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Sample records for kinases regulate astrocyte

  1. Extracellular microvesicles from astrocytes contain functional glutamate transporters: regulation by protein kinase C and cell activation

    PubMed Central

    Gosselin, Romain-Daniel; Meylan, Patrick; Decosterd, Isabelle

    2013-01-01

    Glutamate transport through astrocytic excitatory amino-acid transporters (EAAT)-1 and EAAT-2 is paramount for neural homeostasis. EAAT-1 has been reported in secreted extracellular microvesicles (eMV, such as exosomes) and because the protein kinase C (PKC) family controls the sub-cellular distribution of EAATs, we have explored whether PKCs drive EAATs into eMV. Using rat primary astrocytes, confocal immunofluorescence and ultracentrifugation on sucrose gradient we here report that PKC activation by phorbol myristate acetate (PMA) reorganizes EAAT-1 distribution and reduces functional [3H]-aspartate reuptake. Western-blots show that EAAT-1 is present in eMV from astrocyte conditioned medium, together with NaK ATPase and glutamine synthetase all being further increased after PMA treatment. However, nanoparticle tracking analysis reveals that PKC activation did not change particle concentration. Functional analysis indicates that eMV have the capacity to reuptake [3H]-aspartate. In vivo, we demonstrate that spinal astrocytic reaction induced by peripheral nerve lesion (spared nerve injury, SNI) is associated with a phosphorylation of PKC δ together with a shift of EAAT distribution ipsilaterally. Ex vivo, spinal explants from SNI rats release eMV with an increased content of NaK ATPase, EAAT-1 and EAAT-2. These data indicate PKC and cell activation as important regulators of EAAT-1 incorporation in eMV, and raise the possibility that microvesicular EAAT-1 may exert extracellular functions. Beyond a putative role in neuropathic pain, this phenomenon may be important for understanding neural homeostasis and a wide range of neurological diseases associated with astrocytic reaction as well as non-neurological diseases linked to eMV release. PMID:24368897

  2. Glucose Regulates Hypothalamic Long-chain Fatty Acid Metabolism via AMP-activated Kinase (AMPK) in Neurons and Astrocytes*

    PubMed Central

    Taïb, Bouchra; Bouyakdan, Khalil; Hryhorczuk, Cécile; Rodaros, Demetra; Fulton, Stephanie; Alquier, Thierry

    2013-01-01

    Hypothalamic controls of energy balance rely on the detection of circulating nutrients such as glucose and long-chain fatty acids (LCFA) by the mediobasal hypothalamus (MBH). LCFA metabolism in the MBH plays a key role in the control of food intake and glucose homeostasis, yet it is not known if glucose regulates LCFA oxidation and esterification in the MBH and, if so, which hypothalamic cell type(s) and intracellular signaling mechanisms are involved. The aim of this study was to determine the impact of glucose on LCFA metabolism, assess the role of AMP-activated Kinase (AMPK), and to establish if changes in LCFA metabolism and its regulation by glucose vary as a function of the kind of LCFA, cell type, and brain region. We show that glucose inhibits palmitate oxidation via AMPK in hypothalamic neuronal cell lines, primary hypothalamic astrocyte cultures, and MBH slices ex vivo but not in cortical astrocytes and slice preparations. In contrast, oleate oxidation was not affected by glucose or AMPK inhibition in MBH slices. In addition, our results show that glucose increases palmitate, but not oleate, esterification into neutral lipids in neurons and MBH slices but not in hypothalamic astrocytes. These findings reveal for the first time the metabolic fate of different LCFA in the MBH, demonstrate AMPK-dependent glucose regulation of LCFA oxidation in both astrocytes and neurons, and establish metabolic coupling of glucose and LCFA as a distinguishing feature of hypothalamic nuclei critical for the control of energy balance. PMID:24240094

  3. The phosphorylation status of extracellular-regulated kinase 1/2 in astrocytes and neurons from rat hippocampus determines the thrombin-induced calcium release and ROS generation.

    PubMed

    Zündorf, Gregor; Reiser, Georg

    2011-12-01

    Challenge of protease-activated receptors induces cytosolic Ca(2+) concentration ([Ca(2+) ](c)) increase, mitogen-activated protein kinase activation and reactive oxygen species (ROS) formation with a bandwidth of responses in individual cells. We detected in this study in situ the thrombin-induced [Ca(2+) ](c) rise and ROS formation in dissociated hippocampal astrocytes and neurons in a mixed culture. In identified cells, single cell responses were correlated with extracellular-regulated kinase (ERK)1/2 phosphorylation level. On average, in astrocytes, thrombin induced a transient [Ca(2+) ](c) rise with concentration-dependent increase in amplitude and extrusion rate and high ERK1/2 phosphorylation level. Correlation analysis of [Ca(2+) ](c) response characteristics of single astrocytes reveals that astrocytes with nuclear phosphoERK1/2 localization have a smaller Ca(2+) amplitude and extrusion rate compared with cells with a cytosolic phosphoERK1/2 localization. In naive neurons, without thrombin challenge, variable ERK1/2 phosphorylation patterns are observed. ROS were detected by hydroethidine. Only in neurons with increased ERK1/2 phosphorylation level, we see sustained intracellular rise in fluorescence of the dye lasting over several minutes. ROS formation was abolished by pre-incubation with the NADPH oxidase inhibitor apocynin. Additionally, thrombin induced an immediate, transient hydroethidine fluorescence increase. This was interpreted as NADPH oxidase-mediated O(2) (•-) -release into the extracellular milieu, because it was decreased by pre-incubation with apocynin, and could be eluted by superfusion. In conclusion, the phosphorylation status of ERK1/2 determines the thrombin-dependent [Ca(2+) ](c) increase and ROS formation and, thus, influences the capacity of thrombin to regulate neuroprotection or neurodegeneration. PMID:21988180

  4. Lateral regulation of synaptic transmission by astrocytes.

    PubMed

    Covelo, A; Araque, A

    2016-05-26

    Fifteen years ago the concept of the "tripartite synapse" was proposed to conceptualize the functional view that astrocytes are integral elements of synapses. The signaling exchange between astrocytes and neurons within the tripartite synapse results in the synaptic regulation of synaptic transmission and plasticity through an autocrine form of communication. However, recent evidence indicates that the astrocyte synaptic regulation is not restricted to the active tripartite synapse but can be manifested through astrocyte signaling at synapses relatively distant from active synapses, a process termed lateral astrocyte synaptic regulation. This phenomenon resembles the classical heterosynaptic modulation but is mechanistically different because it involves astrocytes and its properties critically depend on the morphological and functional features of astrocytes. Therefore, the functional concept of the tripartite synapse as a fundamental unit must be expanded to include the interaction between tripartite synapses. Through lateral synaptic regulation, astrocytes serve as an active processing bridge for synaptic interaction and crosstalk between synapses with no direct neuronal connectivity, supporting the idea that neural network function results from the coordinated activity of astrocytes and neurons. PMID:25732135

  5. Role of astrocytes in cerebrovascular regulation

    PubMed Central

    Koehler, Raymond C.; Gebremedhin, Debebe; Harder, David R.

    2006-01-01

    Astrocytes send processes to synapses and blood vessels, communicate with other astrocytes through gap junctions and by release of ATP, and thus are an integral component of the neurovascular unit. Electrical field stimulations in brain slices demonstrate an increase in intracellular calcium in astrocyte cell bodies transmitted to perivascular end-feet, followed by a decrease in vascular smooth muscle calcium oscillations and arteriolar dilation. The increase in astrocyte calcium after neuronal activation is mediated, in part, by activation of metabotropic glutamate receptors. Calcium signaling in vitro can also be influenced by adenosine acting on A2B receptors and by epoxyeicosatrienoic acids (EETs) shown to be synthesized in astrocytes. Prostaglandins, EETs, arachidonic acid, and potassium ions are candidate mediators of communication between astrocyte end-feet and vascular smooth muscle. In vivo evidence supports a role for cyclooxygenase-2 metabolites, EETs, adenosine, and neuronally derived nitric oxide in the coupling of increased blood flow to increased neuronal activity. Combined inhibition of the EETs, nitric oxide, and adenosine pathways indicates that signaling is not by parallel, independent pathways. Indirect pharmacological results are consistent with astrocytes acting as intermediaries in neurovascular signaling within the neurovascular unit. For specific stimuli, astrocytes are also capable of transmitting signals to pial arterioles on the brain surface for ensuring adequate inflow pressure to parenchymal feeding arterioles. Therefore, evidence from brain slices and indirect evidence in vivo with pharmacological approaches suggest that astrocytes play a pivotal role in regulating the fundamental physiological response coupling dynamic changes in cerebral blood flow to neuronal synaptic activity. Future work using in vivo imaging and genetic manipulation will be required to provide more direct evidence for a role of astrocytes in neurovascular

  6. Glucocorticoid Regulation of Astrocytic Fate and Function

    PubMed Central

    Yu, Shuang; Yang, Silei; Holsboer, Florian; Sousa, Nuno; Almeida, Osborne F. X.

    2011-01-01

    Glial loss in the hippocampus has been suggested as a factor in the pathogenesis of stress-related brain disorders that are characterized by dysregulated glucocorticoid (GC) secretion. However, little is known about the regulation of astrocytic fate by GC. Here, we show that astrocytes derived from the rat hippocampus undergo growth inhibition and display moderate activation of caspase 3 after exposure to GC. Importantly, the latter event, observed both in situ and in primary astrocytic cultures is not followed by either early- or late-stage apoptosis, as monitored by stage I or stage II DNA fragmentation. Thus, unlike hippocampal granule neurons, astrocytes are resistant to GC-induced apoptosis; this resistance is due to lower production of reactive oxygen species (ROS) and a greater buffering capacity against the cytotoxic actions of ROS. We also show that GC influence hippocampal cell fate by inducing the expression of astrocyte-derived growth factors implicated in the control of neural precursor cell proliferation. Together, our results suggest that GC instigate a hitherto unknown dialog between astrocytes and neural progenitors, adding a new facet to understanding how GC influence the cytoarchitecture of the hippocampus. PMID:21811605

  7. Upregulation of adenosine kinase in astrocytes in experimental and human temporal lobe epilepsy

    PubMed Central

    Aronica, Eleonora; Zurolo, Emanuele; Iyer, Anand; de Groot, Marjolein; Anink, Jasper; Carbonell, Caterina; van Vliet, Erwin A.; Baayen, Johannes C.; Boison, Detlev; Gorter, Jan A.

    2011-01-01

    Purpose Adenosine kinase (ADK) represents the key metabolic enzyme for the regulation of extracellular adenosine levels in the brain. In adult brain, ADK is primarily present in astrocytes. Several lines of experimental evidence support a critical role of ADK in different types of brain injury associated with astrogliosis, which is also a prominent morphological feature of temporal lobe epilepsy (TLE). We hypothesized that dysregulation of ADK is an ubiquitous pathological hallmark of TLE. Methods Using immunocytochemistry and western blot analysis, we investigated ADK protein expression in a rat model of TLE during epileptogenesis and the chronic epileptic phase and compared those findings with tissue resected from TLE patients with mesial temporal sclerosis (MTS). Key findings In rat control hippocampus and cortex, a low baseline expression of ADK was found with mainly nuclear localization. One week after the electrical induction of status epilepticus (SE), prominent up-regulation of ADK became evident in astrocytes with a characteristic cytoplasmic localization. This increase in ADK persisted at least for 3-4 months after SE in rats developing a progressive form of epilepsy. In line with the findings from the rat model, expression of astrocytic ADK was also found to be increased in the hippocampus and temporal cortex of TLE patients. In addition, in vitro experiments in human astrocyte cultures showed that ADK expression was increased by several pro-inflammatory molecules (interleukin-1β and LPS). Significance These results suggest that dysregulation of ADK in astrocytes is a common pathological hallmark of TLE. Moreover, in vitro data suggest the existence of an additional layer of modulatory crosstalk between the astrocyte-based adenosine cycle and inflammation. Whether this interaction also can play role in vivo needs to be further investigated. PMID:21635241

  8. ALBUMIN CAUSES INCREASED MYOSIN LIGHT CHAIN KINASE EXPRESSION IN ASTROCYTES VIA P38 MITOGEN ACTIVATED PROTEIN KINASE

    PubMed Central

    Rossi, Janet L.; Ralay Ranaivo, Hantamala; Patel, Fatima; Chrzaszcz, MaryAnn; Venkatesan, Charu; Wainwright, Mark S.

    2011-01-01

    Myosin light chain kinase (MLCK) plays an important role in the reorganization of the cytoskeleton leading to disruption of vascular barrier integrity in multiple organs including the blood brain barrier (BBB) after traumatic brain injury (TBI). MLCK has been linked to transforming growth factor (TGF) and rho kinase signaling pathways, but the mechanisms regulating MLCK expression following TBI are not well understood. Albumin leaks into the brain parenchyma following TBI, activates glia and has been linked to TGF-β receptor signaling. We investigated the role of albumin in the increase in MLCK in astrocytes and the signaling pathways involved in this increase. Following midline closed-skull TBI in mice, there was a significant increase in MLCK-immunoreactive (IR) cells and albumin extravasation, which was prevented by treatment with the MLCK inhibitor ML-7. Using immunohistochemical methods, we identified the MCLK-IR cells as astrocytes. In primary astrocytes, exposure to albumin increased both isoforms of MLCK, 130 and 210. Inhibition of the TGF-β receptor partially prevented the albumin-induced increase in both isoforms, which was not prevented by inhibition of smad3. Inhibition of p38 MAPK, but not ERK, JNK or rho kinase also prevented this increase. These results are further evidence of a role of MCLK in the mechanisms of BBB compromise following TBI, and identify astrocytes as a cell type, in addition to endothelium in the BBB which express MLCK. These findings implicate albumin, acting through p38 MAPK, in a novel mechanism by which activation of MLCK following TBI may lead to compromise of the BBB. PMID:21360574

  9. Activity-dependent regulation of astrocyte GAT levels during synaptogenesis

    PubMed Central

    Muthukumar, Allie K.; Stork, Tobias; Freeman, Marc R.

    2014-01-01

    Astrocytic uptake of GABA through GABA transporters (GATs) is an important mechanism regulating excitatory/inhibitory balance in the nervous system, however mechanisms by which astrocytes regulate GAT levels are undefined. Here we show at mid-pupal stages the Drosophila CNS neuropil is devoid of astrocyte membranes and synapses. Astrocyte membranes subsequently infiltrate the neuropil coordinate with synaptogenesis and a strocyte ablation reduces synapse numbers by half, indicating that Drosophila astrocytes are pro-synaptogenic. Shortly after synapses form in earnest, the GABA transporter, GAT, is up-regulated in astrocytes. Ablation or silencing of GABAergic neurons or disruption of metabotropic GABA receptor (GABABR1/2) signaling in astrocytes leads to decreased astrocytic GAT levels. Interestingly, developmental depletion of astrocytic GABABR1/2 signaling suppresses mechanosensory-induced seizure activity in mutants with hyperexcitable neurons. These data reveal astrocytes actively modulate GAT expression via metabotropic GABA receptor signaling, and highlight the importance of precise regulation of astrocytic GAT in modulation of seizure activity. PMID:25151265

  10. Circadian regulation of ATP release in astrocytes.

    PubMed

    Marpegan, Luciano; Swanstrom, Adrienne E; Chung, Kevin; Simon, Tatiana; Haydon, Philip G; Khan, Sanjoy K; Liu, Andrew C; Herzog, Erik D; Beaulé, Christian

    2011-06-01

    Circadian clocks sustain daily oscillations in gene expression, physiology, and behavior, relying on transcription-translation feedback loops of clock genes for rhythm generation. Cultured astrocytes display daily oscillations of extracellular ATP, suggesting that ATP release is a circadian output. We hypothesized that the circadian clock modulates ATP release via mechanisms that regulate acute ATP release from glia. To test the molecular basis for circadian ATP release, we developed methods to measure in real-time ATP release and Bmal1::dLuc circadian reporter expression in cortical astrocyte cultures from mice of different genotypes. Daily rhythms of gene expression required functional Clock and Bmal1, both Per1 and Per2, and both Cry1 and Cry2 genes. Similarly, high-level, circadian ATP release also required a functional clock mechanism. Whereas blocking IP(3) signaling significantly disrupted ATP rhythms with no effect on Bmal1::dLuc cycling, blocking vesicular release did not alter circadian ATP release or gene expression. We conclude that astrocytes depend on circadian clock genes and IP(3) signaling to express daily rhythms in ATP release. PMID:21653839

  11. Circadian regulation of ATP release in astrocytes

    PubMed Central

    Marpegan, Luciano; Swanstrom, Adrienne E.; Chung, Kevin; Simon, Tatiana; Haydon, Philip G.; Khan, Sanjoy K.; Liu, Andrew C.; Herzog, Erik D.; Beaulé, Christian

    2011-01-01

    Circadian clocks sustain daily oscillations in gene expression, physiology and behavior, relying on transcription-translation feedback loops of clock genes for rhythm generation. Cultured astrocytes display daily oscillations of extracellular ATP, suggesting that ATP release is a circadian output. We hypothesized that the circadian clock modulates ATP release via mechanisms that regulate acute ATP release from glia. To test the molecular basis for circadian ATP release, we developed methods to measure in real-time ATP release and Bmal1::dLuc circadian reporter expression in cortical astrocyte cultures from mice of different genotypes. Daily rhythms of gene expression required functional Clock and Bmal1, both Per1 and Per2, and both Cry1 and Cry2 genes. Similarly, high level, circadian ATP release also required a functional clock mechanism. Whereas blocking IP3 signaling significantly disrupted ATP rhythms with no effect on Bmal1::dLuc cycling, blocking vesicular release did not alter circadian ATP release or gene expression. We conclude that astrocytes depend on circadian clock genes and IP3 signaling to express daily rhythms in ATP release. PMID:21653839

  12. The Complex of Ciliary Neurotrophic Factor-Ciliary Neurotrophic Factor Receptor α Up-Regulates Connexin43 and Intercellular Coupling in Astrocytes via the Janus Tyrosine Kinase/Signal Transducer and Activator of Transcription PathwayD⃞

    PubMed Central

    Ozog, Mark A.; Bernier, Suzanne M.; Bates, Dave C.; Chatterjee, Bishwanath; Lo, Cecilia W.; Naus, Christian C.G.

