Li, Zhuo-Rong; Si, Shu-Yi; Zhao, Li-Xun; Pan, Huai-Ning; Jiang, Jian-Dong

    2009-01-01

    Natural product berberine (BBR) has been reported to have hypoglycemic and insulin-sensitizing activities; however, its mechanism remains unclear. This study was designed to investigate the molecular mechanism of BBR against insulin resistance. Here, we identify insulin receptor (InsR) as a target of BBR to increase insulin sensitivity. In cultured human liver cells, BBR increased InsR messenger RNA (mRNA) and protein expression in a dose- and time-dependent manner. Berberine increased InsR expression in the L6 rat skeletal muscle cells as well. Berberine-enhanced InsR expression improved cellular glucose consumption only in the presence of insulin. Silencing InsR gene with small interfering RNA or blocking the phosphoinositol-3-kinase diminished this effect. Berberine induced InsR gene expression through a protein kinase C (PKC)-dependent activation of its promoter. Inhibition of PKC abolished BBR-caused InsR promoter activation and InsR mRNA transcription. In animal models, treatment of type 2 diabetes mellitus rats with BBR lowered fasting blood glucose and fasting serum insulin, increased insulin sensitivity, and elevated InsR mRNA as well as PKC activity in the liver. In addition, BBR lowered blood glucose in KK-Ay type 2 but not in NOD/LtJ type 1 diabetes mellitus mice that were insulin deficient. Our results suggest that BBR is a unique natural medicine against insulin resistance in type 2 diabetes mellitus and metabolic syndrome.

  20. Targeting Vascular Endothelial Growth Factor Receptor 2 and Protein Kinase D1 Related Pathways by a Multiple Kinase Inhibitor in Angiogenesis and Inflammation Related Processes In Vitro

    PubMed Central

    Varga, Attila; Gyulavári, Pál; Greff, Zoltán; Futosi, Krisztina; Németh, Tamás; Simon-Szabó, Laura; Kerekes, Krisztina; Szántai-Kis, Csaba; Brauswetter, Diána; Kokas, Márton; Borbély, Gábor; Erdei, Anna; Mócsai, Attila; Kéri, György; Vántus, Tibor

    2015-01-01

    Emerging evidence suggests that the vascular endothelial growth factor receptor 2 (VEGFR2) and protein kinase D1 (PKD1) signaling axis plays a critical role in normal and pathological angiogenesis and inflammation related processes. Despite all efforts, the currently available therapeutic interventions are limited. Prior studies have also proved that a multiple target inhibitor can be more efficient compared to a single target one. Therefore, development of novel inflammatory pathway-specific inhibitors would be of great value. To test this possibility, we screened our molecular library using recombinant kinase assays and identified the previously described compound VCC251801 with strong inhibitory effect on both VEGFR2 and PKD1. We further analyzed the effect of VCC251801 in the endothelium-derived EA.hy926 cell line and in different inflammatory cell types. In EA.hy926 cells, VCC251801 potently inhibited the intracellular activation and signaling of VEGFR2 and PKD1 which inhibition eventually resulted in diminished cell proliferation. In this model, our compound was also an efficient inhibitor of in vitro angiogenesis by interfering with endothelial cell migration and tube formation processes. Our results from functional assays in inflammatory cellular models such as neutrophils and mast cells suggested an anti-inflammatory effect of VCC251801. The neutrophil study showed that VCC251801 specifically blocked the immobilized immune-complex and the adhesion dependent TNF-α -fibrinogen stimulated neutrophil activation. Furthermore, similar results were found in mast cell degranulation assay where VCC251801 caused significant reduction of mast cell response. In summary, we described a novel function of a multiple kinase inhibitor which strongly inhibits the VEGFR2-PKD1 signaling and might be a novel inhibitor of pathological inflammatory pathways. PMID:25874616

  1. Targeting vascular endothelial growth factor receptor 2 and protein kinase D1 related pathways by a multiple kinase inhibitor in angiogenesis and inflammation related processes in vitro.

    PubMed

    Varga, Attila; Gyulavári, Pál; Greff, Zoltán; Futosi, Krisztina; Németh, Tamás; Simon-Szabó, Laura; Kerekes, Krisztina; Szántai-Kis, Csaba; Brauswetter, Diána; Kokas, Márton; Borbély, Gábor; Erdei, Anna; Mócsai, Attila; Kéri, György; Vántus, Tibor

    2015-01-01

    Emerging evidence suggests that the vascular endothelial growth factor receptor 2 (VEGFR2) and protein kinase D1 (PKD1) signaling axis plays a critical role in normal and pathological angiogenesis and inflammation related processes. Despite all efforts, the currently available therapeutic interventions are limited. Prior studies have also proved that a multiple target inhibitor can be more efficient compared to a single target one. Therefore, development of novel inflammatory pathway-specific inhibitors would be of great value. To test this possibility, we screened our molecular library using recombinant kinase assays and identified the previously described compound VCC251801 with strong inhibitory effect on both VEGFR2 and PKD1. We further analyzed the effect of VCC251801 in the endothelium-derived EA.hy926 cell line and in different inflammatory cell types. In EA.hy926 cells, VCC251801 potently inhibited the intracellular activation and signaling of VEGFR2 and PKD1 which inhibition eventually resulted in diminished cell proliferation. In this model, our compound was also an efficient inhibitor of in vitro angiogenesis by interfering with endothelial cell migration and tube formation processes. Our results from functional assays in inflammatory cellular models such as neutrophils and mast cells suggested an anti-inflammatory effect of VCC251801. The neutrophil study showed that VCC251801 specifically blocked the immobilized immune-complex and the adhesion dependent TNF-α -fibrinogen stimulated neutrophil activation. Furthermore, similar results were found in mast cell degranulation assay where VCC251801 caused significant reduction of mast cell response. In summary, we described a novel function of a multiple kinase inhibitor which strongly inhibits the VEGFR2-PKD1 signaling and might be a novel inhibitor of pathological inflammatory pathways.

  2. Protein kinase mediated upregulation of endothelin A, endothelin B and 5-hydroxytryptamine 1B/1D receptors during organ culture in rat basilar artery

    PubMed Central

    Hansen-Schwartz, Jacob; Svensson, Carl-Lennart; Xu, Cang-Bao; Edvinsson, Lars

    2002-01-01

    Organ culture has been shown to upregulate both endothelin (ET) and 5-hydroxytryptamine 1B/1D (5-HT1B/1D) receptors in rat cerebral arteries. The purpose of the present study was to investigate the involvement of protein kinases, especially protein kinases C (PKC) and A (PKA) in this process. The effect of inhibiting protein kinases during organ culture with staurosporine (unspecific protein kinase inhitor), RO 31-7549 (specific inhibitor of classical PKC's) and H 89 (specific inhibitor of PKA) was examined using in vitro pharmacological examination of cultured vessel segments with ET-1 (unspecific ETA and ETB agonist), S6c (specific ETB agonist) and 5-CT (5-HT1 agonist). Levels of mRNA coding for the ETA, ETB, 5-HT1B and 5-HT1D receptors were analysed using real-time RT–PCR. Classical PKC's are critically involved in the appearance of the ETB receptor; co-culture with RO 31-7549 abolished the contractile response (6.9±1.8%) and reduced the ETB receptor mRNA by 44±4% as compared to the cultured control. Correlation between decreased ETB receptor mRNA and abolished contractile function indicates upstream involvement of PKC. Inhibition of PKA generally had an enhancing effect on the induced changes giving rise to a 7–25% increase in Emax in response to ET-1, S6c and 5-CT as compared to the cultured control. Staurosporine inhibited the culture induced upregulation of the response of both the ETA and the 5-HT1B/1D receptors, but had no significant effect on the mRNA levels of these receptors. This lack of correlation indicates an additional downstream involvement of protein kinases. PMID:12183337

  3. Proteolytic activation of proapoptotic kinase protein kinase Cδ by tumor necrosis factor α death receptor signaling in dopaminergic neurons during neuroinflammation

    PubMed Central

    2012-01-01

    Background The mechanisms of progressive dopaminergic neuronal loss in Parkinson’s disease (PD) remain poorly understood, largely due to the complex etiology and multifactorial nature of disease pathogenesis. Several lines of evidence from human studies and experimental models over the last decade have identified neuroinflammation as a potential pathophysiological mechanism contributing to disease progression. Tumor necrosis factor α (TNF) has recently emerged as the primary neuroinflammatory mediator that can elicit dopaminergic cell death in PD. However, the signaling pathways by which TNF mediates dopaminergic cell death have not been completely elucidated. Methods In this study we used a dopaminergic neuronal cell model and recombinant TNF to characterize intracellular signaling pathways activated during TNF-induced dopaminergic neurotoxicity. Etanercept and neutralizing antibodies to tumor necrosis factor receptor 1 (TNFR1) were used to block TNF signaling. We confirmed the results from our mechanistic studies in primary embryonic mesencephalic cultures and in vivo using the stereotaxic lipopolysaccharide (LPS) model of nigral dopaminergic degeneration. Results TNF signaling in dopaminergic neuronal cells triggered the activation of protein kinase Cδ (PKCδ), an isoform of the novel PKC family, by caspase-3 and caspase-8 dependent proteolytic cleavage. Both TNFR1 neutralizing antibodies and the soluble TNF receptor Etanercept blocked TNF-induced PKCδ proteolytic activation. Proteolytic activation of PKCδ was accompanied by translocation of the kinase to the nucleus. Notably, inhibition of PKCδ signaling by small interfering (si)RNA or overexpression of a PKCδ cleavage-resistant mutant protected against TNF-induced dopaminergic neuronal cell death. Further, primary dopaminergic neurons obtained from PKCδ knockout (−/−) mice were resistant to TNF toxicity. The proteolytic activation of PKCδ in the mouse substantia nigra in the neuroinflammatory LPS

  4. Internalization and down-regulation of human muscarinic acetylcholine receptor m2 subtypes. Role of third intracellular m2 loop and G protein-coupled receptor kinase 2.

    PubMed

    Tsuga, H; Kameyama, K; Haga, T; Honma, T; Lameh, J; Sadée, W

    1998-02-27

    Internalization and down-regulation of human muscarinic acetylcholine m2 receptors (hm2 receptors) and a hm2 receptor mutant lacking a central part of the third intracellular loop (I3-del m2 receptor) were examined in Chinese hamster ovary (CHO-K1) cells stably expressing these receptors and G protein-coupled receptor kinase 2 (GRK2). Agonist-induced internalization of up to 80-90% of hm2 receptors was demonstrated by measuring loss of [3H]N-methylscopolamine binding sites from the cell surface, and transfer of [3H]quinuclidinyl benzilate binding sites from the plasma membrane into the light-vesicle fractions separated by sucrose density gradient centrifugation. Additionally, translocation of hm2 receptors with endocytic vesicles were visualized by immunofluorescence confocal microscopy. Agonist-induced down-regulation of up to 60-70% of hm2 receptors was demonstrated by determining the loss of [3H]quinuclidinyl benzilate binding sites in the cells. The half-time (t1/2) of internalization and down-regulation in the presence of 10(-4) M carbamylcholine was estimated to be 9.5 min and 2.3 h, respectively. The rates of both internalization and down-regulation of hm2 receptors in the presence of 10(-6) M or lower concentrations of carbamylcholine were markedly increased by coexpression of GRK2. Agonist-induced internalization of I3-del m2 receptors was barely detectable upon incubation of cells for 1 h, but agonist-induced down-regulation of up to 40-50% of I3-del m2 receptors occurred upon incubation with 10(-4) M carbamylcholine for 16 h. However, the rate of down-regulation was lower compared with wild type receptors (t1/2 = 9.9 versus 2.3 h). These results indicate that rapid internalization of hm2 receptors is facilitated by their phosphorylation with GRK2 and does not occur in the absence of the third intracellular loop, but down-regulation of hm2 receptors may occur through both GRK2-facilitating pathway and third intracellular loop-independent pathways.

  5. Intrathecal injection of adenosine 2A receptor agonists reversed neuropathic allodynia through protein kinase (PK)A/PKC signaling.

    PubMed

    Loram, Lisa C; Taylor, Frederick R; Strand, Keith A; Harrison, Jacqueline A; Rzasalynn, Rachael; Sholar, Paige; Rieger, Jayson; Maier, Steven F; Watkins, Linda R

    2013-10-01

    A single intrathecal dose of adenosine 2A receptor (A2AR) agonist was previously reported to produce a multi-week reversal of allodynia in a chronic constriction injury (CCI) model of neuropathic pain. We aimed to determine if this long-term reversal was induced by A2AR agonism versus more generalized across adenosine receptor subtypes, and begin to explore the intracellular signaling cascades involved. In addition, we sought to identify whether the enduring effect could be extended to other models of neuropathic pain. We tested an A1R and A2BR agonist in CCI and found the same long duration effect with A2BR but not A1R agonism. An A2AR agonist (ATL313) produced a significant long-duration reversal of mechanical allodynia induced by long established CCI (administered 6 weeks after surgery), spinal nerve ligation and sciatic inflammatory neuropathy. To determine if ATL313 had a direct effect on glia, ATL313 was coadministered with lipopolysaccharide to neonatal microglia and astrocytes in vitro. ATL313 significantly attenuated TNFα production in both microglia and astrocytes but had no effect on LPS induced IL-10. Protein kinase C significantly reversed the ATL313 effects on TNFα in vitro in microglia and astrocytes, while a protein kinase A inhibitor only effected microglia. Both intrathecal PKA and PKC inhibitors significantly reversed the effect of the A2AR agonist on neuropathic allodynia. Therefore, A2AR agonists administered IT remain an exciting novel target for the treatment of neuropathic pain.

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

    PubMed Central

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

    1998-01-01

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

  7. MASK, a large ankyrin repeat and KH domain-containing protein involved in Drosophila receptor tyrosine kinase signaling.

    PubMed

    Smith, Rachel K; Carroll, Pamela M; Allard, John D; Simon, Michael A

    2002-01-01

    The receptor tyrosine kinases Sevenless (SEV) and the Epidermal growth factor receptor (EGFR) are required for the proper development of the Drosophila eye. The protein tyrosine phosphatase Corkscrew (CSW) is a common component of many RTK signaling pathways, and is required for signaling downstream of SEV and EGFR. In order to identify additional components of these signaling pathways, mutations that enhanced the phenotype of a dominant negative form of Corkscrew were isolated. This genetic screen identified the novel signaling molecule MASK, a large protein that contains two blocks of ankyrin repeats as well as a KH domain. MASK genetically interacts with known components of these RTK signaling pathways. In the developing eye imaginal disc, loss of MASK function generates phenotypes similar to those generated by loss of other components of the SEV and EGFR pathways. These phenotypes include compromised photoreceptor differentiation, cell survival and proliferation. Although MASK is localized predominantly in the cellular cytoplasm, it is not absolutely required for MAPK activation or nuclear translocation. Based on our results, we propose that MASK is a novel mediator of RTK signaling, and may act either downstream of MAPK or transduce signaling through a parallel branch of the RTK pathway.

  8. cAMP-dependent protein kinase inhibits α7 nicotinic receptor activity in layer 1 cortical interneurons through activation of D1/D5 dopamine receptors

    PubMed Central

    Komal, Pragya; Estakhr, Jasem; Kamran, Melad; Renda, Anthony; Nashmi, Raad

    2015-01-01

    Phosphorylation of ion channels, including nicotinic acetylcholine receptors (nAChRs), by protein kinases plays a key role in the modification of synaptic transmission and neuronal excitability. α7 nAChRs are the second most prevalent nAChR subtype in the CNS following α4β2. Serine 365 in the M3–M4 cytoplasmic loop of the α7 nAChR is a phosphorylation site for protein kinase A (PKA). D1/D5 dopamine receptors signal through the adenylate cyclase–PKA pathway and play a key role in working memory and attention in the prefrontal cortex. Thus, we examined whether the dopaminergic system, mediated through PKA, functionally interacts with the α7-dependent cholinergic neurotransmission. In layer 1 interneurons of mouse prefrontal cortex, α7 nicotinic currents were decreased upon stimulation with 8-Br-cAMP, a PKA activator. In HEK 293T cells, dominant negative PKA abolished 8-Br-cAMP's effect of diminishing α7 nicotinic currents, while a constitutively active PKA catalytic subunit decreased α7 currents. In brain slices, the PKA inhibitor KT-5720 nullified 8-Br-cAMP's effect of attenuating α7 nicotinic responses, while applying a PKA catalytic subunit in the pipette solution decreased α7 currents. 8-Br-cAMP stimulation reduced surface expression of α7 nAChRs, but there was no change in single-channel conductance. The D1/D5 dopamine receptor agonist SKF 83822 similarly attenuated α7 nicotinic currents from layer 1 interneurons and this attenuation of nicotinic current was prevented by KT-5720. These results demonstrate that dopamine receptor-mediated activation of PKA negatively modulates nicotinic neurotransmission in prefrontal cortical interneurons, which may be a contributing mechanism of dopamine modulation of cognitive behaviours such as attention or working memory. PMID:25990637

  9. Testis dysfunction by isoproterenol is mediated by upregulating endothelin receptor A, leptin and protein kinase Cvarepsilon and is attenuated by an endothelin receptor antagonist CPU0213.

    PubMed

    Cheng, Yu-Si; Dai, De-Zai; Dai, Yin

    2010-07-01

    This study has examined whether upregulation of endothelin receptor A, leptin and phosphorylated protein kinase Cvarepsilon contributes to stress-induced testicular damaged and its possible reversal by endothelial (ET) antagonism. Adult male Sprague-Dawley rats were randomly divided into control and isoproterenol (ISO 1mg/kg, subcutaneous (s.c.), 10 days) groups, and intervened with the ET receptor antagonist CPU0213 (20mg/kg, s.c.), on days 6-10. In ISO group, testicular succinate dehydrogenase, lactate dehydrogenase, acid phosphotase, and gamma-glutamyl transpeptidase, and serum testosterone decreased, whereas FSH increased, relative to control. The seminiferous tubules were damaged in association with testicular upregulation of protein abundance of leptin and pPKCvarepsilon, and mRNA and protein expression of leptin receptor (OBRb) and ET(A). CPU0213 was effective in ameliorating these abnormalities. Over-expression of ET(A) and leptin accounting for the testis dysfunction is likely to be mediated by pPKCvarepsilon in the ISO treated rats. The upregulated ET pathway appears to be critical in pathologies of the testis.

  10. Evaluation of the role of g protein-coupled receptor kinase 3 in desensitization of mouse odorant receptors in a Mammalian cell line and in olfactory sensory neurons.

    PubMed

    Kato, Aya; Reisert, Johannes; Ihara, Sayoko; Yoshikawa, Keiichi; Touhara, Kazushige

    2014-11-01

    Thousands of odors are sensed and discriminated by G protein-coupled odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs). G protein-coupled receptor kinases (GRKs) may have a role in desensitization of ORs. However, whether ORs are susceptible to agonist-dependent desensitization and whether GRKs affect odorant responsiveness of OSNs are currently unknown. Here we show that GRK3 attenuated the agonist responsiveness of a specific mouse odorant receptor for eugenol (mOR-EG) upon agonist pretreatment in HEK293 cells, but GRK3 did not affect the response amplitude or the recovery kinetics upon repeated agonist stimulation. We performed electrophysiological recordings of single OSNs which expressed mOR-EG and green fluorescent protein (GFP) in the presence or absence of GRK3. The kinetics and amplitude of agonist responsiveness of individual GFP-labeled mOR-EG neurons were not significantly affected by the absence of GRK3. These results indicate that the role of GRK3 in attenuating ORs responsiveness in OSNs may have been overestimated.

  11. Evaluation of the Role of G Protein-Coupled Receptor Kinase 3 in Desensitization of Mouse Odorant Receptors in a Mammalian Cell Line and in Olfactory Sensory Neurons

    PubMed Central

    Kato, Aya; Reisert, Johannes; Ihara, Sayoko; Yoshikawa, Keiichi

    2014-01-01

    Thousands of odors are sensed and discriminated by G protein-coupled odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs). G protein-coupled receptor kinases (GRKs) may have a role in desensitization of ORs. However, whether ORs are susceptible to agonist-dependent desensitization and whether GRKs affect odorant responsiveness of OSNs are currently unknown. Here we show that GRK3 attenuated the agonist responsiveness of a specific mouse odorant receptor for eugenol (mOR-EG) upon agonist pretreatment in HEK293 cells, but GRK3 did not affect the response amplitude or the recovery kinetics upon repeated agonist stimulation. We performed electrophysiological recordings of single OSNs which expressed mOR-EG and green fluorescent protein (GFP) in the presence or absence of GRK3. The kinetics and amplitude of agonist responsiveness of individual GFP-labeled mOR-EG neurons were not significantly affected by the absence of GRK3. These results indicate that the role of GRK3 in attenuating ORs responsiveness in OSNs may have been overestimated. PMID:25313015

  12. Paroxetine Is a Direct Inhibitor of G Protein-Coupled Receptor Kinase 2 and Increases Myocardial Contractility

    SciTech Connect

    Thal, David M.; Homan, Kristoff T.; Chen, Jun; Wu, Emily K.; Hinkle, Patricia M.; Huang, Z. Maggie; Chuprun, J. Kurt; Song, Jianliang; Gao, Erhe; Cheung, Joseph Y.; Sklar, Larry A.; Koch, Walter J.; Tesmer, John J.G.

    2012-08-10

    G protein-coupled receptor kinase 2 (GRK2) is a well-established therapeutic target for the treatment of heart failure. In this paper we identify the selective serotonin reuptake inhibitor (SSRI) paroxetine as a selective inhibitor of GRK2 activity both in vitro and in living cells. In the crystal structure of the GRK2·paroxetine–Gβγ complex, paroxetine binds in the active site of GRK2 and stabilizes the kinase domain in a novel conformation in which a unique regulatory loop forms part of the ligand binding site. Isolated cardiomyocytes show increased isoproterenol-induced shortening and contraction amplitude in the presence of paroxetine, and pretreatment of mice with paroxetine before isoproterenol significantly increases left ventricular inotropic reserve in vivo with no significant effect on heart rate. Neither is observed in the presence of the SSRI fluoxetine. Our structural and functional results validate a widely available drug as a selective chemical probe for GRK2 and represent a starting point for the rational design of more potent and specific GRK2 inhibitors.

  13. Interleukin-1-receptor-associated kinase 2 (IRAK2)-mediated interleukin-1-dependent nuclear factor kappaB transactivation in Saos2 cells requires the Akt/protein kinase B kinase.

    PubMed Central

    Cenni, Vittoria; Sirri, Alessandra; De Pol, Anto; Maraldi, Nadir Mario; Marmiroli, Sandra

    2003-01-01

    The post-receptor pathway that leads to nuclear factor kappaB (NF-kappaB) activation begins with the assembly of a membrane-proximal complex among the interleukin 1 (IL-1) receptors and the adaptor molecules, myeloid differentiation protein 88 (MyD88), IL-1-receptor-associated kinases (IRAKs) and tumour-necrosis-factor-receptor-associated factor 6. Eventually, phosphorylation of the inhibitor of NF-kappaB (IkappaB) by the IkappaB kinases releases NF-kappaB, which translocates to the nucleus and modulates gene expression. In this paper, we report that IRAK2 and MyD88, but not IRAK1, interact physically with Akt, as demonstrated by co-immunoprecipitation and pull-down experiments. Interestingly, the association of Akt with recombinant IRAK2 is decreased by stimulation with IL-1, and is favoured by pre-treatment with phosphatase. Likewise, Akt association with IRAK2 is increased considerably by overexpression of PTEN (phosphatase and tensin homologue deleted on chromosome 10), while it is completely abrogated by overexpression of phosphoinositide-dependent protein kinase 1. These data indicate that Akt takes part in the formation of the signalling complex that conveys the signal from the IL-1 receptors to NF-kappaB, a step that is much more membrane-proximal than was reported previously. We also demonstrate that Akt activity is necessary for IL-1-dependent NF-kappaB transactivation, since a kinase-defective mutant of Akt impairs IRAK2- and MyD88-dependent, but not IRAK1-dependent, NF-kappaB activity, as monitored by a gene reporter assay. Accordingly, IRAK2 failed to trigger inducible nitric oxide synthase and IL-1beta production in cells expressing dominant-negative Akt. However, NF-kappaB binding to DNA was not affected by inhibition of Akt, indicating that Akt regulates NF-kappaB at a level distinct from the dissociation of p65 from IkappaBalpha and its translocation to the nucleus, possibly involving phosphorylation of the p65 transactivation domain. PMID:12906710

  14. Synergistic regulation of m2 muscarinic acetylcholine receptor desensitization and sequestration by G protein-coupled receptor kinase-2 and beta-arrestin-1.

    PubMed

    Schlador, M L; Nathanson, N M

    1997-07-25

    The m2 muscarinic acetylcholine receptor (m2 mAChR) belongs to the superfamily of G protein-coupled receptors and is regulated by many processes that attenuate signaling following prolonged stimulation by agonist. We used a heterologous expression system to examine the ability of G protein-coupled receptor kinase-2 (GRK2) and beta-arrestin-1 to regulate the phosphorylation state and to promote desensitization and sequestration of the m2 mAChR. Treatment of JEG-3 cells transiently expressing the m2 mAChR with a muscarinic agonist induced an approximately 4- or 8-fold increase in receptor phosphorylation in the absence or presence of cotransfected GRK2, respectively, compared with untreated cells transfected with receptor alone. Using the expression of a cAMP-regulated reporter gene to measure receptor function, we found that transiently transfected m2 mAChRs underwent functional desensitization following exposure to agonist. Transfected GRK2 enhanced agonist-induced functional desensitization in a manner that was synergistically enhanced by cotransfection of beta-arrestin-1, which had no effect on m2 mAChR function when coexpressed in the absence of GRK2. Finally, GRK2 and beta-arrestin-1 synergistically enhanced both the rate and extent of agonist-induced m2 mAChR sequestration. These results are the first to demonstrate that agonist-induced desensitization and sequestration of the m2 mAChR in the intact cell can be enhanced by the presence of GRK2 and beta-arrestin-1 and show that these molecules have multiple actions on the m2 mAChR.

  15. The chemokine receptors CXCR1 and CXCR2 couple to distinct G protein-coupled receptor kinases to mediate and regulate leukocyte functions

    PubMed Central

    Raghuwanshi, Sandeep K.; Su, Yingjun; Singh, Vandana; Hayes, Katherine; Richmond, Ann; Richardson, Ricardo M.

    2012-01-01

    The chemokine receptors, CXCR1 and CXCR2, couple to Gαi to induce leukocyte recruitment and activation at sites of inflammation. Upon activation by CXCL8, these receptors become phosphorylated, desensitized and internalized. In this study we investigated the role of different G protein-coupled receptor kinases (GRKs) in CXCR1- and CXCR2-mediated cellular functions. To that end, shRNA was used to inhibit GRK 2, 3, 5 and 6 in RBL-2H3 cells stably expressing CXCR1 or CXCR2, and CXCL8-mediated receptor activation and regulation were assessed. Inhibition of GRK2 and GRK6, respectively, increased CXCR1 and CXCR2 resistance to phosphorylation, desensitization and internalization, and enhanced CXCL8-induced phosphoinositide hydrolysis and exocytosis in vitro. GRK2 depletion diminished CXCR1-induced ERK1/2 phosphorylation but had no effect in CXCR2-induced ERK1/2 phosphorylation. GRK6 depletion had no significant effect on CXCR1 function. However, peritoneal neutrophils from mice deficient in GRK6 (GRK6−/−) displayed an increase in CXCR2-mediated G-protein activation, but in vitro exhibited a decrease in chemotaxis, receptor desensitization and internalization relative to wild type (GRK6+/+) cells. In contrast, neutrophil recruitment in vivo in GRK6−/− mice was increased in response to delivery of CXCL1 through the air-pouch model. In a wound closure assay, GRK6−/− mice showed enhanced myeloperoxidase activity, suggesting enhanced neutrophil recruitment, and faster wound closure as compared to GRK6+/+ animals. Taken together, the results indicate that CXCR1 and CXCR2 couple to distinct GRK isoforms to mediate and regulate inflammatory responses. CXCR1 predominantly couples to GRK2, whereas CXCR2 interacts with GRK6 to negatively regulate receptor sensitization and trafficking, thus affecting cell signaling and angiogenesis. PMID:22869904

  16. Extracellular Signal-Regulated Protein Kinase, c-Jun N-terminal Protein Kinase, and Calcineurin Regulate Transient Receptor Potential M3 (TRPM3) Induced Activation of AP-1.

    PubMed

    Lesch, Andrea; Rössler, Oliver G; Thiel, Gerald

    2017-01-23

    Stimulation of transient receptor potential M3 (TRPM3) cation channels with pregnenolone sulfate induces an influx of Ca(2+) ions into the cells and a rise in the intracellular Ca(2+) concentration, leading to the activation of the activator protein-1 (AP-1) transcription factor. Here, we show that expression of a constitutively active mutant of the Ca(2+) /calmodulin-dependent protein phosphatase calcineurin attenuated pregnenolone sulfate-induced AP-1 activation in TRPM3-expressing cells. Likewise, expression of the regulatory B subunit of calcineurin reduced AP-1 activity in the cells following stimulation of TRPM3 channels. MAP kinase phosphatase-1 has been shown to attenuate TRPM3-mediated AP-1 activation. Here, we show that pregnenolone sulfate-induced stimulation of TRPM3 triggers the phosphorylation and activation of the MAP kinase extracellular signal-regulated protein kinase (ERK1/2). Pharmacological and genetic experiments revealed that stimulation of ERK1/2 is essential for the activation of AP-1 in cells expressing stimulated TRPM3 channels. ERK1/2 is required for the activation of the transcription factor c-Jun, a key component of the AP-1 transcription factor, and regulates c-Fos promoter activity. In addition, we identified c-Jun N-terminal protein kinase (JNK1/2) as a second signal transducer of activated TRPM3 channels. Together, the data show that calcineurin and the protein kinases ERK1/2 and JNK1/2 are important regulators within the signaling cascade connecting TRPM3 channel stimulation with increased AP-1-regulated transcription. This article is protected by copyright. All rights reserved.

  17. Suppression of Mitochondrial Biogenesis through Toll-Like Receptor 4–Dependent Mitogen-Activated Protein Kinase Kinase/Extracellular Signal-Regulated Kinase Signaling in Endotoxin-Induced Acute Kidney Injury

    PubMed Central

    Smith, Joshua A.; Stallons, L. Jay; Collier, Justin B.; Chavin, Kenneth D.

    2015-01-01

    Although disruption of mitochondrial homeostasis and biogenesis (MB) is a widely accepted pathophysiologic feature of sepsis-induced acute kidney injury (AKI), the molecular mechanisms responsible for this phenomenon are unknown. In this study, we examined the signaling pathways responsible for the suppression of MB in a mouse model of lipopolysaccharide (LPS)-induced AKI. Downregulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of MB, was noted at the mRNA level at 3 hours and protein level at 18 hours in the renal cortex, and was associated with loss of renal function after LPS treatment. LPS-mediated suppression of PGC-1α led to reduced expression of downstream regulators of MB and electron transport chain proteins along with a reduction in renal cortical mitochondrial DNA content. Mechanistically, Toll-like receptor 4 (TLR4) knockout mice were protected from renal injury and disruption of MB after LPS exposure. Immunoblot analysis revealed activation of tumor progression locus 2/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (TPL-2/MEK/ERK) signaling in the renal cortex by LPS. Pharmacologic inhibition of MEK/ERK signaling attenuated renal dysfunction and loss of PGC-1α, and was associated with a reduction in proinflammatory cytokine (e.g., tumor necrosis factor-α [TNF-α], interleukin-1β) expression at 3 hours after LPS exposure. Neutralization of TNF-α also blocked PGC-1α suppression, but not renal dysfunction, after LPS-induced AKI. Finally, systemic administration of recombinant tumor necrosis factor-α alone was sufficient to produce AKI and disrupt mitochondrial homeostasis. These findings indicate an important role for the TLR4/MEK/ERK pathway in both LPS-induced renal dysfunction and suppression of MB. TLR4/MEK/ERK/TNF-α signaling may represent a novel therapeutic target to prevent mitochondrial dysfunction and AKI produced by sepsis. PMID:25503387

  18. Signal-transduction pathways that regulate visceral smooth muscle function. III. Coupling of muscarinic receptors to signaling kinases and effector proteins in gastrointestinal smooth muscles.

    PubMed

    Gerthoffer, William T

    2005-05-01

    Stimulation of muscarinic M3 and M2 receptors on gastrointestinal smooth muscle elicits contraction via activation of G proteins that are coupled to a diverse set of downstream signaling pathways and effector proteins. Many studies suggest a canonical excitation-contraction coupling pathway that includes activation of phospholipases, production of inositol 1,4,5-trisphosphate and diacylglycerol, release of calcium from the sarcoplasmic reticulum, activation of L-type calcium channels, and activation of nonselective cation channels. These events lead to elevated intracellular calcium concentration, which activates myosin light chain kinase to phosphorylate and activate myosin II thus causing contraction. In addition, muscarinic receptors are coupled to signaling pathways that modulate the effect of activator calcium. The Rho/Rho kinase pathway inhibits myosin light chain phosphatase, one of the key steps in sensitization of the contractile proteins to calcium. Phosphatidylinositol 3-kinases and Src family tyrosine kinases are also activated by muscarinic agonists. Src family tyrosine kinases regulate L-type calcium and nonselective cation channels. Src activation also leads to activation of ERK and p38 MAPKs. ERK MAPKs phosphorylate caldesmon, an actin filament binding protein. P38 MAPKs activate phospholipases and MAPKAP kinase 2/3, which phosphorylate HSP27. HSP27 may regulate cross-bridge function, actin filament formation, and actin filament attachment to the cell membrane. In addition to the well-known role of M3 muscarinic receptors to regulate myoplasmic calcium levels, the integrated effect of muscarinic activation probably also includes signaling pathways that modulate phospholipases, cyclic nucleotides, contractile protein function, and cytoskeletal protein function.

  19. Neuroprotective effects of inhibiting N-methyl-D-aspartate receptors, P2X receptors and the mitogen-activated protein kinase cascade: a quantitative analysis in organotypical hippocampal slice cultures subjected to oxygen and glucose deprivation.

    PubMed

    Rundén-Pran, E; Tansø, R; Haug, F M; Ottersen, O P; Ring, A

    2005-01-01

    Cell death was assessed by quantitative analysis of propidium iodide uptake in rat hippocampal slice cultures transiently exposed to oxygen and glucose deprivation, an in vitro model of brain ischemia. The hippocampal subfields CA1 and CA3, and fascia dentata were analyzed at different stages from 0 to 48 h after the insult. Cell death appeared at 3 h and increased steeply toward 12 h. Only a slight additional increase in propidium iodide uptake was seen at later intervals. The mitogen-activated protein kinases extracellular signal-regulated kinase 1 and extracellular signal-regulated kinase 2 were activated immediately after oxygen and glucose deprivation both in CA1 and in CA3/fascia dentata. Inhibition of the specific mitogen-activated protein kinase activator mitogen-activated protein kinase kinase by PD98059 or U0126 offered partial protection against oxygen and glucose deprivation-induced cell damage. The non-selective P2X receptor antagonist suramin gave neuroprotection of the same magnitude as the N-methyl-D-aspartate channel blocker MK-801 (approximately 70%). Neuroprotection was also observed with the P2 receptor blocker PPADS. Immunogold data indicated that hippocampal slice cultures (like intact hippocampi) express several isoforms of P2X receptors at the synaptic level, consistent with the idea that the effects of suramin and PPADS are mediated by P2X receptors. Virtually complete neuroprotection was obtained by combined blockade of N-methyl-D-aspartate receptors, P2X receptors, and mitogen-activated protein kinase kinase. Both P2X receptors and N-methyl-D-aspartate receptors mediate influx of calcium. Our results suggest that inhibition of P2X receptors has a neuroprotective potential similar to that of inhibition of N-methyl-D-aspartate receptors. In contrast, our comparative analysis shows that only partial protection can be achieved by inhibiting the extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase cascade, one of the

  20. Protein kinase Cε is required for spinal analgesic synergy between delta opioid and alpha-2A adrenergic receptor agonist pairs.

    PubMed

    Schuster, Daniel J; Kitto, Kelley F; Overland, Aaron C; Messing, Robert O; Stone, Laura S; Fairbanks, Carolyn A; Wilcox, George L

    2013-08-14

    We recently showed that spinal synergistic interactions between δ opioid receptors (δORs) and α2A adrenergic receptors (α2AARs) require protein kinase C (PKC). To identify which PKC isoforms contribute to analgesic synergy, we evaluated the effects of various PKC-isoform-specific peptide inhibitors on synergy between δORs and α2AARs using the tail flick assay of thermal nociception in mice. Only a PKCε inhibitor abolished synergy between a δOR agonist and an α2AAR agonist. We tested a panel of combinations of opioid and adrenergic agonists in PKCε knock-out mice and found that all four combinations of a δOR agonist and an α2AAR agonist required PKCε for antinociceptive synergy. None of the combinations of a μOR agonist with an α2AR agonist required PKCε. Immunohistochemistry confirmed that PKCε could be found in the population of peptidergic primary afferent nociceptors where δORs and α2AARs have been found to extensively colocalize. Immunoreactivity for PKCε was found in the majority of dorsal root ganglion neurons and intensely labeled laminae I and II of the spinal cord dorsal horn. PKCε is widespread in the spinal nociceptive system and in peptidergic primary afferents it appears to be specifically involved in mediating the synergistic interaction between δORs and α2AARs.

  1. G protein-coupled receptor kinase 6/β-arrestin 2 system in a rat model of dopamine supersensitivity psychosis.

    PubMed

    Oda, Yasunori; Tadokoro, Shigenori; Takase, Masayuki; Kanahara, Nobuhisa; Watanabe, Hiroyuki; Shirayama, Yukihiko; Hashimoto, Kenji; Iyo, Masaomi

    2015-12-01

    In humans, long-term antipsychotic treatment is known to induce movement disorders and a psychosis, called dopamine supersensitivity psychosis (DSP). The mechanism by which chronic administration of antipsychotic(s) causes DSP may be the treatment-induced up-regulation of dopamine D2 receptors (DRD2). G protein-coupled receptor kinase 6 (GRK6) and beta-arrestin 2 (ARRB2) play important roles in the trafficking of DRD2 by phosphorylation and internalization. We investigated the effects of chronic continuous treatment with mini-pump-administered haloperidol (HAL) on the sensitivity of Wistar rats to dopamine, as measured by the locomotor response to methamphetamine (MAP) and the density of striatal DRD2. Chronic continuous treatment with HAL resulted in significantly higher locomotor response to MAP and significantly higher striatal DRD2 density compared with those in rats administered vehicle (VEH). Enzyme-linked immunosorbent assays revealed that striatal ARRB2 in DSP model rats tended to decrease in comparison with that in the VEH group. In addition, the ratio of GRK6/ARRB2 in DSP model rats was significantly higher than that in controls. Our results suggest that alterations of the GRK6 and ARRB2 system could induce both DRD2 up-regulation and impairment of the dopamine signaling pathway, resulting potentially in the development of DSP.

  2. Protein Kinase Cϵ Is Required for Spinal Analgesic Synergy between Delta Opioid and Alpha-2A Adrenergic Receptor Agonist Pairs

    PubMed Central

    Schuster, Daniel J.; Kitto, Kelley F.; Overland, Aaron C.; Messing, Robert O.; Stone, Laura S.; Fairbanks, Carolyn A.

    2013-01-01

    We recently showed that spinal synergistic interactions between δ opioid receptors (δORs) and α2A adrenergic receptors (α2AARs) require protein kinase C (PKC). To identify which PKC isoforms contribute to analgesic synergy, we evaluated the effects of various PKC-isoform-specific peptide inhibitors on synergy between δORs and α2AARs using the tail flick assay of thermal nociception in mice. Only a PKCϵ inhibitor abolished synergy between a δOR agonist and an α2AAR agonist. We tested a panel of combinations of opioid and adrenergic agonists in PKCϵ knock-out mice and found that all four combinations of a δOR agonist and an α2AAR agonist required PKCϵ for antinociceptive synergy. None of the combinations of a μOR agonist with an α2AR agonist required PKCϵ. Immunohistochemistry confirmed that PKCϵ could be found in the population of peptidergic primary afferent nociceptors where δORs and α2AARs have been found to extensively colocalize. Immunoreactivity for PKCϵ was found in the majority of dorsal root ganglion neurons and intensely labeled laminae I and II of the spinal cord dorsal horn. PKCϵ is widespread in the spinal nociceptive system and in peptidergic primary afferents it appears to be specifically involved in mediating the synergistic interaction between δORs and α2AARs. PMID:23946412

  3. Substrate recognition by the Lyn protein-tyrosine kinase. NMR structure of the immunoreceptor tyrosine-based activation motif signaling region of the B cell antigen receptor.

    PubMed

    Gaul, B S; Harrison, M L; Geahlen, R L; Burton, R A; Post, C B

    2000-05-26

    The immunoreceptor tyrosine-based activation motif (ITAM) plays a central role in transmembrane signal transduction in hematopoietic cells by mediating responses leading to proliferation and differentiation. An initial signaling event following activation of the B cell antigen receptor is phosphorylation of the CD79a (Ig-alpha) ITAM by Lyn, a Src family protein-tyrosine kinase. To elucidate the structural basis for recognition between the ITAM substrate and activated Lyn kinase, the structure of an ITAM-derived peptide bound to Lyn was determined using exchange-transferred nuclear Overhauser NMR spectroscopy. The bound substrate structure has an irregular helix-like character. Docking based on the NMR data into the active site of the closely related Lck kinase strongly favors ITAM binding in an orientation similar to binding of cyclic AMP-dependent protein kinase rather than that of insulin receptor tyrosine kinase. The model of the complex provides a rationale for conserved ITAM residues, substrate specificity, and suggests that substrate binds only the active conformation of the Src family tyrosine kinase, unlike the ATP cofactor, which can bind the inactive form.

  4. Physical and functional association of a serine-threonine protein kinase to the cytoplasmic domain of the p80 form of the human tumor necrosis factor receptor in human histiocytic lymphoma U-937 cells.

    PubMed

    Darnay, B G; Reddy, S A; Aggarwal, B B

    1994-08-05

    Tumor necrosis factor (TNF) binds two distinct cell surface receptors designated p60 and p80. Our previous studies indicate that a protein kinase from U-937 cells binds to and phosphorylates the p60 receptor. While the p80 receptor is phosphorylated in vivo, no association of a protein kinase has been described. We employed a fusion protein comprising of glutathione S-transferase and the cytoplasmic domain of the p80 receptor (GST-p80CD) to identify cellular proteins that might associate with this receptor. From 35S- and 32P-labeled cells, a protein of 59 kDa bound specifically to GST-p80CD. In vitro kinase reactions indicated that serine/threonine protein kinase activity associated with GST-p80CD and causes its phosphorylation. Additionally, a 59-kDa phosphoprotein was also identified after kinase reactions of proteins bound to GST-p80CD. This kinase activity required either Mg2+ or Mn2+ for optimal activity, and it phosphorylated myelin basic protein, histone H2B, and also the cytoplasmic domain of the p60 receptor. Treatment of cells with TNF increased the p80 receptor-associated kinase activity by 200%. In summary, our results provide evidence of a novel ligand-activated serine/threonine protein kinase that associates with the cytoplasmic domain of the p80 receptor and causes the phosphorylation of both forms of the TNF receptor. This p80 TNF receptor-associated protein and the associated kinase described here are referred to as p80-TRAP and p80-TRAK, respectively.

  5. CCAAT/enhancer-binding protein β: its role in breast cancer and associations with receptor tyrosine kinases

    PubMed Central

    Zahnow, Cynthia A.

    2011-01-01

    The CCAAT/enhancer-binding proteins (C/EBPs) are a family of leucine-zipper transcription factors that regulate gene expression to control cellular proliferation, differentiation, inflammation and metabolism. Encoded by an intronless gene, C/EBPβ is expressed as several distinct protein isoforms (LAP1, LAP2, LIP) whose expression is regulated by the differential use of several in-frame translation start sites. LAP1 and LAP2 are transcriptional activators and are associated with differentiation, whereas LIP is frequently elevated in proliferative tissue and acts as a dominant-negative inhibitor of transcription. However, emerging evidence suggests that LIP can serve as a transcriptional activator in some cellular contexts, and that LAP1 and LAP2 might also have unique actions. The LIP:LAP ratio is crucial for the maintenance of normal growth and development, and increases in this ratio lead to aggressive forms of breast cancer. This review discusses the regulation of C/EBPβ activity by post-translational modification, the individual actions of LAP1, LAP2 and LIP, and the functions and downstream targets that are unique to each isoform. The role of the C/EBPβ isoforms in breast cancer is discussed and emphasis is placed on their interactions with receptor tyrosine kinases. PMID:19351437

  6. The A-kinase anchoring protein (AKAP)-Lbc-signaling complex mediates alpha1 adrenergic receptor-induced cardiomyocyte hypertrophy.

