Science.gov

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. Protein kinase C directly phosphorylates the insulin receptor in vitro and reduces its protein-tyrosine kinase activity.

    PubMed Central

    Bollag, G E; Roth, R A; Beaudoin, J; Mochly-Rosen, D; Koshland, D E

    1986-01-01

    The beta subunit of purified insulin receptor is phosphorylated on a serine residue by purified preparations of protein kinase C (ATP: protein phosphotransferase, EC 2.7.1.37). This phosphorylation is inhibited by antibodies to protein kinase C and stimulated by phospholipids, diacylglycerol, and Ca2+. The phosphorylation of the receptor by protein kinase C does not affect its insulin-binding activity but does inhibit by 65% the receptor's intrinsic tyrosine-specific protein kinase activity (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112). These results indicate that activators of protein kinase C, such as phorbol esters, desensitize cells to insulin by direct protein kinase C action on the insulin receptor. Images PMID:3526339

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

    PubMed

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

    2008-09-12

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

  5. 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

  6. Activation of GABA(B) receptors inhibits protein kinase B/glycogen synthase kinase 3 signaling.

    PubMed

    Lu, Frances Fangjia; Su, Ping; Liu, Fang; Daskalakis, Zafiris J

    2012-11-28

    Accumulated evidence has suggested that potentiation of cortical GABAergic inhibitory neurotransmission may be a key mechanism in the treatment of schizophrenia. However, the downstream molecular mechanisms related to GABA potentiation remain unexplored. Recent studies have suggested that dopamine D2 receptor antagonists, which are used in the clinical treatment of schizophrenia, modulate protein kinase B (Akt)/glycogen synthase kinase (GSK)-3 signaling. Here we report that activation of GABA(B) receptors significantly inhibits Akt/GSK-3 signaling in a β-arrestin-dependent pathway. Agonist stimulation of GABA(B) receptors enhances the phosphorylation of Akt (Thr-308) and enhances the phosphorylation of GSK-3α (Ser-21)/β (Ser-9) in both HEK-293T cells expressing GABA(B) receptors and rat hippocampal slices. Furthermore, knocking down the expression of β-arrestin2 using siRNA abolishes the GABA(B) receptor-mediated modulation of GSK-3 signaling. Our data may help to identify potentially novel targets through which GABA(B) receptor agents may exert therapeutic effects in the treatment of schizophrenia.

  7. 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

  8. Transduction proteins of olfactory receptor cells: identification of guanine nucleotide binding proteins and protein kinase C

    SciTech Connect

    Anholt, R.R.H.; Mumby, S.M.; Stoffers, D.A.; Girard, P.R.; Kuo, J.F.; Snyder, S.H.

    1987-02-10

    The authors have analyzed guanine nucleotide binding proteins (G-proteins) in the olfactory epithelium of Rana catesbeiana using subunit-specific antisera. The olfactory epithelium contained the ..cap alpha.. subunits of three G-proteins, migrating on polyacrylamide gels in SDS with apparent molecular weights of 45,000, 42,000, and 40,000, corresponding to G/sub s/, G/sub i/, and G/sub o/, respectively. A single ..beta.. subunit with an apparent molecular weight of 36,000 was detected. An antiserum against the ..cap alpha.. subunit of retinal transducin failed to detect immunoreactive proteins in olfactory cilia detached from the epithelium. The olfactory cilia appeared to be enriched in immunoreactive G/sub s..cap alpha../ relative to G/sub ichemically bond/ and G/sub ochemically bond/ when compared to membranes prepared from the olfactory epithelium after detachment of the cilia. Bound antibody was detected by autoradiography after incubation with (/sup 125/I)protein. Immunohistochemical studies using an antiserum against the ..beta.. subunit of G-proteins revealed intense staining of the ciliary surface of the olfactory epithelium and of the axon bundles in the lamina propria. In contrast, an antiserum against a common sequence of the ..cap alpha.. subunits preferentially stained the cell membranes of the olfactory receptor cells and the acinar cells of Bowman's glands and the deep submucosal glands. In addition to G-proteins, they have identified protein kinase C in olfactory cilia via a protein kinase C specific antiserum and via phorbol ester binding. However, in contrast to the G-proteins, protein kinase C occurred also in cilia isolated from respiratory epithelium.

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

    SciTech Connect

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

    1991-03-15

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

  10. Protein kinase C does not phosphorylate the externalized form of the transferrin receptor.

    PubMed Central

    Adam, M A; Johnstone, R M

    1987-01-01

    We have investigated the phosphorylation of transferrin receptors both in intact sheep reticulocytes and in isolated plasma membranes. Phosphorylation of the receptor in intact cells or isolated plasma membranes is stimulated by phorbol diesters, suggesting that protein kinase C may be involved. Identical [32P] phosphopeptide tryptic maps are formed in the presence and absence of phorbol diesters. Using heat-treated membranes (which are devoid of endogenous kinase activity) exogenous protein kinase C phosphorylates the same peptides as the endogenous kinase(s). During maturation of reticulocytes to erythrocytes, the transferrin receptor is released to the medium in vesicular form. In cells labelled with [32P]Pi, the released receptor is not labelled with 32P and the exocytosed vesicles do not phosphorylate receptor with [gamma-32P]ATP. The absence of 32P in the released receptor appears to be due to a change in the receptor, since, even in the presence of exogenous protein kinase C, the exocytosed receptor is phosphorylated to approximately 8% of the level obtained with receptors from the plasma membrane. These data suggest that during maturation and externalization the receptor is altered so that it loses its capacity to act as a substrate for exogenous protein kinase C as well as the endogenous kinase(s). This change may be a signal which segregates the receptor for externalization from the receptor pool remaining for transferrin recycling during the final stages of red cell maturation. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:3593234

  11. 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

  12. Molecular basis for activation of G protein-coupled receptor kinases

    SciTech Connect

    Boguth, Cassandra A.; Singh, Puja; Huang, Chih-chin; Tesmer, John J.G.

    2012-03-16

    G protein-coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N-terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N-terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.

  13. Competing G protein-coupled receptor kinases balance G protein and β-arrestin signaling.

    PubMed

    Heitzler, Domitille; Durand, Guillaume; Gallay, Nathalie; Rizk, Aurélien; Ahn, Seungkirl; Kim, Jihee; Violin, Jonathan D; Dupuy, Laurence; Gauthier, Christophe; Piketty, Vincent; Crépieux, Pascale; Poupon, Anne; Clément, Frédérique; Fages, François; Lefkowitz, Robert J; Reiter, Eric

    2012-01-01

    Seven-transmembrane receptors (7TMRs) are involved in nearly all aspects of chemical communications and represent major drug targets. 7TMRs transmit their signals not only via heterotrimeric G proteins but also through β-arrestins, whose recruitment to the activated receptor is regulated by G protein-coupled receptor kinases (GRKs). In this paper, we combined experimental approaches with computational modeling to decipher the molecular mechanisms as well as the hidden dynamics governing extracellular signal-regulated kinase (ERK) activation by the angiotensin II type 1A receptor (AT(1A)R) in human embryonic kidney (HEK)293 cells. We built an abstracted ordinary differential equations (ODE)-based model that captured the available knowledge and experimental data. We inferred the unknown parameters by simultaneously fitting experimental data generated in both control and perturbed conditions. We demonstrate that, in addition to its well-established function in the desensitization of G-protein activation, GRK2 exerts a strong negative effect on β-arrestin-dependent signaling through its competition with GRK5 and 6 for receptor phosphorylation. Importantly, we experimentally confirmed the validity of this novel GRK2-dependent mechanism in both primary vascular smooth muscle cells naturally expressing the AT(1A)R, and HEK293 cells expressing other 7TMRs.

  14. Second messenger-dependent protein kinases and protein synthesis regulate endogenous secretin receptor responsiveness

    PubMed Central

    Ghadessy, Roxana S; Kelly, Eamonn

    2002-01-01

    The present study investigated the role of second messenger-dependent protein kinase A (PKA) and C (PKC) in the regulation of endogenous secretin receptor responsiveness in NG108-15 mouse neuroblastoma×rat glioma hybrid cells. In whole cell cyclic AMP accumulation studies, activation of PKC either by phorbol 12-myristate 13-acetate (PMA) or by purinoceptor stimulation using uridine 5′-triphosphate (UTP) decreased secretin receptor responsiveness. PKC activation also inhibited forskolin-stimulated cyclic AMP accumulation but did not affect cyclic AMP responses mediated by the prostanoid-IP receptor agonist iloprost, or the A2 adenosine receptor agonist 5′-(N-ethylcarboxamido) adenosine (NECA). In additivity experiments, saturating concentrations of secretin and iloprost were found to be additive in terms of cyclic AMP accumulation, whereas saturating concentrations of NECA and iloprost together were not. This suggests compartmentalization of Gs-coupling components in NG108-15 cells and possible heterologous regulation of secretin receptor responsiveness at the level of adenylyl cyclase activation. Cells exposed to the PKA inhibitor H-89, exhibited a time-dependent increase in secretin receptor responsiveness compared to control cells. This effect was selective since cyclic AMP responses to forskolin, iloprost and NECA were not affected by H-89 treatment. Furthermore, treatment with the protein synthesis inhibitor cycloheximide produced a time-dependent increase in secretin receptor responsiveness. Together these results indicate that endogenous secretin receptor responsiveness is regulated by PKC, PKA and protein neosynthesis in NG108-15 cells. PMID:11959806

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

    PubMed

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

    2015-01-01

    GABAA receptors are important for inhibition in the CNS where neurosteroids and protein kinases are potent endogenous modulators. Acting individually, these can either enhance or depress receptor function, dependent upon the type of neurosteroid or kinase and the receptor subunit combination. However, in vivo, these modulators probably act in concert to fine-tune GABAA receptor activity and thus inhibition, although how this is achieved remains unclear. Therefore, we investigated the relationship between these modulators at synaptic-type α1β3γ2L and extrasynaptic-type α4β3δ GABAA receptors using electrophysiology. For α1β3γ2L, potentiation of GABA responses by tetrahydro-deoxycorticosterone was reduced after inhibiting protein kinase C, and enhanced following its activation, suggesting this kinase regulates neurosteroid modulation. In comparison, neurosteroid potentiation was reduced at α1β3(S408A,S409A)γ2L receptors, and unaltered by PKC inhibitors or activators, indicating that phosphorylation of β3 subunits is important for regulating neurosteroid activity. To determine whether extrasynaptic-type GABAA receptors were similarly modulated, α4β3δ and α4β3(S408A,S409A)δ receptors were investigated. Neurosteroid potentiation was reduced at both receptors by the kinase inhibitor staurosporine. By contrast, neurosteroid-mediated potentiation at α4(S443A)β3(S408A,S409A)δ receptors was unaffected by protein kinase inhibition, strongly suggesting that phosphorylation of α4 and β3 subunits is required for regulating neurosteroid activity at extrasynaptic receptors. Western blot analyses revealed that neurosteroids increased phosphorylation of β3(S408,S409) implying that a reciprocal pathway exists for neurosteroids to modulate phosphorylation of GABAA receptors. Overall, these findings provide important insight into the regulation of GABAA receptors in vivo, and into the mechanisms by which GABAergic inhibitory transmission may be simultaneously

  17. 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.

  18. Expression of a gibberellin-induced leucine-rich repeat receptor-like protein kinase in deepwater rice and its interaction with kinase-associated protein phosphatase

    SciTech Connect

    Knaap, E. van der; Sauter, M.; Kende, H. . DOE Plant Research Lab.); Song, W.Y.; Ruan, D.L.; Ronald, P.C. . Dept. of Plant Pathology)

    1999-06-01

    The authors identified in deepwater rice (Oryza sativa L.) a gene encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK (O. sativa transmembrane kinase). The transcript levels of OsTMK increased in the rice internode in response to gibberellin. Expression of OsTMK was especially high in regions undergoing cell division and elongation. The kinase domain of OsTMK was enzymatically active autophosphorylating on serine and threonine residues. A cDNA encoding a rice ortholog of a kinase-associated type 2C protein phosphatase (OsKAPP) was cloned. KAPPs are putative downstream components in kinase-mediated signal transduction pathways. The kinase interaction domain of OsKAPP was phosphorylated in vitro by the kinase domain of OsTMK. RNA gel-blot analysis indicated that the expression of OsTMK and OsKAPP was similar in different tissues of the rice plant. In protein-binding assays, OsKAPP interacted with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with the protein kinase domains of the rice and maize receptor-like protein kinases Xa21 and ZmPK1, respectively.

  19. INSIGHTS INTO THE REGULATION OF 5-HT2A RECEPTORS BY SCAFFOLDING PROTEINS AND KINASES

    PubMed Central

    Allen, John A.; Yadav, Prem N.

    2008-01-01

    SUMMARY 5-HT2A serotonin receptors are essential molecular targets for the actions of LSD-like hallucinogens and atypical antipsychotic drugs. 5-HT2A 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-HT2A receptors and our recent studies suggest multiple scaffolds exist for 5-HT2A receptors including PSD95, arrestin, and caveolin. In addition, a novel interaction has emerged between p90 ribosomal S6 kinase and 5-HT2A 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-HT2A trafficking, targeting and signaling. PMID:18640136

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

    PubMed

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

    2016-09-16

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

  1. 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

  2. Identification of a molecular weight 43,000 protein kinase in acetylcholine receptor-enriched membranes.

    PubMed Central

    Gordon, A S; Milfay, D; Diamond, I

    1983-01-01

    A photoaffinity ATP ligand is used to identify the protein kinase present in acetylcholine receptor-enriched membranes from Torpedo californica. Incubation of these membranes with 8-azido-[alpha-32P]ATP and subsequent irradiation with UV light resulted in covalent labeling of a major band of Mr 43,000. Alkali-stripped membranes that show a selective reduction in the Mr 43,000 polypeptide also show a corresponding reduction in incorporation of photoaffinity label. In addition, the neutralized alkaline extract also showed one band at Mr 43,000 when labeled with the photoaffinity ligand. After alkali extraction, endogenous protein kinase activity decreased in the membranes in proportion to the loss of Mr 43,000 peptide. Moreover, the alkaline extract was able to phosphorylate casein in an exogenous assay system. These results suggest that a Mr 43,000 polypeptide in acetylcholine receptor-enriched membranes is the acetylcholine receptor kinase. Images PMID:6577458

  3. Mapping the Putative G Protein-coupled Receptor (GPCR) Docking Site on GPCR Kinase 2

    PubMed Central

    Beautrait, Alexandre; Michalski, Kevin R.; Lopez, Thomas S.; Mannix, Katelynn M.; McDonald, Devin J.; Cutter, Amber R.; Medina, Christopher B.; Hebert, Aaron M.; Francis, Charnelle J.; Bouvier, Michel; Tesmer, John J. G.; Sterne-Marr, Rachel

    2014-01-01

    G protein-coupled receptor kinases (GRKs) phosphorylate agonist-occupied receptors initiating the processes of desensitization and β-arrestin-dependent signaling. Interaction of GRKs with activated receptors serves to stimulate their kinase activity. The extreme N-terminal helix (αN), the kinase small lobe, and the active site tether (AST) of the AGC kinase domain have previously been implicated in mediating the allosteric activation. Expanded mutagenesis of the αN and AST allowed us to further assess the role of these two regions in kinase activation and receptor phosphorylation in vitro and in intact cells. We also developed a bioluminescence resonance energy transfer-based assay to monitor the recruitment of GRK2 to activated α2A-adrenergic receptors (α2AARs) in living cells. The bioluminescence resonance energy transfer signal exhibited a biphasic response to norepinephrine concentration, suggesting that GRK2 is recruited to Gβγ and α2AAR with EC50 values of 15 nm and 8 μm, respectively. We show that mutations in αN (L4A, V7E, L8E, V11A, S12A, Y13A, and M17A) and AST (G475I, V477D, and I485A) regions impair or potentiate receptor phosphorylation and/or recruitment. We suggest that a surface of GRK2, including Leu4, Val7, Leu8, Val11, and Ser12, directly interacts with receptors, whereas residues such as Asp10, Tyr13, Ala16, Met17, Gly475, Val477, and Ile485 are more important for kinase domain closure and activation. Taken together with data on GRK1 and GRK6, our data suggest that all three GRK subfamilies make conserved interactions with G protein-coupled receptors, but there may be unique interactions that influence selectivity. PMID:25049229

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

    PubMed Central

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

    2015-01-01

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

  5. 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. PMID:26577786

  6. The origin and evolution of G protein-coupled receptor kinases.

    PubMed

    Mushegian, Arcady; Gurevich, Vsevolod V; Gurevich, Eugenia V

    2012-01-01

    G protein-coupled receptor (GPCR) kinases (GRKs) play key role in homologous desensitization of GPCRs. GRKs phosphorylate activated receptors, promoting high affinity binding of arrestins, which precludes G protein coupling. Direct binding to active GPCRs activates GRKs, so that they selectively phosphorylate only the activated form of the receptor regardless of the accessibility of the substrate peptides within it and their Ser/Thr-containing sequence. Mammalian GRKs were classified into three main lineages, but earlier GRK evolution has not been studied. Here we show that GRKs emerged at the early stages of eukaryotic evolution via an insertion of a kinase similar to ribosomal protein S6 kinase into a loop in RGS domain. GRKs in Metazoa fall into two clades, one including GRK2 and GRK3, and the other consisting of all remaining GRKs, split into GRK1-GRK7 lineage and GRK4-GRK5-GRK6 lineage in vertebrates. One representative of each of the two ancient clades is found as early as placozoan Trichoplax adhaerens. Several protists, two oomycetes and unicellular brown algae have one GRK-like protein, suggesting that the insertion of a kinase domain into the RGS domain preceded the origin of Metazoa. The two GRK families acquired distinct structural units in the N- and C-termini responsible for membrane recruitment and receptor association. Thus, GRKs apparently emerged before animals and rapidly expanded in true Metazoa, most likely due to the need for rapid signalling adjustments in fast-moving animals.

  7. The Origin and Evolution of G Protein-Coupled Receptor Kinases

    PubMed Central

    Mushegian, Arcady; Gurevich, Vsevolod V.; Gurevich, Eugenia V.

    2012-01-01

    G protein-coupled receptor (GPCR) kinases (GRKs) play key role in homologous desensitization of GPCRs. GRKs phosphorylate activated receptors, promoting high affinity binding of arrestins, which precludes G protein coupling. Direct binding to active GPCRs activates GRKs, so that they selectively phosphorylate only the activated form of the receptor regardless of the accessibility of the substrate peptides within it and their Ser/Thr-containing sequence. Mammalian GRKs were classified into three main lineages, but earlier GRK evolution has not been studied. Here we show that GRKs emerged at the early stages of eukaryotic evolution via an insertion of a kinase similar to ribosomal protein S6 kinase into a loop in RGS domain. GRKs in Metazoa fall into two clades, one including GRK2 and GRK3, and the other consisting of all remaining GRKs, split into GRK1-GRK7 lineage and GRK4-GRK5-GRK6 lineage in vertebrates. One representative of each of the two ancient clades is found as early as placozoan Trichoplax adhaerens. Several protists, two oomycetes and unicellular brown algae have one GRK-like protein, suggesting that the insertion of a kinase domain into the RGS domain preceded the origin of Metazoa. The two GRK families acquired distinct structural units in the N- and C-termini responsible for membrane recruitment and receptor association. Thus, GRKs apparently emerged before animals and rapidly expanded in true Metazoa, most likely due to the need for rapid signalling adjustments in fast-moving animals. PMID:22442725

  8. New roles of G protein-coupled receptor kinase 2 (GRK2) in cell migration

    PubMed Central

    Penela, Petronila; Ribas, Catalina; Aymerich, Ivette

    2009-01-01

    G protein-coupled receptor kinase 2 (GRK2) was initially identified as a key player, together with β-arrestins, in the regulation of multiple G protein-coupled receptors (GPCR). Further research has revealed a complex GRK2 interactome, that includes a variety of proteins related to cell motility, and a role for GRK2 kinase activity in inhibiting chemokine-induced immune cell migration. In addition, we have recently reported that GRK2 positively regulates integrin and sphingosine-1-phosphate-dependent motility in epithelial cell types and fibroblasts, acting as a scaffold molecule. We suggest that the positive or negative correlation of GRK2 levels with cell migration would depend on the cell type, specific stimuli acting through plasma membrane receptors, or on the signalling context, leading to differential networks of interaction of GRK2 with cell migration-related signalosomes. PMID:19372742

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

    PubMed

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

    2016-07-01

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

  10. Role of non-receptor protein kinases in spermatid transport during spermatogenesis*

    PubMed Central

    Wan, H. T.; Mruk, Dolores D.; Tang, Elizabeth I.; Xiao, Xiang; Cheng, Yan-ho; Wong, Elissa W.P.; Wong, Chris K. C.; Cheng, C. Yan

    2014-01-01

    Non-receptor protein tyrosine kinases are cytoplasmic kinases that activate proteins by phosphorylating target protein tyrosine residues, in turn affecting multiple functions in eukaryotic cells. Herein, we focus on the role of non-receptor protein tyrosine kinases, most notably, FAK, c-Yes and c-Src, in the transport of spermatids across the seminiferous epithelium during spermatogenesis. Since spermatids, which are formed from spermatocytes via meiosis, are immotile haploid cells, they must be transported by Sertoli cells across the seminiferous epithelium during the epithelial cycle of spermatogenesis. Without the timely transport of spermatids across the epithelium, the release of sperms at spermiation fails to occur, leading to infertility. Thus, the molecular event pertinent to spermatid transport is crucial to spermatogenesis. Herein, we provide a critical discussion based on recent findings in the field. We also provide a hypothetical model on spermatid transport, and the role of non-receptor protein tyrosine kinases in this event. We also highlight areas of research that deserve attention by investigators in the field. PMID:24727349

  11. Protein kinase D1 (PKD1) influences androgen receptor (AR) function in prostate cancer cells

    SciTech Connect

    Mak, Paul; Jaggi, Meena; Chauhan, Subhash C.; Balaji, K.C.

    2008-09-05

    Protein kinase D1 (PKD1), founding member of PKD protein family, is down-regulated in advanced prostate cancer (PCa). We demonstrate that PKD1 and androgen receptor (AR) are present as a protein complex in PCa cells. PKD1 is associated with a transcriptional complex which contains AR and promoter sequence of the Prostate Specific Antigen (PSA) gene. Ectopic expression of wild type PKD1 and the kinase dead mutant PKD1 (K628W) attenuated the ligand-dependent transcriptional activation of AR in prostate cancer cells and yeast cells indicating that PKD1 can affect AR transcription activity, whereas knocking down PKD1 enhanced the ligand-dependent transcriptional activation of AR. Co-expression of kinase dead mutant with AR significantly inhibited androgen-mediated cell proliferation in both LNCaP and DU145 PC cells. Our data demonstrate for the first time that PKD1 can influence AR function in PCa cells.

  12. 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

  13. Increased G Protein-Coupled Receptor Kinase (GRK) Expression in the Anterior Cingulate Cortex in Schizophrenia

    PubMed Central

    Funk, Adam J.; Haroutunian, Vahram; Meador-Woodruff, James H.; McCullumsmith, Robert E.

    2014-01-01

    Background Current pharmacological treatments for schizophrenia target G protein-coupled receptors (GPCRs), including dopamine receptors. Ligand bound GPCRs are regulated by a family of G protein-coupled receptor kinases (GRKs), members of which uncouple the receptor from heterotrimeric G proteins, desensitize the receptor, and induce receptor internalization via the arrestin family of scaffolding and signaling molecules. GRKs initiate the activation of downstream signaling pathways, can regulate receptors and signaling molecules independent of GPCR phosphorylation, and modulate epigenetic regulators like histone deacetylases (HDACs). We hypothesize that expression of GRK proteins are altered in schizophrenia, consistent with previous findings of alterations up and downstream from this family of molecules that facilitate intracellular signaling processes. Methods In this study we measured protein expression via Western blot analysis for GRKs 2, 3, 5, and 6 in the anterior cingulate cortex of patients with schizophrenia (N = 36) and a comparison group (N = 33). To control for antipsychotic treatment we measured these same targets in haloperidol treated vs. untreated rats (N = 10 for both). Results We found increased levels of GRK5 in schizophrenia. No changes were detected in GRK protein expression in rats treated with haloperidol decanoate for 9 months. Conclusion These data suggest that increased GRK5 expression may contribute the the pathophysiology of schizophrenia via abnormal regulation of the cytoskeleton, endocytosis, signaling, GPCRs, and histone modification. PMID:25153362

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

    PubMed

    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

  15. 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

  16. Chapter Three - Ubiquitination and Protein Turnover of G-Protein-Coupled Receptor Kinases in GPCR Signaling and Cellular Regulation.

    PubMed

    Penela, P

    2016-01-01

    G-protein-coupled receptors (GPCRs) are responsible for regulating a wide variety of physiological processes, and distinct mechanisms for GPCR inactivation exist to guarantee correct receptor functionality. One of the widely used mechanisms is receptor phosphorylation by specific G-protein-coupled receptor kinases (GRKs), leading to uncoupling from G proteins (desensitization) and receptor internalization. GRKs and β-arrestins also participate in the assembly of receptor-associated multimolecular complexes, thus initiating alternative G-protein-independent signaling events. In addition, the abundant GRK2 kinase has diverse "effector" functions in cellular migration, proliferation, and metabolism homeostasis by means of the phosphorylation or interaction with non-GPCR partners. Altered expression of GRKs (particularly of GRK2 and GRK5) occurs during pathological conditions characterized by impaired GPCR signaling including inflammatory syndromes, cardiovascular disease, and tumor contexts. It is increasingly appreciated that different pathways governing GRK protein stability play a role in the modulation of kinase levels in normal and pathological conditions. Thus, enhanced GRK2 degradation by the proteasome pathway occurs upon GPCR stimulation, what allows cellular adaptation to chronic stimulation in a physiological setting. β-arrestins participate in this process by facilitating GRK2 phosphorylation by different kinases and by recruiting diverse E3 ubiquitin ligase to the receptor complex. Different proteolytic systems (ubiquitin-proteasome, calpains), chaperone activities and signaling pathways influence the stability of GRKs in different ways, thus endowing specificity to GPCR regulation as protein turnover of GRKs can be differentially affected. Therefore, modulation of protein stability of GRKs emerges as a versatile mechanism for feedback regulation of GPCR signaling and basic cellular processes. PMID:27378756

  17. Chapter Three - Ubiquitination and Protein Turnover of G-Protein-Coupled Receptor Kinases in GPCR Signaling and Cellular Regulation.

    PubMed

    Penela, P

    2016-01-01

    G-protein-coupled receptors (GPCRs) are responsible for regulating a wide variety of physiological processes, and distinct mechanisms for GPCR inactivation exist to guarantee correct receptor functionality. One of the widely used mechanisms is receptor phosphorylation by specific G-protein-coupled receptor kinases (GRKs), leading to uncoupling from G proteins (desensitization) and receptor internalization. GRKs and β-arrestins also participate in the assembly of receptor-associated multimolecular complexes, thus initiating alternative G-protein-independent signaling events. In addition, the abundant GRK2 kinase has diverse "effector" functions in cellular migration, proliferation, and metabolism homeostasis by means of the phosphorylation or interaction with non-GPCR partners. Altered expression of GRKs (particularly of GRK2 and GRK5) occurs during pathological conditions characterized by impaired GPCR signaling including inflammatory syndromes, cardiovascular disease, and tumor contexts. It is increasingly appreciated that different pathways governing GRK protein stability play a role in the modulation of kinase levels in normal and pathological conditions. Thus, enhanced GRK2 degradation by the proteasome pathway occurs upon GPCR stimulation, what allows cellular adaptation to chronic stimulation in a physiological setting. β-arrestins participate in this process by facilitating GRK2 phosphorylation by different kinases and by recruiting diverse E3 ubiquitin ligase to the receptor complex. Different proteolytic systems (ubiquitin-proteasome, calpains), chaperone activities and signaling pathways influence the stability of GRKs in different ways, thus endowing specificity to GPCR regulation as protein turnover of GRKs can be differentially affected. Therefore, modulation of protein stability of GRKs emerges as a versatile mechanism for feedback regulation of GPCR signaling and basic cellular processes.

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

    PubMed Central

    Zhang, Naixin; Kima, Peter E.

    2016-01-01

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

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

    PubMed

    Zhang, Naixin; Kima, Peter E

    2016-01-01

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

  20. Heterotrimeric G proteins interact with defense-related receptor-like kinases in Arabidopsis.

    PubMed

    Aranda-Sicilia, María Nieves; Trusov, Yuri; Maruta, Natsumi; Chakravorty, David; Zhang, Yuelin; Botella, José Ramón

    2015-09-01

    Heterotrimeric G proteins (G-proteins) are versatile signaling elements conserved in Eukaryotes. In animals G-proteins relay signals from 7-transmembrane spanning G protein-coupled receptors (GPCRs) to intracellular downstream effectors; however, the existence of GPCRs in plants is controversial. Contrastingly, a surplus of receptor-like kinases (RLKs) provides signal recognition at the plant cell surface. It is established that G proteins are involved in plant defense and suggested that they relay signals from defense-related RLKs. However, it is unclear how the signaling is conducted, as physical interaction between the RLKs and G proteins has not been demonstrated. Using yeast split-ubiquitin system and Bimolecular Fluorescence Complementation assays, we demonstrate physical interaction between the Gα, Gγ1 and Gγ2 subunits, and the defense-related RD-type receptor like kinases CERK1, BAK1 and BIR1. At the same time, no interaction was detected with the non-RD RLK FLS2. We hypothesize that G-proteins mediate signal transduction immediately downstream of the pathogenesis-related RLKs.

  1. Activation of multiple mitogen-activated protein kinases by recombinant calcitonin gene-related peptide receptor.

    PubMed

    Parameswaran, N; Disa, J; Spielman, W S; Brooks, D P; Nambi, P; Aiyar, N

    2000-02-18

    Calcitonin gene-related peptide is a 37-amino-acid neuropeptide and a potent vasodilator. Although calcitonin gene-related peptide has been shown to have a number of effects in a variety of systems, the mechanisms of action and the intracellular signaling pathways, especially the regulation of mitogen-activated protien kinase (MAPK) pathway, is not known. In the present study we investigated the role of calcitonin gene-related peptide in the regulation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfected with a recombinant porcine calcitonin gene-related peptide-1 receptor. Calcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related peptide(8-37), the calcitonin gene-related peptide-receptor antagonist. Calcitonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activator of adenylyl cyclase also stimulated ERK and P38 activities in these cells suggesting the invovement of cAMP in this process. Calcitonin gene-related peptide-stimulated ERK and P38 MAPK activities were inhibited significantly by calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide-(8-37) suggesting the involvement of calcitonin gene-related peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride¿] inhibited calcitonin gene-related peptide-mediated activation of ERK and p38 kinases. On the other hand, preincubation of the cells with wortmannin ¿[1S-(1alpha,6balpha,9abeta,11alpha, 11bbeta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11, 11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3, 2-de]indeno[4,5-h]-2

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

    PubMed

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

    1990-10-25

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

  3. Activation of p38 Mitogen-Activated Protein Kinase Promotes Epidermal Growth Factor Receptor Internalization

    PubMed Central

    Vergarajauregui, Silvia; Miguel, Anitza San; Puertollano, Rosa

    2006-01-01

    Endocytic trafficking plays an important role in the regulation of the epidermal growth factor receptor (EGFR). To address if cellular kinases regulate EGFR internalization, we used anisomycin, a potent activator of kinase cascades in mammalian cells, especially the stress-activated mitogen-activated protein (MAP) kinase subtypes. Here, we report that activation of p38 MAP kinase by anisomycin is sufficient to induce internalization of EGFR. Anisomycin and EGF employ different mechanisms to promote EGFR endocytosis as anisomycin-induced internalization does not require tyrosine kinase activity or ubiquitination of the receptor. In addition, anisomycin treatment did not result in delivery and degradation of EGFR at lysosomes. Incubation with a specific inhibitor of p38, or depletion of endogenous p38 by small interfering RNAs, abolished anisomycin-induced internalization of EGFR while having no effect on transferrin endocytosis, indicating that the effect of p38 activation on EGFR endocytosis is specific. Interestingly, inhibition of p38 activation also abolished endocytosis of EGFR induced by UV radiation. Our results reveal a novel role for p38 in the regulation of EGFR endocytosis and suggest that stimulation of EGFR internalization by p38 might represent a general mechanism to prevent generation of proliferative or anti-apoptotic signals under stress conditions. PMID:16683917

  4. The Cytoplasmic Adaptor Protein Dok7 Activates the Receptor Tyrosine Kinase MuSK via Dimerization

    SciTech Connect

    Bergamin, E.; Hallock, P; Burden, S; Hubbard, S

    2010-01-01

    Formation of the vertebrate neuromuscular junction requires, among others proteins, Agrin, a neuronally derived ligand, and the following muscle proteins: LRP4, the receptor for Agrin; MuSK, a receptor tyrosine kinase (RTK); and Dok7 (or Dok-7), a cytoplasmic adaptor protein. Dok7 comprises a pleckstrin-homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and C-terminal sites of tyrosine phosphorylation. Unique among adaptor proteins recruited to RTKs, Dok7 is not only a substrate of MuSK, but also an activator of MuSK's kinase activity. Here, we present the crystal structure of the Dok7 PH-PTB domains in complex with a phosphopeptide representing the Dok7-binding site on MuSK. The structure and biochemical data reveal a dimeric arrangement of Dok7 PH-PTB that facilitates trans-autophosphorylation of the kinase activation loop. The structure provides the molecular basis for MuSK activation by Dok7 and for rationalizing several Dok7 loss-of-function mutations found in patients with congenital myasthenic syndromes.

  5. The complex G protein-coupled receptor kinase 2 (GRK2) interactome unveils new physiopathological targets

    PubMed Central

    Penela, Petronila; Murga, Cristina; Ribas, Catalina; Lafarga, Vanesa; Mayor, Federico

    2010-01-01

    GRK2 is a ubiquitous member of the G protein-coupled receptor kinase (GRK) family that appears to play a central, integrative role in signal transduction cascades. GRKs participate together with arrestins in the regulation of G protein-coupled receptors (GPCR), a family of hundreds of membrane proteins of key physiological and pharmacological importance, by triggering receptor desensitization from G proteins and GPCR internalization, and also by helping assemble macromolecular signalosomes in the receptor environment acting as agonist-regulated adaptor scaffolds, thus contributing to signal propagation. In addition, emerging evidence indicates that GRK2 can phosphorylate a growing number of non-GPCR substrates and associate with a variety of proteins related to signal transduction, thus suggesting that this kinase could also have diverse ‘effector’ functions. We discuss herein the increasing complexity of such GRK2 ‘interactome’, with emphasis on the recently reported roles of this kinase in cell migration and cell cycle progression and on the functional impact of the altered GRK2 levels observed in several relevant cardiovascular, inflammatory or tumour pathologies. Deciphering how the different networks of potential GRK2 functional interactions are orchestrated in a stimulus, cell type or context-specific way is critical to unveil the contribution of GRK2 to basic cellular processes, to understand how alterations in GRK2 levels or functionality may participate in the onset or development of several cardiovascular, tumour or inflammatory diseases, and to assess the feasibility of new therapeutic strategies based on the modulation of the activity, levels or specific interactions of GRK2. PMID:20590581

  6. 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.

  7. Differential phosphorylation of the progesterone receptor by insulin, epidermal growth factor, and platelet-derived growth factor receptor tyrosine protein kinases.

    PubMed

    Woo, D D; Fay, S P; Griest, R; Coty, W; Goldfine, I; Fox, C F

    1986-01-01

    Purified preparations of insulin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) receptors were compared for their abilities to phosphorylate purified hen oviduct progesterone receptors. The specific activities of all three peptide hormone-induced receptor kinases were first defined using a synthetic tridecapeptide tyrosine protein kinase substrate. Next, equivalent ligand-activated activities of the three receptor kinases were tested for their abilities to phosphorylate hen oviduct progesterone receptor. Both the insulin and EGF receptors phosphorylated progesterone receptor at high affinity, exclusively at tyrosine residues and with maximal stoichiometries that were near unity. In contrast, the PDGF receptor did not recognize progesterone receptor as a substrate. Insulin decreased the Km of the insulin receptor for progesterone receptor subunits as substrates, but had no significant effect on Vmax values. On the other hand, EGF increased the Vmax of the EGF receptor for progesterone receptor subunits as substrates. Phosphorylation of progesterone receptor by the insulin and EGF receptor kinases differed in two additional ways. 1) EGF-activated receptor phosphorylated the 80- and 105-kDa progesterone receptor subunits to an equal extent, whereas insulin-activated receptor preferentially phosphorylated the 80-kDa subunit. 2) Phosphopeptide fingerprinting analyses revealed that while insulin and EGF receptors phosphorylated one identical major site on both progesterone receptor subunits, they differed in their specificities for other sites. PMID:3001059

  8. Ethanol Regulation of Synaptic GABAA α4 Receptors Is Prevented by Protein Kinase A Activation.

    PubMed

    Carlson, Stephen L; Bohnsack, John Peyton; Morrow, A Leslie

    2016-04-01

    Ethanol alters GABAA receptor trafficking and function through activation of protein kinases, and these changes may underlie ethanol dependence and withdrawal. In this study, we used subsynaptic fraction techniques and patch-clamp electrophysiology to investigate the biochemical and functional effects of protein kinase A (PKA) and protein kinase C (PKC) activation by ethanol on synaptic GABAA α4 receptors, a key target of ethanol-induced changes. Rat cerebral cortical neurons were grown for 18 days in vitro and exposed to ethanol and/or kinase modulators for 4 hours, a paradigm that recapitulates GABAergic changes found after chronic ethanol exposure in vivo. PKA activation by forskolin or rolipram during ethanol exposure prevented increases in P2 fraction α4 subunit abundance, whereas inhibiting PKA had no effect. Similarly, in the synaptic fraction, activation of PKA by rolipram in the presence of ethanol prevented the increase in synaptic α4 subunit abundance, whereas inhibiting PKA in the presence of ethanol was ineffective. Conversely, PKC inhibition in the presence of ethanol prevented the ethanol-induced increases in synaptic α4 subunit abundance. Finally, we found that either activating PKA or inhibiting PKC in the presence of ethanol prevented the ethanol-induced decrease in GABA miniature inhibitory postsynaptic current decay τ1, whereas inhibiting PKA had no effect. We conclude that PKA and PKC have opposing effects in the regulation of synaptic α4 receptors, with PKA activation negatively modulating, and PKC activation positively modulating, synaptic α4 subunit abundance and function. These results suggest potential targets for restoring normal GABAergic functioning in the treatment of alcohol use disorders.

  9. Intrinsic cleavage of receptor-interacting protein kinase-1 by caspase-6.

    PubMed

    van Raam, B J; Ehrnhoefer, D E; Hayden, M R; Salvesen, G S

    2013-01-01

    Necroptosis is a form of programmed cell death that occurs in the absence of caspase activation and depends on the activity of the receptor-interacting protein kinases. Inactivation of these kinases by caspase-mediated cleavage has been shown to be essential for successful embryonic development, survival and activation of certain cell types. The initiator of extrinsic apoptosis, caspase-8, which has a pro-death as well as a pro-life function, has been assigned this role. In the present study we demonstrate that caspase-6, an executioner caspase, performs this role during apoptosis induced through the intrinsic pathway. In addition, we demonstrate that in the absence of caspase activity, intrinsic triggers of apoptosis induce the receptor-interacting-kinase-1-dependent production of pro-inflammatory cytokines. We show that ubiquitously expressed caspase-6 has a supporting role in apoptosis by cleaving this kinase, thus preventing production of inflammatory cytokines as well as inhibiting the necroptotic pathway. These findings shed new light on the regulation of necroptosis as well as cell death in an inflammatory environment wherein cells receive both intrinsic and extrinsic death signals.

  10. Ligand-stimulated downregulation of the alpha interferon receptor: role of protein kinase D2.

    PubMed

    Zheng, Hui; Qian, Juan; Varghese, Bentley; Baker, Darren P; Fuchs, Serge

    2011-02-01

    Alpha interferon (IFN-α) controls homeostasis of hematopoietic stem cells, regulates antiviral resistance, inhibits angiogenesis, and suppresses tumor growth. This cytokine is often used to treat cancers and chronic viral infections. The extent of cellular responses to IFN-α is limited by the IFN-induced ubiquitination and degradation of the IFN-α/β receptor chain 1 (IFNAR1) chain of the cognate receptor. IFNAR1 ubiquitination is facilitated by the βTrcp E3 ubiquitin ligase that is recruited to IFNAR1 upon its degron phosphorylation, which is induced by the ligand. Here we report identification of protein kinase D2 (PKD2) as a kinase that mediates the ligand-inducible phosphorylation of IFNAR1 degron and enables binding of βTrcp to the receptor. Treatment of cells with IFN-α induces catalytic activity of PKD2 and stimulates its interaction with IFNAR1. Expression and kinase activity of PKD2 are required for the ligand-inducible stimulation of IFNAR1 ubiquitination and endocytosis and for accelerated proteolytic turnover of IFNAR1. Furthermore, inhibition or knockdown of PKD2 robustly augments intracellular signaling induced by IFN-α and increases the efficacy of its antiviral effects. The mechanisms of the ligand-inducible elimination of IFNAR1 are discussed, along with the potential medical significance of this regulation. PMID:21173164

  11. 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.

  12. 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

  13. Epidermal growth factor (EGF) promotes phosphorylation at threonine-654 of the EGF receptor: possible role of protein kinase C in homologous regulation of the EGF receptor

    SciTech Connect

    Whiteley, B.; Glaser, L.

    1986-10-01

    Treatment of cells with tumor-promoting phorbol diesters, which causes activation of protein kinase C, leads to phosphorylation of the epidermal growth factor (EGF) receptor at threonine-654. Addition of phorbol diesters to intact cells causes inhibition of the EGF-induced tyrosine-protein kinase activity of the EGF receptor and it has been suggested that this effect of phorbol diesters is mediated by the phosphorylation of the receptor by protein kinase C. The authors measured the activity of protein kinase C in A431 cells by determining the incorporation of (/sup 32/P)phosphate into peptides containing threonine-654 obtained by trypsin digestion of EGF receptors. After 3 h of exposure to serum-free medium, A431 cells had no detectable protein kinase C activity. Addition of EGF to these cells resulted in (/sup 32/P) incorporation into threonine-654 as well as into tyrosine residues. This indicates that EGF promotes the activation of protein kinase C in A431 cells. The phophorylation of threonine-654 induced by EGF was maximal after only 5 min of EGF addition and the (/sup 32/P) incorporation into threonine-654 reached 50% of the (/sup 32/P) in a tyrosine-containing peptide. This indicates that a significant percentage of the total EGF receptors are phosphorylated by protein kinase C. A variety of external stimuli activate Na/sup +//H/sup +/ exchange, including EGF, phorbol diesters, and hypertonicity. To ascertain whether activation of protein kinase C is an intracellular common effector of all of these systems, the authors measured the activity of protein kinase C after exposure of A431 cells to hyperosmotic conditions and observed no effect on phosphorylation of threonine-654, therefore, activation of Na/sup +//H/sup +/ exchange by hypertonic medium is independent of protein kinase C activity.

  14. Receptor-like kinase SOBIR1/EVR interacts with receptor-like proteins in plant immunity against fungal infection.

    PubMed

    Liebrand, Thomas W H; van den Berg, Grardy C M; Zhang, Zhao; Smit, Patrick; Cordewener, Jan H G; America, Antoine H P; America, Antione H P; Sklenar, Jan; Jones, Alexandra M E; Tameling, Wladimir I L; Robatzek, Silke; Thomma, Bart P H J; Joosten, Matthieu H A J

    2013-06-11

    The plant immune system is activated by microbial patterns that are detected as nonself molecules. Such patterns are recognized by immune receptors that are cytoplasmic or localized at the plasma membrane. Cell surface receptors are represented by receptor-like kinases (RLKs) that frequently contain extracellular leucine-rich repeats and an intracellular kinase domain for activation of downstream signaling, as well as receptor-like proteins (RLPs) that lack this signaling domain. It is therefore hypothesized that RLKs are required for RLPs to activate downstream signaling. The RLPs Cf-4 and Ve1 of tomato (Solanum lycopersicum) mediate resistance to the fungal pathogens Cladosporium fulvum and Verticillium dahliae, respectively. Despite their importance, the mechanism by which these immune receptors mediate downstream signaling upon recognition of their matching ligand, Avr4 and Ave1, remained enigmatic. Here we show that the tomato ortholog of the Arabidopsis thaliana RLK Suppressor Of BIR1-1/Evershed (SOBIR1/EVR) and its close homolog S. lycopersicum (Sl)SOBIR1-like interact in planta with both Cf-4 and Ve1 and are required for the Cf-4- and Ve1-mediated hypersensitive response and immunity. Tomato SOBIR1/EVR interacts with most of the tested RLPs, but not with the RLKs FLS2, SERK1, SERK3a, BAK1, and CLV1. SOBIR1/EVR is required for stability of the Cf-4 and Ve1 receptors, supporting our observation that these RLPs are present in a complex with SOBIR1/EVR in planta. We show that SOBIR1/EVR is essential for RLP-mediated immunity and propose that the protein functions as a regulatory RLK of this type of cell-surface receptors.

  15. Insulin receptor substrate 1 is a substrate of the Pim protein kinases

    PubMed Central

    Song, Jin H.; Padi, Sathish K. R.; Luevano, Libia A.; Minden, Mark D.; DeAngelo, Daniel J.; Hardiman, Gary; Ball, Lauren E.; Warfel, Noel A.; Kraft, Andrew S.

    2016-01-01

    The Pim family of serine/threonine protein kinases (Pim 1, 2, and 3) contribute to cellular transformation by regulating glucose metabolism, protein synthesis, and mitochondrial oxidative phosphorylation. Drugs targeting the Pim protein kinases are being tested in phase I/II clinical trials for the treatment of hematopoietic malignancies. The goal of these studies was to identify Pim substrate(s) that could help define the pathway regulated by these enzymes and potentially serve as a biomarker of Pim activity. To identify novel substrates, bioinformatics analysis was carried out to identify proteins containing a consensus Pim phosphorylation site. This analysis identified the insulin receptor substrate 1 and 2 (IRS1/2) as potential Pim substrates. Experiments were carried out in tissue culture, animals, and human samples from phase I trials to validate this observation and define the biologic readout of this phosphorylation. Our study demonstrates in both malignant and normal cells using either genetic or pharmacological inhibition of the Pim kinases or overexpression of this family of enzymes that human IRS1S1101 and IRS2S1149 are Pim substrates. In xenograft tumor experiments and in a human phase I clinical trial, a pan-Pim inhibitor administered in vivo to animals or humans decreased IRS1S1101 phosphorylation in tumor tissues. This phosphorylation was shown to have effects on the half-life of the IRS family of proteins, suggesting a role in insulin or IGF signaling. These results demonstrate that IRS1S1101 is a novel substrate for the Pim kinases and provide a novel marker for evaluation of Pim inhibitor therapy. PMID:26956053

  16. Regulation of Extrasynaptic GABAA α4 Receptors by Ethanol-Induced Protein Kinase A, but Not Protein Kinase C Activation in Cultured Rat Cerebral Cortical Neurons.

    PubMed

    Carlson, Stephen L; Bohnsack, J Peyton; Patel, Vraj; Morrow, A Leslie

    2016-01-01

    Ethanol produces changes in GABAA receptor trafficking and function that contribute to ethanol dependence symptomatology. Extrasynaptic γ-aminobutyric acid A receptors (GABAA-R) mediate inhibitory tonic current and are of particular interest because they are potentiated by physiologically relevant doses of ethanol. Here, we isolate GABAA α4δ receptors by western blotting in subsynaptic fractions to investigate protein kinase A (PKA) and protein kinase C (PKC) modulation of ethanol-induced receptor trafficking, while extrasynaptic receptor function is determined by measurement of tonic inhibition and responses evoked by 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). Rat cerebral cortical neurons were grown for 18 days in vitro and exposed to ethanol and/or PKA/PKC modulators. Ethanol exposure (1 hour) did not alter GABAA α4 receptor abundance, but it increased tonic current amplitude, an effect that was prevented by inhibiting PKA, but not PKC. Direct activation of PKA, but not PKC, increased the abundance and tonic current of extrasynaptic α4δ receptors. In contrast, prolonged ethanol exposure (4 hours) reduced α4δ receptor abundance as well as tonic current, and this effect was also PKA dependent. Finally, PKC activation by ethanol or phorbol-12,13-dibutyrate (PdBu) had no effect on extrasynaptic α4δ subunit abundance or activity. We conclude that ethanol alters extrasynaptic α4δ receptor function and expression in cortical neurons in a PKA-dependent manner, but ethanol activation of PKC does not influence these receptors. These results could have clinical relevance for therapeutic strategies to restore normal GABAergic functioning for the treatment of alcohol use disorders.

  17. Smoothened Signaling in Vertebrates Is Facilitated by a G Protein-coupled Receptor Kinase

    PubMed Central

    Philipp, Melanie; Fralish, Gregory B.; Meloni, Alison R.; Chen, Wei; MacInnes, Alyson W.; Barak, Lawrence S.

    2008-01-01

    Smoothened, a heptahelical membrane protein, functions as the transducer of Hedgehog signaling. The kinases that modulate Smoothened have been thoroughly analyzed in flies. However, little is known about how phosphorylation affects Smoothened in vertebrates, mainly, because the residues, where Smoothened is phosphorylated are not conserved from Drosophila to vertebrates. Given its molecular architecture, Smoothened signaling is likely to be regulated in a manner analogous to G protein–coupled receptors (GPCRs). Previously, it has been shown, that arrestins and GPCR kinases, (GRKs) not only desensitize G protein–dependent receptor signaling but also function as triggers for GPCR trafficking and formation of signaling complexes. Here we describe that a GRK contributes to Smoothened-mediated signaling in vertebrates. Knockdown of the zebrafish homolog of mammalian GRK2/3 results in lowered Hedgehog transcriptional responses, impaired muscle development, and neural patterning. Results obtained in zebrafish are corroborated both in cell culture, where zGRK2/3 phosphorylates Smoothened and promotes Smoothened signal transduction and in mice where deletion of GRK2 interferes with neural tube patterning. Together, these data suggest that a GRK functions as a vertebrate kinase for Smoothened, promoting Hedgehog signal transduction during early development. PMID:18815277

  18. 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

    2005-06-01

    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.

  19. Epidermal Growth Factor Receptor Transactivation Is Required for Mitogen-Activated Protein Kinase Activation by Muscarinic Acetylcholine Receptors in HaCaT Keratinocytes

    PubMed Central

    Ockenga, Wymke; Kühne, Sina; Bocksberger, Simone; Banning, Antje; Tikkanen, Ritva

    2014-01-01

    Non-neuronal acetylcholine plays a substantial role in the human skin by influencing adhesion, migration, proliferation and differentiation of keratinocytes. These processes are regulated by the Mitogen-Activated Protein (MAP) kinase cascade. Here we show that in HaCaT keratinocytes all five muscarinic receptor subtypes are expressed, but M1 and M3 are the subtypes involved in mitogenic signaling. Stimulation with the cholinergic agonist carbachol leads to activation of the MAP kinase extracellular signal regulated kinase, together with the protein kinase Akt. The activation is fully dependent on the transactivation of the epidermal growth factor receptor (EGFR), which even appears to be the sole pathway for the muscarinic receptors to facilitate MAP kinase activation in HaCaT cells. The transactivation pathway involves a triple-membrane-passing process, based on activation of matrix metalloproteases, and extracellular ligand release; whereas phosphatidylinositol 3-kinase, Src family kinases or protein kinase C do not appear to be involved in MAP kinase activation. Furthermore, phosphorylation, ubiquitination and endocytosis of the EGF receptor after cholinergic transactivation are different from that induced by a direct stimulation with EGF, suggesting that ligands other than EGF itself mediate the cholinergic transactivation. PMID:25421240

  20. Effective Targeting of Estrogen Receptor Negative Breast Cancers with the Protein Kinase D inhibitor CRT0066101

    PubMed Central

    Borges, Sahra; Perez, Edith A.; Thompson, E. Aubrey; Radisky, Derek C.; Geiger, Xochiquetzal J.; Storz, Peter

    2015-01-01

    Invasive ductal carcinomas (IDCs) of the breast are associated with altered expression of hormone receptors (HR), amplification or overexpression of HER2, or a triple-negative phenotype. The most aggressive cases of IDC are characterized by a high proliferation rate, a great propensity to metastasize and their ability to resist to standard chemotherapy, hormone therapy or HER2 targeted therapy. Using progression tissue microarrays we here demonstrate that the serine/threonine kinase Protein Kinase D3 (PKD3) is highly up-regulated in estrogen receptor (ER)-negative tumors. We identify direct binding of the estrogen receptor to the PRKD3 gene promoter as a mechanism of inhibition of PKD3 expression. Loss of ER results in upregulation of PKD3 leading to all hallmarks of aggressive IDC, including increased cell proliferation, migration and invasion. This identifies ER-negative breast cancers as ideal for treatment with the PKD inhibitor CRT0066101. We show that similar to a knockdown of PKD3, treatment with this inhibitor targets all tumorigenic processes in vitro and decreases growth of primary tumors and metastasis in vivo. Our data strongly support the development of PKD inhibitors for clinical use for ER-negative breast cancers, including the triple-negative phenotype. PMID:25852060

  1. 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.