    2004-01-01

    Cytokines regulate numerous cell processes, including connexin expression and gap junctional coupling. In this study, we examined the effect of ciliary neurotrophic factor (CNTF) on connexin43 (Cx43) expression and intercellular coupling in astrocytes. Murine cortical astrocytes matured in vitro were treated with CNTF (20 ng/ml), soluble ciliary neurotrophic factor receptor α (CNTFRα) (200 ng/ml), or CNTF-CNTFRα. Although CNTF and CNTFRα alone had no effect on Cx43 expression, the heterodimer CNTF-CNTFRα significantly increased both Cx43 mRNA and protein levels. Cx43 immunostaining correlated with increased intercellular coupling as determined by dye transfer analysis. By using the pharmacological inhibitor α-cyano-(3,4-dihydroxy)-N-benzylcinnamide (AG490), the increase in Cx43 was found to be dependent on the Janus tyrosine kinase/signal transducer and activator of transcription (JAK/STAT) pathway. Immunocytochemical analysis revealed that CNTF-CNTFRα treatment produced nuclear localization of phosphorylated STAT3, whereas CNTF treatment alone did not. Transient transfection of constructs containing various sequences of the Cx43 promoter tagged to a LacZ reporter into ROS 17/2.8 cells confirmed that the promoter region between -838 to -1693 was deemed necessary for CNTF-CNTFRα to induce heightened expression. CNTF-CNTFRα did not alter Cx30 mRNA levels, suggesting selectivity of CNTF-CNTFRα for connexin signaling. Together in the presence of soluble receptor, CNTF activates the JAK/STAT pathway leading to enhanced Cx43 expression and intercellular coupling. PMID:15342787

  13. Astrocyte transforming growth factor beta 1 promotes inhibitory synapse formation via CaM kinase II signaling.

    PubMed

    Diniz, Luan Pereira; Tortelli, Vanessa; Garcia, Matheus Nunes; Araújo, Ana Paula Bérgamo; Melo, Helen M; Silva, Gisele S Seixas da; Felice, Fernanda G De; Alves-Leon, Soniza Vieira; Souza, Jorge Marcondes de; Romão, Luciana Ferreira; Castro, Newton Gonçalves; Gomes, Flávia Carvalho Alcantara

    2014-12-01

    The balance between excitatory and inhibitory synaptic inputs is critical for the control of brain function. Astrocytes play important role in the development and maintenance of neuronal circuitry. Whereas astrocytes-derived molecules involved in excitatory synapses are recognized, molecules and molecular mechanisms underlying astrocyte-induced inhibitory synapses remain unknown. Here, we identified transforming growth factor beta 1 (TGF-β1), derived from human and murine astrocytes, as regulator of inhibitory synapse in vitro and in vivo. Conditioned media derived from human and murine astrocytes induce inhibitory synapse formation in cerebral cortex neurons, an event inhibited by pharmacologic and genetic manipulation of the TGF-β pathway. TGF-β1-induction of inhibitory synapse depends on glutamatergic activity and activation of CaM kinase II, which thus induces localization and cluster formation of the synaptic adhesion protein, Neuroligin 2, in inhibitory postsynaptic terminals. Additionally, intraventricular injection of TGF-β1 enhanced inhibitory synapse number in the cerebral cortex. Our results identify TGF-β1/CaMKII pathway as a novel molecular mechanism underlying astrocyte control of inhibitory synapse formation. We propose here that the balance between excitatory and inhibitory inputs might be provided by astrocyte signals, at least partly achieved via TGF-β1 downstream pathways. Our work contributes to the understanding of the GABAergic synapse formation and may be of relevance to further the current knowledge on the mechanisms underlying the development of various neurological disorders, which commonly involve impairment of inhibitory synapse transmission. PMID:25042347

  14. Astrocytic glutamate transport regulates a Drosophila CNS synapse that lacks astrocyte ensheathment.

    PubMed

    MacNamee, Sarah E; Liu, Kendra E; Gerhard, Stephan; Tran, Cathy T; Fetter, Richard D; Cardona, Albert; Tolbert, Leslie P; Oland, Lynne A

    2016-07-01

    Anatomical, molecular, and physiological interactions between astrocytes and neuronal synapses regulate information processing in the brain. The fruit fly Drosophila melanogaster has become a valuable experimental system for genetic manipulation of the nervous system and has enormous potential for elucidating mechanisms that mediate neuron-glia interactions. Here, we show the first electrophysiological recordings from Drosophila astrocytes and characterize their spatial and physiological relationship with particular synapses. Astrocyte intrinsic properties were found to be strongly analogous to those of vertebrate astrocytes, including a passive current-voltage relationship, low membrane resistance, high capacitance, and dye-coupling to local astrocytes. Responses to optogenetic stimulation of glutamatergic premotor neurons were correlated directly with anatomy using serial electron microscopy reconstructions of homologous identified neurons and surrounding astrocytic processes. Robust bidirectional communication was present: neuronal activation triggered astrocytic glutamate transport via excitatory amino acid transporter 1 (Eaat1), and blocking Eaat1 extended glutamatergic interneuron-evoked inhibitory postsynaptic currents in motor neurons. The neuronal synapses were always located within 1 μm of an astrocytic process, but none were ensheathed by those processes. Thus, fly astrocytes can modulate fast synaptic transmission via neurotransmitter transport within these anatomical parameters. J. Comp. Neurol. 524:1979-1998, 2016. © 2016 Wiley Periodicals, Inc. PMID:27073064

  15. The Neurogenic Potential of Astrocytes Is Regulated by Inflammatory Signals.

    PubMed

    Michelucci, Alessandro; Bithell, Angela; Burney, Matthew J; Johnston, Caroline E; Wong, Kee-Yew; Teng, Siaw-Wei; Desai, Jyaysi; Gumbleton, Nigel; Anderson, Gregory; Stanton, Lawrence W; Williams, Brenda P; Buckley, Noel J

    2016-08-01

    Although the adult brain contains neural stem cells (NSCs) that generate new neurons throughout life, these astrocyte-like populations are restricted to two discrete niches. Despite their terminally differentiated phenotype, adult parenchymal astrocytes can re-acquire NSC-like characteristics following injury, and as such, these 'reactive' astrocytes offer an alternative source of cells for central nervous system (CNS) repair following injury or disease. At present, the mechanisms that regulate the potential of different types of astrocytes are poorly understood. We used in vitro and ex vivo astrocytes to identify candidate pathways important for regulation of astrocyte potential. Using in vitro neural progenitor cell (NPC)-derived astrocytes, we found that exposure of more lineage-restricted astrocytes to either tumor necrosis factor alpha (TNF-α) (via nuclear factor-κB (NFκB)) or the bone morphogenetic protein (BMP) inhibitor, noggin, led to re-acquisition of NPC properties accompanied by transcriptomic and epigenetic changes consistent with a more neurogenic, NPC-like state. Comparative analyses of microarray data from in vitro-derived and ex vivo postnatal parenchymal astrocytes identified several common pathways and upstream regulators associated with inflammation (including transforming growth factor (TGF)-β1 and peroxisome proliferator-activated receptor gamma (PPARγ)) and cell cycle control (including TP53) as candidate regulators of astrocyte phenotype and potential. We propose that inflammatory signalling may control the normal, progressive restriction in potential of differentiating astrocytes as well as under reactive conditions and represent future targets for therapies to harness the latent neurogenic capacity of parenchymal astrocytes. PMID:26138449

  16. Multiple Protein Kinases via Activation of Transcription Factors NF-κB, AP-1 and C/EBP-δ Regulate the IL-6/IL-8 Production by HIV-1 Vpr in Astrocytes

    PubMed Central

    Gangwani, Mohitkumar R.; Kumar, Anil

    2015-01-01

    Neurocognitive impairments affect a substantial population of HIV-1 infected individuals despite the success of anti-retroviral therapy in controlling viral replication. Astrocytes are emerging as a crucial cell type that might be playing a very important role in the persistence of neuroinflammation seen in patients suffering from HIV-1 associated neurocognitive disorders. HIV-1 viral proteins including Vpr exert neurotoxicity through direct and indirect mechanisms. Induction of IL-8 in microglial cells has been shown as one of the indirect mechanism through which Vpr reduces neuronal survival. We show that HIV-1 Vpr induces IL-6 and IL-8 in astrocytes in a time-dependent manner. Additional experiments utilizing chemical inhibitors and siRNA revealed that HIV-1 Vpr activates transcription factors NF-κB, AP-1 and C/EBP-δ via upstream protein kinases PI3K/Akt, p38-MAPK and Jnk-MAPK leading to the induction of IL-6 and IL-8 in astrocytes. We demonstrate that one of the mechanism for neuroinflammation seen in HIV-1 infected individuals involves induction of IL-6 and IL-8 by Vpr in astrocytes. Understanding the molecular pathways involved in the HIV-1 neuroinflammation would be helpful in the design of adjunct therapy to ameliorate some of the symptoms associated with HIV-1 neuropathogenesis. PMID:26270987

  17. Megalencephalic leukoencephalopathy with subcortical cysts protein-1 regulates epidermal growth factor receptor signaling in astrocytes.

    PubMed

    Lanciotti, Angela; Brignone, Maria Stefania; Visentin, Sergio; De Nuccio, Chiara; Catacuzzeno, Luigi; Mallozzi, Cinzia; Petrini, Stefania; Caramia, Martino; Veroni, Caterina; Minnone, Gaetana; Bernardo, Antonietta; Franciolini, Fabio; Pessia, Mauro; Bertini, Enrico; Petrucci, Tamara Corinna; Ambrosini, Elena

    2016-04-15

    Mutations in the MLC1 gene, which encodes a protein expressed in brain astrocytes, are the leading cause of MLC, a rare leukodystrophy characterized by macrocephaly, brain edema, subcortical cysts, myelin and astrocyte vacuolation. Although recent studies indicate that MLC1 protein is implicated in the regulation of cell volume changes, the exact role of MLC1 in brain physiology and in the pathogenesis of MLC disease remains to be clarified. In preliminary experiments, we observed that MLC1 was poorly expressed in highly proliferating astrocytoma cells when compared with primary astrocytes, and that modulation of MLC1 expression influenced astrocyte growth. Because volume changes are key events in cell proliferation and during brain development MLC1 expression is inversely correlated to astrocyte progenitor proliferation levels, we investigated the possible role for MLC1 in the control of astrocyte proliferation. We found that overexpression of wild type but not mutant MLC1 in human astrocytoma cells hampered cell growth by favoring epidermal growth factor receptor (EGFR) degradation and by inhibiting EGF-induced Ca(+) entry, ERK1/2 and PLCγ1 activation, and calcium-activated KCa3.1 potassium channel function, all molecular pathways involved in astrocyte proliferation stimulation. Interestingly, MLC1 did not influence AKT, an EGFR-stimulated kinase involved in cell survival. Moreover, EGFR expression was higher in macrophages derived from MLC patients than from healthy individuals. Since reactive astrocytes proliferate and re-express EGFR in response to different pathological stimuli, the present findings provide new information on MLC pathogenesis and unravel an important role for MLC1 in other brain pathological conditions where astrocyte activation occurs. PMID:26908604

  18. C/EBPβ regulates multiple IL-1β-induced human astrocyte inflammatory genes

    PubMed Central

    2012-01-01

    Background CCAAT enhancer-binding protein (C/EBP)β regulates gene expression in multiple organ systems and cell types, including astrocytes in the central nervous system (CNS). Inflammatory stimuli, interleukin (IL)-1β, tumor necrosis factor-α, human immunodeficiency virus (HIV)-1 and lipopolysaccharide induce astrocyte C/EBPβ expression. C/EBPβ is detectable in brains of Alzheimer’s disease (AD), Parkinson’s disease (PD) and HIV-1-associated dementia (HAD) patients, yet little is known about how C/EBPβ contributes to astrocyte gene regulation during neuroinflammation. Methods The expression of 92 human inflammation genes was compared between IL-1β-treated primary human astrocytes and astrocytes transfected with C/EBPβ-specific small interfering (si)RNA prior to IL-1β treatment for 12 h. Transcripts altered by > two-fold compared to control were subjected to one-way analysis of variance and Newman-Keuls post-test for multiple comparisons. Expression of two genes, cyclooxygenase-2 (COX-2) and bradykinin receptor B2 (BDKRB2) was further confirmed in additional human astrocyte donors. Astrocytes were treated with mitogen-activated protein kinase-selective inhibitors, then with IL-1β for 12 or 24 h followed by COX-2 and BDKRB2, expression analyses. Results IL-1β altered expression of 29 of 92 human inflammation genes by at least two-fold in primary human astrocytes in 12 h. C/EBPβ knockdown affected expression of 17 out of 29 IL-1β-regulated genes by > 25%. Two genes relevant to neuroinflammation, COX-2 and BDKRB2, were robustly decreased and increased, respectively, in response to C/EBPβ knockdown, and expression was confirmed in two additional donors. COX-2 and BDKRB2 mRNA remained altered in siRNA-transfected astrocytes at 12, 24 or 72 h. Inhibiting p38 kinase (p38K) activation blocked IL-1β-induced astrocyte COX-2 mRNA and protein expression, but not IL-1β-induced astrocyte BDKRB2 expression. Inhibiting extracellular-regulated

  19. Astrocytes regulate cortical state switching in vivo

    PubMed Central

    Poskanzer, Kira E.; Yuste, Rafael

    2016-01-01

    The role of astrocytes in neuronal function has received increasing recognition, but disagreement remains about their function at the circuit level. Here we use in vivo two-photon calcium imaging of neocortical astrocytes while monitoring the activity state of the local neuronal circuit electrophysiologically and optically. We find that astrocytic calcium activity precedes spontaneous circuit shifts to the slow-oscillation–dominated state, a neocortical rhythm characterized by synchronized neuronal firing and important for sleep and memory. Further, we show that optogenetic activation of astrocytes switches the local neuronal circuit to this slow-oscillation state. Finally, using two-photon imaging of extracellular glutamate, we find that astrocytic transients in glutamate co-occur with shifts to the synchronized state and that optogenetically activated astrocytes can generate these glutamate transients. We conclude that astrocytes can indeed trigger the low-frequency state of a cortical circuit by altering extracellular glutamate, and therefore play a causal role in the control of cortical synchronizations. PMID:27122314

  20. Astrocytes regulate cortical state switching in vivo.

    PubMed

    Poskanzer, Kira E; Yuste, Rafael

    2016-05-10

    The role of astrocytes in neuronal function has received increasing recognition, but disagreement remains about their function at the circuit level. Here we use in vivo two-photon calcium imaging of neocortical astrocytes while monitoring the activity state of the local neuronal circuit electrophysiologically and optically. We find that astrocytic calcium activity precedes spontaneous circuit shifts to the slow-oscillation-dominated state, a neocortical rhythm characterized by synchronized neuronal firing and important for sleep and memory. Further, we show that optogenetic activation of astrocytes switches the local neuronal circuit to this slow-oscillation state. Finally, using two-photon imaging of extracellular glutamate, we find that astrocytic transients in glutamate co-occur with shifts to the synchronized state and that optogenetically activated astrocytes can generate these glutamate transients. We conclude that astrocytes can indeed trigger the low-frequency state of a cortical circuit by altering extracellular glutamate, and therefore play a causal role in the control of cortical synchronizations. PMID:27122314

  1. Ghrelin Regulates Glucose and Glutamate Transporters in Hypothalamic Astrocytes.

    PubMed

    Fuente-Martín, Esther; García-Cáceres, Cristina; Argente-Arizón, Pilar; Díaz, Francisca; Granado, Miriam; Freire-Regatillo, Alejandra; Castro-González, David; Ceballos, María L; Frago, Laura M; Dickson, Suzanne L; Argente, Jesús; Chowen, Julie A

    2016-01-01

    Hypothalamic astrocytes can respond to metabolic signals, such as leptin and insulin, to modulate adjacent neuronal circuits and systemic metabolism. Ghrelin regulates appetite, adiposity and glucose metabolism, but little is known regarding the response of astrocytes to this orexigenic hormone. We have used both in vivo and in vitro approaches to demonstrate that acylated ghrelin (acyl-ghrelin) rapidly stimulates glutamate transporter expression and glutamate uptake by astrocytes. Moreover, acyl-ghrelin rapidly reduces glucose transporter (GLUT) 2 levels and glucose uptake by these glial cells. Glutamine synthetase and lactate dehydrogenase decrease, while glycogen phosphorylase and lactate transporters increase in response to acyl-ghrelin, suggesting a change in glutamate and glucose metabolism, as well as glycogen storage by astrocytes. These effects are partially mediated through ghrelin receptor 1A (GHSR-1A) as astrocytes do not respond equally to desacyl-ghrelin, an isoform that does not activate GHSR-1A. Moreover, primary astrocyte cultures from GHSR-1A knock-out mice do not change glutamate transporter or GLUT2 levels in response to acyl-ghrelin. Our results indicate that acyl-ghrelin may mediate part of its metabolic actions through modulation of hypothalamic astrocytes and that this effect could involve astrocyte mediated changes in local glucose and glutamate metabolism that alter the signals/nutrients reaching neighboring neurons. PMID:27026049

  2. Ghrelin Regulates Glucose and Glutamate Transporters in Hypothalamic Astrocytes

    PubMed Central

    Fuente-Martín, Esther; García-Cáceres, Cristina; Argente-Arizón, Pilar; Díaz, Francisca; Granado, Miriam; Freire-Regatillo, Alejandra; Castro-González, David; Ceballos, María L.; Frago, Laura M.; Dickson, Suzanne L.; Argente, Jesús; Chowen, Julie A.