    PubMed

    Appert-Collin, Aline; Cotecchia, Susanna; Nenniger-Tosato, Monique; Pedrazzini, Thierry; Diviani, Dario

    2007-06-12

    In response to various pathological stresses, the heart undergoes a pathological remodeling process that is associated with cardiomyocyte hypertrophy. Because cardiac hypertrophy can progress to heart failure, a major cause of lethality worldwide, the intracellular signaling pathways that control cardiomyocyte growth have been the subject of intensive investigation. It has been known for more than a decade that the small molecular weight GTPase RhoA is involved in the signaling pathways leading to cardiomyocyte hypertrophy. Although some of the hypertrophic pathways activated by RhoA have now been identified, the identity of the exchange factors that modulate its activity in cardiomyocytes is currently unknown. In this study, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor activity, is critical for activating RhoA and transducing hypertrophic signals downstream of alpha1-adrenergic receptors (ARs). In particular, our results indicate that suppression of AKAP-Lbc expression by infecting rat neonatal ventricular cardiomyocytes with lentiviruses encoding AKAP-Lbc-specific short hairpin RNAs strongly reduces both alpha1-AR-mediated RhoA activation and hypertrophic responses. Interestingly, alpha1-ARs promote AKAP-Lbc activation via a pathway that requires the alpha subunit of the heterotrimeric G protein G12. These findings identify AKAP-Lbc as the first Rho-guanine nucleotide exchange factor (GEF) involved in the signaling pathways leading to cardiomyocytes hypertrophy.

  7. Receptor for Activated Protein Kinase C: Requirement for Efficient MicroRNA Function and Reduced Expression in Hepatocellular Carcinoma

    PubMed Central

    Otsuka, Motoyuki; Takata, Akemi; Yoshikawa, Takeshi; Kojima, Kentaro; Kishikawa, Takahiro; Shibata, Chikako; Takekawa, Mutsuhiro; Yoshida, Haruhiko; Omata, Masao; Koike, Kazuhiko

    2011-01-01

    MicroRNAs (miRNAs) are important regulators of gene expression that control physiological and pathological processes. A global reduction in miRNA abundance and function is a general trait of human cancers, playing a causal role in the transformed phenotype. Here, we sought to newly identify genes involved in the regulation of miRNA function by performing a genetic screen using reporter constructs that measure miRNA function and retrovirus-based random gene disruption. Of the six genes identified, RACK1, which encodes “receptor for activated protein kinase C” (RACK1), was confirmed to be necessary for full miRNA function. RACK1 binds to KH-type splicing regulatory protein (KSRP), a member of the Dicer complex, and is required for the recruitment of mature miRNAs to the RNA-induced silencing complex (RISC). In addition, RACK1 expression was frequently found to be reduced in hepatocellular carcinoma. These findings suggest the involvement of RACK1 in miRNA function and indicate that reduced miRNA function, due to decreased expression of RACK1, may have pathologically relevant roles in liver cancers. PMID:21935400

  8. Overproduction of the membrane-bound receptor-like protein kinase 1, RPK1, enhances abiotic stress tolerance in Arabidopsis.

    PubMed

    Osakabe, Yuriko; Mizuno, Shinji; Tanaka, Hidenori; Maruyama, Kyonoshin; Osakabe, Keishi; Todaka, Daisuke; Fujita, Yasunari; Kobayashi, Masatomo; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2010-03-19

    RPK1 (receptor-like protein kinase 1) localizes to the plasma membrane and functions as a regulator of abscisic acid (ABA) signaling in Arabidopsis. In our current study, we investigated the effect of RPK1 disruption and overproduction upon plant responses to drought stress. Transgenic Arabidopsis overexpressing the RPK1 protein showed increased ABA sensitivity in their root growth and stomatal closure and also displayed less transpirational water loss. In contrast, a mutant lacking RPK1 function, rpk1-1, was found to be resistant to ABA during these processes and showed increased water loss. RPK1 overproduction in these transgenic plants thus increased their tolerance to drought stress. We performed microarray analysis of RPK1 transgenic plants and observed enhanced expression of several stress-responsive genes, such as Cor15a, Cor15b, and rd29A, in addition to H(2)O(2)-responsive genes. Consistently, the expression levels of ABA/stress-responsive genes in rpk1-1 had decreased compared with wild type. The results suggest that the overproduction of RPK1 enhances both the ABA and drought stress signaling pathways. Furthermore, the leaves of the rpk1-1 plants exhibit higher sensitivity to oxidative stress upon ABA-pretreatment, whereas transgenic plants overproducing RPK1 manifest increased tolerance to this stress. Our current data suggest therefore that RPK1 overproduction controls reactive oxygen species homeostasis and enhances both water and oxidative stress tolerance in Arabidopsis.

  9. Protein kinase C involvement in homologous desensitization of delta-opioid receptor coupled to Gi1-phospholipase C activation in Xenopus oocytes.

    PubMed

    Ueda, H; Miyamae, T; Hayashi, C; Watanabe, S; Fukushima, N; Sasaki, Y; Iwamura, T; Misu, Y

    1995-11-01

    We have developed the coexpression system of both delta-opioid receptor (DOR1) and M2-muscarinic receptor (M2) which mediate agonist-evoked currents due to common post-receptor mechanisms including Gi1 and phospholipase C (PLC) activation in Xenopus oocytes reconstituted with Gi1 alpha. The DOR1-currents by 100 nM D-Ser2-leu-enkephalin-Thr6 (DSLET) were selectively desensitized by 10 nM phorbol 12-myristate 13-acetate (PMA). The PMA-desensitization of DSLET-currents was abolished in the presence of calphostin C, a protein kinase C inhibitor, or reversed by an intracellular injection of calcineurin, a protein phosphatase 2B. When a higher concentration (3 microM) of DSLET was used, DSLET-currents were rapidly desensitized by repeated challenges of DSLET itself. However, repeated challenges of 10 microM ACh caused no influence on such DSLET- or M2-currents. The desensitization of DSLET-currents was selectively reversed by protein kinase C inhibitors. Similar results were also obtained with various delta-opioid agonists. These results suggest that protein kinase C is involved in the homologous desensitization of delta-opioid receptors.

  10. Prenatal testosterone exposure induces hypertension in adult females via androgen receptor-dependent protein kinase Cδ-mediated mechanism.

    PubMed

    Blesson, Chellakkan S; Chinnathambi, Vijayakumar; Hankins, Gary D; Yallampalli, Chandra; Sathishkumar, Kunju

    2015-03-01

    Prenatal exposure to excess testosterone induces hyperandrogenism in adult females and predisposes them to hypertension. We tested whether androgens induce hypertension through transcriptional regulation and signaling of protein kinase C (PKC) in the mesenteric arteries. Pregnant Sprague-Dawley rats were injected with vehicle or testosterone propionate (0.5 mg/kg per day from gestation days 15 to 19, SC) and their 6-month-old adult female offspring were examined. Plasma testosterone levels (0.84±0.04 versus 0.42±0.09 ng/mL) and blood pressures (111.6±1.3 versus 104.5±2.4 mm Hg) were significantly higher in prenatal testosterone-exposed rats compared with controls. This was accompanied with enhanced expression of PKCδ mRNA (1.5-fold) and protein (1.7-fold) in the mesenteric arteries of prenatal testosterone-exposed rats. In addition, mesenteric artery contractile responses to PKC activator, phorbol-12,13-dibutyrate, was significantly greater in prenatal testosterone-exposed rats. Treatment with androgen receptor antagonist flutamide (10 mg/kg, SC, BID for 10 days) significantly attenuated hypertension, PKCδ expression, and the exaggerated vasoconstriction in prenatal testosterone-exposed rats. In vitro exposure of testosterone to cultured mesenteric artery smooth muscle cells dose dependently upregulated PKCδ expression. Analysis of PKCδ gene revealed a putative androgen responsive element in the promoter upstream to the transcription start site and an enhancer element in intron-1. Chromatin immunoprecipitation assays showed that androgen receptors bind to these elements in response to testosterone stimulation. Furthermore, luciferase reporter assays showed that the enhancer element is highly responsive to androgens and treatment with flutamide reverses reporter activity. Our studies identified a novel androgen-mediated mechanism for the control of PKCδ expression via transcriptional regulation that controls vasoconstriction and blood pressure.

  11. Protein kinase C-mediated inhibition of transmembrane signalling through CCK(A) and CCK(B) receptors.

    PubMed

    Smeets, R L; Fouraux, M A; van Emst-de Vries, S E; De Pont, J J; Willems, P H

    1998-03-01

    1. The rat CCK(A) and CCK(B) receptors were stably expressed in Chinese hamster ovary (CHO-09) cells in order to compare modes of signal transduction and effects of protein kinase C (PKC) thereupon. 2. Spectrofluorophotometry of Fura-2-loaded cells revealed that both receptors retained their pharmacological characteristics following expression in CHO cells. Sulphated cholecystokinin-(26-33)-peptide amide (CCK-8-S) increased the cytosolic Ca2+ concentration ([Ca2+]i) in CCK(A) cells, measured as an increase in Fura-2 fluorescence emission ratio, 1000 fold more potently than its non-sulphated form (CCK-8-NS) (EC50 values of 0.19 nM and 0.18 microM, respectively). By contrast, CCK-8-S and CCK-8-NS were equally potent in CCK(B) cells (EC50 values of 0.86 nM and 1.18 nM, respectively). The CCK(A) receptor agonist JMV-180 increased [Ca2+]i only in CCK(A) cells. Likewise, pentagastrin increased [Ca2+]i only in CCK(B) cells. Finally, CCK-8-S-induced Ca2+ signalling through the CCK(A) receptor was most potently inhibited by the CCK(A) receptor antagonist L364,718, whereas the CCK(B) receptor antagonist L365,260 was more potent in CCK(B) cells. 3. Receptor-mediated activation of adenylyl cyclase was measured in the presence of the inhibitor of cyclic nucleotide phosphodiesterase activity, 3-isobutyl-1-methylxanthine. CCK-8-S and, to a lesser extent, CCK-8-NS, but not JMV-180 or pentagastrin, stimulated the accumulation of cyclicAMP in CCK(A) cells. By contrast, none of these agonists increased cyclicAMP in CCK(B) cells. 4. Short-term (3 min) pretreatment with the PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA) evoked a rightward shift of the dose-response curve for the Ca2+ mobilizing effect of CCK-8-S in both cell lines. In addition, short-term TPA pretreatment markedly reduced CCK-8-S-induced cyclicAMP accumulation in CCK(A) cells. In both cases, the inhibitory effect of TPA was abolished by the PKC inhibitors, GF-109203X and staurosporine, whereas no inhibition

  12. Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: Essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase

    SciTech Connect

    Hien, Tran Thi; Kim, Nak Doo; Pokharel, Yuba Raj; Oh, Seok Jeong; Lee, Moo Yeol; Kang, Keon Wook

    2010-08-01

    We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10 {mu}g/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

  13. Ginsenoside Rg3 increases nitric oxide production via increases in phosphorylation and expression of endothelial nitric oxide synthase: essential roles of estrogen receptor-dependent PI3-kinase and AMP-activated protein kinase.

    PubMed

    Hien, Tran Thi; Kim, Nak Doo; Pokharel, Yuba Raj; Oh, Seok Jeong; Lee, Moo Yeol; Kang, Keon Wook

    2010-08-01

    We previously showed that ginsenosides increase nitric oxide (NO) production in vascular endothelium and that ginsenoside Rg3 (Rg3) is the most active one among ginseng saponins. However, the mechanism for Rg3-mediated nitric oxide production is still uncertain. In this study, we determined whether Rg3 affects phosphorylation and expression of endothelial nitric oxide synthase (eNOS) in ECV 304 human endothelial cells. Rg3 increased both the phosphorylation and the expression of eNOS in a concentration-dependent manner and a maximal effect was found at 10μg/ml of Rg3. The enzyme activities of phosphatidylinositol 3-kinase (PI3-kinase), c-Jun N-terminal kinase (JNK), and p38 kinase were enhanced as were estrogen receptor (ER)- and glucocorticoid receptor (GR)-dependent reporter gene transcriptions in Rg3-treated endothelial cells. Rg3-induced eNOS phosphorylation required the ER-mediated PI3-kinase/Akt pathway. Moreover, Rg3 activates AMP-activated protein kinase (AMPK) through up-regulation of CaM kinase II and Rg3-stimulated eNOS phosphorylation was reversed by AMPK inhibition. The present results provide a mechanism for Rg3-stimulated endothelial NO production.

  14. Identification of TIFA as an adapter protein that links tumor necrosis factor receptor-associated factor 6 (TRAF6) to interleukin-1 (IL-1) receptor-associated kinase-1 (IRAK-1) in IL-1 receptor signaling.

    PubMed

    Takatsuna, Hiroshi; Kato, Hiroki; Gohda, Jin; Akiyama, Taishin; Moriya, Ayaka; Okamoto, Yoshinari; Yamagata, Yuriko; Otsuka, Masami; Umezawa, Kazuo; Semba, Kentaro; Inoue, Jun-Ichiro

    2003-04-04

    Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals from members of the Toll/interleukin-1 (IL-1) receptor family by interacting with IL-1 receptor-associated kinase-1 (IRAK-1) after IRAK-1 is released from the receptor-MyD88 complex upon IL-1 stimulation. However, the molecular mechanisms underlying regulation of the IRAK-1/TRAF6 interaction are largely unknown. We have identified TIFA, a TRAF-interacting protein with a forkhead-associated (FHA) domain. The FHA domain is a motif known to bind directly to phosphothreonine and phosphoserine. In transient transfection assays, TIFA activates NFkappaBeta and c-Jun amino-terminal kinase. However, TIFA carrying a mutation that abolishes TRAF6 binding or mutations in the FHA domain that are known to abolish FHA domain binding to phosphopeptide fails to activate NFkappaBeta and c-Jun amino-terminal kinase. TIFA, when overexpressed, binds both TRAF6 and IRAK-1 and significantly enhances the IRAK-1/TRAF6 interaction. Furthermore, analysis of endogenous proteins indicates that TIFA associates with TRAF6 constitutively, whereas it associates with IRAK-1 in an IL-1 stimulation-dependent manner in vivo. Thus, TIFA is likely to mediate IRAK-1/TRAF6 interaction upon IL-1 stimulation.

  15. Protein kinase A mediates adenosine A2a receptor modulation of neurotransmitter release via synapsin I phosphorylation in cultured cells from medulla oblongata.

    PubMed

    Matsumoto, Joao Paulo Pontes; Almeida, Marina Gomes; Castilho-Martins, Emerson Augusto; Costa, Maisa Aparecida; Fior-Chadi, Debora Rejane

    2014-08-01

    Synaptic transmission is an essential process for neuron physiology. Such process is enabled in part due to modulation of neurotransmitter release. Adenosine is a synaptic modulator of neurotransmitter release in the Central Nervous System, including neurons of medulla oblongata, where several nuclei are involved with neurovegetative reflexes. Adenosine modulates different neurotransmitter systems in medulla oblongata, specially glutamate and noradrenaline in the nucleus tractussolitarii, which are involved in hypotensive responses. However, the intracellular mechanisms involved in this modulation remain unknown. The adenosine A2a receptor modulates neurotransmitter release by activating two cAMP protein effectors, the protein kinase A and the exchange protein activated by cAMP. Therefore, an in vitro approach (cultured cells) was carried out to evaluate modulation of neurotransmission by adenosine A2a receptor and the signaling intracellular pathway involved. Results show that the adenosine A2a receptor agonist, CGS 21680, increases neurotransmitter release, in particular, glutamate and noradrenaline and such response is mediated by protein kinase A activation, which in turn increased synapsin I phosphorylation. This suggests a mechanism of A2aR modulation of neurotransmitter release in cultured cells from medulla oblongata of Wistar rats and suggest that protein kinase A mediates this modulation of neurotransmitter release via synapsin I phosphorylation.

  16. SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE Directly Interacts with the Cytoplasmic Domain of SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE and Negatively Regulates Leaf Senescence in Arabidopsis1[OPEN

    PubMed Central

    Xiao, Dong; Cui, Yanjiao; Xu, Fan; Xu, Xinxin; Gao, Guanxiao; Wang, Yaxin; Guo, Zhaoxia; Wang, Dan; Wang, Ning Ning

    2015-01-01

    Reversible protein phosphorylation mediated by protein kinases and phosphatases plays an important role in the regulation of leaf senescence. We previously reported that the leucine-rich repeat receptor-like kinase SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE (AtSARK) positively regulates leaf senescence in Arabidopsis (Arabidopsis thaliana). Here, we report the involvement of a protein serine/threonine phosphatase 2C-type protein phosphatase, SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE (SSPP), in the negative regulation of Arabidopsis leaf senescence. SSPP transcript levels decreased greatly during both natural senescence and SARK-induced precocious senescence. Overexpression of SSPP significantly delayed leaf senescence in Arabidopsis. Protein pull-down and bimolecular fluorescence complementation assays demonstrated that the cytosol-localized SSPP could interact with the cytoplasmic domain of the plasma membrane-localized AtSARK. In vitro assays showed that SSPP has protein phosphatase function and can dephosphorylate the cytosolic domain of AtSARK. Consistent with these observations, overexpression of SSPP effectively rescued AtSARK-induced precocious leaf senescence and changes in hormonal responses. All our results suggested that SSPP functions in sustaining proper leaf longevity and preventing early senescence by suppressing or perturbing SARK-mediated senescence signal transduction. PMID:26304848

  17. Imiquimod directly inhibits Hedgehog signalling by stimulating adenosine receptor/protein kinase A-mediated GLI phosphorylation

    PubMed Central

    Wolff, F; Loipetzberger, A; Gruber, W; Esterbauer, H; Aberger, F; Frischauf, A M

    2013-01-01

    Imiquimod (IMQ), a nucleoside analogue of the imidazoquinoline family, is used in the topical treatment of basal cell carcinoma (BCC) and other skin diseases. It is reported to be a TLR7 and TLR8 agonist and, as such, initiates a Th1 immune response by activating sentinel cells in the vicinity of the tumour. BCC is a hedgehog (HH)-driven malignancy with oncogenic glioma-associated oncogene (GLI) signalling activated in a ligand-independent manner. Here we show that IMQ can also directly repress HH signalling by negatively modulating GLI activity in BCC and medulloblastoma cells. Further, we provide evidence that the repressive effect of IMQ on HH signalling is not dependent on TLR/MYD88 signalling. Our results suggest a mechanism for IMQ engaging adenosine receptors (ADORAs) to control GLI signalling. Pharmacological activation of ADORA with either an ADORA agonist or IMQ resulted in a protein kinase A (PKA)-mediated GLI phosphorylation and reduction in GLI activator levels. The activation of PKA and HH pathway target gene downregulation in response to IMQ were abrogated by ADORA inhibition. Furthermore, activated Smoothened signalling, which positively signals to GLI transcription factors, could be effectively counteracted by IMQ. These results reveal a previously unknown mode of action of IMQ in the treatment of BCC and also suggest a role for ADORAs in the regulation of oncogenic HH signalling. PMID:23995793

  18. Overlapping and Opposing Functions of G Protein-coupled Receptor Kinase 2 (GRK2) and GRK5 during Heart Development*

    PubMed Central

    Philipp, Melanie; Berger, Ina M.; Just, Steffen; Caron, Marc G.

    2014-01-01

    G protein-coupled receptor kinases 2 (GRK2) and 5 (GRK5) are fundamental regulators of cardiac performance in adults but are less well characterized for their function in the hearts of embryos. GRK2 and -5 belong to different subfamilies and function as competitors in the control of certain receptors and signaling pathways. In this study, we used zebrafish to investigate whether the fish homologs of GRK2 and -5, Grk2/3 and Grk5, also have unique, complementary, or competitive roles during heart development. We found that they differentially regulate the heart rate of early embryos and equally facilitate heart function in older embryos and that both are required to develop proper cardiac morphology. A loss of Grk2/3 results in dilated atria and hypoplastic ventricles, and the hearts of embryos depleted in Grk5 present with a generalized atrophy. This Grk5 morphant phenotype was associated with an overall decrease of early cardiac progenitors as well as a reduction in the area occupied by myocardial progenitor cells. In the case of Grk2/3, the progenitor decrease was confined to a subset of precursor cells with a committed ventricular fate. We attempted to rescue the GRK loss-of-function heart phenotypes by downstream activation of Hedgehog signaling. The Grk2/3 loss-of-function embryos were rescued by this approach, but Grk5 embryos failed to respond. In summary, we found that GRK2 and GRK5 control cardiac function as well as morphogenesis during development although with different morphological outcomes. PMID:25104355

  19. A Peptide Hormone and Its Receptor Protein Kinase Regulate Plant Cell Expansion

    PubMed Central

    Haruta, Miyoshi; Sabat, Grzegorz; Stecker, Kelly; Minkoff, Benjamin B.; Sussman, Michael R.

    2015-01-01

    Plant cells are immobile; thus, plant growth and development depend on cell expansion rather than cell migration. The molecular mechanism by which the plasma membrane initiates changes in the cell expansion rate remains elusive. We found that a secreted peptide, RALF (rapid alkalinization factor), suppresses cell elongation of the primary root by activating the cell surface receptor FERONIA in Arabidopsis thaliana. A direct peptide-receptor interaction is supported by specific binding of RALF to FERONIA and reduced binding and insensitivity to RALF-induced growth inhibition in feronia mutants. Phosphoproteome measurements demonstrate that the RALF-FERONIA interaction causes phosphorylation of plasma membrane H+–adenosine triphosphatase 2 at Ser899, mediating the inhibition of proton transport. The results reveal a molecular mechanism for RALF-induced extracellular alkalinization and a signaling pathway that regulates cell expansion. PMID:24458638

  20. Roles of receptor for activated protein kinase C1 for modulating immune responses in white shrimp Litopenaeus vannamei.

    PubMed

    Chang, Zhong-Wen; Chang, Chin-Chyuan

    2015-10-01

    Complementary (c)DNA encoding a receptor for activated protein kinase C1 (RACK1) messenger (m)RNA of the white shrimp Litopenaeus vannamei, designated LvRACK1, consisted a 1136-bp cDNA containing an open reading frame (ORF) of 954 bp, a 111-bp 5'-untranslated region (UTR), and a 71-bp 3'-UTR, which is a 36 kDa cytosolic protein, belonging to the Trp-Asp40 (WD40) family of proteins, characterized by containing seven highly conserved Trp-Asp40 (WD40) internal repeats, and a poly A tail. The WD repeat of LvRACK1 can be predicted to form a seven-bladed propeller structure with each WD repeat composed of four antiparallel β-sheets. The WD40 domains have been implicated in protein-protein interactions. A comparison of amino acid sequences showed that LvRACK1 was closely related to arthropods RACK1. LvRACK1 cDNA was synthesized in all tested tissues detected with real-time PCR including haemocytes, hepatopancreas, gills, muscles, subcuticular epithelium, intestines, abdominal nervous ganglia, thoracic nervous ganglia, lymphoid organ, stomach, heart, and antennal gland, especially in subcuticular epithelium and gill. LvRACK1 mRNA transcription in haemocytes of L. vannamei injected with Vibrio alginolyticus decreased. The depletion of LvRACK1 of haemocytes in L. vannamei received its dsRNA revealed the increased respiratory bursts per haemocyte, superoxide dismutase (SOD), activity, glutathione peroxidase (GPx) activity, and clotting time, but showed the decreased total haemocyte count (THC), hyaline cells (HCs), phagocytic activity, and transglutaminase (TG) activity. LvRACK1 silenced shrimp showed the upregulated gene expressions of cyMnSOD, mtMnSOD, peroxinectin (PE), and TGI, and showed the downregulated α2-macroglobulin (α2-M), clottable protein (CP), lysozyme, and crustin gene expressions. It is therefore concluded that LvRACK1 is involved in immune defense and signaling transduction in haemocytes of L. vannamei infected with V. alginolyticus.

  1. Protein kinase A is part of a mechanism that regulates nuclear reimport of the nuclear tRNA export receptors Los1p and Msn5p.

    PubMed

    Pierce, Jacqueline B; van der Merwe, George; Mangroo, Dev

    2014-02-01

    The two main signal transduction mechanisms that allow eukaryotes to sense and respond to changes in glucose availability in the environment are the cyclic AMP (cAMP)/protein kinase A (PKA) and AMP-activated protein kinase (AMPK)/Snf1 kinase-dependent pathways. Previous studies have shown that the nuclear tRNA export process is inhibited in Saccharomyces cerevisiae deprived of glucose. However, the signal transduction pathway involved and the mechanism by which glucose availability regulates nuclear-cytoplasmic tRNA trafficking are not understood. Here, we show that inhibition of nuclear tRNA export is caused by a block in nuclear reimport of the tRNA export receptors during glucose deprivation. Cytoplasmic accumulation of the tRNA export receptors during glucose deprivation is not caused by activation of Snf1p. Evidence obtained suggests that PKA is part of the mechanism that regulates nuclear reimport of the tRNA export receptors in response to glucose availability. This mechanism does not appear to involve phosphorylation of the nuclear tRNA export receptors by PKA. The block in nuclear reimport of the tRNA export receptors appears to be caused by activation of an unidentified mechanism when PKA is turned off during glucose deprivation. Taken together, the data suggest that PKA facilitates return of the tRNA export receptors to the nucleus by inhibiting an unidentified activity that facilitates cytoplasmic accumulation of the tRNA export receptors when glucose in the environment is limiting. A PKA-independent mechanism was also found to regulate nuclear tRNA export in response to glucose availability. This mechanism, however, does not regulate nuclear reimport of the tRNA export receptors.

  2. Analysis of mitogen-activated protein kinase activation by naturally occurring splice variants of TrkC, the receptor for neurotrophin-3.

    PubMed

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

    1997-02-15

    TrkC is a receptor tyrosine kinase that binds neurotrophin-3 (NT-3) with high affinity. A number of naturally occurring splice variants of TrkC exist, including one (TrkC kil4) with a 14 amino acid insertion between subdomains VII and VIII of the tyrosine kinase domain. This kinase insert blocks the ability of NT-3 to stimulate neurite outgrowth in PC12 cells and proliferation in fibroblasts. The inserts also block the ability of TrkC to form a high-affinity complex with Shc and phospholipase C gamma (PLC gamma) and the activation of PtdIns 3-kinase, and attenuates the sustained activation of mitogen-activated protein kinase (MAPK). In the current study we set out to determine whether the attenuation of the activation of MAPK by the insert was the result of the inability of TrkC to activate the Shc-Ras pathway, PtdIns 3-kinase activation, PLC gamma activation, or a combination thereof. Experiments with the use of cell-permeant inhibitors argue against a major role for PLC gamma and PtdIns 3-kinase in the activation of MAPK by TrkC. The introduction of the 14 amino acid kinase insert appeared to slow the kinetics of NT-3-stimulated Shc phosphorylation and Shc-Grb2 association and reduce their magnitude; an effect which was associated with a delayed, and only transient, activation of MAPK. Taken together, our data suggest that the apparent defect in MAPK activation caused by the kinase insert may result predominantly from an inhibition of high-affinity Shc binding, although a role for PLC gamma and PtdIns 3-kinase cannot be completely excluded.

  3. GPCR responses in vascular smooth muscle can occur predominantly through dual transactivation of kinase receptors and not classical Gαq protein signalling pathways.

    PubMed

    Little, Peter J

    2013-05-30

    GPCR signalling is well known to proceed through several linear pathways involving activation of G proteins and their downstream signalling pathways such as activation of phospholipase C. In addition, GPCRs signal via transactivation of Protein Tyrosine Kinase receptors such as that for Epidermal Growth Factor (EGF) and Platelet-Derived Growth Factor (PDGF) where GPCR agonists mediate increase levels of phosphorylated Erk (pErk) the immediate downstream product of the activation of EGF receptor. It has recently been shown that this paradigm can be extended to include the GPCR transactivation of a Protein Serine/Threonine Kinase receptor, specifically the Transforming Growth Factor β Type I receptor (also known as Alk V) (TβRI) in which case GPCR activation leads to the formation of carboxy terminal polyphosphorylated Smad2 (phosphoSmad2) being the immediate downstream product of the activation of TβRI. Growth factor and hormone regulation of proteoglycan synthesis in vascular smooth muscle cells represent one component of an in vitro model of atherosclerosis because modified proteoglycans show enhanced binding to lipoproteins as the initiating step in atherosclerosis. In the example of proteoglycan synthesis stimulated by GPCR agonists such as thrombin and endothelin-1, the transactivation pathways for the EGF receptor and TβRI are both active and together account for essentially all of the response to the GPCRs. In contrast, signalling downstream of GPCRs such as increased inositol 1,4,5 trisphosphate (IP3) and intracellular calcium do not have any effect on GPCR stimulated proteoglycan synthesis. These data lead to the conclusion that dual transactivation pathways for protein tyrosine and serine/threonine kinase receptors may play a far greater role in GPCR signalling than currently recognised.

  4. A Receptor-associated Protein/Phosphatidylinositol 3-Kinase Pathway Controls Pseudopod Formation

    PubMed Central

    Kortholt, Arjan; Bolourani, Parvin; Rehmann, Holger; Keizer-Gunnink, Ineke; Weeks, Gerald; Wittinghofer, Alfred

    2010-01-01

    GbpD, a Dictyostelium discoideum guanine exchange factor specific for Rap1, has been implicated in adhesion, cell polarity, and chemotaxis. Cells overexpressing GbpD are flat, exhibit strongly increased cell-substrate attachment, and extend many bifurcated and lateral pseudopodia. Phg2, a serine/threonine-specific kinase, mediates Rap1-regulated cell-substrate adhesion, but not cell polarity or chemotaxis. In this study we demonstrate that overexpression of GbpD in pi3k1/2-null cells does not induce the adhesion and cell morphology phenotype. Furthermore we show that Rap1 directly binds to the Ras binding domain of PI3K, and overexpression of GbpD leads to strongly enhanced PIP3 levels. Consistently, upon overexpression of the PIP3-degradating enzyme PTEN in GbpD-overexpressing cells, the strong adhesion and cell morphology phenotype is largely lost. These results indicate that a GbpD/Rap/PI3K pathway helps control pseudopod formation and cell polarity. As in Rap-regulated pseudopod formation in Dictyostelium, mammalian Rap and PI3K are essential for determining neuronal polarity, suggesting that the Rap/PI3K pathway is a conserved module regulating the establishment of cell polarity. PMID:20089846

  5. Arabidopsis ERECTA-family receptor kinases mediate morphological alterations stimulated by activation of NB-LRR-type UNI proteins.

    PubMed

    Uchida, Naoyuki; Igari, Kadunari; Bogenschutz, Naomi L; Torii, Keiko U; Tasaka, Masao

    2011-05-01

    Shoot apical meristems (SAMs), which maintain stem cells at the tips of stems, and axillary meristems (AMs), which arise at leaf axils for branch formation, play significant roles in the establishment of plant architecture. Previously, we showed that, in Arabidopsis thaliana, activation of NB-LRR (nucleotide-binding site-leucine-rich repeat)-type UNI proteins affects plant morphology through modulation of the regulation of meristems. However, information about genes involved in the processes was still lacking. Here, we report that ERECTA (ER) receptor kinase family members cooperatively mediate the morphological alterations that are stimulated by activation of UNI proteins. uni-1D is a gain-of-function mutation in the UNI gene and uni-1D mutants exhibit early termination of inflorescence stem growth and also formation of extra AMs at leaf axils. The former defect involves modulation of the SAM activity and is suppressed by er mutation. Though the AM phenotype is not affected by a single er mutation, it is suppressed by simultaneous mutations of ER-family members. It was previously shown that trans-zeatin (tZ)-type cytokinins were involved in the morphological phenotypes of uni-1D mutants and that expression of CYP735A2, which is essential for biosynthesis of tZ-type cytokinins, was modulated in uni-1D mutants. We show that this modulation of CYP735A2 expression requires activities of ER-family members. Moreover, the ER activity in UNI-expressing cells contributes to all morphological phenotypes of uni-1D mutants, suggesting that a cross-talk between ER-family-dependent and UNI-triggered signaling pathways plays a significant role in the morphological alterations observed in uni-1D mutants.

  6. PDGF-induced receptor phosphorylation and phosphoinositide hydrolysis are unaffected by protein kinase C activation in mouse swiss 3T3 and human skin fibroblasts

    SciTech Connect

    Sturani, E.; Vicentini, L.M.; Zippel, R.; Toschi, L.; Pandiella-Alonso, A.; Comoglio, P.M.; Meldolesi, J.

    1986-05-29

    Short (1-10 min) pretreatment of intact cells with activators of protein kinase C (e.g. phorbol-12 myristate, 13-acetate, PMA) affects the activity of a variety of surface receptors (for growth factors, hormones and neurotransmitters), with inhibition of transmembrane signal generation. In two types of fibroblasts it is demonstrated that the PDGF receptor is unaffected by PMA. Exposure to PMA at concentrations up to 100 nM for 10 min failed to inhibit either one of the agonist-induced, receptor-coupled responses of PDGF: the autophosphorylation of receptor molecules at tyrosine residues, and the hydrolysis of membrane polyphosphoinositides. In contrast, the EGF receptor autophosphorylation (in A 431 cells) and the bombesin-induced phosphoinositide hydrolysis were readily inhibited by PMA.

  7. Increases in transient receptor potential vanilloid-1 mRNA and protein in primary afferent neurons stimulated by protein kinase C and their possible role in neurogenic inflammation

    PubMed Central

    Xu, Xijin; Wang, Peng; Zou, Xiaoju; Li, Dingge; Fang, Li; Lin, Qing

    2008-01-01

    A recent study by our group demonstrates pharmacologically that the transient receptor potential vanilloid-1 (TRPV1) is activated by intradermal injection of capsaicin to initiate neurogenic inflammation by the release of neuropeptides in the periphery. In this study, expression of TRPV1, phosphorylated protein kinase C (p-PKC) and calcitonin gene-related peptide (CGRP) in dorsal root ganglion (DRG) neurons were visualized using immunofluorescence, real-time PCR and Western blots to examine whether increases in TRPV1 mRNA and protein levels evoked by capsaicin injection are subject to modulation by the activation of PKC and to analyze the role of this process in the pathogenesis of neurogenic inflammation. Capsaicin injection into the hindpaw skin of anesthetized rats evoked increases in the expression of TRPV1, CGRP and p-PKC in mRNA and/or protein levels and in the number of single labeled TRPV1, p-PKC and CGRP neurons in ipsilateral L4–5 DRGs. Co-expressions of TRPV1 with p-PKC and/or CGRP in DRG neurons were also significantly increased after CAP injection. These evoked expressions both at molecular and cellular levels were significantly inhibited after TRPV1 receptors were blocked by 5′-iodoresiniferatoxin (5 μg) or PKC was inhibited by chelerythrine chloride (5 μg). Taken together, these results provide evidence that up-regulation of TRPV1 mRNA and protein levels under inflammatory conditions evoked by capsaicin injection is subject to modulation by the PKC cascade in which increased CGRP level in DRG neurons may be related to the initiation of neurogenic inflammation. Thus, up-regulation of TRPV1 receptors in DRG neurons seems critical for initiating acute neurogenic inflammation. PMID:18752301

  8. Cardiomyocyte-restricted inhibition of G protein-coupled receptor kinase-3 attenuates cardiac dysfunction after chronic pressure overload.

    PubMed

    von Lueder, Thomas G; Gravning, Jørgen; How, Ole-Jakob; Vinge, Leif E; Ahmed, Mohammed Shakil; Krobert, Kurt A; Levy, Finn Olav; Larsen, Terje S; Smiseth, Otto A; Aasum, Ellen; Attramadal, Håvard

    2012-07-01

    Transgenic mice with cardiac-specific expression of a peptide inhibitor of G protein-coupled receptor kinase (GRK)3 [transgenic COOH-terminal GRK3 (GRK3ct) mice] display myocardial hypercontractility without hypertrophy and enhanced α(1)-adrenergic receptor signaling. A role for GRK3 in the pathogenesis of heart failure (HF) has not been investigated, but inhibition of its isozyme, GRK2, has been beneficial in several HF models. Here, we tested whether inhibition of GRK3 modulated evolving cardiac hypertrophy and dysfunction after pressure overload. Weight-matched male GRK3ct transgenic and nontransgenic littermate control (NLC) mice subjected to chronic pressure overload by abdominal aortic banding (AB) were compared with sham-operated (SH) mice. At 6 wk after AB, a significant increase of cardiac mass consistent with induction of hypertrophy was found, but no differences between GRK3ct-AB and NLC-AB mice were discerned. Simultaneous left ventricular (LV) pressure-volume analysis of electrically paced, ex vivo perfused working hearts revealed substantially reduced systolic and diastolic function in NLC-AB mice (n = 7), which was completely preserved in GRK3ct-AB mice (n = 7). An additional cohort was subjected to in vivo cardiac catheterization and LV pressure-volume analysis at 12 wk after AB. NLC-AB mice (n = 11) displayed elevated end-diastolic pressure (8.5 ± 3.1 vs. 2.9 ± 1.2 mmHg, P < 0.05), reduced cardiac output (3,448 ± 323 vs. 4,488 ± 342 μl/min, P < 0.05), and reduced dP/dt(max) and dP/dt(min) (both P < 0.05) compared with GRK3ct-AB mice (n = 16), corroborating the preserved cardiac structure and function observed in GRK3ct-AB hearts assessed ex vivo. Increased cardiac mass and myocardial mRNA expression of β-myosin heavy chain confirmed the similar induction of cardiac hypertrophy in both AB groups, but only NLC-AB hearts displayed significantly elevated mRNA levels of brain natriuretic peptide and myocardial collagen contents as well as reduced

  9. Fulvestrant-induced cell death and proteasomal degradation of estrogen receptor α protein in MCF-7 cells require the CSK c-Src tyrosine kinase.

    PubMed

    Yeh, Wei-Lan; Shioda, Keiko; Coser, Kathryn R; Rivizzigno, Danielle; McSweeney, Kristen R; Shioda, Toshi

    2013-01-01

    Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.

  10. JAK1 kinase forms complexes with interleukin-4 receptor and 4PS/insulin receptor substrate-1-like protein and is activated by interleukin-4 and interleukin-9 in T lymphocytes.

    PubMed

    Yin, T; Tsang, M L; Yang, Y C

    1994-10-28

    Interleukin (IL)-4 and IL-9 regulate the proliferation of T lymphocytes through interactions with their receptors. Previous studies have shown that unknown tyrosine kinases are involved in the proliferative signaling triggered by IL-4 and IL-9. Here we show that IL-4 and IL-9 induce overlapping (170, 130, and 125 kilodalton (kDa)) and distinct (45 and 88/90 kDa, respectively) protein tyrosine phosphorylation in T lymphocytes. We further identify the 170-kDa tyrosine-phosphorylated protein as 4PS/insulin receptor substrate-1-like (IRS-1L) protein and 130-kDa protein as JAK1 kinase. Furthermore, we demonstrate for the first time that JAK1 forms complexes with the IL-4 receptor and 4PS/IRS-1L protein following ligand-receptor interaction. In addition, we demonstrate that IL-9, but not IL-4, induced tyrosine phosphorylation of Stat 91 transcriptional factor. The overlapping and distinct protein tyrosine phosphorylation and activation of the same JAK1 kinase in T lymphocytes strongly suggests that IL-4 and IL-9 share the common signal transduction pathways and that the specificity for each cytokine could be achieved through the unique tyrosine-phosphorylated proteins triggered by individual cytokines.

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  13. Protein kinase C activators selectively inhibit insulin-stimulated system A transport activity in skeletal muscle at a post-receptor level.

    PubMed Central

    Gumà, A; Camps, M; Palacín, M; Testar, X; Zorzano, A

    1990-01-01

    We have investigated the role of phorbol esters on different biological effects induced by insulin in muscle, such as activation of system A transport activity, glucose utilization and insulin receptor function. System A transport activity was measured by monitoring the uptake of the system A-specific analogue alpha-(methyl)aminoisobutyric acid (MeAIB), by intact rat extensor digitorum longus muscle. The addition of 12-O-tetradecanoylphorbol 13-acetate (TPA, 0.5 microM) for 60 or 180 min did not modify basal MeAIB uptake by muscle, suggesting that insulin signalling required to stimulate MeAIB transport does not involve protein kinase C activation. However, TPA added 30 min before insulin (100 nM) markedly inhibited insulin-stimulated MeAIB uptake. The addition of polymyxin B (0.1 mM) or H-7 (1 mM), protein kinase C inhibitors, alone or in combination with TPA leads to impairment of insulin-stimulated MeAIB uptake. This paradoxical pattern is incompatible with a unique action of Polymyxin B or H-7 on protein kinase C activity. Therefore these agents are not suitable tools with which to investigate whether a certain insulin effect is mediated by protein kinase C. TPA did not cause a generalized inhibition of insulin action. Thus both TPA and insulin increased 3-O-methylglucose uptake by muscle, and their effects were not additive. Furthermore, TPA did not modify insulin-stimulated lactate production by muscle. In keeping with this selective modification of insulin action, treatment of muscles with TPA did not modify insulin receptor binding or kinase activities. In conclusion, phorbol esters do not mimic insulin action on system A transport activity; however, they markedly inhibit insulin-stimulated amino acid transport, with no modification of insulin receptor function in rat skeletal muscle. It is suggested that protein kinase C activation causes a selective post-receptor modification on the biochemical pathway by which insulin activates system A amino acid

  14. High glucose induces inflammatory cytokine through protein kinase C-induced toll-like receptor 2 pathway in gingival fibroblasts

    SciTech Connect

    Jiang, Shao-Yun; Wei, Cong-Cong; Shang, Ting-Ting; Lian, Qi; Wu, Chen-Xuan; Deng, Jia-Yin

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer High glucose significantly induced TLR2 expression in gingival fibroblasts. Black-Right-Pointing-Pointer High glucose increased NF-{kappa}B p65 nuclear activity, IL-1{beta} and TNF-{alpha} levels. Black-Right-Pointing-Pointer PKC-{alpha}/{delta}-TLR2 pathway is involved in periodontal inflammation under high glucose. -- Abstract: Toll-like receptors (TLRs) play a key role in innate immune response and inflammation, especially in periodontitis. Meanwhile, hyperglycemia can induce inflammation in diabetes complications. However, the activity of TLRs in periodontitis complicated with hyperglycemia is still unclear. In the present study, high glucose (25 mmol/l) significantly induced TLR2 expression in gingival fibroblasts (p < 0.05). Also, high glucose increased nuclear factor kappa B (NF-{kappa}B) p65 nuclear activity, tumor necrosis factor-{alpha} (TNF-{alpha}) and interleukin-l{beta} (IL-1{beta}) levels. Protein kinase C (PKC)-{alpha} and {delta} knockdown with siRNA significantly decreased TLR2 and NF-{kappa}B p65 expression (p < 0.05), whereas inhibition of PKC-{beta} had no effect on TLR2 and NF-{kappa}B p65 under high glucose (p < 0.05). Additional studies revealed that TLR2 knockdown significantly abrogated high-glucose-induced NF-{kappa}B expression and inflammatory cytokine secretion. Collectively, these data suggest that high glucose stimulates TNF-{alpha} and IL-1{beta} secretion via inducing TLR2 through PKC-{alpha} and PKC-{delta} in human gingival fibroblasts.

  15. Altered protein kinase C regulation of pulmonary endothelial store- and receptor-operated Ca2+ entry after chronic hypoxia.

    PubMed

    Paffett, Michael L; Riddle, Melissa A; Kanagy, Nancy L; Resta, Thomas C; Walker, Benjimen R

    2010-09-01

    Chronic hypoxia (CH)-induced pulmonary hypertension is associated with decreased basal pulmonary artery endothelial cell (EC) Ca(2+), which correlates with reduced store-operated Ca(2+) (SOC) entry. Protein kinase C (PKC) attenuates SOC entry in ECs. Therefore, we hypothesized that PKC has a greater inhibitory effect on EC SOC and receptor-operated Ca(2+) entry after CH. To test this hypothesis, we assessed SOC in the presence or absence of the nonselective PKC inhibitor GF109203X [2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)maleimide] in freshly isolated, Fura-2-loaded ECs obtained from intrapulmonary arteries of control and CH rats (4 weeks at 0.5 atm). We found that SOC entry and 1-oleoyl-2-acetyl-sn-glycerol (OAG)- and ATP-induced Ca(2+) influx were attenuated in ECs from CH rats versus controls, and GF109203X restored SOC and OAG responses to the level of controls. In contrast, nonselective PKC inhibition with GF109203X or the selective PKC(epsilon) inhibitor myristoylated V1-2 attenuated ATP-induced Ca(2+) entry in ECs from control but not CH pulmonary arteries. ATP-induced Ca(2+) entry was also attenuated by the T-type voltage-gated Ca(2+) channel (VGCC) inhibitor mibefradil in control cells. Consistent with the presence of endothelial T-type VGCC, we observed depolarization-induced Ca(2+) influx in control cells that was inhibited by mibefradil. This response was largely absent in ECs from CH arteries. We conclude that CH enhances PKC-dependent inhibition of SOC- and OAG-induced Ca(2+) entry. Furthermore, these data suggest that CH may reduce the ATP-dependent Ca(2+) entry that is mediated, in part, by PKCepsilon and mibefradil-sensitive Ca(2+) channels in control cells.

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

    PubMed Central

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

    2011-01-01

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

  17. Receptor Tyrosine Kinases in Drosophila Development

    PubMed Central

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

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

  18. A-kinase anchoring protein (AKAP)-Lbc anchors a PKN-based signaling complex involved in α1-adrenergic receptor-induced p38 activation.