  2. Identification and structure-function analysis of subfamily selective G protein-coupled receptor kinase inhibitors.

    PubMed

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

    2015-01-16

    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.

  3. Bioluminescence resonance energy transfer methods to study G protein-coupled receptor-receptor tyrosine kinase heteroreceptor complexes.

    PubMed

    Borroto-Escuela, Dasiel O; Flajolet, Marc; Agnati, Luigi F; Greengard, Paul; Fuxe, Kjell

    2013-01-01

    A large body of evidence indicates that G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) can form heteroreceptor complexes. In these complexes, the signaling from each interacting protomer is modulated to produce an integrated and therefore novel response upon agonist(s) activation. In the GPCR-RTK heteroreceptor complexes, GPCRs can activate RTK in the absence of added growth factor through the use of RTK signaling molecules. This integrative phenomenon is reciprocal and can place also RTK signaling downstream of GPCR. Formation of either stable or transient complexes by these two important classes of membrane receptors is involved in regulating all aspects of receptor function, from ligand binding to signal transduction, trafficking, desensitization, and downregulation among others. Functional phenomena can be modulated with conformation-specific inhibitors that stabilize defined GPCR states to abrogate both GPCR agonist- and growth factor-stimulated cell responses or by means of small interfering heteroreceptor complex interface peptides. The bioluminescence resonance energy transfer (BRET) technology has emerged as a powerful method to study the structure of heteroreceptor complexes closely associated with the study of receptor-receptor interactions in such complexes. In this chapter, we provide an overview of different BRET(2) assays that can be used to study the structure of GPCR-RTK heteroreceptor complexes and their functions. Various experimental designs for optimization of these experiments are also described.

  4. BIOLUMINISCENCE RESONANCE ENERGY TRANSFER (BRET) METHODS TO STUDY G PROTEIN-COUPLED RECEPTOR - RECEPTOR TYROSINE KINASE HETERORECEPTOR COMPLEXES

    PubMed Central

    Borroto-Escuela, Dasiel O.; Flajolet, Marc; Agnati, Luigi F.; Greengard, Paul; Fuxe, Kjell

    2014-01-01

    A large body of evidence indicates that G protein-coupled receptors (GPCRs) and Receptor tyrosine kinases (RTKs) can form heteroreceptor complexes. In these complexes, the signalling from each interacting protomer is modulated to produce an integrated and therefore novel response upon agonist(s) activation. In the GPCR-RTK heteroreceptor complexes, GPCRs can activate RTK in the absence of added growth factor through the use of RTK signalling molecules. This integrative phenomenon is reciprocal, and can place also RTK signalling downstream of GPCR. Formation of either stable or transient complexes by these two important classes of membrane receptors is involved in regulating all aspects of receptor function, from ligand binding to signal transduction, trafficking, desensitization and down regulation among others. Functional phenomena can be modulated with conformation-specific inhibitors that stabilize defined GPCR states to abrogate both GPCR agonist- and growth factor-stimulated cell responses or by means of small interfering heteroreceptor complex interface peptides. The bioluminescence resonance energy transfer (BRET) technology has emerged as a powerful method to study the structure of heteroreceptor complexes closely associated with the study of receptor-receptor interactions in such complexes. In this work we provide an overview of different BRET2 assays that can be used to study the structure of GPCR-RTK heteroreceptor complexes and their functions. Various experimental designs for optimization of these experiments are also described. PMID:24143976

  5. The inducible G protein-coupled receptor edg-1 signals via the G(i)/mitogen-activated protein kinase pathway.

    PubMed

    Lee, M J; Evans, M; Hla, T

    1996-05-10

    The edg-1 gene encodes an inducible G protein-coupled receptor (GPR) homologue that is induced during the in vitro differentiation of human endothelial cells. The aim of this study was to investigate the G protein-coupling and -signaling properties of the edg-1 polypeptide. The third cytosolic loop (i3) of edg-1 associates with G(i) alpha and G(o) alpha polypeptides in a guanosine 5'-O-(thiotriphosphate)-sensitive manner. Immunoprecipitation of the edg-1 polypeptide in transfected cells results in the co-precipitation of G(i) alpha 1 and G(i) alpha 3 polypeptides. These data strongly suggest that edg-1 is capable of coupling to the Gi pathway. Overexpression of the edg-1 GPR in human embryonic kidney 293 cells results in the sustained activation of the MAP kinase activity that is blocked by pertussis toxin treatment. Moreover, NIH3T3 cells permanently transfected with edg-1 exhibit enhanced MAP kinase and phospholipase A2 activities. These data suggest that the G(i)/mitogen-activated protein kinase pathway is a major signaling pathway regulated by the orphan receptor edg-1. PMID:8626678

  6. [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.

  7. Oestrogen receptors interact with the α-catalytic subunit of AMP-activated protein kinase

    PubMed Central

    Lipovka, Yulia; Chen, Hao; Vagner, Josef; Price, Theodore J.; Tsao, Tsu-Shuen; Konhilas, John P.

    2015-01-01

    Normal and pathological stressors engage the AMP-activated protein kinase (AMPK) signalling axis to protect the cell from energetic pressures. Sex steroid hormones also play a critical role in energy metabolism and significantly modify pathological progression of cardiac disease, diabetes/obesity and cancer. AMPK is targeted by 17β-oestradiol (E2), the main circulating oestrogen, but the mechanism by which E2 activates AMPK is currently unknown. Using an oestrogen receptor α/β (ERα/β) positive (T47D) breast cancer cell line, we validated E2-dependent activation of AMPK that was mediated through ERα (not ERβ) by using three experimental strategies. A series of co-immunoprecipitation experiments showed that both ERs associated with AMPK in cancer and striated (skeletal and cardiac) muscle cells. We further demonstrated direct binding of ERs to the α-catalytic subunit of AMPK within the βγ-subunit-binding domain. Finally, both ERs interacted with the upstream liver kinase B 1 (LKB1) kinase complex, which is required for E2-dependent activation of AMPK. We conclude that E2 activates AMPK through ERα by direct interaction with the βγ-binding domain of AMPKα. PMID:26374855

  8. Regulation of group II metabotropic glutamate receptors by G protein-coupled receptor kinases: mGlu2 receptors are resistant to homologous desensitization.

    PubMed

    Iacovelli, L; Molinaro, G; Battaglia, G; Motolese, M; Di Menna, L; Alfiero, M; Blahos, J; Matrisciano, F; Corsi, M; Corti, C; Bruno, V; De Blasi, A; Nicoletti, F

    2009-04-01

    We examined the regulation of mGlu2 and mGlu3 metabotropic glutamate receptor signaling prompted by the emerging role of these receptor subtypes as therapeutic targets for psychiatric disorders, such as anxiety and schizophrenia. In transfected human embryonic kidney 293 cells, G-protein-coupled receptor kinase (GRK) 2 and GRK3 fully desensitized the agonist-dependent inhibition of cAMP formation mediated by mGlu3 receptors. In contrast, GRK2 or other GRKs did not desensitize the cAMP response to mGlu2 receptor activation. Desensitization of mGlu3 receptors by GRK2 required an intact kinase activity, as shown by the use of the kinase-dead mutant GRK2-K220R or the recombinant GRK2 C-terminal domain. Overexpression of beta-arrestin1 also desensitized mGlu3 receptors and did not affect the cAMP signaling mediated by mGlu2 receptors. The difference in the regulation of mGlu2 and mGlu3 receptors was signal-dependent because GRK2 desensitized the activation of the mitogen-activated protein kinase pathway mediated by both mGlu2 and mGlu3 receptors. In vivo studies confirmed the resistance of mGlu2 receptor-mediated cAMP signaling to homologous desensitization. Wild-type, mGlu2(-/-), or mGlu3(-/-) mice were treated intraperitoneally with saline or the mixed mGlu2/3 receptor agonist (-)-2-oxa-4-aminobicyclo[3.1.0]-exhane-4,6-dicarboxylic acid (LY379268; 1 mg/kg) once daily for 7 days. Inhibition of forskolin-stimulated cAMP formation by LY379268 was measured in cortical slices prepared 24 h after the last injection. Agonist pretreatment fully desensitized the cAMP response in wild-type and mGlu2(-/-) mice but had no effect in mGlu3(-/-) mice, in which LY379268 could only activate the mGlu2 receptor. We predict the lack of tolerance when mixed mGlu2/3 receptor agonists or selective mGlu2 enhancers are used continually in patients. PMID:19164443

  9. 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.

  10. 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

  11. 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.

  12. Molecular mechanism for inhibition of G protein-coupled receptor kinase 2 by a selective RNA aptamer

    PubMed Central

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

    2012-01-01

    SUMMARY 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 αF-αG 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. PMID:22727813

  13. Roles of mitogen activated protein kinases and EGF receptor in arsenite-stimulated matrix metalloproteinase-9 production

    SciTech Connect

    Cooper, Karen L.; Myers, Terrance Alix; Rosenberg, Martina; Chavez, Miquella; Hudson, Laurie G. . E-mail: lghudson@unm.edu

    2004-11-01

    The dermatotoxicity of arsenic is well established and epidemiological studies identify an increased incidence of keratinocytic tumors (basal cell and squamous cell carcinoma) associated with arsenic exposure. Little is known about the underlying mechanisms of arsenic-mediated skin carcinogenesis, but activation of mitogen-activated protein (MAP) kinases and subsequent regulation of downstream target genes may contribute to tumor promotion and progression. In this study, we investigated activation of the extracellular signal regulated kinase (ERK) and the stress-associated kinase p38 by arsenite in HaCat cells, a spontaneously immortalized human keratinocyte cell line. Arsenite concentrations {>=}100 {mu}M stimulate rapid activation of p38 and ERK MAP kinases. However, upon extended exposure (24 h), persistent stimulation of p38 and ERK MAP kinases was detected at low micromolar concentrations of arsenite. Although ERK and p38 were activated with similar time and concentration dependence, the mechanism of activation differed for these two MAP kinases. ERK activation by arsenite was fully dependent on the catalytic activity of the epidermal growth factor (EGF) receptor and partially dependent on Src-family kinase activity. In contrast, p38 activation was independent of EGF receptor or Src-family kinase activity. Arsenite-stimulated MAP kinase signal transduction resulted in increased production of matrix metalloproteinase (MMP)-9, an AP-1 regulated gene product. MMP-9 induction by arsenite was prevented when EGF receptor or MAP kinase signaling was inhibited. These studies indicate that EGF receptor activation is a component of arsenite-mediated signal transduction and gene expression in keratinocytes and that low micromolar concentrations of arsenite stimulate key signaling pathways upon extended exposure. Stimulation of MAP kinase cascades by arsenic and subsequent regulation of genes including c-fos, c-jun, and the matrix degrading proteases may play an important

  14. 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

  15. Resveratrol up-regulates AMPA receptor expression via AMP-activated protein kinase-mediated protein translation.

    PubMed

    Wang, Guan; Amato, Stephen; Gilbert, James; Man, Heng-Ye

    2015-08-01

    Resveratrol is a phytoalexin that confers overall health benefits including positive regulation in brain function such as learning and cognition. However, whether and how resveratrol affects synaptic activity remains largely unknown. α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are glutamatergic receptors that mediate the majority of fast excitatory transmission and synaptic plasticity, and thus play a critical role in higher brain functions, including learning and memory. We find that in rat primary neurons, resveratrol can rapidly increase AMPAR protein level, AMPAR synaptic accumulation and the strength of excitatory synaptic transmission. The resveratrol effect on AMPAR protein expression is independent of sirtuin 1 (SIRT1), the conventional downstream target of resveratrol, but rather is mediated by AMP-activated protein kinase (AMPK) and subsequent downstream phosphoinositide 3-kinase (PI3K)/Akt signaling. Application of the AMPK specific activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) mimics the effects of resveratrol on both signaling and AMPAR expression. The resveratrol-induced increase in AMPAR expression results from elevated protein synthesis via regulation of the eukaryotic initiation factor (eIF) 4E/4G complex. Disruption of the translation initiation complex completely blocks resveratrol-dependent AMPAR up-regulation. These findings indicate that resveratrol may regulate brain function through facilitation of AMPAR biogenesis and synaptic transmission.

  16. Binding Affinity Prediction for Ligands and Receptors Forming Tautomers and Ionization Species: Inhibition of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2)

    PubMed Central

    2012-01-01

    Treatment of ionization and tautomerism of ligands and receptors is one of the unresolved issues in structure-based prediction of binding affinities. Our solution utilizes the thermodynamic master equation, expressing the experimentally observed association constant as the sum of products, each valid for a specific ligand–receptor species pair, consisting of the association microconstant and the fractions of the involved ligand and receptor species. The microconstants are characterized by structure-based simulations, which are run for individual species pairs. Here we incorporated the multispecies approach into the QM/MM linear response method and used it for structural correlation of published inhibition data on mitogen-activated protein kinase (MAPK)-activated protein kinase (MK2) by 66 benzothiophene and pyrrolopyridine analogues, forming up to five tautomers and seven ionization species under experimental conditions. Extensive cross-validation showed that the resulting models were stable and predictive. Inclusion of all tautomers and ionization ligand species was essential: the explained variance increased to 90% from 66% for the single-species model. PMID:22280316

  17. G protein-coupled receptor kinase 2 positively regulates epithelial cell migration

    PubMed Central

    Penela, Petronila; Ribas, Catalina; Aymerich, Ivette; Eijkelkamp, Niels; Barreiro, Olga; Heijnen, Cobi J; Kavelaars, Annemieke; Sánchez-Madrid, Francisco; Mayor, Federico

    2008-01-01

    Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a sphingosine-1-phosphate (S1P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration. PMID:18369319

  18. Plasmodesmal receptor-like kinases identified through analysis of rice cell wall extracted proteins.

    PubMed

    Jo, Yeonhwa; Cho, Won Kyong; Rim, Yeonggil; Moon, Juyeon; Chen, Xiong-Yan; Chu, Hyosub; Kim, Cha Young; Park, Zee-Yong; Lucas, William J; Kim, Jae-Yean

    2011-01-01

    In plants, plasmodesmata (PD) are intercellular channels that function in both metabolite exchange and the transport of proteins and RNAs. Currently, many of the PD structural and regulatory components remain to be elucidated. Receptor-like kinases (RLKs) belonging to a notably expanded protein family in plants compared to the animal kingdom have been shown to play important roles in plant growth, development, pathogen resistance, and cell death. In this study, cell biological approaches were used to identify potential PD-associated RLK proteins among proteins contained within cell walls isolated from rice callus cultured cells. A total of 15 rice RLKs were investigated to determine their subcellular localization, using an Agrobacterium-mediated transient expression system. Of these six PD-associated RLKs were identified based on their co-localization with a viral movement protein that served as a PD marker, plasmolysis experiments, and subcellular localization at points of wall contact between spongy mesophyll cells. These findings suggest potential PD functions in apoplasmic signaling in response to environmental stimuli and developmental inputs. PMID:21161304

  19. 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.

  20. Phosphorylation of farnesoid X receptor by protein kinase C promotes its transcriptional activity.

    PubMed

    Gineste, Romain; Sirvent, Audrey; Paumelle, Réjane; Helleboid, Stéphane; Aquilina, Alexis; Darteil, Raphaël; Hum, Dean W; Fruchart, Jean-Charles; Staels, Bart

    2008-11-01

    The farnesoid X receptor (FXR, NR1H4) belongs to the nuclear receptor superfamily and is activated by bile acids such as chenodeoxycholic acid, or synthetic ligands such as GW4064. FXR is implicated in the regulation of bile acid, lipid, and carbohydrate metabolism. Posttranslational modifications regulating its activity have not been investigated yet. Here, we demonstrate that calcium-dependent protein kinase C (PKC) inhibition impairs ligand-mediated regulation of FXR target genes. Moreover, in a transactivation assay, we show that FXR transcriptional activity is modulated by PKC. Furthermore, phorbol 12-myristate 13-acetate , a PKC activator, induces the phosphorylation of endogenous FXR in HepG2 cells and PKCalpha phosphorylates in vitro FXR in its DNA-binding domain on S135 and S154. Mutation of S135 and S154 to alanine residues reduces in cell FXR phosphorylation. In contrast to wild-type FXR, mutant FXRS135AS154A displays an impaired PKCalpha-induced transactivation and a decreased ligand-dependent FXR transactivation. Finally, phosphorylation of FXR by PKC promotes the recruitment of peroxisomal proliferator-activated receptor gamma coactivator 1alpha. In conclusion, these findings show that the phosphorylation of FXR induced by PKCalpha directly modulates the ability of agonists to activate FXR.

  1. TLR4 Signaling augments monocyte chemotaxis by regulating G protein-coupled receptor kinase 2 translocation.

    PubMed

    Liu, Zheng; Jiang, Yong; Li, Yuehua; Wang, Juan; Fan, Liyan; Scott, Melanie J; Xiao, Guozhi; Li, Song; Billiar, Timothy R; Wilson, Mark A; Fan, Jie

    2013-07-15

    Monocytes are critical effector cells of the innate immune system that protect the host by migrating to inflammatory sites, differentiating to macrophages and dendritic cells, eliciting immune responses, and killing pathogenic microbes. MCP-1, also known as CCL2, plays an important role in monocyte activation and migration. The chemotactic function of MCP-1 is mediated by binding to the CCR2 receptor, a member of the G protein-coupled receptor (GPCR) family. Desensitization of GPCR chemokine receptors is an important regulator of the intensity and duration of chemokine stimulation. GPCR kinases (GRKs) induce GPCR phosphorylation, and this leads to GPCR desensitization. Regulation of subcellular localization of GRKs is considered an important early regulatory mechanism of GRK function and subsequent GPCR desensitization. Chemokines and LPS are both present during Gram-negative bacterial infection, and LPS often synergistically exaggerates leukocyte migration in response to chemokines. In this study, we investigated the role and mechanism of LPS-TLR4 signaling on the regulation of monocyte chemotaxis. We demonstrate that LPS augments MCP-1-induced monocyte migration. We also show that LPS, through p38 MAPK signaling, induces phosphorylation of GRK2 at serine 670, which, in turn, suppresses GRK2 translocation to the membrane, thereby preventing GRK2-initiated internalization and desensitization of CCR2 in response to MCP-1. This results in enhanced monocyte migration. These findings reveal a novel function for TLR4 signaling in promoting innate immune cell migration.

  2. Dopamine D1 receptor and protein kinase C isoforms in spontaneously hypertensive rats.

    PubMed

    Yao, L P; Li, X X; Yu, P Y; Xu, J; Asico, L D; Jose, P A

    1998-12-01

    -Dopamine, via D1-like receptors, stimulates the activity of both protein kinase A (PKA) and protein kinase C (PKC), which results in inhibition of renal sodium transport. Since D1-like receptors differentially regulate sodium transport in normotensive and hypertensive rats, they may also differentially regulate PKC expression in these rat strains. Thus, 2 different D1-like agonists (fenoldopam or SKF 38393) were infused into the renal artery of anesthetized normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) (n=5 to 6/drug/strain). Ten or 60 minutes after starting the D1-like agonist infusion, both the infused kidney and the noninfused kidney that served as control were prepared for analysis. The D1-like agonists produced a greater diuresis and natriuresis and inhibited Na+,K+-ATPase activity in proximal tubule (PT) and medullary thick ascending limb (mTAL) to a greater extent in WKY (Delta20+/-1%) than in SHR (Delta7+/-1%, P<0.001). D1-like agonists had no effect on PKC-alpha or PKC-lambda expression in either membrane or cytosol but increased PKC-theta expression in PT in both WKY and SHR at 10 minutes but not at 60 minutes. However, membranous PKC-delta expression in PT and mTAL decreased in WKY but increased in SHR with either 10 or 60 minutes of D1-like agonist infusion. D1-like agonists also decreased membranous PKC-zeta expression in PT and mTAL in WKY but increased it in PT but not in mTAL in SHR. We conclude that there is differential regulation of PKC isoform expression by D1-like agonists that inhibits membranous PKC-delta and PKC-zeta in WKY but stimulates them in SHR; this effect in SHR is similar to the stimulatory effect of norepinephrine and angiotensin II and may be a mechanism for their differential effects on sodium transport.

  3. 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...

  4. δ-Opioid receptors stimulate the metabolic sensor AMP-activated protein kinase through coincident signaling with G(q/11)-coupled receptors.

    PubMed

    Olianas, Maria C; Dedoni, Simona; Olianas, Alessandra; Onali, Pierluigi

    2012-02-01

    AMP-activated protein kinase (AMPK) and δ-opioid receptors (DORs) are both involved in controlling cell survival, energy metabolism, and food intake, but little is known on the interaction between these two signaling molecules. Here we show that activation of human DORs stably expressed in Chinese hamster ovary (CHO) cells increased AMPK activity and AMPK phosphorylation on Thr172. DOR-induced AMPK phosphorylation was prevented by pertussis toxin, reduced by protein kinase A (PKA) activators, and unaffected by PKA, transforming growth factor-β-activated kinase 1, mitogen-activated protein kinase, and protein kinase C inhibitors. Conversely, the DOR effect was reduced by Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) inhibition, apyrase treatment, G(q/11) antagonism, and blockade of P2 purinergic receptors. Apyrase treatment also depressed DOR stimulation of intracellular Ca(2+) concentration, whereas P2 receptor antagonism blocked DOR stimulation of inositol phosphate accumulation. In SH-SY5Y neuroblastoma cells and primary olfactory bulb neurons, DOR activation failed to affect AMPK phosphorylation per se but potentiated the stimulation by either muscarinic agonists or 2-methyl-thio-ADP. Sequestration of G protein βγ subunits (Gβγ) blocked the DOR potentiation of AMPK phosphorylation induced by oxotremorine-M. In CHO cells, the AMPK activator 5-aminoimidazole-4-carboxamide1-β-D-ribonucleoside stimulated AMPK phosphorylation and glucose uptake, whereas pharmacological inhibition of AMPK, expression of a dominant-negative mutant of AMPKα1, and P2Y receptor blockade reduced DOR-stimulated glucose uptake. The data indicate that in different cell systems, DOR activation up-regulates AMPK through a Gβγ-dependent synergistic interaction with G(q/11)-coupled receptors, potentiating Ca(2+) release and CaMKKβ-dependent AMPK phosphorylation. In CHO cells, this coincident signaling mechanism is involved in DOR-induced glucose uptake. PMID:22031472

  5. Downregulation of the Ras–Mitogen-Activated Protein Kinase Pathway by the EphB2 Receptor Tyrosine Kinase Is Required for Ephrin-Induced Neurite Retraction

    PubMed Central

    Elowe, Sabine; Holland, Sacha J.; Kulkarni, Sarang; Pawson, Tony

    2001-01-01

    Activation of the EphB2 receptor tyrosine kinase by clustered ephrin-B1 induces growth cone collapse and neurite retraction in differentiated NG108 neuronal cells. We have investigated the cytoplasmic signaling events associated with EphB2-induced cytoskeletal reorganization in these neuronal cells. We find that unlike other receptor tyrosine kinases, EphB2 induces a pronounced downregulation of GTP-bound Ras and consequently of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. A similar inhibition of the Ras-MAPK pathway was observed on stimulation of endogenous EphB2 in COS-1 cells. Inactivation of Ras, induced by ephrin B1 stimulation of NG108 neuronal cells, requires EphB2 tyrosine kinase activity and is blocked by a truncated form of p120-Ras GTPase-activating protein (p120-RasGAP), suggesting that EphB2 signals through the SH2 domain protein p120-RasGAP to inhibit the Ras-MAPK pathway. Suppression of Ras activity appears functionally important, since expression of a constitutively active variant of Ras impaired the ability of EphB2 to induce neurite retraction. In addition, EphB2 attenuated the elevation in ERK activation induced by attachment of NG108 cells to fibronectin, indicating that the EphB2 receptor can modulate integrin signaling to the Ras GTPase. These results suggest that a primary function of EphB2, a member of the most populous family of receptor tyrosine kinases, is to inactivate the Ras-MAPK pathway in a fashion that contributes to cytoskeletal reorganization and adhesion responses in neuronal growth cones. PMID:11585923

  6. A systematic investigation of the protein kinases involved in NMDA receptor-dependent LTD: evidence for a role of GSK-3 but not other serine/threonine kinases

    PubMed Central

    Peineau, Stéphane; Nicolas, Céline S; Bortolotto, Zuner A; Bhat, Ratan V; Ryves, W Jonathan; Harwood, Adrian J; Dournaud, Pascal; Fitzjohn, Stephen M; Collingridge, Graham L

    2009-01-01

    Background The signalling mechanisms involved in the induction of N-methyl-D-aspartate (NMDA) receptor-dependent long-term depression (LTD) in the hippocampus are poorly understood. Numerous studies have presented evidence both for and against a variety of second messengers systems being involved in LTD induction. Here we provide the first systematic investigation of the involvement of serine/threonine (ser/thr) protein kinases in NMDAR-LTD, using whole-cell recordings from CA1 pyramidal neurons. Results Using a panel of 23 inhibitors individually loaded into the recorded neurons, we can discount the involvement of at least 57 kinases, including PKA, PKC, CaMKII, p38 MAPK and DYRK1A. However, we have been able to confirm a role for the ser/thr protein kinase, glycogen synthase kinase 3 (GSK-3). Conclusion The present study is the first to investigate the role of 58 ser/thr protein kinases in LTD in the same study. Of these 58 protein kinases, we have found evidence for the involvement of only one, GSK-3, in LTD. PMID:19583853

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

    PubMed

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

    1999-01-15

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

  8. G-protein Receptor Kinase 5 Regulates the Cannabinoid Receptor 2-induced Up-regulation of Serotonin 2A Receptors*

    PubMed Central

    Franklin, Jade M.; Carrasco, Gonzalo A.

    2013-01-01

    We have recently reported that cannabinoid agonists can up-regulate and enhance the activity of serotonin 2A (5-HT2A) receptors in the prefrontal cortex (PFCx). Increased expression and activity of cortical 5-HT2A receptors has been associated with neuropsychiatric disorders, such as anxiety and schizophrenia. Here we report that repeated CP55940 exposure selectively up-regulates GRK5 proteins in rat PFCx and in a neuronal cell culture model. We sought to examine the mechanism underlying the regulation of GRK5 and to identify the role of GRK5 in the cannabinoid agonist-induced up-regulation and enhanced activity of 5-HT2A receptors. Interestingly, we found that cannabinoid agonist-induced up-regulation of GRK5 involves CB2 receptors, β-arrestin 2, and ERK1/2 signaling because treatment with CB2 shRNA lentiviral particles, β-arrestin 2 shRNA lentiviral particles, or ERK1/2 inhibitor prevented the cannabinoid agonist-induced up-regulation of GRK5. Most importantly, we found that GRK5 shRNA lentiviral particle treatment prevented the cannabinoid agonist-induced up-regulation and enhanced 5-HT2A receptor-mediated calcium release. Repeated cannabinoid exposure was also associated with enhanced phosphorylation of CB2 receptors and increased interaction between β-arrestin 2 and ERK1/2. These latter phenomena were also significantly inhibited by GRK5 shRNA lentiviral treatment. Our results suggest that sustained activation of CB2 receptors, which up-regulates 5-HT2A receptor signaling, enhances GRK5 expression; the phosphorylation of CB2 receptors; and the β-arrestin 2/ERK interactions. These data could provide a rationale for some of the adverse effects associated with repeated cannabinoid agonist exposure. PMID:23592773

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

    PubMed Central

    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

    2015-01-01

    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. DOI: http://dx.doi.org/10.7554/eLife.06587.001 PMID:26052747

  10. 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

    2015-01-01

    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. PMID:26052747

  11. 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.

  12. Activation of the Ras/Mitogen-Activated Protein Kinase Pathway by Kinase-Defective Epidermal Growth Factor Receptors Results in Cell Survival but Not Proliferation

    PubMed Central

    Walker, Francesca; Kato, Akiko; Gonez, L. Jorge; Hibbs, Margaret L.; Pouliot, Normand; Levitzki, Alexander; Burgess, Antony W.

    1998-01-01

    Signalling by the epidermal growth factor (EGF) receptor (EGFR) has been studied intensively, but for most cell types the analysis is complicated by the fact that EGFR not only homodimerizes but can also form heterodimers with other EGFR family members. Heterodimerization is a particular problem in the study of EGFR mutants, where the true phenotype of the mutants is confounded by the contribution of the heterodimer partner to signal transduction. We have made use of the murine hemopoietic cell line BaF/3, which does not express EGFR family members, to express wild-type (WT) EGFR, three kinase-defective EGFR mutants (V741G, Y740F, and K721R), or a C-terminally truncated EGFR (CT957) and have measured their responses to EGF. We found that under the appropriate conditions EGF can stimulate cell proliferation of BaF/3 cells expressing WT or CT957 EGFRs but not that of cells expressing the kinase-defective mutants. However, EGF promotes the survival of BaF/3 cells expressing either of the kinase-defective receptors (V741G and Y740F), indicating that these receptors can still transmit a survival signal. Analysis of the early signalling events by the WT, V741G, and Y740F mutant EGF receptors indicated that EGF stimulates comparable levels of Shc phosphorylation, Shc–GRB-2 association, and activation of Ras, B-Raf, and Erk-1. Blocking the mitogen-activated protein kinase (MAPK) signalling pathway with the specific inhibitor PD98059 abrogates completely the EGF-dependent survival of cells expressing the kinase-defective EGFR mutants but has no effect on the EGF-dependent proliferation mediated by WT and CT957 EGFRs. Similarly, the Src family kinase inhibitor PP1 abrogates EGF-dependent survival without affecting proliferation. However blocking phosphatidylinositol-3-kinase or JAK-2 kinase with specific inhibitors does arrest growth factor-dependent cell proliferation. Thus, EGFR-mediated mitogenic signalling in BaF/3 cells requires an intact EGFR tyrosine kinase activity

  13. 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

  14. Free fatty acids and protein kinase C activation induce GPR120 (free fatty acid receptor 4) phosphorylation.

    PubMed

    Sánchez-Reyes, Omar B; Romero-Ávila, M Teresa; Castillo-Badillo, Jean A; Takei, Yoshinori; Hirasawa, Akira; Tsujimoto, Gozoh; Villalobos-Molina, Rafael; García-Sáinz, J Adolfo

    2014-01-15

    GPR120, free fatty acid receptor 4, is a recently deorphanized G protein-coupled receptor that seems to play cardinal roles in the regulation of metabolism and in the pathophysiology of inflammatory and metabolic disorders. In the present work a GPR120-Venus fusion protein was expressed in HEK293 Flp-In T-REx cells and its function (increase in intracellular calcium) and phosphorylation were studied. It was observed that the fusion protein migrated in sodium dodecyl sulfate-polyacrylamide gels as a band with a mass of ≈70-75kDa, although other bands of higher apparent weight (>130kDa) were also detected. Cell stimulation with docosahexaenoic acid or α-linolenic acid induced concentration-dependent increases in intracellular calcium and GPR120 phosphorylation. Activation of protein kinase C with phorbol esters also induced a marked receptor phosphorylation but did not alter the ability of 1µM docosahexaenoic acid to increase the intracellular calcium concentration. Phorbol ester-induced GPR120 phosphorylation, but not that induced with docosahexaenoic acid, was blocked by protein kinase C inhibitors (bis-indolyl-maleimide I and Gö 6976) suggesting that conventional kinase isoforms mediate this action. The absence of effect of protein kinase C inhibitors on agonist-induced GPR120 phosphorylation indicates that this kinase does not play a major role in agonist-induced receptor phosphorylation. Docosahexaenoic acid action was associated with marked GPR120 internalization whereas that induced with phorbol esters was smaller at early times. PMID:24239485

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

    PubMed

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

    2005-09-01

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

  16. The Evolving Impact of G Protein-Coupled Receptor Kinases in Cardiac Health and Disease

    PubMed Central

    Sato, Priscila Y.; Chuprun, J. Kurt; Schwartz, Mathew; Koch, Walter J.

    2015-01-01

    G protein-coupled receptors (GPCRs) are important regulators of various cellular functions via activation of intracellular signaling events. Active GPCR signaling is shut down by GPCR kinases (GRKs) and subsequent β-arrestin-mediated mechanisms including phosphorylation, internalization, and either receptor degradation or resensitization. The seven-member GRK family varies in their structural composition, cellular localization, function, and mechanism of action (see sect. II). Here, we focus our attention on GRKs in particular canonical and novel roles of the GRKs found in the cardiovascular system (see sects. III and IV). Paramount to overall cardiac function is GPCR-mediated signaling provided by the adrenergic system. Overstimulation of the adrenergic system has been highly implicated in various etiologies of cardiovascular disease including hypertension and heart failure. GRKs acting downstream of heightened adrenergic signaling appear to be key players in cardiac homeostasis and disease progression, and herein we review the current data on GRKs related to cardiac disease and discuss their potential in the development of novel therapeutic strategies in cardiac diseases including heart failure. PMID:25834229

  17. 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.

  18. Activated protein kinase C binds to intracellular receptors in rat hepatocytes.

    PubMed Central

    Robles-Flores, M; García-Sáinz, J A

    1993-01-01

    The aim of this study was to identify in rat hepatocytes cellular polypeptides that bind protein kinase C (PKC) and may influence its activity and its compartmentation. At least seven proteins, with apparent M(r) values between 12,000 and 36,000, that behave like Receptors for Activated C-Kinase (RACKs) were found in the Triton-X-100-insoluble fraction of these cells; i.e. PKC bound to these polypeptides when it was in its active form. RACKS seem to be PKC substrates. Studies using isotype-specific PKC antibodies suggested some selectivity of RACKs, i.e. RACKs in the M(r) approximately 28,000-36,000 region bound PKC-alpha and PKC-beta in the presence of phosphatidylserine, diolein and Ca2+, whereas those of M(r) approximately 12,000-14,000 bound all isoforms studied, and, in contrast with the other RACKs, they did this even in the absence of Ca2+. Peptide I (KGDYEKILVALCGGN), which has a sequence suggested to be involved in the PKC-RACKs interaction [Mochly-Rosen, Khaner, Lopez and Smith (1991) J. Biol. Chem. 266, 14866-14868], inhibited PKC activity. Preincubation of RACKs with antisera directed against peptide I prevented PKC binding to them. The data suggest that peptide I blocks PKC binding to RACKs by two mechanisms: inhibition of PKC activity and competition with a putative binding site. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8257439

  19. Antagonist-induced micro-opioid receptor up-regulation decreases G-protein receptor kinase-2 and dynamin-2 abundance in mouse spinal cord.

    PubMed

    Patel, Minesh; Gomes, Benedict; Patel, Chintan; Yoburn, Byron C

    2002-06-20

    Chronic treatment with opioid receptor antagonists has been shown to increase the density of micro-, delta- and kappa-opioid receptors in cell culture and in the intact animal. Although opioid receptor antagonist-induced up-regulation is a robust phenomenon, the mechanisms responsible for the increase in receptor density remain unclear. In the present study, changes in a kinase and a GTPase that have been implicated in G-protein-coupled receptor regulation were examined following opioid receptor antagonist treatment. Mice were implanted s.c. with a naltrexone pellet or placebo pellet. On the eighth day following implantation, spinal cord was removed and G-protein receptor kinase-2 (GRK-2) and dynamin-2 abundance were determined using a quantitative immunoblot approach. Changes in micro-opioid receptor density were also determined. Naltrexone treatment produced a significant (145%) increase in micro-opioid receptor density. Naltrexone treatment was associated with a significant 36% decrease in GRK-2 and 30% decrease in dynamin-2 abundance in spinal cord. These data raise the possibility that opioid receptor antagonist-induced micro-opioid receptor up-regulation in the intact animal may be due to a reduction in constitutive internalization of opioid receptors.

  20. Phosphorylation of the human leukemia inhibitory factor (LIF) receptor by mitogen-activated protein kinase and the regulation of LIF receptor function by heterologous receptor activation.

    PubMed Central

    Schiemann, W P; Graves, L M; Baumann, H; Morella, K K; Gearing, D P; Nielsen, M D; Krebs, E G; Nathanson, N M

    1995-01-01

    We used a bacterially expressed fusion protein containing the entire cytoplasmic domain of the human leukemia inhibitory factor (LIF) receptor to study its phosphorylation in response to LIF stimulation. The dose- and time-dependent relationships for phosphorylation of this construct in extracts of LIF-stimulated 3T3-L1 cells were superimposable with those for the stimulation of mitogen-activated protein kinase (MAPK). Indeed, phosphorylation of the cytoplasmic domain of the low-affinity LIF receptor alpha-subunit (LIFR) in Mono Q-fractionated, LIF-stimulated 3T3-L1 extracts occurred only in those fractions containing activated MAPK; Ser-1044 served as the major phosphorylation site in the human LIFR for MAPK both in agonist-stimulated 3T3-L1 lysates and by recombinant extracellular signal-regulated kinase 2 in vitro. Expression in rat H-35 hepatoma cells of LIFR or chimeric granulocyte-colony-stimulating factor receptor (G-CSFR)-LIFR mutants lacking Ser-1044 failed to affect cytokine-stimulated expression of a reporter gene under the control of the beta-fibrinogen gene promoter but eliminated the insulin-induced attenuation of cytokine-stimulated gene expression. Thus, our results identify the human LIFR as a substrate for MAPK and suggest a mechanism of heterologous receptor regulation of LIFR signaling occurring at Ser-1044. Images Fig. 2 Fig. 4 PMID:7777512

  1. Protein kinase C regulates tonic GABAA receptor-mediated inhibition in the hippocampus and thalamus

    PubMed Central

    Bright, Damian P; Smart, Trevor G

    2013-01-01

    Tonic inhibition mediated by extrasynaptic GABAA receptors (GABAARs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABAARs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole-cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC-mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABAAR-mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABAAR activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABAARs, which represent a key extrasynaptic GABAAR isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABAARs. The inhibitory effects of PKC activation on α4β2δ GABAAR activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC-mediated phosphorylation can be an important physiological regulator of tonic GABAAR-mediated inhibition. PMID:24102973

  2. 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

  3. Receptor-Interacting Protein Kinase-2 Inhibition by CYLD Impairs Antibacterial Immune Responses in Macrophages

    PubMed Central

    Wex, Katharina; Schmid, Ursula; Just, Sissy; Wang, Xu; Wurm, Rebecca; Naumann, Michael; Schlüter, Dirk; Nishanth, Gopala

    2016-01-01

    Upon infection with intracellular bacteria, nucleotide oligomerization domain protein 2 recognizes bacterial muramyl dipeptide and binds, subsequently, to receptor-interacting serine/threonine kinase 2 (RIPK2), which activates immune responses via the nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB) and extracellular signal-regulated kinase (ERK) pathways. Activation of RIPK2 depends on its K63 ubiquitination by E3 ligases, whereas the deubiquitinating enzyme A20 counter regulates RIPK2 activity by cleaving K63-polyubiquitin chains from RIPK2. Here, we newly identify the deubiquitinating enzyme CYLD as a new inhibitor of RIPK2. We show that CYLD binds to and removes K63-polyubiquitin chains from RIPK2 in Listeria monocytogenes (Lm) infected murine bone marrow-derived macrophages. CYLD-mediated K63 deubiquitination of RIPK2 resulted in an impaired activation of both NF-κB and ERK1/2 pathways, reduced production of proinflammatory cytokines interleukin-6 (IL-6), IL-12, anti-listerial reactive oxygen species (ROS) and nitric oxide (NO), and, finally, impaired pathogen control. In turn, RIPK2 inhibition by siRNA prevented activation of NF-κB and ERK1/2 and completely abolished the protective effect of CYLD deficiency with respect to the production of IL-6, NO, ROS, and pathogen control. Noteworthy, CYLD also inhibited autophagy of Listeria in a RIPK2-ERK1/2-dependent manner. The protective function of CYLD deficiency was dependent on interferon gamma (IFN-γ) prestimulation of infected macrophages. Interestingly, the reduced NF-κB activation in CYLD-expressing macrophages limited the protective effect of IFN-γ by reducing NF-κB-dependent signal transducers and activators of transcription-1 (STAT1) activation. Taken together, our study identifies CYLD as an important inhibitor of RIPK2-dependent antibacterial immune responses in macrophages. PMID:26834734

  4. Src-Like adaptor protein (SLAP) binds to the receptor tyrosine kinase Flt3 and modulates receptor stability and downstream signaling.

    PubMed

    Kazi, Julhash U; Rönnstrand, Lars

    2012-01-01

    Fms-like tyrosine kinase 3 (Flt3) is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML). Src-like adaptor protein (SLAP) is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient samples suggests that SLAP mRNA is differentially expressed in different cancers and its expression was significantly increased in patients carrying the Flt3-ITD mutation. Thus, our data suggest a novel role of SLAP in different cancers and in modulation of receptor tyrosine kinase signaling apart from its conventional role in regulation of receptor stability.

  5. Intraplantar PGE2 causes nociceptive behaviour and mechanical allodynia: the role of prostanoid E receptors and protein kinases

    PubMed Central

    Kassuya, C A L; Ferreira, J; Claudino, R F; Calixto, J B

    2007-01-01

    Background and purpose: Receptor subtypes involved in PGE2-induced nociception are still controversial. The present study investigated the prostanoid E receptor (EP) subtypes and the protein kinase (PK) pathways involved in the nociception induced by PGE2 injection in the mouse paw. Experimental approach: Paw-licking and mechanical allodynia were measured in vivo and protein kinase activation ex vivo by Western blots of extracts of paw skin. Key results: Intraplantar (i.pl.) injection of PGE2 into the mouse paw caused nociceptive behaviour of short duration with mean ED50 of 1.43 nmol. PGE2 produced a longer-lasting mechanical allodynia, with an ED50 of 0.05 nmol. Intraplantar injection of antagonists at EP3 or EP4, but not at EP1 or EP2 receptors inhibited PGE2-induced paw-licking. Paw-licking caused by PGE2 was blocked by an inhibitor of PKA but only partially decreased by inhibition of the extracellular-regulated kinase (ERK). Selective inhibitors of PKC, c-Jun N-terminal kinase (JNK) or p38, all failed to affect PGE2-induced paw-licking. An EP3 antagonist inhibited PGE2-induced mechanical allodynia. However, inhibitors of PKA, PKC or ERK, but not p38 or JNK, also partially inhibited PGE2-induced mechanical allodynia. Western blot analyses confirmed that i.pl. injection of PGE2 activated PKA, PKCα, and mitogen activated kinases (MAPKs) in the paw. Co-treatment with EP3 or EP4 receptor antagonists reduced PGE2-induced PKA and ERK, but not PKCα activation. Conclusions and Implications: The present results indicate that the nociceptive behaviour and mechanical allodynia caused by i.pl. PGE2 are mediated through activation of distinct EP receptors and PK-dependent mechanisms. PMID:17310141

  6. Atomic Structure of GRK5 Reveals Distinct Structural Features Novel for G Protein-coupled Receptor Kinases*♦

    PubMed Central

    Komolov, Konstantin E.; Bhardwaj, Anshul; Benovic, Jeffrey L.

    2015-01-01

    G protein-coupled receptor kinases (GRKs) are members of the protein kinase A, G, and C families (AGC) and play a central role in mediating G protein-coupled receptor phosphorylation and desensitization. One member of the family, GRK5, has been implicated in several human pathologies, including heart failure, hypertension, cancer, diabetes, and Alzheimer disease. To gain mechanistic insight into GRK5 function, we determined a crystal structure of full-length human GRK5 at 1.8 Å resolution. GRK5 in complex with the ATP analog 5′-adenylyl β,γ-imidodiphosphate or the nucleoside sangivamycin crystallized as a monomer. The C-terminal tail (C-tail) of AGC kinase domains is a highly conserved feature that is divided into three segments as follows: the C-lobe tether, the active-site tether (AST), and the N-lobe tether (NLT). This domain is fully resolved in GRK5 and reveals novel interactions with the nucleotide and N-lobe. Similar to other AGC kinases, the GRK5 AST is an integral part of the nucleotide-binding pocket, a feature not observed in other GRKs. The AST also mediates contact between the kinase N- and C-lobes facilitating closure of the kinase domain. The GRK5 NLT is largely displaced from its previously observed position in other GRKs. Moreover, although the autophosphorylation sites in the NLT are >20 Å away from the catalytic cleft, they are capable of rapid cis-autophosphorylation suggesting high mobility of this region. In summary, we provide a snapshot of GRK5 in a partially closed state, where structural elements of the kinase domain C-tail are aligned to form novel interactions to the nucleotide and N-lobe not previously observed in other GRKs. PMID:26032409

  7. A MAPK cascade downstream of ERECTA receptor-like protein kinase regulates Arabidopsis inflorescence architecture by promoting localized cell proliferation.

    PubMed

    Meng, Xiangzong; Wang, Huachun; He, Yunxia; Liu, Yidong; Walker, John C; Torii, Keiko U; Zhang, Shuqun

    2012-12-01

    Spatiotemporal-specific cell proliferation and cell differentiation are critical to the formation of normal tissues, organs, and organisms. The highly coordinated cell differentiation and proliferation events illustrate the importance of cell-cell communication during growth and development. In Arabidopsis thaliana, ERECTA (ER), a receptor-like protein kinase, plays important roles in promoting localized cell proliferation, which determines inflorescence architecture, organ shape, and size. However, the downstream signaling components remain unidentified. Here, we report a mitogen-activated protein kinase (MAPK; or MPK) cascade that functions downstream of ER in regulating localized cell proliferation. Similar to an er mutant, loss of function of MPK3/MPK6 or their upstream MAPK kinases (MAPKKs; or MKKs), MKK4/MKK5, resulted in shortened pedicels and clustered inflorescences. Epistasis analysis demonstrated that the gain of function of MKK4 and MKK5 transgenes could rescue the loss-of-function er mutant phenotype at both morphological and cellular levels, suggesting that the MPK3/MPK6 cascade functions downstream of the ER receptor. Furthermore, YODA (YDA), a MAPKK kinase, was shown to be upstream of MKK4/MKK5 and downstream of ER in regulating inflorescence architecture based on both gain- and loss-of-function data. Taken together, these results suggest that the YDA-MKK4/MKK5-MPK3/MPK6 cascade functions downstream of the ER receptor in regulating localized cell proliferation, which further shapes the morphology of plant organs. PMID:23263767

  8. 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. PMID:27057269

  9. Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling

    PubMed Central

    Jung, Seung-Ryoung; Seo, Jong Bae; Deng, Yi; Asbury, Charles L.; Hille, Bertil

    2016-01-01

    Activated Gq protein–coupled receptors (GqPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein–tagged optical probes to monitor several consequences of PAR2 signaling, including PIP2 depletion and β-arrestin translocation in real time. During continuous activation of PAR2, PIP2 was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of β-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP2 restoration significantly and even adding a delayed secondary phase of further PIP2 depletion. These effects of β-arrestin knockdown on PIP2 recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient β-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein–coupled receptor kinase potentiated the PIP2 depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited β-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by β-arrestin to explain why knockdown of β-arrestin led to

  10. 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.

  11. B-type receptor for platelet-derived growth factor mediates a chemotactic response by means of ligand-induced activation of the receptor protein-tyrosine kinase.

    PubMed Central

    Westermark, B; Siegbahn, A; Heldin, C H; Claesson-Welsh, L

    1990-01-01

    Porcine aorta endothelial cells are devoid of receptors for platelet-derived growth factor (PDGF). We have transfected such cells with cDNA for the PDGF B-type receptor, both the wild-type receptor and a mutant form of the receptor (K634A), in which the putative nucleotide-binding lysine of the protein-tyrosine domain has been changed to alanine. Immunoprecipitation studies of metabolically labeled cells showed that both types of receptors were synthesized and processed to the mature form of Mr 190,000. In cells expressing the wild-type receptor, PDGF-BB, the natural ligand for the B-type receptor, induced membrane ruffling and reorganization of actin. Such a response has previously been seen in cells expressing the natural PDGF B-type receptor in response to PDGF-BB. No such effect was induced in nontransfected cells or in cells expressing the K634A mutant receptor. PDGF was also shown to be chemotactic for cells expressing the wild-type receptor, whereas no chemotactic response was elicited in control cells or in cells expressing the K634A mutant receptor. Our study thus provides formal evidence that the PDGF B-type receptor mediates a motility response including actin reorganization and chemotaxis. Furthermore, the results establish a role for the receptor-associated protein-tyrosine kinase in the transduction of the chemotactic signal. Images PMID:2153283

  12. TRPC3 amplifies B-cell receptor-induced ERK signalling via protein kinase D-dependent Rap1 activation.

    PubMed

    Numaga-Tomita, Takuro; Nishida, Motohiro; Putney, James W; Mori, Yasuo

    2016-01-15

    Sustained activation of extracellular-signal-regulated kinase (ERK) has an important role in the decision regarding the cell fate of B-lymphocytes. Recently, we demonstrated that the diacylglycerol-activated non-selective cation channel canonical transient receptor potential 3 (TRPC3) is required for the sustained ERK activation induced by the B-cell receptor. However, the signalling mechanism underlying TRPC3-mediated ERK activation remains elusive. In the present study, we have shown that TRPC3 mediates Ca(2+) influx to sustain activation of protein kinase D (PKD) in a protein kinase C-dependent manner in DT40 B-lymphocytes. The later phase of ERK activation depends on the small G-protein Rap1, known as a downstream target of PKD, whereas the earlier phase of ERK activation depends on the Ras protein. It is of interest that sustained ERK phosphorylation is required for the full induction of the immediate early gene Egr-1 (early growth response 1). These results suggest that TRPC3 reorganizes the BCR signalling complex by switching the subtype of small G-proteins to sustain ERK activation in B-lymphocytes.

  13. Effects of inhibitors of vascular endothelial growth factor receptor 2 and downstream pathways of receptor tyrosine kinases involving phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin or mitogen-activated protein kinase in canine hemangiosarcoma cell lines.

    PubMed

    Adachi, Mami; Hoshino, Yuki; Izumi, Yusuke; Sakai, Hiroki; Takagi, Satoshi

    2016-07-01

    Canine hemangiosarcoma (HSA) is a progressive malignant neoplasm with no current effective treatment. Previous studies showed that receptor tyrosine kinases and molecules within their downstream pathways involving phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (m-TOR) or mitogen-activated protein kinase (MAPK) were overexpressed in canine, human, and murine tumors, including HSA. The present study investigated the effects of inhibitors of these pathways in canine splenic and hepatic HSA cell lines using assays of cell viability and apoptosis. Inhibitors of the MAPK pathway did not affect canine HSA cell viability. However, cell viability was significantly reduced by exposure to inhibitors of vascular endothelial growth factor receptor 2 and the PI3K/Akt/m-TOR pathway; these inhibitors also induced apoptosis in these cell lines. These results suggest that these inhibitors reduce the proliferation of canine HSA cells by inducing apoptosis. Further study of these inhibitors, using xenograft mouse models of canine HSA, are warranted to explore their potential for clinical application. PMID:27408334

  14. Induction of G protein-coupled receptor kinases 2 and 3 contributes to the cross-talk between mu and ORL1 receptors following prolonged agonist exposure.

    PubMed

    Thakker, D R; Standifer, K M

    2002-11-01

    The molecular mechanism(s) underlying cross-tolerance between mu and opioid receptor-like 1 (ORL1) receptor agonists were investigated using two human neuroblastoma cell lines endogenously expressing these receptors and G protein-coupled receptor kinases (GRKs). Prolonged (24 h) activation of the mu receptor desensitized both mu and ORL1 receptor-mediated inhibition of forskolin-stimulated cAMP accumulation and upregulated GRK2 levels in SH-SY5Y and BE(2)-C cells. Prolonged ORL1 activation increased GRK2 levels and desensitized both receptors in SH-SY5Y cells. Upregulation of GRK2 correlated with increases in levels of transcription factors Sp1 or AP-2. PD98059, an upstream inhibitor of extracellular signal-regulated kinases 1 and 2 (ERK1/2), reversed all these events. Pretreatment with orphanin FQ/nociceptin (OFQ/N) also upregulated GRK3 levels in both cell lines, and desensitized both receptors in BE(2)-C cells. Protein kinase C (PKC), but not ERK1/2, inhibition blocked OFQ/N-mediated GRK3 induction and mu and ORL1 receptor desensitization in BE(2)-C cells. Antisense DNA treatment confirmed the involvement of GRK2/3 in mu and ORL1 desensitization. Here, we demonstrate for the first time a role for ERK1/2-mediated GRK2 induction in the development of tolerance to mu agonists, as well as cross-tolerance to OFQ/N. We also demonstrate that chronic OFQ/N-mediated desensitization of ORL1 and mu receptors occurs via cell-specific pathways, involving ERK1/2-dependent GRK2, or PKC-dependent and ERK1/2-independent GRK3 induction.