    2016-01-01

    Hypothalamic astrocytes can respond to metabolic signals, such as leptin and insulin, to modulate adjacent neuronal circuits and systemic metabolism. Ghrelin regulates appetite, adiposity and glucose metabolism, but little is known regarding the response of astrocytes to this orexigenic hormone. We have used both in vivo and in vitro approaches to demonstrate that acylated ghrelin (acyl-ghrelin) rapidly stimulates glutamate transporter expression and glutamate uptake by astrocytes. Moreover, acyl-ghrelin rapidly reduces glucose transporter (GLUT) 2 levels and glucose uptake by these glial cells. Glutamine synthetase and lactate dehydrogenase decrease, while glycogen phosphorylase and lactate transporters increase in response to acyl-ghrelin, suggesting a change in glutamate and glucose metabolism, as well as glycogen storage by astrocytes. These effects are partially mediated through ghrelin receptor 1A (GHSR-1A) as astrocytes do not respond equally to desacyl-ghrelin, an isoform that does not activate GHSR-1A. Moreover, primary astrocyte cultures from GHSR-1A knock-out mice do not change glutamate transporter or GLUT2 levels in response to acyl-ghrelin. Our results indicate that acyl-ghrelin may mediate part of its metabolic actions through modulation of hypothalamic astrocytes and that this effect could involve astrocyte mediated changes in local glucose and glutamate metabolism that alter the signals/nutrients reaching neighboring neurons. PMID:27026049

  3. Leptin signaling in astrocytes regulates hypothalamic neuronal circuits and feeding.

    PubMed

    Kim, Jae Geun; Suyama, Shigetomo; Koch, Marco; Jin, Sungho; Argente-Arizon, Pilar; Argente, Jesús; Liu, Zhong-Wu; Zimmer, Marcelo R; Jeong, Jin Kwon; Szigeti-Buck, Klara; Gao, Yuanqing; Garcia-Caceres, Cristina; Yi, Chun-Xia; Salmaso, Natalina; Vaccarino, Flora M; Chowen, Julie; Diano, Sabrina; Dietrich, Marcelo O; Tschöp, Matthias H; Horvath, Tamas L

    2014-07-01

    We found that leptin receptors were expressed in hypothalamic astrocytes and that their conditional deletion led to altered glial morphology and synaptic inputs onto hypothalamic neurons involved in feeding control. Leptin-regulated feeding was diminished, whereas feeding after fasting or ghrelin administration was elevated in mice with astrocyte-specific leptin receptor deficiency. These data reveal an active role of glial cells in hypothalamic synaptic remodeling and control of feeding by leptin. PMID:24880214

  4. p53 isoforms regulate astrocyte-mediated neuroprotection and neurodegeneration.

    PubMed

    Turnquist, C; Horikawa, I; Foran, E; Major, E O; Vojtesek, B; Lane, D P; Lu, X; Harris, B T; Harris, C C

    2016-09-01

    Bidirectional interactions between astrocytes and neurons have physiological roles in the central nervous system and an altered state or dysfunction of such interactions may be associated with neurodegenerative diseases, such as Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS). Astrocytes exert structural, metabolic and functional effects on neurons, which can be either neurotoxic or neuroprotective. Their neurotoxic effect is mediated via the senescence-associated secretory phenotype (SASP) involving pro-inflammatory cytokines (e.g., IL-6), while their neuroprotective effect is attributed to neurotrophic growth factors (e.g., NGF). We here demonstrate that the p53 isoforms Δ133p53 and p53β are expressed in astrocytes and regulate their toxic and protective effects on neurons. Primary human astrocytes undergoing cellular senescence upon serial passaging in vitro showed diminished expression of Δ133p53 and increased p53β, which were attributed to the autophagic degradation and the SRSF3-mediated alternative RNA splicing, respectively. Early-passage astrocytes with Δ133p53 knockdown or p53β overexpression were induced to show SASP and to exert neurotoxicity in co-culture with neurons. Restored expression of Δ133p53 in near-senescent, otherwise neurotoxic astrocytes conferred them with neuroprotective activity through repression of SASP and induction of neurotrophic growth factors. Brain tissues from AD and ALS patients possessed increased numbers of senescent astrocytes and, like senescent astrocytes in vitro, showed decreased Δ133p53 and increased p53β expression, supporting that our in vitro findings recapitulate in vivo pathology of these neurodegenerative diseases. Our finding that Δ133p53 enhances the neuroprotective function of aged and senescent astrocytes suggests that the p53 isoforms and their regulatory mechanisms are potential targets for therapeutic intervention in neurodegenerative diseases. PMID:27104929

  5. Fatty acid-binding protein 7 regulates function of caveolae in astrocytes through expression of caveolin-1.

    PubMed

    Kagawa, Yoshiteru; Yasumoto, Yuki; Sharifi, Kazem; Ebrahimi, Majid; Islam, Ariful; Miyazaki, Hirofumi; Yamamoto, Yui; Sawada, Tomoo; Kishi, Hiroko; Kobayashi, Sei; Maekawa, Motoko; Yoshikawa, Takeo; Takaki, Eiichi; Nakai, Akira; Kogo, Hiroshi; Fujimoto, Toyoshi; Owada, Yuji

    2015-05-01

    Fatty acid-binding proteins (FABPs) bind and solubilize long-chain fatty acids, controlling intracellular lipid dynamics. FABP7 is expressed by astrocytes in the developing brain, and suggested to be involved in the control of astrocyte lipid homeostasis. In this study, we sought to examine the role of FABP7 in astrocytes, focusing on plasma membrane lipid raft function, which is important for receptor-mediated signal transduction in response to extracellular stimuli. In FABP7-knockout (KO) astrocytes, the ligand-dependent accumulation of Toll-like receptor 4 (TLR4) and glial cell-line-derived neurotrophic factor receptor alpha 1 into lipid raft was decreased, and the activation of mitogen-activated protein kinases and nuclear factor-κB was impaired after lipopolysaccharide (LPS) stimulation when compared with wild-type astrocytes. In addition, the expression of caveolin-1, not cavin-1, 2, 3, caveolin-2, and flotillin-1, was found to be decreased at the protein and transcriptional levels. FABP7 re-expression in FABP7-KO astrocytes rescued the decreased level of caveolin-1. Furthermore, caveolin-1-transfection into FABP7-KO astrocytes significantly increased TLR4 recruitment into lipid raft and tumor necrosis factor-α production after LPS stimulation. Taken together, these data suggest that FABP7 controls lipid raft function through the regulation of caveolin-1 expression and is involved in the response of astrocytes to the external stimuli. GLIA 2015;63:780-794. PMID:25601031

  6. NEURONAL ACTIVITY REGULATES GLUTAMATE TRANSPORTER DYNAMICS IN DEVELOPING ASTROCYTES

    PubMed Central

    Benediktsson, A.M.; Marrs, G.S.; Tu, J.C.; Worley, P.F.; Rothstein, J.D.; Bergles, D.E.; Dailey, M.E.

    2011-01-01

    Glutamate transporters maintain a low ambient level of glutamate in the CNS and shape the activation of glutamate receptors at synapses. Nevertheless, the mechanisms that regulate the trafficking and localization of transporters near sites of glutamate release are poorly understood. Here we examined the subcellular distribution and dynamic remodeling of the predominant glutamate transporter GLT-1 (EAAT2) in developing hippocampal astrocytes. Immunolabeling revealed that endogenous GLT-1 is concentrated into discrete clusters along branches of developing astrocytes that were apposed preferentially to synapsin-1 positive synapses. GFP-GLT-1 fusion proteins expressed in astrocytes also formed distinct clusters that lined the edges of astrocyte processes, as well as the tips of filopodia and spine-like structures. Time-lapse 3D confocal imaging in tissue slices revealed that GFP-GLT-1 clusters were dynamically remodeled on a timescale of minutes. Some transporter clusters moved within developing astrocyte branches as filopodia extended and retracted, while others maintained stable positions at the tips of spine-like structures. Blockade of neuronal activity with tetrodotoxin reduced both the density and perisynaptic localization of GLT-1 clusters. Conversely, enhancement of neuronal activity increased the size of GLT-1 clusters and their proximity to synapses. Together, these findings indicate that neuronal activity influences both the organization of glutamate transporters in developing astrocyte membranes and their position relative to synapses. PMID:22052455

  7. Astrocytes.

    ERIC Educational Resources Information Center

    Kimelberg, Harold K.; Norenberg, Michael D.

    1989-01-01

    Describes the astrocytes' function as equal partners with neurons in both the normal and the abnormal brain. Discusses the developmental scaffolds, inert scar tissue, Huntington's disease, psychiatric disorders, and the identification of these brain cells. (RT)

  8. Astrocyte sodium signaling and the regulation of neurotransmission.

    PubMed

    Kirischuk, Sergei; Héja, László; Kardos, Julianna; Billups, Brian

    2016-10-01

    The transmembrane Na(+) concentration gradient is an important source of energy required not only to enable the generation of action potentials in excitable cells, but also for various transmembrane transporters both in excitable and non-excitable cells, like astrocytes. One of the vital functions of astrocytes in the central nervous system (CNS) is to regulate neurotransmitter concentrations in the extracellular space. Most neurotransmitters in the CNS are removed from the extracellular space by Na(+) -dependent neurotransmitter transporters (NeuTs) expressed both in neurons and astrocytes. Neuronal NeuTs control mainly phasic synaptic transmission, i.e., synaptically induced transient postsynaptic potentials, while astrocytic NeuTs contribute to the termination of phasic neurotransmission and modulate the tonic tone, i.e., the long-lasting activation of extrasynaptic receptors by neurotransmitter that has diffused out of the synaptic cleft. Consequently, local intracellular Na(+) ([Na(+) ]i ) transients occurring in astrocytes, for example via the activation of ionotropic neurotransmitter receptors, can affect the driving force for neurotransmitter uptake, in turn modulating the spatio-temporal profiles of neurotransmitter levels in the extracellular space. As some NeuTs are close to thermodynamic equilibrium under resting conditions, an increase in astrocytic [Na(+) ]i can stimulate the direct release of neurotransmitter via NeuT reversal. In this review we discuss the role of astrocytic [Na(+) ]i changes in the regulation of uptake/release of neurotransmitters. It is emphasized that an activation of one neurotransmitter system, including either its ionotropic receptor or Na(+) -coupled co-transporter, can strongly influence, or even reverse, other Na(+) -dependent NeuTs, with potentially significant consequences for neuronal communication. GLIA 2016;64:1655-1666. PMID:26566753

  9. B4GALT6 regulates astrocyte activation during CNS inflammation

    PubMed Central

    Mayo, Lior; Trauger, Sunia A.; Blain, Manon; Nadeau, Meghan; Patel, Bonny; Alvarez, Jorge I.; Mascanfroni, Ivan D.; Yeste, Ada; Kivisäkk, Pia; Kallas, Keith; Ellezam, Benjamin; Bakshi, Rohit; Prat, Alexandre; Antel, Jack P.; Weiner, Howard L.; Quintana, Francisco J.

    2014-01-01

    Astrocytes play complex roles in the response to trauma, infection or inflammation in the central nervous system (CNS). Thus, it is important to characterize the mechanisms regulating astrocyte function, as well as potential targets for the therapeutic modulation of astrocyte activity. Here we report that lactosylceramide (LacCer) levels are up-regulated in the CNS during chronic experimental autoimmune encephalomyelitis (EAE), an experimental model of multiple sclerosis (MS). We found that LacCer synthesized by β-1,4-galactosyltransferase 6 (B4GALT6) in astrocytes acts in an autocrine manner to trigger transcriptional programs that promote the recruitment and activation of CNS-infiltrating monocytes and microglia, and neurodegeneration. We also detected increased B4GALT6 expression and LacCer levels in CNS MS lesions. Finally, the inhibition of LacCer synthesis suppressed local CNS innate immunity and neurodegeneration in EAE, and interfered with the activation of human astrocytes in vitro. Thus, B4GALT6 is a potential therapeutic target for MS and other neuroinflammatory disorders. PMID:25216636

  10. 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

  11. p38 mitogen-activated protein kinase inhibitor reduces neurocan production in cultured spinal cord astrocytes.

    PubMed

    Yamaoka, Gotaro; Morino, Tadao; Morizane, Kei; Horiuchi, Hideki; Miura, Hiromasa; Ogata, Tadanori

    2012-06-20

    Chondroitin sulfate proteoglycans are formed in scar tissue after a spinal cord injury and inhibit axon regrowth. The production of neurocan, one of these chondroitin sulfate proteoglycans, in cultured spinal cord astrocytes increased after the addition of epidermal growth factor (EGF) in a dose-dependent manner (2-200 ng/ml). In astrocytes stimulated by 20 ng/ml of EGF, neurocan production was inhibited after the addition of the p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580: 3-10 μM) in a dose-dependent manner. These results suggest that the activation of p38 MAPK is one of the mechanisms of neurocan production in EGF-stimulated astrocytes. The p38 MAPK inhibitor may reduce neurocan production and accelerate axonal regrowth after a spinal cord injury. PMID:22525836

  12. Apoptosis signal-regulating kinase 1 mediates striatal degeneration via the regulation of C1q

    PubMed Central

    Cho, Kyoung Joo; Cheon, So Young; Kim, Gyung Whan

    2016-01-01

    Apoptosis signal-regulating kinase-1 (ASK1), an early signaling element in the cell death pathway, has been hypothesized to participate in the pathology of neurodegenerative diseases. The systemic administration of 3-nitropropionic acid (3-NP) facilitates the development of selective striatal lesions. However, it remains unclear whether specific neurons are selectively targeted in 3-NP-infused striatal degeneration. Recently, it has been proposed that complement-mediated synapse elimination may be reactivated aberrantly in the pathology of neurodegenerative diseases. We hypothesized that ASK1 is involved in striatal astrocyte reactivation; reactive astrocyte secretes molecules detrimental to neuron; and striatal neurons are more susceptible to these factors. Our results indicate that striatal astrocyte is reactivated and ASK1 level increases after 3-NP general and chronic infusion. Reactive striatal astrocyte increases TGF-beta differentially to cortex and striatum. ASK1 may be involved in regulation of astrocyte TGF-beta and it is linked to the C1q level in spatial and temporal, and moreover in the earlier stage of progressing striatal neuronal loss. Conclusively the present study suggests that ASK1 mediates 3-NP toxicity and regulates C1q level through the astrocyte TGF-beta. And also it may suggest that C1q level may be a surrogate of prediction marker representing neurodegenerative disease progress before developing behavioral impairment. PMID:26728245

  13. Phosphorylation of Mitogen- and Stress-Activated Protein Kinase-1 in Astrocytic Inflammation: A Possible Role in Inhibiting Production of Inflammatory Cytokines

    PubMed Central

    Shi, Jinlong; Ni, Lanchun; Huang, Qingfeng; Xia, Liang; Nie, Dekang; Lu, Xiaojian; Chen, Jian; Shi, Wei

    2013-01-01

    Purpose It is generally accepted that inflammation has a role in the progression of many central nervous system (CNS) diseases, although the mechanisms through which this occurs remain unclear. Among mitogen-activated protein kinase (MAPK) targets, mitogen- and stress-activated protein kinase (MSK1) has been thought to be involved in the pathology of inflammatory gene expression. In this study, the roles of MSK1 activation in neuroinflammation were investigated. Methods The bacterial lipopolysaccharide (LPS)-induced brain injury model was performed on Sprague-Dawley rats. The dynamic expression changes and the cellular location of p-MSK1 in the brain cortex were detected by Western blot and immunofluorescence staining. The synthesis of inflammatory cytokines in astrocytes was detected by enzyme-linked immunosorbent assay (ELISA). Results Phosphorylated MSK1 (p-MSK1 Thr-581) was induced significantly after intracerebral injection of LPS into the lateral ventricles of the rat brain. Specific upregulation of p-MSK1 in astrocytes was also observed in inflamed cerebral cortex. At 1 day after LPS stimulation, iNOS, TNFα expression, and the astrocyte marker glial fibrillary acidic protein (GFAP) were increased significantly. Also, in vitro studies indicated that the upregulation of p-MSK1 (Thr-581) may be involved in the subsequent astrocyte inflammatory process, following LPS challenge. Using an enzyme-linked immunosorbent assay (ELISA), it was confirmed that treatment with LPS in primary astrocytes stimulated the synthesis of inflammatory cytokines, through MAPKs signaling pathways. In cultured primary astrocytes, both knock-down of total MSK1 by small interfering RNAs (siRNA) or specific mutation of Thr-581 resulted in higher production of certain cytokines, such as TNFα and IL-6. Conclusions Collectively, these results suggest that MSK1 phosphorylation is associated with the regulation of LPS-induced brain injury and possibly acts as a negative regulator of

  14. Metabolic Interplay between Astrocytes and Neurons Regulates Endocannabinoid Action

    PubMed Central

    Viader, Andreu; Blankman, Jacqueline L.; Zhong, Peng; Liu, Xiaojie; Schlosburg, Joel E.; Joslyn, Christopher M.; Liu, Qing-Song; Tomarchio, Aaron J.; Lichtman, Aron H.; Selley, Dana E.; Sim-Selley, Laura J.; Cravatt, Benjamin F.

    2015-01-01

    Summary The endocannabinoid 2-arachidonoylglycerol (2-AG) is a retrograde lipid messenger that modulates synaptic function, neurophysiology, and behavior. 2-AG signaling is terminated by enzymatic hydrolysis—a reaction that is principally performed by monoacylglycerol lipase (MAGL). MAGL is broadly expressed throughout the nervous system, and the contributions of different brain cell types to regulating 2-AG activity have remained unclear. Here, we genetically dissect the cellular anatomy of MAGL-mediated 2-AG metabolism in the brain and show that neurons and astrocytes coordinately regulate 2-AG content and endocannabinoid-dependent forms of synaptic plasticity and behavior. We also find that astrocytic MAGL is mainly responsible for converting 2-AG to neuroinflammatory prostaglandins via a mechanism that may involve transcellular shuttling of lipid substrates. Astrocytic-neuronal interplay thus provides distributed oversight of 2-AG metabolism and function, and, through doing so, protects the nervous system from excessive CB1 receptor activation and promotes endocannabinoid crosstalk with other lipid transmitter systems. PMID:26212325

  15. Astrocytes regulate adult hippocampal neurogenesis through ephrin-B signaling

    PubMed Central

    Ashton, Randolph S.; Conway, Anthony; Pangarkar, Chinmay; Bergen, Jamie; Lim, Kwang-Il; Shah, Priya; Bissell, Mina; Schaffer, David V.

    2012-01-01

    Neurogenesis in the adult hippocampus involves activation of quiescent neural stem cells (NSCs) to yield transiently amplifying NSCs and progenitors, and ultimately neurons that affect learning and memory. This process is tightly controlled by microenvironmental cues, though few endogenous factors are known to regulate neuronal differentiation. While astrocytes have been implicated, their role in juxtacrine (i.e. cell-cell contact-dependent) signaling within NSC niches has not been investigated. We show that ephrin-B2 presented from rodent hippocampal astrocytes regulates neurogenesis in vivo. Furthermore, clonal analysis in NSC fate-mapping studies reveals a novel role for ephrin-B2 in instructing neuronal differentiation. Additionally, ephrin-B2 signaling, transduced by EphB4 receptors on NSCs, activates β-catenin in vitro and in vivo independent of Wnt signaling and upregulates proneural transcription factors. Ephrin-B2+ astrocytes thus promote neuronal differentiation of adult NSCs through juxtacrine signaling, findings that advance our understanding of adult neurogenesis and may have future regenerative medicine implications. PMID:22983209

  16. Radiation induction of the receptor tyrosine kinase gene Ptk-3 in normal rat astrocytes

    SciTech Connect

    Sakuma, S.; Hideyuki, S.; Akihiro, I.