    PubMed

    Cariolato, Luca; Cavin, Sabrina; Diviani, Dario

    2011-03-11

    The mitogen-activated protein kinases (MAPKs) pathways are highly organized signaling systems that transduce extracellular signals into a variety of intracellular responses. In this context, it is currently poorly understood how kinases constituting these signaling cascades are assembled and activated in response to receptor stimulation to generate specific cellular responses. Here, we show that AKAP-Lbc, an A-kinase anchoring protein (AKAP) with an intrinsic Rho-specific guanine nucleotide exchange factor activity, is critically involved in the activation of the p38α MAPK downstream of α(1b)-adrenergic receptors (α(1b)-ARs). Our results indicate that AKAP-Lbc can assemble a novel transduction complex containing the RhoA effector PKNα, MLTK, MKK3, and p38α, which integrates signals from α(1b)-ARs to promote RhoA-dependent activation of p38α. In particular, silencing of AKAP-Lbc expression or disrupting the formation of the AKAP-Lbc·p38α signaling complex specifically reduces α(1)-AR-mediated p38α activation without affecting receptor-mediated activation of other MAPK pathways. These findings provide a novel mechanistic hypothesis explaining how assembly of macromolecular complexes can specify MAPK signaling downstream of α(1)-ARs.

  19. Mitogen-activated protein kinase activity is involved in effector functions triggered by the CD94/NKG2-C NK receptor specific for HLA-E.

    PubMed

    Carretero, M; Llano, M; Navarro, F; Bellón, T; López-Botet, M

    2000-10-01

    The CD94/NKG2C heterodimer constitutes an activating receptor involved in NK cell-mediated recognition of the class lb molecule HLA-E. It transduces the triggering signal through an ITAM-bearing molecule, DAP12/KARAP, coupled non-covalently to the receptor. Here we show that specific engagement of the receptor complex expressed on the surface of an NK clone induced the phosphorylation of mitogen-activated protein kinase (MAPK). By the use of the MEK inhibitor PD098059 we demonstrate that the MAPK pathway participates in the CD94-dependent TNF-alpha production and cytotoxicity. Moreover, we transferred the activating function by transfection of the heterologous RBL cell line with CD94/NKG2-C/DAP12. In this system, cross-linking of the receptor induced calcium mobilization, serotonin release and phosphorylation of MAPK.

  20. Critical role of X-box binding protein 1 in NADPH oxidase 4-triggered cardiac hypertrophy is mediated by receptor interacting protein kinase 1.

    PubMed

    Chen, Li; Zhao, Mingyue; Li, Junli; Wang, Yu; Bao, Qinxue; Wu, Siyuan; Deng, Xueqin; Tang, Xiaoju; Wu, Wenchao; Liu, Xiaojing

    2017-02-16

    NADPH oxidase 4 (NOX4) and the NOX4-related redox signaling are implicated in cardiac hypertrophy. NOX4 is interrelated with endoplasmic reticulum stress (ERS). Spliced X-box binding protein 1 (Xbp1s) is a key mediator of ERS while its role in cardiac hypertrophy is still poorly understood. Recently, receptor interacting protein kinase 1(RIPK1) has been increasingly reported to be associated with ERS. Therefore, we aimed to test the hypothesis that Xbp1s mediates NOX4-triggered cardiac hypertrophy via RIPK1 signaling. In the heart tissue of transverse aortic constriction (TAC) rats and in primary cultured neonatal cardiomyocytes(NCMs) treated with angiotensinII(AngII) or isoproterenol (ISO), NOX4 expression and reactive oxygen species (ROS) generation, and expression of Xbp1s as well as RIPK1-related phosphorylation of P65 subunit of NF-κB were elevated. Gene silencing of NOX4 by specific small interfering RNA (siRNA) significantly blocked the upregulation of NOX4, generation of ROS, splicing of Xbp1 and activation of the RIPK1-related NF-κB signaling, meanwhile attenuated cardiomyocyte hypertrophy. In addition, ROS scavenger (N-acetyl-L-cysteine, NAC) and NOX4 inhibitor GKT137831 reduced ROS generation and alleviated activation of Xbp1 and RIPK1-related NF-κB signaling. Furthermore, splicing of Xbp1 was responsible for the increase in RIPK1 expression in AngII or ISO-treated NCMs. Upregulated RIPK1 in turn activates NF-κB signaling in a kinase activity-independent manner. These findings suggest that Xbp1s plays an important role in NOX4-triggered cardiomyocyte hypertrophy via activating its downstream effector RIPK1, which may prove significant for the development of future therapeutic strategies.

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

    PubMed Central

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

    1992-01-01

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

  2. The activity and stability of the intrinsically disordered Cip/Kip protein family are regulated by non-receptor tyrosine kinases

    PubMed Central

    Otieno, Steve; Lelli, Moreno; Kriwacki, Richard W.

    2014-01-01

    The Cip/Kip family of cyclin-dependent kinase (Cdk) inhibitors includes p21Cip1, p27Kip1 and p57Kip2. Their kinase inhibitory activities are mediated by a homologous N-terminal kinase-inhibitory domain (KID). The Cdk inhibitory activity and stability of p27 have been shown to be regulated by a two-step phosphorylation mechanism involving a tyrosine residue within the KID and a threonine residue within the flexible C-terminus. We show that these residues are conserved in p21 and p57, suggesting that a similar phosphorylation cascade regulates these Cdk inhibitors. However, the presence of a cyclin binding motif within its C-terminus alters the regulatory interplay between p21 and Cdk2/cyclin A, and its responses to tyrosine phosphorylation and altered p21:Cdk2/cyclin A stoichiometry. We also show that the Cip/Kip proteins can be phosphorylated in vitro by representatives of many non-receptor tyrosine kinase (NRTK) sub-families, suggesting that NRTKs may generally regulate the activity and stability of these Cdk inhibitors. Our results further suggest that the Cip/Kip proteins integrate signals from various NRTK pathways and cell cycle regulation. PMID:25463440

  3. Epigenetic Activation of μ-Opioid Receptor Gene via Increased Expression and Function of Mitogen- and Stress-Activated Protein Kinase 1.

    PubMed

    Wagley, Yadav; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H

    2017-04-01

    Since the discovery of μ-opioid receptor (MOR) gene two decades ago, various regulatory factors have been shown to interact with the MOR promoter and modulate transcript levels. However, the majority of early transcriptional studies on MOR gene have not addressed how intracellular signaling pathways mediate extracellular modulators. In this study, we demonstrate that MOR epigenetic regulation requires multiple coordinated signals converging at the MOR promoter, involving mitogen-activated protein kinase (MAPK) activation and mitogen- and stress-activated protein kinase 1 (MSK1)-ranges of intracellular signaling pathways similar to those activated by opioid agonists. Inhibiting p38 MAPK or extracellular signal-regulated kinase (ERK) 1/2 MAPK (upstream activators of MSK1) reduced MOR expression levels; accordingly, the functional role of MSK1, but not MSK2, was demonstrated using genetic approaches. However, for maximal MSK1 effect, an open chromatin configuration was required, because in vitro CpG methylation of the MOR promoter abolished MSK1 activity. Finally, endogenous MSK1 levels concomitantly increased to regulate MOR gene expression during neuronal differentiation of P19 cells, suggesting a conserved role of this kinase in the epigenic activation of MOR in neurons. Taken together, our findings indicate that the expression of MOR gene requires the activity of intracellular signaling pathways that have been implicated in the behavioral outcomes of opioid drugs, which suggests that an autoregulatory mechanism may function in opioid systems.

  4. Molecular cloning and characterization of two novel genes from hexaploid wheat that encode double PR-1 domains coupled with a receptor-like protein kinase.

    PubMed

    Lu, Shunwen; Faris, Justin D; Edwards, Michael C

    2017-04-01

    Hexaploid wheat (Triticum aestivum L.) contains at least 23 TaPr-1 genes encoding the group 1 pathogenesis-related (PR-1) proteins as identified in our previous work. Here, we report the cloning and characterization of TaPr-1-rk1 and TaPr-1-rk2, two novel genes closely related to the wheat PR-1 family. The two TaPr-1-rk genes are located on homoeologous chromosomes 3D and 3A, respectively, and each contains a large open reading frame (7385 or 6060 bp) that is interrupted by seven introns and subjected to alternative splicing (AS) with five or six isoforms of mRNA transcripts. The deduced full-length TaPR-1-RK1 and TaPR-1-RK2 proteins (95% identity) contain two repeat PR-1 domains, the second of which is fused via a transmembrane helix to a serine/threonine kinase catalytic (STKc) domain characteristic of receptor-like protein kinases. Phylogenetic analysis indicated that the two PR-1 domains of the TaPR-1-RK proteins form sister clades with their homologues identified in other monocot plants and are well separated from stand-alone PR-1 proteins, whereas the STKc domains may have originated from cysteine-rich receptor-like kinases (CRKs). Reverse-transcriptase-PCR analysis revealed that the TaPr-1-rk genes are predominantly expressed in wheat leaves and their expression levels are elevated in response to pathogen attack, such as infection by barley stripe mosaic virus (BSMV), and also to stress conditions, most obviously, to soil salinity. This is the first report of PR-1-CRK hybrid proteins in wheat. The data may shed new insights into the function/evolutionary origin of the PR-1 family and the STKc-mediated defense/stress response pathways in plants.

  5. A critical role for Syk protein tyrosine kinase in Fc receptor-mediated antigen presentation and induction of dendritic cell maturation.

    PubMed

    Sedlik, Christine; Orbach, Daniel; Veron, Philippe; Schweighoffer, Edina; Colucci, Francesco; Gamberale, Romina; Ioan-Facsinay, Andrea; Verbeek, Sjef; Ricciardi-Castagnoli, Paola; Bonnerot, Christian; Tybulewicz, Victor L J; Di Santo, James; Amigorena, Sebastian

    2003-01-15

    Dendritic cells (DCs) are the only APCs capable of initiating adaptive immune responses. The initiation of immune responses requires that DCs 1) internalize and present Ags; and 2) undergo a differentiation process, called "maturation", which transforms DCs into efficient APCs. DC maturation may be initiated by the engagement of different surface receptors, including certain cytokine receptors (such as TNFR), Toll-like receptors, CD40, and FcRs. The early activation events that link receptor engagement and DC maturation are not well characterized. We found that FcR engagement by immune complexes induced the phosphorylation of Syk, a protein tyrosine kinase acting immediately downstream of FcRs. Syk was dispensable for DC differentiation in vitro and in vivo, but was strictly required for immune complexes internalization and subsequent Ag presentation to T lymphocytes. Importantly, Syk was also required for the induction of DC maturation and IL-12 production after FcR engagement, but not after engagement of other surface receptors, such as TNFR or Toll-like receptors. Therefore, protein tyrosine phosphorylation by Syk represents a novel pathway for the induction of DC maturation.

  6. Protein kinase C activation potentiates gating of the vanilloid receptor VR1 by capsaicin, protons, heat and anandamide

    PubMed Central

    Vellani, Vittorio; Mapplebeck, Sarah; Moriondo, Andrea; Davis, John B; McNaughton, Peter A

    2001-01-01

    The effects of activation of protein kinase C (PKC) on membrane currents gated by capsaicin, protons, heat and anandamide were investigated in primary sensory neurones from neonatal rat dorsal root ganglia (DRG) and in HEK293 cells (human embryonic kidney cell line) transiently or stably expressing the human vanilloid receptor hVR1. Maximal activation of PKC by a brief application of phorbol 12-myristate 13-acetate (PMA) increased the mean membrane current activated by a low concentration of capsaicin by 1.65-fold in DRG neurones and 2.18-fold in stably transfected HEK293 cells. Bradykinin, which activates PKC, also enhanced the response to capsaicin in DRG neurones. The specific PKC inhibitor RO31-8220 prevented the enhancement caused by PMA. Activation of PKC did not enhance the membrane current at high concentrations of capsaicin, showing that PKC activation increases the probability of channel opening rather than unmasking channels. Application of PMA alone activated an inward current in HEK293 cells transiently transfected with VR1. The current was suppressed by the VR1 antagonist capsazepine. PMA did not, however, activate a current in the large majority of DRG neurones nor in HEK293 cells stably transfected with VR1. Removing external Ca2+ enhanced the response to a low concentration of capsaicin 2.40-fold in DRG neurones and 3.42-fold in HEK293 cells. Activation of PKC in zero Ca2+ produced no further enhancement of the response to capsaicin in either DRG neurones or HEK293 cells stably transfected with VR1. The effects of PKC activation on the membrane current gated by heat, anandamide and low pH were qualitatively similar to those on the capsaicin-gated current. The absence of a current activated by PMA in most DRG neurones or in stably transfected HEK293 cells suggests that activation of PKC does not directly open VR1 channels, but instead increases the probability that they will be activated by capsaicin, heat, low pH or anandamide. Removal of calcium

  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. Tobacco Translationally Controlled Tumor Protein Interacts with Ethylene Receptor Tobacco Histidine Kinase1 and Enhances Plant Growth through Promotion of Cell Proliferation1[OPEN

    PubMed Central

    Tao, Jian-Jun; Cao, Yang-Rong; Chen, Hao-Wei; Wei, Wei; Li, Qing-Tian; Ma, Biao; Zhang, Wan-Ke; Chen, Shou-Yi; Zhang, Jin-Song

    2015-01-01

    Ethylene is an important phytohormone in the regulation of plant growth, development, and stress response throughout the lifecycle. Previously, we discovered that a subfamily II ethylene receptor tobacco (Nicotiana tabacum) Histidine Kinase1 (NTHK1) promotes seedling growth. Here, we identified an NTHK1-interacting protein translationally controlled tumor protein (NtTCTP) by the yeast (Saccharomyces cerevisiae) two-hybrid assay and further characterized its roles in plant growth. The interaction was further confirmed by in vitro glutathione S-transferase pull down and in vivo coimmunoprecipitation and bimolecular fluorescence complementation assays, and the kinase domain of NTHK1 mediates the interaction with NtTCTP. The NtTCTP protein is induced by ethylene treatment and colocalizes with NTHK1 at the endoplasmic reticulum. Overexpression of NtTCTP or NTHK1 reduces plant response to ethylene and promotes seedling growth, mainly through acceleration of cell proliferation. Genetic analysis suggests that NtTCTP is required for the function of NTHK1. Furthermore, association of NtTCTP prevents NTHK1 from proteasome-mediated protein degradation. Our data suggest that plant growth inhibition triggered by ethylene is regulated by a unique feedback mechanism, in which ethylene-induced NtTCTP associates with and stabilizes ethylene receptor NTHK1 to reduce plant response to ethylene and promote plant growth through acceleration of cell proliferation. PMID:25941315

  9. Tobacco Translationally Controlled Tumor Protein Interacts with Ethylene Receptor Tobacco Histidine Kinase1 and Enhances Plant Growth through Promotion of Cell Proliferation.

    PubMed

    Tao, Jian-Jun; Cao, Yang-Rong; Chen, Hao-Wei; Wei, Wei; Li, Qing-Tian; Ma, Biao; Zhang, Wan-Ke; Chen, Shou-Yi; Zhang, Jin-Song

    2015-09-01

    Ethylene is an important phytohormone in the regulation of plant growth, development, and stress response throughout the lifecycle. Previously, we discovered that a subfamily II ethylene receptor tobacco (Nicotiana tabacum) Histidine Kinase1 (NTHK1) promotes seedling growth. Here, we identified an NTHK1-interacting protein translationally controlled tumor protein (NtTCTP) by the yeast (Saccharomyces cerevisiae) two-hybrid assay and further characterized its roles in plant growth. The interaction was further confirmed by in vitro glutathione S-transferase pull down and in vivo coimmunoprecipitation and bimolecular fluorescence complementation assays, and the kinase domain of NTHK1 mediates the interaction with NtTCTP. The NtTCTP protein is induced by ethylene treatment and colocalizes with NTHK1 at the endoplasmic reticulum. Overexpression of NtTCTP or NTHK1 reduces plant response to ethylene and promotes seedling growth, mainly through acceleration of cell proliferation. Genetic analysis suggests that NtTCTP is required for the function of NTHK1. Furthermore, association of NtTCTP prevents NTHK1 from proteasome-mediated protein degradation. Our data suggest that plant growth inhibition triggered by ethylene is regulated by a unique feedback mechanism, in which ethylene-induced NtTCTP associates with and stabilizes ethylene receptor NTHK1 to reduce plant response to ethylene and promote plant growth through acceleration of cell proliferation.

  10. Microgravity and Signaling Molecules in Rat Osteoblasts: Downstream of Receptor Tyrosine Kinase, G-Protein-Coupled Receptor, and Small GTP-Binding Proteins

    NASA Technical Reports Server (NTRS)

    Kumel, Yasuhiro; Shimokawa, Hitoyata; Morita, Sadao; Katano, Hisako; Akiyama, Hideo; Hirano, Masahiko; Ohya, Keiichi; Sams, Clarence F.; Whitson, Peggy A.

    2005-01-01

    Rat osteoblasts were cultured for 4 and 5 days aboard Space Shuttle and solubilized on board. The mRNA levels of the post-receptor signaling molecules were analyzed by quantitative RT-PCR. The G-protein alpha subunit G(alpha)q mRNA levels were elevated 3-fold by microgravity. G(alpha)q stimulates PLC(beta), and then PKC. PKC(delta) and PKC(theta) mRNA levels were increased 2- to 5-fold by microgravity The mRNA levels of SOS and Ras GRF were increased 4 to 5-fold by microgravity, while Ras GAP was not altered. Spaceflight-induced bone loss might be attributed to microgravity modulation of the signaling pathway in osteoblasts.

  11. The interplay between G protein-coupled receptor kinase 2 (GRK2) and histone deacetylase 6 (HDAC6) at the crossroads of epithelial cell motility

    PubMed Central

    Lafarga, Vanesa; Mayor, Jr, Federico; Penela, Petronila

    2012-01-01

    G protein-coupled receptor kinase 2 (GRK2) is emerging as a key integrative node in cell migration control. In addition to its canonical role in the desensitization of G protein-coupled receptors involved in chemotaxis, novel recently identified GRK2 substrates and interacting partners appear to mediate the GRK2-dependent modulation of diverse molecular processes involved in motility, such as gradient sensing, cell polarity or cytoskeletal reorganization. We have recently identified an interaction between GRK2 and histone deacetylase 6 (HDAC6), a major cytoplasmic α-tubulin deacetylase involved in cell motility and adhesion. GRK2 dynamically associates with and phosphorylates HDAC6 to stimulate its α-tubulin deacetylase activity at specific cellular localizations such as the leading edge of migrating cells, thus promoting local tubulin deacetylation and enhanced motility. This GRK2-HDAC6 functional interaction may have important implications in pathological contexts related to aberrant epithelial cell migration. PMID:23076141

  12. Fps/Fes protein-tyrosine kinase regulates mast cell adhesion and migration downstream of Kit and beta1 integrin receptors.

    PubMed

    Smith, Julie A; Samayawardhena, Lionel A; Craig, Andrew W B

    2010-03-01

    Activation of Kit receptor protein-tyrosine kinase (PTK) by its ligand Stem Cell Factor (SCF) is required for the development of mast cells, and for the regulation of mast cell proliferation, migration and modulation of inflammatory mediator release. Recent studies have implicated the non-receptor PTK Fps/Fes (hereafter referred to as Fes) in signaling downstream of oncogenic Kit, however, the potential role of Fes in regulating Kit signaling is not well defined. In this study, we show that SCF induces transient tyrosine phosphorylation of wild-type Fes as well as kinase-dead Fes in bone marrow-derived mast cells (BMMCs). The latter finding implicates an upstream kinase acting on Fes, which we identified as Fyn PTK. SCF treatment of BMMCs promoted recruitment of Fes to Kit, potentially via direct interaction of the Fes SH2 domain with phosphorylated Kit. While Fes was not required for SCF-induced signaling to Akt and Erk kinases, Fes-deficient (fes-/-) BMMCs displayed a defect in sustained p38 kinase activation, compared to control cells. SCF-treated Fes-deficient BMMCs also displayed elevated beta1 integrin-mediated cell adhesion and spreading on fibronectin, compared to control cells, and a reduction in cell polarization at later times of SCF treatment. Restoring Fes expression in fes-/- BMMCs by retroviral transduction was sufficient to rescue cell spreading and polarization defects. Interestingly, SCF-induced chemotaxis of BMMCs was also defective in Fes-deficient BMMCs, and restored in Fes-rescue BMMCs. Overall, these results implicate Fes in regulating cross-talk between Kit and beta1 integrins to promote cytoskeletal reorganization and motility of mast cells.

  13. Transferrin receptor 2 and HFE regulate furin expression via mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) signaling. Implications for transferrin-dependent hepcidin regulation

    PubMed Central

    Poli, Maura; Luscieti, Sara; Gandini, Valentina; Maccarinelli, Federica; Finazzi, Dario; Silvestri, Laura; Roetto, Antonella; Arosio, Paolo

    2010-01-01

    Background Impaired regulation of hepcidin in response to iron is the cause of genetic hemochromatosis associated with defects of HFE and transferrin receptor 2. However, the role of these proteins in the regulation of hepcidin expression is unclear. Design and Methods Hepcidin expression, SMAD and extracellular signal-regulated kinase (Erk) phosphorylation and furin expression were analyzed in hepatic HepG2 cells in which HFE and transferrin receptor 2 were down-regulated or expressed, or furin activity specifically inhibited. Furin expression was also analyzed in the liver of transferrin receptor 2 null mice. Results We showed that the silencing of HFE and transferrin receptor 2 reduced both Erk phosphorylation and furin expression, that the exogenous expression of the two enhanced the induction of phosphoErk1/2 and furin by holotransferrin, but that this did not occur when the pathogenic HFE mutant C282Y was expressed. Furin, phosphoErk1/2 and phosphoSMAD1/5/8 were down-regulated also in transferrin receptor 2-null mice. Treatment of HepG2 cells with an inhibitor of furin activity caused a strong suppression of hepcidin mRNA, probably due to the inhibition of bone morphogenic protein maturation. Conclusions The data indicate that transferrin receptor 2 and HFE are involved in holotransferrin-dependent signaling for the regulation of furin which involved Erk phosphorylation. Furin in turn may control hepcidin expression. PMID:20634490

  14. Effects of the activated mitogen-activated protein kinase pathway via the c-ros receptor tyrosine kinase on the T47D breast cancer cell line following alcohol exposure.

    PubMed

    Lee, Hyung Tae; Kim, Se Kye; Choi, Mi Ran; Park, Ji Hyun; Jung, Kyoung Hwa; Chai, Young Gyu

    2013-03-01

    Compared to other cancers affecting women, breast cancer is significantly associated with alcohol consumption. However, the principles underlying the carcinogenesis of alcohol-induced breast cancer and the related metastatic mechanisms have yet to be established. To observe the effect of alcohol on the growth regulation in breast cancer cells, we identified differentially expressed proteins in alcohol-exposed human breast cancer T47D cells using gel-based proteomics analysis. The expression of c-ros receptor tyrosine kinase (ROS1) was increased and activated by autophosphorylation, thereby activating mitogen- and stress-activated protein kinase 1 (MSK1) through the mitogen‑activated protein kinase (MAPK) pathway; activated MSK1, in turn, phosphorylated histone 3 serine 10 (H3S10p) residues in the nucleus. The increase in H3S10 phosphorylation consequently increased the level of expression of immediate-early gene such as c-fos. This study demonstrated that when breast cancer cells are exposed to alcohol, phosphorylated ROS1 activates MSK1 via Erk1/2 in the MAPK pathway, which then induces modifications to histone residues that regulate gene expression by 14-3-3 protein recruitment, leading to a lack of control of breast cancer cell proliferation.

  15. GLYCINE-RICH RNA-BINDING PROTEIN1 interacts with RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 and suppresses cell death and defense responses in pepper (Capsicum annuum).

    PubMed

    Kim, Dae Sung; Kim, Nak Hyun; Hwang, Byung Kook

    2015-01-01

    Plants use a variety of innate immune regulators to trigger cell death and defense responses against pathogen attack. We identified pepper (Capsicum annuum) GLYCINE-RICH RNA-BINDING PROTEIN1 (CaGRP1) as a RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 (CaPIK1)-interacting partner, based on bimolecular fluorescence complementation and coimmunoprecipitation analyses as well as gene silencing and transient expression analysis. CaGRP1 contains an N-terminal RNA recognition motif and a glycine-rich region at the C-terminus. The CaGRP1 protein had DNA- and RNA-binding activity in vitro. CaGRP1 interacted with CaPIK1 in planta. CaGRP1 and CaGRP1-CaPIK1 complexes were localized to the nucleus in plant cells. CaPIK1 phosphorylated CaGRP1 in vitro and in planta. Transient coexpression of CaGRP1 with CaPIK1 suppressed the CaPIK1-triggered cell death response, accompanied by a reduced CaPIK1-triggered reactive oxygen species (ROS) burst. The RNA recognition motif region of CaGRP1 was responsible for the nuclear localization of CaGRP1 as well as the suppression of the CaPIK1-triggered cell death response. CaGRP1 silencing in pepper conferred enhanced resistance to Xanthomonas campestris pv vesicatoria (Xcv) infection; however, CaPIK1-silenced plants were more susceptible to Xcv. CaGRP1 interacts with CaPIK1 and negatively regulates CaPIK1-triggered cell death and defense responses by suppressing ROS accumulation.

  16. Role of protein kinase C in caerulein induced expression of substance P and neurokinin-1-receptors in murine pancreatic acinar cells

    PubMed Central

    Koh, Yung-Hua; Tamizhselvi, Ramasamy; Moochhala, Shabbir; Bian, Jin-Song; Bhatia, Madhav

    2011-01-01

    Substance P (SP) is involved in the pathophysiology of acute pancreatitis (AP) via binding to its high-affinity receptor, neurokinin-1-receptor (NK1R). An up-regulation of SP and NK1R expression was observed in experimental AP and in caerulein-stimulated pancreatic acinar cells. However, the mechanisms that lead to this up-regulation are not fully understood. In this study, we showed the role of protein kinase C (PKC) in caerulein-induced SP and NK1R production in isolated mouse pancreatic acinar cells. Caerulein (10−7 M) stimulation rapidly activated the conventional PKC-α and novel PKC-δ as observed by the phosphorylation of these molecules. Pre-treatment of pancreatic acinar cells with Gö6976 (1–10 nM) and rottlerin (1–10 μM) inhibited PKC-α and PKC-δ phosphorylation, respectively, but not the other way round. At these concentrations used, PKC-α and PKC-δ inhibition reversed the caerulein-induced up-regulation of SP and NK1R, indicating an important role of PKCs in the modulation of SP and NK1R expression. Further experiments looking into signalling mechanisms showed that treatment of pancreatic acinar cells with both Gö6976 and rottlerin inhibited the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Inhibition of PKC-α or PKC-δ also affected caerulein-induced transcription factor activation, as represented by nuclear factor-κB and AP-1 DNA-binding activity. The findings in this study suggested that PKC is upstream of the mitogen-activated protein kinases and transcription factors, which then lead to the up-regulation of SP/NK1R expression in caerulein-treated mouse pancreatic acinar cells. PMID:20973912

  17. Role of protein kinase C in caerulein induced expression of substance P and neurokinin-1-receptors in murine pancreatic acinar cells.

    PubMed

    Koh, Yung-Hua; Tamizhselvi, Ramasamy; Moochhala, Shabbir; Bian, Jin-Song; Bhatia, Madhav

    2011-10-01

    Substance P (SP) is involved in the pathophysiology of acute pancreatitis (AP) via binding to its high-affinity receptor, neurokinin-1-receptor (NK1R). An up-regulation of SP and NK1R expression was observed in experimental AP and in caerulein-stimulated pancreatic acinar cells. However, the mechanisms that lead to this up-regulation are not fully understood. In this study, we showed the role of protein kinase C (PKC) in caerulein-induced SP and NK1R production in isolated mouse pancreatic acinar cells. Caerulein (10(-7) M) stimulation rapidly activated the conventional PKC-α and novel PKC-δ as observed by the phosphorylation of these molecules. Pre-treatment of pancreatic acinar cells with Gö6976 (1-10 nM) and rottlerin (1-10 μM) inhibited PKC-α and PKC-δ phosphorylation, respectively, but not the other way round. At these concentrations used, PKC-α and PKC-δ inhibition reversed the caerulein-induced up-regulation of SP and NK1R, indicating an important role of PKCs in the modulation of SP and NK1R expression. Further experiments looking into signalling mechanisms showed that treatment of pancreatic acinar cells with both Gö6976 and rottlerin inhibited the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Inhibition of PKC-α or PKC-δ also affected caerulein-induced transcription factor activation, as represented by nuclear factor-κB and AP-1 DNA-binding activity. The findings in this study suggested that PKC is upstream of the mitogen-activated protein kinases and transcription factors, which then lead to the up-regulation of SP/NK1R expression in caerulein-treated mouse pancreatic acinar cells.

  18. Overexpression of an Arabidopsis cysteine-rich receptor-like protein kinase, CRK5, enhances abscisic acid sensitivity and confers drought tolerance

    PubMed Central

    Lu, Kai; Liang, Shan; Wu, Zhen; Bi, Chao; Yu, Yong-Tao; Wang, Xiao-Fang; Zhang, Da-Peng

    2016-01-01

    Receptor-like kinases (RLKs) have been reported to regulate many developmental and defense process, but only a few members have been functionally characterized. In the present study, our observations suggest that one of the RLKs, a membrane-localized cysteine-rich receptor-like protein kinase, CRK5, is involved in abscisic acid (ABA) signaling in Arabidopsis thaliana. Overexpression of CRK5 increases ABA sensitivity in ABA-induced early seedling growth arrest and promotion of stomatal closure and inhibition of stomatal opening. Interestingly, and importantly, overexpression of CRK5 enhances plant drought tolerance without affecting plant growth at the mature stages and plant productivity. Transgenic lines overexpressing a mutated form of CRK5, CRK5 K372E with the change of the 372nd conserved amino acid residue from lysine to glutamic acid in its kinase domain, result in wild-type ABA and drought responses, supporting the role of CRK5 in ABA signaling. The loss-of-function mutation of the CRK5 gene does not affect the ABA response, while overexpression of two homologs of CRK5, CRK4 and CRK19, confers ABA responses, suggesting that these CRK members function redundantly. We further showed that WRKY18, WRKY40 and WRKY60 transcription factors repress the expression of CRK5, and that CRK5 likely functions upstream of ABI2 in ABA signaling. These findings help in understanding the complex ABA signaling network. PMID:27406784

  19. Insulin receptor substrate-2 phosphorylation is necessary for protein kinase C zeta activation by insulin in L6hIR cells.

    PubMed

    Oriente, F; Formisano, P; Miele, C; Fiory, F; Maitan, M A; Vigliotta, G; Trencia, A; Santopietro, S; Caruso, M; Van Obberghen, E; Beguinot, F

    2001-10-05

    We have investigated glycogen synthase (GS) activation in L6hIR cells expressing a peptide corresponding to the kinase regulatory loop binding domain of insulin receptor substrate-2 (IRS-2) (KRLB). In several clones of these cells (B2, F4), insulin-dependent binding of the KRLB to insulin receptors was accompanied by a block of IRS-2, but not IRS-1, phosphorylation, and insulin receptor binding. GS activation by insulin was also inhibited by >70% in these cells (p < 0.001). The impairment of GS activation was paralleled by a similarly sized inhibition of glycogen synthase kinase 3 alpha (GSK3 alpha) and GSK3 beta inactivation by insulin with no change in protein phosphatase 1 activity. PDK1 (a phosphatidylinositol trisphosphate-dependent kinase) and Akt/protein kinase B (PKB) activation by insulin showed no difference in B2, F4, and in control L6hIR cells. At variance, insulin did not activate PKC zeta in B2 and F4 cells. In L6hIR, inhibition of PKC zeta activity by either a PKC zeta antisense or a dominant negative mutant also reduced by 75% insulin inactivation of GSK3 alpha and -beta (p < 0.001) and insulin stimulation of GS (p < 0.002), similar to Akt/PKB inhibition. In L6hIR, insulin induced protein kinase C zeta (PKC zeta) co-precipitation with GSK3 alpha and beta. PKC zeta also phosphorylated GSK3 alpha and -beta. Alone, these events did not significantly affect GSK3 alpha and -beta activities. Inhibition of PKC zeta activity, however, reduced Akt/PKB phosphorylation of the key serine sites on GSK3 alpha and -beta by >80% (p < 0.001) and prevented full GSK3 inactivation by insulin. Thus, IRS-2, not IRS-1, signals insulin activation of GS in the L6hIR skeletal muscle cells. In these cells, insulin inhibition of GSK3 alpha and -beta requires dual phosphorylation by both Akt/PKB and PKC zeta.

  20. Co-active receptor tyrosine kinases mitigate the effect of FGFR inhibitors in FGFR1-amplified lung cancers with low FGFR1 protein expression.

    PubMed

    Kotani, H; Ebi, H; Kitai, H; Nanjo, S; Kita, K; Huynh, T G; Ooi, A; Faber, A C; Mino-Kenudson, M; Yano, S

    2016-07-07

    Targeted therapies are effective in subsets of lung cancers with EGFR mutations and anaplastic lymphoma kinase (ALK) translocations. Large-scale genomics have recently expanded the lung cancer landscape with FGFR1 amplification found in 10-20% of squamous cell carcinomas (SCCs). However, the response rates have been low for biomarker-directed fibroblast growth factor receptor (FGFR) inhibitor therapy in SCC, which contrasts to the relatively high rates of response seen in EGFR mutant and ALK-translocated lung cancers treated with epidermal growth factor receptor (EGFR) inhibitors and ALK inhibitors, respectively. In order to better understand the low response rates of FGFR1-amplified lung cancers to FGFR inhibitors, relationships between gene copy number, mRNA expression and protein expression of FGFR1 were assessed in cell lines, tumor specimens and data from The Cancer Genome Atlas. The importance of these factors for the sensitivity to FGFR inhibitors was determined by analyzing drug screen data and conducting in vitro and in vivo experiments. We report that there was a discrepancy between FGFR1 amplification level and FGFR1 protein expression in a number of these cell lines, and the cancers with unexpectedly low FGFR1 expression were uniformly resistant to the different FGFR inhibitors. Further interrogation of the receptor tyrosine kinase activity in these discordant cell lines revealed co-activation of HER2 and platelet-derived growth factor receptor-α (PDGFRα) caused by gene amplification or ligand overexpression maintained phosphoinositide 3-kinase (PI3K) and MEK/ERK signaling even in the presence of FGFR inhibitor. Accordingly, co-inhibition of FGFR1 and HER2 or PDGFRα led to enhanced drug responses. In contrast, FGFR1-amplified high FGFR1 protein-expressing lung cancers are sensitive to FGFR inhibitor monotherapy by downregulating ERK signaling. Addition of a PI3K inhibitor to these high FGFR1 protein-expressing cancers further sensitized them to FGFR

  1. Neuromedin U type 1 receptor stimulation of A-type K+ current requires the βγ subunits of Go protein, protein kinase A, and extracellular signal-regulated kinase 1/2 (ERK1/2) in sensory neurons.

    PubMed

    Zhang, Yiming; Jiang, Dongsheng; Zhang, Yuan; Jiang, Xinghong; Wang, Fen; Tao, Jin

    2012-05-25

    Although neuromedin U (NMU) has been implicated in analgesia, the detailed mechanisms still remain unclear. In this study, we identify a novel functional role of NMU type 1 receptor (NMUR1) in regulating the transient outward K(+) currents (I(A)) in small dorsal root ganglion (DRG) neurons. We found that NMU reversibly increased I(A) in a dose-dependent manner, instead the sustained delayed rectifier K(+) current (I(DR)) was not affected. This NMU-induced I(A) increase was pertussis toxin-sensitive and was totally reversed by NMUR1 knockdown. Intracellular application of GDPβS (guanosine 5'-O-(2-thiodiphosphate)), QEHA peptide, or a selective antibody raised against the Gα(o) or Gβ blocked the stimulatory effects of NMU. Pretreatment of the cells with the protein kinase A (PKA) inhibitor or ERK inhibitor abolished the NMU-induced I(A) response, whereas inhibition of phosphatidylinositol 3-kinase or PKC had no such effects. Exposure of DRG neurons to NMU markedly induced the phosphorylation of ERK (p-ERK), whereas p-JNK or p-p38 was not affected. Moreover, the NMU-induced p-ERK increase was attenuated by PKA inhibition and activation of PKA by foskolin would mimic the NMU-induced I(A) increase. Functionally, we observed a significant decrease of the firing rate of neuronal action potential induced by NMU and pretreatment of DRG neurons with 4-AP could abolish this effect. In summary, these results suggested that NMU increases I(A) via activation of NMUR1 that couples sequentially to the downstream activities of Gβγ of the G(o) protein, PKA, and ERK, which could contribute to its physiological functions including neuronal hypoexcitability in DRG neurons.

  2. CUL3 and protein kinases

    PubMed Central

    Metzger, Thibaud; Kleiss, Charlotte; Sumara, Izabela

    2013-01-01

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

  3. The regulator of G protein signaling (RGS) domain of G protein-coupled receptor kinase 5 (GRK5) regulates plasma membrane localization and function.

    PubMed

    Xu, Hua; Jiang, Xiaoshan; Shen, Ke; Fischer, Christopher C; Wedegaertner, Philip B

    2014-07-01

    The G protein-coupled receptor (GPCR) kinases (GRKs) phosphorylate activated GPCRs at the plasma membrane (PM). Here GRK5/GRK4 chimeras and point mutations in GRK5 identify a short sequence within the regulator of G protein signaling (RGS) domain in GRK5 that is critical for GRK5 PM localization. This region of the RGS domain of GRK5 coincides with a region of GRK6 and GRK1 shown to form a hydrophobic dimeric interface (HDI) in crystal structures. Coimmunoprecipitation (coIP) and acceptor photobleaching fluorescence resonance energy transfer assays show that expressed GRK5 self-associates in cells, whereas GRK5-M165E/F166E (GRK5-EE), containing hydrophilic mutations in the HDI region of the RGS domain, displays greatly decreased coIP interactions. Both forcing dimerization of GRK5-EE, via fusion to leucine zipper motifs, and appending an extra C-terminal membrane-binding region to GRK5-EE (GRK5-EE-CT) recover PM localization. In addition, GRK5-EE displays a decreased ability to inhibit PAR1-induced calcium release compared with GRK5 wild type (wt). In contrast, PM-localized GRK5-EE-CaaX (appending a C-terminal prenylation and polybasic motif from K-ras) or GRK5-EE-CT shows comparable ability to GRK5 wt to inhibit PAR1-induced calcium release. The results suggest a novel model in which GRK5 dimerization is important for its plasma membrane localization and function.

  4. G Protein-Coupled Receptor Kinase 3 and Protein Kinase C Phosphorylate the Distal C-Terminal Tail of the Chemokine Receptor CXCR4 and Mediate Recruitment of Beta-Arrestin.

    PubMed

    Luo, Jiansong; Busillo, John M; Stumm, Ralf; Benovic, Jeffrey L

    2017-03-22

    Phosphorylation of G protein-coupled receptors (GPCRs) is a key event for cell signaling and regulation of receptor function. Previously, using tandem mass spectrometry, we identified two phosphorylation sites at the distal C-terminal tail of the chemokine receptor CXCR4, but were unable to determine which specific residues were phosphorylated. Here, we demonstrate that serines 346 and/or 347 (Ser-346/7) of CXCR4 are phosphorylated upon stimulation with the agonist CXCL12 as well as a CXCR4 pepducin, ATI-2341. ATI-2341, a Gi-biased CXCR4 agonist, induced more robust phosphorylation of Ser-346/7 compared to CXCL12. Knockdown of GRK2, GRK3 or GRK6 reduced CXCL12-induced phosphorylation of Ser-346/7 with GRK3 knockdown having the strongest effect, while inhibition of the conventional PKC isoforms reduced phosphorylation of Ser-346/7 induced by either CXCL12 or ATI-2341. The loss of GRK3- or PKC-mediated phosphorylation of Ser-346/7 impaired the recruitment of β-arrestin to CXCR4. We also found that a pseudo-substrate peptide inhibitor for PKCζ effectively inhibited CXCR4 phosphorylation and signaling, most likely by functioning as a non-specific CXCR4 antagonist. Together, these studies demonstrate the role Ser-346/7 plays in arrestin recruitment and initiation of the process of receptor desensitization and provide insight into the dysregulation of CXCR4 observed in patients with various forms of WHIM syndrome.

  5. Duplication and Divergence of Leucine-Rich Repeat Receptor-Like Protein Kinase (LRR-RLK) Genes in Basal Angiosperm Amborella trichopoda

    PubMed Central

    Liu, Ping-Li; Xie, Lu-Lu; Li, Peng-Wei; Mao, Jian-Feng; Liu, Hui; Gao, Shu-Min; Shi, Peng-Hao; Gong, Jun-Qing

    2016-01-01

    Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are the largest group of receptor-like kinases, which are one of the largest protein superfamilies in plants, and play crucial roles in development and stress responses. Although the evolution of LRR-RLK families has been investigated in some eudicot and monocot plants, no comprehensive evolutionary studies have been performed for these genes in basal angiosperms like Amborella trichopoda. In this study, we identified 94 LRR-RLK genes in the genome of A. trichopoda. The number of LRR-RLK genes in the genome of A. trichopoda is only 17–50% of that of several eudicot and monocot species. Tandem duplication and whole-genome duplication have made limited contributions to the expansion of LRR-RLK genes in A. trichopoda. According to the phylogenetic analysis, all A. trichopoda LRR-RLK genes can be organized into 18 subfamilies, which roughly correspond to the LRR-RLK subfamilies defined in Arabidopsis thaliana. Most LRR-RLK subfamilies are characterized by highly conserved protein structures, motif compositions, and gene structures. The unique gene structure, protein structures, and protein motif compositions of each subfamily provide evidence for functional divergence among LRR-RLK subfamilies. Moreover, the expression data of LRR-RLK genes provided further evidence for the functional diversification of them. In addition, selection analyses showed that most LRR-RLK protein sites are subject to purifying selection. Our results contribute to a better understanding of the evolution of LRR-RLK gene family in angiosperm and provide a framework for further functional investigation on A. trichopoda LRR-RLKs. PMID:28066499

  6. Duplication and Divergence of Leucine-Rich Repeat Receptor-Like Protein Kinase (LRR-RLK) Genes in Basal Angiosperm Amborella trichopoda.

    PubMed

    Liu, Ping-Li; Xie, Lu-Lu; Li, Peng-Wei; Mao, Jian-Feng; Liu, Hui; Gao, Shu-Min; Shi, Peng-Hao; Gong, Jun-Qing

    2016-01-01

    Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) are the largest group of receptor-like kinases, which are one of the largest protein superfamilies in plants, and play crucial roles in development and stress responses. Although the evolution of LRR-RLK families has been investigated in some eudicot and monocot plants, no comprehensive evolutionary studies have been performed for these genes in basal angiosperms like Amborella trichopoda. In this study, we identified 94 LRR-RLK genes in the genome of A. trichopoda. The number of LRR-RLK genes in the genome of A. trichopoda is only 17-50% of that of several eudicot and monocot species. Tandem duplication and whole-genome duplication have made limited contributions to the expansion of LRR-RLK genes in A. trichopoda. According to the phylogenetic analysis, all A. trichopoda LRR-RLK genes can be organized into 18 subfamilies, which roughly correspond to the LRR-RLK subfamilies defined in Arabidopsis thaliana. Most LRR-RLK subfamilies are characterized by highly conserved protein structures, motif compositions, and gene structures. The unique gene structure, protein structures, and protein motif compositions of each subfamily provide evidence for functional divergence among LRR-RLK subfamilies. Moreover, the expression data of LRR-RLK genes provided further evidence for the functional diversification of them. In addition, selection analyses showed that most LRR-RLK protein sites are subject to purifying selection. Our results contribute to a better understanding of the evolution of LRR-RLK gene family in angiosperm and provide a framework for further functional investigation on A. trichopoda LRR-RLKs.

  7. G protein-coupled receptors (GPCRs) That Signal via Protein Kinase A (PKA) Cross-talk at Insulin Receptor Substrate 1 (IRS1) to Activate the phosphatidylinositol 3-kinase (PI3K)/AKT Pathway.

    PubMed

    Law, Nathan C; White, Morris F; Hunzicker-Dunn, Mary E

    2016-12-30

    G protein-coupled receptors (GPCRs) activate PI3K/v-AKT thymoma viral oncoprotein (AKT) to regulate many cellular functions that promote cell survival, proliferation, and growth. However, the mechanism by which GPCRs activate PI3K/AKT remains poorly understood. We used ovarian preantral granulosa cells (GCs) to elucidate the mechanism by which the GPCR agonist FSH via PKA activates the PI3K/AKT cascade. Insulin-like growth factor 1 (IGF1) is secreted in an autocrine/paracrine manner by GCs and activates the IGF1 receptor (IGF1R) but, in the absence of FSH, fails to stimulate YXXM phosphorylation of IRS1 (insulin receptor substrate 1) required for PI3K/AKT activation. We show that PKA directly phosphorylates the protein phosphatase 1 (PP1) regulatory subunit myosin phosphatase targeting subunit 1 (MYPT1) to activate PP1 associated with the IGF1R-IRS1 complex. Activated PP1 is sufficient to dephosphorylate at least four IRS1 Ser residues, Ser(318), Ser(346), Ser(612), and Ser(789), and promotes IRS1 YXXM phosphorylation by the IGF1R to activate the PI3K/AKT cascade. Additional experiments indicate that this mechanism also occurs in breast cancer, thyroid, and preovulatory granulosa cells, suggesting that the PKA-dependent dephosphorylation of IRS1 Ser/Thr residues is a conserved mechanism by which GPCRs signal to activate the PI3K/AKT pathway downstream of the IGF1R.