  15. Induction of G protein-coupled receptor kinases 2 and 3 contributes to the cross-talk between mu and ORL1 receptors following prolonged agonist exposure.

    PubMed

    Thakker, D R; Standifer, K M

    2002-11-01

    The molecular mechanism(s) underlying cross-tolerance between mu and opioid receptor-like 1 (ORL1) receptor agonists were investigated using two human neuroblastoma cell lines endogenously expressing these receptors and G protein-coupled receptor kinases (GRKs). Prolonged (24 h) activation of the mu receptor desensitized both mu and ORL1 receptor-mediated inhibition of forskolin-stimulated cAMP accumulation and upregulated GRK2 levels in SH-SY5Y and BE(2)-C cells. Prolonged ORL1 activation increased GRK2 levels and desensitized both receptors in SH-SY5Y cells. Upregulation of GRK2 correlated with increases in levels of transcription factors Sp1 or AP-2. PD98059, an upstream inhibitor of extracellular signal-regulated kinases 1 and 2 (ERK1/2), reversed all these events. Pretreatment with orphanin FQ/nociceptin (OFQ/N) also upregulated GRK3 levels in both cell lines, and desensitized both receptors in BE(2)-C cells. Protein kinase C (PKC), but not ERK1/2, inhibition blocked OFQ/N-mediated GRK3 induction and mu and ORL1 receptor desensitization in BE(2)-C cells. Antisense DNA treatment confirmed the involvement of GRK2/3 in mu and ORL1 desensitization. Here, we demonstrate for the first time a role for ERK1/2-mediated GRK2 induction in the development of tolerance to mu agonists, as well as cross-tolerance to OFQ/N. We also demonstrate that chronic OFQ/N-mediated desensitization of ORL1 and mu receptors occurs via cell-specific pathways, involving ERK1/2-dependent GRK2, or PKC-dependent and ERK1/2-independent GRK3 induction. PMID:12423667

  16. RhoA/phosphatidylinositol 3-kinase/protein kinase B/mitogen-activated protein kinase signaling after growth arrest-specific protein 6/mer receptor tyrosine kinase engagement promotes epithelial cell growth and wound repair via upregulation of hepatocyte growth factor in macrophages.

    PubMed

    Lee, Ye-Ji; Park, Hyun-Jung; Woo, So-Youn; Park, Eun-Mi; Kang, Jihee Lee

    2014-09-01

    Growth arrest-specific protein 6 (Gas6)/Mer receptor tyrosine kinase (Mer) signaling modulates cytokine secretion and helps to regulate the immune response and apoptotic cell clearance. Signaling pathways that activate an epithelial growth program in macrophages are still poorly defined. We report that Gas6/Mer/RhoA signaling can induce the production of epithelial growth factor hepatic growth factor (HGF) in macrophages, which ultimately promotes epithelial cell proliferation and wound repair. The RhoA/protein kinase B (Akt)/mitogen-activated protein (MAP) kinases, including p38 MAP kinase, extracellular signal-regulated protein kinase, and Jun NH2-terminal kinase axis in RAW 264.7 cells, was identified as Gas6/Mer downstream signaling pathway for the upregulation of HGF mRNA and protein. Conditioned medium from RAW 264.7 cells that had been exposed to Gas6 or apoptotic cells enhanced epithelial cell proliferation of the epithelial cell line LA-4 and wound closure. Cotreatment with an HGF receptor-blocking antibody or c-Met antagonist downregulated this enhancement. Inhibition of Mer with small interfering RNA (siRNA) or the RhoA/Rho kinase pathway by RhoA siRNA or Rho kinase pharmacologic inhibitor suppressed Gas6-induced HGF mRNA and protein expression in macrophages and blocked epithelial cell proliferation and wound closure induced by the conditioned medium. Our data provide evidence that macrophages can be reprogrammed by Gas6 to promote epithelial proliferation and wound repair via HGF, which is induced by the Mer/RhoA/Akt/MAP kinase pathway. Thus, defects in Gas6/Mer/RhoA signaling in macrophages may delay tissue repair after injury to the alveolar epithelium.

  17. Gravin orchestrates protein kinase A and β2-adrenergic receptor signaling critical for synaptic plasticity and memory.

    PubMed

    Havekes, Robbert; Canton, David A; Park, Alan J; Huang, Ted; Nie, Ting; Day, Jonathan P; Guercio, Leonardo A; Grimes, Quinn; Luczak, Vincent; Gelman, Irwin H; Baillie, George S; Scott, John D; Abel, Ted

    2012-12-12

    A kinase-anchoring proteins (AKAPs) organize compartmentalized pools of protein kinase A (PKA) to enable localized signaling events within neurons. However, it is unclear which of the many expressed AKAPs in neurons target PKA to signaling complexes important for long-lasting forms of synaptic plasticity and memory storage. In the forebrain, the anchoring protein gravin recruits a signaling complex containing PKA, PKC, calmodulin, and PDE4D (phosphodiesterase 4D) to the β2-adrenergic receptor. Here, we show that mice lacking the α-isoform of gravin have deficits in PKA-dependent long-lasting forms of hippocampal synaptic plasticity including β2-adrenergic receptor-mediated plasticity, and selective impairments of long-term memory storage. Furthermore, both hippocampal β2-adrenergic receptor phosphorylation by PKA, and learning-induced activation of ERK in the CA1 region of the hippocampus are attenuated in mice lacking gravin-α. We conclude that gravin compartmentalizes a significant pool of PKA that regulates learning-induced β2-adrenergic receptor signaling and ERK activation in the hippocampus in vivo, thereby organizing molecular interactions between glutamatergic and noradrenergic signaling pathways for long-lasting synaptic plasticity, and memory storage.

  18. Multiple Scaffolding Functions of β-Arrestins in the Degradation of G Protein-coupled Receptor Kinase 2*

    PubMed Central

    Nogués, Laura; Salcedo, Alicia; Mayor, Federico; Penela, Petronila

    2011-01-01

    G protein-coupled receptor kinase 2 (GRK2) plays a fundamental role in the regulation of G protein-coupled receptors (GPCRs), and changes in GRK2 expression levels can have an important impact on cell functions. GRK2 is known to be degraded by the proteasome pathway. We have shown previously that β-arrestins participate in enhanced kinase turnover upon GPCR stimulation by facilitating GRK2 phosphorylation by c-Src or by MAPK or by recruiting the Mdm2 E3 ubiquitin ligase to the receptor complex. In this report, we have investigated how such diverse β-arrestin scaffold functions are integrated to modulate GRK2 degradation. Interestingly, we found that in the absence of GPCR activation, β-arrestins do not perform an adaptor role for GRK2/Mdm2 association, but rather compete with GRK2 for direct Mdm2 binding to regulate basal kinase turnover. Upon agonist stimulation, β-arrestins-mediated phosphorylation of GRK2 at serine 670 by MAPK facilitates Mdm2-mediated GRK2 degradation, whereas c-Src-dependent phosphorylation would support the action of an undetermined β-arrestin-recruited ligase in the absence of GPCR activation. The ability of β-arrestins to play different scaffold functions would allow coordination of both Mdm2-dependent and -independent processes aimed at the specific modulation of GRK2 turnover in different signaling contexts. PMID:21081496

  19. [The contribution of protein kinase C and Rho-kinase to the control of the receptor-dependent artery contraction decreases with age independently of sympathetic innervation].

    PubMed

    Mochalov, S V; Kalenchuk, V U; Gaĭnullina, D K; Vorotnikov, A V; Tarasova, O S

    2008-01-01

    The age-related dynamics of the activity of signalling pathways coupled to alpha1-adrenergic receptors and their dependence on the sympathetic innervation of arterial smooth muscle have been studied. The effects of the protein kinase C inhibitor (GF109203X, 10(-6) M) and the Rho-kinase inhibitor (Y27632, 10(-5) M) on the isometric contraction of the rat saphenous artery, induced by the alpha1-adrenoceptor agonist methoxamine, were examined. It was shown that the sensitivity to methoxamine of arteries from 2-week-old rats that are partially innervated was reduced as compared to adults, but the effects of both inhibitors were more prominent. The denervation induced by the excision of sympathetic ganglia increased the arterial sensitivity to methoxamine but was not accompanied by changes in sensitivity to the inhibitors. Therefore, the postnatal development of the arterial smooth muscle is characterized by a decrease in the contribution of protein kinase C and Rho-kinase to the regulation of contraction; however, these changes do not correlate with changes in the sensitivity of arteries to methoxamine and development of sympathetic innervation. PMID:19137699

  20. G-protein-coupled receptor kinase-interacting proteins inhibit apoptosis by inositol 1,4,5-triphosphate receptor-mediated Ca2+ signal regulation.

    PubMed

    Zhang, Songbai; Hisatsune, Chihiro; Matsu-Ura, Toru; Mikoshiba, Katsuhiko

    2009-10-16

    The inositol 1,4,5-trisphosphate (IP(3)) receptor (IP(3)R) is an intracellular IP(3)-gated calcium (Ca(2+)) release channel and plays important roles in regulation of numerous Ca(2+)-dependent cellular responses. Many intracellular modulators and IP(3)R-binding proteins regulate the IP(3)R channel function. Here we identified G-protein-coupled receptor kinase-interacting proteins (GIT), GIT1 and GIT2, as novel IP(3)R-binding proteins. We found that both GIT1 and GIT2 directly bind to all three subtypes of IP(3)R. The interaction was favored by the cytosolic Ca(2+) concentration and it functionally inhibited IP(3)R activity. Knockdown of GIT induced and accelerated caspase-dependent apoptosis in both unstimulated and staurosporine-treated cells, which was attenuated by wild-type GIT1 overexpression or pharmacological inhibitors of IP(3)R, but not by a mutant form of GIT1 that abrogates the interaction. Thus, we conclude that GIT inhibits apoptosis by modulating the IP(3)R-mediated Ca(2+) signal through a direct interaction with IP(3)R in a cytosolic Ca(2+)-dependent manner.

  1. 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

  2. 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. PMID:27636711

  3. 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.

  4. 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

  5. Phosphorylation of the Drosophila Transient Receptor Potential Ion Channel Is Regulated by the Phototransduction Cascade and Involves Several Protein Kinases and Phosphatases

    PubMed Central

    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. PMID:24040070

  6. Receptor-Interacting Protein Kinase 3 Deficiency Delays Cutaneous Wound Healing.

    PubMed

    Godwin, Andrew; Sharma, Archna; Yang, Weng-Lang; Wang, Zhimin; Nicastro, Jeffrey; Coppa, Gene F; Wang, Ping

    2015-01-01

    Wound healing consists of a complex, dynamic and overlapping process involving inflammation, proliferation and tissue remodeling. A better understanding of wound healing process at the molecular level is needed for the development of novel therapeutic strategies. Receptor-interacting protein kinase 3 (RIPK3) controls programmed necrosis in response to TNF-α during inflammation and has been shown to be highly induced during cutaneous wound repair. However, its role in wound healing remains to be demonstrated. To study this, we created dorsal cutaneous wounds on male wild-type (WT) and RIPK3-deficient (Ripk3-/-) mice. Wound area was measured daily until day 14 post-wound and skin tissues were collected from wound sites at various days for analysis. The wound healing rate in Ripk3-/- mice was slower than the WT mice over the 14-day course; especially, at day 7, the wound size in Ripk3-/- mice was 53% larger than that of WT mice. H&E and Masson-Trichrome staining analysis showed impaired quality of wound closure in Ripk3-/- wounds with delayed re-epithelialization and angiogenesis and defected granulation tissue formation and collagen deposition compared to WT. The neutrophil infiltration pattern was altered in Ripk3-/- wounds with less neutrophils at day 1 and more neutrophils at day 3. This altered pattern was also reflected in the differential expression of IL-6, KC, IL-1β and TNF-α between WT and Ripk3-/- wounds. MMP-9 protein expression was decreased with increased Timp-1 mRNA in the Ripk3-/- wounds compared to WT. The microvascular density along with the intensity and timing of induction of proangiogenic growth factors VEGF and TGF-β1 were also decreased or delayed in the Ripk3-/- wounds. Furthermore, mouse embryonic fibroblasts (MEFs) from Ripk3-/- mice migrated less towards chemoattractants TGF-β1 and PDGF than MEFs from WT mice. These results clearly demonstrate that RIPK3 is an essential molecule to maintain the temporal manner of the normal progression

  7. Receptor-Interacting Protein Kinase 3 Deficiency Delays Cutaneous Wound Healing

    PubMed Central

    Yang, Weng-Lang; Wang, Zhimin; Nicastro, Jeffrey; Coppa, Gene F.; Wang, Ping

    2015-01-01

    Wound healing consists of a complex, dynamic and overlapping process involving inflammation, proliferation and tissue remodeling. A better understanding of wound healing process at the molecular level is needed for the development of novel therapeutic strategies. Receptor-interacting protein kinase 3 (RIPK3) controls programmed necrosis in response to TNF-α during inflammation and has been shown to be highly induced during cutaneous wound repair. However, its role in wound healing remains to be demonstrated. To study this, we created dorsal cutaneous wounds on male wild-type (WT) and RIPK3-deficient (Ripk3-/-) mice. Wound area was measured daily until day 14 post-wound and skin tissues were collected from wound sites at various days for analysis. The wound healing rate in Ripk3-/- mice was slower than the WT mice over the 14-day course; especially, at day 7, the wound size in Ripk3-/- mice was 53% larger than that of WT mice. H&E and Masson-Trichrome staining analysis showed impaired quality of wound closure in Ripk3-/- wounds with delayed re-epithelialization and angiogenesis and defected granulation tissue formation and collagen deposition compared to WT. The neutrophil infiltration pattern was altered in Ripk3-/- wounds with less neutrophils at day 1 and more neutrophils at day 3. This altered pattern was also reflected in the differential expression of IL-6, KC, IL-1β and TNF-α between WT and Ripk3-/- wounds. MMP-9 protein expression was decreased with increased Timp-1 mRNA in the Ripk3-/- wounds compared to WT. The microvascular density along with the intensity and timing of induction of proangiogenic growth factors VEGF and TGF-β1 were also decreased or delayed in the Ripk3-/- wounds. Furthermore, mouse embryonic fibroblasts (MEFs) from Ripk3-/- mice migrated less towards chemoattractants TGF-β1 and PDGF than MEFs from WT mice. These results clearly demonstrate that RIPK3 is an essential molecule to maintain the temporal manner of the normal progression

  8. 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

  9. 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.

  10. Endocytosis of Receptor Tyrosine Kinases

    PubMed Central

    Goh, Lai Kuan

    2013-01-01

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

  11. The Syk protein tyrosine kinase can function independently of CD45 or Lck in T cell antigen receptor signaling.

    PubMed Central

    Chu, D H; Spits, H; Peyron, J F; Rowley, R B; Bolen, J B; Weiss, A

    1996-01-01

    The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs. However, there is a CD45-deficient cell line that can signal through its TCR. We have studied this cell line to identify a TCR signaling pathway that is independent of CD45 regulation. In the course of these experiments, we found that the Syk PTK, but not the ZAP-70 PTK, is able to mediate TCR signaling independently of CD45 and of Lck. For this function, Syk requires functional kinase and SH2 domains, as well as intact phosphorylation sites in the regulatory loop of its kinase domain. Thus, differential expression of Syk is likely to explain the paradoxical phenotypes of different CD45-deficient T cells. Finally, these results suggest differences in activation requirements between two closely related PTK family members, Syk and ZAP-70. The differential activities of these two kinases suggest that they may play distinct, rather than completely redundant, roles in lymphocyte signaling. Images PMID:8947048

  12. Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases.

    PubMed

    Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J; Nie, Guangjun

    2016-04-15

    Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment.

  13. A Network of Mitogen-Activated Protein Kinases Links G Protein-Coupled Receptors to the c-jun Promoter: a Role for c-Jun NH2-Terminal Kinase, p38s, and Extracellular Signal-Regulated Kinase 5

    PubMed Central

    Marinissen, Maria Julia; Chiariello, Mario; Pallante, Michael; Gutkind, J. Silvio

    1999-01-01

    The expression of the c-jun proto-oncogene is rapidly induced in response to mitogens acting on a large variety of cell surface receptors. The resulting functional activity of c-Jun proteins appears to be critical for cell proliferation. Recently, we have shown that a large family of G protein-coupled receptors (GPCRs), represented by the m1 muscarinic receptor, can initiate intracellular signaling cascades that result in the activation of mitogen-activated protein kinases (MAPK) and c-Jun NH2-terminal kinases (JNK) and that the activation of JNK but not of MAPK correlated with a remarkable increase in the expression of c-jun mRNA. Subsequently, however, we obtained evidence that GPCRs can potently stimulate the activity of the c-jun promoter through MEF2 transcription factors, which do not act downstream from JNK. In view of these observations, we set out to investigate further the nature of the signaling pathway linking GPCRs to the c-jun promoter. Utilizing NIH 3T3 cells, we found that GPCRs can activate the c-jun promoter in a JNK-independent manner. Additionally, we demonstrated that these GPCRs can elevate the activity of novel members of the MAPK family, including ERK5, p38α, p38γ, and p38δ, and that the activation of certain kinases acting downstream from MEK5 (ERK5) and MKK6 (p38α and p38γ) is necessary to fully activate the c-jun promoter. Moreover, in addition to JNK, ERK5, p38α, and p38γ were found to stimulate the c-jun promoter by acting on distinct responsive elements. Taken together, these results suggest that the pathway linking GPCRs to the c-jun promoter involves the integration of numerous signals transduced by a highly complex network of MAPK, rather than resulting from the stimulation of a single linear protein kinase cascade. Furthermore, our findings suggest that each signaling pathway affects one or more regulatory elements on the c-jun promoter and that the transcriptional response most likely results from the temporal integration

  14. Overexpression of human insulin receptor substrate 1 induces cellular transformation with activation of mitogen-activated protein kinases.

    PubMed Central

    Ito, T; Sasaki, Y; Wands, J R

    1996-01-01

    The receptor insulin substrate 1 protein (IRS-1) is a specific substrate for insulin receptor tyrosine kinase. Expression and tyrosyl phosphorylation of IRS-1 play an important role during normal hepatocyte growth, and the gene is overexpressed in hepatocellular carcinoma tissue. We determined if IRS-1 overexpression directly contributes to cellular transformation. The human IRS-1 gene was subcloned into a mammalian expression vector driven by the cytomegalovirus early promoter. NIH 3T3 cells transiently transfected with this vector subsequently developed transformed foci. Several stably transfected cell lines were established, and they grew efficiently under low-serum conditions and formed colonies when plated in soft agar. Cell lines overexpressing IRS-1 displayed increased tyrosyl phosphorylation of IRS-1 and association with Grb2 but not with the p85 subunit of phosphatidylinositol 3' kinase. Since Grb2 is a component of the son-of-sevenless-Ras pathway and upstream in the mitogen-activated protein kinase (MAPK) cascade, enzymatic activities of the major components of this cascade, such as MAPK kinase and MAPK were evaluated and found to be substantially increased in three independent cell lines with IRS-1 protein overexpression. Such cells, when injected into nude mice, were highly tumorigenic, and there may be a correlation between the degree of MAPK activation and tumor growth rate. This report describes the generation of a transformed phenotype by overexpression of a molecule without a catalytic domain far upstream in the signal transduction cascade and suggests that prolonged activation of MAPKs by this mechanism may be one of the molecular events related to hepatocellular transformation. PMID:8622697

  15. Vascular endothelial growth factor-related protein: a ligand and specific activator of the tyrosine kinase receptor Flt4.

    PubMed Central

    Lee, J; Gray, A; Yuan, J; Luoh, S M; Avraham, H; Wood, W I

    1996-01-01

    The tyrosine kinases Flt4, Flt1, and Flk1 (or KDR) constitute a family of endothelial cell-specific receptors with seven immunoglobulin-like domains and a split kinase domain. Flt1 and Flk1 have been shown to play key roles in vascular development; these two receptors bind and are activated by vascular endothelial growth factor (VEGF). No ligand has been identified for Flt4, whose expression becomes restricted during development to the lymphatic endothelium. We have identified cDNA clones from a human glioma cell line that encode a secreted protein with 32% amino acid identity to VEGF. This protein, designated VEGF-related protein (VRP), specifically binds to the extracellular domain of Flt4, stimulates the tyrosine phosphorylation of Flt4 expressed in mammalian cells, and promotes the mitogenesis of human lung endothelial cells. VRP fails to bind appreciably to the extracellular domain of Flt1 or Flk1. The protein contains a C-terminal, cysteine-rich region of about 180 amino acids that is not found in VEGF. A 2.4-kb VRP mRNA is found in several human tissues including adult heart, placenta, ovary, and small intestine and in fetal lung and kidney. Images Fig. 1 Fig. 2 Fig. 3 Fig. 6 PMID:8700872

  16. Rip1 (receptor-interacting protein kinase 1) mediates necroptosis and contributes to renal ischemia/reperfusion injury.

    PubMed

    Linkermann, Andreas; Bräsen, Jan H; Himmerkus, Nina; Liu, Shuya; Huber, Tobias B; Kunzendorf, Ulrich; Krautwald, Stefan

    2012-04-01

    Loss of kidney function in renal ischemia/reperfusion injury is due to programmed cell death, but the contribution of necroptosis, a newly discovered form of programmed necrosis, has not been evaluated. Here, we identified the presence of death receptor-mediated but caspase-independent cell death in murine tubular cells and characterized it as necroptosis by the addition of necrostatin-1, a highly specific receptor-interacting protein kinase 1 inhibitor. The detection of receptor-interacting protein kinase 1 and 3 in whole-kidney lysates and freshly isolated murine proximal tubules led us to investigate the contribution of necroptosis in a mouse model of renal ischemia/reperfusion injury. Treatment with necrostatin-1 reduced organ damage and renal failure, even when administered after reperfusion, resulting in a significant survival benefit in a model of lethal renal ischemia/reperfusion injury. Unexpectedly, specific blockade of apoptosis by zVAD, a pan-caspase inhibitor, did not prevent the organ damage or the increase in urea and creatinine in vivo in renal ischemia/reperfusion injury. Thus, necroptosis is present and has functional relevance in the pathophysiological course of ischemic kidney injury and shows the predominance of necroptosis over apoptosis in this setting. Necrostatin-1 may have therapeutic potential to prevent and treat renal ischemia/reperfusion injury. PMID:22237751

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

    PubMed Central

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

    1996-01-01

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

  18. 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.

  19. Specificity of g protein-coupled receptor kinase 6-mediated phosphorylation and regulation of single-cell m3 muscarinic acetylcholine receptor signaling.

    PubMed

    Willets, Jonathon M; Mistry, Rajendra; Nahorski, Stefan R; Challiss, R A John

    2003-11-01

    Previously we have shown that G protein-coupled receptor kinase (GRK) 6 plays a major role in the regulation of the human M3 muscarinic acetylcholine receptor (M3 mAChR) in the human neuroblastoma SH-SY5Y. However, 30-fold overexpression of the catalytically inactive, dominant-negative K215RGRK6 produced only a 50% suppression of M3 mAChR phosphorylation and desensitization. Here, we have attempted to determine whether other endogenous kinases play a role in the regulation of M3 mAChR signaling. In contrast to the clear attenuating effect of K215RGRK6 expression on M3 mAChR regulation, dominant-negative forms of GRKs (K220RGRK2, K220RGRK3, K215RGRK5) and casein kinase 1alpha (K46RCK1alpha) were without effect. In addition, inhibition of a variety of second-messenger-regulated kinases and the tyrosine kinase Src also had no effect upon agonist-stimulated M3 mAChR regulation. To investigate further the desensitization process we have followed changes in inositol 1,4,5-trisphosphate in single SHSY5Y cells using the pleckstrin homology domain of PLCdelta1 tagged with green fluorescent protein (eGFP-PHPLCdelta1). Stimulation of cells with approximate EC50 concentrations of agonist before and after a desensitizing period of agonist exposure resulted in a marked attenuation of the latter response. Altered GRK6 activity, through overexpression of wild-type GRK6 or K215RGRK6, enhanced or reduced the degree of M3 mAChR desensitization, respectively. Taken together, our data indicate that M3 mAChR desensitization is mediated by GRK6 in human SH-SY5Y cells, and we show that receptor desensitization of phospholipase C signaling can be monitored in 'real-time' in single, living cells. PMID:14573754

  20. Regulation of extracellular-signal regulated kinase and c-Jun N-terminal kinase by G-protein-linked muscarinic acetylcholine receptors.

    PubMed Central

    Wylie, P G; Challiss, R A; Blank, J L

    1999-01-01

    Extracellular signal-regulated kinases (ERKs) and c-Jun N-terminal kinases (JNKs, or stress-activated protein kinases) are activated by diverse extracellular signals and mediate a variety of cellular responses, including mitogenesis, differentiation, hypertrophy, inflammatory reactions and apoptosis. We have examined the involvement of Ca2+ and protein kinase C (PKC) in ERK and JNK activation by the human G-protein-coupled m2 and m3 muscarinic acetylcholine receptors (mAChR) expressed in Chinese hamster ovary (CHO) cells. We show that the Ca2+-mobilizing m3 AChR is efficiently coupled to JNK and ERK activation, whereas the m2 AChR activates ERK but not JNK. Activation of JNK in CHO-m3 cells by the agonist methacholine (MCh) was delayed in onset and more sustained relative to that of ERK in either CHO-m2 or CHO-m3 cells. The EC50 values for MCh-induced ERK activation in both cell types were essentially identical and similar to that for JNK activation in CHO-m3 cells, suggesting little amplification of the response. Agonist-stimulated Ins(1,4,5)P3 accumulation in CHO-m3 cells was insensitive to pertussis toxin (PTX), consistent with a Gq/phosphoinositide-specific phospholipase C-beta mediated pathway, whereas a significant component of ERK and JNK activation in CHO-m3 cells was PTX-sensitive, indicating Gi/o involvement. Using manipulations that prevent receptor-mediated extracellular Ca2+ influx and intracellular Ca2+-store release, we also show that ERK activation by m2 and m3 receptors is Ca2+-independent. In contrast, a significant component (>50%) of JNK activation mediated by the m3 AChR was dependent on Ca2+, mainly derived from extracellular influx. PKC inhibition and down-regulation studies suggested that JNK activation was negatively regulated by PKC. Conversely, ERK activation by both m2 and m3 AChRs required PKC, suggesting a novel mechanism for PKC activation by PTX-sensitive m2 AChRs. In summary, mAChRs activate JNK and ERK via divergent mechanisms

  1. The ER structural protein Rtn4A stabilizes and enhances signaling through the receptor tyrosine kinase ErbB3.

    PubMed

    Hatakeyama, Jason; Wald, Jessica H; Rafidi, Hanine; Cuevas, Antonio; Sweeney, Colleen; Carraway, Kermit L

    2016-01-01

    ErbB3 and ErbB4 are receptor tyrosine kinases that are activated by the neuregulin (NRG) family of growth factors. These receptors govern various developmental processes, and their dysregulation contributes to several human disease states. The abundance of ErbB3 and ErbB4, and thus signaling through these receptors, is limited by the E3 ubiquitin ligase Nrdp1, which targets ErbB3 and ErbB4 for degradation. Reticulons are proteins that influence the morphology of the endoplasmic reticulum (ER) by promoting the formation of tubules, a response of cells to some stressors. We found that the ER structural protein reticulon 4A (Rtn4A, also known as Nogo-A) increased ErbB3 abundance and proliferative signaling by suppressing Nrdp1 function. Rtn4A interacted with Nrdp1 and stabilized ErbB3 in an Nrdp1-dependent manner. Rtn4A overexpression induced the redistribution of Nrdp1 from a cytosolic or perinuclear localization to ER tubules. Rtn4A knockdown in human breast tumor cells decreased ErbB3 abundance, NRG-stimulated signaling, and cellular proliferation and migration. Because proteins destined for the plasma membrane are primarily synthesized in the sheet portions of the ER, our observations suggest that Rtn4A counteracts the Nrdp1-mediated degradation of ErbB3 by sequestering the ubiquitin ligase into ER tubules. The involvement of a reticulon suggests a molecular link between ER structure and the sensitivity of cells to receptor tyrosine kinase-mediated survival signals at the cell surface. PMID:27353365

  2. Receptor Tyrosine Kinase and Tyrosine Kinase Inhibitors

    PubMed Central

    Mirshafiey, Abbas; Ghalamfarsa, Ghasem; Asghari, Babak

    2014-01-01

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

  3. Protein kinase B modulates the sensitivity of human neuroblastoma cells to insulin-like growth factor receptor inhibition.

    PubMed

    Guerreiro, Ana S; Boller, Danielle; Shalaby, Tarek; Grotzer, Michael A; Arcaro, Alexandre

    2006-12-01

    The potential of the novel insulin-like growth factor receptor (IGF-IR) inhibitor NVP-AEW541 as an antiproliferative agent in human neuroblastoma was investigated. Proliferation of a panel of neuroblastoma cell lines was inhibited by NVP-AEW541 with IC(50) values ranging from 0.15 to 5 microM. Experiments using an IGF-IR neutralizing antibody confirmed that the IGF-IR was essential to support growth of neuroblastoma cell lines. The expression levels of the IGF-IR in individual neuroblastoma cell lines did not correlate with the sensitivities to NVP-AEW541, while coexpression of the IGF-IR and the insulin receptor (IR) correlated with lower sensitivity to the inhibitor in some cell lines. Intriguingly, high levels of activation of Akt/protein kinase B (PKB) and phosphorylation of the ribosomal S6 protein were observed in neuroblastoma cell lines with decreased sensitivities to NVP-AEW541. Inhibition of Akt/PKB activity restored the sensitivity of neuroblastoma cells to the IGF-IR inhibitor. Transfection of neuroblastoma cells with activated Akt or ribosomal protein S6 kinase (S6K) decreased the sensitivity of the cells to NVP-AEW541. IGF-I-stimulated proliferation of neuroblastoma cell lines was completely blocked by NVP-AEW541, or by a combination of an inhibitor of phosphoinositide 3-kinase and rapamycin. In addition to its antiproliferative effects, NVP-AEW541 sensitized neuroblastoma cells to cisplatin-induced apoptosis. Together, our data demonstrate that NVP-AEW541 in combination with Akt/PKB inhibitors or chemotherapeutic agents may represent a novel approach to target human neuroblastoma cell proliferation.

  4. Rab8 modulates metabotropic glutamate receptor subtype 1 intracellular trafficking and signaling in a protein kinase C-dependent manner.

    PubMed

    Esseltine, Jessica L; Ribeiro, Fabiola M; Ferguson, Stephen S G

    2012-11-21

    Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors (GPCRs) that are activated by glutamate, the primary excitatory neurotransmitter in the CNS. Alterations in glutamate receptor signaling are implicated in neuropathologies such as Alzheimer's disease, ischemia, and Huntington's disease among others. Group 1 mGluRs (mGluR1 and mGluR5) are primarily coupled to Gα(q/11) leading to the activation of phospholipase C and the formation of diacylglycerol and inositol 1,4,5-trisphosphate, which results in the release of intracellular calcium stores and protein kinase C (PKC) activation. Desensitization, endocytosis, and recycling are major mechanisms of GPCR regulation, and the intracellular trafficking of GPCRs is linked to the Rab family of small G proteins. Rab8 is a small GTPase that is specifically involved in the regulation of secretory/recycling vesicles, modulation of the actin cytoskeleton, and cell polarity. Rab8 has been shown to regulate the synaptic delivery of AMPA receptors during long-term potentiation and during constitutive receptor recycling. We show here that Rab8 interacts with the C-terminal tail of mGluR1a in an agonist-dependent manner and plays a role in regulating of mGluR1a signaling and intracellular trafficking in human embryonic kidney 293 cells. Specifically, Rab8 expression attenuates mGluR1a-mediated inositol phosphate formation and calcium release from mouse neurons in a PKC-dependent manner, while increasing cell surface mGluR1a expression via decreased receptor endocytosis. These experiments provide us with an understanding of the role Rabs play in coordinated regulation of mGluR1a and how this impacts mGluR1a signaling.

  5. Activation of protein kinase C or cAMP-dependent protein kinase increases phosphorylation of the c-erbA-encoded thyroid hormone receptor and of the v-erbA-encoded protein.

    PubMed Central

    Goldberg, Y; Glineur, C; Gesquière, J C; Ricouart, A; Sap, J; Vennström, B; Ghysdael, J

    1988-01-01

    The c-erbA proto-oncogene encodes a nuclear receptor for thyroid hormone (T3), which is believed to stimulate transcription from specific target promoters upon binding to cis-acting DNA sequence elements. The v-erbA oncogene of avian erythroblastosis virus (AEV) encodes a ligand-independent version of this nuclear receptor. The v-erbA product inhibits terminal differentiation of avian erythroblasts, presumably by affecting the transcription of specific genes. We show here that the c-erbA-encoded nuclear receptor (p46c-erbA) is phosphorylated on serine residues on two distinct sites. One of these sites, defined by the limit tryptic phosphopeptide 28SSQCLVK, is retained on the v-erbA-encoded P75gag-v-erbA protein. This site is located in the amino-terminal domain of these molecules, 21 amino acids upstream of the DNA-binding region. Phosphorylation of this site in both p46c-erbA and P75gag-v-erbA is enhanced 10-fold following treatment of cells with activators of either protein kinase C or cAMP-dependent protein kinase. Since cAMP-dependent protein kinase phosphorylates both p46c-erbA and P75gag-v-erbA in vitro at the same site as that observed in vivo, at least part of the cAMP-dependent phosphorylation of erbA molecules in cells could result from direct phosphorylation by this enzyme. The possible role phosphorylation may play in the function of the erbA-encoded transcriptional factors is discussed. Images PMID:2903825

  6. Evidence for new homotypic and heterotypic interactions between transmembrane helices of proteins involved in receptor tyrosine kinase and neuropilin signaling.

    PubMed

    Sawma, Paul; Roth, Lise; Blanchard, Cécile; Bagnard, Dominique; Crémel, Gérard; Bouveret, Emmanuelle; Duneau, Jean-Pierre; Sturgis, James N; Hubert, Pierre

    2014-12-12

    Signaling in eukaryotic cells frequently relies on dynamic interactions of single-pass membrane receptors involving their transmembrane (TM) domains. To search for new such interactions, we have developed a bacterial two-hybrid system to screen for both homotypic and heterotypic interactions between TM helices. We have explored the dimerization of TM domains from 16 proteins involved in both receptor tyrosine kinase and neuropilin signaling. This study has revealed several new interactions. We found that the TM domain of Mucin-4, a putative intramembrane ligand for erbB2, dimerizes not only with erbB2 but also with all four members of the erbB family. In the Neuropilin/Plexin family of receptors, we showed that the TM domains of Neuropilins 1 and 2 dimerize with themselves and also with Plexin-A1, Plexin-B1, and L1CAM, but we were unable to observe interactions with several other TM domains notably those of members of the VEGF receptor family. The potentially important Neuropilin 1/Plexin-A1 interaction was confirmed using a surface plasmon resonance assay. This work shows that TM domain interactions can be highly specific. Exploring further the propensities of TM helix-helix association in cell membrane should have important practical implications related to our understanding of the structure-function of bitopic proteins' assembly and subsequent function, especially in the regulation of signal transduction. PMID:25315821

  7. G protein-coupled receptor signaling via Src kinase induces endogenous human transient receptor potential vanilloid type 6 (TRPV6) channel activation.

    PubMed

    Spehr, Jennifer; Gelis, Lian; Osterloh, Markus; Oberland, Sonja; Hatt, Hanns; Spehr, Marc; Neuhaus, Eva M

    2011-04-15

    Ca(2+) homeostasis plays a critical role in a variety of cellular processes. We showed previously that stimulation of the prostate-specific G protein-coupled receptor (PSGR) enhances cytosolic Ca(2+) and inhibits proliferation of prostate cells. Here, we analyzed the signaling mechanisms underlying the PSGR-mediated Ca(2+) increase. Using complementary molecular, biochemical, electrophysiological, and live-cell imaging techniques, we found that endogenous Ca(2+)-selective transient receptor potential vanilloid type 6 (TRPV6) channels are critically involved in the PSGR-induced Ca(2+) signal. Biophysical characterization of the current activated by PSGR stimulation revealed characteristic properties of TRPV6. The molecular identity of the involved channel was confirmed using RNA interference targeting TrpV6. TRPV6-mediated Ca(2+) influx depended on Src kinase activity. Src kinase activation occurred independently of G protein activation, presumably by direct interaction with PSGR. Taken together, we report that endogenous TRPV6 channels are activated downstream of a G protein-coupled receptor and present the first physiological characterization of these channels in situ. PMID:21349844

  8. Identification of four novel phosphorylation sites in estrogen receptor α: impact on receptor-dependent gene expression and phosphorylation by protein kinase CK2

    PubMed Central

    2009-01-01

    Background Estrogen receptor α (ERα) phosphorylation is important for estrogen-dependent transcription of ER-dependent genes, ligand-independent receptor activation and endocrine therapy response in breast cancer. However ERα phosphorylation at the previously identified sites does not fully account for these receptor functions. To determine if additional ERα phosphorylation sites exist, COS-1 cells expressing human ERα were labeled with [32P]H3PO4 in vivo and ERα tryptic phosphopeptides were isolated to identify phosphorylation sites. Results Previously uncharacterized phosphorylation sites at serines 46/47, 282, 294, and 559 were identified by manual Edman degradation and phosphoamino acid analysis and confirmed by mutagenesis and phospho-specific antibodies. Antibodies detected phosphorylation of endogenous ERα in MCF-7, MCF-7-LCC2, and Ishikawa cancer cell lines by immunoblot. Mutation of Ser-282 and Ser-559 to alanine (S282A, S559A) resulted in ligand independent activation of ERα as determined by both ERE-driven reporter gene assays and endogenous pS2 gene expression in transiently transfected HeLa cells. Mutation of Ser-46/47 or Ser-294 to alanine markedly reduced estradiol dependent reporter activation. Additionally protein kinase CK2 was identified as a kinase that phosphorylated ERα at S282 and S559 using motif analysis, in vitro kinase assays, and incubation of cells with CK2 kinase inhibitor. Conclusion These novel ERα phosphorylation sites represent new means for modulation of ERα activity. S559 represents the first phosphorylation site identified in the extreme C-terminus (F domain) of a steroid receptor. PMID:20043841

  9. Crystal Structure of G Protein-coupled Receptor Kinase 5 in Complex with a Rationally Designed Inhibitor.

    PubMed

    Homan, Kristoff T; Waldschmidt, Helen V; Glukhova, Alisa; Cannavo, Alessandro; Song, Jianliang; Cheung, Joseph Y; Koch, Walter J; Larsen, Scott D; Tesmer, John J G

    2015-08-21

    G protein-coupled receptor kinases (GRKs) regulate cell signaling by initiating the desensitization of active G protein-coupled receptors. The two most widely expressed GRKs (GRK2 and GRK5) play a role in cardiovascular disease and thus represent important targets for the development of novel therapeutic drugs. In the course of a GRK2 structure-based drug design campaign, one inhibitor (CCG215022) exhibited nanomolar IC50 values against both GRK2 and GRK5 and good selectivity against other closely related kinases such as GRK1 and PKA. Treatment of murine cardiomyocytes with CCG215022 resulted in significantly increased contractility at 20-fold lower concentrations than paroxetine, an inhibitor with more modest selectivity for GRK2. A 2.4 Å crystal structure of the GRK5·CCG215022 complex was determined and revealed that the inhibitor binds in the active site similarly to its parent compound GSK180736A. As designed, its 2-pyridylmethyl amide side chain occupies the hydrophobic subsite of the active site where it forms three additional hydrogen bonds, including one with the catalytic lysine. The overall conformation of the GRK5 kinase domain is similar to that of a previously determined structure of GRK6 in what is proposed to be its active state, but the C-terminal region of the enzyme adopts a distinct conformation. The kinetic properties of site-directed mutants in this region are consistent with the hypothesis that this novel C-terminal structure is representative of the membrane-bound conformation of the enzyme.

  10. Receptor Interacting Protein 3 Suppresses Vascular Smooth Muscle Cell Growth by Inhibition of the Phosphoinositide 3-Kinase-Akt Axis*

    PubMed Central

    Li, Qian; Li, Geng; Lan, Xiaomei; Zheng, Ming; Chen, Kuang-Hueih; Cao, Chun-Mei; Xiao, Rui-Ping

    2010-01-01

    Proliferation of vascular smooth muscle cells (VSMCs) is a primary mechanism underlying cardiovascular proliferative disorders. Phosphoinositide 3-kinase (PI3K)-Akt (or protein kinase B) axis has been assigned at the center of pathways that regulate cell proliferation. Here we demonstrate that enhanced PI3K-Akt signaling by mitogenic stimulation or arterial injury profoundly elevates expression of receptor interacting protein 3 (RIP3) in primary cultured rat VSMCs and in vivo and that the up-regulation of RIP3 leads to VSMC growth arrest and apoptosis via inhibiting the PI3K-Akt signaling pathway, thereby alleviating balloon injury-induced neointimal formation. Specifically, mitogenic stimulation with platelet-derived growth factor-BB or angiotensin II leads to a profound increase in RIP3 expression, which is abolished by inhibition of PI3K or Akt, and increased PI3K-Akt signaling by expression of a constitutively active PI3K mutant also elevates RIP3 expression. Importantly, adenoviral overexpression of RIP3 not only triggers apoptosis but also causes cell cycle arrest at G1/G0 phases that is associated with suppressed Akt activation. In sharp contrast, RIP3 gene silencing enhances serum- and platelet-derived growth factor-induced cell proliferation and Akt activation. In vivo adenoviral gene delivery of rat RIP3 (rRIP3) increased apoptosis and reduced VSMC proliferation, thus, effectively alleviating balloon injury-induced neointimal formation. The growth-suppressive and pro-apoptotic effects are independent of rRIP3 Ser/Thr kinase activity, because overexpression of a kinase-inactive mutant of rRIP3, similar to its wild type, is sufficient to induce growth arrest and apoptosis. These findings reveal a novel growth-suppressive action of RIP3, marking RIP3 as an important factor to prevent excessive mitogenic stimulation- or injury-induced vascular smooth muscle cells hyperplasia. PMID:20042608

  11. Thromboxane Receptor Activates the AMP-Activated Protein Kinase in Vascular Smooth Muscle Cells via Hydrogen Peroxide

    PubMed Central

    Zhang, Miao; Dong, Yunzhou; Xu, Jian; Xie, Zhonglin; Wu, Yong; Song, Ping; Guzman, Melissa; Wu, Jiliang; Zou, Ming-Hui

    2010-01-01

    Thromboxane A2 receptor (TPr) stimulation induces cellular hypertrophy in vascular smooth muscle cells (VSMCs); however, regulation of VSMC hypertrophy remains poorly understood. Here we show that TPr stimulation activates AMP-activated kinase (AMPK), which in turn limits TPr-induced protein synthesis in VSMCs. Exposure of cultured VSMCs to either TPr agonists, IBOP and U46619, or exogenous hydrogen peroxide (H2O2) caused time- and dose-dependent AMPK activation, as evidenced by increased phosphorylation of both AMPK-Thr172 and acetyl-coenzyme A carboxylase–Ser79, a downstream enzyme of AMPK, whereas SQ29548, a selective TPr antagonist, significantly attenuated TPr-enhanced AMPK activation. In parallel, both IBOP and U46619 significantly increased the production of reactive oxygen species such as H2O2. Furthermore, adenoviral overexpression of catalase (an H2O2 scavenger) abolished, whereas superoxide dismutase (which catalyzes H2O2 formation) enhanced, IBOP-induced AMPK activation, suggesting that TPr-activated AMPK was mediated by H2O2. Consistently, exposure of VSMCs to either TPr agonists or exogenous H2O2 dose-dependently increased the phosphorylation of LKB1 (at serines 428 and 307), an AMPK kinase, as well as coimmunoprecipitation of AMPK with LKB1. In addition, direct mutagenesis of either Ser428 or Ser307 of LKB1 into alanine, like the kinase-dead LKB1 mutant, abolished both TPr-stimulated AMPK activation and coimmunoprecipitation. Finally, genetic inhibition of AMPK significantly accentuated IBOP-enhanced protein synthesis, whereas adenoviral overexpression of constitutively active AMPK abolished IBOP-enhance protein synthesis in VSMCs. We conclude that TPr stimulation triggers reactive oxygen species–mediated LKB1-dependent AMPK activation, which in return inhibits cellular protein synthesis in VSMCs. PMID:18063812

  12. Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement.

    PubMed

    Saouaf, S J; Mahajan, S; Rowley, R B; Kut, S A; Fargnoli, J; Burkhardt, A L; Tsukada, S; Witte, O N; Bolen, J B

    1994-09-27

    We evaluated in WEHI 231 B cells the time-dependent responses of Lyn, Blk, Btk, Syk, and three members of the Jak family of protein tyrosine kinases following antibody-mediated surface engagement of the B-cell antigen receptor. Our results show that the enzyme activities of Lyn and Blk were stimulated within seconds of antigen receptor engagement and correlated with the initial tyrosine phosphorylation of the Ig alpha and Ig beta subunits of the B-cell antigen receptor. Btk enzyme activity was also transiently stimulated and was maximal at approximately 5 min after B-cell receptor surface binding. Syk activity gradually increased to a maximum at 10-30 min following receptor ligation and was found to parallel the association of Syk with the tyrosine phosphorylated Ig alpha and Ig beta subunits of the receptor. While the specific activities of the Jak1, Jak2, and Tyk2 protein tyrosine kinases were unaltered following B-cell receptor ligation, the abundance of Jak1 and Jak2 were increased 3- to 4-fold within 10 min of receptor engagement. These results demonstrate that multiple families of non-transmembrane protein tyrosine kinases are temporally regulated during the process of B-cell antigen receptor-initiated intracellular signal transduction. PMID:7524079

  13. Temporal differences in the activation of three classes of non-transmembrane protein tyrosine kinases following B-cell antigen receptor surface engagement.

    PubMed Central

    Saouaf, S J; Mahajan, S; Rowley, R B; Kut, S A; Fargnoli, J; Burkhardt, A L; Tsukada, S; Witte, O N; Bolen, J B

    1994-01-01

    We evaluated in WEHI 231 B cells the time-dependent responses of Lyn, Blk, Btk, Syk, and three members of the Jak family of protein tyrosine kinases following antibody-mediated surface engagement of the B-cell antigen receptor. Our results show that the enzyme activities of Lyn and Blk were stimulated within seconds of antigen receptor engagement and correlated with the initial tyrosine phosphorylation of the Ig alpha and Ig beta subunits of the B-cell antigen receptor. Btk enzyme activity was also transiently stimulated and was maximal at approximately 5 min after B-cell receptor surface binding. Syk activity gradually increased to a maximum at 10-30 min following receptor ligation and was found to parallel the association of Syk with the tyrosine phosphorylated Ig alpha and Ig beta subunits of the receptor. While the specific activities of the Jak1, Jak2, and Tyk2 protein tyrosine kinases were unaltered following B-cell receptor ligation, the abundance of Jak1 and Jak2 were increased 3- to 4-fold within 10 min of receptor engagement. These results demonstrate that multiple families of non-transmembrane protein tyrosine kinases are temporally regulated during the process of B-cell antigen receptor-initiated intracellular signal transduction. Images PMID:7524079

  14. Spreading depression induces expression of calcium-independent protein kinase C subspecies in ischaemia-sensitive cortical layers: regulation by N-methyl-D-aspartate receptors and glucocorticoids.

    PubMed

    Koponen, S; Keinänen, R; Roivainen, R; Hirvonen, T; Närhi, M; Chan, P H; Koistinaho, J

    1999-01-01

    Spreading depression is a wave of sustained depolarization challenging the energy metabolism of the cells without causing irreversible damage. In the ischaemic brain, sreading depression-like depolarization contributes to the evolution of ischaemia to infarction. The depolarization is propagated by activation of N-methyl-D-aspartate receptors, but changes in signal transduction downstream of the receptors are not known. Because protein phosphorylation is a general mechanism whereby most cellular processes are regulated, and inhibition of N-methyl-D-aspartate receptors or protein kinase C is neuroprotective, the expression of protein kinase C subspecies in spreading depression was examined. Cortical treatment with KCl induced an upregulation of protein kinase Cdelta and zeta messenger RNA at 4 and 8 h, whereas protein kinase Calpha, beta, gamma and epsilon did not show significant changes. The gene induction was the strongest in layers 2 and 3, and was followed by an increased number of protein kinase Cdelta-immunoreactive neurons. Protein kinase Cdelta and zeta inductions were inhibited by pretreatment with an N-methyl-D-aspartate receptor antagonist, dizocilpine maleate, which also blocked spreading depression propagation, and with dexamethasone, which acted without blocking the propagation. Quinacrine, a phospholipase A2 inhibitor, reduced only protein kinase C5 induction. In addition, N(G)(-nitro-L-arginine methyl ester, a nitric oxide synthase inhibitor, did not influence protein kinase Cdelta or zeta induction, whereas 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione, an alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate receptor antagonist, and the cyclo-oxygenase inhibitors indomethacin and diclophenac tended to increase gene expression. The data show that cortical spreading depression induces Ca2(+)-independent protein kinase C subspecies delta and zeta, but not Ca(2+)-dependent subspecies, through activation of N-methyl-D-aspartate receptors and

  15. [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.

  16. 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.

  17. The novel Smad protein Expansion regulates the receptor tyrosine kinase pathway to control Drosophila tracheal tube size.

    PubMed

    Iordanou, Ekaterini; Chandran, Rachana R; Yang, Yonghua; Essak, Mina; Blackstone, Nicholas; Jiang, Lan

    2014-09-01

    Tubes with distinct shapes and sizes are critical for the proper function of many tubular organs. Here we describe a unique phenotype caused by the loss of a novel, evolutionarily-conserved, Drosophila Smad-like protein, Expansion. In expansion mutants, unicellular and intracellular tracheal branches develop bubble-like cysts with enlarged apical membranes. Cysts in unicellular tubes are enlargements of the apical lumen, whereas cysts in intracellular tubes are cytoplasmic vacuole-like compartments. The cyst phenotype in expansion mutants is similar to, but weaker than, that observed in double mutants of Drosophila type III receptor tyrosine phosphatases (RPTPs), Ptp4E and Ptp10D. Ptp4E and Ptp10D negatively regulate the receptor tyrosine kinase (RTK) pathways, especially epithelial growth factor receptor (EGFR) and fibroblast growth factor receptor/breathless (FGFR, Btl) signaling to maintain the proper size of unicellular and intracellular tubes. We show Exp genetically interacts with RTK signaling, the downstream targets of RPTPs. Cyst size and number in expansion mutants is enhanced by increased RTK signaling and suppressed by reduced RTK signaling. Genetic interaction studies strongly suggest that Exp negatively regulates RTK (EGFR, Btl) signaling to ensure proper tube sizes. Smad proteins generally function as intermediate components of the transforming growth factor-β (TGF-β, DPP) signaling pathway. However, no obvious genetic interaction between expansion and TGF-β (DPP) signaling was observed. Therefore, Expansion does not function as a typical Smad protein. The expansion phenotype demonstrates a novel role for Smad-like proteins in epithelial tube formation.

  18. Molecular Characterization and Comparative Sequence Analysis of Defense-Related Gene, Oryza rufipogon Receptor-Like Protein Kinase 1

    PubMed Central

    Law, Yee-Song; Gudimella, Ranganath; Song, Beng-Kah; Ratnam, Wickneswari; Harikrishna, Jennifer Ann

    2012-01-01

    Many of the plant leucine rich repeat receptor-like kinases (LRR-RLKs) have been found to regulate signaling during plant defense processes. In this study, we selected and sequenced an LRR-RLK gene, designated as Oryza rufipogon receptor-like protein kinase 1 (OrufRPK1), located within yield QTL yld1.1 from the wild rice Oryza rufipogon (accession IRGC105491). A 2055 bp coding region and two exons were identified. Southern blotting determined OrufRPK1 to be a single copy gene. Sequence comparison with cultivated rice orthologs (OsI219RPK1, OsI9311RPK1 and OsJNipponRPK1, respectively derived from O. sativa ssp. indica cv. MR219, O. sativa ssp. indica cv. 9311 and O. sativa ssp. japonica cv. Nipponbare) revealed the presence of 12 single nucleotide polymorphisms (SNPs) with five non-synonymous substitutions, and 23 insertion/deletion sites. The biological role of the OrufRPK1 as a defense related LRR-RLK is proposed on the basis of cDNA sequence characterization, domain subfamily classification, structural prediction of extra cellular domains, cluster analysis and comparative gene expression. PMID:22942769

  19. RTKdb: database of Receptor Tyrosine Kinase.

    PubMed

    Grassot, Julien; Mouchiroud, Guy; Perrière, Guy

    2003-01-01

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

  20. Ras-dependent mitogen-activated protein kinase activation by G protein-coupled receptors. Convergence of Gi- and Gq-mediated pathways on calcium/calmodulin, Pyk2, and Src kinase.