    1995-07-01

    Radiation-induced gene expression was examined in rat astrocyte cultures using differential display of mRNA via reverse transcriptase-polymerase chain reaction. A 0.3-kb cDNA that was consistently observed in irradiated cultures but not in unirradiated cultures was cloned and sequenced. It was found to be identical to Ptk-3, a receptor tyrosine kinase gene identified recently. The protein encoded by Ptk-3 is a member of a novel class of receptor tyrosine kinases whose extracellular domain contains regions of homology with coagulation factors V and VIII and complement component C1. Northern blot analysis revealed that the expression of Ptk-3 was increased in rat astrocytes by 0.5 h after exposure to 10 Gy and remained at the same elevated level for at least 24 h. The maximum increase occurred after 5 Gy cloning studies indicated the presence of at least two Ptk-3 mRNA transcripts, which are probable the result of an alternative splicing mechanism. The short isoform lacks a 37 amino acid sequence in the glycine/proline-rich juxtamembrane region. The splicing pattern of the Ptk-3 gene was not altered by radiation. However, the ratios of the longer to the shorter mRNA transcripts differed between adult cortex, neonatal cortex and in vitro astrocyte cultures. 36 refs., 5 figs.

  17. Lactate Produced by Glycogenolysis in Astrocytes Regulates Memory Processing

    PubMed Central

    Newman, Lori A.; Korol, Donna L.; Gold, Paul E.

    2011-01-01

    When administered either systemically or centrally, glucose is a potent enhancer of memory processes. Measures of glucose levels in extracellular fluid in the rat hippocampus during memory tests reveal that these levels are dynamic, decreasing in response to memory tasks and loads; exogenous glucose blocks these decreases and enhances memory. The present experiments test the hypothesis that glucose enhancement of memory is mediated by glycogen storage and then metabolism to lactate in astrocytes, which provide lactate to neurons as an energy substrate. Sensitive bioprobes were used to measure brain glucose and lactate levels in 1-sec samples. Extracellular glucose decreased and lactate increased while rats performed a spatial working memory task. Intrahippocampal infusions of lactate enhanced memory in this task. In addition, pharmacological inhibition of astrocytic glycogenolysis impaired memory and this impairment was reversed by administration of lactate or glucose, both of which can provide lactate to neurons in the absence of glycogenolysis. Pharmacological block of the monocarboxylate transporter responsible for lactate uptake into neurons also impaired memory and this impairment was not reversed by either glucose or lactate. These findings support the view that astrocytes regulate memory formation by controlling the provision of lactate to support neuronal functions. PMID:22180782

  18. Substrate-dependent regulation of ascorbate transport in astrocytes

    SciTech Connect

    Wilson, J.X.; Jaworski, E.M.; Kulaga, A.; Dixon, S.J. )

    1990-02-26

    Astrocytes possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na{sup +}-dependent L-ascorbate transporter in the plasma membrane. The present study examined the effects of ascorbate deprivation and supplementation on the activity of the transport system. Initial rates of L-ascorbate uptake were measured by incubating primary cultures of rat astrocytes with L-({sup 14}C)ascorbate for 1 minute at 37C. They observed that the maximal uptake rate, V{sub max}, rapidly (<3 hours) increased when cultured cells were deprived of L-ascorbate. There was no change in the apparent affinity (K{sub m}) of the transport system for ascorbate. V{sub max} returned to normal following addition of L-ascorbate, but not D-isoascorbate, to the medium. The authors conclude that astrocytes adapt ascorbate transport rates to changes in substrate availability. Furthermore, the data suggest that the transport system located in the astroglial plasma membrane regulates intracellular ascorbate concentration, because changes in transport rate may compensate for regional differences and temporal fluctuations in extracellular ascorbate levels.

  19. Energy-dependent volume regulation in primary cultured cerebral astrocytes.

    PubMed

    Olson, J E; Sankar, R; Holtzman, D; James, A; Fleischhacker, D

    1986-08-01

    Cell volume regulation and energy metabolism were studied in primary cultured cerebral astrocytes during exposure to media of altered osmolarity. Cells suspended in medium containing 1/2 the normal concentration of NaCl (hypoosmotic) swell immediately to a volume 40-50% larger than cells suspended in isoosmotic medium. The cell volume in hypoosmotic medium then decreases over 30 min to a volume approximately 25% larger than cells in isoosmotic medium. In hyperosmotic medium (containing twice the normal concentration of NaCl), astrocytes shrink by 29%. Little volume change occurs following this initial shrinkage. Cells resuspended in isoosmotic medium after a 30 min incubation in hypoosmotic medium shrink immediately to a volume 10% less than the volume of cells incubated continuously in isoosmotic medium. Thus, the regulatory volume decrease (RVD) in hypoosmotic medium involves a net reduction of intracellular osmoles. The RVD is partially blocked by inhibitors of mitochondrial electron transport but is unaffected by an inhibitor of glycolysis or by an uncoupler of oxidative phosphorylation. Inhibition of RVD by these metabolic agents is correlated with decreased cellular ATP levels. Ouabain, added immediately after hypoosmotic induced swelling, completely inhibits RVD, but does not alter cell volume if added after RVD has taken place. Ouabain also inhibits cell respiration 27% more in hypoosmotic medium than in isoosmotic medium indicating that the (Na,K)-ATPase-coupled ion pump is more active in the hypoosmotic medium. These data suggest that the cell volume response of astrocytes in hypoosmotic medium involves the net movement of osmoles by a mechanism dependent on cellular energy and tightly coupled to the (Na,K)-ATPase ion pump. This process may be important in the energy-dependent osmoregulation in the brain, a critical role attributed to the astrocyte in vivo. PMID:3015986

  20. Substrate regulation of ascorbate transport activity in astrocytes

    SciTech Connect

    Wilson, J.X.; Jaworski, E.M.; Kulaga, A.; Dixon, S.J. )

    1990-10-01

    Astrocytes possess a concentrative L-ascorbate (vitamin C) uptake mechanism involving a Na(+)-dependent L-ascorbate transporter located in the plasma membrane. The present experiments examined the effects of deprivation and supplementation of extracellular L-ascorbate on the activity of this transport system. Initial rates of L-ascorbate uptake were measured by incubating primary cultures of rat astrocytes with L-(14C)ascorbate for 1 min at 37 degrees C. We observed that the apparent maximal rate of uptake (Vmax) increased rapidly (less than 1 h) when cultured cells were deprived of L-ascorbate. In contrast, there was no change in the apparent affinity of the transport system for L-(14C)ascorbate. The increase in Vmax was reversed by addition of L-ascorbate, but not D-isoascorbate, to the medium. The effects of external ascorbate on ascorbate transport activity were specific in that preincubation of cultures with L-ascorbate did not affect uptake of 2-deoxy-D-(3H(G))glucose. We conclude that the astroglial ascorbate transport system is modulated by changes in substrate availability. Regulation of transport activity may play a role in intracellular ascorbate homeostasis by compensating for regional differences and temporal fluctuations in external ascorbate levels.

  1. Astrocyte and Macrophage Regulation of YKL-40 Expression and Cellular Response in Neuroinflammation

    PubMed Central

    Bonneh-Barkay, Dafna; Bissel, Stephanie J.; Kofler, Julia; Starkey, Adam; Wang, Guoji; Wiley, Clayton A.

    2013-01-01

    Numerous inflammatory conditions are associated with elevated YKL-40 expression by infiltrating macrophages. Thus, we were surprised to observe minimal macrophage and abundant astrocyte expression of YKL-40 in neuroinflammatory conditions. The aims of the current study were to better delineate this discrepancy, characterize the factors that regulate YKL-40 expression in macrophages and astrocytes and study whether YKL-40 expression correlates with cell morphology and/or activation state. In vitro, macrophages expressed high levels of YKL-40 that was induced by classical activation and inhibited by alternative activation. Cytokines released from macrophages induced YKL-40 transcription in astrocytes that was accompanied by morphological changes and altered astrocytic motility. Because coculturing of astrocytes and macrophages did not reverse this in vitro expression pattern, additional components of the in vivo central nervous system (CNS) milieu must be required to suppress macrophage and induce astrocyte expression of YKL-40. PMID:22074331

  2. Regulation of astrocyte activity via control over stiffness of cellulose acetate electrospun nanofiber.

    PubMed

    Min, Seul Ki; Jung, Sang Myung; Ju, Jung Hyeon; Kwon, Yeo Seon; Yoon, Gwang Heum; Shin, Hwa Sung

    2015-10-01

    Astrocytes are involved in neuron protection following central nervous system (CNS) injury; accordingly, engineered astrocytes have been investigated for their usefulness in cell therapy for CNS injury. Nanofibers have attracted a great deal of attention in neural tissue engineering, but their mechanical properties greatly influence physiology. Cellulose acetate (CA) has been studied for use in scaffolds owing to its biocompatibility, biodegradability, and good thermal stability. In this study, stiffness of CA nanofibers controlled by heat treatment was shown to regulate astrocyte activity. Adhesion and viability increased in culture as substrate became stiffer but showed saturation at greater than 2 MPa of tensile strength. Astrocytes became more active in terms of increasing intermediate filament glial fibrillary acidic protein (GFAP). The results of this study demonstrate the effects of stiffness alone on cellular behaviors in a three-dimensional culture and highlight the efficacy of heat-treated CA for astrocyte culture in that the simple treatment enables control of astrocyte activity. PMID:26091629

  3. Leptin regulates glutamate and glucose transporters in hypothalamic astrocytes

    PubMed Central

    Fuente-Martín, Esther; García-Cáceres, Cristina; Granado, Miriam; de Ceballos, María L.; Sánchez-Garrido, Miguel Ángel; Sarman, Beatrix; Liu, Zhong-Wu; Dietrich, Marcelo O.; Tena-Sempere, Manuel; Argente-Arizón, Pilar; Díaz, Francisca; Argente, Jesús; Horvath, Tamas L.; Chowen, Julie A.

    2012-01-01

    Glial cells perform critical functions that alter the metabolism and activity of neurons, and there is increasing interest in their role in appetite and energy balance. Leptin, a key regulator of appetite and metabolism, has previously been reported to influence glial structural proteins and morphology. Here, we demonstrate that metabolic status and leptin also modify astrocyte-specific glutamate and glucose transporters, indicating that metabolic signals influence synaptic efficacy and glucose uptake and, ultimately, neuronal function. We found that basal and glucose-stimulated electrical activity of hypothalamic proopiomelanocortin (POMC) neurons in mice were altered in the offspring of mothers fed a high-fat diet. In adulthood, increased body weight and fasting also altered the expression of glucose and glutamate transporters. These results demonstrate that whole-organism metabolism alters hypothalamic glial cell activity and suggest that these cells play an important role in the pathology of obesity. PMID:23064363

  4. Antofine-induced connexin43 gap junction disassembly in rat astrocytes involves protein kinase Cβ.

    PubMed

    Huang, Yu-Fang; Liao, Chih-Kai; Lin, Jau-Chen; Jow, Guey-Mei; Wang, Hwai-Shi; Wu, Jiahn-Chun

    2013-03-01

    Antofine, a phenanthroindolizidine alkaloid derived from Cryptocaryachinensis and Ficusseptica in the Asclepiadaceae milkweed family, is cytotoxic for various cancer cell lines. In this study, we demonstrated that treatment of rat primary astrocytes with antofine induced dose-dependent inhibition of gap junction intercellular communication (GJIC), as assessed by scrape-loading 6-carboxyfluorescein dye transfer. Levels of Cx43 protein were also decreased in a dose- and time-dependent manner following antofine treatment. Double-labeling immunofluorescence microscopy showed that antofine (10ng/ml) induced endocytosis of surface gap junctions into the cytoplasm, where Cx43 was co-localized with the early endosome marker EEA1. Inhibition of lysosomes or proteasomes by co-treatment with antofine and their respective specific inhibitors, NH4Cl or MG132, partially inhibited the antofine-induced decrease in Cx43 protein levels, but did not inhibit the antofine-induced inhibition of GJIC. After 30min of treatment, antofine induced a rapid increase in the intracellular Ca(2+) concentration and activation of protein kinase C (PKC)α/βII, which was maintained for at least 6h. Co-treatment of astrocytes with antofine and the intracellular Ca(2+) chelator BAPTA-AM prevented downregulation of Cx43 and inhibition of GJIC. Moreover, co-treatment with antofine and a specific PKCβ inhibitor prevented endocytosis of gap junctions, downregulation of Cx43, and inhibition of GJIC. Taken together, these findings indicate that antofine induces Cx43 gap junction disassembly by the PKCβ signaling pathway. Inhibition of GJIC by antofine may undermine the neuroprotective effect of astrocytes in CNS. PMID:23403203

  5. Treatment of cultured human astrocytes and vascular endothelial cells with protein kinase CK2 inhibitors induces early changes in cell shape and cytoskeleton.

    PubMed

    Kramerov, A A; Golub, A G; Bdzhola, V G; Yarmoluk, S M; Ahmed, K; Bretner, M; Ljubimov, A V

    2011-03-01

    Ubiquitous protein kinase CK2 is a key regulator of cell migration, proliferation and tumor growth. CK2 is abundant in retinal astrocytes, and its inhibition suppresses retinal neovascularization in a mouse retinopathy model. In human astrocytes, CK2 co-distributes with GFAP-containing intermediate filaments, which implies its association with cytoskeleton. Contrary to astrocytes, CK2 is co-localized in microvascular endothelial cells (HBMVEC) with microtubules and actin stress fibers, but not with vimentin-containing intermediate filaments. Specific CK2 inhibitors (TBB, TBI, TBCA and DMAT) and nine novel CK2 inhibiting compounds (TID43, TID46, Quinolone-7, Quinolone-39, FNH28, FNH62, FNH64, FNH68 and FNH74) were tested at 10-200 μM for their ability to induce morphological alterations in cultured human astrocytes (HAST-40), and HBMVEC (For explanation of the inhibitor names, see "Methods" section). CK2 inhibitors caused dramatic changes in shape of cultured cells with effective inhibitor concentrations between 50 and 100 μM. Attached cells retracted, acquired shortened processes, and eventually rounded up and detached. CK2 inhibitor-induced morphological alterations were completely reversible and were not blocked by caspase inhibition. However, longer treatment or higher inhibitor concentration did cause apoptosis. The speed and potency of the CK2 inhibitors effects on cell shape and adhesion were inversely correlated with serum concentration. Western analyses showed that TBB and TBCA elicited a significant (about twofold) increase in the activation of p38 and ERK1/2 MAP kinases that may be involved in cytoskeleton regulation. This novel early biological cell response to CK2 inhibition may underlie the anti-angiogenic effect of CK2 suppression in the retina. PMID:21125314

  6. Phorbol 12-myristate 13-acetate induces protein kinase ceta-specific proliferative response in astrocytic tumor cells.

    PubMed

    Hussaini, I M; Karns, L R; Vinton, G; Carpenter, J E; Redpath, G T; Sando, J J; VandenBerg, S R

    2000-07-21

    Protein kinase C (PKC) activation has been implicated in cellular proliferation in neoplastic astrocytes. The roles for specific PKC isozymes in regulating this glial response, however, are not well understood. The aim of this study was to characterize the expression of PKC isozymes and the role of PKC-eta expression in regulating cellular proliferation in two well characterized astrocytic tumor cell lines (U-1242 MG and U-251 MG) with different properties of growth in cell culture. Both cell lines expressed an array of conventional (alpha, betaI, betaII, and gamma) and novel (theta and epsilon) PKC isozymes that can be activated by phorbol myristate acetate (PMA). Another novel PKC isozyme, PKC-eta, was only expressed by U-251 MG cells. In contrast, PKC-delta was readily detected in U-1242 MG cells but was present only at low levels in U-251 MG cells. PMA (100 nm) treatment for 24 h increased cell proliferation by over 2-fold in the U-251 MG cells, whereas it decreased the mitogenic response in the U-1242 MG cells by over 90%. When PKC-eta was stably transfected into U-1242 MG cells, PMA increased cell proliferation by 2.2-fold, similar to the response of U-251 MG cells. The cell proliferation induced by PMA in both the U-251 MG and U-1242-PKC-eta cells was blocked by the PKC inhibitor bisindolylmaleimide (0.5 micrometer) and the MEK inhibitor, PD 98059 (50 micrometer). Transient transfection of wild type U-251 with PKC-eta antisense oligonucleotide (1 micrometer) also blocked the PMA-induced increase in [(3)H]thymidine incorporation. The data demonstrate that two glioblastoma lines, with functionally distinct proliferative responses to PMA, express different novel PKC isozymes and that the differential expression of PKC-eta plays a determining role in the different proliferative capacity. PMID:10806212

  7. Miro1 Regulates Activity-Driven Positioning of Mitochondria within Astrocytic Processes Apposed to Synapses to Regulate Intracellular Calcium Signaling.

    PubMed

    Stephen, Terri-Leigh; Higgs, Nathalie F; Sheehan, David F; Al Awabdh, Sana; López-Doménech, Guillermo; Arancibia-Carcamo, I Lorena; Kittler, Josef T

    2015-12-01

    It is fast emerging that maintaining mitochondrial function is important for regulating astrocyte function, although the specific mechanisms that govern astrocyte mitochondrial trafficking and positioning remain poorly understood. The mitochondrial Rho-GTPase 1 protein (Miro1) regulates mitochondrial trafficking and detachment from the microtubule transport network to control activity-dependent mitochondrial positioning in neurons. However, whether Miro proteins are important for regulating signaling-dependent mitochondrial dynamics in astrocytic processes remains unclear. Using live-cell confocal microscopy of rat organotypic hippocampal slices, we find that enhancing neuronal activity induces transient mitochondrial remodeling in astrocytes, with a concomitant, transient reduction in mitochondrial trafficking, mediated by elevations in intracellular Ca(2+). Stimulating neuronal activity also induced mitochondrial confinement within astrocytic processes in close proximity to synapses. Furthermore, we show that the Ca(2+)-sensing EF-hand domains of Miro1 are important for regulating mitochondrial trafficking in astrocytes and required for activity-driven mitochondrial confinement near synapses. Additionally, activity-dependent mitochondrial positioning by Miro1 reciprocally regulates the levels of intracellular Ca(2+) in astrocytic processes. Thus, the regulation of intracellular Ca(2+) signaling, dependent on Miro1-mediated mitochondrial positioning, could have important consequences for astrocyte Ca(2+) wave propagation, gliotransmission, and ultimately neuronal function. PMID:26631479

  8. Proteomic analysis of astrocytic secretion that regulates neurogenesis using quantitative amine-specific isobaric tagging

    SciTech Connect

    Yan, Hu; Zhou, Wenhao; Wei, Liming; Zhong, Fan; Yang, Yi

    2010-01-08

    Astrocytes are essential components of neurogenic niches that affect neurogenesis through membrane association and/or the release of soluble factors. To identify factors released from astrocytes that could regulate neural stem cell differentiation and proliferation, we used mild oxygen-glucose deprivation (OGD) to inhibit the secretory capacity of astrocytes. Using the Transwell co-culture system, we found that OGD-treated astrocytes could not promote neural stem cell differentiation and proliferation. Next, isobaric tagging for the relative and absolute quantitation (iTRAQ) proteomics techniques was performed to identify the proteins in the supernatants of astrocytes (with or without OGD). Through a multi-step analysis and gene ontology classification, 130 extracellular proteins were identified, most of which were involved in neuronal development, the inflammatory response, extracellular matrix composition and supportive functions. Of these proteins, 44 had never been reported to be produced by astrocytes. Using ProteinPilot software analysis, we found that 60 extracellular proteins were significantly altered (27 upregulated and 33 downregulated) in the supernatant of OGD-treated astrocytes. Among these proteins, 7 have been reported to be able to regulate neurogenesis, while others may have the potential to regulate neurogenesis. This study profiles the major proteins released by astrocytes, which play important roles in the modulation of neurogenesis.