  8. PTP1B-dependent regulation of receptor tyrosine kinase signaling by the actin-binding protein Mena.

    PubMed

    Hughes, Shannon K; Oudin, Madeleine J; Tadros, Jenny; Neil, Jason; Del Rosario, Amanda; Joughin, Brian A; Ritsma, Laila; Wyckoff, Jeff; Vasile, Eliza; Eddy, Robert; Philippar, Ulrike; Lussiez, Alisha; Condeelis, John S; van Rheenen, Jacco; White, Forest; Lauffenburger, Douglas A; Gertler, Frank B

    2015-11-01

    During breast cancer progression, alternative mRNA splicing produces functionally distinct isoforms of Mena, an actin regulator with roles in cell migration and metastasis. Aggressive tumor cell subpopulations express Mena(INV), which promotes tumor cell invasion by potentiating EGF responses. However, the mechanism by which this occurs is unknown. Here we report that Mena associates constitutively with the tyrosine phosphatase PTP1B and mediates a novel negative feedback mechanism that attenuates receptor tyrosine kinase signaling. On EGF stimulation, complexes containing Mena and PTP1B are recruited to the EGFR, causing receptor dephosphorylation and leading to decreased motility responses. Mena also interacts with the 5' inositol phosphatase SHIP2, which is important for the recruitment of the Mena-PTP1B complex to the EGFR. When Mena(INV) is expressed, PTP1B recruitment to the EGFR is impaired, providing a mechanism for growth factor sensitization to EGF, as well as HGF and IGF, and increased resistance to EGFR and Met inhibitors in signaling and motility assays. In sum, we demonstrate that Mena plays an important role in regulating growth factor-induced signaling. Disruption of this attenuation by Mena(INV) sensitizes tumor cells to low-growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes.

  9. A novel role for c-Myc in G protein-coupled receptor kinase 4 (GRK4) transcriptional regulation in human kidney proximal tubule cells.

    PubMed

    Gildea, John J; Tran, Hanh T; Van Sciver, Robert E; Bigler Wang, Dora; Carlson, Julia M; Felder, Robin A

    2013-05-01

    The G protein-coupled receptor kinase 4 (GRK4) negatively regulates the dopaminergic system by desensitizing the dopamine-1-receptor. The expressional control of GRK4 has not been reported, but here we show that the transcription factor c-Myc binds to the promoter of GRK4 and positively regulates GRK4 protein expression in human renal proximal tubule cells (RPTCs). Addition of phorbol esters to RPTCs not only increased c-Myc binding to the GRK4 promoter but also increased both phospho-c-Myc and GRK4 expression. The phorbol ester-mediated increase in GRK4 expression was completely blocked by the c-Myc inhibitor, 10074-G5, indicating that GRK4 is downstream of phospho-c-Myc. The autocrine production of angiotensin II (Ang II) in RPTCs increased the phosphorylation and activation of c-Myc and subsequently GRK4 expression. 3-Amino-4-thio-butyl sulfonate, an inhibitor of aminopeptidase A, increased RPTC secretion of Ang II. 3-Amino-4-thio-butyl sulfonate or Ang II increased the expression of both phospho-c-Myc and GRK4, which was blocked by 10074-G5. Blockade of the Ang II type 1 receptor with losartan decreased phospho-c-Myc and GRK4 expression. Both inhibition of c-Myc activity and blockade of Ang II type 1 receptor restored the coupling of dopamine-1-receptor to adenylyl cyclase stimulation in uncoupled RPTCs, whereas phorbol esters or Ang II caused the uncoupling of normally coupled RPTCs. We suggest that the Ang II type 1 receptor impairs dopamine-1-receptor function via c-Myc activation of GRK4. This novel pathway may be involved in the increase in blood pressure in hypertension that is mediated by increased activity of the renin-angiotensin system and decreased activity of the renal dopaminergic system.

  10. Inhibition of G-protein-coupled Receptor Kinase 2 Prevents the Dysfunctional Cardiac Substrate Metabolism in Fatty Acid Synthase Transgenic Mice.

    PubMed

    Abd Alla, Joshua; Graemer, Muriel; Fu, Xuebin; Quitterer, Ursula

    2016-02-05

    Impairment of myocardial fatty acid substrate metabolism is characteristic of late-stage heart failure and has limited treatment options. Here, we investigated whether inhibition of G-protein-coupled receptor kinase 2 (GRK2) could counteract the disturbed substrate metabolism of late-stage heart failure. The heart failure-like substrate metabolism was reproduced in a novel transgenic model of myocardium-specific expression of fatty acid synthase (FASN), the major palmitate-synthesizing enzyme. The increased fatty acid utilization of FASN transgenic neonatal cardiomyocytes rapidly switched to a heart failure phenotype in an adult-like lipogenic milieu. Similarly, adult FASN transgenic mice developed signs of heart failure. The development of disturbed substrate utilization of FASN transgenic cardiomyocytes and signs of heart failure were retarded by the transgenic expression of GRKInh, a peptide inhibitor of GRK2. Cardioprotective GRK2 inhibition required an intact ERK axis, which blunted the induction of cardiotoxic transcripts, in part by enhanced serine 273 phosphorylation of Pparg (peroxisome proliferator-activated receptor γ). Conversely, the dual-specific GRK2 and ERK cascade inhibitor, RKIP (Raf kinase inhibitor protein), triggered dysfunctional cardiomyocyte energetics and the expression of heart failure-promoting Pparg-regulated genes. Thus, GRK2 inhibition is a novel approach that targets the dysfunctional substrate metabolism of the failing heart.

  11. Inhibition of G-protein-coupled Receptor Kinase 2 Prevents the Dysfunctional Cardiac Substrate Metabolism in Fatty Acid Synthase Transgenic Mice*♦

    PubMed Central

    Abd Alla, Joshua; Graemer, Muriel; Fu, Xuebin; Quitterer, Ursula

    2016-01-01

    Impairment of myocardial fatty acid substrate metabolism is characteristic of late-stage heart failure and has limited treatment options. Here, we investigated whether inhibition of G-protein-coupled receptor kinase 2 (GRK2) could counteract the disturbed substrate metabolism of late-stage heart failure. The heart failure-like substrate metabolism was reproduced in a novel transgenic model of myocardium-specific expression of fatty acid synthase (FASN), the major palmitate-synthesizing enzyme. The increased fatty acid utilization of FASN transgenic neonatal cardiomyocytes rapidly switched to a heart failure phenotype in an adult-like lipogenic milieu. Similarly, adult FASN transgenic mice developed signs of heart failure. The development of disturbed substrate utilization of FASN transgenic cardiomyocytes and signs of heart failure were retarded by the transgenic expression of GRKInh, a peptide inhibitor of GRK2. Cardioprotective GRK2 inhibition required an intact ERK axis, which blunted the induction of cardiotoxic transcripts, in part by enhanced serine 273 phosphorylation of Pparg (peroxisome proliferator-activated receptor γ). Conversely, the dual-specific GRK2 and ERK cascade inhibitor, RKIP (Raf kinase inhibitor protein), triggered dysfunctional cardiomyocyte energetics and the expression of heart failure-promoting Pparg-regulated genes. Thus, GRK2 inhibition is a novel approach that targets the dysfunctional substrate metabolism of the failing heart. PMID:26670611

  12. Transient Receptor Potential Channel 1 Deficiency Impairs Host Defense and Proinflammatory Responses to Bacterial Infection by Regulating Protein Kinase Cα Signaling

    PubMed Central

    Zhou, Xikun; Ye, Yan; Sun, Yuyang; Li, Xuefeng; Wang, Wenxue; Privratsky, Breanna; Tan, Shirui; Zhou, Zongguang; Huang, Canhua; Wei, Yu-Quan; Birnbaumer, Lutz

    2015-01-01

    Transient receptor potential channel 1 (TRPC1) is a nonselective cation channel that is required for Ca2+ homeostasis necessary for cellular functions. However, whether TRPC1 is involved in infectious disease remains unknown. Here, we report a novel function for TRPC1 in host defense against Gram-negative bacteria. TRPC1−/− mice exhibited decreased survival, severe lung injury, and systemic bacterial dissemination upon infection. Furthermore, silencing of TRPC1 showed decreased Ca2+ entry, reduced proinflammatory cytokines, and lowered bacterial clearance. Importantly, TRPC1 functioned as an endogenous Ca2+ entry channel critical for proinflammatory cytokine production in both alveolar macrophages and epithelial cells. We further identified that bacterium-mediated activation of TRPC1 was dependent on Toll-like receptor 4 (TLR4), which induced endoplasmic reticulum (ER) store depletion. After activation of phospholipase Cγ (PLC-γ), TRPC1 mediated Ca2+ entry and triggered protein kinase Cα (PKCα) activity to facilitate nuclear translocation of NF-κB/Jun N-terminal protein kinase (JNK) and augment the proinflammatory response, leading to tissue damage and eventually mortality. These findings reveal that TRPC1 is required for host defense against bacterial infections through the TLR4-TRPC1-PKCα signaling circuit. PMID:26031335

  13. Characterization of the specific interaction between the DNA aptamer sgc8c and protein tyrosine kinase-7 receptors at the surface of T-cells by biosensing AFM.

    PubMed

    Leitner, Michael; Poturnayova, Alexandra; Lamprecht, Constanze; Weich, Sabine; Snejdarkova, Maja; Karpisova, Ivana; Hianik, Tibor; Ebner, Andreas

    2017-04-01

    We studied the interaction of the specific DNA aptamer sgc8c immobilized at the AFM tip with its corresponding receptor, the protein tyrosine kinase-7 (PTK7) embedded in the membrane of acute lymphoblastic leukemia (ALL) cells (Jurkat T-cells). Performing single molecule force spectroscopy (SMFS) experiments, we showed that the aptamer sgc8c bound with high probability (38.3 ± 7.48%) and high specificity to PTK7, as demonstrated by receptor blocking experiments and through comparison with the binding behavior of a nonspecific aptamer. The determined kinetic off-rate (koff = 5.16 s(-1)) indicates low dissociation of the sgc8c-PTK7 complex. In addition to the pulling force experiments, simultaneous topography and recognition imaging (TREC) experiments using AFM tips functionalized with sgc8c aptamers were realized on the outer regions surface of surface-immobilized Jurkat cells for the first time. This allowed determination of the distribution of PTK7 without any labeling and at near physiological conditions. As a result, we could show a homogeneous distribution of PTK7 molecules on the outer regions of ALL cells with a surface density of 325 ± 12 PTK7 receptors (or small receptor clusters) per μm(2). Graphical Abstract The specific interaction of the DNA aptamer sgc8c and protein tyrosine kinase-7 (PTK7) on acute lymphoblastic leukemia (ALL) cells was characterized. AFM based single molecule force spectroscopy (SMFS) yielded a kinetic off-rate of 5.16 s(-1) of the complex. Simultaneous topography and recognition imaging (TREC) revealed a PTK7 density of 325 ± 12 molecules or clusters per μm(2) in the cell membrane.

  14. EP4 and EP2 receptor activation of protein kinase A by prostaglandin E2 impairs macrophage phagocytosis of Clostridium sordellii

    PubMed Central

    Rogers, Lisa M.; Thelen, Tennille; Fordyce, Krystle; Bourdonnay, Emilie; Lewis, Casey; Yu, Han; Zhang, Junyong; Xie, Jingli; Serezani, Carlos H.; Peters-Golden, Marc; Aronoff, David M.

    2013-01-01

    Problem Clostridium sordellii causes endometrial infections but little is known regarding host defenses against this pathogen. Method of Study We tested the hypothesis that the immunoregulatory lipid prostaglandin (PG) E2 suppresses human macrophage clearance of C. sordellii through receptor-induced increases in intracellular cAMP. The THP-1 macrophage cell line was used to quantify C. sordellii phagocytosis. Results PGE2 increased cAMP levels, activated protein kinase A (PKA), and inhibited the class A scavenger receptor-dependent phagocytosis of C. sordellii. Activation of the EP2 and EP4 receptors increased intracellular cAMP and inhibited phagocytosis, with evidence favoring a more important role for EP4 over EP2. This was supported by EP receptor expression data and the use of pharmacological receptor antagonists. In addition, the PKA isoform RI appeared to be more important than RII in mediating the suppression of ingestion of C. sordellii. Conclusions The endogenous lipid mediator PGE2 impairs human innate immune responses against C. sordellii. PMID:23902376

  15. Calycosin Promotes Angiogenesis Involving Estrogen Receptor and Mitogen-Activated Protein Kinase (MAPK) Signaling Pathway in Zebrafish and HUVEC

    PubMed Central

    Li, Zhen Hua; Zhang, Zai Jun; Hu, Guang; Cheang, Lorita Chi Veng; Alex, Deepa; Hoi, Maggie Pui Man; Kwan, Yiu Wa; Chan, Shun Wan; Leung, George Pak Heng; Lee, Simon Ming Yuen

    2010-01-01

    Background Angiogenesis plays an important role in a wide range of physiological processes, and many diseases are associated with the dysregulation of angiogenesis. Radix Astragali is a Chinese medicinal herb commonly used for treating cardiovascular disorders and has been shown to possess angiogenic effect in previous studies but its active constituent and underlying mechanism remain unclear. The present study investigates the angiogenic effects of calycosin, a major isoflavonoid isolated from Radix Astragali, in vitro and in vivo. Methodology Tg(fli1:EGFP) and Tg(fli1:nEGFP) transgenic zebrafish embryos were treated with different concentrations of calycosin (10, 30, 100 µM) from 72 hpf to 96 hpf prior morphological observation and angiogenesis phenotypes assessment. Zebrafish embryos were exposed to calycosin (10, 100 µM) from 72 hpf to 78 hpf before gene-expression analysis. The effects of VEGFR tyrosine kinase inhibitor on calycosin-induced angiogenesis were studied using 72 hpf Tg(fli1:EGFP) and Tg(fli1:nEGFP) zebrafish embryos. The pro-angiogenic effects of calycosin were compared with raloxifene and tamoxifen in 72 hpf Tg(fli1:EGFP) zebrafish embryos. The binding affinities of calycosin to estrogen receptors (ERs) were evaluated by cell-free and cell-based estrogen receptor binding assays. Human umbilical vein endothelial cell cultures (HUVEC) were pretreated with different concentrations of calycosin (3, 10, 30, 100 µM) for 48 h then tested for cell viability and tube formation. The role of MAPK signaling in calycosin-induced angiogenesis was evaluated using western blotting. Conclusion Calycosin was shown to induce angiogenesis in human umbilical vein endothelial cell cultures (HUVEC) in vitro and zebrafish embryos in vivo via the up-regulation of vascular endothelial growth factor (VEGF), VEGFR1 and VEGFR2 mRNA expression. It was demonstrated that calycosin acted similar to other selective estrogen receptor modulators (SERMs), such as raloxifene and

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. Oncoprotein protein kinase

    DOEpatents

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

    2005-03-08

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

  2. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1997-01-01

    An isolated polypeptide (JNK) characterized by having a molecular weight of 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.

  3. 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.

  4. 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.

  5. 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.

  6. Protein kinase C-alpha regulates insulin action and degradation by interacting with insulin receptor substrate-1 and 14-3-3 epsilon.

    PubMed

    Oriente, Francesco; Andreozzi, Francesco; Romano, Chiara; Perruolo, Giuseppe; Perfetti, Anna; Fiory, Francesca; Miele, Claudia; Beguinot, Francesco; Formisano, Pietro

    2005-12-09

    Protein kinase C (PKC)-alpha exerts a regulatory function on insulin action. We showed by overlay blot that PKCalpha directly binds a 180-kDa protein, corresponding to IRS-1, and a 30-kDa molecular species, identified as 14-3-3epsilon. In intact NIH-3T3 cells overexpressing insulin receptors (3T3-hIR), insulin selectively increased PKCalpha co-precipitation with IRS-1, but not with IRS-2, and with 14-3-3epsilon, but not with other 14-3-3 isoforms. Overexpression of 14-3-3epsilon in 3T3-hIR cells significantly reduced IRS-1-bound PKCalpha activity, without altering IRS-1/PKCalpha co-precipitation. 14-3-3epsilon overexpression also increased insulin-stimulated insulin receptor and IRS-1 tyrosine phosphorylation, followed by increased activation of Raf1, ERK1/2, and Akt/protein kinase B. Insulin-induced glycogen synthase activity and thymidine incorporation were also augmented. Consistently, selective depletion of 14-3-3epsilon by antisense oligonucleotides caused a 3-fold increase of IRS-1-bound PKCalpha activity and a similarly sized reduction of insulin receptor and IRS-1 tyrosine phosphorylation and signaling. In turn, selective inhibition of PKCalpha expression by antisense oligonucleotides reverted the negative effect of 14-3-3epsilon depletion on insulin signaling. Moreover, PKCalpha inhibition was accompanied by a >2-fold decrease of insulin degradation. Similar results were also obtained by overexpressing 14-3-3epsilon. Thus, in NIH-3T3 cells, insulin induces the formation of multimolecular complexes, including IRS-1, PKCalpha, and 14-3-3epsilon. The presence of 14-3-3epsilon in the complex is not necessary for IRS-1/PKCalpha interaction but modulates PKCalpha activity, thereby regulating insulin signaling and degradation.

  7. Prednisone inhibits the focal adhesion kinase/receptor activator of NF-κB ligand/mitogen-activated protein kinase signaling pathway in rats with adriamycin-induced nephropathy.

    PubMed

    Ye, Minyuan; Zheng, Jing; Chen, Xiaoying; Chen, Xuelan; Wu, Xinhong; Lin, Xiuqin; Liu, Yafang

    2015-11-01

    The aim of the present study was to investigate the mechanisms underlying the effects of prednisone on adriamycin-induced nephritic rat kidney damage via the focal adhesion kinase (FAK)/receptor activator of nuclear factor-κB ligand (RANKL)/mitogen‑activated protein kinase (MAPK) signaling pathway. An adriamycin‑induced nephritic rat model was established to investigate these mechanisms. A total of 30 healthy male Sprague‑Dawley rats were randomly assigned to the normal, model or prednisone group. Samples of urine were collected over the course of 24 h at days 7, 14, and 28, and renal cortex tissue samples were harvested at days 14, and 28 following nephritic rat model establishment. The total urinary protein content was measured by biuret colorimetry. Pathological changes in the kidney tissue samples were observed using an electron microscope. The mRNA expressions levels of FAK, RANKL, p38, extracellular signal‑regulated kinase (ERK), c‑Jun N‑terminal kinase (JNK), and nephrin were then quantified by reverse transcription‑quantitative polymerase chain reaction. In addition, the protein expressions levels of FAK, RANKL, p38, ERK, JNK, phosphorylated (p)‑FAK, p‑ERK, and p‑JNK were quantified by western blotting. As compared with the normal group, the protein expression levels of FAK, RANKL, p-FAK, p38 and p-ERK in the model group were increased. In the prednisone group, the protein expression levels of p-ERK decreased, as compared with the normal group. In the prednisone group, the urinary protein levels, the protein expression levels of FAK, RANKL, p38, p-FAK, p-p38 and the mRNA expression levels of FAK, p38, RANKL, ERK, JNK decreased, as compared with the model group. In the prednisone group, the mRNA and protein expression levels of nephrin and the serum expression levels of RANKL increased, the serum expression levels of osteoprotegerin (OPG) were decreased, as compared with the model group. No significant changes in the protein expression

  8. Fc gamma receptor cross-linking activates p42, p38, and JNK/SAPK mitogen-activated protein kinases in murine macrophages: role for p42MAPK in Fc gamma receptor-stimulated TNF-alpha synthesis.

    PubMed

    Rose, D M; Winston, B W; Chan, E D; Riches, D W; Gerwins, P; Johnson, G L; Henson, P M

    1997-04-01

    Fc gamma R cross-linking on murine macrophages resulted in the activation of mitogen-activated protein kinase (MAPK) family members p42MAPK, p38, and c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase (SAPK). The temporal pattern of activation was distinct for each kinase. p42MAPK activation peaked at 5 min after receptor cross-linking, while peak p38 activity occurred 5 to 10 min later. Maximal JNK/SAPK activation occurred 20 min after Fc gamma R cross-linking. The selective MAPK/extracellular signal-regulated kinase-1 (MEK-1) inhibitor PD 098059 inhibited activation of p42MAPK induced by Fc gamma R cross-linking, but not p38 or JNK/SAPK activation. PD 098059 also inhibited the synthesis of TNF-alpha induced by Fc gamma R cross-linking (IC50 approximately 0.1 microM). Together, these results suggest that 1) the activation of MAPKs may play a role in Fc gammaR signal transduction, and 2) the activation of p42MAPK is necessary for Fc gamma R cross-linking-induced TNF-alpha synthesis.

  9. Receptor-interacting protein kinase 2 promotes triple-negative breast cancer cell migration and invasion via activation of nuclear factor-kappaB and c-Jun N-terminal kinase pathways

    PubMed Central

    2014-01-01

    Introduction Metastasis is the main cause of breast cancer morbidity and mortality. Processes that allow for tumor cell migration and invasion are important therapeutic targets. Here we demonstrate that receptor-interacting protein kinase 2 (RIP2), a kinase known to be involved in inflammatory processes, also has novel roles in cancer cell migration and invasion. Methods A total of six breast cancer expression databases, including The Cancer Genome Atlas, were assessed for RIP2 expression among various clinical subtypes and its role as a prognostic biomarker. mRNA fluorescence in situ hybridization (FISH) for RIP2 was performed on 17 stage III breast cancers to determine if there was a correlation between RIP2 expression and lymph node involvement. RNA-interference was used to knock-down RIP2 expression in MDA-MB-231, Htb126, SUM149PT, MCF7, T47D, and HCC1428 cells. Cell migration and invasion were measured in vitro by scratch/wound healing and transwell migration assays. A xenograft mouse model was used to assess tumor growth and chemosensitivity to docetaxel in vivo in MDA-MB-231 cells with and without RIP2 small hairpin RNA knockdown. Western blot and immunofluorescence imaging were used to evaluate protein expressions. Results Interrogation of expression databases showed that RIP2 expression is significantly over-expressed in triple-negative breast cancers (TNBC: estrogen-receptor (ER) negative, progesterone-receptor (PR) negative, Her2/neu- (Her2) negative), compared to other clinical subtypes. High RIP2 expression correlates with worse progression-free survival using a combined breast cancer expression array dataset consisting of 946 patients. Multivariate analysis shows RIP2 as an independent prognostic biomarker. Knock-down of RIP2 significantly decreases migration in both scratch/wound healing and transwell migration assays in MDA-MB-231, Htb126, SUM149PT, MCF7, and T47D cells and is correlated with decreased Nuclear Factor-kappaB and c-Jun N

  10. Genome-wide identification, characterization and expression analysis of populus leucine-rich repeat receptor-like protein kinase genes

    PubMed Central

    2013-01-01

    Background Leucine-rich repeat receptor-like kinases (LRR-RLKs) comprise the largest group within the receptor-like kinase (RLK) superfamily in plants. This gene family plays critical and diverse roles in plant growth, development and stress response. Although the LRR-RLK families in Arabidopsis and rice have been previously analyzed, no comprehensive studies have been performed on this gene family in tree species. Results In this work, 379 LRR-RLK genes were retrieved from the Populus trichocarpa genome and further grouped into 14 subfamilies based on their structural and sequence similarities. Approximately 82% (312 out of 379) of the PtLRR-RLK genes are located in segmental duplication blocks indicating the role of duplication process in the expansion of this gene family. The conservation and variation in motif composition and intron/exon arrangement among PtLRR-RLK subfamilies were analyzed to provide additional support for their phylogenetic relationship and more importantly to indicate the potential divergence in their functions. Expression profiling of PtLRR-RLKs showed that they were differentially expressed in different organs and tissues and some PtLRR-RLKs were specifically expressed in meristem tissues, which indicated their potential involvement in tissue development and differentiation. For most AtLRR-RLKs with defined functions, Populus homologues exhibiting similar expression patterns could be identified, which might indicate the functional conservation during evolution. Among 12 types of environmental cues analyzed by the genome-wide microarray data, PtLRR-RLKs showed specific responses to shoot organogenesis, wounding, low ammonium feeding, hypoxia and seasonal dormancy, but not to drought, re-watering after drought, flooding, AlCl3 treatment and bacteria or fungi treatments. Conclusions This study provides the first comprehensive genomic analysis of the Populus LRR-RLK gene family. Segmental duplication contributes significantly to the expansion

  11. Drug design for protein kinases and phosphatases: flexible-receptor docking, binding affinity and specificity, and drug-binding kinetics.

    PubMed

    Wong, Chung F; Bairy, Sneha

    2013-01-01

    This article reviews some of our experiences on applying computational techniques to aid the design of drugs targeting protein kinases and phosphatases. It is not a comprehensive review. Rather, it focuses on several less explored approaches or ideas that we have experiences on. It reviews some recent improvements on the Poisson-Boltzmann/Surface Area model for calculating binding affinity and discusses ways to perform calculations that are more tolerant to statistical and systematic errors. Several new ways to incorporate protein flexibility in molecular docking and estimating binding affinity are also discussed. Its discussions also go beyond binding affinity to considering drug-binding kinetics, not only on investigating protein-ligand interactions in isolation, but also on accounting for upstream and downstream influences that can occur in cells, through kinetic modeling of cell signaling. This review also describes a quick molecular simulation method for understanding drug-binding kinetics at the molecular level, with the hope of generating guiding principles for designing drugs with the desired kinetic properties. Sources of drug-binding selectivity that appear obvious but often overlooked are also discussed.

  12. SRPK1 and Akt Protein Kinases Phosphorylate the RS Domain of Lamin B Receptor with Distinct Specificity: A Combined Biochemical and In Silico Approach

    PubMed Central

    Nikolakaki, Eleni; Vlassi, Metaxia; Giannakouros, Thomas

    2016-01-01

    Activated Akt has been previously implicated in acting on RS domain-containing proteins. However, it has been questioned whether its action is direct or it is mediated by co-existing SR kinase activity. To address this issue we studied in detail the phosphorylation of Lamin B Receptor (LBR) by Akt. Using synthetic peptides and a set of recombinant proteins expressing mutants of the LBR RS domain we now demonstrate that while all serines of the RS domain represent more or less equal phosphoacceptor sites for SRPK1, Ser80 and Ser82 are mainly targeted by Akt. 3D-modeling combined with molecular dynamics (MD) simulations show that amongst short, overlapping LBR RS-containing peptides complying with the minimum Akt recognition consensus sequence, only those bearing phosphosites either at Ser80 or Ser82 are able to fit into the active site of Akt, at least as effectively as its known substrate, GSK3-β. Combined our results provide evidence that Akt kinases directly phosphorylate an RS domain-containing protein and that both the residues N-terminal the phosphosite and at position +1 are essential for Akt specificity, with the latter substrate position being compatible with the arginine residue of RS-repeats. PMID:27105349

  13. Enhanced Inhibition of Prostate Tumor Growth by Dual Targeting the Androgen Receptor and the Regulatory Subunit Type Iα of Protein Kinase A in Vivo

    PubMed Central

    Eder, Iris E.; Egger, Martina; Neuwirt, Hannes; Seifarth, Christof; Maddalo, Danilo; Desiniotis, Andreas; Schäfer, Georg; Puhr, Martin; Bektic, Jasmin; Cato, Andrew C. B.; Klocker, Helmut

    2013-01-01

    Progression to castration resistance is a major problem in the treatment of advanced prostate cancer and is likely to be driven by activation of several molecular pathways, including androgen receptor (AR) and cyclic AMP-dependent protein kinase A (PKA). In this study, we examined the therapeutic efficacy of a combined inhibition of the AR and the regulatory subunit type Iα (RIα) of protein kinase A with second generation antisense oligonucleotides (ODNs) in androgen-sensitive LNCaP and castration-resistant LNCaPabl tumors in vivo. We found that targeting the AR alone inhibited LNCaP, as well as LNCaPabl tumors. Combined inhibition resulted in an improved response over single targeting and even a complete tumor remission in LNCaPabl. Western blot analysis revealed that both ODNs were effective in reducing their target proteins when administered alone or in combination. In addition, treatment with the ODNs was associated with an induction of apoptosis. Our data suggest that dual targeting of the AR and PKARIα is more effective in inhibiting LNCaP and LNCaPabl tumor growth than single treatment and may give a treatment benefit, especially in castration-resistant prostate cancers. PMID:23736698

  14. DNA-Encoded Library Screening Identifies Benzo[b][1,4]oxazepin-4-ones as Highly Potent and Monoselective Receptor Interacting Protein 1 Kinase Inhibitors.

    PubMed

    Harris, Philip A; King, Bryan W; Bandyopadhyay, Deepak; Berger, Scott B; Campobasso, Nino; Capriotti, Carol A; Cox, Julie A; Dare, Lauren; Dong, Xiaoyang; Finger, Joshua N; Grady, LaShadric C; Hoffman, Sandra J; Jeong, Jae U; Kang, James; Kasparcova, Viera; Lakdawala, Ami S; Lehr, Ruth; McNulty, Dean E; Nagilla, Rakesh; Ouellette, Michael T; Pao, Christina S; Rendina, Alan R; Schaeffer, Michelle C; Summerfield, Jennifer D; Swift, Barbara A; Totoritis, Rachel D; Ward, Paris; Zhang, Aming; Zhang, Daohua; Marquis, Robert W; Bertin, John; Gough, Peter J

    2016-03-10

    The recent discovery of the role of receptor interacting protein 1 (RIP1) kinase in tumor necrosis factor (TNF)-mediated inflammation has led to its emergence as a highly promising target for the treatment of multiple inflammatory diseases. We screened RIP1 against GSK's DNA-encoded small-molecule libraries and identified a novel highly potent benzoxazepinone inhibitor series. We demonstrate that this template possesses complete monokinase selectivity for RIP1 plus unique species selectivity for primate versus nonprimate RIP1. We elucidate the conformation of RIP1 bound to this benzoxazepinone inhibitor driving its high kinase selectivity and design specific mutations in murine RIP1 to restore potency to levels similar to primate RIP1. This series differentiates itself from known RIP1 inhibitors in combining high potency and kinase selectivity with good pharmacokinetic profiles in rodents. The favorable developability profile of this benzoxazepinone template, as exemplified by compound 14 (GSK'481), makes it an excellent starting point for further optimization into a RIP1 clinical candidate.

  15. Protein Kinase D and Gβγ Subunits Mediate Agonist-evoked Translocation of Protease-activated Receptor-2 from the Golgi Apparatus to the Plasma Membrane.

    PubMed

    Jensen, Dane D; Zhao, Peishen; Jimenez-Vargas, Nestor N; Lieu, TinaMarie; Gerges, Marina; Yeatman, Holly R; Canals, Meritxell; Vanner, Stephen J; Poole, Daniel P; Bunnett, Nigel W

    2016-05-20

    Agonist-evoked endocytosis of G protein-coupled receptors has been extensively studied. The mechanisms by which agonists stimulate mobilization and plasma membrane translocation of G protein-coupled receptors from intracellular stores are unexplored. Protease-activated receptor-2 (PAR2) traffics to lysosomes, and sustained protease signaling requires mobilization and plasma membrane trafficking of PAR2 from Golgi stores. We evaluated the contribution of protein kinase D (PKD) and Gβγ to this process. In HEK293 and KNRK cells, the PAR2 agonists trypsin and 2-furoyl-LIGRLO-NH2 activated PKD in the Golgi apparatus, where PKD regulates protein trafficking. PAR2 activation induced translocation of Gβγ, a PKD activator, to the Golgi apparatus, determined by bioluminescence resonance energy transfer between Gγ-Venus and giantin-Rluc8. Inhibitors of PKD (CRT0066101) and Gβγ (gallein) prevented PAR2-stimulated activation of PKD. CRT0066101, PKD1 siRNA, and gallein all inhibited recovery of PAR2-evoked Ca(2+) signaling. PAR2 with a photoconvertible Kaede tag was expressed in KNRK cells to examine receptor translocation from the Golgi apparatus to the plasma membrane. Irradiation of the Golgi region (405 nm) induced green-red photo-conversion of PAR2-Kaede. Trypsin depleted PAR2-Kaede from the Golgi apparatus and repleted PAR2-Kaede at the plasma membrane. CRT0066101 inhibited PAR2-Kaede translocation to the plasma membrane. CRT0066101 also inhibited sustained protease signaling to colonocytes and nociceptive neurons that naturally express PAR2 and mediate protease-evoked inflammation and nociception. Our results reveal a major role for PKD and Gβγ in agonist-evoked mobilization of intracellular PAR2 stores that is required for sustained signaling by extracellular proteases.

  16. Protein Kinase C Is Involved in the Induction of ATP-Binding Cassette Transporter A1 Expression by Liver X Receptor/Retinoid X Receptor Agonist in Human Macrophages.

    PubMed

    Huwait, Etimad A; Singh, Nishi N; Michael, Daryn R; Davies, Thomas S; Moss, Joe W E; Ramji, Dipak P

    2015-05-07

    The transcription of the ATP-binding cassette transporter A1 (ABCA1) gene, which plays a key anti-atherogenic role, is known to be induced by agonists of liver X receptors (LXRs). LXRs form obligate heterodimers with retinoid X receptors (RXRs) and interact with their recognition sequences in the regulatory regions of key genes implicated in the control of cholesterol, fatty acid and glucose homeostasis. We have previously shown a novel role for c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3K) in the LXRs-mediated induction of macrophage gene expression. Protein kinase C (PKC) is often found to regulate the action of nuclear receptors and cross talk between this kinase family and JNK and/or PI3K has been shown in several settings. We have therefore investigated a potential role for PKC in the action of LXR/RXR agonist 22-(R)-hydroxycholesterol (22-(R)-HC)/9-cis-retinoic acid (9cRA) in THP-1 macrophages, including the induction of ABCA1 expression. The pan PKC inhibitor bisindoylmaleimide was found to attenuate the induction of ABCA1 protein expression, the activation of the JNK signaling pathway and the stimulation of activator protein-1 (AP-1) DNA binding activity in macrophages treated with 22-(R)-HC and 9cRA. The role of PKC in the action of these ligands was confirmed further by the use of more isotype-specific inhibitors. These studies therefore reveal a potentially important role for PKC in the action of 22-(R)-HC and 9cRA in human macrophages. This article is protected by copyright. All rights reserved.

  17. Protein Kinase C Is Involved in the Induction of ATP-Binding Cassette Transporter A1 Expression by Liver X Receptor/Retinoid X Receptor Agonist in Human Macrophages.

    PubMed

    Huwait, Etimad A; Singh, Nishi N; Michael, Daryn R; Davies, Thomas S; Moss, Joe W E; Ramji, Dipak P

    2015-09-01

    The transcription of the ATP-binding cassette transporter A1 (ABCA1) gene, which plays a key anti-atherogenic role, is known to be induced by agonists of liver X receptors (LXRs). LXRs form obligate heterodimers with retinoid X receptors (RXRs) and interact with their recognition sequences in the regulatory regions of key genes implicated in the control of cholesterol, fatty acid and glucose homeostasis. We have previously shown a novel role for c-Jun N-terminal kinase (JNK) and phosphoinositide 3-kinase (PI3K) in the LXRs-mediated induction of macrophage gene expression. Protein kinase C (PKC) is often found to regulate the action of nuclear receptors and cross talk between this kinase family and JNK and/or PI3K has been shown in several settings. We have, therefore, investigated a potential role for PKC in the action of LXR/RXR agonist 22-(R)-hydroxycholesterol (22-(R)-HC)/9-cis-retinoic acid (9cRA) in THP-1 macrophages, including the induction of ABCA1 expression. The pan PKC inhibitor bisindoylmaleimide was found to attenuate the induction of ABCA1 protein expression, the activation of the JNK signaling pathway and the stimulation of activator protein-1 (AP-1) DNA binding activity in macrophages treated with 22-(R)-HC and 9cRA. The role of PKC in the action of these ligands was confirmed further by the use of more isotype-specific inhibitors. These studies, therefore, reveal a potentially important role for PKC in the action of 22-(R)-HC and 9cRA in human macrophages.

  18. Food restriction increases NMDA receptor-mediated calcium-calmodulin kinase II and NMDA receptor/extracellular signal-regulated kinase 1/2-mediated cyclic amp response element-binding protein phosphorylation in nucleus accumbens upon D-1 dopamine receptor stimulation in rats.

    PubMed

    Haberny, S L; Carr, K D

    2005-01-01

    Biological drive states exert homeostatic control in part by increasing the reinforcing effects of environmental incentive stimuli. An apparent by-product of this adaptive response is the enhanced acquisition of drug self-administration behavior in food-restricted (FR) animals. While previous research has demonstrated increased central sensitivity to rewarding effects of abused drugs and direct dopamine (DA) receptor agonists in FR subjects, the underlying neurobiology is not well understood. Recently, it was demonstrated that intracerebroventricular (i.c.v.) injection of the D-1 DA receptor agonist, SKF-82958 produces a stronger activation of striatal extracellular signal-regulated kinase (ERK) 1/2 and cyclic AMP response element-binding protein (CREB) in FR relative to ad libitum (AL) fed rats. The main purpose of the present study was to characterize the involvement and mechanisms of interaction between NMDA receptor function and the augmented cellular responses to D-1 DA receptor stimulation in nucleus accumbens (NAc) of FR rats. In experiment 1, Western immunoblotting was used to demonstrate that i.c.v. injection of SKF-82958 (20 microg) produces greater phosphorylation of the NMDA NR1 subunit and calcium-calmodulin kinase II (CaMK II) in NAc of FR as compared with AL rats. In experiment 2, pretreatment of subjects with the NMDA antagonist, MK-801 (1.0 mg/kg, i.p.) decreased SKF-82958-induced activation of CaMK II, ERK1/2 and CREB, and reversed the augmenting effect of FR on activation of all three proteins. In experiment 3, pretreatment with the mitogen-activated protein kinase/ERK kinase inhibitor SL-327 (60 mg/kg, i.p.) suppressed SKF-82958- induced activation of ERK1/2 and reversed the augmenting effect of FR on CREB activation. These results point to specific neuroadaptations in the NAc of FR rats whereby D-1 DA receptor stimulation leads to increased NMDA NR1 subunit phosphorylation and consequent increases in NMDA receptor-dependent CaMK II and ERK1

  19. The immunomodulatory adapter proteins DAP12 and Fc receptor γ-chain (FcRγ) regulate development of functional osteoclasts through the Syk tyrosine kinase

    PubMed Central

    Mócsai, Attila; Humphrey, Mary Beth; Van Ziffle, Jessica A. G.; Hu, Yongmei; Burghardt, Andrew; Spusta, Steven C.; Majumdar, Sharmila; Lanier, Lewis L.; Lowell, Clifford A.; Nakamura, Mary C.

    2004-01-01

    Osteoclasts, the only bone-resorbing cells, are central to the pathogenesis of osteoporosis, yet their development and regulation are incompletely understood. Multiple receptors of the immune system use a common signaling paradigm whereby phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) within receptor-associated adapter proteins recruit the Syk tyrosine kinase. Here we demonstrate that a similar mechanism is required for development of functional osteoclasts. Mice lacking two ITAM-bearing adapters, DAP12 and the Fc receptor γ-chain (FcRγ), are severely osteopetrotic. DAP12-/-FcRγ-/- bone marrow cells fail to differentiate into multinucleated osteoclasts or resorb bone in vitro and show impaired phosphorylation of the Syk tyrosine kinase. syk-/- progenitors are similarly defective in osteoclast development and bone resorption. Intact SH2-domains of Syk, introduced by retroviral transduction, are required for functional reconstitution of syk-/- osteoclasts, whereas intact ITAM-domains on DAP12 are required for reconstitution of DAP12-/- FcRγ-/- cells. These data indicate that recruitment of Syk to phosphorylated ITAMs is critical for osteoclastogenesis. Although DAP12 appears to be primarily responsible for osteoclast differentiation in cultures directly stimulated with macrophage-colony stimulating factor and receptor activator of NF-κB ligand cytokines, DAP12 and FcRγ have overlapping roles in supporting osteoclast development in osteoblast–osteoclast cocultures, which mirrors their overlapping functions in vivo. These results provide new insight into the biology of osteoclasts and suggest novel therapeutic targets in diseases of bony remodeling. PMID:15073337

  20. Protein kinase C mediates the synergistic interaction between agonists acting at alpha2-adrenergic and delta-opioid receptors in spinal cord.

    PubMed

    Overland, Aaron C; Kitto, Kelley F; Chabot-Doré, Anne-Julie; Rothwell, Patrick E; Fairbanks, Carolyn A; Stone, Laura S; Wilcox, George L

    2009-10-21

    Coactivation of spinal alpha(2)-adrenergic receptors (ARs) and opioid receptors produces antinociceptive synergy. Antinociceptive synergy between intrathecally administered alpha(2)AR and opioid agonists is well documented, but the mechanism underlying this synergy remains unclear. The delta-opioid receptor (DOP) and the alpha(2A)ARs are coexpressed on the terminals of primary afferent fibers in the spinal cord where they may mediate this phenomenon. We evaluated the ability of the DOP-selective agonist deltorphin II (DELT), the alpha(2)AR agonist clonidine (CLON) or their combination to inhibit calcitonin gene-related peptide (CGRP) release from spinal cord slices. We then examined the possible underlying signaling mechanisms involved through coadministration of inhibitors of phospholipase C (PLC), protein kinase C (PKC) or protein kinase A (PKA). Potassium-evoked depolarization of spinal cord slices caused concentration-dependent release of CGRP. Coadministration of DELT and CLON inhibited the release of CGRP in a synergistic manner as confirmed statistically by isobolograpic analysis. Synergy was dependent on the activation of PLC and PKC, but not PKA, whereas the effect of agonist administration alone was only dependent on PLC. The importance of these findings was confirmed in vivo, using a thermal nociceptive test, demonstrating the PKC dependence of CLON-DELT antinociceptive synergy in mice. That inhibition of CGRP release by the combination was maintained in the presence of tetrodotoxin in spinal cord slices suggests that synergy does not rely on interneuronal signaling and may occur within single subcellular compartments. The present study reveals a novel signaling pathway underlying the synergistic analgesic interaction between DOP and alpha(2)AR agonists in the spinal cord.

  1. Methamphetamine-induced short-term increase and long-term decrease in spatial working memory affects protein Kinase M zeta (PKMζ), dopamine, and glutamate receptors

    PubMed Central

    Braren, Stephen H.; Drapala, Damian; Tulloch, Ingrid K.; Serrano, Peter A.

    2014-01-01

    Methamphetamine (MA) is a toxic, addictive drug shown to modulate learning and memory, yet the neural mechanisms are not fully understood. We investigated the effects of 2 weekly injections of MA (30 mg/kg) on working memory using the radial 8-arm maze (RAM) across 5 weeks in adolescent-age mice. MA-treated mice show a significant improvement in working memory performance 1 week following the first MA injection compared to saline-injected controls. Following 5 weeks of MA abstinence mice were re-trained on a reference and working memory version of the RAM to assess cognitive flexibility. MA-treated mice show significantly more working memory errors without effects on reference memory performance. The hippocampus and dorsal striatum were assessed for expression of glutamate receptors subunits, GluA2 and GluN2B; dopamine markers, dopamine 1 receptor (D1), dopamine transporter (DAT) and tyrosine hydroxylase (TH); and memory markers, protein kinase M zeta (PKMζ) and protein kinase C zeta (PKCζ). Within the hippocampus, PKMζ and GluA2 are both significantly reduced after MA supporting the poor memory performance. Additionally, a significant increase in GluN2B and decrease in D1 identifies dysregulated synaptic function. In the striatum, MA treatment increased cytosolic DAT and TH levels associated with dopamine hyperfunction. MA treatment significantly reduced GluN2B while increasing both PKMζ and PKCζ within the striatum. We discuss the potential role of PKMζ/PKCζ in modulating dopamine and glutamate receptors after MA treatment. These results identify potential underlying mechanisms for working memory deficits induced by MA. PMID:25566006

  2. Protein Kinase C Mediates the Synergistic Interaction Between Agonists Acting at Alpha-2-Adrenergic and Delta-Opioid Receptors in Spinal Cord

    PubMed Central

    Overland, Aaron C.; Kitto, Kelley F.; Chabot-Doré, Anne-Julie; Rothwell, Patrick E.; Fairbanks, Carolyn A.; Stone, Laura S.; Wilcox, George L.