    PubMed

    Della Rocca, G J; van Biesen, T; Daaka, Y; Luttrell, D K; Luttrell, L M; Lefkowitz, R J

    1997-08-01

    Many receptors that couple to heterotrimeric guanine-nucleotide binding proteins (G proteins) have been shown to mediate rapid activation of the mitogen-activated protein kinases Erk1 and Erk2. In different cell types, the signaling pathways employed appear to be a function of the available repertoire of receptors, G proteins, and effectors. In HEK-293 cells, stimulation of either alpha1B- or alpha2A-adrenergic receptors (ARs) leads to rapid 5-10-fold increases in Erk1/2 phosphorylation. Phosphorylation of Erk1/2 in response to stimulation of the alpha2A-AR is effectively attenuated by pretreatment with pertussis toxin or by coexpression of a Gbetagamma subunit complex sequestrant peptide (betaARK1ct) and dominant-negative mutants of Ras (N17-Ras), mSOS1 (SOS-Pro), and Raf (DeltaN-Raf). Erk1/2 phosphorylation in response to alpha1B-AR stimulation is also attenuated by coexpression of N17-Ras, SOS-Pro, or DeltaN-Raf, but not by coexpression of betaARK1ct or by pretreatment with pertussis toxin. The alpha1B- and alpha2A-AR signals are both blocked by phospholipase C inhibition, intracellular Ca2+ chelation, and inhibitors of protein-tyrosine kinases. Overexpression of a dominant-negative mutant of c-Src or of the negative regulator of c-Src function, Csk, results in attenuation of the alpha1B-AR- and alpha2A-AR-mediated Erk1/2 signals. Chemical inhibitors of calmodulin, but not of PKC, and overexpression of a dominant-negative mutant of the protein-tyrosine kinase Pyk2 also attenuate mitogen-activated protein kinase phosphorylation after both alpha1B- and alpha2A-AR stimulation. Erk1/2 activation, then, proceeds via a common Ras-, calcium-, and tyrosine kinase-dependent pathway for both Gi- and Gq/11-coupled receptors. These results indicate that in HEK-293 cells, the Gbetagamma subunit-mediated alpha2A-AR- and the Galphaq/11-mediated alpha1B-AR-coupled Erk1/2 activation pathways converge at the level of phospholipase C. These data suggest that calcium

  1. 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

  2. The protein product of the c-cbl protooncogene is phosphorylated after B cell receptor stimulation and binds the SH3 domain of Bruton's tyrosine kinase

    PubMed Central

    1995-01-01

    X-linked agammaglobulinemia, a B cell immunodeficiency, is caused by mutations in the Bruton's tyrosine kinase (Btk) gene. The absence of a functional Btk protein leads to a failure of B cell differentiation and antibody production. B cell receptor stimulation leads to the phosphorylation of the Btk protein and it is, therefore, likely that Btk is involved in B cell receptor signaling. As a nonreceptor tyrosine kinase, Btk is likely to interact with several proteins within the context of a signal transduction pathway. To understand such interactions, we have generated glutathione S-transferase fusion proteins corresponding to different domains of the human Btk protein. We have identified a 120-kD protein present in human B cells as being bound by the SH3 domain of Btk and which, after B cell receptor stimulation, is one of the major substrates of tyrosine phosphorylation. We have shown that this 120-kD protein is the protein product of c-cbl, a protooncogene, which is known to be phosphorylated in response to T cell receptor stimulation and to interact with several other tyrosine kinases. Association of the SH3 domain of Btk with p120cbl provides evidence for an analogous role for p120cbl in B cell signaling pathways. The p120cbl protein is the first identified ligand of the Btk SH3 domain. PMID:7629518

  3. 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

  4. Dynamic gene and protein expression patterns of the autism-associated Met receptor tyrosine kinase in the developing mouse forebrain

    PubMed Central

    Judson, Matthew C.; Bergman, Mica Y.; Campbell, Daniel B.; Eagleson, Kathie L.; Levitt, Pat

    2009-01-01

    The establishment of appropriate neural circuitry depends upon the coordination of multiple developmental events across space and time. These events include proliferation, migration, differentiation, and survival - all of which can be mediated by hepatocyte growth factor (HGF) signaling through the Met receptor tyrosine kinase. We previously found a functional promoter variant of the MET gene to be associated with autism spectrum disorder, suggesting that forebrain circuits governing social and emotional function may be especially vulnerable to developmental disruptions in HGF/Met signaling. However, little is known about the spatiotemporal distribution of Met expression in the forebrain during the development of such circuits. To advance our understanding of the neurodevelopmental influences of Met activation, we employed complementary Western blotting, in situ hybridization and immunohistochemistry to comprehensively map Met transcript and protein expression throughout perinatal and postnatal development of the mouse forebrain. Our studies reveal complex and dynamic spatiotemporal patterns of expression during this period. Spatially, Met transcript is localized primarily to specific populations of projection neurons within the neocortex and in structures of the limbic system, including the amygdala, hippocampus and septum. Met protein appears to be principally located in axon tracts. Temporally, peak expression of transcript and protein occurs during the second postnatal week. This period is characterized by extensive neurite outgrowth and synaptogenesis, supporting a role for the receptor in these processes. Collectively, these data suggest that Met signaling may be necessary for the appropriate wiring of forebrain circuits with particular relevance to social and emotional dimensions of behavior. PMID:19226509

  5. Protein kinase C α is involved in the regulation of AXL receptor tyrosine kinase expression in triple-negative breast cancer cells.

    PubMed

    Yue, Chia-Herng; Liu, Liang-Chih; Kao, Erl-Shyh; Lin, Ho; Hsu, Li-Sung; Hsu, Chih-Wei; Lin, Yu-Yu; Lin, Yi-Syuan; Liu, Jer-Yuh; Lee, Chia-Jen

    2016-08-01

    AXL receptor tyrosine kinase is overexpressed in triple-negative breast cancer (TNBC), and has a function in cancer progression and metastases. However, the mechanism underlying AXL gene regulation in TNBC remains unknown. In this study, the involvement of protein kinase C α (PKCα) in the expression of AXL was investigated in human TNBC cells. The microarray data from other studies showed that PKCα is significantly correlated with AXL expression in TNBC cell lines. Tissue array analysis also confirmed their correlation in TNBC. The PKCα inhibitor Go6976 was used to treat MDA‑MB‑231 and Hs578T TNBC cells, which resulted in decreased expression of AXL and epithelia-mesenchymal transition-related gene vimentin, and decreased cell proliferation. An MZF‑1 acidic domain fragment (MZF-1 peptide), which was designed to downregulate PKCα expression, was transfected into the cells and resulted in inhibition of AXL expression. This effect was reversed by co‑treatment with the constitutive form of PKCα. Moreover, the downregulation of PKCα was also confirmed by treatment with TAT‑fused MZF‑1 peptide. Thus, the current study proposes that AXL may be correlated with PKCα‑dependent TNBC cells, and could be modulated by MZF‑1 peptides. PMID:27357025

  6. 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

  7. Myocardial pathology induced by aldosterone is dependent on non-canonical activities of G protein-coupled receptor kinases

    PubMed Central

    Cannavo, Alessandro; Liccardo, Daniela; Eguchi, Akito; Elliott, Katherine J.; Traynham, Christopher J.; Ibetti, Jessica; Eguchi, Satoru; Leosco, Dario; Ferrara, Nicola; Rengo, Giuseppe; Koch, Walter J.

    2016-01-01

    Hyper-aldosteronism is associated with myocardial dysfunction including induction of cardiac fibrosis and maladaptive hypertrophy. Mechanisms of these cardiotoxicities are not fully understood. Here we show that mineralocorticoid receptor (MR) activation by aldosterone leads to pathological myocardial signalling mediated by mitochondrial G protein-coupled receptor kinase 2 (GRK2) pro-death activity and GRK5 pro-hypertrophic action. Moreover, these MR-dependent GRK2 and GRK5 non-canonical activities appear to involve cross-talk with the angiotensin II type-1 receptor (AT1R). Most importantly, we show that ventricular dysfunction caused by chronic hyper-aldosteronism in vivo is completely prevented in cardiac Grk2 knockout mice (KO) and to a lesser extent in Grk5 KO mice. However, aldosterone-induced cardiac hypertrophy is totally prevented in Grk5 KO mice. We also show human data consistent with MR activation status in heart failure influencing GRK2 levels. Therefore, our study uncovers GRKs as targets for ameliorating pathological cardiac effects associated with high-aldosterone levels. PMID:26932512

  8. The effect of G protein-coupled receptor kinase 2 (GRK2) on lactation and on proliferation of mammary epithelial cells from dairy cows.

    PubMed

    Hou, Xiaoming; Hu, Hongliu; Lin, Ye; Qu, Bo; Gao, Xuejun; Li, Qingzhang

    2016-07-01

    Milk protein is an important component of milk and a nutritional source for human consumption. To better understand the molecular events underlying synthesis of milk proteins, the global gene expression patterns in mammary glands of dairy cow with high-quality milk (>3% milk protein; >3.5% milk fat) and low-quality milk (<3% milk protein; <3.5% milk fat) were examined via digital gene expression study. A total of 139 upregulated and 66 downregulated genes were detected in the mammary tissues of lactating cows with high-quality milk compared with the tissues of cows with low-quality milk. A pathway enrichment study of these genes revealed that the top 5 pathways that were differentially affected in the tissues of cows with high- versus low-quality milk involved metabolic pathways, cancer, cytokine-cytokine receptor interactions, regulation of the actin cytoskeleton, and insulin signaling. We also found that the G protein-coupled receptor kinase 2 (GRK2) was one of the most highly upregulated genes in lactating mammary tissue with low-quality milk compared with tissue with high-quality milk. The knockdown of GRK2 in cultured bovine mammary epithelial cells enhanced CSN2 expression and activated signaling molecules related to translation, including protein kinase B, mammalian target of rapamycin, and p70 ribosomal protein S6 kinase 1 (S6K1), whereas overexpression of GRK2 had the opposite effects. However, expression of genes involved in the mitogen-activated protein kinase pathway was positively regulated by GRK2. Therefore, GRK2 seems to act as a negative mediator of milk-protein synthesis via the protein kinase B-mammalian target of rapamycin signaling axis. Furthermore, GRK2 may negatively control milk-protein synthesis by activating the mitogen-activated protein kinase pathway in dairy cow mammary epithelial cells. PMID:27132107

  9. Enhanced Uterine Contractility and Stillbirth in Mice Lacking G Protein-Coupled Receptor Kinase 6 (GRK6): Implications for Oxytocin Receptor Desensitization.

    PubMed

    Grotegut, Chad A; Mao, Lan; Pierce, Stephanie L; Swamy, Geeta K; Heine, R Phillips; Murtha, Amy P

    2016-04-01

    Oxytocin is a potent uterotonic agent and is used clinically for induction and augmentation of labor, as well as for prevention and treatment of postpartum hemorrhage. Oxytocin increases uterine contractility by activating the oxytocin receptor (OXTR), a member of the G protein-coupled receptor family, which is prone to molecular desensitization. After oxytocin binding, the OXTR is phosphorylated by a member of the G protein-coupled receptor kinase (GRK) family, which allows for recruitment of β-arrestin, receptor internalization, and desensitization. According to previous in vitro analyses, desensitization of calcium signaling by the OXTR is mediated by GRK6. The objective of this study was to determine the role of GRK6 in mediating uterine contractility. Here, we demonstrate that uterine GRK6 levels increase in pregnancy and using a telemetry device to measure changes in uterine contractility in live mice during labor, show that mice lacking GRK6 produce a phenotype of enhanced uterine contractility during both spontaneous and oxytocin-induced labor compared with wild-type or GRK5 knockout mice. In addition, the observed enhanced contractility was associated with high rates of term stillbirth. Lastly, using a heterologous in vitro model, we show that β-arrestin recruitment to the OXTR, which is necessary for homologous OXTR desensitization, is dependent on GRK6. Our findings suggest that GRK6-mediated OXTR desensitization in labor is necessary for normal uterine contractile patterns and optimal fetal outcome. PMID:26886170

  10. Oncogenic activation of the Met receptor tyrosine kinase fusion protein, Tpr-Met, involves exclusion from the endocytic degradative pathway.

    PubMed

    Mak, H H L; Peschard, P; Lin, T; Naujokas, M A; Zuo, D; Park, M

    2007-11-01

    Multiple mechanisms of dysregulation of receptor tyrosine kinases (RTKs) are observed in human cancers. In addition to gain-of-function, loss of negative regulation also contributes to oncogenic activation of RTKs. Negative regulation of many RTKs involves their internalization and degradation in the lysosome, a process regulated through ubiquitination. RTK oncoproteins activated following chromosomal translocation, are no longer transmembrane proteins, and are predicted to escape lysosomal degradation. To test this, we used the Tpr-Met oncogene, generated following chromosomal translocation of the hepatocyte growth factor receptor (Met). Unlike Met, Tpr-Met is localized in the cytoplasm and also lacks the binding site for Cbl ubiquitin ligases. We determined whether subcellular localization of Tpr-Met, and/or loss of its Cbl-binding site, is important for oncogenic activity. Presence of a Cbl-binding site and ubiquitination of cytosolic Tpr-Met oncoproteins does not alter their transforming activity. In contrast, plasma membrane targeting allows Tpr-Met to enter the endocytic pathway, and Tpr-Met transforming activity as well as protein stability are decreased in a Cbl-dependent manner. We show that transformation by Tpr-Met is in part dependent on its ability to escape normal downregulatory mechanisms. This provides a paradigm for many RTK oncoproteins activated following chromosomal translocation.

  11. 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.

  12. Protein Kinase D1-Dependent Phosphorylation of Dopamine D1 Receptor Regulates Cocaine-Induced Behavioral Responses

    PubMed Central

    Wang, Ning; Su, Ping; Zhang, Ying; Lu, Jie; Xing, Baoming; Kang, Kai; Li, Wenqi; Wang, Yun

    2014-01-01

    The dopamine (DA) D1 receptor (D1R) is critically involved in reward and drug addiction. Phosphorylation-mediated desensitization or internalization of D1R has been extensively investigated. However, the potential for upregulation of D1R function through phosphorylation remains to be determined. Here we report that acute cocaine exposure induces protein kinase D1 (PKD1) activation in the rat striatum, and knockdown of PKD1 in the rat dorsal striatum attenuates cocaine-induced locomotor hyperactivity. Moreover, PKD1-mediated phosphorylation of serine 421 (S421) of D1R promotes surface localization of D1R and enhances downstream extracellular signal-regulated kinase signaling in D1R-transfected HEK 293 cells. Importantly, injection of the peptide Tat-S421, an engineered Tat fusion-peptide targeting S421 (Tat-S421), into the rat dorsal striatum inhibits cocaine-induced locomotor hyperactivity and injection of Tat-S421 into the rat hippocampus or the shell of the nucleus accumbens (NAc) also inhibits cocaine-induced conditioned place preference (CPP). However, injection of Tat-S421 into the rat NAc shell does not establish CPP by itself and injection of Tat-S421 into the hippocampus does not influence spatial learning and memory. Thus, targeting S421 of D1R represents a promising strategy for the development of pharmacotherapeutic treatments for drug addiction and other disorders that result from DA imbalances. PMID:24362306

  13. G protein-coupled receptor kinase-2 is a novel regulator of collagen synthesis in adult human cardiac fibroblasts.

    PubMed

    D'Souza, Karen M; Malhotra, Ricky; Philip, Jennifer L; Staron, Michelle L; Theccanat, Tiju; Jeevanandam, Valluvan; Akhter, Shahab A

    2011-04-29

    Cardiac fibroblasts (CF) make up 60-70% of the total cell number in the heart and play a critical role in regulating normal myocardial function and in adverse remodeling following myocardial infarction and the transition to heart failure. Recent studies have shown that increased intracellular cAMP can inhibit CF transformation and collagen synthesis in adult rat CF; however, mechanisms by which cAMP production is regulated in CF have not been elucidated. We investigated the potential role of G protein-coupled receptor kinase-2 (GRK2) in modulating collagen synthesis by adult human CF isolated from normal and failing left ventricles. Baseline collagen synthesis was elevated in failing CF and was not inhibited by β-agonist stimulation in contrast to normal controls. β-adrenergic receptor (β-AR) signaling was markedly uncoupled in the failing CF, and expression and activity of GRK2 were increased 3-fold. Overexpression of GRK2 in normal CF recapitulated a heart failure phenotype with minimal inhibition of collagen synthesis following β-agonist stimulation. In contrast, knockdown of GRK2 expression in normal CF enhanced cAMP production and led to greater β-agonist-mediated inhibition of basal and TGFβ-stimulated collagen synthesis versus control. Inhibition of GRK2 activity in failing CF by expression of the GRK2 inhibitor, GRK2ct, or siRNA-mediated knockdown restored β-agonist-stimulated inhibition of collagen synthesis and decreased collagen synthesis in response to TGFβ stimulation. GRK2 appears to play a significant role in regulating collagen synthesis in adult human CF, and increased activity of this kinase may be an important mechanism of maladaptive ventricular remodeling as mediated by cardiac fibroblasts.

  14. Phosphorylation of the human 1,25-dihydroxyvitamin D3 receptor by cAMP-dependent protein kinase, in vitro, and in transfected COS-7 cells.

    PubMed

    Jurutka, P W; Hsieh, J C; Haussler, M R

    1993-03-31

    We report that the human 1,25-dihydroxyvitamin D3 receptor is an efficient substrate for cAMP-dependent protein kinase, in vitro. This phosphorylation reaction is rapid and neither dependent upon nor significantly affected by the presence of the 1,25-dihydroxyvitamin D3 ligand. Preliminary mapping experiments utilizing C-terminal truncation mutants reveal that the primary site(s) of phosphorylation, in vitro, is localized between amino acids 133 and 201. Cotransfection of the catalytic subunit of murine cAMP-dependent protein kinase and the human 1,25-dihydroxyvitamin D3 receptor into monkey kidney (COS-7) cells not only results in a dramatic kinase-dependent increase in receptor phosphorylation but also elicits an attenuation in 1,25-dihydroxyvitamin D3-dependent transcriptional activation of a reporter gene. These observations suggest a potential role for cAMP-dependent protein kinase in the modulation of 1,25-dihydroxyvitamin D3 receptor-mediated gene regulation. PMID:8385450

  15. 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

  16. The heterotrimeric G q protein-coupled angiotensin II receptor activates p21 ras via the tyrosine kinase-Shc-Grb2-Sos pathway in cardiac myocytes.

    PubMed Central

    Sadoshima, J; Izumo, S

    1996-01-01

    p21 ras plays as important role in cell proliferation, transformation and differentiation. Recently, the requirement of p21 ras has been suggested for cellular responses induced by stimulation of heterotrimeric G protein-coupled receptors. However, it remains to be determined how agonists for G protein-coupled receptors activate p21 ras in metazoans. We show here that stimulation of the G q protein-coupled angiotensin II (Ang II) receptor causes activation of p21 ras in cardiac myocytes. The p21 ras activation by Ang II is mediated by an increase in the guanine nucleotide exchange activity, but not by an inhibition of the GTPase-activating protein. Ang II causes rapid tyrosine phosphorylation of Shc and its association with Grb2 and mSos-1, a guanine nucleotide exchange factor of p21 ras. This leads to translocation of mSos-1 to the membrane fraction. Shc associates with the SH3 domain of Fyn whose tyrosine kinase activity is activated by Ang II with a similar time course as that of tyrosine phosphorylation of Shc. Ang II-induced increase in the guanine nucleotide exchange activity was inhibited by a peptide ligand specific to the SH3 domain of the Src family tyrosine kinases. These results suggest that an agonist for a pertussis toxin-insensitive G protein-coupled receptor may initiate the cross-talk with non-receptor-type tyrosine kinases, thereby activating p21 ras using a similar mechanism as receptor tyrosine kinase-induced p21 ras activation. Images PMID:8631299

  17. G protein receptor kinase 4 polymorphisms: β-blocker pharmacogenetics and treatment-related outcomes in hypertension.

    PubMed

    Vandell, Alexander G; Lobmeyer, Maximilian T; Gawronski, Brian E; Langaee, Taimour Y; Gong, Yan; Gums, John G; Beitelshees, Amber L; Turner, Stephen T; Chapman, Arlene B; Cooper-DeHoff, Rhonda M; Bailey, Kent R; Boerwinkle, Eric; Pepine, Carl J; Liggett, Stephen B; Johnson, Julie A

    2012-10-01

    G protein-coupled receptor kinases (GRKs) are important regulatory proteins for many G protein-coupled receptors, but little is known about GRK4 pharmacogenetics. We hypothesized that 3 nonsynonymous GRK4 single-nucleotide polymorphisms, R65L (rs2960306), A142V (rs1024323), and A486V (rs1801058), would be associated with blood pressure response to atenolol, but not hydrochlorothiazide, and would be associated with long-term cardiovascular outcomes (all-cause death, nonfatal myocardial infarction, nonfatal stroke) in participants treated with an atenolol-based versus verapamil-SR-based antihypertensive strategy. GRK4 single-nucleotide polymorphisms were genotyped in 768 hypertensive participants from the Pharmacogenomic Evaluation of Antihypertensive Responses (PEAR) trial. In whites and blacks, increasing copies of the variant 65L-142V haplotype were associated with significantly reduced atenolol-induced diastolic blood pressure lowering (-9.1±6.8 versus -6.8±7.1 versus -5.3±6.4 mm Hg in participants with 0, 1, and 2 copies of 65L-142V, respectively; P=0.0088). One thousand four hundred sixty participants with hypertension and coronary artery disease from the INternational VErapamil SR/Trandolapril STudy (INVEST) were genotyped, and variant alleles of all 3 GRK4 single-nucleotide polymorphisms were associated with increased risk for adverse cardiovascular outcomes in an additive fashion, with 486V homozygotes reaching statistical significance (odds ratio, 2.29 [1.48-3.55]; P=0.0002). These effects on adverse cardiovascular outcomes were independent of antihypertensive treatment. These results suggest that the presence of GRK4 variant alleles may be important determinants of blood pressure response to atenolol and risk for adverse cardiovascular events. The associations with GRK4 variant alleles were stronger in patients who were also ADRB1 389R homozygotes, suggesting a potential interaction between these 2 genes.

  18. Protein kinase activity associated with Fc. gamma. /sub 2a/ receptor of a murine macrophage like cell line, P388D/sub 1/

    SciTech Connect

    Hirata, Y.; Suzuki, T.

    1987-12-15

    The properties of protein kinase activity associated with Fc receptor specific for IgG/sub 2a/(Fc..gamma../sub 2a/R) of a murine macrophage like cell line, P388D/sub 1/, were investigated. IgG/sub 2a/-binding protein isolated from the detergent lysate of P388D/sub 1/ cells by affinity chromatography of IgG-Sepharose was found to contain four distinct proteins of M/sub r/ 50,000, 43,000, 37,000, and 17,000, which could be autophosphorylated upon incubation with (..gamma..-/sup 32/P)ATP. The autophosphorylation of Fc..gamma../sub 2a/ receptor complex ceased when exogenous phosphate acceptors (casein or histone) were added in the reaction mixture. Phosphorylation of casein catalyzed by Fc..gamma../sub 2a/ receptor complex was dependent on casein concentration, increased with time or temperature, was dependent on the concentration of ATP and Mg/sup 2 +/, and was maximum at pH near 8. Casein phosphorylation was significantly inhibited by a high concentration of Mn/sup 2 +/ or KCl or by a small amount of heparin and was enhanced about 2-fold by protamine. Casein kinase activity associated with Fc..gamma../sub 2a/ receptor used ATP as substrate with an apparent K/sub m/ of 2 ..mu..M as well as GTP with an apparent K/sub m/ of 10 ..mu..M. Prior heating (60/sup 0/C for 15 min) or treatment with protease (trypsin or Pronase) of Fc..gamma../sub 2a/ receptor complex almost totally abolished casein kinase activity. Thin-layer chromatography of a partial acid hydrolysate of the phosphorylated casein showed that the site of phosphorylation is at a seryl residue. These results suggest that Fc..gamma../sub 2//sub a/ receptor forms a molecule complex with protein kinase, whose characteristics resemble those of type II casein kinase but are different from those of cyclic nucleotide dependent protein kinase or from those of C protein kinase.

  19. Protein kinase A regulation of P2X(4) receptors: requirement for a specific motif in the C-terminus.

    PubMed

    Brown, David A; Yule, David I

    2010-02-01

    The P2X purinergic receptor sub-family of ligand-gated ion channels are subject to protein kinase modulation. We have previously demonstrated that P2X(4)R signaling can be positively regulated by increasing intracellular cAMP levels. The molecular mechanism underlying this effect was, however, unknown. The present study initially addressed whether protein kinase A (PKA) activation was required. Subsequently a mutational approach was utilized to determine which region of the receptor was required for this potentiation. In both DT-40 3KO and HEK-293 cells transiently expressing P2X(4)R, forskolin treatment enhanced ATP-mediated signaling. Specific PKA inhibitors prevented the forskolin-induced enhancement of ATP-mediated inward currents in P2X(4)R expressing HEK-293 cells. To define which region of the P2X(4)R was required for the potentiation, mutations were generated in the cytoplasmic C-terminal tail. It was determined that a limited region of the C-terminus, consisting of a non-canonical tyrosine based sorting motif, was required for the effects of PKA. Of note, this region does not harbor any recognizable PKA phosphorylation motifs, and no direct phosphorylation of P2X(4)R was detected, suggesting that PKA phosphorylation of an accessory protein interacts with the endocytosis motif in the C-terminus of the P2X(4)R. In support of this notion, using Total Internal Reflection Fluorescence Microscopy (TIRF)\\ P2X(4)-EGFP was shown to accumulate at/near the plasma membrane following forskolin treatment. In addition, disrupting the endocytosis machinery using a dominant-negative dynamin construct also prevented the PKA-mediated enhancement of ATP-stimulated Ca(2+) signals. Our results are consistent with a novel mechanism of P2XR regulation, whereby PKA activity, without directly phosphorylating P2X(4)R, markedly enhances ATP-stimulated P2X(4)R currents and hence cytosolic Ca(2+) signals. This may occur at least in part, by altering the trafficking of a population of

  20. PhosphoThr Peptide Binding Globally Rigidifies much of the FHA Domain from Arabidopsis Receptor Kinase-Associated Protein Phosphatase

    PubMed Central

    Ding, Zhaofeng; Lee, Gui-in; Liang, Xiangyang; Gallazzi, Fabio; Arunima, A.; Van Doren, Steven R.

    2008-01-01

    A net increase in the backbone rigidity of the kinase-interacting FHA domain (KI-FHA) from the Arabidopsis receptor kinase-associated protein phosphatase (KAPP) accompanies the binding of a phosphoThr peptide from its CLV1 receptor-like kinase partner, according to 15N NMR relaxation at 11.7 and 14.1 T. All of the loops of free KI-FHA display evidence of nsec-scale motions. Many of these same residues have residual dipolar couplings that deviate from structural predictions. Binding of the CLV1 pT868 peptide seems to reduce nsec-scale fluctuations of all loops, including half of the residues of recognition loops. Residues important for affinity are found to be rigid, i.e. conserved residues and residues of the subsite for the key pT+3 peptide position. –This behavior parallels SH2 and PTB domain recognition of pTyr peptides. PhosphoThr peptide binding increases KI-FHA backbone rigidity (S2) of three recognition loops, a loop nearby, seven strands from the β-sandwich, and a distal loop. Compensating the trend of increased rigidity, binding enhances fast mobility at a few sites in four loops on the periphery of the recognition surface and in two loops on the far side of the β-sandwich. Line broadening evidence of µsec to msec-scale fluctuations occurs across the six-stranded β-sheet and nearby edges of the β-sandwich; this forms a network connected by packing of interior side chains and H-bonding. A patch of the slowly fluctuating residues coincides with the site of segment-swapped dimerization in crystals of the FHA domain of human Chfr. Phosphopeptide binding introduces µsec to msec-scale fluctuations to more residues of the long 8/9 recognition loop of KI-FHA. The rigidity of this FHA domain appears to couple as a whole to pThr peptide binding. PMID:16042389

  1. 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

  2. Protein kinase C regulates tonic GABA(A) receptor-mediated inhibition in the hippocampus and thalamus.

    PubMed

    Bright, Damian P; Smart, Trevor G

    2013-11-01

    Tonic inhibition mediated by extrasynaptic GABA(A) receptors (GABA(A) Rs) is an important regulator of neuronal excitability. Phosphorylation by protein kinase C (PKC) provides a key mode of regulation for synaptic GABA(A) Rs underlying phasic inhibition; however, less attention has been focused on the plasticity of tonic inhibition and whether this can also be modulated by receptor phosphorylation. To address this issue, we used whole-cell patch clamp recording in acute murine brain slices at both room and physiological temperatures to examine the effects of PKC-mediated phosphorylation on tonic inhibition. Recordings from dentate gyrus granule cells in the hippocampus and dorsal lateral geniculate relay neurons in the thalamus demonstrated that PKC activation caused downregulation of tonic GABA(A) R-mediated inhibition. Conversely, inhibition of PKC resulted in an increase in tonic GABA(A) R activity. These findings were corroborated by experiments on human embryonic kidney 293 cells expressing recombinant α4β2δ GABA(A) Rs, which represent a key extrasynaptic GABA(A) R isoform in the hippocampus and thalamus. Using bath application of low GABA concentrations to mimic activation by ambient neurotransmitter, we demonstrated a similar inhibition of receptor function following PKC activation at physiological temperature. Live cell imaging revealed that this was correlated with a loss of cell surface GABA(A) Rs. The inhibitory effects of PKC activation on α4β2δ GABA(A) R activity appeared to be mediated by direct phosphorylation at a previously identified site on the β2 subunit, serine 410. These results indicate that PKC-mediated phosphorylation can be an important physiological regulator of tonic GABA(A) R-mediated inhibition.

  3. In vivo and in vitro specificity of protein tyrosine kinases for immunoglobulin G receptor (FcgammaRII) phosphorylation.

    PubMed Central

    Bewarder, N; Weinrich, V; Budde, P; Hartmann, D; Flaswinkel, H; Reth, M; Frey, J

    1996-01-01

    Human B cells express four immunoglobulin G receptors, FcgammaRIIa, FcgammaRIIb1, FcgammaRIIb2, and FcgammaRIIc. Coligation of either FcgammaRII isoform with the B-cell antigen receptor (BCR) results in the abrogation of B-cell activation, but only the FcgammaRIIa/c and FcgammaIIb1 isoforms become phosphorylated. To identify the FcgammaRII-phosphorylating protein tyrosine kinase (PTK), we used the combination of an in vitro and an in vivo approach. In an in vitro assay using recombinant cytoplasmic tails of the different FcgammaRII isoforms as well as tyrosine exchange mutants, we show that each of the BCR-associated PTKs (Lyn, Blk, Fyn, and Syk) shows different phosphorylation patterns with regard to the different FcgammaR isoforms and point mutants. While each PTK phosphorylated FcgammaRIIa/c, FcgammaRIIb1 was phosphorylated by Lyn and Blk whereas FcgammaRIIb2 became phosphorylated only by Blk. Mutants lacking both tyrosine residues of the immune receptor tyrosine-based activation motif (ITAM) of FcgammaRIIa/c were not phosphorylated by Blk and Fyn, while Lyn-mediated phosphorylation was dependent on the presence of the C-terminal tyrosine of the ITAM. Results obtained in assays using an FcgammaR- B-cell line transfected with wild-type or mutated FcgammaRIIa demonstrated that exchange of the C-terminal tyrosine of the ITAM of FcgammaRIIa/c was sufficient to abolish FcgammaRIIa/c phosphorylation in B cells. Additionally, we could show that Lyn and Fyn bind to FcgammaRIIa/c, with the ITAM being necessary for association. Comparison of the phosphorylation pattern of each PTK observed in vitro with the phosphorylation pattern observed in vivo suggests that Lyn is the most likely candidate for FcgammaRIIa/c and FcgammaRIIb1 phosphorylation in vivo. PMID:8756631

  4. Protein tyrosine kinase regulates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking induced by acute hypoxia in cultured brainstem neurons.

    PubMed

    Wang, H; Yu, L C; Li, Y C

    2016-01-01

    This study was performed to investigate the modulation effect of protein tyrosine kinase on postsynaptic a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking induced by acute hypoxia in cultured brainstem neurons. The cultured neurons were exposed to 1% O2 and the expression of AMPA receptor subunit GluR2 on the cell surface was significantly increased, while total GluR2 was not markedly changed. Furthermore, the hypoxia-induced increase in GluR2 expression on the cell surface was partially blocked by the protein tyrosine kinase membrane-permeable inhibitor genistein. In contrast, both the protein tyrosine kinase agonist nerve growth factor and protein tyrosine phosphatase inhibitor vanadate promoted the hypoxia-induced increase of GluR2 expression on cell surface. Moreover, GluR2 could be phosphorylated by tyrosine under normoxia and hypoxia conditions in vitro on brainstem neurons, and tyrosine phosphorylation of GluR2 was significantly stronger under hypoxia conditions. Our results indicate that acute hypoxia induces the AMPA receptor subunit GluR2 to rapidly migrate to the cell membrane to modify the strength of the synapse. This study indicates that tyrosine phosphorylation of the receptor is an important pathway regulating the rapid migration of GluR2 in the postsynaptic domain induced by hypoxia. PMID:27525851

  5. 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.

  6. Ameliorating Endothelial Mitochondrial Dysfunction Restores Coronary Function via Transient Receptor Potential Vanilloid 1-Mediated Protein Kinase A/Uncoupling Protein 2 Pathway.

    PubMed

    Xiong, Shiqiang; Wang, Peijian; Ma, Liqun; Gao, Peng; Gong, Liuping; Li, Li; Li, Qiang; Sun, Fang; Zhou, Xunmei; He, Hongbo; Chen, Jing; Yan, Zhencheng; Liu, Daoyan; Zhu, Zhiming

    2016-02-01

    Coronary heart disease arising from atherosclerosis is a leading cause of cardiogenic death worldwide. Mitochondria are the principal source of reactive oxygen species (ROS), and defective oxidative phosphorylation by the mitochondrial respiratory chain contributes to ROS generation. Uncoupling protein 2 (UCP2), an adaptive antioxidant defense factor, protects against mitochondrial ROS-induced endothelial dysfunction in atherosclerosis. The activation of transient receptor potential vanilloid 1 (TRPV1) attenuates vascular dysfunction. Therefore, whether TRPV1 activation antagonizes coronary lesions by alleviating endothelial mitochondrial dysfunction and enhancing the activity of the protein kinase A/UCP2 pathway warrants examination. ApoE(-/-), ApoE(-/-)/TRPV1(-/-), and ApoE(-/-)/UCP2(-/-) mice were fed standard chow, a high-fat diet (HFD), or the HFD plus 0.01% capsaicin. HFD intake profoundly impaired coronary vasodilatation and myocardial perfusion and shortened the survival duration of ApoE(-/-) mice. TRPV1 or UCP2 deficiency exacerbated HFD-induced coronary dysfunction and was associated with increased ROS generation and reduced nitric oxide production in the endothelium. The activation of TRPV1 by capsaicin upregulated UCP2 expression via protein kinase A phosphorylation, thereby alleviating endothelial mitochondrial dysfunction and inhibiting mitochondrial ROS generation. In vivo, dietary capsaicin supplementation enhanced coronary relaxation and prolonged the survival duration of HFD-fed ApoE(-/-) mice. These effects were not observed in ApoE(-/-) mice lacking the TRPV1 or UCP2 gene. The upregulation of protein kinase A /UCP2 via TRPV1 activation ameliorates coronary dysfunction and prolongs the lifespan of atherosclerotic mice by ameliorating endothelial mitochondrial dysfunction. Dietary capsaicin supplementation may represent a promising intervention for the primary prevention of coronary heart disease. PMID:26667415

  7. SENESCENCE-SUPPRESSED PROTEIN PHOSPHATASE Directly Interacts with the Cytoplasmic Domain of SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE and Negatively Regulates Leaf Senescence in Arabidopsis.

    PubMed

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

    2015-10-01

    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.

  8. 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

  9. Microbial Protein-tyrosine Kinases*

    PubMed Central

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

    2014-01-01

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

  10. Protein kinase A increases type-2 inositol 1,4,5-trisphosphate receptor activity by phosphorylation of serine 937.

    PubMed

    Betzenhauser, Matthew J; Fike, Jenna L; Wagner, Larry E; Yule, David I

    2009-09-11

    Protein kinase A (PKA) phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) represents a mechanism for shaping intracellular Ca(2+) signals following a concomitant elevation in cAMP. Activation of PKA results in enhanced Ca(2+) release in cells that express predominantly InsP(3)R2. PKA is known to phosphorylate InsP(3)R2, but the molecular determinants of this effect are not known. We have expressed mouse InsP(3)R2 in DT40-3KO cells that are devoid of endogenous InsP(3)R and examined the effects of PKA phosphorylation on this isoform in unambiguous isolation. Activation of PKA increased Ca(2+) signals and augmented the single channel open probability of InsP(3)R2. A PKA phosphorylation site unique to the InsP(3)R2 was identified at Ser(937). The enhancing effects of PKA activation on this isoform required the phosphorylation of Ser(937), since replacing this residue with alanine eliminated the positive effects of PKA activation. These results provide a mechanism responsible for the enhanced Ca(2+) signaling following PKA activation in cells that express predominantly InsP(3)R2.

  11. Protein kinase Cβ is critical for the metabolic switch to glycolysis following B-cell antigen receptor engagement.

    PubMed

    Blair, Derek; Dufort, Fay J; Chiles, Thomas C

    2012-11-15

    Signals derived from the BCR (B-cell antigen receptor) control survival, development and antigenic responses. One mechanism by which BCR signals may mediate these responses is by regulating cell metabolism. Indeed, the bioenergetic demands of naïve B-cells increase following BCR engagement and are characterized by a metabolic switch to aerobic glycolysis; however, the signalling pathways involved in this metabolic reprogramming are poorly defined. The PKC (protein kinase C) family plays an integral role in B-cell survival and antigenic responses. Using pharmacological inhibition and mice deficient in PKCβ, we demonstrate an essential role of PKCβ in BCR-induced glycolysis in B-cells. In contrast, mice deficient in PKCδ exhibit glycolytic rates comparable with those of wild-type B-cells following BCR cross-linking. The induction of several glycolytic genes following BCR engagement is impaired in PKCβ-deficient B-cells. Moreover, blocking glycolysis results in decreased survival of B-cells despite BCR engagement. The results establish a definitive role for PKCβ in the metabolic switch to glycolysis following BCR engagement of naïve B-cells.

  12. RTK SLAP down: the emerging role of Src-like adaptor protein as a key player in receptor tyrosine kinase signaling.

    PubMed

    Wybenga-Groot, Leanne E; McGlade, C Jane

    2015-02-01

    SLAP (Src like adaptor protein) contains adjacent Src homology 3 (SH3) and Src homology 2 (SH2) domains closely related in sequence to that of cytoplasmic Src family tyrosine kinases. Expressed most abundantly in the immune system, SLAP function has been predominantly studied in the context of lymphocyte signaling, where it functions in the Cbl dependent downregulation of antigen receptor signaling. However, accumulating evidence suggests that SLAP plays a role in the regulation of a broad range of membrane receptors including members of the receptor tyrosine kinase (RTK) family. In this review we highlight the role of SLAP in the ubiquitin dependent regulation of type III RTKs PDGFR, CSF-1R, KIT and Flt3, as well as Eph family RTKs. SLAP appears to bind activated type III and Eph RTKs via a conserved autophosphorylated juxtamembrane tyrosine motif in an SH2-dependent manner, suggesting that SLAP is important in regulating RTK signaling.

  13. Rapid activation of sodium-proton exchange and extracellular signal-regulated protein kinase in fibroblasts by G protein-coupled 5-HT1A receptor involves distinct signalling cascades.

    PubMed Central

    Garnovskaya, M N; Mukhin, Y; Raymond, J R

    1998-01-01

    These experiments tested the hypothesis that signalling elements involved in the activation of the extracellular signal-regulated protein kinase (ERK) mediate rapid activation of sodium-proton exchange (NHE) in fibroblasts when both signals are initiated by a single G protein-coupled receptor, the 5-HT1A receptor. Similarities between the two processes were comparable concentration-response curves and time-courses, and overlapping sensitivity to some pharmacological inhibitors of tyrosine kinases (staurosporine and genistein), and phosphoinositide 3'-kinase (wortmannin and LY204002). Activation of NHE was much more sensitive to the phosphatidylcholine-specific phospholipase inhibitor (D609) than was ERK. Neither pathway was sensitive to manoeuvres designed to block PKC. In contrast, Src or related kinases appear to be required to activate ERK, but not NHE. Transfection of cDNA constructs encoding inactive mutant phosphoinositide 3'-kinase, Grb2, Sos, Ras, and Raf molecules were successful in attenuating ERK, but had essentially no effect upon NHE activation. Finally, PD98059, an inhibitor of mitogen activated/extracellular signal regulated kinase kinase, blocked ERK but not NHE activation. Thus, in CHO fibroblast cells, activation by the 5-HT1A receptor of ERK and NHE share a number of overlapping features. However, our studies do not support a major role for ERK, when activated by the 5-HT1A receptor, as a short-term upstream regulator of NHE activity. PMID:9461547

  14. The viral G protein-coupled receptor ORF74 unmasks phospholipase C signaling of the receptor tyrosine kinase IGF-1R.

    PubMed

    de Munnik, Sabrina M; van der Lee, Rosan; Velders, Daniëlle M; van Offenbeek, Jody; Smits-de Vries, Laura; Leurs, Rob; Smit, Martine J; Vischer, Henry F

    2016-06-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) encodes the constitutively active G protein-coupled receptor ORF74, which is expressed on the surface of infected host cells and has been linked to the development of the angioproliferative tumor Kaposi's sarcoma. Furthermore, the insulin-like growth factor (IGF)-1 receptor, a receptor tyrosine kinase, also plays an essential role in Kaposi's sarcoma growth and survival. In this study we examined the effect of the constitutively active viral receptor ORF74 on human IGF-1R signaling. Constitutive and CXCL1-induced ORF74 signaling did not transactivate IGF-1R. In contrast, IGF-1 stimulated phospholipase C (PLC) activation in an ORF74-dependent manner without affecting chemokine binding to ORF74. Inhibition of constitutive ORF74 activity by mutagenesis or the inverse agonist CXCL10, or neutralizing IGF-1R with an antibody or silencing IGF-1R expression using siRNA inhibited PLC activation by IGF-1. Transactivation of ORF74 in response to IGF-1 occurred independently of Src, PI3K, and secreted ORF74 ligands. Furthermore, tyrosine residues in the carboxyl-terminus and intracellular loop 2 of ORF74 are not essential for IGF-1-induced PLC activation. Interestingly, PLC activation in response to IGF-1 is specific for ORF74 as IGF-1 was unable to activate PLC in cells expressing the constitutively active human cytomegalovirus (HCMV)-encoded GPCR US28. Interestingly, IGF-1 does not induce β-arrestin recruitment to ORF74. The proximity ligation assay revealed close proximity between ORF74 and IGF-1R on the cell surface, but a physical interaction was not confirmed by co-immunoprecipitation. Unmasking IGF-1R signaling to PLC in response to IGF-1 is a previously unrecognized action of ORF74. PMID:26931381

  15. Protein Kinase A Is Part of a Mechanism That Regulates Nuclear Reimport of the Nuclear tRNA Export Receptors Los1p and Msn5p

    PubMed Central

    Pierce, Jacqueline B.; van der Merwe, George

    2014-01-01

    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. PMID:24297441

  16. 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.

  17. Signaling from the Human Melanocortin 1 Receptor to ERK1 and ERK2 Mitogen-Activated Protein Kinases Involves Transactivation of cKIT

    PubMed Central

    Herraiz, Cecilia; Journé, Fabrice; Abdel-Malek, Zalfa; Ghanem, Ghanem; Jiménez-Cervantes, Celia; García-Borrón, José C.

    2011-01-01

    Melanocortin 1 receptor (MC1R), a Gs protein-coupled receptor expressed in melanocytes, is a major determinant of skin pigmentation, phototype and cancer risk. Upon stimulation by αMSH, MC1R triggers the cAMP and ERK1/ERK2 MAPK pathways. In mouse melanocytes, ERK activation by αMSH binding to Mc1r depends on cAMP, and melanocytes are considered a paradigm for cAMP-dependent ERK activation. However, human MC1R variants associated with red hair, fair skin [red hair color (RHC) phenotype], and increased skin cancer risk display reduced cAMP signaling but activate ERKs as efficiently as wild type in heterologous cells, suggesting independent signaling to ERKs and cAMP in human melanocytes. We show that MC1R signaling activated the ERK pathway in normal human melanocytes and melanoma cells expressing physiological levels of endogenous RHC variants. ERK activation was comparable for wild-type and mutant MC1R and was independent on cAMP because it was neither triggered by stimulation of cAMP synthesis with forskolin nor blocked by the adenylyl cyclase inhibitor 2′,5′-dideoxyadenosine. Stimulation of MC1R with αMSH did not lead to protein kinase C activation and ERK activation was unaffected by protein kinase C inhibitors. Conversely, pharmacological interference, small interfering RNA studies, expression profiles, and functional reconstitution experiments showed that αMSH-induced ERK activation resulted from Src tyrosine kinase-mediated transactivation of the stem cell factor receptor, a receptor tyrosine kinase essential for proliferation, differentiation, and survival of melanocyte precursors, thus demonstrating a functional link between the stem cell factor receptor and MC1R. Moreover, this transactivation phenomenon is unique because it is unaffected by natural mutations impairing canonical MC1R signaling through the cAMP pathway. PMID:21084381

  18. Kainate receptor-mediated apoptosis in primary cultures of cerebellar granule cells is attenuated by mitogen-activated protein and cyclin-dependent kinase inhibitors

    PubMed Central

    Giardina, Sarah F; Beart, Philip M

    2002-01-01

    Previous studies have suggested that neuronal apoptosis is the result of an abortive attempt to re-enter the cell cycle, and more recently the cyclin-dependent (CDKs) and the mitogen-activated protein (MAP) kinases, two superfamilies of kinases that influence and control cell cycle progression, have been implicated in neuronal apoptosis. Here, to examine whether CDK/MAPK related pathways are involved in excitotoxicity, we studied the actions of various kinase inhibitors on apoptosis induced by the ionotropic glutamate (Glu) receptor agonist, kainate (KA), in primary cultures of murine cerebellar granule cells (CGCs). KA-mediated neurotoxicity was concentration-dependent, as determined by a cell viability assay monitoring the reduction of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and largely apoptotic in nature, as shown by morphological examination and labelling of DNA fragmentation in situ using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP digoxigenin nick-end labelling (TUNEL). KA-mediated neurotoxicity and apoptosis was completely attenuated by the mixed CDK and MAP kinase inhibitor, olomoucine, in a concentration-dependent manner (50 – 600 μM), and partially by roscovitine (1 – 100 μM), a more selective CDK inihibitor. The p38 MAP kinase inhibitor, SB203580 (1 – 100 μM), partially attenuated KA receptor-mediated apoptosis, as did the MAP kinase kinase inhibitors PD98509 (1 – 100 μM) and U0126 (1 – 100 μM). These findings provide new evidence for a complex network of interacting pathways involving CDK/MAPK that control apoptosis downstream of KA receptor activation in excitotoxic neuronal cell death. PMID:11934814

  19. Molecular Basis of the Interaction of the Human Protein Tyrosine Phosphatase Non-receptor Type 4 (PTPN4) with the Mitogen-activated Protein Kinase p38γ.

    PubMed

    Maisonneuve, Pierre; Caillet-Saguy, Célia; Vaney, Marie-Christine; Bibi-Zainab, Edoo; Sawyer, Kristi; Raynal, Bertrand; Haouz, Ahmed; Delepierre, Muriel; Lafon, Monique; Cordier, Florence; Wolff, Nicolas

    2016-08-01

    The human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cell death induction in neuroblastoma and glioblastoma cell lines in a PDZ·PDZ binding motifs-dependent manner, but the cellular partners of PTPN4 involved in cell protection are unknown. Here, we described the mitogen-activated protein kinase p38γ as a cellular partner of PTPN4. The main contribution to the p38γ·PTPN4 complex formation is the tight interaction between the C terminus of p38γ and the PDZ domain of PTPN4. We solved the crystal structure of the PDZ domain of PTPN4 bound to the p38γ C terminus. We identified the molecular basis of recognition of the C-terminal sequence of p38γ that displays the highest affinity among all endogenous partners of PTPN4. We showed that the p38γ C terminus is also an efficient inducer of cell death after its intracellular delivery. In addition to recruiting the kinase, the binding of the C-terminal sequence of p38γ to PTPN4 abolishes the catalytic autoinhibition of PTPN4 and thus activates the phosphatase, which can efficiently dephosphorylate the activation loop of p38γ. We presume that the p38γ·PTPN4 interaction promotes cellular signaling, preventing cell death induction.

  20. 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.

  1. Identification of a receptor-like protein kinase gene rapidly induced by abscisic acid, dehydration, high salt, and cold treatments in Arabidopsis thaliana.

    PubMed Central

    Hong, S W; Jon, J H; Kwak, J M; Nam, H G

    1997-01-01

    A cDNA clone for a receptor-like protein kinase gene (RPK1) was isolated from Arabidopsis thaliana. The clone is 1952 bp long with 1623 bp of an open reading frame encoding a peptide of 540 amino acids. The deduced peptide (RPK1) contains four distinctive domains characteristic of receptor kinases: (a) a putative amino-terminal signal sequence domain; (b) a domain with five extracellular leucine-rich repeat sequences; (c) a membrane-spanning domain; and (d) a cytoplasmic protein kinase domain that contains all of the 11 subdomains conserved among protein kinases. The RPK1 gene is expressed in flowers, stems, leaves, and roots. Expression of the RPK1 gene is induced within 1 h after treatment with abscisic acid (ABA). The gene is also rapidly induced by several environmental stresses such as dehydration, high salt, and low temperature, suggesting that the gene is involved in a general stress response. The dehydration-induced expression is not impaired in aba-1, abi1-1, abi2-1, and abi3-1 mutants, suggesting that the dehydration-induced expression of the RPK1 gene is ABA-independent. A possible role of this gene in the signal transduction pathway of ABA and the environmental stresses is discussed. PMID:9112773

  2. Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions.

    PubMed

    Pandalaneni, Sravan; Karuppiah, Vijaykumar; Saleem, Muhammad; Haynes, Lee P; Burgoyne, Robert D; Mayans, Olga; Derrick, Jeremy P; Lian, Lu-Yun

    2015-07-24

    Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca(2+)-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca(2+)/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca(2+)/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178-Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca(2+)/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178-Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1.

  3. Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions.

    PubMed

    Pandalaneni, Sravan; Karuppiah, Vijaykumar; Saleem, Muhammad; Haynes, Lee P; Burgoyne, Robert D; Mayans, Olga; Derrick, Jeremy P; Lian, Lu-Yun

    2015-07-24

    Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca(2+)-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca(2+)/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca(2+)/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178-Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca(2+)/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178-Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1. PMID:25979333

  4. Characterization of 5' end of human thromboxane receptor gene. Organizational analysis and mapping of protein kinase C--responsive elements regulating expression in platelets.

    PubMed

    D'Angelo, D D; Davis, M G; Houser, W A; Eubank, J J; Ritchie, M E; Dorn, G W

    1995-09-01

    Platelet thromboxane receptors are acutely and reversibly upregulated after acute myocardial infarction. To determine if platelet thromboxane receptors are under transcriptional control, we isolated and characterized human genomic DNA clones containing the 5' flanking region of the thromboxane receptor gene. The exon-intron structure of the 5' portion of the thromboxane receptor gene was determined initially by comparing the nucleotide sequence of the 5' flanking genomic clone with that of a novel human uterine thromboxane receptor cDNA that extended the mRNA 141 bp further upstream than the previously identified human placental cDNA. A major transcription initiation site was located in three human tissues approximately 560 bp upstream from the translation initiation codon and 380 bp upstream from any previously identified transcription initiation site. The thromboxane receptor gene has neither a TATA nor a CAAT consensus site. Promoter function of the 5' flanking region of the thromboxane receptor gene was evaluated by transfection of thromboxane receptor gene promoter/chloramphenicol acetyltransferase (CAT) chimera plasmids into platelet-like K562 cells. Thromboxane receptor promoter activity, as assessed by CAT expression, was relatively weak but was significantly enhanced by phorbol ester treatment. Functional analysis of 5' deletion constructs in transfected K562 cells and gel mobility shift localized the major phorbol ester-responsive motifs in the thromboxane receptor gene promoter to a cluster of activator protein-2 (AP-2) binding consensus sites located approximately 1.8 kb 5' from the transcription initiation site. These studies are the first to determine the structure and organization of the 5' end of the thromboxane receptor gene and demonstrate that thromboxane receptor gene expression can be regulated by activation of protein kinase C via induction of an AP-2-like nuclear factor binding to upstream promoter elements. These findings strongly suggest

  5. 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.