  9. Microglial Janus kinase/signal transduction and activator of transcription 3 pathway activity directly impacts astrocyte and spinal neuron characteristics.

    PubMed

    Molet, Jenny; Mauborgne, Annie; Diallo, Mickael; Armand, Vincent; Geny, David; Villanueva, Luis; Boucher, Yves; Pohl, Michel

    2016-01-01

    After peripheral nerve injury microglial reactivity change in the spinal cord is associated with an early activation of Janus kinase (JAK)/STAT3 transduction pathway whose blockade attenuates local inflammation and pain hypersensitivity. However, the consequences of microglial JAK/STAT3-mediated signaling on neighboring cells are unknown. Using an in vitro paradigm we assessed the impact of microglial JAK/STAT3 activity on functional characteristics of astrocytes and spinal cord neurons. Purified rat primary microglia was stimulated with JAK/STAT3 classical activator interleukin-6 in the presence or absence of a selective STAT3 inhibitor and rat primary astrocytes or spinal cord neurons were exposed to microglia conditioned media (CM). JAK/STAT3 activity-generated microglial CM modulated both astrocyte and neuron characteristics. Beyond inducing mRNA expression changes in various targets of interest in astrocytes and neurons, microglia CM activated c-Jun N-terminal kinase, STAT3 and NF-κB intracellular pathways in astrocytes and promoted their proliferation. Without modifying neuronal excitability or survival, CM affected the nerve processes morphology and distribution of the post-synaptic density protein 95, a marker of glutamatergic synaptic contacts. These findings show that JAK/STAT3 activity in microglia impacts the functional characteristics of astrocytes and neurons. This suggests its participation in spinal cord tissue plasticity and remodeling occurring after peripheral nerve injury. We show that the activity of JAK/STAT3 pathway in microglial cells confers them a specific signaling modality toward neighboring cells, promoting astrocyte proliferation and changes in neuronal morphology. These in vitro data suggest that the early JAK/STAT3 activation in spinal cord microglia, associated with peripheral nerve injury, participates in functional alteration of various cell populations and in spinal tissue remodeling. PMID:26440453

  10. Astrocytic laminin regulates pericyte differentiation and maintains blood brain barrier integrity

    NASA Astrophysics Data System (ADS)

    Yao, Yao; Chen, Zu-Lin; Norris, Erin H.; Strickland, Sidney

    2014-03-01

    Blood brain barrier (BBB) breakdown is not only a consequence of but also contributes to many neurological disorders, including stroke and Alzheimer’s disease. How the basement membrane (BM) contributes to the normal functioning of the BBB remains elusive. Here we use conditional knockout mice and an acute adenovirus-mediated knockdown model to show that lack of astrocytic laminin, a brain-specific BM component, induces BBB breakdown. Using functional blocking antibody and RNAi, we further demonstrate that astrocytic laminin, by binding to integrin α2 receptor, prevents pericyte differentiation from the BBB-stabilizing resting stage to the BBB-disrupting contractile stage, and thus maintains the integrity of BBB. Additionally, loss of astrocytic laminin decreases aquaporin-4 (AQP4) and tight junction protein expression. Altogether, we report a critical role for astrocytic laminin in BBB regulation and pericyte differentiation. These results indicate that astrocytic laminin maintains the integrity of BBB through, at least in part, regulation of pericyte differentiation.

  11. Astrocytic Ephrin-B1 Regulates Synapse Remodeling Following Traumatic Brain Injury

    PubMed Central

    Nikolakopoulou, Angeliki M.; Koeppen, Jordan; Garcia, Michael; Leish, Joshua; Obenaus, Andre

    2016-01-01

    Traumatic brain injury (TBI) can result in tissue alterations distant from the site of the initial injury, which can trigger pathological changes within hippocampal circuits and are thought to contribute to long-term cognitive and neuropsychological impairments. However, our understanding of secondary injury mechanisms is limited. Astrocytes play an important role in brain repair after injury and astrocyte-mediated mechanisms that are implicated in synapse development are likely important in injury-induced synapse remodeling. Our studies suggest a new role of ephrin-B1, which is known to regulate synapse development in neurons, in astrocyte-mediated synapse remodeling following TBI. Indeed, we observed a transient upregulation of ephrin-B1 immunoreactivity in hippocampal astrocytes following moderate controlled cortical impact model of TBI. The upregulation of ephrin-B1 levels in hippocampal astrocytes coincided with a decline in the number of vGlut1-positive glutamatergic input to CA1 neurons at 3 days post injury even in the absence of hippocampal neuron loss. In contrast, tamoxifen-induced ablation of ephrin-B1 from adult astrocytes in ephrin-B1loxP/yERT2-CreGFAP mice accelerated the recovery of vGlut1-positive glutamatergic input to CA1 neurons after TBI. Finally, our studies suggest that astrocytic ephrin-B1 may play an active role in injury-induced synapse remodeling through the activation of STAT3-mediated signaling in astrocytes. TBI-induced upregulation of STAT3 phosphorylation within the hippocampus was suppressed by astrocyte-specific ablation of ephrin-B1 in vivo, whereas the activation of ephrin-B1 in astrocytes triggered an increase in STAT3 phosphorylation in vitro. Thus, regulation of ephrin-B1 signaling in astrocytes may provide new therapeutic opportunities to aid functional recovery after TBI. PMID:26928051

  12. Regulation of the pituitary tumor transforming gene by insulin-like-growth factor-I and insulin differs between malignant and non-neoplastic astrocytes

    SciTech Connect

    Chamaon, Kathrin; Kirches, Elmar; Kanakis, Dimitrios; Braeuninger, Stefan; Dietzmann, Knut; Mawrin, Christian . E-mail: christian.mawrin@medizin.uni-magdeburg.de

    2005-05-27

    The reasons for overexpression of the oncogene pituitary tumor transforming gene (PTTG) in tumors are still not fully understood. A possible influence of the insulin-like growth factor I (Igf-I) may be of interest, since enhanced Igf-I signalling was reported in various human tumors. We examined the influence of Igf-I and insulin on PTTG expression in human astrocytoma cells in comparison to proliferating non-neoplastic rat embryonal astrocytes. PTTG mRNA expression and protein levels were increased in malignant astrocytes treated with Igf-I or insulin, whereas in rat embryonic astrocytes PTTG expression and protein levels increased only when cells were exposed to Igf-I. Enhanced transcription did not occur after treatment with inhibitors of phosphoinositol-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK), blocking the two basic signalling pathways of Igf-I and insulin. In addition to this transcriptional regulation, both kinases directly bind to PTTG, suggesting a second regulatory route by phosphorylation. However, the interaction of endogenous PTTG with MAPK and PI3K, as well as PTTG phosphorylation were independent from Igf-I or insulin. The latter results were also found in human testis, which contains high PTTG levels as well as in nonneoplastic astrocytes. This suggest, that PI3K and MAPK signalling is involved in PTTG regulation not only in malignant astrocytomas but also in non-tumorous cells.

  13. Pathogenesis of Lyme neuroborreliosis: mitogen-activated protein kinases Erk1, Erk2, and p38 in the response of astrocytes to Borrelia burgdorferi lipoproteins.

    PubMed

    Ramesh, Geeta; Philipp, Mario T

    Lyme borreliosis, which is prevalent both in the US and in Europe, is an infectious disease that may cause local inflammation in numerous organs. We have hypothesized that, as with some neurodegenerative diseases, the pathogenesis of the neurocognitive deficiencies associated with Lyme neuroborreliosis of the central nervous system also has an inflammatory component. Dysregulated production of pro-inflammatory cytokines such as IL-6 and TNF-alpha can lead to neuronal damage. Mitogen-activated protein kinases (MAPK) play a key role in the regulation of neuronal development, growth, and survival, as well as that of pro-inflammatory cytokine production. As a model, we explored the possibility that MAPK-mediated lipoprotein-induced apoptosis and gliosis of rhesus monkey astrocytes stimulated in vitro. Lipoproteins are the key inflammatory molecule type of Borrelia burgdorferi, the spirochete that causes Lyme disease, and we had previously shown that lipoprotein-induced TNF-alpha production in astrocytes caused astrocyte apoptosis, and IL-6 enhanced proliferation of these cells. Lipoproteins readily activated p38 and Erk1/2 MAPK, thus enlisting these pathways among the kinase pathways that spirochetes may address as they invade the central nervous system. We also investigated whether specific inhibition of p38 and Erk1/2 MAPK would inhibit TNF-alpha and IL-6 production and thus astrocyte apoptosis, and proliferation, respectively. Lipoprotein-stimulated IL-6 production was unaffected by the MAPK inhibitors. In contrast, inhibition of both p38 and Erk1/2 significantly diminished TNF-alpha production, and totally abrogated production of this cytokine when both MAPK pathways were inhibited simultaneously. MAPK inhibition thus may be considered as a strategy to control inflammation and apoptosis in Lyme neuroborreliosis. PMID:15893422

  14. Intracellular Na(+) inhibits volume-regulated anion channel in rat cortical astrocytes.

    PubMed

    Minieri, Laura; Pivonkova, Helena; Harantova, Lenka; Anderova, Miroslava; Ferroni, Stefano

    2015-02-01

    Accumulating evidence indicates that increased intracellular Na(+) concentration ([Na(+) ]i ) in astroglial cells is associated with the development of brain edema under ischemic conditions, but the underlying mechanisms are still elusive. Here, we report that in primary cultured rat cortical astrocytes, elevations of [Na(+) ]i reflecting those achieved during ischemia cause a marked decrease in hypotonicity-evoked current mediated by volume-regulated anion channel (VRAC). Pharmacological manipulations revealed that VRAC inhibition was not due to the reverse mode of the plasma membrane sodium/calcium exchanger. The negative modulation of VRAC was also observed in an astrocytic cell line lacking the predominant astrocyte water channel aquaporin 4, indicating that [Na(+) ]i effect was not mediated by the regulation of aquaporin 4 activity. The inward rectifier Cl(-) current, which is also expressed by cultured astrocytes, was not affected by [Na(+) ]i increase. VRAC depression by high [Na(+) ]i was confirmed in adult astrocytes, suggesting that it was not developmentally regulated. Altogether, these results provide the first evidence that intracellular Na(+) dynamics can modulate astrocytic membrane conductance that controls functional processes linked to cell volume regulation and add further support to the concept that limiting astrocyte intracellular Na(+) accumulation might be a favorable strategy to counteract the development of brain edema. PMID:25279950

  15. Cleavage of Hyaluronan and CD44 Adhesion Molecule Regulate Astrocyte Morphology via Rac1 Signalling

    PubMed Central

    Konopka, Anna; Zeug, Andre; Skupien, Anna; Kaza, Beata; Mueller, Franziska; Chwedorowicz, Agnieszka; Ponimaskin, Evgeni; Wilczynski, Grzegorz M.; Dzwonek, Joanna

    2016-01-01

    Communication of cells with their extracellular environment is crucial to fulfill their function in physiological and pathophysiological conditions. The literature data provide evidence that such a communication is also important in case of astrocytes. Mechanisms that contribute to the interaction between astrocytes and extracellular matrix (ECM) proteins are still poorly understood. Hyaluronan is the main component of ECM in the brain, where its major receptor protein CD44 is expressed by a subset of astrocytes. Considering the fact that functions of astrocytes are tightly coupled with changes in their morphology (e.g.: glutamate clearance in the synaptic cleft, migration, astrogliosis), we investigated the influence of hyaluronan cleavage by hyaluronidase, knockdown of CD44 by specific shRNA and CD44 overexpression on astrocyte morphology. Our results show that hyaluronidase treatment, as well as knockdown of CD44, in astrocytes result in a “stellate”-like morphology, whereas overexpression of CD44 causes an increase in cell body size and changes the shape of astrocytes into flattened cells. Moreover, as a dynamic reorganization of the actin cytoskeleton is supposed to be responsible for morphological changes of cells, and this reorganization is controlled by small GTPases of the Rho family, we hypothesized that GTPase Rac1 acts as a downstream effector for hyaluronan and CD44 in astrocytes. We used FRET-based biosensor and a dominant negative mutant of Rac1 to investigate the involvement of Rac1 activity in hyaluronidase- and CD44-dependent morphological changes of astrocytes. Both, hyaluronidase treatment and knockdown of CD44, enhances Rac1 activity while overexpression of CD44 reduces the activity state in astrocytes. Furthermore, morphological changes were blocked by specific inhibition of Rac1 activity. These findings indicate for the first time that regulation of Rac1 activity is responsible for hyaluronidase and CD44-driven morphological changes of

  16. Control of the neurovascular coupling by nitric oxide-dependent regulation of astrocytic Ca2+ signaling

    PubMed Central

    Muñoz, Manuel F.; Puebla, Mariela; Figueroa, Xavier F.

    2015-01-01

    Neuronal activity must be tightly coordinated with blood flow to keep proper brain function, which is achieved by a mechanism known as neurovascular coupling. Then, an increase in synaptic activity leads to a dilation of local parenchymal arterioles that matches the enhanced metabolic demand. Neurovascular coupling is orchestrated by astrocytes. These glial cells are located between neurons and the microvasculature, with the astrocytic endfeet ensheathing the vessels, which allows fine intercellular communication. The neurotransmitters released during neuronal activity reach astrocytic receptors and trigger a Ca2+ signaling that propagates to the endfeet, activating the release of vasoactive factors and arteriolar dilation. The astrocyte Ca2+ signaling is coordinated by gap junction channels and hemichannels formed by connexins (Cx43 and Cx30) and channels formed by pannexins (Panx-1). The neuronal activity-initiated Ca2+ waves are propagated among neighboring astrocytes directly via gap junctions or through ATP release via connexin hemichannels or pannexin channels. In addition, Ca2+ entry via connexin hemichannels or pannexin channels may participate in the regulation of the astrocyte signaling-mediated neurovascular coupling. Interestingly, nitric oxide (NO) can activate connexin hemichannel by S-nitrosylation and the Ca2+-dependent NO-synthesizing enzymes endothelial NO synthase (eNOS) and neuronal NOS (nNOS) are expressed in astrocytes. Therefore, the astrocytic Ca2+ signaling triggered in neurovascular coupling may activate NO production, which, in turn, may lead to Ca2+ influx through hemichannel activation. Furthermore, NO release from the hemichannels located at astrocytic endfeet may contribute to the vasodilation of parenchymal arterioles. In this review, we discuss the mechanisms involved in the regulation of the astrocytic Ca2+ signaling that mediates neurovascular coupling, with a special emphasis in the possible participation of NO in this process

  17. AGGRECAN IS EXPRESSED BY EMBRYONIC BRAIN GLIA AND REGULATES ASTROCYTE DEVELOPMENT

    PubMed Central

    Domowicz, Miriam S.; Sanders, Timothy A.; Ragsdale, Clifton W.; Schwartz, Nancy B.

    2008-01-01

    Determination of the molecules that regulate astrocyte development has been hindered by the paucity of markers that identify astrocytic precursors in vivo. Here we report that the chondroitin sulfate proteoglycan aggrecan both regulates astrocyte development and is expressed by embryonic glial precursors. During chick brain development, the onset of aggrecan expression precedes that of the astrocytic marker GFAP and is concomitant with detection of the early glial markers GLAST and glutamine synthetase. In co-expression studies, we established that aggrecan-rich cells contain the radial glial markers nestin, BLBP and GLAST and later in embryogenesis, the astroglial marker GFAP. Parallel in vitro studies showed that ventricular zone cultures, enriched in aggrecan-expressing cells, could be directed to a GFAP -positive fate in G5-supplemented differentiation media. Analysis of the chick aggrecan mutant nanomelia revealed marked increases in expression of the astrocyte differentiation genes GFAP, GLAST and GS in the absence of extracellular aggrecan. These increases in astrocytic marker gene expression could not be accounted for by changes in precursor proliferation or cell death, suggesting that aggrecan regulates the rate of astrocyte differentiation. Taken together, these results indicate a major role for aggrecan in the control of glial cell maturation during brain development. PMID:18207138

  18. Astrocyte regulation of sleep circuits: experimental and modeling perspectives

    PubMed Central

    Fellin, Tommaso; Ellenbogen, Jeffery M.; De Pittà, Maurizio; Ben-Jacob, Eshel; Halassa, Michael M.