    2009-01-01

    Co-activation of spinal α2-adrenergic receptors (AR) and opioid receptors (OR) produces antinociceptive synergy. Antinociceptive synergy between intrathecally (i.t.) administered α2AR and OR agonists is well documented, but the mechanism underlying this synergy remains unclear. The delta-opioid receptor (DOP) and the α2AAR are co-expressed on the terminals of primary afferent fibers in the spinal cord where they may mediate this phenomenon. We evaluated the ability of the DOP-selective agonist deltorphin II (DELT), the α2AR agonist clonidine (CLON) or their combination to inhibit calcitonin gene-related peptide (CGRP) release from spinal cord slices. We then examined the possible underlying signaling mechanisms involved through co-administration of inhibitors of phospholipase C (PLC), protein kinase C (PKC) or protein kinase A (PKA). Potassium-evoked depolarization of spinal cord slices caused concentration-dependent release of CGRP. Co-administration of DELT and CLON inhibited the release of CGRP in a synergistic manner as confirmed statistically by isobolograpic analysis. Synergy was dependent on the activation of PLC and PKC, but not PKA, while the effect of agonist administration alone was only dependent on PLC. The importance of these findings was confirmed in vivo, demonstrating the PKC-dependence on CLON-DELT antinociceptive synergy in mice. That inhibition of CGRP release by the combination was maintained in the presence of tetrodotoxin in spinal cord slices suggests that synergy does not rely on interneuronal signaling and may occur within single subcellular compartments. The present study reveals a novel signaling pathway underlying the synergistic analgesic interaction between DOP and α2AR agonists in the spinal cord. PMID:19846714

  3. Differential effects of protein kinase C activation on 5-HT1A receptor coupling to Ca2+ and K+ currents in rat serotonergic neurones.

    PubMed Central

    Chen, Y; Penington, N J

    1996-01-01

    1. Activation of the enzyme protein kinase C (PKC) partially uncouples receptors from the inhibition of Ca2+ current. We have studied the effect of PKC activation on 5-HT1A receptor coupling of Ca2+ currents and 5-HT-induced K+ current (IK,5-HT) in acutely isolated adult rat dorsal raphe neurones. 2. The phorbol ester 4 beta-phorbol 12-myristate, 13-acetate (PMA; 1 microM) did not significantly alter the peak Ca2+ current. A maximal dose of 5-HT inhibited Ca2+ current on average by 52%; after application of PMA, the inhibition was only 30% and the effect was irreversible for the duration of the experiment. 3. The inactive phorbol ester 4 alpha-phorbol (1 microM) did not reduce the effectiveness of 5-HT. When the kinase inhibitor staurosporine (ST; 200 nM) was added, PMA reduced the effect of 5-HT by only 13.9%. ST partially prevented or reversed the effect of PMA, depending on the order of addition. 4. The voltage-dependent rate or re-inhibition by 5-HT was reduced by PMA, suggesting that fewer activated G-protein subunits are available to interact with Ca2+ channel after the action of PMA. 5. In contrast, PMA (1 microM) did not have a significant effect on IK,5-HT. 6. PKC activation has an inhibitory effect on one branch of the 5-HT1A receptor transduction fork, namely inhibition of Ca2+ influx, but not on the activation of IK,5-HT. PMID:8910201

  4. Sequestration of muscarinic acetylcholine receptor m2 subtypes. Facilitation by G protein-coupled receptor kinase (GRK2) and attenuation by a dominant-negative mutant of GRK2.

    PubMed

    Tsuga, H; Kameyama, K; Haga, T; Kurose, H; Nagao, T

    1994-12-23

    Sequestration of m2 receptors (muscarinic acetylcholine receptor m2 subtypes), which was assessed as loss of N-[3H]methylscopolamine ([3H]NMS) binding activity from the cell surface, was examined in COS 7 and BHK-21 cells that had been transfected with expression vectors encoding the m2 receptor and, independently, vectors encoding a G protein-coupled receptor kinase (GRK2) (beta-adrenergic receptor kinase 1) or a GRK2 dominant-negative mutant (DN-GRK2). The sequestration of m2 receptors became apparent when the cells were treated with 10(-5) M or higher concentrations of carbamylcholine. In this case, approximately 40% or 20-25% of the [3H]NMS binding sites on COS 7 or BHK-21 cells, respectively, were sequestered with a half-life of 15-25 min. In cells in which GRK2 was also expressed, the sequestration became apparent in the presence of 10(-7) M carbamylcholine. Approximately 40% of the [3H]NMS binding sites on both COS 7 and BHK-21 cells were sequestered in the presence of 10(-6) M or higher concentrations of carbamylcholine. When DN-GRK2 was expressed in COS 7 cells, the proportion of [3H]NMS binding sites sequestered in the presence of 10(-5) M or higher concentrations of carbamylcholine was reduced to 20-30%. These results indicate that the phosphorylation of m2 receptors by GRK2 facilitates their sequestration. These results are in contrast with the absence of a correlation between sequestration and the phosphorylation of beta-adrenergic receptors by the GRK2 and suggests that the consequences of phosphorylation by GRK2 are different for different receptors.

  5. Nobiletin, a citrus flavonoid with neurotrophic action, augments protein kinase A-mediated phosphorylation of the AMPA receptor subunit, GluR1, and the postsynaptic receptor response to glutamate in murine hippocampus.

    PubMed

    Matsuzaki, Kentaro; Miyazaki, Kenichi; Sakai, Seiichiro; Yawo, Hiromu; Nakata, Norihito; Moriguchi, Shigeki; Fukunaga, Kohji; Yokosuka, Akihito; Sashida, Yutaka; Mimaki, Yoshihiro; Yamakuni, Tohru; Ohizumi, Yasushi

    2008-01-14

    Nobiletin isolated from citrus peels prevents bulbectomy- and amyloid-beta protein-induced memory impairment in rodents. In the present study, using combined methods of biochemistry and electrophysiology, we examined the effects of nobiletin on phosphorylation of GluR1 receptor, the subunit of alpha-amino-3-hydroxy-5-methyl-D-aspartate (AMPA) receptors, and the receptor-mediated synaptic transmission in the hippocampus, a region implicated in memory formation, in culture and/or in slices. Western blot analysis showed that nobiletin-stimulated phosphorylation of multiple protein kinase A (PKA) substrates at 10 min following the treatment in cultured hippocampal neurons. In the cultured neurons, this natural compound also increased not only PKA activity, but also phosphorylation of GluR1 receptor at a PKA phosphorylation site, Ser 845, which has been demonstrated to be critical for synaptic plasticity, including enhancement of postsynaptic glutamate response, and important for spatial memory in vivo. The increased phosphorylation of GluR1 receptor at Ser 845 was abolished by H89 (N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride), the PKA inhibitor, but not U0126 (1,4-diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene), the mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, in the cultured neurons. An increment of the phosphorylation of GluR1 receptor at Ser 845 was induced by nobiletin in the hippocampal slices as well. Furthermore, our electrophysiological analysis showed that nobiletin potentiated the AMPA receptor-mediated synaptic transmission at Schaffer collateral-CA1 pyramidal cell synapses in the hippocampal slices. This potentiation induced by the natural compound was not accompanied by the changes in paired-pulse ratio, and partially occluded the long-term potentiation, indicating the possible involvement of the postsynaptic mechanism. These findings suggest that nobiletin probably up-regulates synaptic transmission

  6. Transcriptional activation of peroxisome proliferator-activated receptor-{gamma} requires activation of both protein kinase A and Akt during adipocyte differentiation

    SciTech Connect

    Kim, Sang-pil; Ha, Jung Min; Yun, Sung Ji; Kim, Eun Kyoung; Chung, Sung Woon; Hong, Ki Whan; Kim, Chi Dae; Bae, Sun Sik

    2010-08-13

    Research highlights: {yields} Elevated cAMP activates both PKA and Epac. {yields} PKA activates CREB transcriptional factor and Epac activates PI3K/Akt pathway via Rap1. {yields} Akt modulates PPAR-{gamma} transcriptional activity in concert with CREB. -- Abstract: Peroxisome proliferator-activated receptor-{gamma} (PPAR-{gamma}) is required for the conversion of pre-adipocytes. However, the mechanism underlying activation of PPAR-{gamma} is unclear. Here we showed that cAMP-induced activation of protein kinase A (PKA) and Akt is essential for the transcriptional activation of PPAR-{gamma}. Hormonal induction of adipogenesis was blocked by a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), by a protein kinase A (PKA) inhibitor (H89), and by a Rap1 inhibitor (GGTI-298). Transcriptional activity of PPAR-{gamma} was markedly enhanced by 3-isobutyl-1-methylxanthine (IBMX), but not insulin and dexamethasone. In addition, IBMX-induced PPAR-{gamma} transcriptional activity was blocked by PI3K/Akt, PKA, or Rap1 inhibitors. 8-(4-Chlorophenylthio)-2'-O-methyl-cAMP (8-pCPT-2'-O-Me-cAMP) which is a specific agonist for exchanger protein directly activated by cAMP (Epac) significantly induced the activation of Akt. Furthermore, knock-down of Akt1 markedly attenuated PPAR-{gamma} transcriptional activity. These results indicate that both PKA and Akt signaling pathways are required for transcriptional activation of PPAR-{gamma}, suggesting post-translational activation of PPAR-{gamma} might be critical step for adipogenic gene expression.

  7. Neuronal angiotensin II type 1 receptor upregulation in heart failure: activation of activator protein 1 and Jun N-terminal kinase.

    PubMed

    Liu, Dongmei; Gao, Lie; Roy, Shyamal K; Cornish, Kurtis G; Zucker, Irving H

    2006-10-27

    Chronic heart failure (CHF) is a leading cause of mortality in developed countries. Angiotensin II (Ang II) plays an important role in the development and progression of CHF. Many of the important functions of Ang II are mediated by the Ang II type 1 receptor (AT(1)R), including the increase in sympathetic nerve activity in CHF. However, the central regulation of the AT(1)R in the setting of CHF is not well understood. This study investigated the AT(1)R in the rostral ventrolateral medulla (RVLM) of rabbits with CHF, its downstream pathway, and its gene regulation by the transcription factor activator protein 1 (AP-1). Studies were performed in 5 groups of rabbits: sham (n=5), pacing-induced (3 to 4 weeks) CHF (n=5), CHF with intracerebroventricular (ICV) losartan treatment (n=5), normal with ICV Ang II treatment (n=5), and normal with ICV Ang II plus losartan treatment (n=5). AT(1)R mRNA and protein expressions, plasma Ang II, and AP-1-DNA binding activity were significantly higher in RVLM of CHF compared with Sham rabbits (240.4+/-30.2%, P<0.01; 206.6+/-25.8%, P<0.01; 280+/-36.5%, P<0.05; 207+/-16.4%, P<0.01, respectively). Analysis of the stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) pathway showed that phosphorylated c-Jun proteins, phosphorylated JNK proteins, and JNK activity increased significantly in RVLM of CHF compared with sham (262.9+/-48.1%, 213.8+/-27.7%, 148.2+/-10.1% of control, respectively). Importantly, ICV losartan in CHF rabbits attenuated these increases. ICV Ang II in normal rabbits simulated the molecular changes seen in CHF. This effect was blocked by concomitant ICV losartan. In addition, Ang II-induced AT(1)R expression was blocked by losartan and a JNK inhibitor, but not by extracellular signal-regulated kinase or p38 MAP kinase inhibitors in a neuronal cell culture. These data suggest that central Ang II activates the AT(1)R, SAPK/JNK pathway. AP-1 may further regulate gene expression in RVLM in the CHF state.

  8. Mitogen-activated protein kinase signaling pathways promote low-density lipoprotein receptor-related protein 1-mediated internalization of beta-amyloid protein in primary cortical neurons.

    PubMed

    Yang, Wei-Na; Ma, Kai-Ge; Qian, Yi-Hua; Zhang, Jian-Shui; Feng, Gai-Feng; Shi, Li-Li; Zhang, Zhi-Chao; Liu, Zhao-Hui

    2015-07-01

    Mounting evidence suggests that the pathological hallmarks of Alzheimer's disease (AD) are caused by the intraneuronal accumulation of beta-amyloid protein (Aβ). Reuptake of extracellular Aβ is believed to contribute significantly to the intraneuronal Aβ pool in the early stages of AD. Published reports have claimed that the low-density lipoprotein receptor-related protein 1 (LRP1) mediates Aβ1-42 uptake and lysosomal trafficking in GT1-7 neuronal cells and mouse embryonic fibroblast non-neuronal cells. However, there is no direct evidence supporting the role of LRP1 in Aβ internalization in primary neurons. Our recent study indicated that p38 MAPK and ERK1/2 signaling pathways are involved in regulating α7 nicotinic acetylcholine receptor (α7nAChR)-mediated Aβ1-42 uptake in SH-SY5Y cells. This study was designed to explore the regulation of MAPK signaling pathways on LRP1-mediated Aβ internalization in neurons. We found that extracellular Aβ1-42 oligomers could be internalized into endosomes/lysosomes and mitochondria in cortical neurons. Aβ1-42 and LRP1 were also found co-localized in neurons during Aβ1-42 internalization, and they could form Aβ1-42-LRP1 complex. Knockdown of LRP1 expression significantly decreased neuronal Aβ1-42 internalization. Finally, we identified that p38 MAPK and ERK1/2 signaling pathways regulated the internalization of Aβ1-42 via LRP1. Therefore, these results demonstrated that LRP1, p38 MAPK and ERK1/2 mediated the internalization of Aβ1-42 in neurons and provided evidence that blockade of LRP1 or inhibitions of MAPK signaling pathways might be a potential approach to lowering brain Aβ levels and served a potential therapeutic target for AD.

  9. G protein-coupled receptor kinase-2 (GRK-2) regulates serotonin metabolism through the monoamine oxidase AMX-2 in Caenorhabditis elegans.

    PubMed

    Wang, Jianjun; Luo, Jiansong; Aryal, Dipendra K; Wetzel, William C; Nass, Richard; Benovic, Jeffrey L

    2017-02-17

    G protein-coupled receptors (GPCRs) regulate many animal behaviors. GPCR signaling is mediated by agonist-promoted interactions of GPCRs with heterotrimeric G proteins, GPCR kinases (GRKs) and arrestins. To further elucidate the role of GRKs in regulating GPCR-mediated behaviors, we utilized the genetic model system Caenorhabditis elegans. Our studies demonstrate that grk-2 loss-of-function strains are egg-laying defective and contain low levels of serotonin (5-HT) and high levels of the 5-HT metabolite 5-hydroxyindole acetic acid (5-HIAA). The egg-laying defect could be rescued by the expression of wild type, but not by catalytically inactive grk-2 or by the selective expression of grk-2 in hermaphrodite-specific neurons. The addition of 5-HT or inhibition of 5-HT metabolism also rescued the egg-laying defect. Furthermore, we demonstrate that AMX-2 is the primary monoamine oxidase that metabolizes 5-HT in Caenorhabditis elegans and we found also that grk-2 loss-of-function strains have abnormally high levels of AMX-2 compared to wild-type nematodes. Interestingly, GRK-2 was also found to interact with and promote the phosphorylation of AMX-2. Additional studies reveal that 5-HIAA functions to inhibit egg laying in a manner dependent on the 5-HT receptor SER-1 and the G protein GOA-1. These results demonstrate that GRK-2 modulates 5-HT metabolism by regulating AMX-2 function and that 5-HIAA may function in the SER-1 signaling pathway.

  10. Decreased interactions in protein kinase A-glucocorticoid receptor signaling in the hippocampus after selective removal of the basal forebrain cholinergic input.

    PubMed

    Lim, Chol Seung; Kim, Youn Jung; Hwang, Yoo Kyeong; Bañuelos, Christina; Bizon, Jennifer L; Han, Jung-Soo

    2012-03-01

    Removal of the cholinergic innervation to the hippocampus via selective immunolesions of septohippocampal cholinergic neurons induces dysfunction of the hypothalamic-pituitary-adrenocortical (HPA) axis and decreases glucocorticoid receptor (GR) mRNA. This study examined whether removal of the cholinergic innervation decreased GR protein levels and induced changes in the interaction between GR and the cytoplasmic catalytic subunit of protein kinase A (PKAc) in the hippocampus. In lesioned animals, GR protein levels were markedly decreased in the nucleus, but not in the cytosol of hippocampal neurons, whereas mineralocorticoid receptor (MR) levels remained unchanged in both the nucleus and cytosol. PKAc levels did not differ between lesioned and control groups, but PKAc activity was reduced in lesion tissue compared with the controls. The interaction between GR and PKAc was also decreased in the hippocampus without cholinergic input. These results indicate that degeneration of septohippocampal cholinergic neurons leads to reduced PKAc activity in the hippocampus which, in turn, alters GR signaling. The altered GR signaling induced by the degeneration of basal forebrain cholinergic neurons may contribute to dysfunction of the HPA axis in aged animals and patients with Alzheimer's disease (AD) and lead to neuropsychiatric symptoms that occur throughout the course of AD.

  11. Free cholesterol accumulation in macrophage membranes activates Toll-like receptors and p38 mitogen-activated protein kinase and induces cathepsin K.

    PubMed

    Sun, Yu; Ishibashi, Minako; Seimon, Tracie; Lee, Mingsum; Sharma, Sudarshana M; Fitzgerald, Katherine A; Samokhin, Andriy O; Wang, Yibin; Sayers, Scott; Aikawa, Masanori; Jerome, W Gray; Ostrowski, Michael C; Bromme, Dieter; Libby, Peter; Tabas, Ira A; Welch, Carrie L; Tall, Alan R

    2009-02-27

    The molecular events linking lipid accumulation in atherosclerotic plaques to complications such as aneurysm formation and plaque disruption are poorly understood. BALB/c-Apoe(-/-) mice bearing a null mutation in the Npc1 gene display prominent medial erosion and atherothrombosis, whereas their macrophages accumulate free cholesterol in late endosomes and show increased cathepsin K (Ctsk) expression. We now show increased cathepsin K immunostaining and increased cysteinyl proteinase activity using near infrared fluorescence imaging over proximal aortas of Apoe(-/-), Npc1(-/-) mice. In mechanistic studies, cholesterol loading of macrophage plasma membranes (cyclodextrin-cholesterol) or endosomal system (AcLDL+U18666A or Npc1 null mutation) activated Toll-like receptor (TLR) signaling, leading to sustained phosphorylation of p38 mitogen-activated protein kinase and induction of p38 targets, including Ctsk, S100a8, Mmp8, and Mmp14. Studies in macrophages from knockout mice showed major roles for TLR4, following plasma membrane cholesterol loading, and for TLR3, after late endosomal loading. TLR signaling via p38 led to phosphorylation and activation of the transcription factor Microphthalmia transcription factor, acting at E-box elements in the Ctsk promoter. These studies suggest that free cholesterol enrichment of either plasma or endosomal membranes in macrophages leads to activation of signaling via various TLRs, prolonged p38 mitogen-activated protein kinase activation, and induction of Mmps, Ctsk, and S100a8, potentially contributing to plaque complications.

  12. Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis.

    PubMed

    Liu, Qian; Tao, Bo; Liu, Guizhu; Chen, Guilin; Zhu, Qian; Yu, Ying; Yu, Yu; Xiong, Hong

    2016-02-26

    Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy.

  13. Thromboxane A2 Receptor Inhibition Suppresses Multiple Myeloma Cell Proliferation by Inducing p38/c-Jun N-terminal Kinase (JNK) Mitogen-activated Protein Kinase (MAPK)-mediated G2/M Progression Delay and Cell Apoptosis*

    PubMed Central

    Liu, Qian; Tao, Bo; Liu, Guizhu; Chen, Guilin; Zhu, Qian; Yu, Ying; Yu, Yu; Xiong, Hong

    2016-01-01

    Multiple myeloma (MM) is a plasma cell malignancy without effective therapeutics. Thromboxane A2 (TxA2)/TxA2 receptor (T prostanoid receptor (TP)) modulates the progression of some carcinomas; however, its effects on MM cell proliferation remain unclear. In this study, we evaluated cyclooxygenase (COX) enzymes and downstream prostaglandin profiles in human myeloma cell lines RPMI-8226 and U-266 and analyzed the effects of COX-1/-2 inhibitors SC-560 and NS-398 on MM cell proliferation. Our observations implicate COX-2 as being involved in modulating cell proliferation. We further incubated MM cells with prostaglandin receptor antagonists or agonists and found that only the TP antagonist, SQ29548, suppressed MM cell proliferation. TP silencing and the TP agonist, U46619, further confirmed this finding. Moreover, SQ29548 and TP silencing promoted MM cell G2/M phase delay accompanied by reducing cyclin B1/cyclin-dependent kinase-1 (CDK1) mRNA and protein expression. Notably, cyclin B1 overexpression rescued MM cells from G2/M arrest. We also found that the TP agonist activated JNK and p38 MAPK phosphorylation, and inhibitors of JNK and p38 MAPK depressed U46619-induced proliferation and cyclin B1/CDK1 protein expression. In addition, SQ29548 and TP silencing led to the MM cell apoptotic rate increasing with improving caspase 3 activity. The knockdown of caspase 3 reversed the apoptotic rate. Taken together, our results suggest that TxA2/TP promotes MM cell proliferation by reducing cell delay at G2/M phase via elevating p38 MAPK/JNK-mediated cyclin B1/CDK1 expression and hindering cell apoptosis. The TP inhibitor has potential as a novel agent to target kinase cascades for MM therapy. PMID:26724804

  14. Nck-2, a Novel Src Homology2/3-containing Adaptor Protein That Interacts with the LIM-only Protein PINCH and Components of Growth Factor Receptor Kinase-signaling Pathways

    PubMed Central

    Tu, Yizeng; Li, Fugang; Wu, Chuanyue

    1998-01-01

    Many of the protein–protein interactions that are essential for eukaryotic intracellular signal transduction are mediated by protein binding modules including SH2, SH3, and LIM domains. Nck is a SH3- and SH2-containing adaptor protein implicated in coordinating various signaling pathways, including those of growth factor receptors and cell adhesion receptors. We report here the identification, cloning, and characterization of a widely expressed, Nck-related adaptor protein termed Nck-2. Nck-2 comprises primarily three N-terminal SH3 domains and one C-terminal SH2 domain. We show that Nck-2 interacts with PINCH, a LIM-only protein implicated in integrin-linked kinase signaling. The PINCH-Nck-2 interaction is mediated by the fourth LIM domain of PINCH and the third SH3 domain of Nck-2. Furthermore, we show that Nck-2 is capable of recognizing several key components of growth factor receptor kinase-signaling pathways including EGF receptors, PDGF receptor-β, and IRS-1. The association of Nck-2 with EGF receptors was regulated by EGF stimulation and involved largely the SH2 domain of Nck-2, although the SH3 domains of Nck-2 also contributed to the complex formation. The association of Nck-2 with PDGF receptor-β was dependent on PDGF activation and was mediated solely by the SH2 domain of Nck-2. Additionally, we have detected a stable association between Nck-2 and IRS-1 that was mediated primarily via the second and third SH3 domain of Nck-2. Thus, Nck-2 associates with PINCH and components of different growth factor receptor-signaling pathways via distinct mechanisms. Finally, we provide evidence indicating that a fraction of the Nck-2 and/or Nck-1 proteins are associated with the cytoskeleton. These results identify a novel Nck-related SH2- and SH3-domain–containing protein and suggest that it may function as an adaptor protein connecting the growth factor receptor-signaling pathways with the integrin-signaling pathways. PMID:9843575

  15. Immune-Mediated Nephropathy and Systemic Autoimmunity in Mice Does Not Require Receptor Interacting Protein Kinase 3 (RIPK3)

    PubMed Central

    Corradetti, Chelsea; Jog, Neelakshi R.; Gallucci, Stefania; Madaio, Michael; Balachandran, Siddharth

    2016-01-01

    Immune mediated nephropathy is one of the most serious manifestations of lupus and is characterized by severe inflammation and necrosis that, if untreated, eventually leads to renal failure. Although lupus has a higher incidence in women, both sexes can develop lupus glomerulonephritis; nephritis in men develops earlier and is more severe than in women. It is therefore important to understand the cellular and molecular mechanisms mediating nephritis in each sex. Previous work by our lab found that the absence or pharmacological inhibition of Poly [ADP-ribose] polymerase 1 (PARP-1), an enzyme involved in DNA repair and necrotic cell death, affects only male mice and results in milder nephritis, with less in situ inflammation, and diminished incidence of necrotic lesions, allowing for higher survival rates. A second pathway mediating necrosis involves Receptor-Interacting Serine-Threonine Kinase 3 (RIPK3); in this study we sought to investigate the impact of RIPK3 on the development of lupus and nephritis in both sexes. To this end, we used two inducible murine models of lupus: chronic graft versus host disease (cGvHD) and pristane-induced lupus; and nephrotoxic serum (NTS)-induced nephritis as a model of immune mediated nephropathy. We found that the absence of RIPK3 has neither positive nor negative impact on the disease development or progression of lupus and nephritis in all three models, and in both male and female mice. We conclude that RIPK3 is dispensable for the pathogenesis of lupus and immune mediated nephropathy as to accelerate, worsen or ameliorate the disease. PMID:27669412

  16. Downregulation of protease-activated receptor-1 in human lung fibroblasts is specifically mediated by the prostaglandin E receptor EP2 through cAMP elevation and protein kinase A.

    PubMed

    Sokolova, Elena; Hartig, Roland; Reiser, Georg

    2008-07-01

    Many cellular functions of lung fibroblasts are controlled by protease-activated receptors (PARs). In fibrotic diseases, PAR-1 plays a major role in controlling fibroproliferative and inflammatory responses. Therefore, in these diseases, regulation of PAR-1 expression plays an important role. Using the selective prostaglandin EP2 receptor agonist butaprost and cAMP-elevating agents, we show here that prostaglandin (PG)E(2), via the prostanoid receptor EP2 and subsequent cAMP elevation, downregulates mRNA and protein levels of PAR-1 in human lung fibroblasts. Under these conditions, the functional response of PAR-1 in fibroblasts is reduced. These effects are specific for PGE(2). Activation of other receptors coupled to cAMP elevation, such as beta-adrenergic and adenosine receptors, does not reproduce the effects of PGE(2). PGE(2)-mediated downregulation of PAR-1 depends mainly on protein kinase A activity, but does not depend on another cAMP effector, the exchange protein activated by cAMP. PGE(2)-induced reduction of PAR-1 level is not due to a decrease of PAR-1 mRNA stability, but rather to transcriptional regulation. The present results provide further insights into the therapeutic potential of PGE(2) to specifically control fibroblast function in fibrotic diseases.

  17. 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

  18. Specific dephosphorylation of phosphoproteins by protein-serine and -tyrosine kinases.

    PubMed

    Kole, H K; Abdel-Ghany, M; Racker, E

    1988-08-01

    Five protein kinases are shown to serve as specific phosphatases in the absence of ADP. Although the rates of hydrolysis are very slow compared to the forward phosphorylation rates under optimal conditions, they are of the same order as the reverse reaction in the presence of ADP. Because cells contain approximately equal to 3 mM ATP, neither the reverse reaction nor the phosphatase is likely to play a physiological role. beta-casein B phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A) is specifically dephosphorylated by protein kinase A but not by polypeptide-dependent protein kinase (protein kinase P). beta-casein B phosphorylated by protein kinase P is specifically dephosphorylated by protein kinase P but not by protein kinase A. Histone H1 phosphorylated by protein kinase C is dephosphorylated by the same enzyme in the absence of ADP. In all cases tested addition of ADP and F1-ATPase accelerates moderately the rate of dephosphorylation. Native H+-ATPase from yeast plasma membranes is isolated mainly in the phosphorylated form. It is dephosphorylated and rephosphorylated by protein kinase P but not by protein kinase A. Protein-tyrosine kinase of the epidermal growth factor receptor phosphorylates the random synthetic polypeptide poly(Glu80Tyr20). The phosphorylated polymer is specifically dephosphorylated in the absence of ADP by epidermal growth factor receptor preparations but not by insulin receptor preparations. The same polymer phosphorylated by insulin receptor is dephosphorylated by insulin receptor but not by epidermal growth factor receptor preparations. By using a cycle of dephosphorylation-rephosphorylation, it is possible to identify proteins that are phosphorylated by these protein kinases in vivo. Should this method be applicable to additional protein kinases, it should be possible to estimate the quantitative contribution of each protein kinase to a single phosphoprotein.

  19. Specific dephosphorylation of phosphoproteins by protein-serine and -tyrosine kinases.

    PubMed Central

    Kole, H K; Abdel-Ghany, M; Racker, E

    1988-01-01

    Five protein kinases are shown to serve as specific phosphatases in the absence of ADP. Although the rates of hydrolysis are very slow compared to the forward phosphorylation rates under optimal conditions, they are of the same order as the reverse reaction in the presence of ADP. Because cells contain approximately equal to 3 mM ATP, neither the reverse reaction nor the phosphatase is likely to play a physiological role. beta-casein B phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A) is specifically dephosphorylated by protein kinase A but not by polypeptide-dependent protein kinase (protein kinase P). beta-casein B phosphorylated by protein kinase P is specifically dephosphorylated by protein kinase P but not by protein kinase A. Histone H1 phosphorylated by protein kinase C is dephosphorylated by the same enzyme in the absence of ADP. In all cases tested addition of ADP and F1-ATPase accelerates moderately the rate of dephosphorylation. Native H+-ATPase from yeast plasma membranes is isolated mainly in the phosphorylated form. It is dephosphorylated and rephosphorylated by protein kinase P but not by protein kinase A. Protein-tyrosine kinase of the epidermal growth factor receptor phosphorylates the random synthetic polypeptide poly(Glu80Tyr20). The phosphorylated polymer is specifically dephosphorylated in the absence of ADP by epidermal growth factor receptor preparations but not by insulin receptor preparations. The same polymer phosphorylated by insulin receptor is dephosphorylated by insulin receptor but not by epidermal growth factor receptor preparations. By using a cycle of dephosphorylation-rephosphorylation, it is possible to identify proteins that are phosphorylated by these protein kinases in vivo. Should this method be applicable to additional protein kinases, it should be possible to estimate the quantitative contribution of each protein kinase to a single phosphoprotein. Images PMID:2901092

  20. Regulation of protein kinase C-related kinase (PRK) signalling by the TPα and TPβ isoforms of the human thromboxane A2 receptor: Implications for thromboxane- and androgen- dependent neoplastic and epigenetic responses in prostate cancer.

    PubMed

    O'Sullivan, Aine G; Mulvaney, Eamon P; Kinsella, B Therese

    2017-04-01

    The prostanoid thromboxane (TX) A2 and its T Prostanoid receptor (the TP) are increasingly implicated in prostate cancer (PCa). Mechanistically, we recently discovered that both TPα and TPβ form functional signalling complexes with members of the protein kinase C-related kinase (PRK) family, AGC- kinases essential for the epigenetic regulation of androgen receptor (AR)-dependent transcription and promising therapeutic targets for treatment of castrate-resistant prostate cancer (CRPC). Critically, similar to androgens, activation of the PRKs through the TXA2/TP signalling axis induces phosphorylation of histone H3 at Thr11 (H3Thr11), a marker of androgen-induced chromatin remodelling and transcriptional activation, raising the possibility that TXA2-TP signalling can mimic and/or enhance AR-induced cellular changes even in the absence of circulating androgens such as in CRPC. Hence the aim of the current study was to investigate whether TXA2/TP-induced PRK activation can mimic and/or enhance AR-mediated cellular responses in the model androgen-responsive prostate adenocarcinoma LNCaP cell line. We reveal that TXA2/TP signalling can act as a neoplastic- and epigenetic-regulator, promoting and enhancing both AR-associated chromatin remodelling (H3Thr11 phosphorylation, WDR5 recruitment and acetylation of histone H4 at lysine 16) and AR-mediated transcriptional activation (e.g of the KLK3/prostate-specific antigen and TMPRSS2 genes) through mechanisms involving TPα/TPβ mediated-PRK1 and PRK2, but not PRK3, signalling complexes. Overall, these data demonstrate that TPα/TPβ can act as neoplastic and epigenetic regulators by mimicking and/or enhancing the actions of androgens within the prostate and provides further mechanistic insights into the role of the TXA2/TP signalling axis in PCa, including potentially in CRPC.

  1. Mycobacterium tuberculosis triggers apoptosis in peripheral neutrophils involving toll-like receptor 2 and p38 mitogen protein kinase in tuberculosis patients.

    PubMed

    Alemán, Mercedes; Schierloh, Pablo; de la Barrera, Silvia S; Musella, Rosa M; Saab, María A; Baldini, Matías; Abbate, Eduardo; Sasiain, María C

    2004-09-01

    Polymorphonuclear neutrophils (PMN) exposed to Mycobacterium tuberculosis display bactericidal responses and produce inflammatory proteins. This PMN-mediated inflammatory response is regulated by an activation of the apoptotic program, which collaborates to avoid tissue injury. In vitro, circulating PMN from patients with tuberculosis (TB) show an increased spontaneous apoptosis, and M. tuberculosis-induced activation accelerates the PMN apoptosis. In this study, we evaluated the mechanisms involved in spontaneous and M. tuberculosis-induced apoptosis. We demonstrate that apoptosis of PMN is not induced by lipoarabinomannan or by a whole-cell lysate of M. tuberculosis and that neither tumor necrosis factor alpha nor CD11b, CD14, and Fcgamma receptors are involved. Apoptosis of PMN from patients with active TB (TB-PMN) is induced by the interaction with the whole M. tuberculosis via Toll-like receptor 2 (TLR2), and, in contrast to spontaneous apoptosis, it involves the p38 mitogen-activated protein kinase (MAPK) pathway. These results correlate with a high expression of phosphorylated p38 (p-p38) in circulating TB-PMN and with the ability of M. tuberculosis to induce in vitro the expression of p-p38 in PMN. Therefore, when the bacterial burden is low, TB-PMN could be detecting nonopsonized M. tuberculosis via TLR2, leading to the activation of the p38 MAPK pathway, which in turn would induce PMN activation and apoptosis. This mechanism needs further confirmation at the site of infection.

  2. Phosphorylation by p38 Mitogen-Activated Protein Kinase Promotes Estrogen Receptor α Turnover and Functional Activity via the SCFSkp2 Proteasomal Complex

    PubMed Central

    Bhatt, Shweta; Xiao, Zhen; Meng, Zhaojing

    2012-01-01

    The nuclear hormone receptor estrogen receptor α (ERα) mediates the actions of estrogens in target cells and is a master regulator of the gene expression and proliferative programs of breast cancer cells. The presence of ERα in breast cancer cells is crucial for the effectiveness of endocrine therapies, and its loss is a hallmark of endocrine-insensitive breast tumors. However, the molecular mechanisms underlying the regulation of the cellular levels of ERα are not fully understood. Our findings reveal a unique cellular pathway involving the p38 mitogen-activated protein kinase (p38MAPK)-mediated phosphorylation of ERα at Ser-294 that specifies its turnover by the SCFSkp2 proteasome complex. Consistently, we observed an inverse relationship between ERα and Skp2 or active p38MAPK in breast cancer cell lines and human tumors. ERα regulation by Skp2 was cell cycle stage dependent and critical for promoting the mitogenic effects of estradiol via ERα. Interestingly, by the knockdown of Skp2 or the inhibition of p38MAPK, we restored functional ERα protein levels and the control of gene expression and proliferation by estrogen and antiestrogen in ERα-negative breast cancer cells. Our findings highlight a novel pathway with therapeutic potential for restoring ERα and the responsiveness to endocrine therapy in some endocrine-insensitive ERα-negative breast cancers. PMID:22431515

  3. Beta 2 subunit-containing nicotinic receptors mediate acute nicotine-induced activation of calcium/calmodulin-dependent protein kinase II-dependent pathways in vivo.

    PubMed

    Jackson, K J; Walters, C L; Damaj, M I

    2009-08-01

    Nicotine is the addictive component of tobacco, and successful smoking cessation therapies must address the various processes that contribute to nicotine addiction. Thus, understanding the nicotinic acetylcholine receptor (nAChR) subtypes and subsequent molecular cascades activated after nicotine exposure is of the utmost importance in understanding the progression of nicotine dependence. One possible candidate is the calcium/calmodulin-dependent protein kinase II (CaMKII) pathway. Substrates of this kinase include the vesicle-associated protein synapsin I and the transcription factor cAMP response element-binding protein (CREB). The goal of these studies was to examine these postreceptor mechanisms after acute nicotine treatment in vivo. We first show that administration of nicotine increases CaMKII activity in the ventral tegmental area (VTA), nucleus accumbens (NAc), and amygdala. In beta2 nAChR knockout (KO) mice, nicotine does not induce an increase in kinase activity, phosphorylated (p)Synapsin I, or pCREB. In contrast, alpha7 nAChR KO mice show nicotine-induced increases in CaMKII activity and pCREB, similar to their wild-type littermates. Moreover, we show that when animals are pretreated with the CaMKII inhibitors 4-[(2S)-2-[(5-isoquinolinylsulfonyl) methylamino]-3-oxo-3-(4-phenyl-1-piperazinyl)propyl]phenyl isoquinolinesulfonic acid ester (KN-62) and N-[2-[[[3-(4-chlorophenyl)-2 propenyl]methylamino]methyl]phenyl]-N-(2-hydroxyethyl)-4-methoxybenzenesulphonamide (KN-93), nicotine-induced increase in the kinase activity and pCREB was attenuated in the VTA and NAc, whereas pretreatment with (2-[N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine, phosphate) (KN-92), the inactive analog, did not alter the nicotine-induced increase in pCREB. Taken together, these data suggest that the nicotine-induced increase in CaMKII activity may correlate with the nicotine-induced increase in pSynapsin I and pCREB in the VTA and NAc via beta2

  4. Direct interaction of Syk and Lyn protein tyrosine kinases in rat basophilic leukemia cells activated via type I Fc epsilon receptors.

    PubMed

    Amoui, M; Dráberová, L; Tolar, P; Dráber, P

    1997-01-01

    Activation of rat mast cells through the receptor with high affinity for IgE (Fc epsilonRI) requires a complex set of interactions involving transmembrane subunits of the Fc epsilonRI and two classes of nonreceptor protein tyrosine kinase (PTK). the Src family PTK p53/p56(lyn) (Lyn) and the Syk/ZAP-family PTK p72(syk) (Syk). Early activation events involve increased activity of Lyn and Syk kinases and their translocation into membrane domains containing aggregated Fc epsilonRI, but the molecular mechanisms responsible for these changes have remained largely unclear. To determine the role of Fc epsilonRI subunits in this process, we have analyzed Syk- and Lyn-associated proteins in activated rat basophilic leukemia (RBL) cells and their variants deficient in the expression of Fc epsilonRI beta or gamma subunits. Sepharose 4B gel chromatography of postnuclear supernatants from Nonidet-P40-solubilized antigen (Ag)- or pervanadate-activated RBL cells revealed extensive changes in the size of complexes formed by Lyn and Syk kinases and other cellular components. A fusion protein containing Src homology 2 (SH2) and SH3 domains of Lyn bound Syk from lysates of nonactivated RBL cells; an increased binding was observed when lysates from Ag- or pervanadate-activated cells were used. A similar amount of Syk was bound when lysates from pervanadate-activated variant cells deficient in the expression of Fc epsilonRI beta or gamma subunits were used, suggesting that Fc epsilonRI does not function as the only intermediate in the formation of the Syk-Lyn complexes. Further experiments have indicated that Syk-Lyn interactions occur in Ag-activated RBL cells under in vivo conditions and that these interactions could involve direct binding of the Lyn SH2 domain with phosphorylated tyrosine of Syk. The physical association of Lyn and Syk during mast-like cell activation supports the recently proposed functional cooperation of these two tyrosine kinases in Fc epsilonRI signaling.

  5. Degradation of Activated Protein Kinases by Ubiquitination

    PubMed Central

    Lu, Zhimin; Hunter, Tony

    2009-01-01

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

  6. Interaction with receptor for activated C-kinase 1 (RACK1) sensitizes the phosphodiesterase PDE4D5 towards hydrolysis of cAMP and activation by protein kinase C

    PubMed Central

    Bird, Rebecca J.; Baillie, George S.; Yarwood, Stephen J.

    2010-01-01

    We have previously identified the PKC (protein kinase C)-anchoring protein RACK1 (receptor for activated C-kinase 1), as a specific binding partner for the cAMP-specific phosphodiesterase PDE4D5, suggesting a potential site for cross-talk between the PKC and cAMP signalling pathways. In the present study we found that elevation of intracellular cAMP, with the β2-adrenoceptor agonist isoproterenol (isoprenaline), led to activation of PDE4 enzymes in the particulate and soluble fractions of HEK (human embryonic kidney)-293 cells. In contrast activation of PDE4D5, with isoproterenol and the PKC activator PMA, was restricted to the particulate fraction, where it interacts with RACK1; however, RACK1 is dispensable for anchoring PDE4D5 to the particulate fraction. Kinetic studies demonstrated that RACK1 alters the conformation of particulate-associated PDE4D5 so that it more readily interacts with its substrate cAMP and with rolipram, a PDE4 inhibitor that specifically targets the active site of the enzyme. Interaction with RACK1 was also essential for PKC-dependent and ERK (extracellular-signal-regulated kinase)-independent phosphorylation (on Ser126), and activation of PDE4D5 in response to PMA and isoproterenol, both of which trigger the recruitment of PKCα to RACK1. Together these results reveal novel signalling cross-talk, whereby RACK1 mediates PKC-dependent activation of PDE4D5 in the particulate fraction of HEK-293 cells in response to elevations in intracellular cAMP. PMID:20819076

  7. Atorvastatin promotes human monocyte differentiation toward alternative M2 macrophages through p38 mitogen-activated protein kinase-dependent peroxisome proliferator-activated receptor γ activation.

    PubMed

    Zhang, Ou; Zhang, Jinying

    2015-05-01

    M1 and M2 macrophages are detectable in human atherosclerotic lesions, and M2 macrophages are present at locations distant from the lipid core in more stable zones of the plaque and appear to exert anti-inflammatory properties on M1 macrophages. Peroxisome proliferator-activated receptor (PPAR) γ promotes the differentiation of monocytes into anti-inflammatory M2 macrophages. Although both statins and PPARγ ligands have been reported to protect against the progression of atherosclerosis, no data are currently available regarding the implication of statins in the alternative differentiation of human monocytes. In the present study, we hypothesized that atorvastatin may exert novel effects to prime human monocytes toward an anti-inflammatory alternative M2 phenotype. To this aim, we first found that abundant M2 markers were expressed in human circulating monocytes after atorvastatin treatment. Moreover, atorvastatin was able to induce PPARγ expression and activation in human monocytes in vivo and in vitro, resulting in priming primary human monocytes differentiation into M2 macrophages with a more pronounced paracrine anti-inflammatory activity in M1 macrophages. Additional data with molecular approaches revealed that p38 mitogen-activated protein kinase (MAPK) but not extracellular signal-regulated kinase (ERK) 1/2 activation was involved in atorvastatin-mediated PPARγ activation and enhanced alternative M2 macrophage phenotype. Collectively, our data demonstrated that atorvastatin promotes human monocyte differentiation toward alternative M2 macrophages via p38 MAPK-dependent PPARγ activation.

  8. AMP-activated protein kinase is required for exercise-induced peroxisome proliferator-activated receptor co-activator 1 translocation to subsarcolemmal mitochondria in skeletal muscle.

    PubMed

    Smith, Brennan K; Mukai, Kazutaka; Lally, James S; Maher, Amy C; Gurd, Brendon J; Heigenhauser, George J F; Spriet, Lawrence L; Holloway, Graham P

    2013-03-15

    In skeletal muscle, mitochondria exist as two subcellular populations known as subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria. SS mitochondria preferentially respond to exercise training, suggesting divergent transcriptional control of the mitochondrial genomes. The transcriptional co-activator peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α) and mitochondrial transcription factor A (Tfam) have been implicated in the direct regulation of the mitochondrial genome in mice, although SS and IMF differences may exist, and the potential signalling events regulating the mitochondrial content of these proteins have not been elucidated. Therefore, we examined the potential for PGC-1α and Tfam to translocate to SS and IMF mitochondria in human subjects, and performed experiments in rodents to identify signalling mechanisms regulating these translocation events. Acute exercise in humans and rats increased PGC-1α content in SS but not IMF mitochondria. Acute exposure to 5-aminoimidazole-4-carboxamide-1-β-ribofuranoside in rats recapitulated the exercise effect of increased PGC-1α protein within SS mitochondria only, suggesting that AMP-activated protein kinase (AMPK) signalling is involved. In addition, rendering AMPK inactive (AMPK kinase dead mice) prevented exercise-induced PGC-1α translocation to SS mitochondria, further suggesting that AMPK plays an integral role in these translocation events. In contrast to the conserved PGC-1α translocation to SS mitochondria across species (humans, rats and mice), acute exercise only increased mitochondrial Tfam in rats. Nevertheless, in rat resting muscle PGC-1α and Tfam co-immunoprecipate with α-tubulin, suggesting a common cytosolic localization. These data suggest that exercise causes translocation of PGC-1α preferentially to SS mitochondria in an AMPK-dependent manner.