  6. Fulvestrant-Induced Cell Death and Proteasomal Degradation of Estrogen Receptor α Protein in MCF-7 Cells Require the CSK c-Src Tyrosine Kinase

    PubMed Central

    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. PMID:23593342

  7. 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

  8. Serotonin via 5-HT7 receptors activates p38 mitogen-activated protein kinase and protein kinase C epsilon resulting in interleukin-6 synthesis in human U373 MG astrocytoma cells.

    PubMed

    Lieb, Klaus; Biersack, Lisa; Waschbisch, Anne; Orlikowski, Sonja; Akundi, Ravi Shankar; Candelario-Jalil, Eduardo; Hüll, Michael; Fiebich, Bernd L

    2005-05-01

    Serotonin [5-hydroxytryptamine (5-HT)] is a widely distributed neurotransmitter which is involved in neuroimmunomodulatory processes. Previously, it has been demonstrated that 5-HT may induce interleukin (IL)-6 expression in primary rat hippocampal astrocytes. The present study was undertaken to investigate the molecular pathways underlying this induction of IL-6 synthesis. As a model system, we used the human astrocytoma cell line U373 MG, which synthesizes IL-6 upon stimulation with various inducers. 5-HT dose- and time-dependently induced IL-6 protein synthesis. We identified several 5-HT receptors to be expressed on U373 MG cells, including the 5-HT1D, 5-HT2A, 5-HT3 and 5-HT7 receptors. In this report, we show that the 5-HT-induced IL-6 release is mediated by the 5-HT7 receptor based on several agonist/antagonists that were used. 5-HT-induced IL-6 synthesis is inhibited by the partially selective 5-HT7 receptor antagonist, pimozide, and the selective antagonist SB269970. Furthermore, IL-6 synthesis was induced by the 5-HT7 receptor agonist carboxamidotryptamin. In addition, we found p38 MAPKs and protein kinase C (PKC) epsilon to be involved in 5-HT-induced IL-6 synthesis as specific inhibitors of these enzymes (SB202190 and RO-31-8425, respectively) blocked 5-HT-induced IL-6 synthesis. Furthermore, 5-HT mediated the phosphorylation of both p38 MAPK as well as the PKC epsilon isoform. The p42/44 MAPKs, however, were not involved in 5-HT-induced IL-6 synthesis. This study shows, for the first time, a central role of 5-HT7 receptor linked to p38 MAPK and PKC epsilon for the induction of cytokine synthesis in astrocytic cells. PMID:15836614

  9. 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

  10. Protein tyrosine kinase and mitogen-activated protein kinase signalling pathways contribute to differences in heterophil-mediated innate immune responsiveness between two lines of broilers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein tyrosine phosphorylation mediates signal transduction of cellular processes, with protein tyrosine kinases (PTKs) regulating virtually all signaling events. The mitogen-activated protein kinase (MAPK) super-family consists of three conserved pathways that convert receptor activation into ce...

  11. Receptor-like protein kinase HvLysMR1 of barley (Hordeum vulgare L.) is induced during leaf senescence and heavy metal stress.

    PubMed

    Ouelhadj, Akli; Kaminski, Marc; Mittag, Maria; Humbeck, Klaus

    2007-01-01

    The Hordeum vulgare cDNA clone HvLysMR1 that encodes a putative receptor-like protein kinase was identified by restriction fragment differential display-polymerase chain reaction (PCR) comparing cDNA populations derived from mRNAs of primary leaves stressed with chromium for 48 h with controls. The full-length sequence codes for a protein with 622 amino acids which includes characteristic domains of lysine motif receptor-like kinases: an N-terminal signal peptide, two lysine motifs, a transmembrane region, and a serine/threonine kinase domain at the C-terminal end. The expression of HvLysMR1 is induced during exposure to different heavy metals and its transcript accumulates during leaf senescence. Addition of the calcium ionophore A23187 induces HvLysMR1 expression, indicating the involvement of Ca2+ in the regulation of HvLysMR1. In vitro phosphorylation of HvLysMR1 was analysed with [32P]ATP. Using the overexpressed and purified HvLysMR1-kinase domain, the phosphorylation of HvLysMR1 could be confirmed by nano-liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) with neutral loss-triggered MS-MS-MS spectra at amino acids localized at the juxtamembrane region. The involvement of HvLysMR1 during heavy metal stress and leaf senescence is discussed. PMID:17322551

  12. Receptor Tyrosine Kinases in Drosophila Development

    PubMed Central

    Sopko, Richelle; Perrimon, Norbert

    2013-01-01

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

  13. Heterotrimeric G Proteins Serve as a Converging Point in Plant Defense Signaling Activated by Multiple Receptor-Like Kinases1[C][W][OA

    PubMed Central

    Liu, Jinman; Ding, Pingtao; Sun, Tongjun; Nitta, Yukino; Dong, Oliver; Huang, Xingchuan; Yang, Wei; Li, Xin; Botella, José Ramón; Zhang, Yuelin

    2013-01-01

    In fungi and metazoans, extracellular signals are often perceived by G-protein-coupled receptors (GPCRs) and transduced through heterotrimeric G-protein complexes to downstream targets. Plant heterotrimeric G proteins are also involved in diverse biological processes, but little is known about their upstream receptors. Moreover, the presence of bona fide GPCRs in plants is yet to be established. In Arabidopsis (Arabidopsis thaliana), heterotrimeric G protein consists of one Gα subunit (G PROTEIN α-SUBUNIT1), one Gβ subunit (ARABIDOPSIS G PROTEIN β-SUBUNIT1 [AGB1]), and three Gγs subunits (ARABIDOPSIS G PROTEIN γ-SUBUNIT1 [AGG1], AGG2, and AGG3). We identified AGB1 from a suppressor screen of BAK1-interacting receptor-like kinase1-1 (bir1-1), a mutant that activates cell death and defense responses mediated by the receptor-like kinase (RLK) SUPPRESSOR OF BIR1-1. Mutations in AGB1 suppress the cell death and defense responses in bir1-1 and transgenic plants overexpressing SUPPRESSOR OF BIR1-1. In addition, agb1 mutant plants were severely compromised in immunity mediated by three other RLKs, FLAGELLIN-SENSITIVE2 (FLS2), Elongation Factor-TU RECEPTOR (EFR), and CHITIN ELICITOR RECEPTOR KINASE1 (CERK1), respectively. By contrast, G PROTEIN α-SUBUNIT1 is not required for either cell death in bir1-1 or pathogen-associated molecular pattern-triggered immunity mediated by FLS2, EFR, and CERK1. Further analysis of agg1 and agg2 mutant plants indicates that AGG1 and AGG2 are also required for pathogen-associated molecular pattern-triggered immune responses mediated by FLS2, EFR, and CERK1, as well as cell death and defense responses in bir1-1. We hypothesize that the Arabidopsis heterotrimeric G proteins function as a converging point of plant defense signaling by mediating responses initiated by multiple RLKs, which may fulfill equivalent roles to GPCRs in fungi and animals. PMID:23424249

  14. Inhibition of protein kinase C decreases sensitivity of GABA receptor subtype to fipronil insecticide in insect neurosecretory cells.

    PubMed

    Murillo, Laurence; Hamon, Alain; Es-Salah-Lamoureux, Zeineb; Itier, Valérie; Quinchard, Sophie; Lapied, Bruno

    2011-12-01

    Phosphorylation by serine/threonine kinases has been described as a new mechanism for regulating the effects of insecticides on insect neuronal receptors and channels. Although insect GABA receptors are commercially important targets for insecticides (e.g. fipronil), their modulation by kinases is poorly understood and the influence of phosphorylation on insecticide sensitivity is unknown. Using the whole-cell patch-clamp technique, we investigated the modulatory effect of PKC and CaMKinase II on GABA receptor subtypes (GABAR1 and GABAR2) in DUM neurons isolated from the terminal abdominal ganglion (TAG) of Periplaneta americana. Chloride currents through GABAR2 were selectively abolished by PMA and PDBu (the PKC activators) and potentiated by Gö6983, an inhibitor of PKC. Furthermore, using KN-62, a specific CaMKinase II inhibitor, we demonstrated that CaMKinase II activation was also involved in the regulation of GABAR2 function. In addition, using CdCl(2) (the calcium channel blocker) and LOE-908, a blocker of TRPγ, we revealed that calcium influx through TRPγ played an important role in kinase activations. Comparative studies performed with CACA, a selective agonist of GABAR1 in DUM neurons confirmed the involvement of these kinases in the specific regulation of GABAR2. Furthermore, our study reported that GABAR1 was less sensitive than GABAR2 to fipronil. This was demonstrated by the biphasic concentration-response curve and the current-voltage relationship established with both GABA and CACA. Finally, we demonstrated that GABAR2 was 10-fold less sensitive to fipronil following inhibition of PKC, whereas inhibition of CaMKinase II did not alter the effect of fipronil. PMID:21684305

  15. 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.

  16. Toll-Like Receptor 4-Mediated Activation of p38 Mitogen-Activated Protein Kinase Is a Determinant of Respiratory Virus Entry and Tropism▿

    PubMed Central

    Marchant, David; Singhera, Gurpreet K.; Utokaparch, Soraya; Hackett, Tillie L.; Boyd, John H.; Luo, Zongshu; Si, Xiaoning; Dorscheid, Delbert R.; McManus, Bruce M.; Hegele, Richard G.

    2010-01-01

    Respiratory viruses exert a heavy toll of morbidity and mortality worldwide. Despite this burden there are few specific treatments available for respiratory virus infections. Since many viruses utilize host cell enzymatic machinery such as protein kinases for replication, we determined whether pharmacological inhibition of kinases could, in principle, be used as a broad antiviral strategy for common human respiratory virus infections. A panel of green fluorescent protein (GFP)-expressing recombinant respiratory viruses, including an isolate of H1N1 influenza virus (H1N1/Weiss/43), was used to represent a broad range of virus families responsible for common respiratory infections (Adenoviridae, Paramyxoviridae, Picornaviridae, and Orthomyxoviridae). Kinase inhibitors were screened in a high-throughput assay that detected virus infection in human airway epithelial cells (1HAEo-) using a fluorescent plate reader. Inhibition of p38 mitogen-activated protein kinase (MAPK) signaling was able to significantly inhibit replication by all viruses tested. Therefore, the pathways involved in virus-mediated p38 and extracellular signal-regulated kinase (ERK) MAPK activation were investigated using bronchial epithelial cells and primary fibroblasts derived from MyD88 knockout mouse lungs. Influenza virus, which activated p38 MAPK to approximately 10-fold-greater levels than did respiratory syncytial virus (RSV) in 1HAEo- cells, was internalized about 8-fold faster and more completely than RSV. We show for the first time that p38 MAPK is a determinant of virus infection that is dependent upon MyD88 expression and Toll-like receptor 4 (TLR4) ligation. Imaging of virus-TLR4 interactions showed significant clustering of TLR4 at the site of virus-cell interaction, triggering phosphorylation of downstream targets of p38 MAPK, suggesting the need for a signaling receptor to activate virus internalization. PMID:20702616

  17. Protein kinase C-α downregulates estrogen receptor-α by suppressing c-Jun phosphorylation in estrogen receptor-positive breast cancer cells.

    PubMed

    Kim, Sangmin; Lee, Jeongmin; Lee, Se Kyung; Bae, Soo Youn; Kim, Jiyoung; Kim, Minkuk; Kil, Won Ho; Kim, Seok Won; Lee, Jeong Eon; Nam, Seok Jin

    2014-03-01

    Protein kinase C (PKC) activity is elevated in malignant compared with that in normal human breast tissue. In the present study, we investigated the regulatory mechanism and the co-relationship between PKC-α and estrogen receptor-α (ER-α) in ER-α-positive and tamoxifen-resistant (TAMR) breast cancer cells. Our results showed that the level of ER-α expression was significantly decreased in TAMR when compared with that in tamoxifen-sensitive (TAMS) breast cancer cells. However, PKC-α phosphorylation was increased in TAMR breast cancer cells when compared to that in TAMS breast cancer cells. Additionally, ER-α expression was significantly decreased due to the overexpression of constitutively active PKC-α (CA-PKC-α). Next, we investigated the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a reversible activator of PKC, on ER-α expression in ER-α-positive breast cancer cells. TPA decreased the levels of ER-α expression in a time- and dose-dependent manner. In contrast, the TPA-induced downregulation of ER-α was prevented by Go6983, a specific PKC inhibitor. Notably, we found that CA-PKC-α suppressed c-JUN phosphorylation, which is a major activating protein-1 factor, and TPA-induced downregulation of ER-α was prevented by SR11302, a specific activator protein-1 inhibitor. Taken together, we demonstrated that PKC-α activity suppressed the level of ER-α expression by inhibiting c-JUN phosphorylation in ER-α-positive breast cancer cells. Therefore, we suggest that PKC-α may be a potential therapeutic target for treating ER-positive and TAMR breast cancer.

  18. Regulation of epidermal-growth-factor-receptor signal transduction by cis-unsaturated fatty acids. Evidence for a protein kinase C-independent mechanism.

    PubMed Central

    Casabiell, X; Pandiella, A; Casanueva, F F

    1991-01-01

    The effect of acute treatment with non-esterified fatty acids (NEFA) on transmembrane signalling has been investigated in three different cell lines. In EGFR T17 cells, pretreatment with cis-unsaturated (oleic and palmitoleic acids) NEFA, but not with saturated or trans-unsaturated NEFA, inhibited the epidermal-growth-factor (EGF)-induced increases in cytosolic [Ca2+], membrane potential and Ins(1,4,5)P3 generation. The blocking effect was found to be time- and dose-dependent and rapidly reversible after washout. However, oleic acid treatment did not block either binding of 125I-EGF to its receptor or EGF-induced autophosphorylation of the EGF receptor. The mechanism of action of NEFA could not be attributed to protein kinase C activation, since (i) down-regulation of the enzyme by long-term treatment with phorbol esters did not prevent blockade by oleic acid, and (ii) the effects of acutely administered phorbol ester and oleic acid were additive. In this cell line, signalling at bradykinin and bombesin receptors was also impaired by oleic acid. In A431 cells, oleic acid also blocked signal transduction at the EGF and B2 bradykinin receptors. Finally, in PC12 cells, oleic acid blocked the Ca2+ influx mediated by the activation of B2 bradykinin receptors. In conclusion: (1) NEFA block signal transduction by interfering with receptor-phospholipase C or phospholipase C-substrate interaction without preventing ligand binding; (2) NEFA do not act by a protein kinase C-mediated mechanism; (3) the effect of NEFA is dependent on their configuration rather than hydrophobicity or chain length; (4) this effect is evident in several different cell lines and receptor systems. Images Fig. 4. PMID:1898356

  19. Phosphorylation of protein phosphatase inhibitor-1 by protein kinase C.

    PubMed

    Sahin, Bogachan; Shu, Hongjun; Fernandez, Joseph; El-Armouche, Ali; Molkentin, Jeffery D; Nairn, Angus C; Bibb, James A

    2006-08-25

    Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr(35). Moreover, Ser(67) of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser(67) inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser(65) in vitro. In contrast, Ser(67) phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser(65). Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser(65) and Ser(67), but not Ser(65) alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser(65) inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser(67) protects phospho-Ser(65) inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser(65)/Ser(67) inhibitor-1 in this tissue. In contrast, the activation of N-methyl-d-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser(65)/Ser(67) inhibitor-1 levels. Phosphomimetic mutation of Ser(65) and/or Ser(67) did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser(65)/Ser(67) inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser(67) and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.

  20. Targeting of TGF-β-activated protein kinase 1 inhibits chemokine (C-C motif) receptor 7 expression, tumor growth and metastasis in breast cancer

    PubMed Central

    Hung, Wen-Chun; Hou, Ming-Feng

    2015-01-01

    TGF-β-activated protein kinase 1 (TAK1) is a critical mediator in inflammation, immune response and cancer development. Our previous study demonstrated that activation of TAK1 increases the expression of chemokine (C-C motif) receptor 7 (CCR7) and promotes lymphatic invasion ability of breast cancer cells. However, the expression and association of activated TAK1 and CCR7 in breast tumor tissues is unknown and the therapeutic effect by targeting TAK1 is also unclear. We showed that activated TAK1 (as indicated by phospho-TAK1) and its binding protein TAB1 are strongly expressed in breast tumor tissues (77% and 74% respectively). In addition, increase of phospho-TAK1 or TAB1 is strongly associated with over-expression of CCR7. TAK1 inhibitor 5Z-7-Oxozeaenol (5Z-O) inhibited TAK1 activity, suppressed downstream signaling pathways including p38, IκB kinase (IKK) and c-Jun N-terminal kinase (JNK) and reduced CCR7 expression in metastatic MDA-MB-231 cells. In addition, 5Z-O repressed NF-κB- and c-JUN-mediated transcription of CCR7 gene. Knockdown of TAB1 attenuated CCR7 expression and tumor growth in an orthotopic animal study. More importantly, lymphatic invasion and lung metastasis were suppressed. Collectively, our results demonstrate that constitutive activation of TAK1 is frequently found in human breast cancer and this kinase is a potential therapeutic target for this cancer. PMID:25557171

  1. 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

  2. Transcriptional regulation through glutamate receptors: Involvement of tyrosine kinases.

    PubMed

    López-Bayghen, Esther; Aguirre, Adán; Ortega, Arturo

    2003-12-01

    Glutamate receptors play a key role in neuronal plasticity, learning and memory, and in several neuropathologies. Short-term and long-term changes in synaptic efficacy are triggered by glutamate. Although an enhanced glutamate-dependent tyrosine phosphorylation has been described in several systems, its role in membrane-to-nuclei signaling is unclear. Taking advantage of the fact that the gene encoding the chick kainate-binding protein undergoes a glutamate-dependent transcriptional regulation via an activator protein-1 (AP-1) site, we evaluated the involvement of tyrosine kinases in this process. We describe here the participation of receptor and non-receptor tyrosine kinases in the signaling cascade triggered by glutamate. Our results suggest that in Bergmann glia cells, glutamate receptors transactivate receptor tyrosine kinases, favoring the idea of a complex network of signals activated by this excitatory neurotransmitter that results in regulation of gene expression.

  3. 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

  4. 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.

  5. [Regulation of G protein-coupled receptor kinase 5 mRNA and protein level in rat brain by addictive drugs].

    PubMed

    Zhu, Min; Fan, Xue-Liang; Yang, Wei-Lin; Jiang, Yan; Ma, Lan

    2004-10-25

    G protein-coupled receptor kinase 5 (GRK5) plays an important role in the regulation of GPCR-transduced signals. Our previous study showed that acute administration of morphine could significantly increase GRK5 mRNA level in the cerebral cortex and hippocampus of the rat brain. The current study investigated the potential effects of acute administration of addictive drugs including morphine, heroine and cocaine on GRK5 mRNA level in the rat brain using in situ hybridization and analyzed the effects of acute and chronic morphine treatments on GRK5 protein level in the rat brain using Western blotting assay. Our results showed that 2 h after the initial morphine (10 mg/kg), cocaine (15 mg/kg) and heroine (1 mg/kg) treatment, the mRNA level of GRK5 in the parietal cortex increased about 110% (P<0.01), 70% (P<0.05) and 100% (P<0.01), respectively. In the temporal cortex, GRK5 mRNA level increased about 90% (P<0.01), 40% (P<0.05) and 80.0% (P<0.01), respectively . In the hippocampus, the mRNA level of GRK5 increased about 60% (P<0.01), 30% (P<0.05) and 80% (P<0.01). However, the mRNA level of GRK5 remained unchanged after acute morphine, cocaine or heroine treatment. In the cerebral cortex of the rat brain, the acute administration of morphine (NS-Mor) increased GRK5 protein level by about 60% while the chronic morphine treatment (Mor-Mor) increased GRK5 protein level even higher [about 130% compared with the control group (chronic saline treatment, NS-NS) group, P<0.01]. In the hippocampus, GRK5 protein level remained unchanged after acute administration of morphine (P>0.1),while the level of GRK5 protein tended to decrease after chronic morphine treatment (P=0.098). In the thalamus, acute morphine treatment caused no change in GRK5 protein level (P>0.1) while after chronic morphine treatment, GRK5 protein level decreased significantly (more than 90%, P<0.01), Taken together, our results indicate that addictive drugs can regulate GRK5 in the rat brain on protein level

  6. 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.

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

    PubMed

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

    2015-12-01

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

  8. Killer Cell Lectin-like Receptor G1 Inhibits NK Cell Function through Activation of Adenosine 5'-Monophosphate-Activated Protein Kinase.

    PubMed

    Müller-Durovic, Bojana; Lanna, Alessio; Polaco Covre, Luciana; Mills, Rachel S; Henson, Sian M; Akbar, Arne N

    2016-10-01

    NK cells are the first line of defense against infected and transformed cells. Defective NK cell activity was shown to increase susceptibility for viral infections and reduce tumor immune-surveillance. With age, the incidence of infectious diseases and malignancy rises dramatically, suggesting that impaired NK cell function might contribute to disease in these individuals. We found an increased frequency of NK cells with high expression of the inhibitory killer cell lectin-like receptor G1 (KLRG1) in individuals >70 y. The role of KLRG1 in ageing is not known, and the mechanism of KLRG1-induced inhibition of NK cell function is not fully understood. We report that NK cells with high KLRG1 expression spontaneously activate the metabolic sensor AMP-activated protein kinase (AMPK) and that activation of AMPK negatively regulates NK cell function. Pre-existing AMPK activity is further amplified by ligation of KLRG1 in these cells, which leads to internalization of the receptor and allows interaction with AMPK. We show that KLRG1 activates AMPK by preventing its inhibitory dephosphorylation by protein phosphatase-2C rather than inducing de novo kinase activation. Finally, inhibition of KLRG1 or AMPK prevented KLRG1-induced activation of AMPK and reductions in NK cell cytotoxicity, cytokine secretion, proliferation, and telomerase expression. This novel signaling pathway links metabolic sensing, effector function, and cell differentiation with inhibitory receptor signaling that may be exploited to enhance NK cell activity during ageing.

  9. Endosulfan Induces CYP1A1 Expression Mediated through Aryl Hydrocarbon Receptor Signal Transduction by Protein Kinase C

    PubMed Central

    Han, Eun Hee; Kim, Hyung Gyun; Lee, Eun Ji

    2015-01-01

    CYP1A1 is a phase I xenobiotic-metabolizing enzyme whose expression is mainly driven by AhR. Endosulfan is an organochlorine pesticide used agriculturally for a wide range of crops. In this study, we investigated the effect of endosulfan on CYP1A1 expression and regulation. Endosulfan significantly increased CYP1A1 enzyme activity as well as mRNA and protein levels. In addition, endosulfan markedly induced XRE transcriptional activity. CH-223191, an AhR antagonist, blocked the endosulfan-induced increase in CYP1A1 mRNA and protein expression. Moreover, endosulfan did not induce CYP1A1 gene expression in AhR-deficient mutant cells. Furthermore, endosulfan enhanced the phosphorylation of calcium calmodulin (CaM)-dependent protein kinase (CaMK) and protein kinase C (PKC). In conclusion, endosulfan-induced up-regulation of CYP1A1 is associated with AhR activation, which may be mediated by PKC-dependent pathways. PMID:26877836

  10. 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.

  11. 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.

  12. Structural characterization and immunomodulatory activity of Grifola frondosa polysaccharide via toll-like receptor 4-mitogen-activated protein kinases-nuclear factor κB pathways.

    PubMed

    Ma, Xiaolei; Meng, Meng; Han, Lirong; Cheng, Dai; Cao, Xiaohong; Wang, Chunling

    2016-06-15

    We isolated a neutral polysaccharide from the fruiting body of a mushroom Grifola frondosa (GFP-A). The aim of this study was to characterize a neutral α-d-polysaccharide derived from G. frondosa and evaluate its immunomodulatory effect on toll-like receptor 4, mitogen-activated protein kinases and nuclear factor κB pathways of protein expression in macrophages. The structural features of GFP-A were characterized by physicochemical and instrumental analyses. Its molecular weight was found to be 8.48 × 10(2) kDa. The main chain of GFP-A consisted of (1 → 4)-linked and (1 → 6)-linked α-d-glucopyranosyl, and (1 → 3,6)-linked α-d-mannopyranosyl residues, which branched at C-3. The branches consisted of (1 → 6)-linked α-d-galactopyranosyl and t-l-rhamnopyranosyl residues. An in vitro immunomodulatory assay for pro-inflammatory cytokines (interleukin-1β, interleukin-2, tumor necrosis factor alpha, etc.) using the macrophage cell line, RAW 264.7, revealed that GFP-A exhibited significant immunomodulatory activity by stimulating the toll-like receptor 4, mitogen-activated protein kinases to nuclear factor κB/pathway. PMID:27220562

  13. 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

  14. 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

    2016-07-01

    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

  15. Dopamine D4 receptors linked to protein kinase G are required for changes in dopamine release followed by locomotor activity after repeated cocaine administration.

    PubMed

    Oh, Jeong Hwan; Lee, Dong Kun; Shim, Yoon-Bo; Ryu, In Soo; Seo, Su Yeon; Kim, Jieun; Yang, Ju Hwan; Cho, Hyun-Wook; Choe, Eun Sang

    2015-05-01

    We previously found that the dopamine D2-type receptors (D2 and D3 receptors), coupled to protein kinase G (PKG), upregulate locomotor activity after repeated cocaine administration. In this study, D4 receptors, another type of D2 receptor also coupled to PKG, were examined to determine their requirement in the regulation of locomotor activity after repeated cocaine administration. The results demonstrated that repeated injections of cocaine (20 mg/kg), given once a day for seven consecutive days, significantly increased extracellular dopamine concentrations. Intra-caudate infusion of the D4 receptor agonist, PD168077 (10 nmol), and the PKG inhibitor, KT5823 (2 nmol), significantly decreased the repeated cocaine-induced increase in dopamine levels and locomotor activity. However, intra-caudate infusion of KT5823, but not PD168077, decreased ∆FosB immunoreactivity elevated by repeated cocaine administration. These findings suggest that D4 receptors linked to PKG could be a key modulator for dopamine release required for changes in locomotor activity caused by repeated cocaine exposure. PMID:25702161

  16. Calcium-Permeable AMPA Receptors Mediate the Induction of the Protein Kinase A-Dependent Component of Long-Term Potentiation in the Hippocampus

    PubMed Central

    Park, Pojeong; Sanderson, Thomas M.; Amici, Mascia; Choi, Sun-Lim; Bortolotto, Zuner A.; Zhuo, Min

    2016-01-01

    Two forms of NMDA receptor (NMDAR)-dependent long-term potentiation (LTP) at hippocampal CA1 synapses can be distinguished based on their sensitivity to inhibitors of protein kinase A (PKA). The PKA-dependent form requires multiple episodes of high-frequency stimulation (HFS) or theta burst stimuli (TBS) with a spacing between episodes in the order of minutes. To investigate the mechanism by which spaced episodes induce the PKA-dependent form of LTP, we have compared, in interleaved experiments, spaced (s) and compressed (c) TBS protocols in the rat CA1 synapses. We find that LTP induced by sTBS, but not that induced by cTBS, involves the insertion of calcium-permeable (CP) AMPARs, as assessed using pharmacological and electrophysiological criteria. Furthermore, a single TBS when paired with rolipram [4-(3-(cyclopentyloxy)-4-methoxyphenyl)pyrrolidin-2-one], to activate PKA, generates an LTP that also involves the insertion of CP-AMPARs. These data demonstrate that the involvement of CP-AMPARs in LTP is critically determined by the timing of the induction trigger and is associated specifically with the PKA-dependent form of LTP. SIGNIFICANCE STATEMENT Long-term potentiation is a family of synaptic mechanisms that are believed to be important for learning and memory. Two of the most extensively studied forms are triggered by the synaptic activation of NMDA receptors and expressed by changes in AMPA receptor function. They can be distinguished on the basis of their requirement for activation of a protein kinase, PKA. We show that the PKA-dependent form also involves the transient insertion of calcium-permeable AMPA receptors. These results have implications for relating synaptic plasticity to learning and memory and suggest a specific linkage between PKA activation and the rapid synaptic insertion of calcium-permeable AMPA receptors during long-term potentiation. PMID:26758849

  17. Protein Crystals of Raf Kinase

    NASA Technical Reports Server (NTRS)

    1995-01-01

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

  18. Glutamate receptor δ2 associates with metabotropic glutamate receptor 1 (mGluR1), protein kinase Cγ, and canonical transient receptor potential 3 and regulates mGluR1-mediated synaptic transmission in cerebellar Purkinje neurons.

    PubMed

    Kato, Akihiko S; Knierman, Michael D; Siuda, Edward R; Isaac, John T R; Nisenbaum, Eric S; Bredt, David S

    2012-10-31

    Cerebellar motor coordination and cerebellar Purkinje cell synaptic function require metabotropic glutamate receptor 1 (mGluR1, Grm1). We used an unbiased proteomic approach to identify protein partners for mGluR1 in cerebellum and discovered glutamate receptor δ2 (GluRδ2, Grid2, GluΔ2) and protein kinase Cγ (PKCγ) as major interactors. We also found canonical transient receptor potential 3 (TRPC3), which is also needed for mGluR1-dependent slow EPSCs and motor coordination and associates with mGluR1, GluRδ2, and PKCγ. Mutation of GluRδ2 changes subcellular fractionation of mGluR1 and TRPC3 to increase their surface expression. Fitting with this, mGluR1-evoked inward currents are increased in GluRδ2 mutant mice. Moreover, loss of GluRδ2 disrupts the time course of mGluR1-dependent synaptic transmission at parallel fiber-Purkinje cells synapses. Thus, GluRδ2 is part of the mGluR1 signaling complex needed for cerebellar synaptic function and motor coordination, explaining the shared cerebellar motor phenotype that manifests in mutants of the mGluR1 and GluRδ2 signaling pathways.

  19. Bacillus bombysepticus α-Toxin Binding to G Protein-Coupled Receptor Kinase 2 Regulates cAMP/PKA Signaling Pathway to Induce Host Death

    PubMed Central

    Lin, Ping; Cheng, Tingcai; Ma, Sanyuan; Gao, Junping; Jin, Shengkai; Jiang, Liang; Xia, Qingyou

    2016-01-01

    Bacterial pathogens and their toxins target host receptors, leading to aberrant behavior or host death by changing signaling events through subversion of host intracellular cAMP level. This is an efficient and widespread mechanism of microbial pathogenesis. Previous studies describe toxins that increase cAMP in host cells, resulting in death through G protein-coupled receptor (GPCR) signaling pathways by influencing adenylyl cyclase or G protein activity. G protein-coupled receptor kinase 2 (GRK2) has a central role in regulation of GPCR desensitization. However, little information is available about the pathogenic mechanisms of toxins associated with GRK2. Here, we reported a new bacterial toxin-Bacillus bombysepticus (Bb) α-toxin that was lethal to host. We showed that Bb α-toxin interacted with BmGRK2. The data demonstrated that Bb α-toxin directly bound to BmGRK2 to promote death by affecting GPCR signaling pathways. This mechanism involved stimulation of Gαs, increase level of cAMP and activation of protein kinase A (PKA). Activated cAMP/PKA signal transduction altered downstream effectors that affected homeostasis and fundamental biological processes, disturbing the structural and functional integrity of cells, resulting in death. Preventing cAMP/PKA signaling transduction by inhibitions (NF449 or H-89) substantially reduced the pathogenicity of Bb α-toxin. The discovery of a toxin-induced host death specifically linked to GRK2 mediated signaling pathway suggested a new model for bacterial toxin action. Characterization of host genes whose expression and function are regulated by Bb α-toxin and GRK2 will offer a deeper understanding of the pathogenesis of infectious diseases caused by pathogens that elevate cAMP. PMID:27022742

  20. A YAC contig spanning a cluster of human type III receptor protein tyrosine kinase genes (PDGFRA-KIT-KDR) in chromosome segment 4q12

    SciTech Connect

    Spritz, R.A.; Strunk, K.M.; Lee, S.T.

    1994-07-15

    The authors have mapped five genes encoding protein tyrosine kinases (PTKs) to the pericentromeric region of human chromosome 4. PTK4 and TYRO4, which encode nonreceptor intracellular PTKs, are located at 4p12 and 4q13, respectively. The other three genes, PDGFRA, KIT, and KDR, encode type III transmembrane receptor PTKs for known ligands. The authors have developed a contig of 29 yeast artificial chromosomes (YACs) spanning approximately 2 Mb of DNA at 4q12 that includes PDGFRA, KIT, and KDR, and have used this YAC contig to map 12 different sequence-tagged sites in this region. PDGFRA, KIT, and KDR thus constitute a cluster of genes at 4q12 encoding closely related type III receptor PTKs. Mutations of the human KIT gene result in piebaldism, an autosomal dominant disorder of melanocyte development. 42 refs., 3 figs., 2 tabs.

  1. Activation of protein kinase C and inositol 1,4,5-triphosphate receptors antagonistically modulate voltage-gated sodium channels in striatal neurons.

    PubMed

    Hourez, Raphaël; Azdad, Karima; Vanwalleghem, Gilles; Roussel, Céline; Gall, David; Schiffmann, Serge N

    2005-10-19

    Regulation of voltage-gated sodium channels is crucial to firing patterns that constitute the output of medium spiny neurons (MSN), projecting neurons of the striatum. This modulation is thus critical for the final integration of information processed within the striatum. It has been shown that the adenylate cyclase pathway reduces sodium currents in MSN through channel phosphorylation by cAMP-dependent protein kinase. However, it is unknown whether a phospholipase C (PLC)-mediated signaling cascade could also modulate voltage-gated sodium channels within MSN. Using the whole-cell patch clamp technique, we investigated the effects of activation of two key components in PLC-mediated signaling cascades: protein kinase C (PKC) and inositol-1,4,5-triphosphate (IP(3)) receptors on voltage-dependent sodium current. Cellular dialysis with phorbol 12-myristate 13-acetate, an activator of PKC, significantly reduced peak sodium current amplitude, while adenophostin A, an activator of IP(3) receptors, significantly increased peak sodium current amplitude. This effect of adenophostin was abolished by calcium chelation or by FK506, an inhibitor of calcineurin. These results suggest an antagonistic role of PKC and IP(3) in the modulation of striatal voltage-gated sodium channels, peak current amplitude being decreased through phosphorylation by PKC and increased through dephosphorylation by calcineurin.

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

    PubMed Central

    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.

    2015-01-01

    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 MenaINV, 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 MenaINV 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 MenaINV sensitizes tumor cells to low–growth factor concentrations, thereby increasing the migration and invasion responses that contribute to aggressive, malignant cell phenotypes. PMID:26337385

  3. 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.

  4. Protein kinase C-mediated changes in synaptic efficacy at the neuromuscular junction in vitro: the role of postsynaptic acetylcholine receptors.

    PubMed

    Lanuza, M A; Li, M X; Jia, M; Kim, S; Davenport, R; Dunlap, V; Nelson, P G

    2000-09-15

    Activation of a mouse in vitro neuromuscular synapse produces a reduction in synaptic efficacy which is greater for nonactivated than for activated inputs to the myotubes. This has been shown to require thrombin and thrombin receptor activation and to involve a protein kinase C (PKC)-mediated step. We show in the present work that phorbol ester activation of PKC produces physiological loss of synapses in a time- and dose-related manner. We observe, using quantitative imaging methods, a parallel loss of acetylcholine receptors (AChR) from synaptically functional neurite-associated receptor aggregates in nerve-muscle cocultures. Biochemical measurements of total AChR show that PKC activation reduces both AChR stability (increases receptor loss) and receptor insertion into the surface membrane. Taken together, the data suggest that PKC activation decreases the stability of AChR aggregates in the muscle surface membrane. We conclude that PKC plays a crucial role in activity-dependent synapse reduction and does so, at least in part, by altering AChR stability. PMID:10972958

  5. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Linn, Anning

    1996-01-01

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

  6. 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

  7. Two distinct forms of M-locus protein kinase localize to the plasma membrane and interact directly with S-locus receptor kinase to transduce self-incompatibility signaling in Brassica rapa.

    PubMed

    Kakita, Mitsuru; Murase, Kohji; Iwano, Megumi; Matsumoto, Tomohito; Watanabe, Masao; Shiba, Hiroshi; Isogai, Akira; Takayama, Seiji

    2007-12-01

    Many flowering plants possess systems of self-incompatibility (SI) to prevent inbreeding. In Brassica, SI recognition is controlled by the multiallelic gene complex (S-haplotypes) at the S-locus, which encodes both the male determinant S-locus protein 11 (SP11/SCR) and the female determinant S-receptor kinase (SRK). Upon self-pollination, the S-haplotype-specific interaction between the pollen-borne SP11 and the cognate stigmatic SRK receptor induces SI signaling in the stigmatic papilla cell and results in rejection of the self-pollen. Our genetic analysis of a self-compatible mutant revealed the involvement of a cytoplasmic protein kinase, M-locus protein kinase (MLPK), in the SI signaling, but its exact physiological function remains unknown. In this study, we identified two different MLPK transcripts, MLPKf1 and MLPKf2, which are produced using alternative transcriptional initiation sites and encode two isoforms that differ only at the N termini. While MLPKf1 and MLPKf2 exhibited distinct expression profiles, both were expressed in papilla cells. MLPKf1 localizes to the plasma membrane through its N-terminal myristoylation motif, while MLPKf2 localizes to the plasma membrane through its N-terminal hydrophobic region. Although both MLPKf1 and MLPKf2 could independently complement the mlpk/mlpk mutation, their mutant forms that lack the plasma membrane localization motifs failed to complement the mutation. Furthermore, a bimolecular fluorescence complementation assay revealed direct interactions between SRK and the MLPK isoforms in planta. These results suggest that MLPK isoforms localize to the papilla cell membrane and interact directly with SRK to transduce SI signaling.

  8. GsSRK, a G-type lectin S-receptor-like serine/threonine protein kinase, is a positive regulator of plant tolerance to salt stress.

    PubMed

    Sun, Xiao-Li; Yu, Qing-Yue; Tang, Li-Li; Ji, Wei; Bai, Xi; Cai, Hua; Liu, Xiao-Fei; Ding, Xiao-Dong; Zhu, Yan-Ming

    2013-03-15

    Receptor-like protein kinases (RLKs) play vital roles in sensing outside signals, yet little is known about RLKs functions and roles in stress signal perception and transduction in plants, especially in wild soybean. Through the microarray analysis, GsSRK was identified as an alkaline (NaHCO3)-responsive gene, and was subsequently isolated from Glycine soja by homologous cloning. GsSRK encodes a 93.22kDa protein with a highly conserved serine/threonine protein kinase catalytic domain, a G-type lectin region, and an S-locus region. Real-time PCR results showed that the expression levels of GsSRK were largely induced by ABA, salt, and drought stresses. Over expression of GsSRK in Arabidopsis promoted seed germination, as well as primary root and rosette leaf growth during the early stages of salt stress. Compared to the wild type Arabidopsis, GsSRK overexpressors exhibited enhanced salt tolerance and higher yields under salt stress, with higher chlorophyll content, lower ion leakage, higher plant height, and more siliques at the adult developmental stage. Our studies suggest that GsSRK plays a crucial role in plant response to salt stress.

  9. Differential Roles of Smad1 and p38 Kinase in Regulation of Peroxisome Proliferator-activating Receptor γ during Bone Morphogenetic Protein 2-induced Adipogenesis

    PubMed Central

    Hata, Kenji; Nishimura, Riko; Ikeda, Fumiyo; Yamashita, Kenji; Matsubara, Takuma; Nokubi, Takashi; Yoneda, Toshiyuki

    2003-01-01

    Bone morphogenetic protein 2 (BMP2) promotes the differentiation of undifferentiated mesenchymal cells into adipocytes. To investigate the molecular mechanisms that regulate this differentiation process, we studied the relationship between BMP2 signaling and peroxisome proliferator-activating receptor γ (PPARγ) during adipogenesis of mesenchymal cells by using pluripotent mesenchymal cell line C3H10T1/2. In C3H10T1/2 cells, BMP2 induced expression of PPARγ along with adipogenesis. Overexpression of Smad6, a natural antagonist for Smad1, blocked PPARγ expression and adipocytic differentiation induced by BMP2. Overexpression of dominant-negative PPARγ also diminished adipocytic differentiation of C3H10T1/2 cells, suggesting the central role of PPARγ in BMP2-induced adipocytic differentiation. Specific inhibitors for p38 kinase inhibited BMP2-induced adipocytic differentiation and transcriptional activation of PPARγ, whereas overexpression of Smad6 had no effect on transcriptional activity of PPARγ. Furthermore, activation of p38 kinase by overexpression of TAK1 and TAB1, without affecting PPARγ expression, led the up-regulation of transcriptional activity of PPARγ. These results suggest that both Smad and p38 kinase signaling are concomitantly activated and responsible for BMP2-induced adipocytic differentiation by inducing and up-regulating PPARγ, respectively. Thus, BMP2 controls adipocytic differentiation by using two distinct signaling pathways that play differential roles in this process in C3H10T1/2 cells. PMID:12589053

  10. 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. PMID:27030010

  11. Oncoprotein protein kinase

    DOEpatents

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

    1997-02-25

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

  12. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    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.

  13. 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.

  14. 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.

  15. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning

    1999-01-01

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

  16. Oncoprotein protein kinase

    DOEpatents

    Karin, Michael; Hibi, Masahiko; Lin, Anning; 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.

  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; 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.

  19. 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.

  20. 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.

  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

    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. Receptor tyrosine kinase targeting in multicellular spheroids.

    PubMed

    Breslin, Susan; O'Driscoll, Lorraine

    2015-01-01

    While growing cells as a monolayer is the traditional method for cell culture, the incorporation of multicellular spheroids into experimental design is becoming increasingly popular. This is due to the understanding that cells grown as spheroids tend to replicate the in vivo situation more reliably than monolayer cells. Thus, the use of multicellular spheroids may be more clinically relevant than monolayer cell cultures. Here, we describe methods for multicellular 3D spheroid generation that may be used to provide samples for receptor tyrosine kinase (and other protein) detection. Methods described include the forced-floating poly-HEMA method, the hanging-drop method, and the use of ECM to form multicellular 3D spheroids. PMID:25319898

  3. Bivalent Inhibitors of Protein Kinases

    PubMed Central

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

    2015-01-01

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

  4. Farnesoid X receptor activation by chenodeoxycholic acid induces detoxifying enzymes through AMP-activated protein kinase and extracellular signal-regulated kinase 1/2-mediated phosphorylation of CCAAT/enhancer binding protein β.

    PubMed

    Noh, Kyoung; Kim, Young Mi; Kim, Young Woo; Kim, Sang Geon

    2011-08-01

    Farnesoid X receptor (FXR) regulates redox homeostasis and elicits a cytoprotective effect. CCAAT/enhancer binding protein-β (C/EBPβ) plays a role in regulating the expression of hepatocyte-specific genes and contributes to hepatocyte protection and liver regeneration. In view of the role of FXR in xenobiotic metabolism and hepatocyte survival, this study investigated the potential of FXR to activate C/EBPβ for the induction of detoxifying enzymes and the responsible regulatory pathway. Chenodeoxycholic acid (CDCA), a major component in bile acids, activates FXR. In HepG2 cells, CDCA treatment activated C/EBPβ, as shown by increases in its phosphorylation, nuclear accumulation, and expression. 3-(2,6-Dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl-)oxymethyl-5-isopropyl-isoxazole (GW4064), a synthetic FXR ligand, had similar effects. In addition, CDCA enhanced luciferase gene transcription from the construct containing -1.65-kb GSTA2 promoter, which contained C/EBP response element (pGL-1651). Moreover, CDCA treatment activated AMP-activated protein kinase (AMPK), which led to extracellular signal-regulated kinase 1/2 (ERK1/2) activation, as evidenced by the results of experiments using a dominant-negative mutant of AMPKα and chemical inhibitor. The activation of ERK1/2 was responsible for the activating phosphorylation of C/EBPβ. FXR knockdown attenuated the ability of CDCA to activate AMPK and ERK1/2 and phosphorylate C/EBPβ. Consistently, enforced expression of FXR promoted the phosphorylation of AMPKα, ERK1/2, and C/EBPβ, verifying that C/EBPβ phosphorylation elicited by CDCA results from the activation of AMPK and ERK1/2 by FXR. In mice, CDCA treatment activated C/EBPβ with the induction of detoxifying enzymes in the liver. Our results demonstrate that CDCA induces antioxidant and xenobiotic-metabolizing enzymes by activating C/EBPβ through AMPK-dependent ERK1/2 pathway downstream of FXR.

  5. Cardiac-specific ablation of G-protein receptor kinase 2 redefines its roles in heart development and beta-adrenergic signaling.

    PubMed

    Matkovich, Scot J; Diwan, Abhinav; Klanke, Justin L; Hammer, Daniel J; Marreez, Yehia; Odley, Amy M; Brunskill, Eric W; Koch, Walter J; Schwartz, Robert J; Dorn, Gerald W

    2006-10-27

    G-protein receptor kinase 2 (GRK2) is 1 of 7 mammalian GRKs that phosphorylate ligand-bound 7-transmembrane receptors, causing receptor uncoupling from G proteins and potentially activating non-G-protein signaling pathways. GRK2 is unique among members of the GRK family in that its genetic ablation causes embryonic lethality. Cardiac abnormalities in GRK2 null embryos implicated GRK2 in cardiac development but prevented studies of the knockout phenotype in adult hearts. Here, we created GRK2-loxP-targeted mice and used Cre recombination to generate germline and cardiac-specific GRK2 knockouts. GRK2 deletion in the preimplantation embryo with EIIa-Cre (germline null) resulted in developmental retardation and embryonic lethality between embryonic day 10.5 (E10.5) and E11.5. At E9.5, cardiac myocyte specification and cardiac looping were normal, but ventricular development was delayed. Cardiomyocyte-specific ablation of GRK2 in the embryo with Nkx2.5-driven Cre (cardiac-specific GRK2 knockout) produced viable mice with normal heart structure, function, and cardiac gene expression. Cardiac-specific GRK2 knockout mice exhibited enhanced inotropic sensitivity to the beta-adrenergic receptor agonist isoproterenol, with impairment of normal inotropic and lusitropic tachyphylaxis, and exhibited accelerated development of catecholamine toxicity with chronic isoproterenol treatment. These findings show that cardiomyocyte autonomous GRK2 is not essential for myocardial development after cardiac specification, suggesting that embryonic developmental abnormalities may be attributable to extracardiac effects of GRK2 ablation. In the adult heart, cardiac GRK2 is a major factor regulating inotropic and lusitropic tachyphylaxis to beta-adrenergic agonist, which likely contributes to its protective effects in catecholamine cardiomyopathy. PMID:17008600

  6. Factors influencing the inhibition of protein kinases.

    PubMed

    Brockhoff, Marielle; Hau, Jean-Christophe; Fontana, Patrizia; Zimmermann, Catherine; Pover, Alain De; Erdmann, Dirk; Chène, Patrick

    2012-04-01

    The protein kinase field is a very active research area in the pharmaceutical industry and many activities are ongoing to identify inhibitors of these proteins. The design of new chemical entities with improved pharmacological properties requires a deeper understanding of the factors that modulate inhibitor-kinase interactions. In this report, we studied the effect of two of these factors--the magnesium ion cofactor and the protein substrate--on inhibitors of the type I insulin-like growth factor receptor. Our results show that the concentration of magnesium ion influences the potency of adenosine triphosphate (ATP) competitive inhibitors, suggesting an explanation for the observation that such compounds retain their nanomolar potency in cells despite the presence of millimolar levels of ATP. We also showed that the peptidic substrate affects the potency of these inhibitors in a different manner, suggesting that the influence of this substrate on compound potency should be taken into consideration during drug discovery.

  7. Phosphorylation of the Deubiquitinase USP20 by Protein Kinase A Regulates Post-endocytic Trafficking of β2 Adrenergic Receptors to Autophagosomes during Physiological Stress*

    PubMed Central

    Kommaddi, Reddy Peera; Jean-Charles, Pierre-Yves; Shenoy, Sudha K.

    2015-01-01

    Ubiquitination by the E3 ligase Nedd4 and deubiquitination by the deubiquitinases USP20 and USP33 have been shown to regulate the lysosomal trafficking and recycling of agonist-activated β2 adrenergic receptors (β2ARs). In this work, we demonstrate that, in cells subjected to physiological stress by nutrient starvation, agonist-activated ubiquitinated β2ARs traffic to autophagosomes to colocalize with the autophagy marker protein LC3-II. Furthermore, this trafficking is synchronized by dynamic posttranslational modifications of USP20 that, in turn, are induced in a β2AR-dependent manner. Upon β2AR activation, a specific isoform of the second messenger cAMP-dependent protein kinase A (PKAα) rapidly phosphorylates USP20 on serine 333 located in its unique insertion domain. This phosphorylation of USP20 correlates with a characteristic SDS-PAGE mobility shift of the protein, blocks its deubiquitinase activity, promotes its dissociation from the activated β2AR complex, and facilitates trafficking of the ubiquitinated β2AR to autophagosomes, which fuse with lysosomes to form autolysosomes where receptors are degraded. Dephosphorylation of USP20 has reciprocal effects and blocks trafficking of the β2AR to autophagosomes while promoting plasma membrane recycling of internalized β2ARs. Our findings reveal a dynamic regulation of USP20 by site-specific phosphorylation as well as the interdependence of signal transduction and trafficking pathways in balancing adrenergic stimulation and maintaining cellular homeostasis. PMID:25666616

  8. 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

  9. Constitutive tyrosine phosphorylation of the T-cell receptor (TCR) zeta subunit: regulation of TCR-associated protein tyrosine kinase activity by TCR zeta.

    PubMed Central

    van Oers, N S; Tao, W; Watts, J D; Johnson, P; Aebersold, R; Teh, H S

    1993-01-01

    The T-cell receptor (TCR) zeta subunit is an important component of the TCR complex, involved in signal transduction events following TCR engagement. In this study, we showed that the TCR zeta chain is constitutively tyrosine phosphorylated to similar extents in thymocytes and lymph node T cells. Approximately 35% of the tyrosine-phosphorylated TCR zeta (phospho zeta) precipitated from total cell lysates appeared to be surface associated. Furthermore, constitutive phosphorylation of TCR zeta in T cells occurred independently of antigen stimulation and did not require CD4 or CD8 coreceptor expression. In lymph node T cells that constitutively express tyrosine-phosphorylated TCR zeta, there was a direct correlation between surface TCR-associated protein tyrosine kinase (PTK) activity and expression of phospho zeta. TCR stimulation of these cells resulted in an increase in PTK activity that coprecipitated with the surface TCR complex and a corresponding increase in the levels of phospho zeta. TCR ligations also contributed to the detection of several additional phosphoproteins that coprecipitated with surface TCR complexes, including a 72-kDa tyrosine-phosphorylated protein. The presence of TCR-associated PTK activity also correlated with the binding of a 72-kDa protein, which became tyrosine phosphorylated in vitro kinase assays, to tyrosine phosphorylated TCR zeta. The cytoplasmic region of the TCR zeta chain was synthesized, tyrosine phosphorylated, and conjugated to Sepharose beads. Only tyrosine-phosphorylated, not nonphosphorylated, TCR zeta beads were capable of immunoprecipitating the 72-kDa protein from total cell lysates. This 72-kDa protein is likely the murine equivalent of human PTK ZAP-70, which has been shown to associate specifically with phospho zeta. These results suggest that TCR-associated PTK activity is regulated, at least in part, by the tyrosine phosphorylation status of TCR zeta. Images PMID:7689151

  10. 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

  11. Regulation of GluA1 α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Receptor Function by Protein Kinase C at Serine-818 and Threonine-840

    PubMed Central

    Jenkins, Meagan A.; Wells, Gordon; Bachman, Julia; Snyder, James P.; Jenkins, Andrew; Huganir, Richard L.; Oswald, Robert E.