    2012-01-01

    Integrated within neural circuits, astrocytes have recently been shown to modulate brain rhythms thought to mediate sleep function. Experimental evidence suggests that local impact of astrocytes on single synapses translates into global modulation of neuronal networks and behavior. We discuss these findings in the context of current conceptual models of sleep generation and function, each of which have historically focused on neural mechanisms. We highlight the implications and the challenges introduced by these results from a conceptual and computational perspective. We further provide modeling directions on how these data might extend our knowledge of astrocytic properties and sleep function. Given our evolving understanding of how local cellular activities during sleep lead to functional outcomes for the brain, further mechanistic and theoretical understanding of astrocytic contribution to these dynamics will undoubtedly be of great basic and translational benefit. PMID:22973222

  19. Astrocyte Elevated Gene-1 (AEG-1) Regulates Lipid Homeostasis*

    PubMed Central

    Robertson, Chadia L.; Srivastava, Jyoti; Siddiq, Ayesha; Gredler, Rachel; Emdad, Luni; Rajasekaran, Devaraja; Akiel, Maaged; Shen, Xue-Ning; Corwin, Frank; Sundaresan, Gobalakrishnan; Zweit, Jamal; Croniger, Colleen; Gao, Xiaoli; Ghosh, Shobha; Hylemon, Philip B.; Subler, Mark A.; Windle, Jolene J.; Fisher, Paul B.; Sarkar, Devanand

    2015-01-01

    Astrocyte elevated gene-1 (AEG-1), also known as MTDH (metadherin) or LYRIC, is an established oncogene. However, the physiological function of AEG-1 is not known. To address this question, we generated an AEG-1 knock-out mouse (AEG-1KO) and characterized it. Although AEG-1KO mice were viable and fertile, they were significantly leaner with prominently less body fat and lived significantly longer compared with wild type (WT). When fed a high fat and cholesterol diet (HFD), WT mice rapidly gained weight, whereas AEG-1KO mice did not gain weight at all. This phenotype of AEG-1KO mice is due to decreased fat absorption from the intestines, not because of decreased fat synthesis or increased fat consumption. AEG-1 interacts with retinoid X receptor (RXR) and inhibits RXR function. In enterocytes of AEG-1KO mice, we observed increased activity of RXR heterodimer partners, liver X receptor and peroxisome proliferator-activated receptor-α, key inhibitors of intestinal fat absorption. Inhibition of fat absorption in AEG-1KO mice was further augmented when fed an HFD providing ligands to liver X receptor and peroxisome proliferator-activated receptor-α. Our studies reveal a novel role of AEG-1 in regulating nuclear receptors controlling lipid metabolism. AEG-1 may significantly modulate the effects of HFD and thereby function as a unique determinant of obesity. PMID:26070567

  20. Astrocyte Elevated Gene-1 (AEG-1) Regulates Lipid Homeostasis.

    PubMed

    Robertson, Chadia L; Srivastava, Jyoti; Siddiq, Ayesha; Gredler, Rachel; Emdad, Luni; Rajasekaran, Devaraja; Akiel, Maaged; Shen, Xue-Ning; Corwin, Frank; Sundaresan, Gobalakrishnan; Zweit, Jamal; Croniger, Colleen; Gao, Xiaoli; Ghosh, Shobha; Hylemon, Philip B; Subler, Mark A; Windle, Jolene J; Fisher, Paul B; Sarkar, Devanand

    2015-07-17

    Astrocyte elevated gene-1 (AEG-1), also known as MTDH (metadherin) or LYRIC, is an established oncogene. However, the physiological function of AEG-1 is not known. To address this question, we generated an AEG-1 knock-out mouse (AEG-1KO) and characterized it. Although AEG-1KO mice were viable and fertile, they were significantly leaner with prominently less body fat and lived significantly longer compared with wild type (WT). When fed a high fat and cholesterol diet (HFD), WT mice rapidly gained weight, whereas AEG-1KO mice did not gain weight at all. This phenotype of AEG-1KO mice is due to decreased fat absorption from the intestines, not because of decreased fat synthesis or increased fat consumption. AEG-1 interacts with retinoid X receptor (RXR) and inhibits RXR function. In enterocytes of AEG-1KO mice, we observed increased activity of RXR heterodimer partners, liver X receptor and peroxisome proliferator-activated receptor-α, key inhibitors of intestinal fat absorption. Inhibition of fat absorption in AEG-1KO mice was further augmented when fed an HFD providing ligands to liver X receptor and peroxisome proliferator-activated receptor-α. Our studies reveal a novel role of AEG-1 in regulating nuclear receptors controlling lipid metabolism. AEG-1 may significantly modulate the effects of HFD and thereby function as a unique determinant of obesity. PMID:26070567

  1. Modulating Astrocyte Transition after Stroke to Promote Brain Rescue and Functional Recovery: Emerging Targets Include Rho Kinase

    PubMed Central

    Abeysinghe, Hima Charika S.; Phillips, Ellie L.; Chin-Cheng, Heung; Beart, Philip M.; Roulston, Carli L.

    2016-01-01

    Stroke is a common and serious condition, with few therapies. Whilst previous focus has been directed towards biochemical events within neurons, none have successfully prevented the progression of injury that occurs in the acute phase. New targeted treatments that promote recovery after stroke might be a better strategy and are desperately needed for the majority of stroke survivors. Cells comprising the neurovascular unit, including blood vessels and astrocytes, present an alternative target for supporting brain rescue and recovery in the late phase of stroke, since alteration in the unit also occurs in regions outside of the lesion. One of the major changes in the unit involves extensive morphological transition of astrocytes resulting in altered energy metabolism, decreased glutamate reuptake and recycling, and retraction of astrocyte end feed from both blood vessels and neurons. Whilst globally inhibiting transitional change in astrocytes after stroke is reported to result in further damage and functional loss, we discuss the available evidence to suggest that the transitional activation of astrocytes after stroke can be modulated for improved outcomes. In particular, we review the role of Rho-kinase (ROCK) in reactive gliosis and show that inhibiting ROCK after stroke results in reduced scar formation and improved functional recovery. PMID:26927079

  2. Modulating Astrocyte Transition after Stroke to Promote Brain Rescue and Functional Recovery: Emerging Targets Include Rho Kinase.

    PubMed

    Abeysinghe, Hima Charika S; Phillips, Ellie L; Chin-Cheng, Heung; Beart, Philip M; Roulston, Carli L

    2016-01-01

    Stroke is a common and serious condition, with few therapies. Whilst previous focus has been directed towards biochemical events within neurons, none have successfully prevented the progression of injury that occurs in the acute phase. New targeted treatments that promote recovery after stroke might be a better strategy and are desperately needed for the majority of stroke survivors. Cells comprising the neurovascular unit, including blood vessels and astrocytes, present an alternative target for supporting brain rescue and recovery in the late phase of stroke, since alteration in the unit also occurs in regions outside of the lesion. One of the major changes in the unit involves extensive morphological transition of astrocytes resulting in altered energy metabolism, decreased glutamate reuptake and recycling, and retraction of astrocyte end feed from both blood vessels and neurons. Whilst globally inhibiting transitional change in astrocytes after stroke is reported to result in further damage and functional loss, we discuss the available evidence to suggest that the transitional activation of astrocytes after stroke can be modulated for improved outcomes. In particular, we review the role of Rho-kinase (ROCK) in reactive gliosis and show that inhibiting ROCK after stroke results in reduced scar formation and improved functional recovery. PMID:26927079

  3. Regulation and function of yeast PAS kinase

    PubMed Central

    Grose, Julianne H.; Sundwall, Eleanor; Rutter, Jared

    2016-01-01

    The inability to coordinate cellular metabolic processes with the cellular and organismal nutrient environment leads to a variety of disorders, including diabetes and obesity. Nutrient-sensing protein kinases, such as AMPK and mTOR, play a pivotal role in metabolic regulation and are promising therapeutic targets for the treatment of disease. In this Extra View, we describe another member of the nutrient-sensing protein kinase group, PAS kinase, which plays a role in the regulation of glucose utilization in both mammals and yeast. PAS kinase deficient mice are resistant to high fat diet-induced weight gain, insulin resistance and hepatic triglyceride hyperaccumulation, suggesting a role for PAS kinase in the regulation of glucose and lipid metabolism in mammals. Likewise, PAS kinase deficient yeast display altered glucose partitioning, favoring glycogen biosynthesis at the expense of cell wall biosynthesis. As a result, PAS kinase deficient yeast are sensitive to cell wall perturbing agents. This partitioning of glucose in response to PAS kinase activation is due to phosphorylation of Ugp1, the enzyme primarily responsible for UDP-glucose production. The two yeast PAS kinase homologs, Psk1 and Psk2, are activated by two stimuli, cell integrity stress and nonfermentative carbon sources. We review what is known about yeast PAS kinase and describe a genetic screen that may help elucidate pathways involved in PAS kinase activation and function. PMID:19440050

  4. Regulation of nociceptin/orphanin FQ gene expression by neuropoietic cytokines and neurotrophic factors in neurons and astrocytes.

    PubMed

    Buzas, B; Symes, A J; Cox, B M

    1999-05-01

    We have identified the gene encoding nociceptin/orphanin FQ (N/OFQ), the novel opioid-like neuropeptide, as responsive to ciliary neurotrophic factor (CNTF). N/OFQ mRNA levels were induced five- and ninefold by CNTF in striatal and cortical neurons. In primary astrocytes CNTF also increased N/OFQ mRNA levels. CNTF is a multifunctional cytokine that mediates the development and differentiation of both neurons and astrocytes and supports the survival of various neurons. CNTF is also an injury-induced factor in the brain playing a crucial role in astrogliosis. The mechanism by which CNTF elicits these effects is not well understood, but it is likely to involve regulation of specific genes. CNTF regulation of N/OFQ expression was sensitive to the kinase inhibitors H-7 and genistein but not to inhibition of protein synthesis. This pharmacological profile is consistent with CNTF activating the Janus protein tyrosine kinase (JAK)/ signal transducers and activators of transcription (STAT) pathway to induce N/OFQ transcription. In nuclear extracts of CNTF-treated striatal neurons DNA binding of STAT proteins was increased. Radioimmunoassays revealed elevated N/OFQ immunoreactivity in striatal neurons after CNTF treatment. Expression of the related proenkephalin gene was not affected by CNTF in either neuronal or glial cultures. Regulation of N/OFQ expression by CNTF might point to a possible function of N/OFQ during development and after neural injury. PMID:10217264

  5. Astrocytes regulate heterogeneity of presynaptic strengths in hippocampal networks.

    PubMed

    Letellier, Mathieu; Park, Yun Kyung; Chater, Thomas E; Chipman, Peter H; Gautam, Sunita Ghimire; Oshima-Takago, Tomoko; Goda, Yukiko

    2016-05-10

    Dendrites are neuronal structures specialized for receiving and processing information through their many synaptic inputs. How input strengths are modified across dendrites in ways that are crucial for synaptic integration and plasticity remains unclear. We examined in single hippocampal neurons the mechanism of heterosynaptic interactions and the heterogeneity of synaptic strengths of pyramidal cell inputs. Heterosynaptic presynaptic plasticity that counterbalances input strengths requires N-methyl-d-aspartate receptors (NMDARs) and astrocytes. Importantly, this mechanism is shared with the mechanism for maintaining highly heterogeneous basal presynaptic strengths, which requires astrocyte Ca(2+) signaling involving NMDAR activation, astrocyte membrane depolarization, and L-type Ca(2+) channels. Intracellular infusion of NMDARs or Ca(2+)-channel blockers into astrocytes, conditionally ablating the GluN1 NMDAR subunit, or optogenetically hyperpolarizing astrocytes with archaerhodopsin promotes homogenization of convergent presynaptic inputs. Our findings support the presence of an astrocyte-dependent cellular mechanism that enhances the heterogeneity of presynaptic strengths of convergent connections, which may help boost the computational power of dendrites. PMID:27118849

  6. Pituitary Adenylate cyclase-activating polypeptide orchestrates neuronal regulation of the astrocytic glutamate-releasing mechanism system xc (.).

    PubMed

    Kong, Linghai; Albano, Rebecca; Madayag, Aric; Raddatz, Nicholas; Mantsch, John R; Choi, SuJean; Lobner, Doug; Baker, David A

    2016-05-01

    Glutamate signaling is achieved by an elaborate network involving neurons and astrocytes. Hence, it is critical to better understand how neurons and astrocytes interact to coordinate the cellular regulation of glutamate signaling. In these studies, we used rat cortical cell cultures to examine whether neurons or releasable neuronal factors were capable of regulating system xc (-) (Sxc), a glutamate-releasing mechanism that is expressed primarily by astrocytes and has been shown to regulate synaptic transmission. We found that astrocytes cultured with neurons or exposed to neuronal-conditioned media displayed significantly higher levels of Sxc activity. Next, we demonstrated that the pituitary adenylate cyclase-activating polypeptide (PACAP) may be a neuronal factor capable of regulating astrocytes. In support, we found that PACAP expression was restricted to neurons, and that PACAP receptors were expressed in astrocytes. Interestingly, blockade of PACAP receptors in cultures comprised of astrocytes and neurons significantly decreased Sxc activity to the level observed in purified astrocytes, whereas application of PACAP to purified astrocytes increased Sxc activity to the level observed in cultures comprised of neurons and astrocytes. Collectively, these data reveal that neurons coordinate the actions of glutamate-related mechanisms expressed by astrocytes, such as Sxc, a process that likely involves PACAP. A critical gap in modeling excitatory signaling is how distinct components of the glutamate system expressed by neurons and astrocytes are coordinated. In these studies, we found that system xc (-) (Sxc), a glutamate release mechanism expressed by astrocytes, is regulated by releasable neuronal factors including PACAP. This represents a novel form of neuron-astrocyte communication, and highlights the possibility that pathological changes involving astrocytic Sxc may stem from altered neuronal activity. PMID:26851652

  7. HIV-1, Methamphetamine and Astrocyte Glutamate Regulation: Combined Excitotoxic Implications for Neuro-AIDS

    PubMed Central

    Cisneros, Irma E; Ghorpade, Anuja

    2012-01-01

    Glutamate, the most abundant excitatory transmitter in the brain can lead to neurotoxicity when not properly regulated. Excitotoxicity is a direct result of abnormal regulation of glutamate concentrations in the synapse, and is a common neurotoxic mediator associated with neurodegenerative disorders. It is well accepted that methamphetamine (METH), a potent central nervous stimulant with high abuse potential, and human immunodeficiency virus (HIV)-1 are implicated in the progression of neurocognitive malfunction. Both have been shown to induce common neurodegenerative effects such as astrogliosis, compromised blood brain barrier integrity, and excitotoxicity in the brain. Reduced glutamate uptake from neuronal synapses likely leads to the accumulation of glutamate in the extracellular spaces. Astrocytes express the glutamate transporters responsible for majority of the glutamate uptake from the synapse, as well as for vesicular glutamate release. However, the cellular and molecular mechanisms of astrocyte-mediated excitotoxicity in the context of METH and HIV-1 are undefined. Topics reviewed include dysregulation of the glutamate transporters, specifically excitatory amino acid transporter-2, metabotropic glutamate receptor(s) expression and the release of glutamate by vesicular exocytosis. We also discuss glutamate concentration dysregulation through astrocytic expression of enzymes for glutamate synthesis and metabolism. Lastly, we discuss recent evidence of various astrocyte and neuron crosstalk mechanisms implicated in glutamate regulation. Astrocytes play an essential role in the neuropathologies associated with METH/HIV-1-induced excitotoxicity. We hope to shed light on common cellular and molecular pathways astrocytes share in glutamate regulation during drug abuse and HIV-1 infection. PMID:22591363

  8. A Novel Subtype of Astrocytes Expressing TRPV4 (Transient Receptor Potential Vanilloid 4) Regulates Neuronal Excitability via Release of Gliotransmitters*

    PubMed Central

    Shibasaki, Koji; Ikenaka, Kazuhiro; Tamalu, Fuminobu; Tominaga, Makoto; Ishizaki, Yasuki

    2014-01-01

    Astrocytes play active roles in the regulation of synaptic transmission. Neuronal excitation can evoke Ca2+ transients in astrocytes, and these Ca2+ transients can modulate neuronal excitability. Although only a subset of astrocytes appears to communicate with neurons, the types of astrocytes that can regulate neuronal excitability are poorly characterized. We found that ∼30% of astrocytes in the brain express transient receptor potential vanilloid 4 (TRPV4), indicating that astrocytic subtypes can be classified on the basis of their expression patterns. When TRPV4+ astrocytes are activated by ligands such as arachidonic acid, the activation propagates to neighboring astrocytes through gap junctions and by ATP release from the TRPV4+ astrocytes. After activation, both TRPV4+ and TRPV4− astrocytes release glutamate, which acts as an excitatory gliotransmitter to increase synaptic transmission through type 1 metabotropic glutamate receptor (mGluR). Our results indicate that TRPV4+ astrocytes constitute a novel subtype of the population and are solely responsible for initiating excitatory gliotransmitter release to enhance synaptic transmission. We propose that TRPV4+ astrocytes form a core of excitatory glial assembly in the brain and function to efficiently increase neuronal excitation in response to endogenous TRPV4 ligands. PMID:24737318

  9. Differential regulation of neuregulin 1 expression by progesterone in astrocytes and neurons

    PubMed Central

    LACROIX-FRALISH, MICHAEL L.; TAWFIK, VIVIANNE L.; NUTILE-MCMENEMY, NANCY; HARRIS, BRENT T.