  9. Cloning of a Conserved Receptor-Like Protein Kinase Gene and Its Use as a Functional Marker for Homoeologous Group-2 Chromosomes of the Triticeae Species

    PubMed Central

    Qin, Bi; Chen, Tingting; Cao, Aizhong; Wang, Haiyan; Xing, Liping; Ling, Hongqing; Wang, Daowen; Yu, Chunmei; Xiao, Jin; Ji, Jianhui; Chen, Xueluan; Chen, Peidu; Liu, Dajun; Wang, Xiue

    2012-01-01

    Receptor-like kinases (RLKs) play broad biological roles in plants. We report on a conserved receptor-like protein kinase (RPK) gene from wheat and other Triticeae species. The TaRPK1 was isolated from the Triticum aestivum cv. Prins - Triticum timopheevii introgression line IGVI-465 carrying the powdery mildew resistance gene Pm6. The TaRPK1 was mapped to homoeologous chromosomes 2A (TaRPK1-2A), 2D (TaRPK1-2D) and the Pm6-carrier chromosome 2G (TaRPK1-2G) of IGVI-465. Under the tested conditions, only the TaRPK1-2G allele was actively transcribed, producing two distinct transcripts via alternative splicing. The predicted 424-amino acid protein of TaRPK1-2G contained a signal peptide, a transmembrane domain and an intracellular serine/threonine kinase domain, but lacked a typical extracellular domain. The expression of TaRPK1-2G gene was up-regulated upon the infection by Blumeria graminis f.sp. tritici (Bgt) and treatment with methyl jasmonate (MeJA), but down-regulated in response to treatments of SA and ABA. Over-expression of TaRPK1-2G in the powdery mildew susceptible wheat variety Prins by a transient expression assay showed that it slightly reduced the haustorium index of the infected Bgt. These data indicated that TaRPK1-2G participated in the defense response to Bgt infection and in the JA signaling pathway. Phylogenetic analysis indicated that TaRPK1-2G was highly conserved among plant species, and the amino acid sequence similarity of TaRPK1-2G among grass species was more than 86%. Based on its conservation, the RPK gene-based STS primers were designed, and used to amplify the RPK orthologs from the homoeologous group-2 chromosomes of all the tested Triticeae species, such as chromosome 2G of T. timopheevii, 2R of Secale cereale, 2H of Hordeum vulgare, 2S of Aegilops speltoides, 2Sl of Ae. longissima, 2Mg of Ae. geniculata, 2Sp and 2Up of Ae. peregrina. The developed STS markers serve as conserved functional markers for the identification of

  10. Estrogen Receptor β Signaling through Phosphatase and Tensin Homolog/Phosphoinositide 3-Kinase/Akt/Glycogen Synthase Kinase 3 Down-Regulates Blood-Brain Barrier Breast Cancer Resistance Protein

    PubMed Central

    Hartz, A. M. S.; Madole, E. K.; Miller, D. S.

    2010-01-01

    Breast cancer resistance protein (BCRP) is an ATP-driven efflux pump at the blood-brain barrier that limits central nervous system pharmacotherapy. Our previous studies showed rapid loss of BCRP transport activity in rat brain capillaries exposed to low concentrations of 17-β-estradiol (E2); this occurred without acute change in BCRP protein expression. Here, we describe a pathway through which sustained, extended exposure to E2 signals down-regulation of BCRP at the blood-brain barrier. Six-hour exposure of isolated rat and mouse brain capillaries to E2 reduced BCRP transport activity and BCRP monomer and dimer expression. Experiments with brain capillaries from estrogen receptor (ER)α and ERβ knockout mice and with ER agonists and antagonists showed that E2 signaled through ERβ to down-regulate BCRP expression. In rat brain capillaries, E2 increased unphosphorylated, active phosphatase and tensin homolog (PTEN); decreased phosphorylated, active Akt; and increased phosphorylated, active glycogen synthase kinase (GSK)3. Consistent with this, inhibition of phosphoinositide 3-kinase (PI3K) or Akt decreased BCRP activity and protein expression, and inhibition of PTEN or GSK3 reversed the E2 effect on BCRP. Lactacystin, a proteasome inhibitor, abolished E2-mediated BCRP down-regulation, suggesting internalization followed by transporter degradation. Dosing mice with E2 reduced BCRP activity in brain capillaries within 1 h; this reduction persisted for 24 h. BCRP protein expression in brain capillaries was unchanged 1 h after E2 dosing but was substantially reduced 6 and 24 h after dosing. Thus, E2 signals through ERβ, PTEN/PI3K/Akt/GSK3 to stimulate proteasomal degradation of BCRP. These in vitro and in vivo findings imply that E2-mediated down-regulation of blood-brain barrier BCRP has the potential to increase brain uptake of chemotherapeutics that are BCRP substrates. PMID:20460386

  11. Continuous delta opioid receptor activation reduces neuronal voltage gated sodium channel (NaV1.7) levels through activation of protein kinase C in painful diabetic neuropathy

    PubMed Central

    Chattopadhyay, Munmun; Mata, Marina; Fink, David J.

    2012-01-01

    The NaV1.7 tetrodotoxin-sensitive voltage-gated sodium channel isoform plays a critical role in nociception. In rodent models of diabetic neuropathy, increased NaV1.7 in dorsal root ganglion (DRG) neurons correlates with the emergence of pain-related behaviors characteristic of painful diabetic neuropathy (PDN). We examined the effect of transgene-mediated expression of enkephalin on pain-related behaviors and their biochemical correlates in DRG neurons. Transfection of DRG neurons by subcutaneous inoculation of a herpes simplex virus (HSV)-based vector expressing proenkephalin (PE) reversed nocisponsive behavioral responses to heat, cold, and mechanical pressure characteristic of PDN. Vector-mediated enkephalin production in vivo prevented the increase in DRG NaV1.7 observed in PDN, an effect that correlated with inhibition of phosphorylation of p38 MAP kinase and protein kinase C (PKC). Primary DRG neurons in vitro exposed to 45 mM glucose for 18 hrs also demonstrated an increase in NaV1.7 and increased phosphorylation of p38 and PKC; these changes were prevented by transfection in vitro with the enkephalin-expressing vector. The effect of hyperglycemia on NaV1.7 production in vitro was mimicked by exposure to PMA, and blocked by the myristolated PKC inhibitor 20–28 or the p38 inhibitor SB202190; the effect of vector-mediated enkephalin on NaV1.7 levels was prevented by naltrindole. The results of these studies suggest that activation of the presynaptic delta opioid receptor by enkephalin prevents the increase in neuronal NaV1.7 in DRG through inhibition of PKC and p38. These results establish a novel interaction between the delta opioid receptor and voltage gated sodium channels. PMID:18579738

  12. GABAB receptor transduction mechanisms, and cross-talk between protein kinases A and C, in GABAergic terminals synapsing onto neurons of the rat nucleus basalis of Meynert

    PubMed Central

    Kubota, Hisahiko; Katsurabayashi, Shutaro; Moorhouse, Andrew J; Murakami, Nobuya; Koga, Hitoshi; Akaike, Norio

    2003-01-01

    The transduction mechanisms underlying presynaptic GABAB receptor-mediated inhibition of transmitter release have been characterized for a variety of synapses in the central nervous system (CNS). These studies have suggested a range of transduction mechanisms, including a role for second messengers such as protein kinases A (PKA) and C (PKC). In the present study, we have examined the intracellular signalling pathways underlying baclofen-induced inhibition of GABA release from terminals synapsing onto rat basalis of Meynert neurons using patch-clamp recordings. Baclofen, a selective GABAB receptor agonist, reversibly decreased both evoked and spontaneous, miniature, GABAergic inhibitory postsynaptic currents (eIPSCs and mIPSCs, respectively). Such baclofen actions were completely abolished by CGP55845A, a selective GABAB receptor antagonist, and by staurosporine, a non-selective PKA and PKC inhibitor. The mIPSC frequency was still decreased by baclofen even in the presence of 4 AP, a K+ channel blocker, and Cd2+, a voltage-dependent calcium channel blocker. Pharmacological activation or inhibition of PKC activity affected basal GABA release and mildly affected the response to baclofen. Inhibition of the cAMP/PKA cascade also affected basal GABA release and, in a subset of neurons, occluded the effects of baclofen, suggesting that the GABAB receptor-mediated inhibitory action on GABA release was mediated via decreases in PKA activity. In addition, PKA inhibition occluded the effects of PKC modulation on both basal GABA release and on the response to baclofen. Our results characterize the transduction pathway of baclofen at these nucleus basalis of Maynert (nBM) synapses and show, for the first time, some cross-talk between the cAMP/PKA and PKC pathways in mammalian presynaptic nerve terminals. PMID:12815184

  13. Glucocorticoid receptor activation inhibits p53-induced apoptosis of MCF10Amyc cells via induction of protein kinase Cε.

    PubMed

    Aziz, Moammir H; Shen, Hong; Maki, Carl G

    2012-08-24

    Glucocorticoid receptor (GR) is a ligand-dependent transcription factor that can promote apoptosis or survival in a cell-specific manner. Activated GR has been reported to inhibit apoptosis in mammary epithelial cells and breast cancer cells by increasing pro-survival gene expression. In this study, activated GR inhibited p53-dependent apoptosis in MCF10A cells and human mammary epithelial cells that overexpress the MYC oncogene. Specifically, GR agonists hydrocortisone or dexamethasone inhibited p53-dependent apoptosis induced by cisplatin, ionizing radiation, or the MDM2 antagonist Nutlin-3. In contrast, the GR antagonist RU486 sensitized the cells to apoptosis by these agents. Apoptosis inhibition was associated with maintenance of mitochondrial membrane potential, diminished caspase-3 and -7 activation, and increased expression at both the mRNA and protein level of the anti-apoptotic PKC family member PKCε. Knockdown of PKCε via siRNA targeting reversed the protective effect of dexamethasone and restored apoptosis sensitivity. These data provide evidence that activated GR can inhibit p53-dependent apoptosis through induction of the anti-apoptotic factor PKCε.

  14. Serine 157, a retinoic acid receptor alpha residue phosphorylated by protein kinase C in vitro, is involved in RXR.RARalpha heterodimerization and transcriptional activity.

    PubMed

    Delmotte, M H; Tahayato, A; Formstecher, P; Lefebvre, P

    1999-12-31

    Retinoic acid (RA) regulation of cellular proliferation and differentiation is mediated, at least in part, through two related nuclear receptors, RAR and RXR. RA-induced modulation of gene expression leads generally to cellular differentiation, whereas stimulation of the protein kinase C (PKC) signaling pathway is associated with cellular proliferation. Pursuant to our discovery that prolonged activation of PKCs induced a strong decrease in RA responsiveness of a retinoid-inducible reporter gene, we have further investigated the connections between these two signaling pathways. We demonstrate that PKC isoforms alpha and gamma are able to phosphorylate human RARalpha (hRARalpha) in vitro on a single serine residue located in the extended DNA binding domain (T box). The introduction of a negative charge at this position (serine 157) strongly decreased hRARalpha transcriptional activity, whereas a similar mutation at other PKC consensus phosphorylation sites had no effect. The effect on transcriptional activation was correlated with a decrease in the capacity of hRARalpha to heterodimerize with hRXRalpha. Thus hRARalpha is a direct target for PKCalpha and gamma, which may control retinoid receptor transcriptional activities during cellular proliferation and differentiation.

  15. Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway.

    PubMed

    Murthy, Karnam S; Zhou, Huiping; Grider, John R; Brautigan, David L; Eto, Masumi; Makhlouf, Gabriel M

    2003-08-15

    Signalling via m3 and m2 receptors in smooth muscles involved activation of two G-protein-dependent pathways by each receptor. m2 receptors were coupled via Gbetagammai3 with activation of phospholipase C-beta3, phosphoinositide 3-kinase and Cdc42/Rac1 (where Cdc stands for cell division cycle) and p21-activated kinase 1 (PAK1), resulting in phosphorylation and inactivation of myosin light chain kinase (MLCK). Each step was inhibited by methoctramine and pertussis toxin. PAK1 activity was abolished in cells expressing both Cdc42-DN (where DN stands for dominant negative) and Rac1-DN. MLCK phosphorylation was inhibited by PAK1 antibody, and in cells expressing Cdc42-DN and Rac1-DN. m3 receptors were coupled via Galpha(q/11) with activation of phospholipase C-beta1 and via RhoA with activation of Rho-associated kinase (Rho kinase), phospholipase D and protein kinase C (PKC). Rho kinase and phospholipase D activities were inhibited by C3 exoenzyme and in cells expressing RhoA-DN. PKC activity was inhibited by bisindolylmaleimide, and in cells expressing RhoA-DN; PKC activity was also inhibited partly by Y27632 (44+/-5%). PKC-induced phosphorylation of PKC-activated 17 kDa inhibitor protein of type 1 phosphatase (CPI-17) at Thr38 was abolished by bisindolylmaleimide and inhibited partly by Y27632 (28+/-3%). Rho-kinase-induced phosphorylation of myosin phosphatase targeting subunit (MYPT1) and was abolished by Y27632. Sustained phosphorylation of 20 kDa regulatory light chain of myosin II (MLC20) and contraction were abolished by bisindolylmaleimide Y27632 and C3 exoenzyme and in cells expressing RhoA-DN. The results suggest that Rho-kinase-dependent phosphorylation of MYPT1 and PKC-dependent phosphorylation and enhancement of CPI-17 binding to the catalytic subunit of MLC phosphatase (MLCP) act co-operatively to inhibit MLCP activity, leading to sustained stimulation of MLC20 phosphorylation and contraction. Because Y27632 inhibited both Rho kinase and PKC activities

  16. Differential signalling by muscarinic receptors in smooth muscle: m2-mediated inactivation of myosin light chain kinase via Gi3, Cdc42/Rac1 and p21-activated kinase 1 pathway, and m3-mediated MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation via Rho-associated kinase/myosin phosphatase targeting subunit 1 and protein kinase C/CPI-17 pathway.

    PubMed Central

    Murthy, Karnam S; Zhou, Huiping; Grider, John R; Brautigan, David L; Eto, Masumi; Makhlouf, Gabriel M

    2003-01-01

    Signalling via m3 and m2 receptors in smooth muscles involved activation of two G-protein-dependent pathways by each receptor. m2 receptors were coupled via Gbetagammai3 with activation of phospholipase C-beta3, phosphoinositide 3-kinase and Cdc42/Rac1 (where Cdc stands for cell division cycle) and p21-activated kinase 1 (PAK1), resulting in phosphorylation and inactivation of myosin light chain kinase (MLCK). Each step was inhibited by methoctramine and pertussis toxin. PAK1 activity was abolished in cells expressing both Cdc42-DN (where DN stands for dominant negative) and Rac1-DN. MLCK phosphorylation was inhibited by PAK1 antibody, and in cells expressing Cdc42-DN and Rac1-DN. m3 receptors were coupled via Galpha(q/11) with activation of phospholipase C-beta1 and via RhoA with activation of Rho-associated kinase (Rho kinase), phospholipase D and protein kinase C (PKC). Rho kinase and phospholipase D activities were inhibited by C3 exoenzyme and in cells expressing RhoA-DN. PKC activity was inhibited by bisindolylmaleimide, and in cells expressing RhoA-DN; PKC activity was also inhibited partly by Y27632 (44+/-5%). PKC-induced phosphorylation of PKC-activated 17 kDa inhibitor protein of type 1 phosphatase (CPI-17) at Thr38 was abolished by bisindolylmaleimide and inhibited partly by Y27632 (28+/-3%). Rho-kinase-induced phosphorylation of myosin phosphatase targeting subunit (MYPT1) and was abolished by Y27632. Sustained phosphorylation of 20 kDa regulatory light chain of myosin II (MLC20) and contraction were abolished by bisindolylmaleimide Y27632 and C3 exoenzyme and in cells expressing RhoA-DN. The results suggest that Rho-kinase-dependent phosphorylation of MYPT1 and PKC-dependent phosphorylation and enhancement of CPI-17 binding to the catalytic subunit of MLC phosphatase (MLCP) act co-operatively to inhibit MLCP activity, leading to sustained stimulation of MLC20 phosphorylation and contraction. Because Y27632 inhibited both Rho kinase and PKC activities

  17. Negative regulation of ErbB family receptor tyrosine kinases.

    PubMed

    Sweeney, C; Carraway, K L

    2004-01-26

    Receptors of the EGF receptor or ErbB family of growth factor receptor tyrosine kinases are frequently overexpressed in a variety of solid tumours, and the aberrant activation of their tyrosine kinase activities is thought to contribute to tumour growth and progression. Much effort has been put into developing inhibitors of ErbB receptors, and both antibody and small-molecule approaches have exhibited clinical success. Recently, a number of endogenous negative regulatory proteins have been identified that suppress the signalling activity of ErbB receptors in cells. These include intracellular RING finger E3 ubiquitin ligases such as cbl and Nrdp1 that mediate ErbB receptor degradation, and may include a wide variety of secreted and transmembrane proteins that suppress receptor activation by growth factor ligands. It will be of interest to determine the extent to which tumour cells suppress these pathways to promote their progression, and whether restoration of endogenous receptor-negative regulatory pathways may be exploited for therapeutic benefit.

  18. Catalytic control in the EGF Receptor and its connection to general kinase regulatory mechanisms

    PubMed Central

    Jura, Natalia; Zhang, Xuewu; Endres, Nicholas F.; Seeliger, Markus A.; Schindler, Thomas; Kuriyan, John

    2011-01-01

    Summary In contrast to the active conformations of protein kinases, which are essentially the same for all kinases, inactive kinase conformations are structurally diverse. Some inactive conformations are, however, observed repeatedly in different kinases, perhaps reflecting an important role in catalysis. In this review, we analyze one of these recurring conformations, first identified in CDK and Src kinases, which turned out to be central to understanding of how kinase domain of the EGF receptor is activated. This mechanism, which involves the stabilization of the active conformation of an α helix, has features in common with mechanisms operative in several other kinases. PMID:21474065

  19. Regulation of FcϵRI Signaling in Mast Cells by G Protein-coupled Receptor Kinase 2 and Its RH Domain*

    PubMed Central

    Subramanian, Hariharan; Gupta, Kshitij; Parameswaran, Narayanan; Ali, Hydar

    2014-01-01

    Agonist-induced phosphorylation of G protein-coupled receptors (GPCRs) by GPCR kinases (GRKs) promotes their desensitization and internalization. Here, we sought to determine the role of GRK2 on FcϵRI signaling and mediator release in mast cells. The strategies utilized included lentiviral shRNA-mediated GRK2 knockdown, GRK2 gene deletion (GRK2flox/flox/cre recombinase) and overexpression of GRK2 and its regulator of G protein signaling homology (RH) domain (GRK2-RH). We found that silencing GRK2 expression caused ∼50% decrease in antigen-induced Ca2+ mobilization and degranulation but resulted in ablation of cytokine (IL-6 and IL-13) generation. The effect of GRK2 on cytokine generation does not require its catalytic activity but is mediated via the phosphorylation of p38 and Akt. Overexpression of GRK2 or its RH domain (GRK2-RH) enhanced antigen-induced mast cell degranulation and cytokine generation without affecting the expression levels of any of the FcϵRI subunits (α, β, and γ). GRK2 or GRK2-RH had no effect on antigen-induced phosphorylation of FcϵRIγ or Src but enhanced tyrosine phosphorylation of Syk. These data demonstrate that GRK2 modulates FcϵRI signaling in mast cells via at least two mechanisms. One involves GRK2-RH and modulates tyrosine phosphorylation of Syk, and the other is mediated via the phosphorylation of p38 and Akt. PMID:24904059

  20. Reconstruction of the Chemotaxis Receptor-Kinase Assembly

    SciTech Connect

    Park,S.; Borbat, P.; Gonzalez-Bonet, G.; Bhatnagar, J.; Pollard, A.; Freed, J.; Bilwes, A.; Crane, B.

    2006-01-01

    In bacterial chemotaxis, an assembly of transmembrane receptors, the CheA histidine kinase and the adaptor protein CheW processes environmental stimuli to regulate motility. The structure of a Thermotoga maritima receptor cytoplasmic domain defines CheA interaction regions and metal ion-coordinating charge centers that undergo chemical modification to tune receptor response. Dimeric CheA-CheW, defined by crystallography and pulsed ESR, positions two CheWs to form a cleft that is lined with residues important for receptor interactions and sized to clamp one receptor dimer. CheW residues involved in kinase activation map to interfaces that orient the CheW clamps. CheA regulatory domains associate in crystals through conserved hydrophobic surfaces. Such CheA self-contacts align the CheW receptor clamps for binding receptor tips. Linking layers of ternary complexes with close-packed receptors generates a lattice with reasonable component ratios, cooperative interactions among receptors and accessible sites for modification enzymes.

  1. Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

    SciTech Connect

    Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do

    2012-03-01

    Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells

  2. Venus kinase receptors: prospects in signaling and biological functions of these invertebrate kinases.

    PubMed

    Dissous, Colette; Morel, Marion; Vanderstraete, Mathieu

    2014-01-01

    Venus kinase receptors (VKRs) form a family of invertebrate receptor tyrosine kinases (RTKs) initially discovered in the parasitic platyhelminth Schistosoma mansoni. VKRs are single transmembrane receptors that contain an extracellular venus fly trap structure similar to the ligand-binding domain of G protein-coupled receptors of class C, and an intracellular tyrosine kinase domain close to that of insulin receptors. VKRs are found in a large variety of invertebrates from cnidarians to echinoderms and are highly expressed in larval stages and in gonads, suggesting a role of these proteins in embryonic and larval development as well as in reproduction. VKR gene silencing could demonstrate the function of these receptors in oogenesis as well as in spermatogenesis in S. mansoni. VKRs are activated by amino acids and are highly responsive to arginine. As many other RTKs, they form dimers when activated by ligands and induce intracellular pathways involved in protein synthesis and cellular growth, such as MAPK and PI3K/Akt/S6K pathways. VKRs are not present in vertebrates or in some invertebrate species. Questions remain open about the origin of this little-known RTK family in evolution and its role in emergence and specialization of Metazoa. What is the meaning of maintenance or loss of VKR in some phyla or species in terms of development and physiological functions? The presence of VKRs in invertebrates of economical and medical importance, such as pests, vectors of pathogens, and platyhelminth parasites, and the implication of these RTKs in gametogenesis and reproduction processes are valuable reasons to consider VKRs as interesting targets in new programs for eradication/control of pests and infectious diseases, with the main advantage in the case of parasite targeting that VKR counterparts are absent from the vertebrate host kinase panel.

  3. Venus Kinase Receptors: Prospects in Signaling and Biological Functions of These Invertebrate Kinases

    PubMed Central

    Dissous, Colette; Morel, Marion; Vanderstraete, Mathieu

    2014-01-01

    Venus kinase receptors (VKRs) form a family of invertebrate receptor tyrosine kinases (RTKs) initially discovered in the parasitic platyhelminth Schistosoma mansoni. VKRs are single transmembrane receptors that contain an extracellular venus fly trap structure similar to the ligand-binding domain of G protein-coupled receptors of class C, and an intracellular tyrosine kinase domain close to that of insulin receptors. VKRs are found in a large variety of invertebrates from cnidarians to echinoderms and are highly expressed in larval stages and in gonads, suggesting a role of these proteins in embryonic and larval development as well as in reproduction. VKR gene silencing could demonstrate the function of these receptors in oogenesis as well as in spermatogenesis in S. mansoni. VKRs are activated by amino acids and are highly responsive to arginine. As many other RTKs, they form dimers when activated by ligands and induce intracellular pathways involved in protein synthesis and cellular growth, such as MAPK and PI3K/Akt/S6K pathways. VKRs are not present in vertebrates or in some invertebrate species. Questions remain open about the origin of this little-known RTK family in evolution and its role in emergence and specialization of Metazoa. What is the meaning of maintenance or loss of VKR in some phyla or species in terms of development and physiological functions? The presence of VKRs in invertebrates of economical and medical importance, such as pests, vectors of pathogens, and platyhelminth parasites, and the implication of these RTKs in gametogenesis and reproduction processes are valuable reasons to consider VKRs as interesting targets in new programs for eradication/control of pests and infectious diseases, with the main advantage in the case of parasite targeting that VKR counterparts are absent from the vertebrate host kinase panel. PMID:24860549

  4. Cinnamaldehyde prevents adipocyte differentiation and adipogenesis via regulation of peroxisome proliferator-activated receptor-γ (PPARγ) and AMP-activated protein kinase (AMPK) pathways.

    PubMed

    Huang, Bo; Yuan, Hai Dan; Kim, Do Yeon; Quan, Hai Yan; Chung, Sung Hyun

    2011-04-27

    Cinnamaldehyde (CA), one of the active components of cinnamon, has been known to exert several pharmacological effects such as anti-inflammatory, antioxidant, antitumor, and antidiabetic activities. However, its antiobesity effect has not been reported yet. This study investigated the antidifferentiation effect of CA on 3T3-L1 preadipocytes, and the antiobesity activity of CA was further explored using high-fat-diet-induced obese ICR mice. During 3T3-L1 preadipocytes were differentiated into adipocytes, 10-40 μM CA was treated and lipid contents were quantified by Oil Red O staining, along with changes in the expression of genes and proteins associated with adipocyte differentiation and adipogenesis. It was found that CA significantly reduced lipid accumulation and down-regulated the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), CCAAT/enhancer-binding proteins α (C/EBPα), and sterol regulatory element-binding protein 1 (SREBP1) in concentration-dependent manners. Moreover, CA markedly up-regulated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), and these effects were blunted in the presence of AMPK inhibitor, compound C. In the animal study, weight gains, insulin resistance index, plasma triglyceride (TG), nonesterified fatty acid (NEFA), and cholesterol levels in the 40 mg/kg of CA-administered group were significantly decreased by 67.3, 55, 39, 31, and 23%, respectively, when compared to the high-fat diet control group. In summary, these results suggest that CA exerts antiadipogenic effects through modulation of the PPAR-γ and AMPK signaling pathways.

  5. Pathway illuminated: visualizing protein kinase C signaling.

    PubMed

    Violin, Jonathan D; Newton, Alexandra C

    2003-12-01

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

  6. The insulin and IGF1 receptor kinase domains are functional dimers in the activated state

    NASA Astrophysics Data System (ADS)

    Cabail, M. Zulema; Li, Shiqing; Lemmon, Eric; Bowen, Mark E.; Hubbard, Stevan R.; Miller, W. Todd

    2015-03-01

    The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of α-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.

  7. The p38 mitogen-activated protein kinase signaling pathway is involved in regulating low-density lipoprotein receptor-related protein 1-mediated β-amyloid protein internalization in mouse brain.

    PubMed

    Ma, Kai-Ge; Lv, Jia; Hu, Xiao-Dan; Shi, Li-Li; Chang, Ke-Wei; Chen, Xin-Lin; Qian, Yi-Hua; Yang, Wei-Na; Qu, Qiu-Min

    2016-07-01

    Alzheimer's disease (AD) is one of the most common neurodegenerative diseases. Recently, increasing evidence suggests that intracellular β-amyloid protein (Aβ) alone plays a pivotal role in the progression of AD. Therefore, understanding the signaling pathway and proteins that control Aβ internalization may provide new insight for regulating Aβ levels. In the present study, the regulation of Aβ internalization by p38 mitogen-activated protein kinases (MAPK) through low-density lipoprotein receptor-related protein 1 (LRP1) was analyzed in vivo. The data derived from this investigation revealed that Aβ1-42 were internalized by neurons and astrocytes in mouse brain, and were largely deposited in mitochondria and lysosomes, with some also being found in the endoplasmic reticulum. Aβ1-42-LRP1 complex was formed during Aβ1-42 internalization, and the p38 MAPK signaling pathway was activated by Aβ1-42 via LRP1. Aβ1-42 and LRP1 were co- localized in the cells of parietal cortex and hippocampus. Furthermore, the level of LRP1-mRNA and LRP1 protein involved in Aβ1-42 internalization in mouse brain. The results of this investigation demonstrated that Aβ1-42 induced an LRP1-dependent pathway that related to the activation of p38 MAPK resulting in internalization of Aβ1-42. These results provide evidence supporting a key role for the p38 MAPK signaling pathway which is involved in the regulation of Aβ1-42 internalization in the parietal cortex and hippocampus of mouse through LRP1 in vivo.

  8. Hypertonicity regulates the function of human neutrophils by modulating chemoattractant receptor signaling and activating mitogen-activated protein kinase p38.

    PubMed Central

    Junger, W G; Hoyt, D B; Davis, R E; Herdon-Remelius, C; Namiki, S; Junger, H; Loomis, W; Altman, A

    1998-01-01

    Excessive neutrophil activation causes posttraumatic complications, which may be reduced with hypertonic saline (HS) resuscitation. We tested if this is because of modulated neutrophil function by HS. Clinically relevant hypertonicity (10-25 mM) suppressed degranulation and superoxide formation in response to fMLP and blocked the activation of the mitogen activated protein kinases (MAPK) ERK1/2 and p38, but did not affect Ca2+ mobilization. HS did not suppress oxidative burst in response to phorbol myristate acetate (PMA). This indicates that HS suppresses neutrophil function by intercepting signal pathways upstream of or apart from PKC. HS activated p38 by itself and enhanced degranulation in response to PKC activation. This enhancement was reduced by inhibition of p38 with SB203580, suggesting that p38 up-regulation participates in HS-induced enhancements of degranulation. HS had similar effects on the degranulation of cells that were previously stimulated with fMLP, but had no effect on its own, suggesting that HS enhancement of degranulation requires another signal. We conclude that depending on other stimuli, HS can suppress neutrophil activation by intercepting multiple receptor signals or augment degranulation by enhancing p38 signaling. In patients HS resuscitation may reduce posttraumatic complications by preventing neutrophil activation via chemotactic factors released during reperfusion. PMID:9637711

  9. Activation of platelet-activating factor (PAF) receptor stimulates nitric oxide (NO) release via protein kinase C-alpha in HEC-1B human endometrial epithelial cell line.

    PubMed Central

    Dearn, S.; Rahman, M.; Lewis, A.; Ahmed, Z.; Eggo, M. C.; Ahmed, A.

    2000-01-01

    BACKGROUND: Impairment of the fertility in the platelet-activating factor (PAF) receptor transgenic female mice suggests changes in PAF functions can influence uterine receptivity. We hypothesized that vasodilatory actions of PAF in the uterus was exerted by PAF-mediated nitric oxide (NO) release via activation of isoenzyme-specific protein kinase C (PKC). MATERIALS AND METHODS: Inducible and endothelial NOS was shown by Reverse transcription polymerase chain reaction RT-PCR in cDNA synthesized from RNA extract of proliferative and secretory endometrium as well endometrial epithelial cell lines HEC-1B. The effect of WEB2170, N(G)-monomethyl-L-arginine (L-NMMA) and Ro31-8220 on PAF mediated NO release by HEC-1B cell was determined. PAF induced translocation of PKCalpha in HEC-1B cell and its antagonist effect by Ro 31-8220 was studied by Western immunoblot analysis. PKC isoenzyme regulated by PAF was determined in HEC-1B cell lysate by immunoprecipitation. RESULTS: PAF-evoked a rapid and concentration-dependent biphasic increase in total NO in human HEC-1B endometrial epithelial cell line [as measured by a Sievers NOA 280A NO Chemiluminescent Analyser.] This increase in NO release was attenuated by the PAF receptor antagonist, WEB2170. Inhibition of NO synthesis by N(G)-monomethyl-L-arginine produced marked dose-dependent attenuation of PAF-mediated NO release, indicating nitric oxide synthase (NOS) activation. PAF-mediated NO release was also inhibited by the PKC inhibitor Ro 31-8220 and by the removal of extracellular calcium, suggesting a dependency on PKC and calcium, respectively. RT-PCR analysis showed expression of inducible NOS and endothelial NOS in human endometrium, myometrium and HEC-1B cells. Western immunoblot analysis showed PKCalpha, betaII and iota were the principal isozymes present in the HEC-1B cell line and normal endometrium, suggesting that both HEC-1B cells and normal endometrium have similar PKC isozymes. PAF induced the translocation of

  10. Cross-talk from β-adrenergic receptors modulates α2A-adrenergic receptor endocytosis in sympathetic neurons via protein kinase A and spinophilin.

    PubMed

    Cottingham, Christopher; Lu, Roujian; Jiao, Kai; Wang, Qin

    2013-10-04

    Inter-regulation of adrenergic receptors (ARs) via cross-talk is a long appreciated but mechanistically unclear physiological phenomenon. Evidence from the AR literature and our own extensive studies on regulation of α2AARs by the scaffolding protein spinophilin have illuminated a potential novel mechanism for cross-talk from β to α2ARs. In the present study, we have characterized a mode of endogenous AR cross-talk in native adrenergic neurons whereby canonical βAR-mediated signaling modulates spinophilin-regulated α2AAR endocytosis through PKA. Our findings demonstrate that co-activation of β and α2AARs, either by application of endogenous agonist or by simultaneous stimulation with distinct selective agonists, results in acceleration of endogenous α2AAR endocytosis in native neurons. We show that receptor-independent PKA activation by forskolin is sufficient to accelerate α2AAR endocytosis and that α2AAR stimulation alone drives accelerated endocytosis in spinophilin-null neurons. Endocytic response acceleration by β/α2AAR co-activation is blocked by PKA inhibition and lost in spinophilin-null neurons, consistent with our previous finding that spinophilin is a substrate for phosphorylation by PKA that disrupts its interaction with α2AARs. Importantly, we show that α2AR agonist-mediated α2AAR/spinophilin interaction is blocked by βAR co-activation in a PKA-dependent fashion. We therefore propose a novel mechanism for cross-talk from β to α2ARs, whereby canonical βAR-mediated signaling coupled to PKA activation results in phosphorylation of spinophilin, disrupting its interaction with α2AARs and accelerating α2AAR endocytic responses. This mechanism of cross-talk has significant implications for endogenous adrenergic physiology and for therapeutic targeting of β and α2AARs.

  11. Synergistic interaction of CLAVATA1, CLAVATA2, and RECEPTOR-LIKE PROTEIN KINASE 2 in cyst nematode parasitism of Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant-parasitic cyst nematodes secrete CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (ESR) (CLE)-like effector proteins. These proteins act as ligand mimics of plant CLE peptides and are required for successful nematode infection. Previously, we showed that CLV2 and CORYNE (CRN), a heterodimer recept...

  12. A novel role of G protein-coupled receptor kinase 5 in urotensin II-stimulated cellular hypertrophy in H9c2UT cells.

    PubMed

    Park, Cheon Ho; Lee, Ju Hee; Lee, Mi Young; Lee, Jeong Hyun; Lee, Byung Ho; Oh, Kwang-Seok

    2016-11-01

    Urotensin II (UII) is a neural hormone that induces cardiac hypertrophy and may be involved in the pathogenesis of cardiac remodeling and heart failure. Hypertrophy has been linked to histone deacetylase 5 (HDAC5) phosphorylation and nuclear factor κB (NF-κB) translocation, both of which are predominantly mediated by G protein-coupled receptor kinase 5 (GRK5). In the present study, we found that UII rapidly and strongly stimulated nuclear export of HDAC5 and nuclear import of NF-κB in H9c2 cells overexpressing the urotensin II receptor (H9c2UT). Hence, we hypothesized that GRK5 and its signaling pathway may play a role in UII-mediated cellular hypertrophy. H9c2UT cells were transduced with a GRK5 small hairpin RNA interference recombinant lentivirus, resulting in the down-regulation of GRK5. Under UII stimulation, reduced levels of GRK5 in H9c2UT cells led to suppression of UII-mediated HDAC5 phosphorylation and activation of the NF-κB signaling pathway. In contrast, UII-mediated activations of ERK1/2 and GSK3α/β were not affected by down-regulation of GRK5. In a cellular hypertrophy assay, down-regulation of GRK5 significantly suppressed UII-mediated hypertrophy of H9c2UT cells. Furthermore, UII-mediated cellular hypertrophy was inhibited by amlexanox, a selective GRK5 inhibitor, in H9c2UT cells and neonatal cardiomyocytes. Our results suggest that GRK5 may be involved in a UII-mediated hypertrophic response via activation of NF-κB and HDAC5 at least in part by ERK1/2 and GSK3α/β-independent pathways.

  13. Novel type of receptor-like protein kinase from a higher plant (Catharanthus roseus). cDNA, gene, intramolecular autophosphorylation, and identification of a threonine important for auto- and substrate phosphorylation.

    PubMed

    Schulze-Muth, P; Irmler, S; Schröder, G; Schröder, J

    1996-10-25

    We characterize CrRLK1, a novel type of receptor-like kinase (RLK), from the plant Catharanthus roseus (Madagascar periwinkle). The protein (90.2 kDa) deduced from the complete genomic and cDNA sequences is a RLK by predicting a N-terminal signal peptide, a large extracytoplasmic domain, a membrane-spanning hydrophobic region followed by a transfer-stop signal, and a C-terminal cytoplasmic protein kinase with all 11 conserved subdomains. It is a novel RLK type because the predicted extracytoplasmic region shares no similarity with other RLKs. The autophosphorylation was investigated with affinity-purified proteins expressed in Escherichia coli. The activity was higher with Mn2+ than with Mg2+ and achieved half-maximal rates at 2-2.5 microM ATP. The phosphorylation was predominantly on Thr, less on Ser, and not on Tyr. In contrast to other plant RLK, the kinase used an intra- rather than an intermolecular phosphorylation mechanism. After protein cleavage with formic acid, most of the radioactivity was in a 14.1-kDa peptide located at the end of the kinase domain. Mutagenesis of the four Thr residues in this peptide identified Thr-720 in the subdomain XI as important for autophosphorylation and for phosphorylation of beta-casein. This Thr is conserved in other related kinases, suggesting a subfamily sharing common autophosphorylation mechanisms.

  14. Light-assisted small molecule screening against protein kinases

    PubMed Central

    Inglés-Prieto, Álvaro; Reichhart, Eva; Muellner, Markus K.; Nowak, Matthias; Nijman, Sebastian M.; Grusch, Michael; Janovjak, Harald

    2015-01-01

    High-throughput live-cell screens are intricate elements of systems biology studies and drug discovery pipelines. Here, we demonstrate an optogenetics-assisted method that obviates the addition of chemical activators and reporters, reduces the number of operational steps and increases information content in a cell-based small molecule screen against human protein kinases including an orphan receptor tyrosine kinase. This blueprint for all-optical screening can be adapted to many drug targets and cellular processes. PMID:26457372

  15. Role of protein kinase A in phosphorylation of NMDA receptor 1 subunits in dorsal horn and spinothalamic tract neurons after intradermal injection of capsaicin in rats.

    PubMed

    Zou, X; Lin, Q; Willis, W D

    2002-01-01

    Protein phosphorylation is a major mechanism for regulation of N-methyl-D-aspartate (NMDA) receptor function. The NMDA receptor 1 subunit (NR1) is phosphorylated by protein kinase A (PKA) on serine 890 and 897. We have recently reported that there is enhanced phosphorylation of NR1 on serine 897 in dorsal horn and spinothalamic tract (STT) neurons after intradermal injection of capsaicin (CAP) in rats [Zou et al. (2000) J. Neurosci. 20, 6989-6997]. Whether or not this phosphorylation, which develops during central sensitization following CAP injection, is mediated by PKA remains to be determined. In this study, western blots and immunofluorescence staining were employed to observe if pretreatment with a PKA inhibitor, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, HCl (H89), blocks the enhanced phosphorylation of NR1 on serine 897 following injection of CAP into the glabrous skin of one hind paw of anesthetized rats. Western blots showed that pretreatment with H89 caused a decrease in CAP-induced phosphorylation of NR1 protein in spinal cord segments L(4)-S(1). In experiments using immunofluorescence staining, the numbers of phospho-NR1-like immunoreactive (p-NR1-LI) neurons seen after CAP injection were significantly decreased in the dorsal horn of the L(4)-L(5) segments on the side ipsilateral to the injection after PKA was inhibited. When STT cells were labeled by microinjection of the retrograde tracer, fluorogold, we found that the proportion of p-NR1-LI STT cells on the side ipsilateral to the injection in the superficial laminae of spinal cord segments L(4)-L(5) was markedly reduced when H89 was administered intrathecally before CAP injection. However, the proportion of p-NR1-LI STT cells in deep laminae was unchanged unless the PKC inhibitor, chelerythrine chloride, was co-administered with H89. Combined with our previous findings, the present results indicate that NR1 in spinal dorsal horn neurons, including the superficial dorsal horn STT

  16. Inhibitory effects of omega-3 fatty acids on early brain injury after subarachnoid hemorrhage in rats: Possible involvement of G protein-coupled receptor 120/β-arrestin2/TGF-β activated kinase-1 binding protein-1 signaling pathway.

    PubMed

    Yin, Jia; Li, Haiying; Meng, Chengjie; Chen, Dongdong; Chen, Zhouqing; Wang, Yibin; Wang, Zhong; Chen, Gang

    2016-06-01

    Omega-3 fatty acids have been reported to improve neuron functions during aging and in patients affected by mild cognitive impairment, and mediate potent anti-inflammatory via G protein-coupled receptor 120 (GPR120) signal pathway. Neuron dysfunction and inflammatory response also contributed to the progression of subarachnoid hemorrhage (SAH)-induced early brain injury (EBI). This study was to examine the effects of omega-3 fatty acids on SAH-induced EBI. Two weeks before SAH, 30% Omega-3 fatty acids was administered by oral gavage at 1g/kg body weight once every 24h. Specific siRNA for GPR120 was exploited. Terminal deoxynucleotidyl transferase dUTP nick end labeling, fluoro-Jade B staining, and neurobehavioral scores and brain water content test showed that omega-3 fatty acids effectively suppressed SAH-induced brain cell apoptosis and neuronal degradation, behavioral impairment, and brain edema. Western blot, immunoprecipitation, and electrophoretic mobility shift assays results showed that omega-3 fatty acids effectively suppressed SAH-induced elevation of inflammatory factors, including cyclooxygenase-2, monocyte chemoattractant protein-1, and inducible nitric oxide synthase. In addition, omega-3 fatty acids could inhibit phosphorylation of transforming growth factor β activated kinase-1 (TAK1), MEK4, c-Jun N-terminal kinase, and IkappaB kinase as well as activation of nuclear factor kappa B through regulating GPR120/β-arrestin2/TAK1 binding protein-1 pathway. Furthermore, siRNA-induced GPR120 silencing blocked the protective effects of omega-3 fatty acids. Here, we show that stimulation of GPR120 with omega-3 fatty acids pretreatment causes anti-apoptosis and anti-inflammatory effects via β-arrestin2/TAK1 binding protein-1/TAK1 pathway in the brains of SAH rats. Fish omega-3 fatty acids as part of a daily diet may reduce EBI in an experimental rat model of SAH.

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

    PubMed

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

    2012-02-01

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

  18. GsLRPK, a novel cold-activated leucine-rich repeat receptor-like protein kinase from Glycine soja, is a positive regulator to cold stress tolerance.

    PubMed

    Yang, Liang; Wu, Kangcheng; Gao, Peng; Liu, Xiaojuan; Li, Guangpu; Wu, Zujian

    2014-02-01

    Plant LRR-RLKs serve as protein interaction platforms, and as regulatory modules of protein activation. Here, we report the isolation of a novel plant-specific LRR-RLK from Glycine soja (termed GsLRPK) by differential screening. GsLRPK expression was cold-inducible and shows Ser/Thr protein kinase activity. Subcellular localization studies using GFP fusion protein indicated that GsLRPK is localized in the plasma membrane. Real-time PCR analysis indicated that temperature, salt, drought, and ABA treatment can alter GsLRPK gene transcription in G. soja. However, just protein induced by cold stress not by salinity and ABA treatment in tobacco was found to possess kinase activity. Furthermore, we found that overexpression of GsLRPK in yeast and Arabidopsis can enhance resistance to cold stress and increase the expression of a number of cold responsive gene markers.

  19. Role of aberrant striatal dopamine D1 receptor/cAMP/protein kinase A/DARPP32 signaling in the paradoxical calming effect of amphetamine.

    PubMed

    Napolitano, Francesco; Bonito-Oliva, Alessandra; Federici, Mauro; Carta, Manolo; Errico, Francesco; Magara, Salvatore; Martella, Giuseppina; Nisticò, Robert; Centonze, Diego; Pisani, Antonio; Gu, Howard H; Mercuri, Nicola B; Usiello, Alessandro

    2010-08-18

    Attention deficit/hyperactivity disorder (ADHD) is characterized by inattention, impulsivity, and motor hyperactivity. Several lines of research support a crucial role for the dopamine transporter (DAT) gene in this psychiatric disease. Consistently, the most commonly prescribed medications in ADHD treatment are stimulant drugs, known to preferentially act on DAT. Recently, a knock-in mouse [DAT-cocaine insensitive (DAT-CI)] has been generated carrying a cocaine-insensitive DAT that is functional but with reduced dopamine uptake function. DAT-CI mutants display enhanced striatal extracellular dopamine levels and basal motor hyperactivity. Herein, we showed that DAT-CI animals present higher striatal dopamine turnover, altered basal phosphorylation state of dopamine and cAMP-regulated phosphoprotein 32 kDa (DARPP32) at Thr75 residue, but preserved D(2) receptor (D(2)R) function. However, although we demonstrated that striatal D(1) receptor (D(1)R) is physiologically responsive under basal conditions, its stimulus-induced activation strikingly resulted in paradoxical electrophysiological, behavioral, and biochemical responses. Indeed, in DAT-CI animals, (1) striatal LTP was completely disrupted, (2) R-(+)-6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF 81297) treatment induced paradoxical motor calming effects, and (3) SKF 81297 administration failed to increase cAMP/protein kinase A (PKA)/DARPP32 signaling. Such biochemical alteration selectively affected dopamine D(1)Rs since haloperidol, by blocking the tonic inhibition of D(2)R, unmasked a normal activation of striatal adenosine A(2A) receptor-mediated cAMP/PKA/DARPP32 cascade in mutants. Most importantly, our studies highlighted that amphetamine, nomifensine, and bupropion, through increased striatal dopaminergic transmission, are able to revert motor hyperactivity of DAT-CI animals. Overall, our results suggest that the paradoxical motor calming effect induced by these

  20. Biphasic Regulation of Yes-associated Protein (YAP) Cellular Localization, Phosphorylation, and Activity by G Protein-coupled Receptor Agonists in Intestinal Epithelial Cells: A NOVEL ROLE FOR PROTEIN KINASE D (PKD).