    2014-01-01

    Three residues within the AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor subunit GluA1 C terminus (Ser818, Ser831, Thr840) can be phosphorylated by Ca2+/phospholipid-dependent protein kinase (PKC). Here, we show that PKC phosphorylation of GluA1 Ser818 or Thr840 enhances the weighted mean channel conductance without altering the response time course or agonist potency. These data support the idea that these residues constitute a hyper-regulatory domain for the AMPA receptor. Introduction of phosphomimetic mutations increases conductance only at these three sites within the proximal C terminus, consistent with a structural model with a flexible linker connecting the distal C-terminal domain to the more proximal domain containing a helix bracketed by Ser831 and Thr840. NMR spectra support this model and raise the possibility that phosphorylation can alter the configuration of this domain. Our findings provide insight into the structure and function of the C-terminal domain of GluA1, which controls AMPA receptor function and trafficking during synaptic plasticity in the central nervous system. PMID:24452473

  12. A role for the CaM Kinase II related anchoring protein (αkap) in maintaining the stability of nicotinic Acetylcholine Receptors

    PubMed Central

    Mouslim, Chakib; Aittaleb, Mohamed; Hume, Richard I.; Akaaboune, Mohammed

    2012-01-01

    αkap, a muscle specific anchoring protein encoded within the Camk2a gene is thought to play a role in targeting multiple calcium/calmodulin kinase II isoforms to specific subcellular locations. Here we demonstrate a novel function of αkap in stabilizing nicotinic acetylcholine receptors (AChR). Knockdown of αkap expression with shRNA significantly enhanced the degradation of AChR α-subunits (AChRα), leading to fewer and smaller AChR clusters on the surface of differentiated C2C12 myotubes. Mutagenesis and biochemical studies in HEK293T cells revealed that αkap promoted AChRα stability by a ubiquitin-dependent mechanism. In the absence of αkap, AChRα was heavily ubiquitinated and the number of AChRα was increased by proteasome inhibitors. However, in the presence of αkap, AChRα was less ubiquitinated and proteasome inhibitors had almost no effect on AChRα accumulation. The major sites of AChRα ubiquitination reside within the large intracellular loop and mutations of critical lysine residues in this loop to arginine increased AChRα stability in the absence of αkap. These results provide an unexpected mechanism by which αkap controls receptor trafficking onto the surface of muscle cells, and thus the maintenance of postsynaptic receptor density and synaptic function. PMID:22496563

  13. Therapeutic implications of a human neutralizing antibody to the macrophage-stimulating protein receptor tyrosine kinase (RON), a c-MET family member.

    PubMed

    O'Toole, Jennifer M; Rabenau, Karen E; Burns, Kerri; Lu, Dan; Mangalampalli, Venkat; Balderes, Paul; Covino, Nicole; Bassi, Rajiv; Prewett, Marie; Gottfredsen, Kimberly J; Thobe, Megan N; Cheng, Yuan; Li, Yiwen; Hicklin, Daniel J; Zhu, Zhenping; Waltz, Susan E; Hayman, Michael J; Ludwig, Dale L; Pereira, Daniel S

    2006-09-15

    RON is a member of the c-MET receptor tyrosine kinase family. Like c-MET, RON is expressed by a variety of epithelial-derived tumors and cancer cell lines and it is thought to play a functional role in tumorigenesis. To date, antagonists of RON activity have not been tested in vivo to validate RON as a potential cancer target. In this report, we used an antibody phage display library to generate IMC-41A10, a human immunoglobulin G1 (IgG1) antibody that binds with high affinity (ED50 = 0.15 nmol/L) to RON and effectively blocks interaction with its ligand, macrophage-stimulating protein (MSP; IC50 = 2 nmol/L). We found IMC-41A10 to be a potent inhibitor of receptor and downstream signaling, cell migration, and tumorigenesis. It antagonized MSP-induced phosphorylation of RON, mitogen-activated protein kinase (MAPK), and AKT in several cancer cell lines. In HT-29 colon, NCI-H292 lung, and BXPC-3 pancreatic cancer xenograft tumor models, IMC-41A10 inhibited tumor growth by 50% to 60% as a single agent, and in BXPC-3 xenografts, it led to tumor regressions when combined with Erbitux. Western blot analyses of HT-29 and NCI-H292 xenograft tumors treated with IMC-41A10 revealed a decrease in MAPK phosphorylation compared with control IgG-treated tumors, suggesting that inhibition of MAPK activity may be required for the antitumor activity of IMC-41A10. To our knowledge, this is the first demonstration that a RON antagonist and specifically an inhibitory antibody of RON negatively affects tumorigenesis. Another major contribution of this report is an extensive analysis of RON expression in approximately 100 cancer cell lines and approximately 300 patient tumor samples representing 10 major cancer types. Taken together, our results highlight the potential therapeutic usefulness of RON activity inhibition in human cancers.

  14. Alpha-Ca2+/calmodulin-dependent protein kinase II contributes to the developmental programming of anxiety in serotonin receptor 1A knock-out mice.

    PubMed

    Lo Iacono, Luisa; Gross, Cornelius

    2008-06-11

    Mice lacking the serotonin receptor 1A [Htr1aknock-out (Htr1a(KO))] display increased innate and conditioned anxiety-related behavior. Expression of the receptor in the mouse forebrain during development is sufficient to restore normal anxiety-related behavior to knock-out mice, demonstrating a role for serotonin in the developmental programming of anxiety circuits. However, the precise developmental period as well as the signaling pathways and neural substrates involved in this phenomenon are unknown. Here, we show that pharmacological blockade of the receptor from postnatal day 13 (P13)-P34 is sufficient to reproduce the knock-out phenotype in adulthood, thus defining a role for serotonin in the maturation and refinement of anxiety circuits during a limited postnatal period. Furthermore, we identify increases in the phosphorylation of alpha-Ca(2+)/calmodulin-dependent protein kinase II (alphaCaMKII) at threonine 286 in the hippocampus of young Htr1a(KO) mice under anxiety-provoking conditions. Increases in alphaCaMKII phosphorylation were most pronounced in the CA1 region of the hippocampus and were localized to the extrasynaptic compartment, consistent with a tissue-specific effect of the receptor. No changes in alphaCaMKII phosphorylation were found in adult knock-out mice, suggesting a transient role of alphaCaMKII as a downstream target of the receptor. Finally, the anxiety phenotype was abolished when knock-out mice were crossed to mice in which alphaCaMKII phosphorylation was compromised by the heterozygous mutation of threonine 286 into alanine. These findings suggest that modulation of alphaCaMKII function by serotonin during a restricted postnatal period contributes to the developmental programming of anxiety-related behavior. PMID:18550767

  15. Mycobacterium tuberculosis Serine/Threonine Protein Kinases

    PubMed Central

    PRISIC, SLADJANA; HUSSON, ROBERT N.

    2014-01-01

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

  16. Increased hepatic receptor interacting protein kinase 3 expression due to impaired proteasomal functions contributes to alcohol-induced steatosis and liver injury

    PubMed Central

    Wang, Shaogui; Ni, Hong-Min; Dorko, Kenneth; Kumer, Sean C.; Schmitt, Timothy M.; Nawabi, Atta; Komatsu, Masaaki; Huang, Heqing; Ding, Wen-Xing

    2016-01-01

    Chronic alcohol exposure increased hepatic receptor-interacting protein kinase (RIP) 3 expression and necroptosis in the liver but its mechanisms are unclear. In the present study, we demonstrated that chronic alcohol feeding plus binge (Gao-binge) increased RIP3 but not RIP1 protein levels in mouse livers. RIP3 knockout mice had decreased serum alanine amino transferase activity and hepatic steatosis but had no effect on hepatic neutrophil infiltration compared with wild type mice after Gao-binge alcohol treatment. The hepatic mRNA levels of RIP3 did not change between Gao-binge and control mice, suggesting that alcohol-induced hepatic RIP3 proteins are regulated at the posttranslational level. We found that Gao-binge treatment decreased the levels of proteasome subunit alpha type-2 (PSMA2) and proteasome 26S subunit, ATPase 1 (PSMC1) and impaired hepatic proteasome function. Pharmacological or genetic inhibition of proteasome resulted in the accumulation of RIP3 in mouse livers. More importantly, human alcoholics had decreased expression of PSMA2 and PSMC1 but increased protein levels of RIP3 compared with healthy human livers. Moreover, pharmacological inhibition of RIP1 decreased Gao-binge-induced hepatic inflammation, neutrophil infiltration and NF-κB subunit (p65) nuclear translocation but failed to protect against steatosis and liver injury induced by Gao-binge alcohol. In conclusion, results from this study suggest that impaired hepatic proteasome function by alcohol exposure may contribute to hepatic accumulation of RIP3 resulting in necroptosis and steatosis while RIP1 kinase activity is important for alcohol-induced inflammation. PMID:26769846

  17. 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

  18. Heterodimerization of human orexin receptor 1 and kappa opioid receptor promotes protein kinase A/cAMP-response element binding protein signaling via a Gαs-mediated mechanism.

    PubMed

    Chen, Jing; Zhang, Rumin; Chen, Xiaoyu; Wang, Chunmei; Cai, Xin; Liu, Haiqing; Jiang, Yunlu; Liu, Chuanxin; Bai, Bo

    2015-07-01

    Orexin and dynorphin are co-expressed in the same synaptic vesicles of hypothalamic neurons and play opposing roles in cocaine self-administration, brain stimulation reward, and impulsivity in ventral tegmental area (VTA), where dopamine neurons express both OX1R and KORs. However, detailed mechanisms of how the coreleased peptides and both receptors fine-tune their signalings and physiological/behavioral effects together remain unclear. Here we explore the possibility of heterodimerization between OX1R and KOR and reveal novel signal transduction mechanisms. First, we demonstrated co-expression of OX1R and KOR in rat hippocampal neurons by single-cell PCR. Furthermore, heterodimerization between OX1R and KOR was examined using bioluminescence and fluorescence resonance energy transfer (BRET and FRET). Our data revealed that human OX1R and KOR heterodimerize, and this heterodimer associates with Gαs, leading to increased protein kinase A (PKA) signaling pathway activity, including upregulation of intracellular cAMP levels and cAMP-response element (CRE) luciferase reporter activity, resulting in increased cAMP-response element binding protein (CREB) phosphorylation. These results support the view that OX1R and KOR heterodimerization might have an anti-depressive role.

  19. Presynaptic N-Methyl-d-aspartate (NMDA) Receptor Activity Is Increased Through Protein Kinase C in Paclitaxel-induced Neuropathic Pain.

    PubMed

    Xie, Jing-Dun; Chen, Shao-Rui; Chen, Hong; Zeng, Wei-An; Pan, Hui-Lin

    2016-09-01

    Painful peripheral neuropathy is a severe adverse effect of chemotherapeutic drugs such as paclitaxel (Taxol). The glutamate N-methyl-d-aspartate receptors (NMDARs) are critically involved in the synaptic plasticity associated with neuropathic pain. However, paclitaxel treatment does not alter the postsynaptic NMDAR activity of spinal dorsal horn neurons. In this study, we determined whether paclitaxel affects presynaptic NMDAR activity by recording excitatory postsynaptic currents (EPSCs) of dorsal horn neurons in spinal cord slices. In paclitaxel-treated rats, the baseline frequency of miniature EPSCs (mEPSCs) was significantly increased; the NMDAR antagonist 2-amino-5-phosphonopentanoic acid (AP5) completely normalized this frequency. Also, AP5 significantly reduced the amplitude of monosynaptic EPSCs evoked by dorsal root stimulation and reversed the reduction in the paired-pulse ratio of evoked EPSCs in paclitaxel-treated rats. Blocking GluN2A-containing, but not GluN2B-containing, NMDARs largely decreased the frequency of mEPSCs and the amplitude of evoked EPSCs of dorsal horn neurons in paclitaxel-treated rats. Furthermore, inhibition of protein kinase C fully reversed the increased frequency of mEPSCs and the amplitude of evoked EPSCs in paclitaxel-treated rats. Paclitaxel treatment significantly increased the protein level of GluN2A and phosphorylated GluN1 in the dorsal root ganglion. In addition, intrathecal injection of AP5 or systemic administration of memantine profoundly attenuated pain hypersensitivity induced by paclitaxel. Our findings indicate that paclitaxel treatment induces tonic activation of presynaptic NMDARs in the spinal cord through protein kinase C to potentiate nociceptive input from primary afferent nerves. Targeting presynaptic NMDARs at the spinal cord level may be an effective strategy for treating chemotherapy-induced neuropathic pain. PMID:27458019

  20. 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

  1. Involvement of OST1 Protein Kinase and PYR/PYL/RCAR Receptors in Methyl Jasmonate-Induced Stomatal Closure in Arabidopsis Guard Cells.

    PubMed

    Yin, Ye; Adachi, Yuji; Nakamura, Yoshimasa; Munemasa, Shintaro; Mori, Izumi C; Murata, Yoshiyuki

    2016-08-01

    Methyl jasmonate (MeJA) induces stomatal closure. It has been shown that stomata of many ABA-insensitive mutants are also insensitive to MeJA, and a low amount of ABA is a prerequisite for the MeJA response. However, the molecular mechanisms of the interaction between ABA and MeJA signaling remain to be elucidated. Here we studied the interplay of signaling of the two hormones in guard cells using the quadruple ABA receptor mutant pyr1 pyl1 pyl2 pyl4 and ABA-activated protein kinase mutants ost1-2 and srk2e. In the quadruple mutant, MeJA-induced stomatal closure, H2O2 production, nitric oxide (NO) production, cytosolic alkalization and plasma membrane Ca(2+)-permeable current (ICa) activation were not impaired. At the same time, the inactivation of the inward-rectifying K(+) current was impaired. In contrast to the quadruple mutant, MeJA-induced stomatal closure, H2O2 production, NO production and cytosolic alkalization were impaired in ost1-2 and srk2e as well as in aba2-2, the ABA-deficient mutant. The activation of ICa was also impaired in srk2e. Collectively, these results indicated that OST1 was essential for MeJA-induced stomatal closure, while PYR1, PYL1, PYL2 and PYL4 ABA receptors were not sufficient factors. MeJA did not appear to activate OST1 kinase activity. This implies that OST1 mediates MeJA signaling through an undetectable level of activity or a non-enzymatic action. MeJA induced the expression of an ABA synthesis gene, NCED3, and increased ABA contents only modestly. Taken together with previous reports, this study suggests that MeJA signaling in guard cells is primed by ABA and is not brought about through the pathway mediated by PYR1, PYL1 PYL2 and PYL4.

  2. Involvement of OST1 Protein Kinase and PYR/PYL/RCAR Receptors in Methyl Jasmonate-Induced Stomatal Closure in Arabidopsis Guard Cells.

    PubMed

    Yin, Ye; Adachi, Yuji; Nakamura, Yoshimasa; Munemasa, Shintaro; Mori, Izumi C; Murata, Yoshiyuki

    2016-08-01

    Methyl jasmonate (MeJA) induces stomatal closure. It has been shown that stomata of many ABA-insensitive mutants are also insensitive to MeJA, and a low amount of ABA is a prerequisite for the MeJA response. However, the molecular mechanisms of the interaction between ABA and MeJA signaling remain to be elucidated. Here we studied the interplay of signaling of the two hormones in guard cells using the quadruple ABA receptor mutant pyr1 pyl1 pyl2 pyl4 and ABA-activated protein kinase mutants ost1-2 and srk2e. In the quadruple mutant, MeJA-induced stomatal closure, H2O2 production, nitric oxide (NO) production, cytosolic alkalization and plasma membrane Ca(2+)-permeable current (ICa) activation were not impaired. At the same time, the inactivation of the inward-rectifying K(+) current was impaired. In contrast to the quadruple mutant, MeJA-induced stomatal closure, H2O2 production, NO production and cytosolic alkalization were impaired in ost1-2 and srk2e as well as in aba2-2, the ABA-deficient mutant. The activation of ICa was also impaired in srk2e. Collectively, these results indicated that OST1 was essential for MeJA-induced stomatal closure, while PYR1, PYL1, PYL2 and PYL4 ABA receptors were not sufficient factors. MeJA did not appear to activate OST1 kinase activity. This implies that OST1 mediates MeJA signaling through an undetectable level of activity or a non-enzymatic action. MeJA induced the expression of an ABA synthesis gene, NCED3, and increased ABA contents only modestly. Taken together with previous reports, this study suggests that MeJA signaling in guard cells is primed by ABA and is not brought about through the pathway mediated by PYR1, PYL1 PYL2 and PYL4. PMID:27354421

  3. Ionotrophic 5-hydroxytryptamine type 3 receptor activates the protein kinase C-dependent phospholipase D pathway in human T-cells.

    PubMed Central

    Khan, N A; Hichami, A

    1999-01-01

    The present study was undertaken to investigate the role of the 5-hydroxytryptamine (5-HT) ionotrophic receptor 5-HT(3) in the activation of human Jurkat T-cells. 5-HT and 2-methyl-5-HT (2Me-5-HT), an agonist of the 5-HT(3) receptor, induced increases in intracellular free Na(+) concentrations, [Na(+)](i), via opening of the ionotrophic receptor in these cells. These two serotonergic (5-hydroxytryptaminergic) agents potentiated phytohaemagglutinin (PHA)-induced T-cell activation. However, they failed to potentiate dioctanoglycerol-plus-ionomycin-stimulated T-cell blastogenesis. Interestingly, an inhibitor of protein kinase C (PKC), GF 109203X, curtailed significantly 5-HT and 2Me-5-HT-potentiated T-cell activation. These results demonstrate that the opening of the 5-HT(3) ionotrophic receptor is implicated in T-cell activation via the PKC pathway. Furthermore, 5-HT and 2Me-5-HT stimulated phospholipase D (PLD) activity, as measured by the production of phosphatidylethanol and phosphatidylbutanol at the expense of phosphatidic acid (PA). GF 109203X significantly curtailed the 5-HT- and 2Me-5-HT-induced PLD activity and T-cell activation. The PLD/PA pathway stimulated by these two serotonergic agents resulted in the production of 1,2-diacylglycerol (DAG) mass in Jurkat T-cells. These results altogether suggest that 5-HT and 2Me-5-HT potentiate T-cell activation via increases in [Na(+)](i) and the activation of the PKC-dependent PLD pathway. PMID:10548551

  4. Ionotrophic 5-hydroxytryptamine type 3 receptor activates the protein kinase C-dependent phospholipase D pathway in human T-cells.

    PubMed

    Khan, N A; Hichami, A

    1999-11-15

    The present study was undertaken to investigate the role of the 5-hydroxytryptamine (5-HT) ionotrophic receptor 5-HT(3) in the activation of human Jurkat T-cells. 5-HT and 2-methyl-5-HT (2Me-5-HT), an agonist of the 5-HT(3) receptor, induced increases in intracellular free Na(+) concentrations, [Na(+)](i), via opening of the ionotrophic receptor in these cells. These two serotonergic (5-hydroxytryptaminergic) agents potentiated phytohaemagglutinin (PHA)-induced T-cell activation. However, they failed to potentiate dioctanoglycerol-plus-ionomycin-stimulated T-cell blastogenesis. Interestingly, an inhibitor of protein kinase C (PKC), GF 109203X, curtailed significantly 5-HT and 2Me-5-HT-potentiated T-cell activation. These results demonstrate that the opening of the 5-HT(3) ionotrophic receptor is implicated in T-cell activation via the PKC pathway. Furthermore, 5-HT and 2Me-5-HT stimulated phospholipase D (PLD) activity, as measured by the production of phosphatidylethanol and phosphatidylbutanol at the expense of phosphatidic acid (PA). GF 109203X significantly curtailed the 5-HT- and 2Me-5-HT-induced PLD activity and T-cell activation. The PLD/PA pathway stimulated by these two serotonergic agents resulted in the production of 1,2-diacylglycerol (DAG) mass in Jurkat T-cells. These results altogether suggest that 5-HT and 2Me-5-HT potentiate T-cell activation via increases in [Na(+)](i) and the activation of the PKC-dependent PLD pathway.

  5. A plant receptor-like gene, the S-locus receptor kinase of Brassica oleracea L. , encodes a functional serine/threonine kinase

    SciTech Connect

    Stein, J.C.; Nasrallah, J.B. )

    1993-03-01

    To investigate the catalytic properties of the Brassica oleracea S-locus receptor kinase (SRK), the authors have expressed the domain that is homologous to protein kinases as a fusion protein in Escherichia coli. Following in vivo labeling of cultures with [sup 32]P-labeled inorganic phosphate, they observed phosphorylation of the fusion protein on serine and threonine, but not on tyrosine. In contrast, labeling was not observed when lysine-524, a residue conserved among all protein kinases, was mutated to arginine, thus confirmed that SRK phosphorylation was the result of intrinsic serine/threonine kinase activity. 26 refs., 3 figs.

  6. Chemotaxis and oospore formation in Phytophthora sojae are controlled by G-protein-coupled receptors with a phosphatidylinositol phosphate kinase domain.

    PubMed

    Yang, X; Zhao, W; Hua, C; Zheng, X; Jing, M; Li, D; Govers, F; Meijer, H J G; Wang, Y

    2013-04-01

    G-protein-coupled receptors (GPCRs) are key cellular components that mediate extracellular signals into intracellular responses. Genome mining revealed that Phytophthora spp. have over 60 GPCR genes among which a prominent class of 12 encoding novel proteins with an N-terminal GPCR domain fused to a C-terminal phosphatidylinositol phosphate kinase (PIPK) domain. This study focuses on two GPCR-PIPKs (GKs) in Phytophthora sojae. PsGK4 and PsGK5 are differentially expressed during the life cycle with the highest expression in cysts and during cyst germination, and at late infection stages. In P. sojae transformants that constitutively express RFP-tagged PsGK4 and PsGK5, the fusion proteins in hyphae reside in small, rapidly moving vesicular-like structures. Functional analysis using gene silencing showed that PsGK4-silenced transformants displayed higher levels of encystment and a reduced cyst germination rate when compared with the recipient strain. Moreover, GK4 deficiency (or reduction) resulted in severe defects in zoospore chemotaxis towards isoflavones and soybean roots. In contrast, PsGK5-silenced transformants exhibited no obvious defects in asexual development but oospore production was severely impaired. Both, PsGK4- and PsGK5-silenced transformants showed reduced pathogenicity. These results point to involvement of GKs in zoospore behaviour, chemotaxis and oospore development, and suggest that PsGK4 and PsGK5 each head independent signalling pathways. PMID:23448757

  7. Chemotaxis and oospore formation in Phytophthora sojae are controlled by G-protein-coupled receptors with a phosphatidylinositol phosphate kinase domain.

    PubMed

    Yang, X; Zhao, W; Hua, C; Zheng, X; Jing, M; Li, D; Govers, F; Meijer, H J G; Wang, Y

    2013-04-01

    G-protein-coupled receptors (GPCRs) are key cellular components that mediate extracellular signals into intracellular responses. Genome mining revealed that Phytophthora spp. have over 60 GPCR genes among which a prominent class of 12 encoding novel proteins with an N-terminal GPCR domain fused to a C-terminal phosphatidylinositol phosphate kinase (PIPK) domain. This study focuses on two GPCR-PIPKs (GKs) in Phytophthora sojae. PsGK4 and PsGK5 are differentially expressed during the life cycle with the highest expression in cysts and during cyst germination, and at late infection stages. In P. sojae transformants that constitutively express RFP-tagged PsGK4 and PsGK5, the fusion proteins in hyphae reside in small, rapidly moving vesicular-like structures. Functional analysis using gene silencing showed that PsGK4-silenced transformants displayed higher levels of encystment and a reduced cyst germination rate when compared with the recipient strain. Moreover, GK4 deficiency (or reduction) resulted in severe defects in zoospore chemotaxis towards isoflavones and soybean roots. In contrast, PsGK5-silenced transformants exhibited no obvious defects in asexual development but oospore production was severely impaired. Both, PsGK4- and PsGK5-silenced transformants showed reduced pathogenicity. These results point to involvement of GKs in zoospore behaviour, chemotaxis and oospore development, and suggest that PsGK4 and PsGK5 each head independent signalling pathways.

  8. 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.

  9. Modulation of adrenal catecholamine secretion by in vivo gene transfer and manipulation of G protein-coupled receptor kinase-2 activity.

    PubMed

    Lymperopoulos, Anastasios; Rengo, Giuseppe; Zincarelli, Carmela; Soltys, Stephen; Koch, Walter J

    2008-02-01

    We recently reported that the upregulation of adrenal G protein-coupled receptor kinase-2 (GRK2) causes enhanced catecholamine (CA) secretion by desensitizing sympatho-inhibitory alpha (2)-adrenergic receptors (alpha (2)ARs) of chromaffin cells, and thereby aggravating heart failure (HF). In this study, we sought to develop an efficient and reproducible in vivo adrenal gene transfer method to determine whether manipulation of adrenal GRK2 levels/activity regulates physiological CA secretion in rats. We specifically investigated two different in vivo gene delivery methods: direct injection into the suprarenal glands, and retrograde delivery through the suprarenal veins. We delivered adenoviral (Ad) vectors containing either GRK2 or an inhibitor of GRK2 activity, the beta ARKct. We found both delivery approaches equally effective at supporting robust (>80% of the whole organ) and adrenal-restricted transgene expression, in the cortical region as well as in the medullar region. Additionally, rats with AdGRK2-infected adrenals exhibit enhanced plasma CA levels when compared with control rats (AdGFP-injected adrenals), whereas plasma CA levels after Ad beta ARKct infection were significantly lower. Finally, in isolated chromaffin cells, alpha (2)ARs of AdGRK2-infected cells failed to inhibit CA secretion whereas Ad beta ARKct-infected cells showed normal alpha (2)AR responsiveness. These results not only indicate that in vivo adrenal gene transfer is an effective way of manipulating adrenal gland signalling, but also identify GRK2 as a critically important molecule involved in CA secretion.

  10. Energy-sensing factors coactivator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and AMP-activated protein kinase control expression of inflammatory mediators in liver: induction of interleukin 1 receptor antagonist.

    PubMed

    Buler, Marcin; Aatsinki, Sanna-Mari; Skoumal, Réka; Komka, Zsolt; Tóth, Miklós; Kerkelä, Risto; Georgiadi, Anastasia; Kersten, Sander; Hakkola, Jukka

    2012-01-13

    Obesity and insulin resistance are associated with chronic, low grade inflammation. Moreover, regulation of energy metabolism and immunity are highly integrated. We hypothesized that energy-sensitive coactivator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and AMP-activated protein kinase (AMPK) may modulate inflammatory gene expression in liver. Microarray analysis revealed that PGC-1α up-regulated expression of several cytokines and cytokine receptors, including interleukin 15 receptor α (IL15Rα) and, even more importantly, anti-inflammatory interleukin 1 receptor antagonist (IL1Rn). Overexpression of PGC-1α and induction of PGC-1α by fasting, physical exercise, glucagon, or cAMP was associated with increased IL1Rn mRNA and protein expression in hepatocytes. Knockdown of PGC-1α by siRNA down-regulated cAMP-induced expression of IL1Rn in mouse hepatocytes. Furthermore, knockdown of peroxisome proliferator-activated receptor α (PPARα) attenuated IL1Rn induction by PGC-1α. Overexpression of PGC-1α, at least partially through IL1Rn, suppressed interleukin 1β-induced expression of acute phase proteins, C-reactive protein, and haptoglobin. Fasting and exercise also induced IL15Rα expression, whereas glucagon and cAMP resulted in reduction in IL15Rα mRNA levels. Finally, AMPK activator metformin and adenoviral overexpression of AMPK up-regulated IL1Rn and down-regulated IL15Rα in primary hepatocytes. We conclude that PGC-1α and AMPK alter inflammatory gene expression in liver and thus integrate energy homeostasis and inflammation. Induction of IL1Rn by PGC-1α and AMPK may be involved in the beneficial effects of exercise and caloric restriction and putative anti-inflammatory effects of metformin.

  11. Targeting receptor tyrosine kinases in gastric cancer

    PubMed Central

    Morishita, Asahiro; Gong, Jian; Masaki, Tsutomu

    2014-01-01

    Molecularly targeted therapeutic agents are constantly being developed and have been shown to be effective in various clinical trials. One group of representative targeted oncogenic kinases, the receptor tyrosine kinases (RTKs), has been associated with gastric cancer development. Trastuzumab, an inhibitor of ERBB2, has been approved for the treatment of gastric cancer, although other receptor tyrosine kinases, such as epidermal growth factor receptor, vascular endothelial growth factor, platelet-derived growth factor receptor, c-Met, IGF-1R and fibroblast growth factor receptor 2, are also activated in gastric cancer. The promising results of the trastuzumab clinical trial for gastric cancer resulted in the approval of trastuzumab-based therapy as a first-line treatment for human epidermal growth factor receptor 2-positive patients. On the other hand, the trial examining bevacizumab in combination with conventional chemotherapy did not meet its primary goal of increasing the overall survival time of gastric cancer patients; however, a significantly higher response rate and a longer progression-free survival were observed in the bevacizumab arm of the trial. Other clinical trials, especially phase III trials that have tested drugs targeting RTKs, such as cetuximab, panitumumab, gefitinib, erlotinib, figitumumab, sorafenib, sunitinib and lapatinib, have shown that these drugs have modest effects against gastric cancer. This review summarizes the recent results from the clinical trials of molecularly targeted drugs and suggests that further improvements in the treatment of advanced gastric cancer can be achieved through the combination of conventional drugs with the new molecularly targeted therapies. PMID:24782606

  12. Chronic hypoxia reduces adenosine A2A receptor-mediated inhibition of calcium current in rat PC12 cells via downregulation of protein kinase A

    PubMed Central

    Kobayashi, Shuichi; Beitner-Johnson, Dana; Conforti, Laura; Millhorn, David E

    1998-01-01

    Adenosine has been shown to decrease Ca2+ current (ICa) and attenuate the hypoxia-induced enhancement of intracellular free Ca2+ ([Ca2+]i) in oxygen-sensitive rat phaeochromocytoma (PC12) cells. These effects are mediated via the adenosine A2A receptor and protein kinase A (PKA). The current study was undertaken to determine the effects of adenosine on Ca2+ current and hypoxia-induced change in [Ca2+]i during chronic hypoxia.Whole cell patch-clamp studies revealed that the effect of adenosine on ICa was significantly reduced when PC12 cells were exposed to hypoxia (10 % O2) for 24 and 48 h.Ca2+ imaging studies using fura-2 revealed that the anoxia-induced increase in [Ca2+]i was significantly enhanced when PC12 cells were exposed to 10 % O2 for up to 48 h. In contrast, the inhibitory effects of adenosine on anoxia-induced elevation of [Ca2+]i was significantly blunted in PC12 cells exposed to hypoxia for 48 h.Northern blot analysis revealed that mRNA for the A2A receptor, which is the only adenosine receptor subtype expressed in PC12 cells, was significantly upregulated by hypoxia. Radioligand binding analysis with [3H]CGS21680, a selective A2A receptor ligand, showed that the number of adenosine A2A receptor binding sites was similarly increased during exposure to 10 % O2 for 48 h.PKA enzyme activity was significantly inhibited when PC12 cells were exposed to 10 % O2 for 24 and 48 h. However, we found that hypoxia failed to induce change in adenosine- and forskolin-stimulated adenylate cyclase enzyme activity. Chronic hypoxia also did not alter the immunoreactivity level of the G protein Gsα, an effector of the A2 signalling pathway.Whole cell patch-clamp analysis showed that the effect of 8-bromo-cAMP, an activator of PKA, on ICa was significantly attenuated during 48 h exposure to 10 % O2.We conclude therefore that the reduced effect of adenosine on ICa and [Ca2+]i in PC12 cells exposed to chronic hypoxia is due to hypoxia-induced downregulation of PKA. This

  13. Chronic hypoxia reduces adenosine A2A receptor-mediated inhibition of calcium current in rat PC12 cells via downregulation of protein kinase A.

    PubMed

    Kobayashi, S; Beitner-Johnson, D; Conforti, L; Millhorn, D E

    1998-10-15

    1. Adenosine has been shown to decrease Ca2+ current (ICa) and attenuate the hypoxia-induced enhancement of intracellular free Ca2+ ([Ca2+]i) in oxygen-sensitive rat phaeochromocytoma (PC12) cells. These effects are mediated via the adenosine A2A receptor and protein kinase A (PKA). The current study was undertaken to determine the effects of adenosine on Ca2+ current and hypoxia-induced change in [Ca2+]i during chronic hypoxia. 2. Whole cell patch-clamp studies revealed that the effect of adenosine on ICa was significantly reduced when PC12 cells were exposed to hypoxia (10 % O2) for 24 and 48 h. 3. Ca2+ imaging studies using fura-2 revealed that the anoxia-induced increase in [Ca2+]i was significantly enhanced when PC12 cells were exposed to 10 % O2 for up to 48 h. In contrast, the inhibitory effects of adenosine on anoxia-induced elevation of [Ca2+]i was significantly blunted in PC12 cells exposed to hypoxia for 48 h. 4. Northern blot analysis revealed that mRNA for the A2A receptor, which is the only adenosine receptor subtype expressed in PC12 cells, was significantly upregulated by hypoxia. Radioligand binding analysis with [3H]CGS21680, a selective A2A receptor ligand, showed that the number of adenosine A2A receptor binding sites was similarly increased during exposure to 10% O2 for 48 h. 5. PKA enzyme activity was significantly inhibited when PC12 cells were exposed to 10% O2 for 24 and 48 h. However, we found that hypoxia failed to induce change in adenosine- and forskolin-stimulated adenylate cyclase enzyme activity. Chronic hypoxia also did not alter the immunoreactivity level of the G protein Gsalpha, an effector of the A2 signalling pathway. 6. Whole cell patch-clamp analysis showed that the effect of 8-bromo-cAMP, an activator of PKA, on ICa was significantly attenuated during 48 h exposure to 10% O2.7. We conclude therefore that the reduced effect of adenosine on ICa and [Ca2+]i in PC12 cells exposed to chronic hypoxia is due to hypoxia

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

    PubMed Central

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

    2012-01-01

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

  15. 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

  16. 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.

  17. The orphan nuclear receptor NUR77 regulates hormone-induced StAR transcription in Leydig cells through cooperation with Ca2+/calmodulin-dependent protein kinase I.

    PubMed

    Martin, Luc J; Boucher, Nicolas; Brousseau, Catherine; Tremblay, Jacques J

    2008-09-01

    Cholesterol transport in the mitochondrial membrane, an essential step of steroid biosynthesis, is mediated by a protein complex containing the steroidogenic acute regulatory (StAR) protein. The importance of this transporter is underscored by mutations in the human StAR gene that cause lipoid congenital adrenal hyperplasia, male pseudohermaphroditism, and adrenal insufficiency. StAR transcription in steroidogenic cells is hormonally regulated and involves several transcription factors. The nuclear receptor NUR77 is present in steroidogenic cells, and its expression is induced by hormones known to activate StAR expression. We have now established that StAR transcription in cAMP-stimulated Leydig cells requires de novo protein synthesis and involves NUR77. We found that cAMP-induced NUR77 expression precedes that of StAR both at the mRNA and protein levels in Leydig cells. In these cells, small interfering RNA-mediated NUR77 knockdown reduces cAMP-induced StAR expression. Chromatin immunoprecipitation assays revealed a cAMP-dependent increase in NUR77 recruitment to the proximal StAR promoter, whereas transient transfections in MA-10 Leydig cells confirmed that NUR77 can activate the StAR promoter and that this requires an element located at -95 bp. cAMP-induced StAR and NUR77 expression in Leydig cells was found to require a Ca2+/calmodulin-dependent protein kinase (CaMK)-dependent signaling pathway. Consistent with this, we show that within the testis, CaMKI is specifically expressed in Leydig cells. Finally, we report that CaMKI transcriptionally cooperates with NUR77, but not steroidogenic factor 1, to further enhance StAR promoter activity in Leydig cells. All together, our results implicate NUR77 as a mediator of cAMP action on StAR transcription in steroidogenic Leydig cells and identify a role for CaMKI in this process.

  18. Very large G protein-coupled receptor 1 regulates myelin-associated glycoprotein via Gαs/Gαq-mediated protein kinases A/C

    PubMed Central

    Shin, Daesung; Lin, Shu-Ting; Fu, Ying-Hui; Ptáček, Louis J.

    2013-01-01

    VLGR1 (very large G protein-coupled receptor 1), also known as MASS1 (monogenic audiogenic seizure susceptible 1), is an orphan G protein-coupled receptor that contains a large extracellular N terminus with 35 calcium-binding domains. A truncating mutation in the Mass1 gene causes autosomal recessive, sound-induced seizures in the Frings mouse. However, the function of MASS1 and the mechanism underlying Frings mouse epilepsy are not known. Here, we found that MASS1 protein is enriched in the myelinated regions of the superior and inferior colliculi, critical areas for the initiation and propagation of audiogenic seizures. Using a panel of myelin antibodies, we discovered that myelin-associated glycoprotein (MAG) expression is dramatically decreased in Frings mice. MASS1 inhibits the ubiquitylation of MAG, thus enhancing the stability of this protein, and the calcium-binding domains of MASS1 are essential for this regulation. Furthermore, MASS1 interacts with Gαs/Gαq and activates PKA and PKC in response to extracellular calcium. Suppression of signaling by MASS1 RNAi or a specific inhibitor abrogates MAG up-regulation. We postulate that MASS1 senses extracellular calcium and activates cytosolic PKA/PKC pathways to regulate myelination by means of MAG protein stability in myelin-forming cells of the auditory pathway. Further work is required to determine whether MAG dysregulation is a cause or consequence of audiogenic epilepsy and whether there are other pathways regulated by MASS1. PMID:24191038

  19. Knocking down the transcript of protein kinase C-lambda modulates hypothalamic glutathione peroxidase, melanocortin receptor and neuropeptide Y gene expression in amphetamine-treated rats.

    PubMed

    Hsieh, Yih-Shou; Yang, Shun-Fa; Chen, Pei-Ni; Chu, Shu-Chen; Chen, Chin-Hsiu; Kuo, Dong-Yih

    2011-07-01

    It has been reported that neuropeptide Y (NPY) contributes to the behavioral response of amphetamine (AMPH), a psychostimulant. The present study examined whether protein kinase C (PKC)-λ signaling was involved in this action. Moreover, possible roles of glutathione peroxidase (GP) and melanocortin receptor 4 (MC4R) were also examined. Rats were given AMPH daily for 4 days. Hypothalamic NPY, PKCλ, GP and MC4R were determined and compared. Pretreatment with α-methyl-para-tyrosine could block AMPH-induced anorexia, revealing that endogenous catecholamine was involved in regulating AMPH anorexia. PKCλ, GP and MC4R were increased with maximal response on Day 2 during AMPH treatment, which were concomitant with the decreases in NPY. cAMP response element binding protein (CREB) DNA binding activity was increased during AMPH treatment, revealing the involvement of CREB-dependent gene transcription. An interruption of cerebral PKCλ transcript could partly block AMPH-induced anorexia and partly reverse NPY, MC4R and GP mRNA levels to normal. These results suggest that PKCλ participates in regulating AMPH-induced anorexia via a modulation of hypothalamic NPY gene expression and that increases of GP and MC4R may contribute to this modulation. Our results provided molecular evidence for the regulation of AMPH-induced behavioral response.

  20. 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

  1. UV-induced signal transduction in epidermal cells: from surface receptors to protein kinase C: a mathematical model

    NASA Astrophysics Data System (ADS)

    Stolnitz, Mikhail M.; Peshkova, Anna Y.

    2002-05-01

    In the paper the mathematical model of UV-induced PKC activation is presented. Phosphorylation of membrane receptors, activation of phospholipases and phospholipids turnover, diacylglycerol, inositol trisphosphate and arachidonic acid production, calcium releasing are taken into account.

  2. 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

  3. 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.

  4. A Residue Quartet in the Extracellular Domain of the Prolactin Receptor Selectively Controls Mitogen-activated Protein Kinase Signaling.

    PubMed

    Zhang, Chi; Nygaard, Mads; Haxholm, Gitte W; Boutillon, Florence; Bernadet, Marie; Hoos, Sylviane; England, Patrick; Broutin, Isabelle; Kragelund, Birthe B; Goffin, Vincent

    2015-05-01

    Cytokine receptors elicit several signaling pathways, but it is poorly understood how they select and discriminate between them. We have scrutinized the prolactin receptor as an archetype model of homodimeric cytokine receptors to address the role of the extracellular membrane proximal domain in signal transfer and pathway selection. Structure-guided manipulation of residues involved in the receptor dimerization interface identified one residue (position 170) that in cell-based assays profoundly altered pathway selectivity and species-specific bio-characteristics. Subsequent in vitro spectroscopic and nuclear magnetic resonance analyses revealed that this residue was part of a residue quartet responsible for specific local structural changes underlying these effects. This included alteration of a novel aromatic T-stack within the membrane proximal domain, which promoted selective signaling affecting primarily the MAPK (ERK1/2) pathway. Importantly, activation of the MAPK pathway correlated with in vitro stabilities of ternary ligand·receptor complexes, suggesting a threshold mean lifetime of the complex necessary to achieve maximal activation. No such dependence was observed for STAT5 signaling. Thus, this study establishes a residue quartet in the extracellular membrane proximal domain of homodimeric cytokine receptors as a key regulator of intracellular signaling discrimination.

  5. Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

    PubMed Central

    Mahajan, S; Fargnoli, J; Burkhardt, A L; Kut, S A; Saouaf, S J; Bolen, J B

    1995-01-01

    Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation. PMID:7565679

  6. Src family protein tyrosine kinases induce autoactivation of Bruton's tyrosine kinase.

    PubMed

    Mahajan, S; Fargnoli, J; Burkhardt, A L; Kut, S A; Saouaf, S J; Bolen, J B

    1995-10-01

    Bruton's tyrosine kinase (Btk) is tyrosine phosphorylated and enzymatically activated following ligation of the B-cell antigen receptor. These events are temporally regulated, and Btk activation follows that of various members of the Src family of protein tyrosine kinases, thus raising the possibility that Src kinases participate in the Btk activation process. We have evaluated the mechanism underlying Btk enzyme activation and have explored the potential regulatory relationship between Btk and Src protein kinases. We demonstrate in COS transient-expression assays that Btk can be activated through intramolecular autophosphorylation at tyrosine 551 and that Btk autophosphorylation is required for Btk catalytic functions. Coexpression of Btk with members of the Src family of protein tyrosine kinases, but not Syk, led to Btk tyrosine phosphorylation and activation. Using a series of point mutations in Blk (a representative Src protein kinase) and Btk, we show that Src kinases activate Btk through an indirect mechanism that requires membrane association of the Src enzymes as well as functional Btk SH3 and SH2 domains. Our results are compatible with the idea that Src protein tyrosine kinases contribute to Btk activation by indirectly stimulating Btk intramolecular autophosphorylation. PMID:7565679

  7. Src-protein tyrosine kinases are required for cocaine-induced increase in the expression and function of the NMDA receptor in the ventral tegmental area.

    PubMed

    Schumann, Johanna; Michaeli, Avner; Yaka, Rami

    2009-02-01

    Cocaine-induced long-term potentiation of glutamatergic synapses in the ventral tegmental area (VTA) has been proposed as a key process that contributes to the development of addictive behaviors. In particular, the activation of ionotrophic glutamate NMDA receptor (NMDAR) in the VTA is critical for the initiation of cocaine sensitization. Here we show that application of cocaine both in slices and in vivo induced an increase in tyrosine phosphorylation of the NR2A, but not the NR2B subunit of the NMDAR in juvenile rats. Cocaine induced an increase in the activity of both Fyn and Src kinases, and the Src-protein tyrosine kinase (Src-PTKs) inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), abolished both cocaine-induced increase in tyrosine phosphorylation of the NR2A subunit and the increase in the expression of NR1, NR2A, and NR2B in the VTA. Moreover, cocaine-induced enhancement in NMDAR-mediated excitatory post-synaptic currents was completely abolished by PP2. Taken together, these results suggest that acute cocaine induced an increase in the expression of NMDAR subunits and enhanced tyrosine phosphorylation of NR2A-containing NMDAR through members of the Src-PTKs. This in turn, increased NMDAR-mediated currents in VTA dopamine neurons. These results provide a potential cellular mechanism by which cocaine triggers NMDAR-dependent synaptic plasticity of VTA neurons that may underlie the development of behavioral sensitization. PMID:19046409

  8. The Three Receptor Tyrosine Kinases c-KIT, VEGFR2 and PDGFRα, Closely Spaced at 4q12, Show Increased Protein Expression in Triple-Negative Breast Cancer

    PubMed Central

    Jansson, Sara; Bendahl, Pär-Ola; Grabau, Dorthe Aamand; Falck, Anna-Karin; Fernö, Mårten; Aaltonen, Kristina; Rydén, Lisa

    2014-01-01

    Background Triple-negative breast cancer (TNBC) is a heterogeneous subgroup of breast cancer with poor prognosis and no targeted therapy available. Receptor tyrosine kinases (RTKs) are emerging targets in anticancer therapy and many RTK-inhibiting drugs are currently being developed. The aim of this study was to elucidate if there is a correlation between the protein expression of three RTKs c-KIT, VEGFR2 and PDGFRα, their gene copy number, and prognosis in TNBC compared to non-TNBC. Methods Tumor tissue samples from patients diagnosed with primary breast cancer were stained with immunohistochemistry (IHC) for protein assessment, and with fluorescence in situ hybridization (FISH) for gene copy number determination. Breast cancer mortality (BCM), measured from the date of surgery to death, was used as endpoint. Results The cohort included 464 patients, out of which 34 (7.3%) had a TNBC. High expression of the three RTKs was more common in TNBC compared to non-TNBC: c-KIT 49% vs. 10% (P<0.001), PDGFRα 32% vs. 19% (P = 0.07) and VEGFR2 32% vs. 6% (P<0.001). The odds ratio (OR) of c-KIT, VEGFR2 and PDGFRα positivity, adjusted for tumor characteristics, was 6.8, 3.6 and 1.3 times higher for TNBC than for non-TNBC. 73.5% of the TNBC had high expression of at least one of the three investigated receptors, compared to 30.0% of the non-TNBC (P<0.001). Survival analysis showed no significant difference in BCM for TNBC patients with high vs. low c-KIT, PDGFRα or VEGFR2 protein expression. 193 (42%) tumors were evaluated with FISH. No correlation was seen between increased gene copy number and TNBC, or between increased gene copy number and high protein expression of the RTK. Conclusion c-KIT, VEGFR2 and PDGFRα show higher protein expression in TNBC compared to non-TNBC. Further investigation clarifying the importance of these RTKs in TNBC is encouraged, as they are possible targets for anticancer therapy. PMID:25025175

  9. Cell signaling by receptor-tyrosine kinases

    PubMed Central

    Lemmon, Mark A.; Schlessinger, Joseph

    2010-01-01

    Recent structural studies of receptor tyrosine kinases (RTKs) have revealed unexpected diversity in the mechanisms of their activation by growth factor ligands. Strategies for inducing dimerization by ligand binding are surprisingly diverse, as are mechanisms that couple this event to activation of the intracellular tyrosine kinase domains. As our understanding of these details becomes increasingly sophisticated, it provides an important context for therapeutically countering the effects of pathogenic RTK mutations in cancer and other diseases. Much remains to be learned, however, about the complex signaling networks downstream from RTKs and how alterations in these networks are translated into cellular responses. PMID:20602996

  10. 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.

  11. μ-Opioid and 5-HT1A receptors heterodimerize and show signalling crosstalk via G protein and MAP-kinase pathways.

    PubMed

    Cussac, Didier; Rauly-Lestienne, Isabelle; Heusler, Peter; Finana, Frédéric; Cathala, Claudie; Bernois, Sophie; De Vries, Luc

    2012-08-01

    μ-opioid receptors have been shown to form heterodimers with several G protein coupled receptors involved in pain regulation such as α(2A)-adrenergic and neurokinin 1 receptors. Because the 5-HT(1A) receptor is also involved in pain control, we investigated whether it can interact with the μ-opioid receptor in cell lines. Using epitope-tagged μ-opioid and 5-HT(1A) receptors, we show that both receptors can co-immunoprecipate when expressed in the same cells. This physical interaction was corroborated by a Bioluminescence Resonance Energy Transfer signal between the μ-opioid receptor fused to Renilla luciferase and the 5-HT(1A) receptor fused to the Green Fluorescent Protein. Consistent with the presence of functional heterodimers, the μ-opioid receptor activated a Gα(o) protein covalently fused to the 5-HT(1A) receptor in membrane preparations as well as a Gα(15) protein fused to the 5-HT(1A) receptor in living cells. We demonstrate that both receptors can coexerce control of the ERK1/2 pathway: for example, μ-opioid receptor-induced ERK1/2 phosphorylation was selectively desensitized by 5-HT(1A) receptor activation. Although 5-HT(1A) and μ-opioid receptors were capable to internalize in response to their own activation, they were ineffective to induce the co-internalization of their partners. Thus, we show a functional heterodimerization of μ-opioid and 5-HT(1A) receptors in cell lines, a complex that might play a role in the control of pain in vivo. These results also support the potential therapeutic action of 5-HT(1A) agonists against nociceptive processes.

  12. Phosphatidylinositol 4-phosphate 5-kinase α facilitates Toll-like receptor 4-mediated microglial inflammation through regulation of the Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) location.

    PubMed

    Nguyen, Tu Thi Ngoc; Kim, Yong Min; Kim, T Doohun; Le, Oanh Thi Tu; Kim, Jae Jin; Kang, Ho Chul; Hasegawa, Hiroshi; Kanaho, Yasunori; Jou, Ilo; Lee, Sang Yoon

    2013-02-22

    Phosphatidylinositol (PI) 4,5-bisphosphate (PIP(2)), generated by PI 4-phosphate 5-kinase (PIP5K), regulates many critical cellular events. PIP(2) is also known to mediate plasma membrane localization of the Toll/IL-1 receptor domain-containing adaptor protein (TIRAP), required for the MyD88-dependent Toll-like receptor (TLR) 4 signaling pathway. Microglia are the primary immune competent cells in brain tissue, and TLR4 is important for microglial activation. However, a functional role for PIP5K and PIP(2) in TLR4-dependent microglial activation remains unclear. Here, we knocked down PIP5Kα, a PIP5K isoform, in a BV2 microglial cell line using stable expression of lentiviral shRNA constructs or siRNA transfection. PIP5Kα knockdown significantly suppressed induction of inflammatory mediators, including IL-6, IL-1β, and nitric oxide, by lipopolysaccharide. PIP5Kα knockdown also attenuated signaling events downstream of TLR4 activation, including p38 MAPK and JNK phosphorylation, NF-κB p65 nuclear translocation, and IκB-α degradation. Complementation of the PIP5Kα knockdown cells with wild type but not kinase-dead PIP5Kα effectively restored the LPS-mediated inflammatory response. We found that PIP5Kα and TIRAP colocalized at the cell surface and interacted with each other, whereas kinase-dead PIP5Kα rendered TIRAP soluble. Furthermore, in LPS-stimulated control cells, plasma membrane PIP(2) increased and subsequently declined, and TIRAP underwent bi-directional translocation between the membrane and cytosol, which temporally correlated with the changes in PIP(2). In contrast, PIP5Kα knockdown that reduced PIP(2) levels disrupted TIRAP membrane targeting by LPS. Together, our results suggest that PIP5Kα promotes TLR4-associated microglial inflammation by mediating PIP(2)-dependent recruitment of TIRAP to the plasma membrane.

  13. Impact of protein tyrosine kinase 6 (PTK6) on human epidermal growth factor receptor (HER) signalling in breast cancer.

    PubMed

    Ludyga, Natalie; Anastasov, Nataša; Gonzalez-Vasconcellos, Iria; Ram, Manuela; Höfler, Heinz; Aubele, Michaela

    2011-05-01

    PTK6, also known as Brk, is highly expressed in over 80% of breast cancers. In the last decade several substrates and interaction partners were identified localising PTK6 downstream of HER receptors. PTK6 seems to be involved in progression of breast tumours, in particular in HER receptor signalling. Here, we show the down-regulation effects of PTK6 in the T47D, BT474 and JIMT-1 breast cancer cell lines. PTK6 knockdown leads to a decreased phosphorylation of HER2, PTEN, MAPK (ERK), p38 MAPK, STAT3 and to a reduced expression of cyclin E. Our findings show that silencing PTK6 impairs the downstream targets of HER receptors and consequently the activation of signalling molecules. Furthermore, lower levels of PTK6 result in reduced migration of T47D and JIMT-1 breast cancer cells. Due to decreased migration, the PTK6 RNA interference might contribute to reduced metastasis and malignant potential of breast cancer cells. Since PTK6 plays an important role in HER receptor signal transduction, its down-regulation might be suitable for future therapy approaches in breast cancer.

  14. In planta assessment of the role of thioredoxin h proteins in the regulation of S-locus receptor kinase signaling in transgenic Arabidopsis.