    2007-01-01

    Glial–neuronal interactions are crucial processes in neuromodulation and synaptic plasticity. The neuregulin 1 family of growth and differentiation factors have been implicated as bidirectional signaling molecules that are involved in mediating some of these interactions. We have shown previously that neuregulin 1 expression is regulated by the gonadal hormones progesterone and 17β-estradiol in the CNS, which might represent a novel, indirect mechanism of the neuromodulatory actions of these gonadal hormones. In the present study, we sought to determine the effects of progesterone and 17β-estradiol on neuregulin 1 expression in rat cortical astrocytes and neurons in vitro. We observed that progesterone increased the expression of neuregulin 1 mRNA and protein in a dose-dependent manner in cultured astrocytes, which was blocked by the progesterone receptor antagonist RU-486. In contrast, 17β-estradiol did not increase either neuregulin 1 mRNA or protein in astrocytes. We observed no effect of either progesterone or 17β-estradiol on neuregulin 1 mRNA and protein in rat cortical neurons in vitro. Finally, we observed that treatment of cortical neurons with recombinant NRG1-β1 caused PSD-95 to localize in puncta similar to that observed following treatment with astrocyte-conditioned medium. These results demonstrate that progesterone regulates neuregulin 1 expression, principally in astrocytes. This might represent a novel mechanism of progesterone-mediated modulation of neurotransmission through the regulation of astrocyte-derived neuregulin 1. PMID:18049715

  10. Regulation of neuron-astrocyte metabolic coupling across the sleep-wake cycle.

    PubMed

    Petit, J-M; Magistretti, P J

    2016-05-26

    Over the last thirty years, a growing number of studies showed that astrocytes play a pivotal role in the energy support to synapses. More precisely, astrocytes adjust energy production to neuronal energy needs through different mechanisms grouped under the term "neurometabolic coupling" (NMC). In this review we describe these mechanisms of coupling and how they involve astrocytes. From a physiological point of view, these mechanisms of coupling are particularly important to ensure normal synaptic functioning when neurons undergo rapid and repetitive changes in the firing rate such as during the sleep/wake transitions. Investigations into brain energy metabolism during the sleep/wake cycle have been mainly focused on glucose (Gluc) consumption and on glycogen metabolism. However, the recent development of substrate-specific biosensors allowed measurements of the variation in extracellular levels of glutamate, Gluc and lactate (Lac) with a time resolution compatible with sleep stage duration. Together with gene expression data these experiments allowed to better define the variations of energy metabolite regulation across the sleep/wake cycle. The aim of this review is to bring into perspective the role of astrocytes and NMC in the regulation of the sleep/wake cycle. The data reviewed also suggest an important role of the astrocytic network. In addition, the role of astrocytes in NMC mechanisms is consistent with the "local and use dependent" sleep hypothesis. PMID:26704637

  11. Mitotic regulation by NIMA-related kinases

    PubMed Central

    O'Regan, Laura; Blot, Joelle; Fry, Andrew M

    2007-01-01

    The NIMA-related kinases represent a family of serine/threonine kinases implicated in cell cycle control. The founding member of this family, the NIMA kinase of Aspergillus nidulans, as well as the fission yeast homologue Fin1, contribute to multiple aspects of mitotic progression including the timing of mitotic entry, chromatin condensation, spindle organization and cytokinesis. Mammals contain a large family of eleven NIMA-related kinases, named Nek1 to Nek11. Of these, there is now substantial evidence that Nek2, Nek6, Nek7 and Nek9 also regulate mitotic events. At least three of these kinases, as well as NIMA and Fin1, have been localized to the microtubule organizing centre of their respective species, namely the centrosome or spindle pole body. Here, they have important functions in microtubule organization and mitotic spindle assembly. Other Nek kinases have been proposed to play microtubule-dependent roles in non-dividing cells, most notably in regulating the axonemal microtubules of cilia and flagella. In this review, we discuss the evidence that NIMA-related kinases make a significant contribution to the orchestration of mitotic progression and thereby protect cells from chromosome instability. Furthermore, we highlight their potential as novel chemotherapeutic targets. PMID:17727698

  12. Regulation of Kir4.1 expression in astrocytes and astrocytic tumors: a role for interleukin-1 β

    PubMed Central

    2012-01-01

    Objective Decreased expression of inwardly rectifying potassium (Kir) channels in astrocytes and glioma cells may contribute to impaired K+ buffering and increased propensity for seizures. Here, we evaluated the potential effect of inflammatory molecules, such as interleukin-1β (IL-1β) on Kir4.1 mRNA and protein expression. Methods We investigated Kir4.1 (Kcnj10) and IL-1β mRNA expression in the temporal cortex in a rat model of temporal lobe epilepsy 24 h and 1 week after induction of status epilepticus (SE), using real-time PCR and western blot analysis. The U373 glioblastoma cell line and human fetal astrocytes were used to study the regulation of Kir4.1 expression in response to pro-inflammatory cytokines. Expression of Kir4.1 protein was also evaluated by means of immunohistochemistry in surgical specimens of patients with astrocytic tumors (n = 64), comparing the expression in tumor patients with (n = 38) and without epilepsy (n = 26). Results Twenty-four hours after onset of SE, Kir4.1 mRNA and protein were significantly down-regulated in temporal cortex of epileptic rats. This decrease in expression was followed by a return to control level at 1 week after SE. The transient downregulation of Kir4.1 corresponded to the time of prominent upregulation of IL-1β mRNA. Expression of Kir4.1 mRNA and protein in glial cells in culture was downregulated after exposure to IL-1β. Evaluation of Kir4.1 in tumor specimens showed a significantly lower Kir4.1 expression in the specimens of patients with epilepsy compared to patients without epilepsy. This paralleled the increased presence of activated microglial cells, as well as the increased expression of IL-1β and the cytoplasmic translocation of high mobility group box 1 (HMGB1). Conclusions Taken together, these findings indicate that alterations in expression of Kir4.1 occurring in epilepsy-associated lesions are possibly influenced by the local inflammatory environment and in particular by the

  13. Reciprocal Regulation of Mitochondrial Dynamics and Calcium Signaling in Astrocyte Processes

    PubMed Central

    Jackson, Joshua G.

    2015-01-01

    We recently showed that inhibition of neuronal activity, glutamate uptake, or reversed-Na+/Ca2+-exchange with TTX, TFB-TBOA, or YM-244769, respectively, increases mitochondrial mobility in astrocytic processes. In the present study, we examined the interrelationships between mitochondrial mobility and Ca2+ signaling in astrocyte processes in organotypic cultures of rat hippocampus. All of the treatments that increase mitochondrial mobility decreased basal Ca2+. As recently reported, we observed spontaneous Ca2+ spikes with half-lives of ∼1 s that spread ∼6 μm and are almost abolished by a TRPA1 channel antagonist. Virtually all of these Ca2+ spikes overlap mitochondria (98%), and 62% of mitochondria are overlapped by these spikes. Although tetrodotoxin, TFB-TBOA, or YM-244769 increased Ca2+ signaling, the specific effects on peak, decay time, and/or frequency were different. To more specifically manipulate mitochondrial mobility, we explored the effects of Miro motor adaptor proteins. We show that Miro1 and Miro2 are both expressed in astrocytes and that exogenous expression of Ca2+-insensitive Miro mutants (KK) nearly doubles the percentage of mobile mitochondria. Expression of Miro1KK had a modest effect on the frequency of these Ca2+ spikes but nearly doubled the decay half-life. The mitochondrial proton ionophore, FCCP, caused a large, prolonged increase in cytosolic Ca2+ followed by an increase in the decay time and the spread of the spontaneous Ca2+ spikes. Photo-ablation of mitochondria in individual astrocyte processes has similar effects on Ca2+. Together, these studies show that Ca2+ regulates mitochondrial mobility, and mitochondria in turn regulate Ca2+ signals in astrocyte processes. SIGNIFICANCE STATEMENT In neurons, the movement and positioning of mitochondria at sites of elevated activity are important for matching local energy and Ca2+ buffering capacity. Previously, we demonstrated that mitochondria are immobilized in astrocytes in response

  14. Sodium Phenylbutyrate Enhances Astrocytic Neurotrophin Synthesis via Protein Kinase C (PKC)-mediated Activation of cAMP-response Element-binding Protein (CREB)

    PubMed Central

    Corbett, Grant T.; Roy, Avik; Pahan, Kalipada

    2013-01-01

    Neurotrophins, such as brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), are believed to be genuine molecular mediators of neuronal growth and homeostatic synapse activity. However, levels of these neurotrophic factors decrease in different brain regions of patients with Alzheimer disease (AD). Induction of astrocytic neurotrophin synthesis is a poorly understood phenomenon but represents a plausible therapeutic target because neuronal neurotrophin production is aberrant in AD and other neurodegenerative diseases. Here, we delineate that sodium phenylbutyrate (NaPB), a Food and Drug Administration-approved oral medication for hyperammonemia, induces astrocytic BDNF and NT-3 expression via the protein kinase C (PKC)-cAMP-response element-binding protein (CREB) pathway. NaPB treatment increased the direct association between PKC and CREB followed by phosphorylation of CREB (Ser133) and induction of DNA binding and transcriptional activation of CREB. Up-regulation of markers for synaptic function and plasticity in cultured hippocampal neurons by NaPB-treated astroglial supernatants and its abrogation by anti-TrkB blocking antibody suggest that NaPB-induced astroglial neurotrophins are functionally active. Moreover, oral administration of NaPB increased the levels of BDNF and NT-3 in the CNS and improved spatial learning and memory in a mouse model of AD. Our results highlight a novel neurotrophic property of NaPB that may be used to augment neurotrophins in the CNS and improve synaptic function in disease states such as AD. PMID:23404502

  15. Sphingosine kinase regulation and cardioprotection

    PubMed Central

    Karliner, Joel S.

    2009-01-01

    Activation of sphingosine kinase/sphingosine-1-phosphate (SK/S1P)-mediated signalling has been recognized as critical for cardioprotection in response to acute ischaemia/reperfusion injury. Incubation of S1P with cultured cardiac myocytes subjected to hypoxia or treatment of isolated hearts either before ischaemia or at the onset of reperfusion (pharmacologic pre- or postconditioning) results in reduced myocyte injury. Synthetic agonists active at S1P receptors mimic these responses. Gene-targeted mice null for the SK1 isoform whose hearts are subjected to ischaemia/reperfusion injury exhibit increased infarct size and respond poorly either to ischaemic pre- or postconditioning. Measurements of cardiac SK activity and S1P parallel these observations. Ischaemic postconditioning combined with sphingosine and S1P rescues the heart from prolonged ischaemia. These observations may have considerable relevance for future therapeutic approaches to acute and chronic myocardial injury. PMID:19017750

  16. Genes Involved in the Astrocyte-Neuron Lactate Shuttle (ANLS) Are Specifically Regulated in Cortical Astrocytes Following Sleep Deprivation in Mice

    PubMed Central

    Petit, Jean-Marie; Gyger, Joël; Burlet-Godinot, Sophie; Fiumelli, Hubert; Martin, Jean-Luc; Magistretti, Pierre J.

    2013-01-01

    Study Objectives: There is growing evidence indicating that in order to meet the neuronal energy demands, astrocytes provide lactate as an energy substrate for neurons through a mechanism called “astrocyte-neuron lactate shuttle” (ANLS). Since neuronal activity changes dramatically during vigilance states, we hypothesized that the ANLS may be regulated during the sleep-wake cycle. To test this hypothesis we investigated the expression of genes associated with the ANLS specifically in astrocytes following sleep deprivation. Astrocytes were purified by fluorescence-activated cell sorting from transgenic mice expressing the green fluorescent protein (GFP) under the control of the human astrocytic GFAP-promoter. Design: 6-hour instrumental sleep deprivation (TSD). Setting: Animal sleep research laboratory. Participants: Young (P23-P27) FVB/N-Tg (GFAP-GFP) 14Mes/J (Tg) mice of both sexes and 7-8 week male Tg and FVB/Nj mice. Interventions: Basal sleep recordings and sleep deprivation achieved using a modified cage where animals were gently forced to move. Measurements and Results: Since Tg and FVB/Nj mice displayed a similar sleep-wake pattern, we performed a TSD in young Tg mice. Total RNA was extracted from the GFP-positive and GFP-negative cells sorted from cerebral cortex. Quantitative RT-PCR analysis showed that levels of Glut1, α-2-Na/K pump, Glt1, and Ldha mRNAs were significantly increased following TSD in GFP-positive cells. In GFP-negative cells, a tendency to increase, although not significant, was observed for Ldha, Mct2, and α-3-Na/K pump mRNAs. Conclusions: This study shows that TSD induces the expression of genes associated with ANLS specifically in astrocytes, underlying the important role of astrocytes in the maintenance of the neuro-metabolic coupling across the sleep-wake cycle. Citation: Petit JM; Gyger J; Burlet-Godinot S; Fiumelli H; Martin JL; Magistretti PJ. Genes involved in the astrocyte-neuron lactate shuttle (ANLS) are specifically

  17. Astrocyte-derived phosphatidic acid promotes dendritic branching

    PubMed Central

    Zhu, Yan-Bing; Gao, Weizhen; Zhang, Yongbo; Jia, Feng; Zhang, Hai-Long; Liu, Ying-Zi; Sun, Xue-Fang; Yin, Yuhua; Yin, Dong-Min

    2016-01-01

    Astrocytes play critical roles in neural circuit formation and function. Recent studies have revealed several secreted and contact-mediated signals from astrocytes which are essential for neurite outgrowth and synapse formation. However, the mechanisms underlying the regulation of dendritic branching by astrocytes remain elusive. Phospholipase D1 (PLD1), which catalyzes the hydrolysis of phosphatidylcholine (PC) to generate phosphatidic acid (PA) and choline, has been implicated in the regulation of neurite outgrowth. Here we showed that knockdown of PLD1 selectively in astrocytes reduced dendritic branching of neurons in neuron-glia mixed culture. Further studies from sandwich-like cocultures and astrocyte conditioned medium suggested that astrocyte PLD1 regulated dendritic branching through secreted signals. We later demonstrated that PA was the key mediator for astrocyte PLD1 to regulate dendritic branching. Moreover, PA itself was sufficient to promote dendritic branching of neurons. Lastly, we showed that PA could activate protein kinase A (PKA) in neurons and promote dendritic branching through PKA signaling. Taken together, our results demonstrate that astrocyte PLD1 and its lipid product PA are essential regulators of dendritic branching in neurons. These results may provide new insight into mechanisms underlying how astrocytes regulate dendrite growth of neurons. PMID:26883475

  18. Structure and Dynamic Regulation of Abl Kinases*

    PubMed Central

    Panjarian, Shoghag; Iacob, Roxana E.; Chen, Shugui; Engen, John R.; Smithgall, Thomas E.

    2013-01-01

    The c-abl proto-oncogene encodes a unique protein-tyrosine kinase (Abl) distinct from c-Src, c-Fes, and other cytoplasmic tyrosine kinases. In normal cells, Abl plays prominent roles in cellular responses to genotoxic stress as well as in the regulation of the actin cytoskeleton. Abl is also well known in the context of Bcr-Abl, the oncogenic fusion protein characteristic of chronic myelogenous leukemia. Selective inhibitors of Bcr-Abl, of which imatinib is the prototype, have had a tremendous impact on clinical outcomes in chronic myelogenous leukemia and revolutionized the field of targeted cancer therapy. In this minireview, we focus on the structural organization and dynamics of Abl kinases and how these features influence inhibitor sensitivity. PMID:23316053

  19. Regulation of Axonal Transport by Protein Kinases.

    PubMed

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

    2015-10-01

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

  20. Src Kinase Regulation in Progressively Invasive Cancer

    PubMed Central

    Xu, Weichen; Allbritton, Nancy; Lawrence, David S.

    2012-01-01

    Metastatic progression is a multistep process that involves tumor growth and survival, motility and invasion, and subsequent proliferation in an inappropriate environment. The Src protein tyrosine kinase has been implicated in many of the biochemical pathways that drive these behaviors. Although Src itself is only rarely mutated in human tumors, its aberrant activity has been noted in various cancers and suggested to serve as a barometer of metastatic potential. With these features in mind, we examined Src kinase regulation at the structural, enzymatic, and expression levels as a function of progressively invasive prostate cancer cell lines. Surprisingly, both total Src content and kinase activity decrease with increasing cell line aggressiveness, an observation that appears to be inconsistent with the well-documented role of Src in the signaling pathways that drive growth and invasion. However, we do observe a direct correlation between Src kinase specific activity (total Src kinase activity/total Src content) and metastatic aggressiveness, possibly suggesting that in highly aggressive cell lines, key signaling enzymes are globally recruited to drive the cancerous phenotype. In addition, although the expected enhanced phosphorylation of Src at Tyr-416 (activation site) is present in the most aggressive prostate cancer cell lines, unexpectedly high phosphorylation levels at the Tyr-527 inhibitory site are observed as well. The latter, rather than representative of inhibited enzyme, is more indicative of primed Src responsive to local phosphorylated binding partners. PMID:23145001

  1. Angiotensin II regulation of angiotensin-converting enzymes in spontaneously hypertensive rat primary astrocyte cultures.

    PubMed

    Gowrisankar, Yugandhar V; Clark, Michelle A

    2016-07-01

    Angiotensin (Ang) II plays a critical role in cardiovascular and blood pressure regulation. Ang II is produced by angiotensin-converting enzyme (ACE) and it interacts with the Ang AT1 receptor to cause much of its well-known cardiovascular effects. Ang-(1-7) is another active peptide produced by the rennin-angiotensin system. This peptide is produced from Ang I or Ang II by the catalytic activity of ACE2. Ang-(1-7) interacts with the Mas receptor to counteract many of the effects of Ang II. Thus, the ACE2/Ang-(1-7)/Mas axis acts opposite of the ACE/Ang II/AT1 axis. In this study we investigated how Ang II regulates the key enzymes of these axes, ACE and its homolog ACE2, and determined whether they are dysregulated in the hypertensive condition. Brainstem and cerebellum astrocytes isolated from the spontaneously hypertensive rat (SHR) were used in these studies. Ang II effect on the enzymes' mRNA and protein levels was measured using quantitative PCR and western blotting techniques, respectively. Results from this study showed that Ang II up-regulated ACE protein levels, but down-regulated ACE mRNA levels in brainstem and cerebellum astrocytes in both models. Ang II also reduced ACE2 mRNA expression in SHR and Wistar astrocytes isolated from both brain regions. Ang II effects on ACE2 protein were biphasic. In SHR astrocytes, Ang II-mediated ACE2 protein initially increased then decreased at later time points. In contrast, in Wistar astrocytes, Ang II initially decreased ACE2 protein expression, but up-regulated the protein at later time points. The findings of these studies suggest that Ang II has a differential effect on ACE and ACE2 expression. Furthermore, in the SHR model there may be alteration in the ACE/ACE2 balance in a manner that favors increased Ang II generation and decreased Ang-(1-7) production contributing to the hypertensive phenotype observed in this model. The levels of angiotensin (Ang) II depend on the actions of angiotensin-converting enzyme

  2. Hdac3 Interaction with p300 Histone Acetyltransferase Regulates the Oligodendrocyte and Astrocyte Lineage Fate Switch.