    PubMed

    Wang, Jia; Sinnett-Smith, James; Stevens, Jan V; Young, Steven H; Rozengurt, Enrique

    2016-08-19

    We examined the regulation of Yes-associated protein (YAP) localization, phosphorylation, and transcriptional activity in intestinal epithelial cells. Our results show that stimulation of intestinal epithelial IEC-18 cells with the G protein-coupled receptor (GPCR) agonist angiotensin II, a potent mitogen for these cells, induced rapid translocation of YAP from the nucleus to the cytoplasm (within 15 min) and a concomitant increase in YAP phosphorylation at Ser(127) and Ser(397) Angiotensin II elicited YAP phosphorylation and cytoplasmic accumulation in a dose-dependent manner (ED50 = 0.3 nm). Similar YAP responses were provoked by stimulation with vasopressin or serum. Treatment of the cells with the protein kinase D (PKD) family inhibitors CRT0066101 and kb NB 142-70 prevented the increase in YAP phosphorylation on Ser(127) and Ser(397) via Lats2, YAP cytoplasmic accumulation, and increase in the mRNA levels of YAP/TEAD-regulated genes (Ctgf and Areg). Furthermore, siRNA-mediated knockdown of PKD1, PKD2, and PKD3 markedly attenuated YAP nuclear-cytoplasmic shuttling, phosphorylation at Ser(127), and induction of Ctgf and Areg expression in response to GPCR activation. These results identify a novel role for the PKD family in the control of biphasic localization, phosphorylation, and transcriptional activity of YAP in intestinal epithelial cells. In turn, YAP and TAZ are necessary for the stimulation of the proliferative response of intestinal epithelial cells to GPCR agonists that act via PKD. The discovery of interaction between YAP and PKD pathways identifies a novel cross-talk in signal transduction and demonstrates, for the first time, that the PKDs feed into the YAP pathway.

  1. Food extracts consumed in Mediterranean countries and East Asia reduce protein concentrations of androgen receptor, phospho-protein kinase B, and phospho-cytosolic phospholipase A(2)alpha in human prostate cancer cells.

    PubMed

    Singh, Jaskirat; Xie, Chanlu; Yao, Mu; Hua, Sheng; Vignarajan, Soma; Jardine, Greg; Hambly, Brett D; Sved, Paul; Dong, Qihan

    2010-04-01

    Active surveillance is an emerging management option for the rising number of men with low-grade, clinically localized prostate cancer. However, 30-40% of men on active surveillance will progress to high-grade disease over 5 y. With the ultimate aim of developing a food-based chemoprevention strategy to retard cancer progression in these otherwise healthy men, we have developed a blend of food extracts commonly consumed in Mediterranean countries and East Asia. The effect of the food extracts known as Blueberry Punch (BBP) on prostate cancer cell growth and key signaling pathways were examined in vitro and in vivo. BBP reduced prostate cancer cell growth in a dose-dependent manner (0.08-2.5%) at 72 h in vitro due to the reduction in cell proliferation and viability. Prostate cancer cell xenograft-bearing mice, administered 10% BBP in drinking water for 2 wk, had a 25% reduction in tumor volume compared with the control (water only). In vitro, BBP reduced protein concentrations in 3 signaling pathways necessary for the proliferation and survival of prostate cancer cells, namely androgen receptor, phospho-protein kinase B/protein kinase B, and phospho-cytosolic phospholipase A(2)alpha. The downstream effectors of these pathways, including prostate-specific antigen and glycogen synthase kinase 3beta, were also reduced. Thus, this palatable food supplement is a potential candidate for testing in clinical trials and may ultimately prove effective in retarding the progression of low-grade, early-stage prostate cancer in men managed by active surveillance.

  2. Fatty acid represses insulin receptor gene expression by impairing HMGA1 through protein kinase C{epsilon}

    SciTech Connect

    Dey, Debleena; Bhattacharya, Anirban; Roy, SibSankar; Bhattacharya, Samir . E-mail: smrbhattacharya@gmail.com

    2007-06-01

    It is known that free fatty acid (FFA) contributes to the development of insulin resistance and type2 diabetes. However, the underlying mechanism in FFA-induced insulin resistance is still unclear. In the present investigation we have demonstrated that palmitate significantly (p < 0.001) inhibited insulin-stimulated phosphorylation of PDK1, the key insulin signaling molecule. Consequently, PDK1 phosphorylation of plasma membrane bound PKC{epsilon} was also inhibited. Surprisingly, phosphorylation of cytosolic PKC{epsilon} was greatly stimulated by palmitate; this was then translocated to the nuclear region and associated with the inhibition of insulin receptor (IR) gene transcription. A PKC{epsilon} translocation inhibitor peptide, {epsilon}V1, suppressed this inhibitory effect of palmitate, suggesting requirement of phospho-PKC{epsilon} migration to implement palmitate effect. Experimental evidences indicate that phospho-PKC{epsilon} adversely affected HMGA1. Since HMGA1 regulates IR promoter activity, expression of IR gene was impaired causing reduction of IR on cell surface and that compromises with insulin sensitivity.

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

    DTIC Science & Technology

    2014-10-01

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

  4. Beta2-containing nicotinic acetylcholine receptors mediate calcium/calmodulin-dependent protein kinase-II and synapsin I protein levels in the nucleus accumbens after nicotine withdrawal in mice.

    PubMed

    Jackson, Kia J; Imad Damaj, M

    2013-02-15

    Nicotinic acetylcholine receptors are calcium-permeable and the initial targets for nicotine. Studies suggest that calcium-dependent mechanisms mediate some behavioral responses to nicotine; however, the post-receptor calcium-dependent mechanisms associated with chronic nicotine and nicotine withdrawal remain unclear. The proteins calcium/calmodulin-dependent protein kinase II (CaMKII) and synapsin I are essential for neurotransmitter release and were shown to be involved in drug dependence. In the current study, using pharmacological techniques, we sought to (a) complement previously published behavioral findings from our lab indicating a role for calcium-dependent signaling in nicotine dependence and (b) expand on previously published acute biochemical and pharmacological findings indicating the relevance of calcium-dependent mechanisms in acute nicotine responses by evaluating the function of CaMKII and synapsin I after chronic nicotine and withdrawal in the nucleus accumbens, a brain region implicated in drug dependence. Male mice were chronically infused with nicotine for 14 days, and treated with the β2-selective antagonist dihydro-β-erythroidine (DHβE), or the α7 antagonist, methyllycaconitine citrate (MLA) 20min prior to dissection of the nucleus accumbens. Results show that phosphorylated and total CaMKII and synapsin I protein levels were significantly increased in the nucleus accumbens after chronic nicotine infusion, and reduced after treatment with DHβE, but not MLA. A spontaneous nicotine withdrawal assessment also revealed significant reductions in phosphorylated CaMKII and synapsin I levels 24h after cessation of nicotine treatment. Our findings suggest that post-receptor calcium-dependent mechanisms associated with nicotine withdrawal are mediated through β2-containing nicotinic receptors.

  5. Protein kinase biochemistry and drug discovery.

    PubMed

    Schwartz, Phillip A; Murray, Brion W

    2011-12-01

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

  6. Prostaglandin E2 negatively regulates AMP-activated protein kinase via protein kinase A signaling pathway.

    PubMed

    Funahashi, Koji; Cao, Xia; Yamauchi, Masako; Kozaki, Yasuko; Ishiguro, Naoki; Kambe, Fukushi

    2009-01-01

    We investigated possible involvement of prostaglandin (PG) E2 in regulation of AMP-activated protein kinase (AMPK). When osteoblastic MG63 cells were cultured in serum-deprived media, Thr-172 phosphorylation of AMPK alpha-subunit was markedly increased. Treatment of the cells with PGE2 significantly reduced the phosphorylation. Ser-79 phosphorylation of acetyl-CoA carboxylase, a direct target for AMPK, was also reduced by PGE2. On the other hand, PGE2 reciprocally increased Ser-485 phosphorylation of the alpha-subunit that could be associated with inhibition of AMPK activity. These effects of PGE2 were mimicked by PGE2 receptor EP2 and EP4 agonists and forskolin, but not by EP1 and EP3 agonists, and the effects were suppressed by an adenylate cyclase inhibitor SQ22536 and a protein kinase A inhibitor H89. Additionally, the PGE2 effects were duplicated in primary calvarial osteoblasts. Together, the present study demonstrates that PGE2 negatively regulates AMPK activity via activation of protein kinase A signaling pathway.

  7. Inhibition of Receptor-Interacting Protein Kinase 1 with Necrostatin–1s ameliorates disease progression in elastase-induced mouse abdominal aortic aneurysm model

    PubMed Central

    Wang, Qiwei; Zhou, Ting; Liu, Zhenjie; Ren, Jun; Phan, Noel; Gupta, Kartik; Stewart, Danielle M.; Morgan, Stephanie; Assa, Carmel; Kent, K. Craig; Liu, Bo

    2017-01-01

    Abdominal aortic aneurysm (AAA) is a common aortic disease with a progressive nature. There is no approved pharmacological treatment to effectively slow aneurysm growth or prevent rupture. Necroptosis is a form of programmed necrosis that is regulated by receptor-interacting protein kinases (RIPs). We have recently demonstrated that the lack of RIP3 in mice prevented aneurysm formation. The goal of the current study is to test whether perturbing necroptosis affects progression of existing aneurysm using the RIP1 inhibitors Necrostatin-1 (Nec-1) and an optimized form of Nec-1, 7-Cl-O-Nec-1 (Nec-1s). Seven days after aneurysm induction by elastase perfusion, mice were randomly administered DMSO, Nec-1 (3.2 mg/kg/day) and Nec-1s (1.6 mg/kg/day) via intraperitoneal injection. Upon sacrifice on day 14 postaneurysm induction, the aortic expansion in the Nec-1s group (64.12 ± 4.80%) was significantly smaller than that of the DMSO group (172.80 ± 13.68%) (P < 0.05). The mean aortic diameter of Nec-1 treated mice appeared to be smaller (121.60 ± 10.40%) than the DMSO group, though the difference was not statistically significant (P = 0.1). Histologically, the aortic structure of Nec-1s-treated mice appeared normal, with continuous and organized elastin laminae and abundant αActin-expressing SMCs. Moreover, Nect-1s treatment diminished macrophage infiltration and MMP9 accumulation and increased aortic levels of tropoelastin and lysyl oxidase. Together, our data suggest that pharmacological inhibition of necroptosis with Nec-1s stabilizes pre-existing aneurysms by diminishing inflammation and promoting connective tissue repair. PMID:28186202

  8. Inhibition of Receptor-Interacting Protein Kinase 1 with Necrostatin-1s ameliorates disease progression in elastase-induced mouse abdominal aortic aneurysm model.

    PubMed

    Wang, Qiwei; Zhou, Ting; Liu, Zhenjie; Ren, Jun; Phan, Noel; Gupta, Kartik; Stewart, Danielle M; Morgan, Stephanie; Assa, Carmel; Kent, K Craig; Liu, Bo

    2017-02-10

    Abdominal aortic aneurysm (AAA) is a common aortic disease with a progressive nature. There is no approved pharmacological treatment to effectively slow aneurysm growth or prevent rupture. Necroptosis is a form of programmed necrosis that is regulated by receptor-interacting protein kinases (RIPs). We have recently demonstrated that the lack of RIP3 in mice prevented aneurysm formation. The goal of the current study is to test whether perturbing necroptosis affects progression of existing aneurysm using the RIP1 inhibitors Necrostatin-1 (Nec-1) and an optimized form of Nec-1, 7-Cl-O-Nec-1 (Nec-1s). Seven days after aneurysm induction by elastase perfusion, mice were randomly administered DMSO, Nec-1 (3.2 mg/kg/day) and Nec-1s (1.6 mg/kg/day) via intraperitoneal injection. Upon sacrifice on day 14 postaneurysm induction, the aortic expansion in the Nec-1s group (64.12 ± 4.80%) was significantly smaller than that of the DMSO group (172.80 ± 13.68%) (P < 0.05). The mean aortic diameter of Nec-1 treated mice appeared to be smaller (121.60 ± 10.40%) than the DMSO group, though the difference was not statistically significant (P = 0.1). Histologically, the aortic structure of Nec-1s-treated mice appeared normal, with continuous and organized elastin laminae and abundant αActin-expressing SMCs. Moreover, Nect-1s treatment diminished macrophage infiltration and MMP9 accumulation and increased aortic levels of tropoelastin and lysyl oxidase. Together, our data suggest that pharmacological inhibition of necroptosis with Nec-1s stabilizes pre-existing aneurysms by diminishing inflammation and promoting connective tissue repair.

  9. Histamine H4-Receptors Inhibit Mast Cell Renin Release in Ischemia/Reperfusion via Protein Kinase Cε-Dependent Aldehyde Dehydrogenase Type-2 Activation

    PubMed Central

    Aldi, Silvia; Takano, Ken-ichi; Tomita, Kengo; Koda, Kenichiro; Chan, Noel Y.-K.; Marino, Alice; Salazar-Rodriguez, Mariselis; Thurmond, Robin L.

    2014-01-01

    Renin released by ischemia/reperfusion (I/R) from cardiac mast cells (MCs) activates a local renin-angiotensin system (RAS) causing arrhythmic dysfunction. Ischemic preconditioning (IPC) inhibits MC renin release and consequent activation of this local RAS. We postulated that MC histamine H4-receptors (H4Rs), being Gαi/o-coupled, might activate a protein kinase C isotype–ε (PKCε)–aldehyde dehydrogenase type-2 (ALDH2) cascade, ultimately eliminating MC-degranulating and renin-releasing effects of aldehydes formed in I/R and associated arrhythmias. We tested this hypothesis in ex vivo hearts, human mastocytoma cells, and bone marrow–derived MCs from wild-type and H4R knockout mice. We found that activation of MC H4Rs mimics the cardioprotective anti-RAS effects of IPC and that protection depends on the sequential activation of PKCε and ALDH2 in MCs, reducing aldehyde-induced MC degranulation and renin release and alleviating reperfusion arrhythmias. These cardioprotective effects are mimicked by selective H4R agonists and disappear when H4Rs are pharmacologically blocked or genetically deleted. Our results uncover a novel cardioprotective pathway in I/R, whereby activation of H4Rs on the MC membrane, possibly by MC-derived histamine, leads sequentially to PKCε and ALDH2 activation, reduction of toxic aldehyde-induced MC renin release, prevention of RAS activation, reduction of norepinephrine release, and ultimately to alleviation of reperfusion arrhythmias. This newly discovered protective pathway suggests that MC H4Rs may represent a new pharmacologic and therapeutic target for the direct alleviation of RAS-induced cardiac dysfunctions, including ischemic heart disease and congestive heart failure. PMID:24696042

  10. Glucocorticoid Receptor, C/EBP, HNF3, and Protein Kinase A Coordinately Activate the Glucocorticoid Response Unit of the Carbamoylphosphate Synthetase I Gene

    PubMed Central

    Christoffels, Vincent M.; Grange, Thierry; Kaestner, Klaus H.; Cole, Timothy J.; Darlington, Gretchen J.; Croniger, Colleen M.; Lamers, Wouter H.

    1998-01-01

    A single far-upstream enhancer is sufficient to confer hepatocyte-specific, glucocorticoid- and cyclic AMP-inducible periportal expression to the carbamoylphosphate synthetase I (CPS) gene. To identify the mechanism of hormone-dependent activation, the composition and function of the enhancer have been analyzed. DNase I protection and gel mobility shift assays revealed the presence of a cyclic AMP response element, a glucocorticoid response element (GRE), and several sites for the liver-enriched transcription factor families HNF3 and C/EBP. The in vivo relevance of the transcription factors interacting with the enhancer in the regulation of CPS expression in the liver was assessed by the analysis of knockout mice. A strong reduction of CPS mRNA levels was observed in glucocorticoid receptor- and C/EBPα-deficient mice, whereas the CPS mRNA was normally expressed in C/EBPβ knockout mice and in HNF3α and -γ double-knockout mice. (The role of HNFβ could not be assessed, because the corresponding knockout mice die at embryonic day 10). In hepatoma cells, most of the activity of the enhancer is contained within a 103-bp fragment, which depends for its activity on the simultaneous occupation of the GRE, HNF3, and C/EBP sites, thus meeting the requirement of a glucocorticoid response unit. In fibroblast-like CHO cells, on the other hand, the GRE in the CPS enhancer does not cooperate with the C/EBP and HNF3 elements in transactivation of the CPS promoter. In both hepatoma and CHO cells, stimulation of expression by cyclic AMP depends mainly on the integrity of the glucocorticoid pathway, demonstrating cross talk between this pathway and the cyclic AMP (protein kinase A) pathway. PMID:9774647

  11. The regulator of G protein signaling (RGS) domain of G protein–coupled receptor kinase 5 (GRK5) regulates plasma membrane localization and function

    PubMed Central

    Xu, Hua; Jiang, Xiaoshan; Shen, Ke; Fischer, Christopher C.; Wedegaertner, Philip B.

    2014-01-01

    The G protein–coupled receptor (GPCR) kinases (GRKs) phosphorylate activated GPCRs at the plasma membrane (PM). Here GRK5/GRK4 chimeras and point mutations in GRK5 identify a short sequence within the regulator of G protein signaling (RGS) domain in GRK5 that is critical for GRK5 PM localization. This region of the RGS domain of GRK5 coincides with a region of GRK6 and GRK1 shown to form a hydrophobic dimeric interface (HDI) in crystal structures. Coimmunoprecipitation (coIP) and acceptor photobleaching fluorescence resonance energy transfer assays show that expressed GRK5 self-associates in cells, whereas GRK5-M165E/F166E (GRK5-EE), containing hydrophilic mutations in the HDI region of the RGS domain, displays greatly decreased coIP interactions. Both forcing dimerization of GRK5-EE, via fusion to leucine zipper motifs, and appending an extra C-terminal membrane-binding region to GRK5-EE (GRK5-EE-CT) recover PM localization. In addition, GRK5-EE displays a decreased ability to inhibit PAR1-induced calcium release compared with GRK5 wild type (wt). In contrast, PM-localized GRK5-EE-CaaX (appending a C-terminal prenylation and polybasic motif from K-ras) or GRK5-EE-CT shows comparable ability to GRK5 wt to inhibit PAR1-induced calcium release. The results suggest a novel model in which GRK5 dimerization is important for its plasma membrane localization and function. PMID:24807909

  12. G1 cell cycle arrest due to the inhibition of erbB family receptor tyrosine kinases does not require the retinoblastoma protein

    SciTech Connect

    Gonzales, Andrea J. . E-mail: Andrea.Gonzales@pfizer.com; Fry, David W.

    2005-02-01

    The erbB receptor family (EGFr, erbB-2, erbB-3, and erbB-4) consists of transmembrane glycoproteins that transduce extracellular signals to the nucleus when activated. erbB family members are widely expressed in epithelial, mesenchymal, and neuronal cells and contribute to the proliferation, differentiation, migration, and survival of these cell types. The present study evaluates the effects of erbB family signaling on cell cycle progression and the role that pRB plays in regulating this process. ErbB family RTK activity was inhibited by PD 158780 in the breast epithelial cell line MCF10A. PD 158780 (0.5 {mu}M) inhibited EGF-stimulated and heregulin-stimulated autophosphorylation and caused a G1 cell cycle arrest within 24 h, which correlated with hypophosporylation of pRB. MCF10A cells lacking functional pRB retained the ability to arrest in G1 when treated with PD 158780. Both cell lines showed induction of p27{sup KIP1} protein when treated with PD 158780 and increased association of p27{sup KIP1} with cyclin E-CDK2. Furthermore, CDK2 kinase activity was dramatically inhibited with drug treatment. Changes in other pRB family members were noted with drug treatment, namely a decrease in p107 and an increase in p130. These findings show that the G1 arrest induced through inhibition of erbB family RTK activity does not require functional pRB.

  13. G-Protein-Coupled Receptor Kinase 4 Polymorphisms and Blood Pressure Response to Metoprolol Among African Americans: Sex-Specificity and Interactions

    PubMed Central

    Bhatnagar, Vibha; O’Connor, Daniel T.; Brophy, Victoria H.; Schork, Nicholas J.; Richard, Erin; Salem, Rany M.; Nievergelt, Caroline M.; Bakris, George L.; Middleton, John P.; Norris, Keith C.; Wright, Jackson; Hiremath, Leena; Contreras, Gabriel; Appel, Lawrence J.; Lipkowitz, Michael S.

    2009-01-01

    BACKGROUND African Americans have a disproportionate burden of hypertension and comorbid disease. Pharmacogenetic markers of blood pressure response have yet to be defined clearly. This study explores the association between G-protein-coupled receptor kinase type 4 (GRK4) variants and blood pressure response to metoprolol among African Americans with early hypertensive nephrosclerosis. METHODS Participants from the African American Study of Kidney Disease and Hypertension (AASK) trial were genotyped at three GRK4 polymorphisms: R65L, A142V, and A486V. A Cox proportional hazards model, stratified by gender, was used to determine the relationship between GRK4 variants and time to reach a mean arterial pressure (MAP) of 107 mm Hg, adjusted for other predictors of blood pressure response. Potential interactions between the three polymorphisms were explored by analyzing the effects of gene haplotypes and by stratifying the analysis by neighboring sites. RESULTS The hazard ratio with 95% confidence interval by A142V among men randomized to a usual MAP (102–107 mm Hg) was 1.54 (1.11–2.44; P = 0.0009). The hazard ratio by A142V with R65/L65 or L65/L65 was 2.14 (1.35–3.39; P = 0.001). Haplotype analyses were consistent but inconclusive. There was no association between A142V and blood pressure response among women. CONCLUSIONS Results suggest a sex-specific relationship between GRK4 A142V and blood pressure response among African-American men with early hypertensive nephrosclerosis. Men with a GRK4 A142 were less responsive to metoprolol if they had a GRK4 L65 variant. The effect of GRK4 variants and blood pressure response to metoprolol should be studied in larger clinical trials. PMID:19119263

  14. Central Rho kinase inhibition restores baroreflex sensitivity and angiotensin II type 1 receptor protein imbalance in conscious rabbits with chronic heart failure.

    PubMed

    Haack, Karla K V; Gao, Lie; Schiller, Alicia M; Curry, Pamela L; Pellegrino, Peter R; Zucker, Irving H

    2013-03-01

    The small GTPase RhoA and its associated kinase ROCKII are involved in vascular smooth muscle cell contraction and endothelial NO synthase mRNA destabilization. Overactivation of the RhoA/ROCKII pathway is implicated in several pathologies, including chronic heart failure (CHF), and may contribute to the enhanced sympathetic outflow seen in CHF as a result of decreased NO availability. Thus, we hypothesized that central ROCKII blockade would improve the sympathovagal imbalance in a pacing rabbit model of CHF in an NO-dependent manner. CHF was induced by rapid ventricular pacing and characterized by an ejection fraction of ≤45%. Animals were implanted with an intracerbroventricular cannula and osmotic minipump (rate, 1 μL/h) containing sterile saline, 1.5 µg/kg per day fasudil (Fas, a ROCKII inhibitor) for 4 days or Fas+100 µg/kg per day Nω-Nitro-l-arginine methyl ester hydrochloride, a NO synthase inhibitor. Arterial baroreflex control was assessed by intravenous infusion of sodium nitroprusside and phenylephrine. Fas infusion significantly lowered resting heart rate by decreasing sympathetic and increasing vagal tone. Furthermore, Fas improved baroreflex gain in CHF in an NO-dependent manner. In CHF Fas animals, the decrease in heart rate in response to intravenous metoprolol was similar to Sham and was reversed by Nω-Nitro-l-arginine methyl ester hydrochloride. Fas decreased angiotensin II type 1 receptor and phospho-ERM protein expression and increased endothelial NO synthase expression in the brain stem of CHF animals. These data strongly suggest that central ROCKII activation contributes to cardiac sympathoexcitation in the setting of CHF and that central Fas restores vagal and sympathetic tone in an NO-dependent manner. ROCKII may be a new central therapeutic target in the setting of CHF.

  15. From pure compounds to complex exposure: Effects of dietary cadmium and lignans on estrogen, epidermal growth factor receptor, and mitogen activated protein kinase signaling in vivo.

    PubMed

    Ali, Imran; Hurmerinta, Teija; Nurmi, Tarja; Berglund, Marika; Rüegg, Joelle; Poutanen, Matti; Halldin, Krister; Mäkelä, Sari; Damdimopoulou, Pauliina

    2016-06-24

    Exposure to environmental endocrine active compounds correlates with altered susceptibility to disease in human populations. Chemical risk assessment is single compound based, although exposure often takes place as heterogeneous mixtures of man-made and natural substances within complex matrices like diet. Here we studied whether the effects of cadmium and enterolactone on endocrine endpoints in dietary exposure can be predicted based on pure compound effects. Ovariectomized estrogen reporter ERE-luciferase (ERE-luc) mice were maintained on diets that intrinsically contain increasing concentrations of cadmium and enterolactone precursors for three and 21 days. The activation of the ERE-luc, epidermal growth factor receptor (EGFR), mitogen activated protein kinase (MAPK)-ERK1/2, and classical estrogen responses were measured. Interactions between the diets and endogenous hormone were evaluated by challenging the animals with 17β-estradiol. Compared to animals on basal purified diet, mice consuming experimental diets were exposed to significantly higher levels of cadmium and enterolactone, yet the exposure remained comparable to typical human dietary intake. Surprisingly, we could not detect effects on endpoints regulated by pure enterolactone, such as ERE-luc activation. However, cadmium accumulation in the liver was accompanied with activation of EGFR and MAPK-ERK1/2 in line with our earlier CdCl2 studies. Further, attenuation of 17β-estradiol-induced ERE-luc response in liver by experimental diets was observed. Our findings indicate that the exposure context can have substantial effects on the activity of endocrine active compounds in vivo. Thus, whenever possible, a context that mimics human exposure should be tested along with pure compounds.

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

    PubMed

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

    1998-12-01

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

  17. 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.

  18. Phosphorylation of the mRNA cap binding protein and eIF-4A by different protein kinases

    SciTech Connect

    Hagedorn, C.H.

    1987-05-01

    These studies were done to determine the identity of a protein kinase that phosphorylates the mRNA cap binding protein (CBP). Two chromatographic steps (dye and ligand and ion exchange HPLC) produced a 500x purification of an enzyme activity in rabbit reticulocytes that phosphorylated CBP at serine residues. Isoelectric focusing analysis of kinase treated CBP demonstrated 5 isoelectric species of which the 2 most anodic species were phosphorylated (contained /sup 32/P). This kinase activity phosphorylated CBP when it was isolated or in the eIF-4F complex. Purified protein kinase C, cAMP or cGMP dependent protein kinase, casein kinase I or II, myosin light chain kinase or insulin receptor kinase did not significantly phosphorylate isolated CBP or CBP in the eIF-4F complex. However, cAMP and cGMP dependent protein kinases and casein kinase II phosphorylated eIF-4A but did not phosphorylate the 46 kDa component of eIF-4F. cAMP dependent protein kinase phosphorylated a approx. 220 kDa protein doublet in eIF-4F preparations. These studies indicate that CBP kinase activity probably represents a previously unidentified protein kinase. In addition, eIF-4A appears to be phosphorylated by several protein kinases whereas the 46 kDa component of the eIF-4F complex was not.

  19. Probing activation/deactivation of the BRASSINOSTEROID INSENSITIVE1 receptor kinase by immunoprecipitation

    PubMed Central

    Martins, Sara; Vert, Grégory; Jaillais, Yvon

    2017-01-01

    Summary Brassinosteroids are plant sterol-derived hormones that control plant growth and development. The BR receptor complex is encoded by the BRASSINOSTEROID INSENSITIVE1 (BRI1) and members of the SOMATIC EMBRYOGENESIS RECEPTOR KINASE family. BR receptor complex activation and deactivation uses different post-translational modifications and recruitment of partner proteins. In this chapter, we describe optimized immunoprecipitation protocols and variants for biochemical analyses of BRI1 post-translational modification and protein-protein interaction. PMID:28124254

  20. 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

  1. Tannic acid, a potent inhibitor of epidermal growth factor receptor tyrosine kinase.

    PubMed

    Yang, Er Bin; Wei, Liu; Zhang, Kai; Chen, Yu Zong; Chen, Wei Ning

    2006-03-01

    Increasing evidence supports the hypothesis that tannic acid, a plant polyphenol, exerts anticarcinogenic activity in chemically induced cancers. In the present study, tannic acid was found to strongly inhibit tyrosine kinase activity of epidermal growth factor receptor (EGFr) in vitro (IC50 = 323 nM). In contrast, the inhibition by tannic acid of p60(c-src) tyrosine kinase (IC50 = 14 microM) and insulin receptor tyrosine kinase (IC50 = 5 microM) was much weaker. The inhibition of EGFr tyrosine kinase by tannic acid was competitive with respect to ATP and non-competitive with respect to peptide substrate. In cultured cells, growth factor-induced tyrosine phosphorylation of growth factor receptors, including EGFr, platelet-derived growth factor receptor, and basic fibroblast growth factor receptor, was inhibited by tannic acid. No inhibition of insulin-induced tyrosine phosphorylation of insulin receptor and insulin-receptor substrate-1 was observed. EGF-stimulated growth of HepG2 cells was inhibited in the presence of tannic acid. The inhibition of serine/threonine-specific protein kinases, including cAMP-dependent protein kinase, protein kinase C and mitogen-activated protein kinase, by tannic acid was only detected at relatively high concentration, IC50 being 3, 325 and 142 microM respectively. The molecular modeling study suggested that tannic acid could be docked into the ATP binding pockets of either EGFr or insulin receptor. These results demonstrate that tannic acid is an in vitro potent inhibitor of EGFr tyrosine kinase.

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

    PubMed Central

    Lai, Shenshen; Safaei, Javad

    2016-01-01

    The reversible phosphorylation of proteins catalyzed by protein kinases in eukaryotes supports an important role for eukaryotic protein kinases (ePKs) in the emergence of nucleated cells in the third superkingdom of life. Choline kinases (ChKs) could also be critical in the early evolution of eukaryotes, because of their function in the biosynthesis of phosphatidylcholine, which is unique to eukaryotic membranes. However, the genomic origins of ePKs and ChKs are unclear. The high degeneracy of protein sequences and broad expansion of ePK families have made this fundamental question difficult to answer. In this study, we identified two class-I aminoacyl-tRNA synthetases with high similarities to consensus amino acid sequences of human protein-serine/threonine kinases. Comparisons of primary and tertiary structures supported that ePKs and ChKs evolved from a common ancestor related to glutaminyl aminoacyl-tRNA synthetases, which may have been one of the key factors in the successful of emergence of ancient eukaryotic cells from bacterial colonies. PMID:26742849

  3. Retinoic acids acting through retinoid receptors protect hippocampal neurons from oxygen-glucose deprivation-mediated cell death by inhibition of c-jun-N-terminal kinase and p38 mitogen-activated protein kinase.

    PubMed

    Shinozaki, Y; Sato, Y; Koizumi, S; Ohno, Y; Nagao, T; Inoue, K

    2007-06-15

    Retinoic acids (RAs), including all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA), play fundamental roles in a variety of physiological events in vertebrates, through their specific nuclear receptors: retinoic acid receptor (RAR) and retinoid X receptor (RXR). Despite the physiological importance of RA, their functional significance under pathological conditions is not well understood. We examined the effect of ATRA on oxygen/glucose-deprivation/reperfusion (OGD/Rep)-induced neuronal damage in cultured rat hippocampal slices, and found that ATRA significantly reduced neuronal death. The cytoprotective effect of ATRA was observed not only in cornu ammonis (CA) 1 but also in CA2 and dentate gyrus (DG), and was attenuated by selective antagonists for RAR or RXR. By contrast, in the CA3 region, no protective effects of ATRA were observed. The OGD/Rep also increased phosphorylated forms of c-jun-N-terminal kinase (P-JNK) and p38 (P-p38) in hippocampus, and specific inhibitors for these kinases protected neurons. ATRA prevented the increases in P-JNK and P-p38 after OGD/Rep, as well as the decrease in NeuN and its shrinkage, all of which were inhibited by antagonists for RAR or RXR. These findings suggest that the ATRA signaling via retinoid receptors results in the inhibition of JNK and p38 activation, leading to the protection of neurons against OGD/Rep-induced damage in the rat hippocampus.

  4. Sesquiterpene dimmer (DSF-27) inhibits the release of neuroinflammatory mediators from microglia by targeting spleen tyrosine kinase (Syk) and Janus kinase 2 (Jak2): Two major non-receptor tyrosine signaling proteins involved in inflammatory events

    SciTech Connect

    Zeng, Ke-Wu; Wang, Shu; Dong, Xin; Jiang, Yong; Jin, Hong-Wei; Tu, Peng-Fei

    2014-03-15

    Non-receptor protein tyrosine kinases (NRPTKs)-dependent inflammatory signal transduction cascades play key roles in immunoregulation. However, drug intervention through NRPTKs-involved immunoregulation mechanism in microglia (the major immune cells of the central nervous system) has not been widely investigated. A main aim of the present study is to elucidate the contribution of two major NRPTKs (Syk and Jak2) in neuroinflammation suppression by a bioactive sesquiterpene dimmer (DSF-27). We found that LPS-stimulated BV-2 cells activated Syk and further initiated Akt/NF-κB inflammatory pathway. This Syk-dependent Akt/NF-κB inflammatory pathway can be effectively ameliorated by DSF-27. Moreover, Jak2 was activated by LPS, which was followed by transcriptional factor Stat3 activation. The Jak2/Stat3 signal was suppressed by DSF-27 through inhibition of Jak2 and Stat3 phosphorylation, promotion of Jak/Stat3 inhibitory factors PIAS3 expression, and down-regulation of ERK and p38 MAPK phosphorylation. Furthermore, DSF-27 protected cortical and mesencephalic dopaminergic neurons against neuroinflammatory injury. Taken together, our findings indicate NRPTK signaling pathways including Syk/NF-κB and Jak2/Stat3 cascades are potential anti-neuroinflammatory targets in microglia, and may also set the basis for the use of sesquiterpene dimmer as a therapeutic approach for neuroinflammation via interruption of these pathways. - Highlights: • Sesquiterpene dimmer DSF-27 inhibits inflammatory mediators' production in microglia. • Syk-dependent Akt/NF-κB pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 pathway is important for DSF-27's anti-inflammation activity. • Jak2/Stat3 signaling pathway is partly regulated by ERK and p38 MAPKs and PIAS3. • DSF-27 protects neurons against microglia-mediated neuroinflammatory injury.

  5. Tyrosine Kinase Receptor Expression in Canine Liposarcoma.

    PubMed

    Avallone, G; Pellegrino, V; Roccabianca, P; Lepri, E; Crippa, L; Beha, G; De Tolla, L; Sarli, G

    2017-03-01

    The expression of tyrosine kinase receptors is attracting major interest in human and veterinary oncological pathology because of their role as targets for adjuvant therapies. Little is known about tyrosine kinase receptor (TKR) expression in canine liposarcoma (LP), a soft tissue sarcoma. The aim of this study was to evaluate the immunohistochemical expression of the TKRs fibroblast growth factor receptor 1 (FGFR1) and platelet-derived growth factor receptor-β (PDGFRβ); their ligands, fibroblast growth factor 2 (FGF2) and platelet-derived growth factor B (PDGFB); and c-kit in canine LP. Immunohistochemical labeling was categorized as high or low expression and compared with the mitotic count and MIB-1-based proliferation index. Fifty canine LPs were examined, classified, and graded. Fourteen cases were classified as well differentiated, 7 as myxoid, 25 as pleomorphic, and 4 as dedifferentiated. Seventeen cases were grade 1, 26 were grade 2, and 7 were grade 3. A high expression of FGF2, FGFR1, PDGFB, and PDGFRβ was identified in 62% (31/50), 68% (34/50), 81.6% (40/49), and 70.8% (34/48) of the cases, respectively. c-kit was expressed in 12.5% (6/48) of the cases. Mitotic count negatively correlated with FGF2 ( R = -0.41; P < .01), being lower in cases with high FGF2 expression, and positively correlated with PDGFRβ ( R = 0.33; P < .01), being higher in cases with high PDGFRβ expression. No other statistically significant correlations were identified. These results suggest that the PDGFRβ-mediated pathway may have a role in the progression of canine LP and may thus represent a promising target for adjuvant cancer therapies.

  6. Unveiling the Membrane-Binding Properties of N-Terminal and C-Terminal Regions of G Protein-Coupled Receptor Kinase 5 by Combined Optical Spectroscopies

    PubMed Central

    2015-01-01

    G protein-coupled receptor kinase 5 (GRK5) is thought to associate with membranes in part via N- and C-terminal segments that are typically disordered in available high-resolution crystal structures. Herein we investigate the interactions of these regions with model cell membrane using combined sum frequency generation (SFG) vibrational spectroscopy and attenuated total reflectance–Fourier transform infrared (ATR-FTIR) spectroscopy. It was found that both regions associate with POPC lipid bilayers but adopt different structures when doing so: GRK5 residues 2–31 (GRK52–31) was in random coil whereas GRK5546–565 was partially helical. When the subphase for the GRK52–31 peptide was changed to 40% TFE/60% 10 mM phosphate pH 7.4 buffer, a large change in the SFG amide I signal indicated that GRK52–31 became partially helical. By inspecting the membrane behavior of two different segments of GRK52–31, namely, GRK52–24 and GRK525–31, we found that residues 25–31 are responsible for membrane binding, whereas the helical character is imparted by residues 2–24. With SFG, we deduced that the orientation angle of the helical segment of GRK52–31 is 46 ± 1° relative to the surface normal in 40% TFE/60% 10 mM phosphate pH = 7.4 buffer but increases to 78 ± 11° with higher ionic strength. We also investigated the effect of PIP2 in the model membrane and concluded that the POPC:PIP2 (9:1) lipid bilayer did not change the behavior of either peptide compared to a pure POPC lipid bilayer. With ATR-FTIR, we also found that Ca2+·calmodulin is able to extract both peptides from the POPC lipid bilayer, consistent with the role of this protein in disrupting GRK5 interactions with the plasma membrane in cells. PMID:24401145

  7. Sprouty-related Ena/Vasodilator-stimulated Phosphoprotein Homology 1-Domain-containing Protein (SPRED1), a Tyrosine-Protein Phosphatase Non-receptor Type 11 (SHP2) Substrate in the Ras/Extracellular Signal-regulated Kinase (ERK) Pathway*

    PubMed Central

    Quintanar-Audelo, Martina; Yusoff, Permeen; Sinniah, Saravanan; Chandramouli, Sumana; Guy, Graeme R.

    2011-01-01

    SHP2 is a tyrosine phosphatase involved in the activation of the Ras/ERK signaling pathway downstream of a number of receptor tyrosine kinases. One of the proposed mechanisms involving SHP2 in this context is to dephosphorylate and inactivate inhibitors of the Ras/ERK pathway. Two protein families bearing a unique, common domain, Sprouty and SPRED proteins, are possible candidates because they have been reported to inhibit the Ras/ERK pathway upon FGF activation. We tested whether any of these proteins are likely substrates of SHP2. Our findings indicate that Sprouty2 binds to the C-terminal tail of SHP2, which is an unlikely substrate binding site, whereas SPRED proteins bind to the tyrosine phosphatase domain that is known to be the binding site for its substrates. Overexpressed SHP2 was able to dephosphorylate SPREDs but not Sprouty2. Finally, we found two tyrosine residues on SPRED1 that are required, when phosphorylated, to inhibit Ras/ERK activation and identified Tyr-420 as a specific dephosphorylation target of SHP2. The evidence obtained indicates that SPRED1 is a likely substrate of SHP2, whose tyrosine dephosphorylation is required to attenuate the inhibitory action of SPRED1 in the Ras/ERK pathway. PMID:21531714

  8. Receptor tyrosine kinase signaling: a view from quantitative proteomics.

    PubMed

    Dengjel, Joern; Kratchmarova, Irina; Blagoev, Blagoy

    2009-10-01

    Growth factor receptor signaling via receptor tyrosine kinases (RTKs) is one of the basic cellular communication principals found in all metazoans. Extracellular signals are transferred via membrane spanning receptors into the cytoplasm, reversible tyrosine phosphorylation being the hallmark of all RTKs. In recent years proteomic approaches have yielded detailed descriptions of cellular signaling events. Quantitative proteomics is able to characterize the exact position and strength of post-translational modifications (PTMs) providing essential information for understanding the molecular basis of signal transduction. Numerous new post-translational modification sites have been identified by quantitative mass spectrometry-based proteomics. In addition, plentiful new players in signal transduction have been identified underlining the complexity and the modular architecture of most signaling networks. In this review, we outline the principles of signal transduction via RTKs and highlight some of the new insights obtained from proteomic approaches such as protein microarrays and quantitative mass spectrometry.

  9. Molecular and biochemical analysis of symbiotic plant receptor kinase complexes

    SciTech Connect

    Cook, Douglas R; Riely, Brendan K

    2010-09-01

    DE-FG02-01ER15200 was a 36-month project, initiated on Sept 1, 2005 and extended with a one-year no cost extension to August 31, 2009. During the project period we published seven manuscripts (2 in review). Including the prior project period (2002-2005) we published 12 manuscripts in journals that include Science, PNAS, The Plant Cell, Plant Journal, Plant Physiology, and MPMI. The primary focus of this work was to further elucidate the function of the Nod factor signaling pathway that is involved in initiation of the legume-rhizobium symbiosis and in particular to explore the relationship between receptor kinase-like proteins and downstream effectors of symbiotic development. During the project period we have map-base cloned two additional players in symbiotic development, including an ERF transcription factor and an ethylene pathway gene (EIN2) that negatively regulates symbiotic signaling; we have also further characterized the subcellular distribution and function of a nuclear-localized symbiosis-specific ion channel, DMI1. The major outcome of the work has been the development of systems for exploring and validating protein-protein interactions that connect symbiotic receptor-like proteins to downstream responses. In this regard, we have developed both homologous (i.e., in planta) and heterologous (i.e., in yeast) systems to test protein interactions. Using yeast 2-hybrid screens we isolated the only known interactor of the nuclear-localized calcium-responsive kinase DMI3. We have also used yeast 2-hybrid methodology to identify interactions between symbiotic signaling proteins and certain RopGTPase/RopGEF proteins that regulate root hair polar growth. More important to the long-term goals of our work, we have established a TAP tagging system that identifies in planta interactions based on co-immuno precipitation and mass spectrometry. The validity of this approach has been shown using known interactors that either co-iummnoprecipate (i.e., remorin) or co

  10. Receptor for bombesin with associated tyrosine kinase activity.

    PubMed Central

    Cirillo, D M; Gaudino, G; Naldini, L; Comoglio, P M

    1986-01-01

    The neuropeptide bombesin is known for its potent mitogenic activity on murine 3T3 fibroblasts and other cells. Recently it has been implicated in the pathogenesis of small cell lung carcinoma, in which it acts through an autocrine loop of growth stimulation. Phosphotyrosine (P-Tyr) antibodies have been successfully used to recognize the autophosphorylated receptors for known growth factors. In Swiss 3T3 fibroblasts, phosphotyrosine antibodies identified a 115,000-Mr cell surface protein (p115) that became phosphorylated on tyrosine as a specific response to bombesin stimulation of quiescent cells. The extent of phosphorylation was dose dependent and correlated with the mitogenic effect induced by bombesin, measured by [3H]thymidine incorporation. Tyrosine phosphorylation of p115 was detectable minutes after the addition of bombesin, and its time course paralleled that described for the binding of bombesin to its receptor. Immunocomplexes of phosphorylated p115 and phosphotyrosine antibodies bound 125I-labeled [Tyr4]bombesin in a specific and saturable manner and displayed an associated tyrosine kinase activity enhanced by bombesin. Furthermore, the 125I-labeled bombesin analog gastrin-releasing peptide, bound to intact live cells, was coprecipitated with p115. These data strongly suggest that p115 participates in the structure and function of the surface receptor for bombesin, a new member of the family of growth factor receptors with associated tyrosine kinase activity. Images PMID:2432404

  11. Genome-wide analysis of lectin receptor-like kinases in Populus

    SciTech Connect

    Yang, Yongil; Labbé, Jessy; Muchero, Wellington; Yang, Xiaohan; Jawdy, Sara S.; Kennedy, Megan; Johnson, Jenifer; Sreedasyam, Avinash; Schmutz, Jeremy; Tuskan, Gerald A.; Chen, Jin -Gui

    2016-09-01

    Receptor-like kinases (RLKs) belong to a large protein family with over 600 members in Arabidopsis and over 1000 in rice. Among RLKs, the lectin receptor-like kinases (LecRLKs) possess a characteristic extracellular carbohydrate-binding lectin domain and play important roles in plant development and innate immunity. In addition, there are 75 and 173 LecRLKs in Arabidopsis and rice, respectively. However, little is known about LecRLKs in perennial woody plants.

  12. Genome-wide analysis of lectin receptor-like kinases in Populus

    DOE PAGES

    Yang, Yongil; Labbé, Jessy; Muchero, Wellington; ...