    PubMed

    Yamamoto, Masaya; Nasrallah, June B

    2013-11-01

    The self-incompatibility (SI) response of the Brassicaceae is mediated by allele-specific interaction between the stigma-localized S-locus receptor kinase (SRK) and its ligand, the pollen coat-localized S-locus cysteine-rich protein (SCR). Based on work in Brassica spp., the thioredoxin h-like proteins THL1 and THL2, which interact with SRK, have been proposed to function as oxidoreductases that negatively regulate SRK catalytic activity. By preventing the spontaneous activation of SRK in the absence of SCR ligand, these thioredoxins are thought to be essential for the success of cross pollinations in self-incompatible plants. However, the in planta role of thioredoxins in the regulation of SI signaling has not been conclusively demonstrated. Here, we addressed this issue using Arabidopsis thaliana plants transformed with the SRKb-SCRb gene pair isolated from self-incompatible Arabidopsis lyrata. These plants express an intense SI response, allowing us to exploit the extensive tools and resources available in A. thaliana for analysis of SI signaling. To test the hypothesis that SRK is redox regulated by thioredoxin h, we expressed a mutant form of SRKb lacking a transmembrane-localized cysteine residue thought to be essential for the SRK-thioredoxin h interaction. We also analyzed transfer DNA insertion mutants in the A. thaliana orthologs of THL1 and THL2. In neither case did we observe an effect on the pollination responses of SRKb-expressing stigmas toward incompatible or compatible pollen. Our results are consistent with the conclusion that, contrary to their proposed role, thioredoxin h proteins are not required to prevent the spontaneous activation of SRK in the A. thaliana stigma. PMID:24077073

  15. Subcellular localization and functions of the barley stem rust resistance receptor-like serine/threonine-specific protein kinase Rpg1.

    PubMed

    Nirmala, Jayaveeramuthu; Brueggeman, Robert; Maier, Christina; Clay, Christine; Rostoks, Nils; Kannangara, C Gamini; von Wettstein, Diter; Steffenson, Brian J; Kleinhofs, Andris

    2006-05-01

    The Rpg1 gene confers resistance to many pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici and has protected barley from serious disease losses for over 60 years. Rpg1 encodes a constitutively expressed protein with two tandem kinase domains. Fractionation by differential centrifugation and aqueous two-phase separation of the microsome proteins located Rpg1 mainly in the cytosol but also in the plasma membrane and intracellular membranes. Recombinant Rpg1 autophosphorylates in vitro intramolecularly only serine and threonine amino acids with a preference for Mn(2+) cations and a K(m) of 0.15 and a V(max) of 0.47 nmol.min(-1).mg(-1) protein. The inability of wild-type Rpg1 to transphosphorylate a recombinant Rpg1 inactivated by site-directed mutation confirmed that Rpg1 autophosphorylation proceeds exclusively via an intramolecular mechanism. Site-directed mutagenesis of the two adjacent lysine residues in the ATP anchor of the two-kinase domains established that the first of the two tandem kinase domains is nonfunctional and that lysine 461 of the second domain is the catalytically active residue. Transgenic barley, expressing Rpg1 mutated in either the kinase 1 or 2 domains, were fully susceptible to P. graminis f. sp. tritici revealing requirement of both kinase domains for resistance. In planta-expressed Rpg1 mutant protein confirmed that mutation in domain 2, but not 1, rendered the protein incapable of autophosphorylation.

  16. Protein Kinase Cδ and Calmodulin Regulate Epidermal Growth Factor Receptor Recycling from Early Endosomes through Arp2/3 Complex and Cortactin

    PubMed Central

    Lladó, Anna; Timpson, Paul; Vilà de Muga, Sandra; Moretó, Jemina; Pol, Albert; Grewal, Thomas; Daly, Roger J.

    2008-01-01

    The intracellular trafficking of the epidermal growth factor receptor (EGFR) is regulated by a cross-talk between calmodulin (CaM) and protein kinase Cδ (PKCδ). On inhibition of CaM, PKCδ promotes the formation of enlarged early endosomes and blocks EGFR recycling and degradation. Here, we show that PKCδ impairs EGFR trafficking due to the formation of an F-actin coat surrounding early endosomes. The PKCδ-induced polymerization of actin is orchestrated by the Arp2/3 complex and requires the interaction of cortactin with PKCδ. Accordingly, inhibition of actin polymerization by using cytochalasin D or by overexpression of active cofilin, restored the normal morphology of the organelle and the recycling of EGFR. Similar results were obtained after down-regulation of cortactin and the sequestration of the Arp2/3 complex. Furthermore we demonstrate an interaction of cortactin with CaM and PKCδ, the latter being dependent on CaM inhibition. In summary, this study provides the first evidence that CaM and PKCδ organize actin dynamics in the early endosomal compartment, thereby regulating the intracellular trafficking of EGFR. PMID:17959830

  17. Altered mitogen-activated protein kinase signal transduction in human skin fibroblasts during in vitro aging: differential expression of low-density lipoprotein receptor.

    PubMed

    Bose, Chhanda; Bhuvaneswaran, Chidambaram; Udupa, Kodetthoor B

    2004-02-01

    The purpose of the study was to investigate the correlation of low-density lipoprotein receptor (LDLr) and mitogen-activated protein kinases (MAPK) in fibroblasts after serial passage in vitro. We used early-passage ( approximately 20 mean population division, MPD) and late-passage ( approximately 60 MPD) human skin fibroblasts to study the LDLr expression and MAPK at basal and after interleukin-1beta (IL-1beta) stimulation. We found a reduced LDLr expression in late-passage fibroblasts in comparison with early-passage fibroblasts, and late-passage fibroblasts showed a delayed induction of MAPK after IL-1beta stimulation, confirmed by the delay in translocation of MAPK from cytoplasmic to nuclear fraction. Using two specific inhibitors of MAPK, we could show a reduced LDLr expression in early-passage fibroblasts, indicating a direct relationship between MAPK signaling and LDLr expression. We conclude that one of the reasons for reduced LDLr gene expression in late passage fibroblast is related to MAPK signaling.

  18. beta2 Adrenergic receptor activation induces microglial NADPH oxidase activation and dopaminergic neurotoxicity through an ERK-dependent/protein kinase A-independent pathway.

    PubMed

    Qian, Li; Hu, Xiaoming; Zhang, Dan; Snyder, Amanda; Wu, Hung-Ming; Li, Yachen; Wilson, Belinda; Lu, Ru-Band; Hong, Jau-Shyong; Flood, Patrick M

    2009-11-15

    Activation of the beta2 adrenergic receptor (beta2AR) on immune cells has been reported to possess anti-inflammatory properties, however, the pro-inflammatory properties of beta2AR activation remain unclear. In this study, using rat primary mesencephalic neuron-glia cultures, we report that salmeterol, a long-acting beta2AR agonist, selectively induces dopaminergic (DA) neurotoxicity through its ability to activate microglia. Salmeterol selectively increased the production of reactive oxygen species (ROS) by NADPH oxidase (PHOX), the major superoxide-producing enzyme in microglia. A key role of PHOX in mediating salmeterol-induced neurotoxicity was demonstrated by the inhibition of DA neurotoxicity in cultures pretreated with diphenylene-iodonium (DPI), an inhibitor of PHOX activity. Mechanistic studies revealed the activation of microglia by salmeterol results in the selective phosphorylation of ERK, a signaling pathway required for the translocation of the PHOX cytosolic subunit p47(phox) to the cell membrane. Furthermore, we found ERK inhibition, but not protein kinase A (PKA) inhibition, significantly abolished salmeterol-induced superoxide production, p47(phox) translocation, and its ability to mediate neurotoxicity. Together, these findings indicate that beta2AR activation induces microglial PHOX activation and DA neurotoxicity through an ERK-dependent/PKA-independent pathway.

  19. Phospholipase C/protein kinase C pathway mediates angiotensin II-dependent apoptosis in neonatal rat cardiac fibroblasts expressing AT1 receptor.

    PubMed

    Vivar, Raul; Soto, Cristian; Copaja, Miguel; Mateluna, Francisca; Aranguiz, Pablo; Muñoz, Juan Pablo; Chiong, Mario; Garcia, Lorena; Letelier, Alan; Thomas, Walter G; Lavandero, Sergio; Díaz-Araya, Guillermo

    2008-08-01

    Cardiac fibroblasts are the major non-myocyte cell constituent in the myocardium, and they are involved in heart remodeling. Angiotensin II type 1 receptor (AT1R) mediates the established actions of angiotensin II (Ang II), and changes in its expression have been reported in cardiac fibroblasts after myocardial infarction. However, the AT1R-dependent signaling pathways involved in cardiac fibroblast death remain unknown. Using adenovirus, we ectopically expressed AT1R in cultured neonatal rat cardiac fibroblasts and investigated the role of the phospholipase (PLC)/protein kinase C (PKC) pathway on Ang II-dependent death. Ang II induced cardiac fibroblast death characterized by an early loss of mitochondrial membrane potential, increased Bax/Bcl-2 ratio, caspase-3 activation, and DNA fragmentation. All these effects were prevented by the AT1R antagonist losartan, PLC inhibitor U73122, and PKC inhibitor Gö6976. We conclude that Ang II stimulates the intrinsic apoptotic pathway in cultured cardiac fibroblasts by the AT1R/PLC/PKC signaling pathway. PMID:18670360

  20. Tyrosine kinase BMX phosphorylates phosphotyrosine-primed motif mediating the activation of multiple receptor tyrosine kinases.

    PubMed

    Chen, Sen; Jiang, Xinnong; Gewinner, Christina A; Asara, John M; Simon, Nicholas I; Cai, Changmeng; Cantley, Lewis C; Balk, Steven P

    2013-05-28

    The nonreceptor tyrosine kinase BMX (bone marrow tyrosine kinase gene on chromosome X) is abundant in various cell types and activated downstream of phosphatidylinositol-3 kinase (PI3K) and the kinase Src, but its substrates are unknown. Positional scanning peptide library screening revealed a marked preference for a priming phosphorylated tyrosine (pY) in the -1 position, indicating that BMX substrates may include multiple tyrosine kinases that are fully activated by pYpY sites in the kinase domain. BMX phosphorylated focal adhesion kinase (FAK) at Tyr⁵⁷⁷ subsequent to its Src-mediated phosphorylation at Tyr⁵⁷⁶. Loss of BMX by RNA interference or by genetic deletion in mouse embryonic fibroblasts (MEFs) markedly impaired FAK activity. Phosphorylation of the insulin receptor in the kinase domain at Tyr¹¹⁸⁹ and Tyr¹¹⁹⁰, as well as Tyr¹¹⁸⁵, and downstream phosphorylation of the kinase AKT at Thr³⁰⁸ were similarly impaired by BMX deficiency. However, insulin-induced phosphorylation of AKT at Ser⁴⁷³ was not impaired in Bmx knockout MEFs or liver tissue from Bmx knockout mice, which also showed increased insulin-stimulated glucose uptake, possibly because of decreased abundance of the phosphatase PHLPP (PH domain leucine-rich repeat protein phosphatase). Thus, by identifying the pYpY motif as a substrate for BMX, our findings suggest that BMX functions as a central regulator among multiple signaling pathways mediated by tyrosine kinases. PMID:23716717

  1. Environmental Enrichment Increases Glucocorticoid Receptors and Decreases GluA2 and Protein Kinase M Zeta (PKMζ) Trafficking During Chronic Stress: A Protective Mechanism?

    PubMed Central

    Zanca, Roseanna M.; Braren, Stephen H.; Maloney, Brigid; Schrott, Lisa M.; Luine, Victoria N.; Serrano, Peter A.

    2015-01-01

    Environmental enrichment (EE) housing paradigms have long been shown beneficial for brain function involving neural growth and activity, learning and memory capacity, and for developing stress resiliency. The expression of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA2, which is important for synaptic plasticity and memory, is increased with corticosterone (CORT), undermining synaptic plasticity and memory. Thus, we determined the effect of EE and stress on modulating GluA2 expression in Sprague-Dawley male rats. Several markers were evaluated which include: plasma CORT, the glucocorticoid receptor (GR), GluA2, and the atypical protein kinase M zeta (PKMζ). For 1 week standard-(ST) or EE-housed animals were treated with one of the following four conditions: (1) no stress; (2) acute stress (forced swim test, FST; on day 7); (3) chronic restraint stress (6 h/day for 7 days); and (4) chronic + acute stress (restraint stress 6 h/day for 7 days + FST on day 7). Hippocampi were collected on day 7. Our results show that EE animals had reduced time immobile on the FST across all conditions. After chronic + acute stress EE animals showed increased GR levels with no change in synaptic GluA2/PKMζ. ST-housed animals showed the reverse pattern with decreased GR levels and a significant increase in synaptic GluA2/PKMζ. These results suggest that EE produces an adaptive response to chronic stress allowing for increased GR levels, which lowers neuronal excitability reducing GluA2/PKMζ trafficking. We discuss this EE adaptive response to stress as a potential underlying mechanism that is protective for retaining synaptic plasticity and memory function. PMID:26617502

  2. Environmental Enrichment Increases Glucocorticoid Receptors and Decreases GluA2 and Protein Kinase M Zeta (PKMζ) Trafficking During Chronic Stress: A Protective Mechanism?

    PubMed

    Zanca, Roseanna M; Braren, Stephen H; Maloney, Brigid; Schrott, Lisa M; Luine, Victoria N; Serrano, Peter A

    2015-01-01

    Environmental enrichment (EE) housing paradigms have long been shown beneficial for brain function involving neural growth and activity, learning and memory capacity, and for developing stress resiliency. The expression of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA2, which is important for synaptic plasticity and memory, is increased with corticosterone (CORT), undermining synaptic plasticity and memory. Thus, we determined the effect of EE and stress on modulating GluA2 expression in Sprague-Dawley male rats. Several markers were evaluated which include: plasma CORT, the glucocorticoid receptor (GR), GluA2, and the atypical protein kinase M zeta (PKMζ). For 1 week standard-(ST) or EE-housed animals were treated with one of the following four conditions: (1) no stress; (2) acute stress (forced swim test, FST; on day 7); (3) chronic restraint stress (6 h/day for 7 days); and (4) chronic + acute stress (restraint stress 6 h/day for 7 days + FST on day 7). Hippocampi were collected on day 7. Our results show that EE animals had reduced time immobile on the FST across all conditions. After chronic + acute stress EE animals showed increased GR levels with no change in synaptic GluA2/PKMζ. ST-housed animals showed the reverse pattern with decreased GR levels and a significant increase in synaptic GluA2/PKMζ. These results suggest that EE produces an adaptive response to chronic stress allowing for increased GR levels, which lowers neuronal excitability reducing GluA2/PKMζ trafficking. We discuss this EE adaptive response to stress as a potential underlying mechanism that is protective for retaining synaptic plasticity and memory function.

  3. Green tea epigallocatechin gallate inhibits insulin stimulation of adipocyte glucose uptake via the 67-kilodalton laminin receptor and AMP-activated protein kinase pathways.

    PubMed

    Hsieh, Chi-Fen; Tsuei, Yi-Wei; Liu, Chi-Wei; Kao, Chung-Cheng; Shih, Li-Jane; Ho, Low-Tone; Wu, Liang-Yi; Wu, Chi-Peng; Tsai, Pei-Hua; Chang, Hsin-Huei; Ku, Hui-Chen; Kao, Yung-Hsi

    2010-10-01

    Insulin and (-)-epigallocatechin gallate (EGCG) are reported to regulate obesity and fat accumulation, respectively. This study investigated the pathways involved in EGCG modulation of insulin-stimulated glucose uptake in 3T3-L1 and C3H10T1/2 adipocytes. EGCG inhibited insulin stimulation of adipocyte glucose uptake in a dose- and time-dependent manner. The concentration of EGCG that decreased insulin-stimulated glucose uptake by 50-60% was approximately 5-10 µM for a period of 2 h. At 10 µM, EGCG and gallic acid were more effective than (-)-epicatechin, (-)-epigallocatechin, and (-)-epicatechin 3-gallate. We identified the EGCG receptor [also known as the 67-kDa laminin receptor (67LR)] in fat cells and extended the findings for this study to clarify whether EGCG-induced changes in insulin-stimulated glucose uptake in adipocytes could be mediated through the 67LR. Pretreatment of adipocytes with a 67LR antibody, but not normal rabbit immunoglobulin, prevented the effects of EGCG on insulin-increased glucose uptake. This suggests that the 67LR mediates the effect of EGCG on insulin-stimulated glucose uptake in adipocytes. Moreover, pretreatment with an AMP-activated protein kinase (AMPK) inhibitor, such as compound C, but not with a glutathione (GSH) activator, such as N-acetyl-L-cysteine (NAC), blocked the antiinsulin effect of EGCG on adipocyte glucose uptake. These data suggest that EGCG exerts its anti-insulin action on adipocyte glucose uptake via the AMPK, but not the GSH, pathway. The results of this study possibly support that EGCG mediates fat content.

  4. 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...

  5. p90 ribosomal S6 kinases play a significant role in early gene regulation in the cardiomyocyte response to G(q)-protein-coupled receptor stimuli, endothelin-1 and α(1)-adrenergic receptor agonists.

    PubMed

    Amirak, Emre; Fuller, Stephen J; Sugden, Peter H; Clerk, Angela

    2013-03-01

    ERK1/2 (extracellular-signal-regulated kinase 1/2) and their substrates RSKs (p90 ribosomal S6 kinases) phosphorylate different transcription factors, contributing differentially to transcriptomic profiles. In cardiomyocytes ERK1/2 are required for >70% of the transcriptomic response to endothelin-1. In the present study we investigated the role of RSKs in the transcriptomic responses to the G(q)-protein-coupled receptor agonists endothelin-1, phenylephrine (a generic α(1)-adrenergic receptor agonist) and A61603 (α(1A)-adrenergic receptor selective). Phospho-ERK1/2 and phospho-RSKs appeared in cardiomyocyte nuclei within 2-3 min of stimulation (endothelin-1>A61603≈phenylephrine). All agonists increased nuclear RSK2, but only endothelin-1 increased the nuclear RSK1 content. PD184352 (inhibits ERK1/2 activation) and BI-D1870 (inhibits RSKs) were used to dissect the contribution of RSKs to the endothelin-1-responsive transcriptome. Of the 213 RNAs up-regulated after 1 h, 51% required RSKs for their up-regulation, whereas 29% required ERK1/2 but not RSKs. The transcriptomic response to phenylephrine overlapped with, but was not identical with, endothelin-1. As with endothelin-1, PD184352 inhibited the up-regulation of most phenylephrine-responsive transcripts, but the greater variation in the effects of BI-D1870 suggests that differential RSK signalling influences global gene expression. A61603 induced similar changes in RNA expression in cardiomyocytes as phenylephrine, indicating that the signal was mediated largely through α(1A)-adrenergic receptors. A61603 also increased expression of immediate early genes in perfused adult rat hearts and, as in cardiomyocytes, up-regulation of the majority of genes was inhibited by PD184352. PD184352 or BI-D1870 prevented the increased surface area induced by endothelin-1 in cardiomyocytes. Thus RSKs play a significant role in regulating cardiomyocyte gene expression and hypertrophy in response to G(q)-protein

  6. 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

  7. 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.

  8. 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. PMID:27000704

  9. Protein kinase A inhibition of macrophage maturation is accompanied by an increase in DNA methylation of the colony-stimulating factor 1 receptor gene.

    PubMed

    Zasłona, Zbigniew; Scruggs, Anne M; Peters-Golden, Marc; Huang, Steven K

    2016-10-01

    Macrophage colony-stimulating factor 1 (CSF-1) plays a critical role in the differentiation of mononuclear phagocytes from bone marrow precursors, and maturing monocytes and macrophages exhibit increased expression of the CSF-1 receptor, CSF1R. The expression of CSF1R is tightly regulated by transcription factors and epigenetic mechanisms. We previously showed that prostaglandin E2 and subsequent activation of protein kinase A (PKA) inhibited CSF1R expression and macrophage maturation. Here, we examine the DNA methylation changes that occur at the Csf1r locus during macrophage maturation in the presence or absence of activated PKA. Murine bone marrow cells were matured to macrophages by incubating cells with CSF-1-containing conditioned medium for up to 6 days in the presence or absence of the PKA agonist 6-bnz-cAMP. DNA methylation of Csf1r promoter and enhancer regions was assayed by bisulphite pyrosequencing. DNA methylation of Csf1r decreased during normal macrophage maturation in concert with an increase in Csf1r mRNA expression. Treatment with the PKA agonist inhibited Csf1r mRNA and protein expression, and increased DNA methylation at the Csf1r promoter. This was associated with decreased binding of the transcription factor PU.1 to the Csf1r promoter. Treatment with the PKA agonist inhibited the responsiveness of macrophages to CSF-1. Levels of endogenous PKA activity decreased during normal macrophage maturation, suggesting that attenuation of this signalling pathway contributes to the increase in CSF1R expression during macrophage maturation. Together, these results demonstrate that macrophage maturation is accompanied by Csf1r hypomethylation, and illustrates for the first time the ability of PKA to increase Csf1r DNA methylation.

  10. Nectandrin B, a lignan isolated from nutmeg, inhibits liver X receptor-α-induced hepatic lipogenesis through AMP-activated protein kinase activation.

    PubMed

    Choi, Du Gon; Kim, Eun Kyung; Yang, Jin Won; Song, Jae Sook; Kim, Young-Mi

    2015-11-01

    Nonalcoholic fatty liver disease is recognized as the most commonly occurring chronic liver disease. Liver X receptor α (LXRα) and sterol regulatory element-binding protein (SREBP)-1c play a central role in de novo fatty acid synthesis. This study investigated pharmacological effects of nectandrin B, a lignan isolated from nutmeg extract, on hepatic lipogenesis stimulated by LXRα-SREBP-1c-mediated pathway and the possible molecular basis. The reporter gene assay revealed that nectandrin B completely represses LXRα activity enhanced by a synthetic LXRα ligand (T0901317) in HepG2 cells. The inhibitory effect was further supported by the suppression of mRNA expression of LXRα target genes, SREBP-1c and LXRα itself. Nectandrin B also inhibited the increase in SREBP-1c expression promoted by insulin plus high glucose, major contributors to hepatic lipid accumulation. LXRα-SREBP-1c-mediated induction of acetyl-CoA carboxylase 1 and fatty acid synthase, major genes for de novo lipogenesis, was suppressed by nectandrin B. Moreover, Oil Red O staining showed that nectandrin B notably attenuates LXRα-induced lipid accumulation. AMP-activated protein kinase (AMPK) inhibits the activities of LXRα and SREBP-1c. Nectandrin B strongly activated AMPK signaling in HepG2 cells. Taken together, the suppressive effects of nectandrin B on lipogenic gene expression and lipid accumulation in hepatocytes may be due to its inhibitory effect on the LXRα-SREBP-1c pathway presumably via AMPK activation. These results suggest the potential of nectandrin B as a therapeutic candidate for fatty liver disease. PMID:26790190

  11. Protein kinase A inhibition of macrophage maturation is accompanied by an increase in DNA methylation of the colony-stimulating factor 1 receptor gene.

    PubMed

    Zasłona, Zbigniew; Scruggs, Anne M; Peters-Golden, Marc; Huang, Steven K

    2016-10-01

    Macrophage colony-stimulating factor 1 (CSF-1) plays a critical role in the differentiation of mononuclear phagocytes from bone marrow precursors, and maturing monocytes and macrophages exhibit increased expression of the CSF-1 receptor, CSF1R. The expression of CSF1R is tightly regulated by transcription factors and epigenetic mechanisms. We previously showed that prostaglandin E2 and subsequent activation of protein kinase A (PKA) inhibited CSF1R expression and macrophage maturation. Here, we examine the DNA methylation changes that occur at the Csf1r locus during macrophage maturation in the presence or absence of activated PKA. Murine bone marrow cells were matured to macrophages by incubating cells with CSF-1-containing conditioned medium for up to 6 days in the presence or absence of the PKA agonist 6-bnz-cAMP. DNA methylation of Csf1r promoter and enhancer regions was assayed by bisulphite pyrosequencing. DNA methylation of Csf1r decreased during normal macrophage maturation in concert with an increase in Csf1r mRNA expression. Treatment with the PKA agonist inhibited Csf1r mRNA and protein expression, and increased DNA methylation at the Csf1r promoter. This was associated with decreased binding of the transcription factor PU.1 to the Csf1r promoter. Treatment with the PKA agonist inhibited the responsiveness of macrophages to CSF-1. Levels of endogenous PKA activity decreased during normal macrophage maturation, suggesting that attenuation of this signalling pathway contributes to the increase in CSF1R expression during macrophage maturation. Together, these results demonstrate that macrophage maturation is accompanied by Csf1r hypomethylation, and illustrates for the first time the ability of PKA to increase Csf1r DNA methylation. PMID:27353657

  12. Multiple regulatory domains on the Byr2 protein kinase.

    PubMed Central

    Tu, H; Barr, M; Dong, D L; Wigler, M

    1997-01-01

    Byr2 protein kinase, a homolog of mammalian mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEKK) and Saccharomyces cerevisiae STE11, is required for pheromone-induced sexual differentiation in the fission yeast Schizosaccharomyces pombe. Byr2 functions downstream of Ste4, Ras1, and the membrane-associated receptor-coupled heterotrimeric G-protein alpha subunit, Gpa1. Byr2 has a distinctive N-terminal kinase regulatory domain and a characteristic C-terminal kinase catalytic domain. Ste4 and Ras1 interact with the regulatory domain of Byr2 directly. Here, we define the domains of Byr2 that bind Ste4 and Ras1 and show that the Byr2 regulatory domain binds to the catalytic domain in the two-hybrid system. Using Byr2 mutants, we demonstrate that these direct physical interactions are all required for proper signaling. In particular, the physical association between Byr2 regulatory and catalytic domains appears to result in autoinhibition, the loss of which results in kinase activation. Furthermore, we provide evidence that Shk1, the S. pombe homolog of the STE20 protein kinase, can directly antagonize the Byr2 intramolecular interaction, possibly by phosphorylating Byr2. PMID:9315645

  13. Activation of G proteins and extracellular signal-regulated kinase 1/2 phosphorylation via human dopamine D4.4 receptors: differential pathway-dependent potencies of receptor agonists.

    PubMed

    Heusler, Peter; Bruins Slot, Liesbeth; Rauly-Lestienne, Isabelle; Palmier, Christiane; Tardif, Stéphanie; Tourette, Amélie; Ailhaud, Marie-Christine; Cussac, Didier

    2009-01-01

    Agonist activity at recombinant human dopamine D4.4 receptors was compared in stably transfected CHO cells using two functional readouts: G protein activation by [35S]GTPgammaS binding and phosphorylation of extracellular signal-regulated kinase 1/2 (pERK1/2). Results with a large series of agonists reveal markedly higher relative agonist efficacy in the pERK1/2 assay compared with [35S]GTPgammaS binding, while potencies were generally higher in the latter readout. Whereas efficacies were highly correlated when comparing both tests, potencies determined using the pERK1/2 assay were neither correlated with those for G protein activation nor with binding affinities. In order to examine if these differences may be attributable to distinct assay conditions (5 min incubation for pERK1/2 compared with binding equilibrium conditions for [35S]GTPgammaS), selected compounds were tested in a modified short-duration [35S]GTPgammaS binding assay. In these experiments, potencies were generally reduced; however, compounds exhibiting comparably high potency in the pERK1/2 assay were not affected by this duration-dependent potency shift. We conclude that assay parameters such as signal amplification and incubation time have to be considered with respect to the appropriate choice of experimental approaches that best reflect agonist activity at dopamine D4 receptors in vivo.

  14. The ubiquitin ligase Mdm2 controls oligodendrocyte maturation by intertwining mTOR with G protein-coupled receptor kinase 2 in the regulation of GPR17 receptor desensitization.

    PubMed

    Fumagalli, Marta; Bonfanti, Elisabetta; Daniele, Simona; Zappelli, Elisa; Lecca, Davide; Martini, Claudia; Trincavelli, Maria L; Abbracchio, Maria P

    2015-12-01

    During oligodendrocyte precursor cell (OPC) differentiation, defective control of the membrane receptor GPR17 has been suggested to block cell maturation and impair remyelination under demyelinating conditions. After the immature oligodendrocyte stage, to enable cells to complete maturation, GPR17 is physiologically down-regulated via phosphorylation/desensitization by G protein-coupled receptor kinases (GRKs); conversely, GRKs are regulated by the "mammalian target of rapamycin" mTOR. However, how GRKs and mTOR are connected to each other in modulating GPR17 function and oligodendrogenesis has remained elusive. Here we show, for the first time, a role for Murine double minute 2 (Mdm2), a ligase previously involved in ubiquitination/degradation of the onco-suppressor p53 protein. In maturing OPCs, both rapamycin and Nutlin-3, a small molecule inhibitor of Mdm2-p53 interactions, increased GRK2 sequestration by Mdm2, leading to impaired GPR17 down-regulation and OPC maturation block. Thus, Mdm2 intertwines mTOR with GRK2 in regulating GPR17 and oligodendrogenesis and represents a novel actor in myelination.

  15. 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.

  16. 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. PMID:27369082

  17. 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.

  18. Recent developments of protein kinase inhibitors as potential AD therapeutics.

    PubMed

    Tell, Volkmar; Hilgeroth, Andreas

    2013-01-01

    Present Alzheimer's disease (AD) therapies suffer from inefficient effects on AD symptoms like memory or cognition, especially in later states of the disease. Used acteylcholine esterase inhibitors or the NMDA receptor antagonist memantine address one target structure which is involved in a complex, multifactorial disease progression. So the benefit for patients is presently poor. A more close insight in the AD progression identified more suggested target structures for drug development. Strategies of AD drug development concentrate on novel target structures combined with the established ones dedicated for combined therapy regimes, preferably by the use of one drug which may address two target structures. Protein kinases have been identified as promising target structures because they are involved in AD progression pathways like pathophysiological tau protein phosphorylations and amyloid β toxicity. The review article will shortly view early inhibitors of single protein kinases like glycogen synthase kinase (gsk3) β and cyclin dependent kinase 5. Novel inhibitors will be discussed which address novel AD relevant protein kinases like dual-specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A). Moreover, multitargeting inhibitors will be presented which target several protein kinases and those which are suspected in influencing other AD relevant processes. Such a multitargeting is the most promising strategy to effectively hamper the multifactorial disease progression and thus gives perspective hopes for a future better patient benefit. PMID:24312003

  19. Recent developments of protein kinase inhibitors as potential AD therapeutics

    PubMed Central

    Tell, Volkmar; Hilgeroth, Andreas

    2013-01-01

    Present Alzheimer’s disease (AD) therapies suffer from inefficient effects on AD symptoms like memory or cognition, especially in later states of the disease. Used acteylcholine esterase inhibitors or the NMDA receptor antagonist memantine address one target structure which is involved in a complex, multifactorial disease progression. So the benefit for patients is presently poor. A more close insight in the AD progression identified more suggested target structures for drug development. Strategies of AD drug development concentrate on novel target structures combined with the established ones dedicated for combined therapy regimes, preferably by the use of one drug which may address two target structures. Protein kinases have been identified as promising target structures because they are involved in AD progression pathways like pathophysiological tau protein phosphorylations and amyloid β toxicity. The review article will shortly view early inhibitors of single protein kinases like glycogen synthase kinase (gsk3) β and cyclin dependent kinase 5. Novel inhibitors will be discussed which address novel AD relevant protein kinases like dual-specificity tyrosine phosphorylation regulated kinase 1A (DYRK1A). Moreover, multitargeting inhibitors will be presented which target several protein kinases and those which are suspected in influencing other AD relevant processes. Such a multitargeting is the most promising strategy to effectively hamper the multifactorial disease progression and thus gives perspective hopes for a future better patient benefit. PMID:24312003

  20. 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.

  1. Plant protein kinase substrates identification using protein microarrays.

    PubMed

    Ma, Shisong; Dinesh-Kumar, Savithramma P

    2015-01-01

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

  2. The venus kinase receptor (VKR) family: structure and evolution

    PubMed Central

    2013-01-01

    Background Receptor tyrosine kinases (RTK) form a family of transmembrane proteins widely conserved in Metazoa, with key functions in cell-to-cell communication and control of multiple cellular processes. A new family of RTK named Venus Kinase Receptor (VKR) has been described in invertebrates. The VKR receptor possesses a Venus Fly Trap (VFT) extracellular module, a bilobate structure that binds small ligands to induce receptor kinase activity. VKR was shown to be highly expressed in the larval stages and gonads of several invertebrates, suggesting that it could have functions in development and/or reproduction. Results Analysis of recent genomic data has allowed us to extend the presence of VKR to five bilaterian phyla (Platyhelminthes, Arthropoda, Annelida, Mollusca, Echinodermata) as well as to the Cnidaria phylum. The presence of NveVKR in the early-branching metazoan Nematostella vectensis suggested that VKR arose before the bilaterian radiation. Phylogenetic and gene structure analyses showed that the 40 receptors identified in 36 animal species grouped monophyletically, and likely evolved from a common ancestor. Multiple alignments of tyrosine kinase (TK) and VFT domains indicated their important level of conservation in all VKRs identified up to date. We showed that VKRs had inducible activity upon binding of extracellular amino-acids and molecular modeling of the VFT domain confirmed the structure of the conserved amino-acid binding site. Conclusions This study highlights the presence of VKR in a large number of invertebrates, including primitive metazoans like cnidarians, but also its absence from nematodes and chordates. This little-known RTK family deserves to be further explored in order to determine its evolutionary origin, its possible interest for the emergence and specialization of Metazoa, and to understand its function in invertebrate development and/or reproductive biology. PMID:23721482

  3. Mechanism of regulation of receptor histidine kinases.

    PubMed

    Ferris, Hedda U; Dunin-Horkawicz, Stanislaw; Hornig, Nora; Hulko, Michael; Martin, Jörg; Schultz, Joachim E; Zeth, Kornelius; Lupas, Andrei N; Coles, Murray

    2012-01-11

    Bacterial transmembrane receptors regulate an intracellular catalytic output in response to extracellular sensory input. To investigate the conformational changes that relay the regulatory signal, we have studied the HAMP domain, a ubiquitous intracellular module connecting input to output domains. HAMP forms a parallel, dimeric, four-helical coiled coil, and rational substitutions in our model domain (Af1503 HAMP) induce a transition in its interhelical packing, characterized by axial rotation of all four helices (the gearbox signaling model). We now illustrate how these conformational changes are propagated to a downstream domain by fusing Af1503 HAMP variants to the DHp domain of EnvZ, a bacterial histidine kinase. Structures of wild-type and mutant constructs are correlated with ligand response in vivo, clearly associating them with distinct signaling states. We propose that altered recognition of the catalytic domain by DHp, rather than a shift in position of the phospho-accepting histidine, forms the basis for regulation of kinase activity.

  4. 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

  5. The coreceptor CD4 is expressed in distinct nanoclusters and does not colocalize with T-cell receptor and active protein tyrosine kinase p56lck.

    PubMed

    Roh, Kyung-Ho; Lillemeier, Björn F; Wang, Feng; Davis, Mark M

    2015-03-31

    CD4 molecules on the surface of T lymphocytes greatly augment the sensitivity and activation process of these cells, but how it functions is not fully understood. Here we studied the spatial organization of CD4, and its relationship to T-cell antigen receptor (TCR) and the active form of Src kinase p56lck (Lck) using single and dual-color photoactivated localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM). In nonactivated T cells, CD4 molecules are clustered in small protein islands, as are TCR and Lck. By dual-color imaging, we find that CD4, TCR, and Lck are localized in their separate clusters with limited interactions in the interfaces between them. Upon T-cell activation, the TCR and CD4 begin clustering together, developing into microclusters, and undergo a larger scale redistribution to form supramolecluar activation clusters (SMACs). CD4 and Lck localize in the inner TCR region of the SMAC, but this redistribution of disparate cluster structures results in enhanced segregation from each other. In nonactivated cells these preclustered structures and the limited interactions between them may serve to limit spontaneous and random activation events. However, the small sizes of these island structures also ensure large interfacial surfaces for potential interactions and signal amplification when activation is initiated. In the later activation stages, the increasingly larger clusters and their segregation from each other reduce the interfacial surfaces and could have a dampening effect. These highly differentiated spatial distributions of TCR, CD4, and Lck and their changes during activation suggest that there is a more complex hierarchy than previously thought. PMID:25829544

  6. 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

  7. Protein kinase Cε-calcineurin cosignaling downstream of toll-like receptor 4 downregulates fibrosis and induces wound healing gene expression in cardiac myofibroblasts.

    PubMed

    Mesquita, Rui F D S; Paul, Margaret A; Valmaseda, Aida; Francois, Asvi; Jabr, Rita; Anjum, Shahzia; Marber, Michael S; Budhram-Mahadeo, Vishwanie; Heads, Richard J

    2014-02-01

    The pathways which regulate resolution of inflammation and contribute to positive remodeling of the myocardium following injury are poorly understood. Here we show that protein kinase C epsilon (PKCε) cooperates with the phosphatase calcineurin (CN) to potentiate induction of cardioprotective gene expression while suppressing expression of fibrosis markers. This was achieved by detailed analysis of the regulation of cyclooxygenase 2 (COX-2) expression as a marker gene and by using gene expression profiling to identify genes regulated by coexpression of CN-Aα/PKCε in adult rat cardiac myofibroblasts (ARVFs) on a larger scale. GeneChip analysis of CN-Aα/PKCε-coexpressing ARVFs showed that COX-2 provides a signature for wound healing and is associated with downregulation of fibrosis markers, including connective tissue growth factor (CTGF), fibronectin, and collagens Col1a1, Col3a1, Col6a3, Col11a1, Col12a1, and Col14a1, with concomitant upregulation of cardioprotection markers, including COX-2 itself, lipocalin 2 (LCN2), tissue inhibitor of metalloproteinase 1 (TIMP-1), interleukin-6 (IL-6), and inducible nitric oxide synthase (iNOS). In primary rat cardiomyocyte cultures Toll-like receptor 4 (TLR4) agonist- or PKCε/CN-dependent COX-2 induction occurred in coresident fibroblasts and was blocked by selective inhibition of CN or PKC α/ε or elimination of fibroblasts. Furthermore, ectopic expression of PKCε and CN in ARVFs showed that the effects on COX-2 expression are mediated by specific NFAT sites within the COX-2 promoter as confirmed by site-directed mutagenesis and chromatin immunoprecipitation (ChIP). Therefore, PKCε may negatively regulate adverse myocardial remodeling by cooperating with CN to downregulate fibrosis and induce transcription of cardioprotective wound healing genes, including COX-2. PMID:24298017

  8. 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. PMID:27108949

  9. Zipper-interacting protein kinase is involved in regulation of ubiquitination of the androgen receptor, thereby contributing to dynamic transcription complex assembly.

    PubMed

    Felten, A; Brinckmann, D; Landsberg, G; Scheidtmann, K H

    2013-10-10

    We have recently identified apoptosis-antagonizing transcription factor (AATF), tumor-susceptibility gene 101 (TSG101) and zipper-interacting protein kinase (ZIPK) as novel coactivators of the androgen receptor (AR). The mechanisms of coactivation remained obscure, however. Here we investigated the interplay and interdependence between these coactivators and the AR using the endogenous prostate specific antigen (PSA) gene as model for AR-target genes. Chromatin immunoprecipitation in combination with siRNA-mediated knockdown revealed that recruitment of AATF and ZIPK to the PSA enhancer was dependent on AR, whereas recruitment of TSG101 was dependent on AATF. Association of AR and its coactivators with the PSA enhancer or promoter occurred in cycles. Dissociation of AR-transcription complexes was due to degradation because inhibition of the proteasome system by MG132 caused accumulation of AR at enhancer/promoter elements. Moreover, inhibition of degradation strongly reduced transcription, indicating that continued and efficient transcription is based on initiation, degradation and reinitiation cycles. Interestingly, knockdown of ZIPK by siRNA had a similar effect as MG132, leading to reduced transcription but enhanced accumulation of AR at androgen-response elements. In addition, knockdown of ZIPK, as well as overexpression of a dominant-negative ZIPK mutant, diminished polyubiquitination of AR. Furthermore, ZIPK cooperated with the E3 ligase Mdm2 in AR-dependent transactivation, assembled into a single complex on chromatin and phosphorylated Mdm2 in vitro. These results suggest that ZIPK has a crucial role in regulation of ubiquitination and degradation of the AR, and hence promoter clearance and efficient transcription.

  10. Mapping of the receptor protein-tyrosine kinase 10 to human chromosome 1q21-q23 and mouse chromosome 1H1-5 by fluorescence in situ hybridization

    SciTech Connect

    Edelhoff, S.; Disteche, C.M.; Lai, C.

    1995-01-01

    Receptor protein-tyrosine kinases (PTKs) play a critical role in the transduction of signals important to cell growth, differentiation, and survival. Mutations affecting the expression of receptor PTK genes have been associated with a number of vertebrate and invertebrate developmental abnormalities, and the aberrant regulation of tyrosine phosphorylation is implicated in a variety of neoplasias. One estimate suggests that approximately 100 receptor PTK genes exist in the mammalian genome, about half of which have been identified. The tyro-10 receptor protein-tyrosine kinase, first identified in a PCR-based survey for novel tyrosine kinases in the rat nervous system, defines a new subfamily of PTKs. It exhibits a catalytic domain most closely related to those found in the trk PTK receptor subfamily, which transduces signals for nerve growth factor and the related molecules brain-derived neurotrophic factor (BDNF), neurotrophin-3, and neurotrophin-4 (NT-3 and NT-4). Trk and the related PTK receptors trkB and trkC play a critical role in the neurotrophin-dependent survival of subsets of sensory and motor neurons. The predicted tyro-10 extracellular region is, however, distinct from that of the trk subfamily and is unique except for a domain shared with the blood coagulation factors V and VIII, thought to be involved in phospholipid binding. Although tyro-10 RNA is most abundant in heart and skeletal muscle in the adult rat, it is expressed in a wide variety of tissues, including the developing and mature brain. Tyro-10 appears identical to the murine TKT sequence reported by Karn et al. and exhibits a high degree of similarity with the CaK, DDR, and Nep PTKs. A ligand for tyro-10 has not yet been identified. 10 refs., 1 fig.

  11. 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.

  12. The role of protein kinase C in transmembrane signaling by the T cell antigen receptor complex. Effects of stimulation with soluble or immobilized CD3 antibodies.

    PubMed

    Manger, B; Weiss, A; Imboden, J; Laing, T; Stobo, J D

    1987-10-15

    Phorbol esters, such as phorbol myristate acetate (PMA), are known to be potent co-stimulants with calcium ionophores for activation of T lymphocytes. The most extensively studied intracellular effect of PMA is its ability to activate the cytoplasmic enzyme protein kinase C (pkC). Herein, we examined the role of pkC activation during T cell activation. During physiologic activation, this enzyme is activated by diacylglycerol which is generated through the hydrolysis of polyphosphoinositides. Therefore, we studied the activation of T lymphocytes induced by a synthetic diacylglycerol, dioctanoylglycerol. In contrast to PMA, this compound can be metabolized in T cells and presumably more closely mimics physiologic activation of pkC. Dioctanoylglycerol together with reagents that induce increases in intracellular free Ca2+ concentration, Ca2+ ionophores, or anti-cluster designation (CD)3 monoclonal antibodies (mAb) were able to induce interleukin 2 receptor expression and proliferation of T lymphocytes. Previous studies have demonstrated that the stimulation of T cells via the CD3/T cell antigen receptor complex by mAb against CD3 leads to an increase in cytoplasmic free Ca2+ and to an activation of pkC. Paradoxically, however, soluble CD3 antibodies do not cause proliferation of resting purified T cells. Inasmuch as immobilization of CD3 mAb has been shown to influence the agonist properties of such antibodies, we compared the ability of soluble and immobilized CD3 mAb to activate pkC. We demonstrated herein that soluble CD3 mAb cause only a very transient activation of pkC in the T cell leukemic line Jurkat. This pkC activation is markedly prolonged when Jurkat cells are stimulated with immobilized rather than soluble CD3 antibodies. These studies suggest that activation of pkC plays a major role in T cell activation and that the activation of pkC is influenced by the form in which CD3 mAb is presented to T cells.

  13. Characterization of protein kinases from Blepharisma intermedium.

    PubMed

    Beyer, J

    1975-12-01

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

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

    DOEpatents

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

    2001-07-03

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

  15. Recent inventions on receptor tyrosine kinase RET modulation.

    PubMed

    Jurvansuu, Jaana M; Goldman, Adrian

    2008-01-01

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

  16. Phosphoinositide 3-kinase mediated signaling in lobster olfactory receptor neurons.

    PubMed

    Corey, Elizabeth A; Bobkov, Yuriy; Pezier, Adeline; Ache, Barry W

    2010-04-01

    In vertebrates and some invertebrates, odorant molecules bind to G protein-coupled receptors on olfactory receptor neurons (ORNs) to initiate signal transduction. Phosphoinositide 3-kinase (PI3K) activity has been implicated physiologically in olfactory signal transduction, suggesting a potential role for a G protein-coupled receptor-activated class I PI3K. Using isoform-specific antibodies, we identified a protein in the olfactory signal transduction compartment of lobster ORNs that is antigenically similar to mammalian PI3Kgamma and cloned a gene for a PI3K with amino acid homology with PI3Kbeta. The lobster olfactory PI3K co-immunoprecipitates with the G protein alpha and beta subunits, and an odorant-evoked increase in phosphatidylinositol (3,4,5)-trisphosphate can be detected in the signal transduction compartment of the ORNs. PI3Kgamma and beta isoform-specific inhibitors reduce the odorant-evoked output of lobster ORNs in vivo. Collectively, these findings provide evidence that PI3K is indeed activated by odorant receptors in lobster ORNs and further support the potential involvement of G protein activated PI3K signaling in olfactory transduction.

  17. Antibodies directed against receptor tyrosine kinases

    PubMed Central

    FAUVEL, Bénédicte; Yasri, Aziz

    2014-01-01

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

  18. 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.

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

    PubMed

    Lai, Shenshen; Safaei, Javad; Pelech, Steven

    2016-03-01

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

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

    PubMed

    Fehér, Attila; Lajkó, Dézi Bianka

    2015-08-01

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

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

    PubMed

    Fehér, Attila; Lajkó, Dézi Bianka

    2015-08-01

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

  2. Activation of Na+/H+ exchanger NHE3 by angiotensin II is mediated by inositol 1,4,5-triphosphate (IP3) receptor-binding protein released with IP3 (IRBIT) and Ca2+/calmodulin-dependent protein kinase II.

    PubMed

    He, Peijian; Klein, Janet; Yun, C Chris

    2010-09-01

    Angiotensin II (ANG II) stimulates renal tubular reabsorption of NaCl by targeting Na(+)/H(+) exchanger NHE3. We have shown previously that inositol 1,4,5-triphosphate receptor-binding protein released with inositol 1,4,5-triphosphate (IRBIT) plays a critical role in stimulation of NHE3 in response to elevated intracellular Ca(2+) concentration ([Ca(2+)](i)). In this study, we investigated the role of IRBIT in mediating NHE3 activation by ANG II. IRBIT is abundantly expressed in the proximal tubules where NHE3 is located. ANG II at physiological concentrations stimulates NHE3 transport activity in a model proximal tubule cell line. ANG II-induced activation of NHE3 was abrogated by knockdown of IRBIT, whereas overexpression of IRBIT enhanced the effect of ANG II on NHE3. ANG II transiently increased binding of IRBIT to NHE3 at 5 min but became dissociated by 45 min. In comparison, it took at least 15 min of ANG II treatment for an increase in NHE3 activity and NHE3 surface expression. The stimulation of NHE3 by ANG II was dependent on changes in [Ca(2+)](i) and Ca(2+)/calmodulin-dependent protein kinases II. Inhibition of CaMKII completely blocked the ANG II-induced binding of IRBIT to NHE3 and the increase in NHE3 surface abundance. Several serine residues of IRBIT are thought to be important for IRBIT binding. Mutations of Ser-68, Ser-71, and Ser-74 of IRBIT decreased binding of IRBIT to NHE3 and its effect on NHE3 activity. In conclusion, our current findings demonstrate that IRBIT is critically involved in mediating activation of NHE3 by ANG II via a Ca(2+)/calmodulin-dependent protein kinases II-dependent pathway.

  3. Genome-wide analysis of lectin receptor-like kinases in Populus

    DOE PAGES

    Yang, Yongil; Labbé, Jessy; Muchero, Wellington; Yang, Xiaohan; Jawdy, Sara S.; Kennedy, Megan; Johnson, Jenifer; Sreedasyam, Avinash; Schmutz, Jeremy; Tuskan, Gerald A.; et al

    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.

  4. 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

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

    SciTech Connect

    S Eathiraj; R Palma; M Hirschi; E Volckova; E Nakuci; J Castro; C Chen; T Chan; D France; M Ashwell

    2011-12-31

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

  6. Trans-Cinnamic Acid Increases Adiponectin and the Phosphorylation of AMP-Activated Protein Kinase through G-Protein-Coupled Receptor Signaling in 3T3-L1 Adipocytes

    PubMed Central

    Kopp, Christina; Singh, Shiva P.; Regenhard, Petra; Müller, Ute; Sauerwein, Helga; Mielenz, Manfred

    2014-01-01

    Adiponectin and intracellular 5′adenosine monophosphate-activated protein kinase (AMPK) are important modulators of glucose and fat metabolism. Cinnamon exerts beneficial effects by improving insulin sensitivity and blood lipids, e.g., through increasing adiponectin concentrations and AMPK activation. The underlying mechanism is unknown. The Gi/Go-protein-coupled receptor (GPR) 109A stimulates adiponectin secretion after binding its ligand niacin. Trans-cinnamic acid (tCA), a compound of cinnamon is another ligand. We hypothesize whether AMPK activation and adiponectin secretion by tCA is transmitted by GPR signaling. Differentiated 3T3-L1 cells were incubated with pertussis toxin (PTX), an inhibitor of Gi/Go-protein-coupling, and treated with different tCA concentrations. Treatment with tCA increased adiponectin and the pAMPK/AMPK ratio (p ≤ 0.001). PTX incubation abolished the increased pAMPK/AMPK ratio and adiponectin secretion. The latter remained increased compared to controls (p ≤ 0.002). tCA treatment stimulated adiponectin secretion and AMPK activation; the inhibitory effect of PTX suggests GPR is involved in tCA stimulated signaling. PMID:24557583

  7. Protein kinase profiling assays: a technology review.

    PubMed

    Wang, Yuren; Ma, Haiching

    2015-11-01

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

  8. 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. PMID:24978055

  9. Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-releasing Hormone*

    PubMed Central

    Higa-Nakamine, Sayomi; Maeda, Noriko; Toku, Seikichi; Yamamoto, Hideyuki

    2015-01-01

    The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1–7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of Gq/11 proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Gö 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer. PMID:26338704

  10. Phosphorylation of Protein Phosphatase Inhibitor-1 by Protein Kinase C*s

    PubMed Central

    Sahin, Bogachan; Shu, Hongjun; Fernandez, Joseph; El-Armouche, Ali; Molkentin, Jeffery D.; Nairn, Angus C.; Bibb, James A.

    2015-01-01

    Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr35. Moreover, Ser67 of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser67 inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser65 in vitro. In contrast, Ser67 phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser65. Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser65 and Ser67, but not Ser65 alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser65 inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser67 protects phospho-Ser65 inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser65/Ser67 inhibitor-1 in this tissue. In contrast, the activation of N-methyl-D-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser65/Ser67 inhibitor-1 levels. Phosphomimetic mutation of Ser65 and/or Ser67 did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser65/Ser67 inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser67 and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation. PMID:16772299

  11. Nicotinic α7 receptor inhibits the acylation stimulating protein-induced production of monocyte chemoattractant protein-1 and keratinocyte-derived chemokine in adipocytes by modulating the p38 kinase and nuclear factor-κB signaling pathways

    PubMed Central

    Jiao, Zhou-Yang; Wu, Jing; Liu, Chao; Wen, Bing; Zhao, Wen-Zeng; Du, Xin-Ling

    2016-01-01

    Obesity is associated with chronic low-grade inflammation, which is characterized by increased infiltration of macrophages into adipose tissue. Acylation stimulating protein (ASP) is an adipokine derived from the immune complement system, which constitutes a link between adipocytes and macrophages, and is involved in energy homeostasis and inflammation. The purpose of the present study was to preliminarily investigate in vitro, whether functional α7nAChR in adipocytes may suppress ASP-induced inflammation and determine the possible signaling mechanism. Studies have reported associations between the expression of α7 nicotinic acetylcholine receptor (α7nAChR) and obesity, insulin resistance and diabetes. Additionally, α7nAChRs are important peripheral mediators of chronic inflammation, which is a key contributor to health problems in obesity. The primary aim of the present study was to evaluate the impact of exogenous ASP and α7nAChR on macrophage infiltration in adipose tissue and to examine the potential underlying molecular mechanism. Western blot analysis revealed that recombinant ASP increased the expression levels of monocyte chemoattractant protein-1 (MCP-1) and keratinocyte-derived chemokine (KC) by 3T3-L1 adipocytes. However, nicotine significantly inhibited the production of ASP-induced cytokines via the stimulation of α7nAChR. It was also found that α7nAChR inhibited the ASP-induced activation of p38 kinase and nuclear factor-κB (NF-κB), and the production of MCP-1 and KC. These data indicated that α7nAChR caused the inhibition of ASP-induced activation of p38 kinase and NF-κB to inhibit the production of MCP-1 and KC. PMID:27572255

  12. Complexity of Receptor Tyrosine Kinase Signal Processing

    PubMed Central

    Volinsky, Natalia; Kholodenko, Boris N.

    2013-01-01

    Our knowledge of molecular mechanisms of receptor tyrosine kinase (RTK) signaling advances with ever-increasing pace. Yet our understanding of how the spatiotemporal dynamics of RTK signaling control specific cellular outcomes has lagged behind. Systems-centered experimental and computational approaches can help reveal how overlapping networks of signal transducers downstream of RTKs orchestrate specific cell-fate decisions. We discuss how RTK network regulatory structures, which involve the immediate posttranslational and delayed transcriptional controls by multiple feed forward and feedback loops together with pathway cross talk, adapt cells to the combinatorial variety of external cues and conditions. This intricate network circuitry endows cells with emerging capabilities for RTK signal processing and decoding. We illustrate how mathematical modeling facilitates our understanding of RTK network behaviors by unraveling specific systems properties, including bistability, oscillations, excitable responses, and generation of intricate landscapes of signaling activities. PMID:23906711

  13. 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

  14. 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...

  15. A threading approach to protein structure prediction: Studies on TNF-like molecules, Rev proteins, and protein kinases

    NASA Astrophysics Data System (ADS)

    Ihm, Yungok

    The main focus of this dissertation is the application of the threading approach to specific biological problems. The threading scheme developed in our group targets incorporating important structural features necessary for detecting structural similarity between the target sequence and the template structure. This enables us to use our threading method to solve problems for which sequence-based methods are not very much useful. We applied our threading method to predict the three-dimensional structures of lentivirus (EIAV, HIV-1, FIV, SIV) Rev proteins. Predicted structures of Rev proteins suggest that they share a structural similarity among themselves (four-helix bundle). Also, the threading approach has been utilized for screening for potential TNF-like molecules in Arabidopsis. The threading approach identified 35 potential TNF-like proteins in Arabidopsis, six of which are particularly interesting to be tested for the receptor kinase ligand activity. Threading method has also been used to identify potentially new protein kinases, which are not included in the protein kinase data base of C. elegans and Arabidopis. We identified eleven potentially new protein kinases and an additional protein worth investigating for protein kinase activity in C. elegans. Further, we identified ten potentially new protein kinases and additional four proteins worth investigating for the protein kinase activity in Arabidopsis.

  16. The histamine H3 receptor antagonist clobenpropit enhances GABA release to protect against NMDA-induced excitotoxicity through the cAMP/protein kinase A pathway in cultured cortical neurons.

    PubMed

    Dai, Haibin; Fu, Qiuli; Shen, Yao; Hu, Weiwei; Zhang, Zhongmiao; Timmerman, Henk; Leurs, Rob; Chen, Zhong

    2007-06-01

    Using the histamine H3 receptor antagonist clobenpropit, the roles of histamine H3 receptors in NMDA-induced necrosis were investigated in rat cultured cortical neurons. Clobenpropit reversed the neurotoxicity in a concentration-dependent manner, and showed peak protection at a concentration of 10(-7) M. This protection was antagonized by the histamine H3 receptor agonist (R)-alpha-methylhistamine, but not by the histamine H1 receptor antagonist pyrilamine or the histamine H2 receptor antagonist cimetidine. In addition, the protection by clobenpropit was inhibited by the GABAA receptor antagonists picrotoxin and bicuculline. Further study demonstrated that the protection by clobenpropit was due to increased GABA release. The inducible GABA release was also inhibited by (R)-alpha-methylhistamine, but not by pyrilamine or cimetidine. Furthermore, both the adenylyl cyclase inhibitor SQ-22536 and the protein kinase A (PKA) inhibitor H-89 reversed the protection and the GABA release by clobenpropit. In addition, clobenpropit reversed the NMDA-induced increase in intracellular calcium level, which was antagonized by (R)-alpha-methylhistamine. These results indicate that clobenpropit enhanced GABA release to protect against NMDA-induced excitotoxicity, which was induced through the cAMP/PKA pathway, and reduction of intracellular calcium level may also be involved.

  17. Endogenous protein phosphorylation and protein kinase activity in winged bean.

    PubMed

    Mukhopadhyay, K; Singh, M

    1997-10-01

    In winged bean (Psophocarpus tetragonolobus) protein kinases (E.C. 2.7.1.37) were found in all tissues studied. There was a significant increase in kinase activity during seed development, with a concomitant enhancement in the phosphorylation of a number of polypeptides; this was reversed in germinating seed cotyledons. Protein phosphorylation was apparently correlated with the increase in the protein content of the developing seed and the growing axis. At least three distinct autophosphorylating proteins could be distinguished in the developing seeds after SDS-PAGE, indicating the presence of different types of protein kinases in winged bean.

  18. 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.

  19. Mechanisms of Activation of Receptor Tyrosine Kinases: Monomers or Dimers

    PubMed Central

    Maruyama, Ichiro N.

    2014-01-01

    Receptor tyrosine kinases (RTKs) play essential roles in cellular processes, including metabolism, cell-cycle control, survival, proliferation, motility and differentiation. RTKs are all synthesized as single-pass transmembrane proteins and bind polypeptide ligands, mainly growth factors. It has long been thought that all RTKs, except for the insulin receptor (IR) family, are activated by ligand-induced dimerization of the receptors. An increasing number of diverse studies, however, indicate that RTKs, previously thought to exist as monomers, are present as pre-formed, yet inactive, dimers prior to ligand binding. The non-covalently associated dimeric structures are reminiscent of those of the IR family, which has a disulfide-linked dimeric structure. Furthermore, recent progress in structural studies has provided insight into the underpinnings of conformational changes during the activation of RTKs. In this review, I discuss two mutually exclusive models for the mechanisms of activation of the epidermal growth factor receptor, the neurotrophin receptor and IR families, based on these new insights. PMID:24758840

  20. Crystallization and preliminary X-ray analysis of ginkbilobin-2 from Ginkgo biloba seeds: a novel antifungal protein with homology to the extracellular domain of plant cysteine-rich receptor-like kinases

    PubMed Central

    Miyakawa, Takuya; Sawano, Yoriko; Miyazono, Ken-ichi; Hatano, Ken-ichi; Tanokura, Masaru

    2007-01-01

    The antifungal protein ginkbilobin-2 (Gnk2) from Ginkgo biloba seeds does not show homology to other pathogenesis-related proteins, but does show homology to the extracellular domain of plant cysteine-rich receptor-like kinases. Native Gnk2 purified from ginkgo nuts and the selenomethionine derivative of recombinant Gnk2 (SeMet-rGnk2) were crystallized by the sitting-drop vapour-diffusion method using different precipitants. X-ray diffraction data were collected from Gnk2 at 2.38 Å resolution and from SeMet-rGnk2 at 2.79 Å resolution using a synchrotron-radiation source. The crystals of both proteins belonged to the primitive cubic space group P213, with unit-cell parameters a = b = c = 143.2 Å. PMID:17768341

  1. Protein kinase activators alter glial cholesterol esterification

    SciTech Connect

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

    1986-05-01

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

  2. Arabidopsis Receptor of Activated C Kinase1 Phosphorylation by WITH NO LYSINE8 KINASE

    DOE PAGES

    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 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

  3. 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.

  4. Energy-sensing Factors Coactivator Peroxisome Proliferator-activated Receptor γ Coactivator 1-α (PGC-1α) and AMP-activated Protein Kinase Control Expression of Inflammatory Mediators in Liver

    PubMed Central

    Buler, Marcin; Aatsinki, Sanna-Mari; Skoumal, Réka; Komka, Zsolt; Tóth, Miklós; Kerkelä, Risto; Georgiadi, Anastasia; Kersten, Sander; Hakkola, Jukka

    2012-01-01

    Obesity and insulin resistance are associated with chronic, low grade inflammation. Moreover, regulation of energy metabolism and immunity are highly integrated. We hypothesized that energy-sensitive coactivator peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α) and AMP-activated protein kinase (AMPK) may modulate inflammatory gene expression in liver. Microarray analysis revealed that PGC-1α up-regulated expression of several cytokines and cytokine receptors, including interleukin 15 receptor α (IL15Rα) and, even more importantly, anti-inflammatory interleukin 1 receptor antagonist (IL1Rn). Overexpression of PGC-1α and induction of PGC-1α by fasting, physical exercise, glucagon, or cAMP was associated with increased IL1Rn mRNA and protein expression in hepatocytes. Knockdown of PGC-1α by siRNA down-regulated cAMP-induced expression of IL1Rn in mouse hepatocytes. Furthermore, knockdown of peroxisome proliferator-activated receptor α (PPARα) attenuated IL1Rn induction by PGC-1α. Overexpression of PGC-1α, at least partially through IL1Rn, suppressed interleukin 1β-induced expression of acute phase proteins, C-reactive protein, and haptoglobin. Fasting and exercise also induced IL15Rα expression, whereas glucagon and cAMP resulted in reduction in IL15Rα mRNA levels. Finally, AMPK activator metformin and adenoviral overexpression of AMPK up-regulated IL1Rn and down-regulated IL15Rα in primary hepatocytes. We conclude that PGC-1α and AMPK alter inflammatory gene expression in liver and thus integrate energy homeostasis and inflammation. Induction of IL1Rn by PGC-1α and AMPK may be involved in the beneficial effects of exercise and caloric restriction and putative anti-inflammatory effects of metformin. PMID:22117073

  5. Protein Connectivity in Chemotaxis Receptor Complexes

    PubMed Central

    Eismann, Stephan; Endres, Robert G.

    2015-01-01

    The chemotaxis sensory system allows bacteria such as Escherichia coli to swim towards nutrients and away from repellents. The underlying pathway is remarkably sensitive in detecting chemical gradients over a wide range of ambient concentrations. Interactions among receptors, which are predominantly clustered at the cell poles, are crucial to this sensitivity. Although it has been suggested that the kinase CheA and the adapter protein CheW are integral for receptor connectivity, the exact coupling mechanism remains unclear. Here, we present a statistical-mechanics approach to model the receptor linkage mechanism itself, building on nanodisc and electron cryotomography experiments. Specifically, we investigate how the sensing behavior of mixed receptor clusters is affected by variations in the expression levels of CheA and CheW at a constant receptor density in the membrane. Our model compares favorably with dose-response curves from in vivo Förster resonance energy transfer (FRET) measurements, demonstrating that the receptor-methylation level has only minor effects on receptor cooperativity. Importantly, our model provides an explanation for the non-intuitive conclusion that the receptor cooperativity decreases with increasing levels of CheA, a core signaling protein associated with the receptors, whereas the receptor cooperativity increases with increasing levels of CheW, a key adapter protein. Finally, we propose an evolutionary advantage as explanation for the recently suggested CheW-only linker structures. PMID:26646441

  6. 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.

  7. 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

  8. Differential protein expression and oncogenic gene network link tyrosine kinase ephrin B4 receptor to aggressive gastric and gastroesophageal junction cancers.

    PubMed

    Liersch-Löhn, Britta; Slavova, Nadia; Buhr, Heinz J; Bennani-Baiti, Idriss M

    2016-03-01

    Transmembrane tyrosine-kinase Ephrin receptors promote tumor progression and/or metastasis of several malignancies including leukemia, follicular lymphoma, glioma, malignant pleural mesothelioma, papillary thyroid carcinoma, sarcomas and ovarian, breast, bladder and non-small cell lung cancers. They also drive intestinal stem cell proliferation and positioning, control intestinal tissue boundaries and are involved in liver, pancreatic and colorectal cancers, indicating involvement in additional digestive system malignancies. We investigated the role of Ephrin-B4 receptor (EPHB4), and its ligand EFNB2, in gastric and gastroesophageal junction cancers in patient cohorts through computational, mathematical, molecular and immunohistochemical analyses. We show that EPHB4 is upregulated in preneoplastic gastroesophageal lesions and its expression further increased in gastroesophageal cancers in several independent cohorts. The closely related EPHB6 receptor, which also binds EFNB2, was downregulated in all tested cohorts, consistent with its tumor-suppressive properties in other cancers. EFNB2 expression is induced in esophageal cells by acidity, suggesting that gastroesophageal reflux disease (GERD) may constitute an early triggering event in activating EFNB2-EPHB4 signaling. Association of EPHB4 to both Barrett's esophagus and to advanced tumor stages, and its overexpression at the tumor invasion front and vascular endothelial cells intimate the notion that EPHB4 may be associated with multiple steps of gastroesophageal tumorigenesis. Analysis of oncogenomic signatures uncovered the first EPHB4-associated gene network (false discovery rate: 7 × 10(-90) ) composed of a five-transcription factor interconnected gene network that drives proliferation, angiogenesis and invasiveness. The EPHB4 oncogenomic network provides a molecular basis for its role in tumor progression and points to EPHB4 as a potential tumor aggressiveness biomarker and drug target in gastroesophageal

  9. 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

  10. Non-degradative Ubiquitination of Protein Kinases.

    PubMed

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

    2016-06-01

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

  11. Physical and functional association of the cbl protooncogen product with an src-family protein tyrosine kinase, p53/56lyn, in the B cell antigen receptor-mediated signaling

    PubMed Central

    1996-01-01

    To identify novel signal transducers involved in signaling mediated by the Src-family protein tyrosine kinases (PTKs), we used a yeast two- hybrid system with a probe corresponding to the regulatory region of p56lyn, a member of Src-family PTKs. One of the isolated clones contained the COOH-terminal 470 amino acid residues of p120c-cbl, the product of the cellular homologue of the v-cbl retroviral oncogene. p120c-cbl is a cytoplasmic protein with nuclear protein-like motifs. Here we show in vivo association of p120c-cbl with p53/56lyn. After stimulation of the B cell antigen receptor (BCR), p120c-cbl was rapidly tyrosine phosphorylated. Studies with lyn- or syk-negative chicken B cells demonstrated that p53/56lyn, but not p72syk, was crucial for tyrosine phosphorylation of p120c-cbl upon stimulation of the BCR. We also show the importance of p59fyn in tyrosine phosphorylation of p120c- cbl in the T-cell receptor-mediated signaling using fyn-overexpressing T cell hybridomas and splenic T cells from fyn-deficient mice. These results suggest that p120c-cbl is an important substrate of Src-family PTKs in the intracellular signaling mediated by the antigen receptors PMID:8627181

  12. Smad2 transduces common signals from receptor serine-threonine and tyrosine kinases.

    PubMed

    de Caestecker, M P; Parks, W T; Frank, C J; Castagnino, P; Bottaro, D P; Roberts, A B; Lechleider, R J

    1998-06-01

    SMAD proteins mediate signals from receptor serine-threonine kinases (RSKs) of the TGF-beta superfamily. We demonstrate here that HGF and EGF, which signal through RTKs, can also mediate SMAD-dependent reporter gene activation and induce rapid phosphorylation of endogenous SMAD proteins by kinase(s) downstream of MEK1. HGF induces phosphorylation and nuclear translocation of epitope-tagged Smad2 and a mutation that blocks TGF-beta signaling also blocks HGF signal transduction. Smad2 may thus act as a common positive effector of TGF-beta- and HGF-induced signals and serve to modulate cross talk between RTK and RSK signaling pathways.

  13. Phosphorylation in vitro of the 85 kDa subunit of phosphatidylinositol 3-kinase and its possible activation by insulin receptor tyrosine kinase.

    PubMed Central

    Hayashi, H; Miyake, N; Kanai, F; Shibasaki, F; Takenawa, T; Ebina, Y

    1991-01-01

    Insulin causes a dramatic and rapid increase in phosphatidylinositol 3-kinase activity in the anti-phosphotyrosine immunoprecipitates of cells overexpressing the human insulin receptor. This enzyme may therefore be a mediator of insulin signal transduction [Endemann, Yonezawa & Roth (1990) J. Biol. Chem. 265, 396-400; Ruderman, Kapeller, White & Cantley (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1411-1415]. At least two questions remain to be elucidated. Firstly, does the insulin receptor tyrosine kinase phosphorylate phosphatidylinositol 3-kinase directly, or does it phosphorylate a protein associated with the 3-kinase? Second, if the enzyme is a direct substrate for the insulin receptor tyrosine kinase, does tyrosine phosphorylation of phosphatidylinositol 3-kinase by the kinase alter the specific enzyme activity, or does the amount of the tyrosine-phosphorylated form of the phosphatidylinositol 3-kinase increase, with no change in the specific activity? We report here evidence that the 85 kDa subunit of highly purified phosphatidylinositol 3-kinase is phosphorylated on the tyrosine residue by the activated normal insulin receptor in vitro, but not by a mutant insulin receptor which lacks tyrosine kinase activity. We found that an increase in enzyme activity was detected in response to insulin not only in the anti-phosphotyrosine immunoprecipitates of the cytosol, but also in the cytosolic fraction before immunoprecipitation. In addition, we partially separated the tyrosine-phosphorylated form from the unphosphorylated form of the enzyme, by using a f.p.l.c. Mono Q column. The insulin-stimulated phosphatidylinositol 3-kinase activity was mainly detected in the fraction containing almost all of the tyrosine-phosphorylated form. This result suggests that tyrosine phosphorylation of phosphatidylinositol 3-kinase by the insulin receptor kinase may increase the specific activity of the former enzyme in vivo. Images Fig. 1. Fig. 2. Fig. 4. PMID:1722393

  14. 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

  15. 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.

  16. Novel receptor-like kinases in cacao contain PR-1 extracellular domains.

    PubMed

    Teixeira, Paulo José Pereira Lima; Costa, Gustavo Gilson Lacerda; Fiorin, Gabriel Lorencini; Pereira, Gonçalo Amarante Guimarães; Mondego, Jorge Maurício Costa

    2013-08-01

    Members of the pathogenesis-related protein 1 (PR-1) family are well-known markers of plant defence responses, forming part of the arsenal of the secreted proteins produced on pathogen recognition. Here, we report the identification of two cacao (Theobroma cacao L.) PR-1s that are fused to transmembrane regions and serine/threonine kinase domains, in a manner characteristic of receptor-like kinases (RLKs). These proteins (TcPR-1f and TcPR-1g) were named PR-1 receptor kinases (PR-1RKs). Phylogenetic analysis of RLKs and PR-1 proteins from cacao indicated that PR-1RKs originated from a fusion between sequences encoding PR-1 and the kinase domain of a LecRLK (Lectin Receptor-Like Kinase). Retrotransposition marks surround TcPR-1f, suggesting that retrotransposition was involved in the origin of PR-1RKs. Genes with a similar domain architecture to cacao PR-1RKs were found in rice (Oryza sativa), barrel medic (Medicago truncatula) and a nonphototrophic bacterium (Herpetosiphon aurantiacus). However, their kinase domains differed from those found in LecRLKs, indicating the occurrence of convergent evolution. TcPR-1g expression was up-regulated in the biotrophic stage of witches' broom disease, suggesting a role for PR-1RKs during cacao defence responses. We hypothesize that PR-1RKs transduce a defence signal by interacting with a PR-1 ligand.

  17. Regulation of polar auxin transport by protein and lipid kinases

    PubMed Central

    Jaillais, Yvon

    2016-01-01

    The directional transport of auxin, known as polar auxin transport, allows asymmetric distribution of this hormone in different cells and tissues. This system creates local auxin maxima, minima and gradients that are instrumental in both organ initiation and shape determination. As such, polar auxin transport is crucial for all aspects of plant development but also for environmental interaction, notably in shaping plant architecture to its environment. Cell-to-cell auxin transport is mediated by a network of auxin carriers that are regulated at the transcriptional and post-translational levels. Here we review our current knowledge on some aspects of the ‘non-genomic’ regulation of auxin transport, putting an emphasis on how phosphorylation by protein and lipid kinases controls the polarity, intracellular trafficking, stability and activity of auxin carriers. We describe the role of several AGC kinases, including PINOID, D6PK and the blue light photoreceptor phot1, in phosphorylating auxin carriers from the PIN and ABCB families. We also highlight the function of some Receptor-Like Kinases (RLK) and two-component histidine kinase receptors in polar auxin transport, noticing that there are likely RLKs involved in coordinating auxin distribution yet to be discovered. In addition, we describe the emerging role of phospholipid phosphorylation in polarity establishment and intracellular trafficking of PIN proteins. We outline these various phosphorylation mechanisms in the context of primary and lateral root development, leaf cell shape acquisition as well as root gravitropism and shoot phototropism. PMID:27242371

  18. Regulation of polar auxin transport by protein and lipid kinases.

    PubMed

    Armengot, Laia; Marquès-Bueno, Maria Mar; Jaillais, Yvon

    2016-07-01

    The directional transport of auxin, known as polar auxin transport (PAT), allows asymmetric distribution of this hormone in different cells and tissues. This system creates local auxin maxima, minima, and gradients that are instrumental in both organ initiation and shape determination. As such, PAT is crucial for all aspects of plant development but also for environmental interaction, notably in shaping plant architecture to its environment. Cell to cell auxin transport is mediated by a network of auxin carriers that are regulated at the transcriptional and post-translational levels. Here we review our current knowledge on some aspects of the 'non-genomic' regulation of auxin transport, placing an emphasis on how phosphorylation by protein and lipid kinases controls the polarity, intracellular trafficking, stability, and activity of auxin carriers. We describe the role of several AGC kinases, including PINOID, D6PK, and the blue light photoreceptor phot1, in phosphorylating auxin carriers from the PIN and ABCB families. We also highlight the function of some receptor-like kinases (RLKs) and two-component histidine kinase receptors in PAT, noting that there are probably RLKs involved in co-ordinating auxin distribution yet to be discovered. In addition, we describe the emerging role of phospholipid phosphorylation in polarity establishment and intracellular trafficking of PIN proteins. We outline these various phosphorylation mechanisms in the context of primary and lateral root development, leaf cell shape acquisition, as well as root gravitropism and shoot phototropism. PMID:27242371

  19. Antenatal betamethasone produces protracted changes in anxiety-like behaviors and in the expression of microtubule-associated protein 2, brain-derived neurotrophic factor and the tyrosine kinase B receptor in the rat cerebellar cortex.

    PubMed

    Pascual, Rodrigo; Valencia, Martina; Bustamante, Carlos

    2015-06-01

    Using classic Golgi staining methods, we previously showed that the administration of synthetic glucocorticoid betamethasone in equivalent doses to those given in cases of human premature birth generates long-term alterations in Purkinje cell dendritic development in the cerebellar cortex. In the present study, we evaluated whether betamethasone alters the immunohistochemical expression of proteins that participate in cerebellar Purkinje cell dendritic development and maintenance, including microtubule-associated protein 2 (MAP2), brain-derived neurotrophic factor (BDNF) and the tyrosine kinase B receptor (TrkB), which are located predominantly in the cerebellar molecular layer where Purkinje cell dendritogenesis occurs. Consistent with our previous Golgi stain studies, we observed that animals prenatally exposed to a single course of betamethasone showed long-term alterations in the expression of MAP2, BDNF and TrkB. Additionally, these protracted molecular changes were accompanied by anxiety-like behaviors in the elevated plus maze and marble burying tests.

  20. Regulation of cholesterol esterification by protein kinase C

    SciTech Connect

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

    1986-03-05

    They have recently identified acyl-CoA cholesterol acyltransferase as the key enzyme for cholesterol esterification in the central nervous system. They found that the activity of glial acyl-CoA cholesterol acyltransferase could be controlled by a phosphorylation-dephosphorylation mechanism. However, repeated attempts to identify cyclic AMP as the bioregulator for this reaction failed. Recently, they have studied the possible involvement of protein kinase C in the regulation of glial cholesterol esterification. Phorbol-12-myristate 13-acetate (PMA) can activate cellular cholesterol esterification in a complex, time-dependent manner. Phorbol analogues inactive toward protein kinase C are also ineffective in this assay. Furthermore, oleoyl-acetyl-glycerol mimics the effect of PMA, confirming the proposal that protein kinase C mediates the effect of these compounds and that the natural bioregulator is probably diacylglycerol. Receptor-mediated polyphosphatidyl-inositol cleavage often produces diacylglycerol and inositol triphosphate. The synergic effects of these two compounds are known to be necessary to elicit other biological responses. Their preliminary studies using calcium ionophore A23187 indicates that Ca/sup + +/ is not required for cellular cholesterol esterification. In sum, glial cholesterol esterification is probably regulated by a calcium-independent and protein kinase C-dependent reaction.

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

    PubMed Central

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

    1993-01-01

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

  2. cGMP/Protein Kinase G Signaling Suppresses Inositol 1,4,5-Trisphosphate Receptor Phosphorylation and Promotes Endoplasmic Reticulum Stress in Photoreceptors of Cyclic Nucleotide-gated Channel-deficient Mice*

    PubMed Central

    Ma, Hongwei; Butler, Michael R.; Thapa, Arjun; Belcher, Josh; Yang, Fan; Baehr, Wolfgang; Biel, Martin; Michalakis, Stylianos; Ding, Xi-Qin

    2015-01-01

    Photoreceptor cyclic nucleotide-gated (CNG) channels play a pivotal role in phototransduction. Mutations in the cone CNG channel subunits CNGA3 and CNGB3 are associated with achromatopsia and cone dystrophies. We have shown endoplasmic reticulum (ER) stress-associated apoptotic cone death and increased phosphorylation of the ER Ca2+ channel inositol 1,4,5-trisphosphate receptor 1 (IP3R1) in CNG channel-deficient mice. We also presented a remarkable elevation of cGMP and an increased activity of the cGMP-dependent protein kinase (protein kinase G, PKG) in CNG channel deficiency. This work investigated whether cGMP/PKG signaling regulates ER stress and IP3R1 phosphorylation in CNG channel-deficient cones. Treatment with PKG inhibitor and deletion of guanylate cyclase-1 (GC1), the enzyme producing cGMP in cones, were used to suppress cGMP/PKG signaling in cone-dominant Cnga3−/−/Nrl−/− mice. We found that treatment with PKG inhibitor or deletion of GC1 effectively reduced apoptotic cone death, increased expression levels of cone proteins, and decreased activation of Müller glial cells. Furthermore, we observed significantly increased phosphorylation of IP3R1 and reduced ER stress. Our findings demonstrate a role of cGMP/PKG signaling in ER stress and ER Ca2+ channel regulation and provide insights into the mechanism of cone degeneration in CNG channel deficiency. PMID:26124274

  3. Compartmentalization role of A-kinase anchoring proteins (AKAPs) in mediating protein kinase A (PKA) signaling and cardiomyocyte hypertrophy.

    PubMed

    Rababa'h, Abeer; Singh, Sonal; Suryavanshi, Santosh V; Altarabsheh, Salah Eldien; Deo, Salil V; McConnell, Bradley K

    2014-12-24

    The Beta-adrenergic receptors (β-ARs) stimulation enhances contractility through protein kinase-A (PKA) substrate phosphorylation. This PKA signaling is conferred in part by PKA binding to A-kinase anchoring proteins (AKAPs). AKAPs coordinate multi-protein signaling networks that are targeted to specific intracellular locations, resulting in the localization of enzyme activity and transmitting intracellular actions of neurotransmitters and hormones to its target substrates. In particular, mAKAP (muscle-selective AKAP) has been shown to be present on the nuclear envelope of cardiomyocytes with various proteins including: PKA-regulatory subunit (RIIα), phosphodiesterase-4D3, protein phosphatase-2A, and ryanodine receptor (RyR2). Therefore, through the coordination of spatial-temporal signaling of proteins and enzymes, mAKAP controls cyclic-adenosine monophosphate (cAMP) levels very tightly and functions as a regulator of PKA-mediated substrate phosphorylation leading to changes in calcium availability and myofilament calcium sensitivity. The goal of this review is to elucidate the critical compartmentalization role of mAKAP in mediating PKA signaling and regulating cardiomyocyte hypertrophy by acting as a scaffolding protein. Based on our literature search and studying the structure-function relationship between AKAP scaffolding protein and its binding partners, we propose possible explanations for the mechanism by which mAKAP promotes cardiac hypertrophy.

  4. Effects of 4-nonylphenol isomers on cell receptors and mitogen-activated protein kinase pathway in mouse Sertoli TM4 cells.

    PubMed

    Liu, Xiaozhen; Nie, Shaoping; Chen, Yangjie; Huang, Danfei; Xie, Mingyong

    2014-12-01

    In the present study, experiments were performed to investigate the effects of nonylphenol (NP) isomers (4-[1,2, 4-trimethylhexyl]-phenol (NP41), 4-[1,2, 5-trimethylhexyl]-phenol (NP42)) on Sertoli TM4 cells. NP41 decreased mRNA expression levels of androgen receptor and toll-like receptor (TLR)-4 in 20-40μM (P<0.05), and increased mRNA levels of estrogen receptor (ER)-α and progesterone receptor in 1-40μM (P<0.05). NP42 treatment only evoked significant decrease in mRNA expression levels of ER-α in 20-40μM (P<0.05). Similarly, NP41 (1-40μM) drastically increased the protein expression of ER-α, which was significantly decreased in 20-40μM NP42 groups (P<0.01). Both NP41 and NP42 showed no effect on the expression of ER-β. Protein levels of follicle stimulating hormone receptor were increased significantly in high concentrations of NP41 (40μM) and NP42 (10-40μM) challenged cells. Furthermore, NP41 and NP42 showed various effects on the expression of junction-associated molecules and inhibin B secretion in TM4 cells. Additionally, activation of JNK1/2 pathway was induced by NP41 and NP42. However, ERK1/2 and p38 pathways were inhibited in TM4 cells exposed to low concentrations of NP41 (0.1-20μM) and NP42 (0.1-1μM), and high concentrations of NP41 (40μM) and NP42 (10-40μM) resulted in a return of p-ERK1/2 and p-p38 to control levels. We proposed that molecular mechanism of reproductive damage in Sertoli cells induced by NPs may be mediated by cell receptors and/or cell signaling pathways, and the effects may be related to the structure of NP isomer.

  5. Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

    SciTech Connect

    Yogi, Alvaro; Callera, Glaucia E.; Mecawi, André S.; Batalhão, Marcelo E.; Carnio, Evelin C.; Antunes-Rodrigues, José; Queiroz, Regina H.; Touyz, Rhian M.; Tirapelli, Carlos R.

    2012-11-01

    Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin–angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective AT{sub 1} receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P)H oxidase‐mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT{sub 1}-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. -- Highlights: ► Acute ethanol intake stimulates RAS activity and vascular oxidative stress. ► RAS plays a role in acute ethanol-induced oxidative damage via AT{sub 1} receptor activation.

  6. CLAVATA1 dominant-negative alleles reveal functional overlap between multiple receptor kinases that regulate meristem and organ development.

    PubMed

    Diévart, Anne; Dalal, Monica; Tax, Frans E; Lacey, Alexzandria D; Huttly, Alison; Li, Jianming; Clark, Steven E

    2003-05-01

    The CLAVATA1 (CLV1) receptor kinase controls stem cell number and differentiation at the Arabidopsis shoot and flower meristems. Other components of the CLV1 signaling pathway include the secreted putative ligand CLV3 and the receptor-like protein CLV2. We report evidence indicating that all intermediate and strong clv1 alleles are dominant negative and likely interfere with the activity of unknown receptor kinase(s) that have functional overlap with CLV1. clv1 dominant-negative alleles show major differences from dominant-negative alleles characterized to date in animal receptor kinase signaling systems, including the lack of a dominant-negative effect of kinase domain truncation and the ability of missense mutations in the extracellular domain to act in a dominant-negative manner. We analyzed chimeric receptor kinases by fusing CLV1 and BRASSINOSTEROID INSENSITIVE1 (BRI1) coding sequences and expressing these in clv1 null backgrounds. Constructs containing the CLV1 extracellular domain and the BRI1 kinase domain were strongly dominant negative in the regulation of meristem development. Furthermore, we show that CLV1 expressed within the pedicel can partially replace the function of the ERECTA receptor kinase. We propose the presence of multiple receptors that regulate meristem development in a functionally related manner whose interactions are driven by the extracellular domains and whose activation requires the kinase domain.

  7. CLAVATA1 Dominant-Negative Alleles Reveal Functional Overlap between Multiple Receptor Kinases That Regulate Meristem and Organ Development

    PubMed Central

    Diévart, Anne; Dalal, Monica; Tax, Frans E.; Lacey, Alexzandria D.; Huttly, Alison; Li, Jianming; Clark, Steven E.

    2003-01-01

    The CLAVATA1 (CLV1) receptor kinase controls stem cell number and differentiation at the Arabidopsis shoot and flower meristems. Other components of the CLV1 signaling pathway include the secreted putative ligand CLV3 and the receptor-like protein CLV2. We report evidence indicating that all intermediate and strong clv1 alleles are dominant negative and likely interfere with the activity of unknown receptor kinase(s) that have functional overlap with CLV1. clv1 dominant-negative alleles show major differences from dominant-negative alleles characterized to date in animal receptor kinase signaling systems, including the lack of a dominant-negative effect of kinase domain truncation and the ability of missense mutations in the extracellular domain to act in a dominant-negative manner. We analyzed chimeric receptor kinases by fusing CLV1 and BRASSINOSTEROID INSENSITIVE1 (BRI1) coding sequences and expressing these in clv1 null backgrounds. Constructs containing the CLV1 extracellular domain and the BRI1 kinase domain were strongly dominant negative in the regulation of meristem development. Furthermore, we show that CLV1 expressed within the pedicel can partially replace the function of the ERECTA receptor kinase. We propose the presence of multiple receptors that regulate meristem development in a functionally related manner whose interactions are driven by the extracellular domains and whose activation requires the kinase domain. PMID:12724544

  8. Profiling Protein Kinases and Other ATP Binding Proteins in Arabidopsis Using Acyl-ATP Probes*

    PubMed Central

    Villamor, Joji Grace; Kaschani, Farnusch; Colby, Tom; Oeljeklaus, Julian; Zhao, David; Kaiser, Markus; Patricelli, Matthew P.; van der Hoorn, Renier A. L.

    2013-01-01

    Many protein activities are driven by ATP binding and hydrolysis. Here, we explore the ATP binding proteome of the model plant Arabidopsis thaliana using acyl-ATP (AcATP)1 probes. These probes target ATP binding sites and covalently label lysine residues in the ATP binding pocket. Gel-based profiling using biotinylated AcATP showed that labeling is dependent on pH and divalent ions and can be competed by nucleotides. The vast majority of these AcATP-labeled proteins are known ATP binding proteins. Our search for labeled peptides upon in-gel digest led to the discovery that the biotin moiety of the labeled peptides is oxidized. The in-gel analysis displayed kinase domains of two receptor-like kinases (RLKs) at a lower than expected molecular weight, indicating that these RLKs lost the extracellular domain, possibly as a result of receptor shedding. Analysis of modified peptides using a gel-free platform identified 242 different labeling sites for AcATP in the Arabidopsis proteome. Examination of each individual labeling site revealed a preference of labeling in ATP binding pockets for a broad diversity of ATP binding proteins. Of these, 24 labeled peptides were from a diverse range of protein kinases, including RLKs, mitogen-activated protein kinases, and calcium-dependent kinases. A significant portion of the labeling sites could not be assigned to known nucleotide binding sites. However, the fact that labeling could be competed with ATP indicates that these labeling sites might represent previously uncharacterized nucleotide binding sites. A plot of spectral counts against expression levels illustrates the high specificity of AcATP probes for protein kinases and known ATP binding proteins. This work introduces profiling of ATP binding activities of a large diversity of proteins in plant proteomes. The data have been deposited in ProteomeXchange with the identifier PXD000188. PMID:23722185

  9. Membrane Topology and Structural Insights into the Peptide Pheromone Receptor ComD, A Quorum-Sensing Histidine Protein Kinase of Streptococcus mutans

    PubMed Central

    Dong, Gaofeng; Tian, Xiao-Lin; Cyr, Kayla; Liu, Tianlei; Lin, William; Tziolas, Geoffrey; Li, Yung-Hua

    2016-01-01

    Quorum sensing activation by signal pheromone (CSP) in Streptococcus mutans depends on the membrane-associated receptor ComD, which senses the signal and triggers the signaling cascade for bacteriocin production and other cell density-dependent activities. However, the mechanism of the signal recognition via the ComD receptor in this species is nearly unexplored. Here, we show that the membrane domain of the ComD protein forms six transmembrane segments with three extracellular loops, loopA, loopB and loopC. By structural and functional analyses of these extracellular loops, we demonstrate that both loopC and loopB are required for CSP recognition, while loopA plays little role in CSP detection. A deletion or substitution mutation of four residues NVIP in loopC abolishes CSP recognition for quorum sensing activities. We conclude that both loopC and loopB are required for forming the receptor and residues NVIP of loopC are essential for CSP recognition and quorum sensing activation in S. mutans. PMID:27199267

  10. Membrane Topology and Structural Insights into the Peptide Pheromone Receptor ComD, A Quorum-Sensing Histidine Protein Kinase of Streptococcus mutans.

    PubMed

    Dong, Gaofeng; Tian, Xiao-Lin; Cyr, Kayla; Liu, Tianlei; Lin, William; Tziolas, Geoffrey; Li, Yung-Hua

    2016-01-01

    Quorum sensing activation by signal pheromone (CSP) in Streptococcus mutans depends on the membrane-associated receptor ComD, which senses the signal and triggers the signaling cascade for bacteriocin production and other cell density-dependent activities. However, the mechanism of the signal recognition via the ComD receptor in this species is nearly unexplored. Here, we show that the membrane domain of the ComD protein forms six transmembrane segments with three extracellular loops, loopA, loopB and loopC. By structural and functional analyses of these extracellular loops, we demonstrate that both loopC and loopB are required for CSP recognition, while loopA plays little role in CSP detection. A deletion or substitution mutation of four residues NVIP in loopC abolishes CSP recognition for quorum sensing activities. We conclude that both loopC and loopB are required for forming the receptor and residues NVIP of loopC are essential for CSP recognition and quorum sensing activation in S. mutans. PMID:27199267

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

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2009-01-01

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

  12. Non-ATP competitive protein kinase inhibitors.

    PubMed

    Garuti, L; Roberti, M; Bottegoni, G

    2010-01-01

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

  13. Regulation of N-Formyl Peptide Receptor Signaling and Trafficking by Arrestin-Src Kinase Interaction.

    PubMed

    Wagener, Brant M; Marjon, Nicole A; Prossnitz, Eric R

    2016-01-01

    Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR)-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2) mutant deficient in Src kinase binding (arr2-P91G/P121E). Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking.

  14. Regulation of N-Formyl Peptide Receptor Signaling and Trafficking by Arrestin-Src Kinase Interaction

    PubMed Central

    Wagener, Brant M.; Marjon, Nicole A.; Prossnitz, Eric R.

    2016-01-01

    Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that N-formyl peptide receptor (FPR)-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2) mutant deficient in Src kinase binding (arr2-P91G/P121E). Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking. PMID:26788723

  15. Studying N-linked glycosylation of receptor tyrosine kinases.

    PubMed

    Itkonen, Harri M; Mills, Ian G

    2015-01-01

    Metabolic alterations have been identified as a frequent event in cancer. This is often associated with increased flux through glycolysis, and also a secondary pathway to glycolysis, hexosamine biosynthetic pathway (HBP). HBP provides substrate for N-linked glycosylation, which occurs in the endoplasmic reticulum and the Golgi apparatus. N-linked glycosylation supports protein folding and correct sorting of proteins to plasma membrane and secretion. This process generates complex glycoforms, which can be recognized by other proteins and glycosylation of receptor tyrosine kinases (RTK) can also regulate their plasma-membrane retention time. Of special interest for experimental biologists, plants produce proteins, termed lectins, which bind with high specificity to glyco-conjugates. For the purposes of molecular biology, plant lectins can be conjugated to different moieties, such as agarose beads, which enable precipitation of specifically glycosylated proteins. In this chapter, we describe in detail how to perform pull-down experiments with commercially available lectins to identify changes in the glycosylation of RTKs. PMID:25319893

  16. Sensitivity and kinase activity of epidermal growth factor receptor (EGFR) exon 19 and others to EGFR-tyrosine kinase inhibitors.

    PubMed

    Furuyama, Kazuto; Harada, Taishi; Iwama, Eiji; Shiraishi, Yoshimasa; Okamura, Kyoko; Ijichi, Kayo; Fujii, Akiko; Ota, Keiichi; Wang, Shuo; Li, Heyan; Takayama, Koichi; Giaccone, Giuseppe; Nakanishi, Yoichi

    2013-05-01

    The presence of epidermal growth factor receptor (EGFR) somatic mutations in non-small-cell lung cancer patients is associated with response to treatment with EGFR-tyrosine kinase inhibitors, such as gefitinib and erlotinib. More than 100 mutations in the kinase domain of EGFR have been identified. In particular there are many variations of deletion mutations in exon 19. In this study, using yellow fluorescent protein-tagged fragments of the EGFR intracellular domain, we examined the differences in sensitivity to gefitinib, erlotinib and afatinib between several exon 19 mutants and other common EGFR mutations. We also used serum of patients undergoing treatment with EGFR-tyrosine kinase inhibitors in this system. In addition, we examined the relative kinase activity of these mutants by measuring relative fluorescent intensity after immunofluorescence staining. We found that both sensitivity to EGFR-tyrosine kinase inhibitors and relative kinase activity differed among several EGFR mutations found in the same region of the kinase domain. This study underscores the importance of reporting the clinical outcome of treatment in relation to different EGFR mutations.

  17. MET Receptor Tyrosine Kinase as an Autism Genetic Risk Factor

    PubMed Central

    Peng, Yun; Huentelman, Matthew; Smith, Christopher; Qiu, Shenfeng

    2014-01-01

    In this chapter, we will briefly discuss recent literature on the role of MET receptor tyrosine kinase (RTK) in brain development and how perturbation of MET signaling may alter normal neurodevelopmental outcomes. Recent human genetic studies have established MET as a risk factor for autism, and the molecular and cellular underpinnings of this genetic risk are only beginning to emerge from obscurity. Unlike many autism risk genes that encode synaptic proteins, the spatial and temporal expression pattern of MET RTK indicates this signaling system is ideally situated to regulate neuronal growth, functional maturation, and establishment of functional brain circuits, particularly in those brain structures involved in higher levels of cognition, social skills, and executive functions. PMID:24290385

  18. Structure and Activation of MuSK, a Receptor Tyrosine Kinase Central to Neuromuscular Junction Formation

    PubMed Central

    Hubbard, Stevan R.; Gnanasambandan, Kavitha

    2014-01-01

    MuSK (muscle-specific kinase) is a receptor tyrosine kinase that plays a central signaling role in formation of neuromuscular junctions (NMJs). MuSK is activated in a complex spatio-temporal manner to cluster acetylcholine receptors on the postsynaptic (muscle) side of the synapse and to induce differentiation of the nerve terminal on the presynaptic side. The ligand for MuSK is LRP4 (low-density lipoprotein receptor-related protein-4), a transmembrane protein in muscle, whose binding affinity for MuSK is potentiated by agrin, a neuronally derived heparan-sulfate proteoglycan. In addition, Dok7, a cytoplasmic adaptor protein, is also required for MuSK activation in vivo. This review focuses on the physical interplay between these proteins and MuSK for activation and downstream signaling, which culminates in NMJ formation. PMID:23467009

  19. A Molecular Brake in the Kinase Hinge Region Regulates the Activity of Receptor Tyrosine Kinases

    SciTech Connect

    Chen,H.; Ma, J.; Li, W.; Eliseenkova, A.; Xu, C.; Neubert, T.; Miller, W.; Mohammadi, M.

    2007-01-01

    Activating mutations in the tyrosine kinase domain of receptor tyrosine kinases (RTKs) cause cancer and skeletal disorders. Comparison of the crystal structures of unphosphorylated and phosphorylated wild-type FGFR2 kinase domains with those of seven unphosphorylated pathogenic mutants reveals an autoinhibitory 'molecular brake' mediated by a triad of residues in the kinase hinge region of all FGFRs. Structural analysis shows that many other RTKs, including PDGFRs, VEGFRs, KIT, CSF1R, FLT3, TEK, and TIE, are also subject to regulation by this brake. Pathogenic mutations activate FGFRs and other RTKs by disengaging the brake either directly or indirectly.

  20. Biochemical and molecular analysis of a transmembrane protein kinase from Arabidopsis thaliana. Progress report, January 1993

    SciTech Connect

    Bleecker, A.B.

    1993-06-01

    We have isolated genomic and cDNA clones encoding a novel receptor-like protein kinase from the higher plant Arabidopsis thaliana. This kinase is being studied by combining biochemical, molecular, and genetic approaches. Domain-specific antibodies immunodecorate a polypeptide with a molecular mass of 120,000 daltons in extracts of Arabidopsis, where it has been found in all portions of the plant examined including root, stem, leaf, flower, and silique. Cytochemical analysis and initial studies using the kinase promoter with the GUS reporter gene system also indicate that the kinase is present throughout the plant. The kinase is glycosylated, like animal receptor kinases, and has been partially purified from Arabidopsis by using lectin columns. The kinase has been expressed in E coli, purified, and found to autophosphorylate on serine and threonine residues, but not on tyrosine residues. As such, it belongs to the small family of receptor-like kinases with serine/threonine specificity. Transgenic plants are now being produced that either overexpress or carry altered forms of the protein kinase gene. These experiments will help determine the natural role the kinase plays in a pathway of signal transduction.

  1. Biochemical and molecular analysis of a transmembrane protein kinase from Arabidopsis thaliana

    SciTech Connect

    Bleecker, A.B.

    1993-01-01

    We have isolated genomic and cDNA clones encoding a novel receptor-like protein kinase from the higher plant Arabidopsis thaliana. This kinase is being studied by combining biochemical, molecular, and genetic approaches. Domain-specific antibodies immunodecorate a polypeptide with a molecular mass of 120,000 daltons in extracts of Arabidopsis, where it has been found in all portions of the plant examined including root, stem, leaf, flower, and silique. Cytochemical analysis and initial studies using the kinase promoter with the GUS reporter gene system also indicate that the kinase is present throughout the plant. The kinase is glycosylated, like animal receptor kinases, and has been partially purified from Arabidopsis by using lectin columns. The kinase has been expressed in E coli, purified, and found to autophosphorylate on serine and threonine residues, but not on tyrosine residues. As such, it belongs to the small family of receptor-like kinases with serine/threonine specificity. Transgenic plants are now being produced that either overexpress or carry altered forms of the protein kinase gene. These experiments will help determine the natural role the kinase plays in a pathway of signal transduction.

  2. Protein kinase C sensitizes olfactory adenylate cyclase

    PubMed Central

    1993-01-01

    Effects of neurotransmitters on cAMP-mediated signal transduction in frog olfactory receptor cells (ORCs) were studied using in situ spike recordings and radioimmunoassays. Carbachol, applied to the mucosal side of olfactory epithelium, amplified the electrical response of ORCs to cAMP-generating odorants, but did not affect unstimulated cells. A similar augmentation of odorant response was observed in the presence of phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC). The electrical response to forskolin, an activator of adenylate cyclase (AC), was also enhanced by PDBu, and it was attenuated by the PKC inhibitor Goe 6983. Forskolin-induced accumulation of cAMP in olfactory tissue was potentiated by carbachol, serotonin, and PDBu to a similar extent. Potentiation was completely suppressed by the PKC inhibitors Goe 6983, staurosporine, and polymyxin B, suggesting that the sensitivity of olfactory AC to stimulation by odorants and forskolin was increased by PKC. Experiments with deciliated olfactory tissue indicated that sensitization of AC was restricted to sensory cilia of ORCs. To study the effects of cell Ca2+ on these mechanisms, the intracellular Ca2+ concentration of olfactory tissue was either increased by ionomycin or decreased by BAPTA/AM. Increasing cell Ca2+ had two effects on cAMP production: (a) the basal cAMP production was enhanced by a mechanism sensitive to inhibitors of calmodulin; and (b) similar to phorbol ester, cell Ca2+ caused sensitization of AC to stimulation by forskolin, an effect sensitive to Goe 6983. Decreasing cell Ca2+ below basal levels rendered AC unresponsive to stimulation by forskolin. These data suggest that a crosstalk mechanism is functional in frog ORCs, linking the sensitivity of AC to the activity of PKC. At increased activity of PKC, olfactory AC becomes more responsive to stimulation by odorants, forskolin, and cell Ca2+. Neurotransmitters appear to use this crosstalk mechanism to regulate olfactory

  3. Activation and inhibition of anaplastic lymphoma kinase receptor tyrosine kinase by monoclonal antibodies and absence of agonist activity of pleiotrophin.

    PubMed

    Moog-Lutz, Christel; Degoutin, Joffrey; Gouzi, Jean Y; Frobert, Yvelyne; Brunet-de Carvalho, Nicole; Bureau, Jocelyne; Créminon, Christophe; Vigny, Marc

    2005-07-15

    Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is transiently expressed in specific regions of the central and peripheral nervous systems, suggesting a role in its normal development and function. The nature of the cognate ligands of ALK in vertebrate is still a matter of debate. We produced a panel of monoclonal antibodies (mAbs) directed against the extracellular domain of the human receptor. Two major species of ALK (220 and 140 kDa) were identified in transfected cells, and the use of our mAbs established that the 140-kDa species results from a cleavage of the 220-kDa form. Two mAbs, in the nm range, induced the differentiation of PC12 cells transiently transfected with ALK. In human embryonic kidney 293 cells stably expressing ALK, these two mAbs strongly activated the receptor and subsequently the mitogen-activated protein kinase pathway. We further showed for the first time that activation of ALK also resulted in a specific activation of STAT3. In contrast, other mAbs presented the characteristics of blocking antibodies. Finally, in these cell systems, a mitogenic form of pleiotrophin, a proposed ligand of ALK, failed to activate this receptor. Thus, in the absence of clearly established ligand(s) in vertebrates, the availability of mAbs allowing the activation or the inhibition of the receptor will be essential for a better understanding of the biological roles of ALK.

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

    PubMed Central

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

    1986-01-01

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

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

    PubMed

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

    1993-12-15

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

  6. A novel human STE20-related protein kinase, HGK, that specifically activates the c-Jun N-terminal kinase signaling pathway.

    PubMed

    Yao, Z; Zhou, G; Wang, X S; Brown, A; Diener, K; Gan, H; Tan, T H

    1999-01-22

    The yeast serine/threonine kinase STE20 activates a signaling cascade that includes STE11 (mitogen-activated protein kinase kinase kinase), STE7 (mitogen-activated protein kinase kinase), and FUS3/KSS1 (mitogen-activated protein kinase) in response to signals from both Cdc42 and the heterotrimeric G proteins associated with transmembrane pheromone receptors. Using degenerate polymerase chain reaction, we have isolated a human cDNA encoding a protein kinase homologous to STE20. This protein kinase, designated HPK/GCK-like kinase (HGK), has nucleotide sequences that encode an open reading frame of 1165 amino acids with 11 kinase subdomains. HGK was a serine/threonine protein kinase that specifically activated the c-Jun N-terminal kinase (JNK) signaling pathway when transfected into 293T cells, but it did not stimulate either the extracellular signal-regulated kinase or p38 kinase pathway. HGK also increased AP-1-mediated transcriptional activity in vivo. HGK-induced JNK activation was inhibited by the dominant-negative MKK4 and MKK7 mutants. The dominant-negative mutant of TAK1, but not MEKK1 or MAPK upstream kinase (MUK), strongly inhibited HGK-induced JNK activation. TNF-alpha activated HGK in 293T cells, as well as the dominant-negative HGK mutants, inhibited TNF-alpha-induced JNK activation. These results indicate that HGK, a novel activator of the JNK pathway, may function through TAK1, and that the HGK --> TAK1 --> MKK4, MKK7 --> JNK kinase cascade may mediate the TNF-alpha signaling pathway. PMID:9890973

  7. Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: a role for angiotensin type 1 receptor.

    PubMed

    Yogi, Alvaro; Callera, Glaucia E; Mecawi, André S; Batalhão, Marcelo E; Carnio, Evelin C; Antunes-Rodrigues, José; Queiroz, Regina H; Touyz, Rhian M; Tirapelli, Carlos R

    2012-11-01

    Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin-angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective AT₁ receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P)H oxidase-mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT₁-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage.

  8. Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase.

    PubMed

    Cai, H; Smola, U; Wixler, V; Eisenmann-Tappe, I; Diaz-Meco, M T; Moscat, J; Rapp, U; Cooper, G M

    1997-02-01

    The Raf protein kinases function downstream of Ras guanine nucleotide-binding proteins to transduce intracellular signals from growth factor receptors. Interaction with Ras recruits Raf to the plasma membrane, but the subsequent mechanism of Raf activation has not been established. Previous studies implicated hydrolysis of phosphatidylcholine (PC) in Raf activation; therefore, we investigated the role of the epsilon isotype of protein kinase C (PKC), which is stimulated