    2016-09-01

    Receptor-like kinases (RLKs) belong to a large protein family with over 600 members in Arabidopsis and over 1000 in rice. Among RLKs, the lectin receptor-like kinases (LecRLKs) possess a characteristic extracellular carbohydrate-binding lectin domain and play important roles in plant development and innate immunity. In addition, there are 75 and 173 LecRLKs in Arabidopsis and rice, respectively. However, little is known about LecRLKs in perennial woody plants.

  13. Differential regulation by agonist and phorbol ester of cloned m1 and m2 muscarinic acetylcholine receptors in mouse Y1 adrenal cells and in Y1 cells deficient in cAMP-dependent protein kinase

    SciTech Connect

    Scherer, N.M.; Nathanson, N.M. )

    1990-09-11

    Cloned muscarinic acetylcholine m1 and m2 receptors were expressed in stably transfected mouse Y1 adrenal cells and in a variant Y1 line, Kin-8, which is deficient in cAMP-dependent protein kinase activity (PKA{sup {minus}}). m1 and m2 receptors were rapidly internalized following exposure of transfected PKA{sup +} or PKA{sup {minus}} cells to the muscarinic agonist carbachol. Thus, agonist-dependent internalization of m1 and m2 did not require PKA activity. A differential effect of PKA on regulation by agonist of the m2 receptor, but not the m1 receptor, was unmasked in PKA{sup {minus}} cells. These data indicate that the basal activity of PKA may modulate the agonist-dependent internalization of the m2 receptor, but not the m1 receptor. The internalization of the m1 and m2 receptors in both PKA{sup +} and PKA{sup {minus}} cells was accompanied by desensitization of functional responses. Exposure of PKA{sup +} cells to 10{sup {minus}7} M phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, resulted in a 30 {plus minus} 9% decrease in the number of m1 receptors on the cell surface. The m2 receptor was not internalized following treatment of either PKA{sup +} or PKA{sup {minus}} cells with PMA. Thus, the m1 and m2 receptors show differential sensitivity to internalization by PMA. Agonist-dependent internalization of the m1 receptor appeared to be independent of activation of PKC because (1) agonist-dependent internalization of m1 was not attenuated in PKA{sup {minus}} cells, (2) the rate and extent of internalization of m1 in cells exposed to PMA were less than those in cells exposed to agonist, and (3) treatment of cells with concanavalin A selectivity blocked internalization of m1 in cells exposed to PMA, but not to agonist. The effects of agonist and PMA on receptor internalization were not additive. Exposure of PKA{sup +} or PKA{sup {minus}} cells to PMA reduced the magnitude of pilocarpine-stimulated PI hydrolysis by about 25%.

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

    PubMed

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

    2003-05-15

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

  15. Receptor for advanced glycation end-products regulates lung fluid balance via protein kinase C-gp91(phox) signaling to epithelial sodium channels.

    PubMed

    Downs, Charles A; Kreiner, Lisa H; Johnson, Nicholle M; Brown, Lou Ann; Helms, My N

    2015-01-01

    The receptor for advanced glycation end-products (RAGE), a multiligand member of the Ig family, may play a crucial role in the regulation of lung fluid balance. We quantified soluble RAGE (sRAGE), a decoy isoform, and advanced glycation end-products (AGEs) from the bronchoalveolar lavage fluid of smokers and nonsmokers, and tested the hypothesis that AGEs regulate lung fluid balance through protein kinase C (PKC)-gp91(phox) signaling to the epithelial sodium channel (ENaC). Human bronchoalveolar lavage samples from smokers showed increased AGEs (9.02 ± 3.03 μg versus 2.48 ± 0.53 μg), lower sRAGE (1,205 ± 292 pg/ml versus 1,910 ± 263 pg/ml), and lower volume(s) of epithelial lining fluid (97 ± 14 ml versus 133 ± 17 ml). sRAGE levels did not predict ELF volumes in nonsmokers; however, in smokers, higher volumes of ELF were predicted with higher levels of sRAGE. Single-channel patch clamp analysis of rat alveolar epithelial type 1 cells showed that AGEs increased ENaC activity measured as the product of the number of channels (N) and the open probability (Po) (NPo) from 0.19 ± 0.08 to 0.83 ± 0.22 (P = 0.017) and the subsequent addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.15 ± 0.07 (P = 0.01). In type 2 cells, human AGEs increased ENaC NPo from 0.12 ± 0.05 to 0.53 ± 0.16 (P = 0.025) and the addition of 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine-N-oxyl decreased ENaC NPo to 0.10 ± 0.03 (P = 0.013). Using molecular and biochemical techniques, we observed that inhibition of RAGE and PKC activity attenuated AGE-induced activation of ENaC. AGEs induced phosphorylation of p47(phox) and increased gp91(phox)-dependent reactive oxygen species production, a response that was abrogated with RAGE or PKC inhibition. Finally, tracheal instillation of AGEs promoted clearance of lung fluid, whereas concomitant inhibition of RAGE, PKC, and gp91(phox) abrogated the response.

  16. Novel protein kinase signaling systems regulating lifespan identified by small molecule library screening using Drosophila.

    PubMed

    Spindler, Stephen R; Li, Rui; Dhahbi, Joseph M; Yamakawa, Amy; Sauer, Frank

    2012-01-01

    Protein kinase signaling cascades control most aspects of cellular function. The ATP binding domains of signaling protein kinases are the targets of most available inhibitors. These domains are highly conserved from mammals to flies. Herein we describe screening of a library of small molecule inhibitors of protein kinases for their ability to increase Drosophila lifespan. We developed an assay system which allowed screening using the small amounts of materials normally present in commercial chemical libraries. The studies identified 17 inhibitors, the majority of which targeted tyrosine kinases associated with the epidermal growth factor receptor (EGFR), platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) receptors, G-protein coupled receptor (GPCR), Janus kinase (JAK)/signal transducer and activator of transcription (STAT), the insulin and insulin-like growth factor (IGFI) receptors. Comparison of the protein kinase signaling effects of the inhibitors in vitro defined a consensus intracellular signaling profile which included decreased signaling by p38MAPK (p38), c-Jun N-terminal kinase (JNK) and protein kinase C (PKC). If confirmed, many of these kinases will be novel additions to the signaling cascades known to regulate metazoan longevity.

  17. Co-transfection with protein kinase D confers phorbol-ester-mediated inhibition on glucagon-stimulated cAMP accumulation in COS cells transfected to overexpress glucagon receptors.

    PubMed Central

    Tobias, E S; Rozengurt, E; Connell, J M; Houslay, M D

    1997-01-01

    Glucagon elicited a profound increase in the intracellular cAMP concentration of COS-7 cells which had been transiently transfected with a cDNA encoding the rat glucagon receptor and under conditions where cAMP phosphodiesterase activity was fully inhibited. This was achieved in a dose-dependent fashion with an EC50 of 1.8+/-0.4 nM glucagon. In contrast with previous observations made using hepatocytes [Heyworth, Whetton, Kinsella and Houslay (1984) FEBS Lett. 170, 38-42], treatment of transfected COS-7 cells with PMA did not inhibit the ability of glucagon to increase intracellular cAMP levels. PMA-mediated inhibition was not conferred by treatment with okadaic acid, nor by co-transfecting cells with cDNAs encoding various protein kinase C isoforms (PKC-alpha, PKC-betaII and PKC-epsilon) or with the PMA-activated G-protein-receptor kinases GRK2 and GRK3. In contrast, PMA induced the marked inhibition of glucagon-stimulated cAMP production in COS-7 cells that had been co-transfected with a cDNA encoding protein kinase D (PKD). Such inhibition was not due to an action on the catalytic unit of adenylate cyclase, as forskolin-stimulated cAMP production was unchanged by PMA treatment of COS cells that had been co-transfected with both the glucagon receptor and PKD. PKD transcripts were detected in RNA isolated from hepatocytes but not from COS-7 cells. Transcripts for GRK2 were present in hepatocytes but not in COS cells, whereas transcripts for GRK3 were not found in either cell type. It is suggested that PKD may play a role in the regulation of glucagon-stimulated adenylate cyclase. PMID:9291130

  18. STRUBBELIG defines a receptor kinase-mediated signaling pathway regulating organ development in Arabidopsis

    PubMed Central

    Chevalier, David; Batoux, Martine; Fulton, Lynette; Pfister, Karen; Yadav, Ram Kishor; Schellenberg, Maja; Schneitz, Kay

    2005-01-01

    An open question remains as to what coordinates cell behavior during organogenesis, permitting organs to reach their appropriate size and shape. The Arabidopsis gene STRUBBELIG (SUB) defines a receptor-mediated signaling pathway in plants. SUB encodes a putative leucine-rich repeat transmembrane receptor-like kinase. The mutant sub phenotype suggests that SUB affects the formation and shape of several organs by influencing cell morphogenesis, the orientation of the division plane, and cell proliferation. Mutational analysis suggests that the kinase domain is important for SUB function. Biochemical assays using bacterially expressed fusion proteins indicate that the SUB kinase domain lacks enzymatic phosphotransfer activity. Furthermore, transgenes encoding WT and different mutant variants of SUB were tested for their ability to rescue the mutant sub phenotype. These genetic data also indicate that SUB carries a catalytically inactive kinase domain. The SUB receptor-like kinase may therefore signal in an atypical fashion. PMID:15951420

  19. Growth factor control of epidermal growth factor receptor kinase activity via an intramolecular mechanism.

    PubMed

    Koland, J G; Cerione, R A

    1988-02-15

    The mechanism by which the protein kinase activity of the epidermal growth factor (EGF) receptor is activated by binding of growth factor was investigated. Detergent-solubilized receptor in monomeric form was isolated by sucrose density gradient centrifugation and both its kinase and autophosphorylation activities monitored. In a low ionic strength medium and with MnCl2 as an activator, the activity of the monomeric receptor was EGF-independent. However, with 0.25 M ammonium sulfate present, the MnCl2-stimulated kinase activity was strikingly EGF-dependent. In contrast, the kinase activity expressed in the presence of MgCl2 showed growth factor control in the absence of added salt. Under the conditions of these experiments there was apparently little tendency for growth factor to induce aggregation of the receptor, indicating that the allosteric activation of the receptor kinase by EGF occurred via an intramolecular mechanism. Whereas detergent-solubilized receptor was the subject of these studies, the kinase activity of cell surface receptors might also be controlled by an intramolecular mechanism. These results indicate that an individual receptor molecule has the potential to function as a transmembrane signal transducer.

  20. Canine pulmonary adenocarcinoma tyrosine kinase receptor expression and phosphorylation

    PubMed Central

    2014-01-01

    Background This study evaluated tyrosine kinase receptor (TKR) expression and activation in canine pulmonary adenocarcinoma (cpAC) biospecimens. As histological similarities exist between human and cpAC, we hypothesized that cpACs will have increased TKR mRNA and protein expression as well as TKR phosphorylation. The molecular profile of cpAC has not been well characterized making the selection of therapeutic targets that would potentially have relevant biological activity impossible. Therefore, the objectives of this study were to define TKR expression and their phosphorylation state in cpAC as well as to evaluate the tumors for the presence of potential epidermal growth factor receptor (EGFR) tyrosine kinase activating mutations in exons 18–21. Immunohistochemistry (IHC) for TKR expression was performed using a tissue microarray (TMA) constructed from twelve canine tumors and companion normal lung samples. Staining intensities of the IHC were quantified by a veterinary pathologist as well as by two different digitalized algorithm image analyses software programs. An antibody array was used to evaluate TKR phosphorylation of the tumor relative to the TKR phosphorylation of normal tissues with the resulting spot intensities quantified using array analysis software. Each EGFR exon PCR product from all of the tumors and non-affected lung tissues were sequenced using sequencing chemistry and the sequencing reactions were run on automated sequencer. Sequence alignments were made to the National Center for Biotechnology Information canine EGFR reference sequence. Results The pro-angiogenic growth factor receptor, PDGFRα, had increased cpAC tumor mRNA, protein expression and phosphorylation when compared to the normal lung tissue biospecimens. Similar to human pulmonary adenocarcinoma, significant increases in cpAC tumor mRNA expression and receptor phosphorylation of the anaplastic lymphoma kinase (ALK) tyrosine receptor were present when compared to the

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

    PubMed

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

    1998-10-06

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

  2. A2A adenosine-receptor-mediated facilitation of noradrenaline release in rat tail artery involves protein kinase C activation and betagamma subunits formed after alpha2-adrenoceptor activation.

    PubMed

    Fresco, Paula; Oliveira, Jorge M A; Kunc, Filip; Soares, Ana Sofia; Rocha-Pereira, Carolina; Gonçalves, Jorge; Diniz, Carmen

    2007-07-01

    This work aimed to investigate the molecular mechanisms involved in the interaction of alpha2-adrenoceptors and adenosine A2A-receptor-mediated facilitation of noradrenaline release in rat tail artery, namely the type of G-protein involved in this effect and the step or steps where the signalling cascades triggered by alpha2-adrenoceptors and A2A-receptors interact. The selective adenosine A2A-receptor agonist 2-p-(2-carboxy ethyl) phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 100 nM) enhanced tritium overflow evoked by trains of 100 pulses at 5 Hz. This effect was abolished by the selective adenosine A2A-receptor antagonist 5-amino-7-(2-phenyl ethyl)-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo [1,5-c]pyrimidine (SCH 58261; 20 nM) and by yohimbine (1 microM). CGS 21680-mediated effects were also abolished by drugs that disrupted G(i/o)-protein coupling with receptors, PTX (2 microg/ml) or NEM (40 microM), by the anti-G(salpha) peptide (2 microg/ml) anti-G(betagamma) peptide (10 microg/ml) indicating coupling of A2A-receptors to G(salpha) and suggesting a crucial role for G(betagamma) subunits in the A(2A)-receptor-mediated enhancement of tritium overflow. Furthermore, phorbol 12-myristate 13-acetate (PMA; 1 microM) or forskolin (1 microM), direct activators of protein kinase C and of adenylyl cyclase, respectively, also enhanced tritium overflow. In addition, PMA-mediated effects were not observed in the presence of either yohimbine or PTX. Results indicate that facilitatory adenosine A2A-receptors couple to G(salpha) subunits which is essential, but not sufficient, for the release facilitation to occur, requiring the involvement of G(i/o)-protein coupling (it disappears after disruption of G(i/o)-protein coupling, PTX or NEM) and/or G(betagamma) subunits (anti-G(betagamma)). We propose a mechanism for the interaction in study suggesting group 2 AC isoforms as a plausible candidate for the interaction site, as these isoforms can integrate inputs from G

  3. The Dictyostelium Kinome—Analysis of the Protein Kinases from a Simple Model Organism

    PubMed Central

    Liu, Allen; Fey, Petra; Pilcher, Karen E; Xu, Yanji; Smith, Janet L

    2006-01-01

    Dictyostelium discoideum is a widely studied model organism with both unicellular and multicellular forms in its developmental cycle. The Dictyostelium genome encodes 285 predicted protein kinases, similar to the count of the much more advanced Drosophila. It contains members of most kinase classes shared by fungi and metazoans, as well as many previously thought to be metazoan specific, indicating that they have been secondarily lost from the fungal lineage. This includes the entire tyrosine kinase–like (TKL) group, which is expanded in Dictyostelium and includes several novel receptor kinases. Dictyostelium lacks tyrosine kinase group kinases, and most tyrosine phosphorylation appears to be mediated by TKL kinases. About half of Dictyostelium kinases occur in subfamilies not present in yeast or metazoa, suggesting that protein kinases have played key roles in the adaptation of Dictyostelium to its habitat. This study offers insights into kinase evolution and provides a focus for signaling analysis in this system. PMID:16596165

  4. 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.

  5. Overexpression of the tomato pollen receptor kinase LePRK1 rewires pollen tube growth to a blebbling mode

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The tubular growth of a pollen tube cell is crucial for the sexual reproduction of flowering plants. LePRK1 is a pollen-specific and plasma membrane–localized receptor-like kinase from tomato (Solanum lycopersicum). LePRK1 interacts with another receptor, LePRK2, and with KINASE PARTNER PROTEIN (KPP...

  6. Mitogen-activated protein kinases regulate expression of neuronal nitric oxide synthase and neurite outgrowth via non-classical retinoic acid receptor signaling in human neuroblastoma SH-SY5Y cells.

    PubMed

    Fujibayashi, Tatsuya; Kurauchi, Yuki; Hisatsune, Akinori; Seki, Takahiro; Shudo, Koichi; Katsuki, Hiroshi

    2015-10-01

    We have previously shown that retinoic acid receptor (RAR) stimulation by an agonist Am80 recruits nitric oxide-dependent signaling via increased expression of neuronal nitric oxide synthase (nNOS) in rat midbrain slice cultures. Using neuroblastoma SH-SY5Y cells, here we investigated the mechanisms of RAR-induced nNOS expression, together with relationship between nNOS expression and neurite outgrowth. Am80 promoted neurite outgrowth, which was attenuated by inhibitors of phosphoinositide 3-kinase (PI3K; LY294002), c-Jun N-terminal kinase (JNK; SP600125) and p38 mitogen-activated protein kinase (p38 MAPK; SB203580). A selective nNOS inhibitor 3-bromo-nitroindazole also suppressed Am80-induced neurite outgrowth. Am80-induced increase in nNOS protein expression was attenuated by LY294002, SP600125 and SB203580, whereas increase in nNOS mRNA expression was attenuated only by LY294002. Am80-induced activation of JNK and p38 MAPK was blocked by LY294002, suggesting that these kinases acted downstream of PI3K. We also confirmed that DAX1, a nuclear receptor reported to regulate nNOS expression, was up-regulated in response to Am80. siRNA-mediated knockdown of DAX1 abrogated Am80-induced nNOS expression and neurite outgrowth. These results reveal for the first time that nNOS expression is crucial for RAR-mediated neurite outgrowth, and that non-genomic signaling such as JNK and p38 MAPK is involved in RAR-mediated nNOS expression.

  7. p38 mitogen-activated protein kinase activates eNOS in endothelial cells by an estrogen receptor alpha-dependent pathway in response to black tea polyphenols.

    PubMed

    Anter, Elad; Chen, Kai; Shapira, Oz M; Karas, Richard H; Keaney, John F

    2005-05-27

    Black tea has been shown to improve endothelial function in patients with coronary artery disease and recent data indicate the polyphenol fraction of black tea enhances endothelial nitric oxide synthase (eNOS) activity through p38 MAP kinase (p38 MAPK) activation. Because the mechanisms for this phenomenon are not yet clear, we sought to elucidate the signaling events in response to black tea polyphenols. Bovine aortic endothelial cells (BAECs) exposed to black tea polyphenols demonstrated eNOS activation that was inhibited by the estrogen receptor (ER) antagonist ICI 182,780, and siRNA-mediated silencing of ER expression. Consistent with this observation, black tea polyphenols induced time-dependent phosphorylation of ERalpha on Ser-118 that was inhibited by ICI 182,780. Phosphorylation of ERalpha on Ser-118 was due to p38 MAP kinase (p38 MAPK) as, it was inhibited by SB203580 and overexpression of dominant-negative p38alpha MAPK. Conversely, constitutively active MKK6 induced p38 MAPK activation that recapitulated the effects of polyphenols by inducing ERalpha phosphorylation and downstream activation of Akt, and eNOS. The key role of ERalpha Ser-118 phosphorylation was confirmed in eNOS-transfected COS-7 cells, as polyphenol-induced eNOS activation required cotransfection with ERalpha subject to phosphorylation at Ser-118. This residue appeared critical for functional association of ERalpha with p38 MAPK as ERalpha with Ser-118 mutated to alanine could not form a complex with p38 MAPK. These findings suggest p38 MAP kinase-mediated eNOS activation requires ERalpha and these data uncover a new mechanism of ERalpha activation that has broad implications for NO bioactivity and endothelial cell phenotype.

  8. Dynamics driven allostery in protein kinases

    PubMed Central

    Kornev, Alexandr P.; Taylor, Susan S.

    2015-01-01

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

  9. The angiotensin receptor type 1-Gq protein-phosphatidyl inositol phospholipase Cbeta-protein kinase C pathway is involved in activation of proximal tubule Na+-ATPase activity by angiotensin(1-7) in pig kidneys.

    PubMed

    Lara, Lucienne S; Correa, Juliana S; Lavelle, Anouchka B; Lopes, Anibal G; Caruso-Neves, Celso

    2008-05-01

    In a previous study, we observed that angiotensin(1-7) (Ang(1-7)) stimulates proximal tubule Na+-ATPase activity through the angiotensin receptor type 1 (AT1R). Here we aimed to study the signalling pathways involved. Our results show that the stimulatory effect of Ang(1-7) on Na+-ATPase activity through AT1R involves a Gq protein-phosphatidyl inositol-phospholipase Cbeta (PI-PLCbeta) pathway because: (1) the effect was reversed by GDPbetaS, a non-hydrolysable GDP analogue, and by a monoclonal Gq protein antibody; (2) the effect was similar and not additive to that of GTPgammaS, a non-hydrolysable GTP analogue; (3) Ang(1-7) induced a rapid decrease (30 s) in phosphatidylinositol 4,5-bisphosphate levels, a PI-PLCbeta substrate; and (4) U73122, a specific inhibitor of PI-PLCbeta, abolished Ang(1-7)-induced stimulation of Na+-ATPase activity. Angiotensin(1-7) increased the protein kinase C (PKC) activity similarly to phorbol-12-myristate-13-acetate (PMA), an activator of PKC. This effect was reversed by losartan, a specific antagonist of AT1R. The stimulatory effects of Ang(1-7) and PMA on Na+-ATPase activity are similar, non-additive and reversed by calphostin C, a specific inhibitor of PKC. A catalytic subunit of PKC (PKC-M) increased the Na+-ATPase activity. These data show that Ang(1-7) stimulates Na+-ATPase activity through the AT1R-Gq protein-PI-PLCbeta-PKC pathway. This effect is similar to that described for angiotensin II, showing for the first time that these compounds could have similar effects in the renal system.

  10. Hemoglobin receptor protein from Porphyromonas gingivalis induces interleukin-8 production in human gingival epithelial cells through stimulation of the mitogen-activated protein kinase and NF-κB signal transduction pathways.

    PubMed

    Fujita, Yuki; Nakayama, Masaaki; Naito, Mariko; Yamachika, Eiki; Inoue, Tetsuyoshi; Nakayama, Koji; Iida, Seiji; Ohara, Naoya

    2014-01-01

    Periodontitis is an inflammatory disease of polymicrobial origin affecting the tissues supporting the tooth. The oral anaerobic bacterium Porphyromonas gingivalis, which is implicated as an important pathogen for chronic periodontitis, triggers a series of host inflammatory responses that promote the destruction of periodontal tissues. Among the virulence factors of P. gingivalis, hemoglobin receptor protein (HbR) is a major protein found in culture supernatants. In this study, we investigated the roles of HbR in the production of inflammatory mediators. We found that HbR induced interleukin-8 (IL-8) production in the human gingival epithelial cell line Ca9-22. p38 mitogen-activated protein kinase (MAPK) and extracellular signal-related kinase 1/2 (Erk1/2) were activated in HbR-stimulated Ca9-22 cells. Inhibitors of p38 MAPK (SB203580) and Erk1/2 (PD98059) blocked HbR-induced IL-8 production. Additionally, HbR stimulated the translocation of NF-κB-p65 to the nucleus, consistent with enhancement of IL-8 expression by activation of the NF-κB pathway. In addition, small interfering RNA (siRNA) targeting activating transcription factor 2 (ATF-2) or cyclic AMP-response element-binding protein (CREB) inhibited HbR-induced IL-8 production. Moreover, pretreatment with SB203580 and PD98059 reduced HbR-induced phosphorylation of CREB and ATF-2, respectively. Combined pretreatment with an inhibitor of NF-κB (BAY11-7082) and SB203580 was more efficient in inhibiting the ability of HbR to induce IL-8 production than pretreatment with either BAY11-7082 or SB203580 alone. Thus, in Ca9-22 cells, the direct activation of p38 MAPK and Erk1/2 by HbR caused the activation of the transcription factors ATF-2, CREB, and NF-κB, thus resulting in the induction of IL-8 production.

  11. 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.

  12. Arabidopsis Receptor of Activated C Kinase1 Phosphorylation by WITH NO LYSINE8 KINASE

    DOE PAGES

    Urano, Daisuke; Czarnecki, Olaf; Wang, Xiaoping; ...

    2014-12-08

    Receptor of activated C kinase1 (RACK1) is a versatile scaffold protein that binds to numerous proteins to regulate diverse cellular pathways in mammals. In Arabidopsis (Arabidopsis thaliana), RACK1 has been shown to regulate plant hormone signaling, stress responses, and multiple processes of growth and development. However, little is known about the molecular mechanism underlying these regulations. In this paper, we show that an atypical serine (Ser)/threonine (Thr) protein kinase, WITH NO LYSINE8 (WNK8), phosphorylates RACK1. WNK8 physically interacted with and phosphorylated RACK1 proteins at two residues: Ser-122 and Thr-162. Genetic epistasis analysis of rack1 wnk8 double mutants indicated that RACK1more » acts downstream of WNK8 in the glucose responsiveness and flowering pathways. The phosphorylation-dead form, RACK1AS122A/T162A, but not the phosphomimetic form, RACK1AS122D/T162E, rescued the rack1a null mutant, implying that phosphorylation at Ser-122 and Thr-162 negatively regulates RACK1A function. The transcript of RACK1AS122D/T162E accumulated at similar levels as those of RACK1S122A/T162A. However, although the steady-state level of the RACK1AS122A/T162A protein was similar to wild-type RACK1A protein, the RACK1AS122D/T162E protein was nearly undetectable, suggesting that phosphorylation affects the stability of RACK1A proteins. In conclusion, these results suggest that RACK1 is phosphorylated by WNK8 and that phosphorylation negatively regulates RACK1 function by influencing its protein stability.« less

  13. Arabidopsis Receptor of Activated C Kinase1 Phosphorylation by WITH NO LYSINE8 KINASE

    SciTech Connect

    Urano, Daisuke; Czarnecki, Olaf; Wang, Xiaoping; Jones, Alan M.; Chen, Jin-Gui

    2014-12-08

    Receptor of activated C kinase1 (RACK1) is a versatile scaffold protein that binds to numerous proteins to regulate diverse cellular pathways in mammals. In Arabidopsis (Arabidopsis thaliana), RACK1 has been shown to regulate plant hormone signaling, stress responses, and multiple processes of growth and development. However, little is known about the molecular mechanism underlying these regulations. In this paper, we show that an atypical serine (Ser)/threonine (Thr) protein kinase, WITH NO LYSINE8 (WNK8), phosphorylates RACK1. WNK8 physically interacted with and phosphorylated RACK1 proteins at two residues: Ser-122 and Thr-162. Genetic epistasis analysis of rack1 wnk8 double mutants indicated that RACK1 acts downstream of WNK8 in the glucose responsiveness and flowering pathways. The phosphorylation-dead form, RACK1AS122A/T162A, but not the phosphomimetic form, RACK1AS122D/T162E, rescued the rack1a null mutant, implying that phosphorylation at Ser-122 and Thr-162 negatively regulates RACK1A function. The transcript of RACK1AS122D/T162E accumulated at similar levels as those of RACK1S122A/T162A. However, although the steady-state level of the RACK1AS122A/T162A protein was similar to wild-type RACK1A protein, the RACK1AS122D/T162E protein was nearly undetectable, suggesting that phosphorylation affects the stability of RACK1A proteins. In conclusion, these results suggest that RACK1 is phosphorylated by WNK8 and that phosphorylation negatively regulates RACK1 function by influencing its protein stability.

  14. Arabidopsis Receptor of Activated C Kinase1 Phosphorylation by WITH NO LYSINE8 KINASE1[OPEN

    PubMed Central

    Urano, Daisuke; Czarnecki, Olaf; Wang, Xiaoping; Chen, Jin-Gui

    2015-01-01

    Receptor of activated C kinase1 (RACK1) is a versatile scaffold protein that binds to numerous proteins to regulate diverse cellular pathways in mammals. In Arabidopsis (Arabidopsis thaliana), RACK1 has been shown to regulate plant hormone signaling, stress responses, and multiple processes of growth and development. However, little is known about the molecular mechanism underlying these regulations. Here, we show that an atypical serine (Ser)/threonine (Thr) protein kinase, WITH NO LYSINE8 (WNK8), phosphorylates RACK1. WNK8 physically interacted with and phosphorylated RACK1 proteins at two residues: Ser-122 and Thr-162. Genetic epistasis analysis of rack1 wnk8 double mutants indicated that RACK1 acts downstream of WNK8 in the glucose responsiveness and flowering pathways. The phosphorylation-dead form, RACK1AS122A/T162A, but not the phosphomimetic form, RACK1AS122D/T162E, rescued the rack1a null mutant, implying that phosphorylation at Ser-122 and Thr-162 negatively regulates RACK1A function. The transcript of RACK1AS122D/T162E accumulated at similar levels as those of RACK1S122A/T162A. However, although the steady-state level of the RACK1AS122A/T162A protein was similar to wild-type RACK1A protein, the RACK1AS122D/T162E protein was nearly undetectable, suggesting that phosphorylation affects the stability of RACK1A proteins. Taken together, these results suggest that RACK1 is phosphorylated by WNK8 and that phosphorylation negatively regulates RACK1 function by influencing its protein stability. PMID:25489024

  15. Arabidopsis receptor of activated C kinase1 phosphorylation by WITH NO LYSINE8 KINASE.

    PubMed

    Urano, Daisuke; Czarnecki, Olaf; Wang, Xiaoping; Jones, Alan M; Chen, Jin-Gui

    2015-02-01

    Receptor of activated C kinase1 (RACK1) is a versatile scaffold protein that binds to numerous proteins to regulate diverse cellular pathways in mammals. In Arabidopsis (Arabidopsis thaliana), RACK1 has been shown to regulate plant hormone signaling, stress responses, and multiple processes of growth and development. However, little is known about the molecular mechanism underlying these regulations. Here, we show that an atypical serine (Ser)/threonine (Thr) protein kinase, WITH NO LYSINE8 (WNK8), phosphorylates RACK1. WNK8 physically interacted with and phosphorylated RACK1 proteins at two residues: Ser-122 and Thr-162. Genetic epistasis analysis of rack1 wnk8 double mutants indicated that RACK1 acts downstream of WNK8 in the glucose responsiveness and flowering pathways. The phosphorylation-dead form, RACK1A(S122A/T162A), but not the phosphomimetic form, RACK1A(S122D/T162E), rescued the rack1a null mutant, implying that phosphorylation at Ser-122 and Thr-162 negatively regulates RACK1A function. The transcript of RACK1A(S122D/T162E) accumulated at similar levels as those of RACK1(S122A/T162A). However, although the steady-state level of the RACK1A(S122A/T162A) protein was similar to wild-type RACK1A protein, the RACK1A(S122D/T162E) protein was nearly undetectable, suggesting that phosphorylation affects the stability of RACK1A proteins. Taken together, these results suggest that RACK1 is phosphorylated by WNK8 and that phosphorylation negatively regulates RACK1 function by influencing its protein stability.

  16. Activation of phosphoinositide 3-kinase by D2 receptor prevents apoptosis in dopaminergic cell lines.

    PubMed

    Nair, Venugopalan D; Olanow, C Warren; Sealfon, Stuart C

    2003-07-01

    Whereas dopamine agonists are known to provide symptomatic benefits for Parkinson's disease, recent clinical trials suggest that they might also be neuroprotective. Laboratory studies demonstrate that dopamine agonists can provide neuroprotective effects in a number of model systems, but the role of receptor-mediated signalling in these effects is controversial. We find that dopamine agonists have robust, concentration-dependent anti-apoptotic activity in PC12 cells that stably express human D(2L) receptors from cell death due to H(2)O(2) or trophic withdrawal and that the protective effects are abolished in the presence of D(2)-receptor antagonists. D(2) agonists are also neuroprotective in the nigral dopamine cell line SN4741, which express endogenous D(2) receptors, whereas no anti-apoptotic activity is observed in native PC12 cells, which do not express detectable D(2) receptors. Notably, the agonists studied differ in their relative efficacy to mediate anti-apoptotic effects and in their capacity to stimulate [(35)S]guanosine 5'-[gamma-thio]triphosphate ([(35)S]GTP[S]) binding, an indicator of G-protein activation. Studies with inhibitors of phosphoinositide 3-kinase (PI 3-kinase), extracellular-signal-regulated kinase or p38 mitogen-activated protein kinase indicate that the PI 3-kinase pathway is required for D(2) receptor-mediated cell survival. These studies indicate that certain dopamine agonists can complex with D(2) receptors to preferentially transactivate neuroprotective signalling pathways and to mediate increased cell survival.

  17. 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

  18. Catalytically defective receptor protein tyrosine kinase PTK7 enhances invasive phenotype by inducing MMP-9 through activation of AP-1 and NF-κB in esophageal squamous cell carcinoma cells

    PubMed Central

    Shin, Won-Sik; Hong, Yuri; Lee, Hae Won; Lee, Seung-Taek

    2016-01-01

    Protein tyrosine kinase 7 (PTK7), a member of the catalytically defective receptor protein tyrosine kinase family, is upregulated in various cancers including esophageal squamous cell carcinoma (ESCC). Here, we have explored the molecular mechanism of PTK7-dependent invasiveness in ESCC cells. PTK7 knockdown reduced gelatin degradation and MMP-9 secretion in cultures of ESCC TE-10 cells, and showed reduced levels of MMP9 mRNA using real-time RT-PCR and luciferase reporter assays. PTK7 knockdown decreased not only phosphorylation of NF-κB, IκB, ERK, and JNK, but also nuclear localization of NF-κB and AP-1 consisting of c-Fos and c-Jun. Activation of AP-1 and NF-κB requires PTK7-mediated activation of tyrosine kinases, including Src. In addition, NF-κB activation by PTK7 involves the PI3K/Akt signaling pathway. PTK7-mediated upregulation of MMP9 was also observed in other ESCC cell lines and in three-dimensional cultures of TE-10 cells. Moreover, MMP-9 expression positively correlated with PTK7 expression in ESCC tumor tissue. These findings demonstrate that PTK7 upregulates MMP9 through activation of AP-1 and NF-κB and, thus increases invasive properties of ESCC cells. PMID:27689325

  19. 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

  20. The S-locus receptor kinase gene in a self-incompatible Brassica napus line encodes a functional serine/threonine kinase.

    PubMed Central

    Goring, D R; Rothstein, S J

    1992-01-01

    An S-receptor kinase (SRK) cDNA, SRK-910, from the active S-locus in a self-incompatible Brassica napus W1 line has been isolated and characterized. The SRK-910 gene is predominantly expressed in pistils and segregates with the W1 self-incompatibility phenotype in an F2 population derived from a cross between the self-incompatible W1 line and a self-compatible Westar line. Analysis of the predicted amino acid sequence demonstrated that the extracellular receptor domain is highly homologous to S-locus glycoproteins, whereas the cytoplasmic kinase domain contains conserved amino acids present in serine/threonine kinases. An SRK-910 kinase protein fusion was produced in Escherichia coli and found to contain kinase activity. Phosphoamino acid analysis confirmed that only serine and threonine residues were phosphorylated. Thus, the SRK-910 gene encodes a functional serine/threonine receptor kinase. PMID:1332796

  1. Targeting protein kinase A in cancer therapy: an update

    PubMed Central

    Sapio, Luigi; Di Maiolo, Francesca; Illiano, Michela; Esposito, Antonietta; Chiosi, Emilio; Spina, Annamaria; Naviglio, Silvio

    2014-01-01

    Protein Kinase A (PKA) is a well known member of the serine-threonin protein kinase superfamily. PKA, also known as cAMP-dependent protein kinase, is a multi-unit protein kinase that mediates signal transduction of G-protein coupled receptors through its activation upon cAMP binding. The widespread expression of PKA subunit genes, and the myriad of mechanisms by which cAMP is regulated within a cell suggest that PKA signaling is one of extreme importance to cellular function. It is involved in the control of a wide variety of cellular processes from metabolism to ion channel activation, cell growth and differentiation, gene expression and apoptosis. Importantly, since it has been implicated in the initiation and progression of many tumors, PKA has been proposed as a novel biomarker for cancer detection, and as a potential molecular target for cancer therapy. Here, we highlight some features of cAMP/PKA signaling that are relevant to cancer biology and present an update on targeting PKA in cancer therapy. PMID:26417307

  2. GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis

    PubMed Central

    Wrzaczek, Michael; Vainonen, Julia P; Stael, Simon; Tsiatsiani, Liana; Help-Rinta-Rahko, Hanna; Gauthier, Adrien; Kaufholdt, David; Bollhöner, Benjamin; Lamminmäki, Airi; Staes, An; Gevaert, Kris; Tuominen, Hannele; Van Breusegem, Frank; Helariutta, Ykä; Kangasjärvi, Jaakko

    2015-01-01

    Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor. PMID:25398910

  3. GRIM REAPER peptide binds to receptor kinase PRK5 to trigger cell death in Arabidopsis.

    PubMed

    Wrzaczek, Michael; Vainonen, Julia P; Stael, Simon; Tsiatsiani, Liana; Help-Rinta-Rahko, Hanna; Gauthier, Adrien; Kaufholdt, David; Bollhöner, Benjamin; Lamminmäki, Airi; Staes, An; Gevaert, Kris; Tuominen, Hannele; Van Breusegem, Frank; Helariutta, Ykä; Kangasjärvi, Jaakko

    2015-01-02

    Recognition of extracellular peptides by plasma membrane-localized receptor proteins is commonly used in signal transduction. In plants, very little is known about how extracellular peptides are processed and activated in order to allow recognition by receptors. Here, we show that induction of cell death in planta by a secreted plant protein GRIM REAPER (GRI) is dependent on the activity of the type II metacaspase METACASPASE-9. GRI is cleaved by METACASPASE-9 in vitro resulting in the release of an 11 amino acid peptide. This peptide bound in vivo to the extracellular domain of the plasma membrane-localized, atypical leucine-rich repeat receptor-like kinase POLLEN-SPECIFIC RECEPTOR-LIKE KINASE 5 (PRK5) and was sufficient to induce oxidative stress/ROS-dependent cell death. This shows a signaling pathway in plants from processing and activation of an extracellular protein to recognition by its receptor.

  4. Characterization of serine 916 as an in vivo autophosphorylation site for protein kinase D/Protein kinase Cmu.

    PubMed

    Matthews, S A; Rozengurt, E; Cantrell, D

    1999-09-10

    Activation of the serine kinase protein kinase D (PKD)/PKCmicro is controlled by the phosphorylation of two serine residues within its activation loop via a PKC-dependent signaling cascade. In this study we have identified the C-terminal serine 916 residue as an in vivo phosphorylation site within active PKD/PKCmu. An antibody that recognized PKD/PKCmu proteins specifically phosphorylated on the serine 916 residue was generated and used to show that phosphorylation of Ser-916 is induced by phorbol ester treatment of cells. Thus, the pS916 antibody is a useful tool to study the regulation of PKD/PKCmu activity in vivo. Antigen receptor ligation of T and B lymphocytes also induced phosphorylation of the serine 916 residue of PKD/PKCmu. Furthermore the regulatory FcgammaRIIB receptor, which mediates vital negative feedback signals to the B cell antigen receptor complex, inhibited the antigen receptor-induced activation and serine 916 phosphorylation of PKD/PKCmu. The degree of serine 916 phosphorylation during lymphocyte activation and inhibition exactly correlated with the activation status of PKD/PKCmu. Moreover, using different mutants of PKD/PKCmu, we show that serine 916 is not trans-phosphorylated by an upstream kinase but is rather an autophosphorylation event that occurs following activation of PKD/PKCmu.

  5. Regulation of the laminin beta 1 (LAMB1), retinoic acid receptor beta, and bone morphogenetic protein 2 genes in mutant F9 teratocarcinoma cell lines partially deficient in cyclic AMP-dependent protein kinase activity.

    PubMed

    Shen, J; Li, C; Gudas, L J

    1997-12-01

    We stably transfected a gene encoding a dominant negative regulatory subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA) into F9 cells and generated cell lines partially deficient in PKA activity (DN16 and DN19). In these cell lines, the retinoic acid (RA) receptor beta and laminin beta(1) chain (LAMB1) genes were regulated normally by RA alone, indicating that in the absence of exogenous modulation of cAMP levels, the PKA signaling pathway does not seem to play a major role in the RA-associated regulation of these genes. However, alterations in gene regulation were observed when the mutant cell lines were treated with a combination of RA and cAMP analogues. Moreover, in the DN16 cell line, which exhibits the lowest PKA activity among the mutant cell lines [22% of wild type (WT) at 1 microM cAMP], there was a significant decrease in the cAMP-associated activation of the LAMB1 gene DNase I hypersensitivity site 2 enhancer, as measured by chloramphenicol acetyl transferase assays. Using electrophoretic mobility shift assays, less protein binding was observed at one of the motifs (C2) within this enhancer region in the DN16 cells as compared to the F9 WT cells after treatment of the cells with RA and cAMP analogues for 24 h. Furthermore, no increase in C2 binding was observed when extracts from RA-treated F9 ST or DN16 cells were subjected to in vitro phosphorylation, suggesting that PKA is involved in the induction of the C2-binding protein in RA-treated cells. In contrast to the results with RA receptor beta and LAMB1, the effects of cAMP analogues on the RA-associated regulation of the bone morphogenetic protein 2 gene were not altered in the cell lines that exhibited reduced PKA activity. These results suggest that a partial reduction in PKA activity is not sufficient to abrogate the effects of cAMP analogues on all of the genes regulated by RA.

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

    PubMed

    Hardie, D G; MacKintosh, R W

    1992-10-01

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

  7. Myocardial ischemia-reperfusion enhances transcriptional expression of endothelin-1 and vasoconstrictor ETB receptors via the protein kinase MEK-ERK1/2 signaling pathway in rat

    PubMed Central

    Kruse, Lars Schack; Berchtold, Lukas Adrian; Grell, Anne-Sofie; Warfvinge, Karin; Edvinsson, Lars

    2017-01-01

    Background Coronary artery remodelling and vasospasm is a complication of acute myocardial ischemia and reperfusion. The underlying mechanisms are complex, but the vasoconstrictor peptide endothelin-1 is suggested to have an important role. This study aimed to determine whether the expression of endothelin-1 and its receptors are regulated in the myocardium and in coronary arteries after experimental ischemia-reperfusion. Furthermore, we evaluated whether treatment with a specific MEK1/2 inhibitor, U0126, modified the expression and function of these proteins. Methods and findings Sprague-Dawley rats were randomly divided into three groups: sham-operated, ischemia-reperfusion with vehicle treatment and ischemia-reperfusion with U0126 treatment. Ischemia was induced by ligating the left anterior descending coronary artery for 30 minutes followed by reperfusion. U0126 was administered before ischemia and repeated 6 hours after start of reperfusion. The contractile properties of isolated coronary arteries to endothelin-1 and sarafotoxin 6c were evaluated using wire-myography. The gene expression of endothelin-1 and endothelin receptors were measured using qPCR. Distribution and localization of proteins (pERK1/2, prepro-endothelin-1, endothelin-1, and endothelin ETA and ETB receptors) were analysed by Western blot and immunohistochemistry. We found that pERK1/2 was significantly augmented in the ischemic area 3 hours after ischemia-reperfusion; this correlated with increased ETB receptor and ET-1 gene expressions in ischemic myocardium and in coronary arteries. ETB receptor-mediated vasoconstriction was observed to be increased in coronary arteries 24 hours after ischemia-reperfusion. Treatment with U0126 reduced pERK1/2, expression of ET-1 and ETB receptor, and ETB receptor-mediated vasoconstriction. Conclusions These findings suggest that the MEK-ERK1/2 signaling pathway is important for regulating endothelin-1 and ETB receptors in myocardium and coronary arteries

  8. GK4, a G-protein-coupled receptor with a phosphatidylinositol phosphate kinase domain in Phytophthora infestans, is involved in sporangia development and virulence.

    PubMed

    Hua, Chenlei; Meijer, Harold J G; de Keijzer, Jeroen; Zhao, Wei; Wang, Yuanchao; Govers, Francine

    2013-04-01

    For dispersal and host infection plant pathogens largely depend on asexual spores. Pathogenesis and sporulation are complex processes that are governed by cellular signalling networks including G-protein and phospholipid signalling. Oomycetes possess a family of novel proteins called GPCR-PIPKs (GKs) that are composed of a seven-transmembrane spanning (7-TM) domain fused to a phosphatidylinositol phosphate kinase (PIPK) domain. Based on this domain structure GKs are anticipated to link G-protein and phospholipid signal pathways; however, their functions are currently unknown. Expression analyses of the 12 GK genes in Phytophthora infestans and their orthologues in Phytophthora sojae, revealed differential expression during asexual development. PiGK1 and PiGK4 were fused to monomeric red fluorescent protein (mRFP) and ectopically expressed in P. infestans. In growing hyphae different subcellular distribution patterns were observed indicating that these two GKs act independently during development. We focused on the functional analyses of PiGK4. Its localization suggested involvement in cell differentiation and elongation and its 7-TM domain showed a canonical GPCR membrane topology. Silencing of GK4 and overexpression of full-length and truncated constructs in P. infestans revealed that PiGK4 is not only involved in spore germination and hyphal elongation but also in sporangia cleavage and infection.

  9. Regulation of polar auxin transport by protein and lipid kinases

    P