    PubMed

    Zhang, Liguo; He, Xuelian; Liu, Lei; Jiang, Minqing; Zhao, Chuntao; Wang, Haibo; He, Danyang; Zheng, Tao; Zhou, Xianyao; Hassan, Aishlin; Ma, Zhixing; Xin, Mei; Sun, Zheng; Lazar, Mitchell A; Goldman, Steven A; Olson, Eric N; Lu, Q Richard

    2016-02-01

    Establishment and maintenance of CNS glial cell identity ensures proper brain development and function, yet the epigenetic mechanisms underlying glial fate control remain poorly understood. Here, we show that the histone deacetylase Hdac3 controls oligodendrocyte-specification gene Olig2 expression and functions as a molecular switch for oligodendrocyte and astrocyte lineage determination. Hdac3 ablation leads to a significant increase of astrocytes with a concomitant loss of oligodendrocytes. Lineage tracing indicates that the ectopic astrocytes originate from oligodendrocyte progenitors. Genome-wide occupancy analysis reveals that Hdac3 interacts with p300 to activate oligodendroglial lineage-specific genes, while suppressing astroglial differentiation genes including NFIA. Furthermore, we find that Hdac3 modulates the acetylation state of Stat3 and competes with Stat3 for p300 binding to antagonize astrogliogenesis. Thus, our data suggest that Hdac3 cooperates with p300 to prime and maintain oligodendrocyte identity while inhibiting NFIA and Stat3-mediated astrogliogenesis, and thereby regulates phenotypic commitment at the point of oligodendrocyte-astrocytic fate decision. PMID:26859354

  3. Zonisamide up-regulated the mRNAs encoding astrocytic anti-oxidative and neurotrophic factors.

    PubMed

    Choudhury, M E; Sugimoto, K; Kubo, M; Iwaki, H; Tsujii, T; Kyaw, W T; Nishikawa, N; Nagai, M; Tanaka, J; Nomoto, M

    2012-08-15

    Zonisamide has been proven as an effective drug for the recovery of degenerating dopaminergic neurons in the animal models of Parkinson's disease. However, several lines of evidence have questioned the neuroprotective capacity of zonisamide in animal models of Parkinson's disease. Although it suppresses dopaminergic neurodegeneration in animal models, the cellular and molecular mechanisms underlying the effectiveness of zonisamide are not fully understood. The current study demonstrates the effects of zonisamide on astrocyte cultures and two 6-hydroxydopamine-induced models of Parkinson's disease. Using primary astrocyte cultures, we showed that zonisamide up-regulated the expression of mRNA encoding mesencephalic astrocyte-derived neurotrophic factor, vascular endothelial growth factor, proliferating cell nuclear antigen, metallothionein-2, copper/zinc superoxide dismutase, and manganese superoxide dismutase. Similar responses to zonisamide were found in substantia nigra where the rats were pre-treated with 6-hydroxydopamine. Notably, pharmacological inhibition of 6-hydroxydopamine-induced toxicity by zonisamide pre-treatment was also confirmed using rat mesencephalic organotypic slice cultures of substantia nigra. In addition to this, zonisamide post-treatment also attenuated the nigral tyrosine hydroxylase-positive neuronal loss induced by 6-hydroxydopamine. Taken together, these studies demonstrate that zonisamide protected dopamine neurons in two Parkinson's disease models through a novel mechanism, namely increasing the expression of some important astrocyte-mediated neurotrophic and anti-oxidative factors. PMID:22659113

  4. SUMOylation regulates the SNF1 protein kinase

    PubMed Central

    Simpson-Lavy, Kobi J.; Johnston, Mark

    2013-01-01

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

  5. Cysteinyl-leukotrienes are released from astrocytes and increase astrocyte proliferation and glial fibrillary acidic protein via cys-LT1 receptors and mitogen-activated protein kinase pathway.

    PubMed

    Ciccarelli, Renata; D'Alimonte, Iolanda; Santavenere, Clara; D'Auro, Mariagrazia; Ballerini, Patrizia; Nargi, Eleonora; Buccella, Silvana; Nicosia, Simonetta; Folco, Giancarlo; Caciagli, Francesco; Di Iorio, Patrizia

    2004-09-01

    Cysteinyl-leukotrienes (cys-LTs), potent mediators in inflammatory diseases, are produced by nervous tissue, but their cellular source and role in the brain are not very well known. In this report we have demonstrated that rat cultured astrocytes express the enzymes (5'-lipoxygenase and LTC(4) synthase) required for cys-LT production, and release cys-LTs in resting condition and, to a greater extent, in response to calcium ionophore A23187, 1 h combined oxygen-glucose deprivation or 2-methyl-thioATP, a selective P2Y(1)/ATP receptor agonist. MK-886, a LT synthesis inhibitor, prevented basal and evoked cys-LT release. In addition, 2-methyl-thioATP-induced cys-LT release was abolished by suramin, a P2 receptor antagonist, or by inhibitors of ATP binding cassette proteins involved in cys-LT release. We also showed that astrocytes express cys-LT(1) and not cys-LT(2) receptors. The stimulation of these receptors by LTD(4) activated the mitogen-activated protein kinase (MAPK) pathway. This effect was: (i) insensitive to inhibitors of receptor-coupled Gi protein (pertussis toxin) or tyrosine kinase receptors (genistein); (ii) abolished by MK-571, a cys-LT(1) selective receptor antagonist, or PD98059, a MAPK inhibitor; (iii) reduced by inhibitors of calcium/calmodulin-dependent kinase II (KN-93), Ca(2+)-dependent and -independent (GF102903X) or Ca(2+)-dependent (Gö6976) protein kinase C isoforms. LTD(4) also increased astrocyte proliferation and glial fibrillary acidic protein content, which are considered hallmarks of reactive astrogliosis. Both effects were counteracted by cell pretreatment with MK-571 or PD98059. Thus, cys-LTs released from astrocytes might play an autocrine role in the induction of reactive astrogliosis that, in brain injuries, contributes to the formation of a reparative glial scar. PMID:15355318

  6. Glutamate Transporter EAAT2 Expression is Up-Regulated in Reactive Astrocytes in Human Periventricular Leukomalacia

    PubMed Central

    DESILVA, TARA M.; BILLIARDS, SARAID S.; BORENSTEIN, NATALIA S.; TRACHTENBERG, FELICIA L.; VOLPE, JOSEPH J.; KINNEY, HANNAH C.; ROSENBERG, PAUL A.

    2010-01-01

    The major neuropathological correlate of cerebral palsy in premature infants is periventricular leukomalacia (PVL), a disorder of the immature cerebral white matter. Cerebral ischemia leading to excitotoxicity is thought to be important in the pathogenesis of this disorder, implying a critical role for glutamate transporters, the major determinants of extracellular glutamate concentration. Previously, we found that EAAT2 expression is limited primarily to premyelinating oligodendrocytes early in development and is rarely observed in astrocytes until >40 weeks. In this study, we analyzed the expression of EAAT2 in cerebral white matter from PVL and control cases. Western blot analysis suggested an up-regulation of EAAT2 in PVL compared with control cases. Single- and double-label immunocytochemistry showed a significantly higher percentage of EAAT2-immunopositive astrocytes in PVL (51.8% ± 5.6%) compared with control white matter (21.4% ± 5.6%; P = 0.004). Macrophages in the necrotic foci in PVL also expressed EAAT2. Premyelinating oligodendrocytes in both PVL and control cases expressed EAAT2, without qualitative difference in expression. The previously unrecognized up-regulation of EAAT2 in reactive astrocytes and its presence in macrophages in PVL reported here may reflect a response to either hypoxic-ischemic injury or inflammation. PMID:18314905

  7. State-dependent changes in astrocyte regulation of extrasynaptic NMDA receptor signalling in neurosecretory neurons.

    PubMed

    Fleming, Tiffany M; Scott, Victoria; Naskar, Krishna; Joe, Natalie; Brown, Colin H; Stern, Javier E

    2011-08-15

    Despite the long-established presence of glutamate NMDA receptors at extrasynaptic sites (eNMDARs), their functional roles remain poorly understood. Factors influencing the concentration and time course of glutamate in the extrasynaptic space, such as the topography of the neuronal–glial microenvironment, as well as glial glutamate transporters, are expected to affect eNMDAR-mediated signalling strength. In this study, we used in vitro and in vivo electrophysiological recordings to assess the properties, functional relevance and modulation of a persistent excitatory current mediated by activation of eNMDARs in hypothalamic supraoptic nucleus (SON) neurons. We found that ambient glutamate of a non-synaptic origin activates eNMDARs to mediate a persistent excitatory current (termed tonic I(NMDA)), which tonically stimulates neuronal activity. Pharmacological blockade of GLT1 astrocyte glutamate transporters, as well as the gliotoxin α-aminodadipic acid, enhanced tonic I(NMDA) and neuronal activity, supporting an astrocyte regulation of tonic I(NMDA) strength. Dehydration, a physiological challenge known to increase SON firing activity and to induce neuroglial remodelling, including reduced neuronal ensheathment by astrocyte processes, resulted in blunted GLT1 efficacy, enhanced tonic I(NMDA) strength, and increased neuronal activity. Taken together, our studies support the view that glial modulation of tonic I(NMDA) activation contributes to regulation of SON neuronal activity, contributing in turn to neuronal homeostatic responses during a physiological challenge. PMID:21690192

  8. Aerobic glycolysis during brain activation: adrenergic regulation and influence of norepinephrine on astrocytic metabolism.

    PubMed

    Dienel, Gerald A; Cruz, Nancy F

    2016-07-01

    Aerobic glycolysis occurs during brain activation and is characterized by preferential up-regulation of glucose utilization compared with oxygen consumption even though oxygen level and delivery are adequate. Aerobic glycolysis is a widespread phenomenon that underlies energetics of diverse brain activities, such as alerting, sensory processing, cognition, memory, and pathophysiological conditions, but specific cellular functions fulfilled by aerobic glycolysis are poorly understood. Evaluation of evidence derived from different disciplines reveals that aerobic glycolysis is a complex, regulated phenomenon that is prevented by propranolol, a non-specific β-adrenoceptor antagonist. The metabolic pathways that contribute to excess utilization of glucose compared with oxygen include glycolysis, the pentose phosphate shunt pathway, the malate-aspartate shuttle, and astrocytic glycogen turnover. Increased lactate production by unidentified cells, and lactate dispersal from activated cells and lactate release from the brain, both facilitated by astrocytes, are major factors underlying aerobic glycolysis in subjects with low blood lactate levels. Astrocyte-neuron lactate shuttling with local oxidation is minor. Blockade of aerobic glycolysis by propranolol implicates adrenergic regulatory processes including adrenal release of epinephrine, signaling to brain via the vagus nerve, and increased norepinephrine release from the locus coeruleus. Norepinephrine has a powerful influence on astrocytic metabolism and glycogen turnover that can stimulate carbohydrate utilization more than oxygen consumption, whereas β-receptor blockade 're-balances' the stoichiometry of oxygen-glucose or -carbohydrate metabolism by suppressing glucose and glycogen utilization more than oxygen consumption. This conceptual framework may be helpful for design of future studies to elucidate functional roles of preferential non-oxidative glucose utilization and glycogen turnover during brain

  9. Up-Regulation of TREK-2 Potassium Channels in Cultured Astrocytes Requires De Novo Protein Synthesis: Relevance to Localization of TREK-2 Channels in Astrocytes after Transient Cerebral Ischemia

    PubMed Central

    Rivera-Pagán, Aixa F.; Rivera-Aponte, David E.; Melnik-Martínez, Katya V.; Zayas-Santiago, Astrid; Kucheryavykh, Lilia Y.; Martins, Antonio H.; Cubano, Luis A.; Skatchkov, Serguei N.; Eaton, Misty J.

    2015-01-01

    Excitotoxicity due to glutamate receptor over-activation is one of the key mediators of neuronal death after an ischemic insult. Therefore, a major function of astrocytes is to maintain low extracellular levels of glutamate. The ability of astrocytic glutamate transporters to regulate the extracellular glutamate concentration depends upon the hyperpolarized membrane potential of astrocytes conferred by the presence of K+ channels in their membranes. We have previously shown that TREK-2 potassium channels in cultured astrocytes are up-regulated by ischemia and may support glutamate clearance by astrocytes during ischemia. Thus, herein we determine the mechanism leading to this up-regulation and assess the localization of TREK-2 channels in astrocytes after transient middle cerebral artery occlusion. By using a cell surface biotinylation assay we confirmed that functional TREK-2 protein is up-regulated in the astrocytic membrane after ischemic conditions. Using real time RT-PCR, we determined that the levels of TREK-2 mRNA were not increased in response to ischemic conditions. By using Western blot and a variety of protein synthesis inhibitors, we demonstrated that the increase of TREK-2 protein expression requires De novo protein synthesis, while protein degradation pathways do not contribute to TREK-2 up-regulation after ischemic conditions. Immunohistochemical studies revealed TREK-2 localization in astrocytes together with increased expression of the selective glial marker, glial fibrillary acidic protein, in brain 24 hours after transient middle cerebral occlusion. Our data indicate that functional TREK-2 channels are up-regulated in the astrocytic membrane during ischemia through a mechanism requiring De novo protein synthesis. This study provides important information about the mechanisms underlying TREK-2 regulation, which has profound implications in neurological diseases such as ischemia where astrocytes play an important role. PMID:25886567

  10. MicroRNA-146a: A Key Regulator of Astrocyte-Mediated Inflammatory Response

    PubMed Central

    Prabowo, Avanita; Fluiter, Kees; Spliet, Wim G. M.; van Rijen, Peter C.; Gorter, Jan A.; Aronica, Eleonora

    2012-01-01

    Increasing evidence supports the involvement of microRNAs (miRNA) in the regulation of inflammation in human neurological disorders. In the present study we investigated the role of miR-146a, a key regulator of the innate immune response, in the modulation of astrocyte-mediated inflammation. Using Taqman PCR and in situ hybridization, we studied the expression of miR-146a in epilepsy-associated glioneuronal lesions which are characterized by prominent activation of the innate immune response. In addition, cultured human astrocytes were used to study the regulation of miR-146a expression in response to proinflammatory cytokines. qPCR and western blot were used to evaluate the effects of overexpression or knockdown of miR-146a on IL-1β signaling. Downstream signaling in the IL-1β pathway, as well as the expression of IL-6 and COX-2 were evaluated by western blot and ELISA. Release several cytokines was evaluated using a human magnetic multiplex cytokine assay on a Luminex® 100™/200™ platform. Increased expression of miR-146a was observed in glioneuronal lesions by Taqman PCR. MiR-146a expression in human glial cell cultures was strongly induced by IL-1β and blocked by IL-1β receptor antagonist. Modulation of miR-146a expression by transfection of astrocytes with anti-miR146a or mimic, regulated the mRNA expression levels of downstream targets of miR-146a (IRAK-1, IRAK-2 and TRAF-6) and the expression of IRAK-1 protein. In addition, the expression of IL-6 and COX-2 upon IL-1β stimulation was suppressed by increased levels of miR-146a and increased by the reduction of miR-146a. Modulation of miR-146a expression affected also the release of several cytokines such as IL-6 and TNF-α. Our observations indicate that in response to inflammatory cues, miR-146a was induced as a negative-feedback regulator of the astrocyte-mediated inflammatory response. This supports an important role of miR-146a in human neurological disorders associated with chronic inflammation

  11. MicroRNA-146a: a key regulator of astrocyte-mediated inflammatory response.

    PubMed

    Iyer, Anand; Zurolo, Emanuele; Prabowo, Avanita; Fluiter, Kees; Spliet, Wim G M; van Rijen, Peter C; Gorter, Jan A; Aronica, Eleonora

    2012-01-01

    Increasing evidence supports the involvement of microRNAs (miRNA) in the regulation of inflammation in human neurological disorders. In the present study we investigated the role of miR-146a, a key regulator of the innate immune response, in the modulation of astrocyte-mediated inflammation. Using Taqman PCR and in situ hybridization, we studied the expression of miR-146a in epilepsy-associated glioneuronal lesions which are characterized by prominent activation of the innate immune response. In addition, cultured human astrocytes were used to study the regulation of miR-146a expression in response to proinflammatory cytokines. qPCR and western blot were used to evaluate the effects of overexpression or knockdown of miR-146a on IL-1β signaling. Downstream signaling in the IL-1β pathway, as well as the expression of IL-6 and COX-2 were evaluated by western blot and ELISA. Release several cytokines was evaluated using a human magnetic multiplex cytokine assay on a Luminex® 100™/200™ platform. Increased expression of miR-146a was observed in glioneuronal lesions by Taqman PCR. MiR-146a expression in human glial cell cultures was strongly induced by IL-1β and blocked by IL-1β receptor antagonist. Modulation of miR-146a expression by transfection of astrocytes with anti-miR146a or mimic, regulated the mRNA expression levels of downstream targets of miR-146a (IRAK-1, IRAK-2 and TRAF-6) and the expression of IRAK-1 protein. In addition, the expression of IL-6 and COX-2 upon IL-1β stimulation was suppressed by increased levels of miR-146a and increased by the reduction of miR-146a. Modulation of miR-146a expression affected also the release of several cytokines such as IL-6 and TNF-α. Our observations indicate that in response to inflammatory cues, miR-146a was induced as a negative-feedback regulator of the astrocyte-mediated inflammatory response. This supports an important role of miR-146a in human neurological disorders associated with chronic inflammation

  12. 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. PMID:22244755

  13. Diacylglycerol kinase θ: regulation and stability.

    PubMed

    Tu-Sekine, Becky; Goldschmidt, Hana; Petro, Elizabeth; Raben, Daniel M

    2013-01-01

    Given the well-established roles of diacylglycerol (DAG) and phosphatidic acid (PtdOH) in a variety of signaling cascades, it is not surprising that there is an increasing interest in understanding their physiological roles and mechanisms that regulate their cellular levels. One class of enzymes capable of coordinately regulating the levels of these two lipids is the diacylglycerol kinases (DGKs). These enzymes catalyze the transfer of the γ-phosphate of ATP to the hydroxyl group of DAG, which generates PtdOH while reducing DAG. As these enzymes reciprocally modulate the relative levels of these two signaling lipids, it is essential to understand the regulation and roles of these enzymes in various tissues. One system where these enzymes play important roles is the nervous system. Of the ten mammalian DGKs, eight of them are readily detected in the mammalian central nervous system (CNS): DGK-α, DGK-β, DGK-γ, DGK-η, DGK-ζ, DGK-ι, DGK-ε, and DGK-θ. Despite the increasing interest in DGKs, little is known about their regulation. We have focused some attention on understanding the enzymology and regulation of one of these DGK isoforms, DGK-θ. We recently showed that DGK-θ is regulated by an accessory protein containing polybasic regions. We now report that this accessory protein is required for the previously reported broadening of the pH profile observed in cell lysates in response to phosphatidylserine (PtdSer). Our data further reveal DGK-θ is regulated by magnesium and zinc, and sensitive to the known DGK inhibitor R599022. These data outline new parameters involved in regulating DGK-θ. PMID:23266086

  14. Mechanism of Plasminogen Activator Inhibitor-1 regulation by Oncostatin M and Interleukin-1 in human astrocytes

    PubMed Central

    Kasza, Aneta; Kiss, Daniel L.; Gopalan, Sunita; Xu, Weili; Rydel, Russell E.; Koj, Aleksander; Kordula, Tomasz

    2015-01-01

    Glial cells that produce and respond to various cytokines mediate inflammatory processes in the brain. Here, we show that oncostatin M (OSM) and interleukin-1 (IL-1) regulate the expression of plasminogen activator inhibitor-1 (PAI-1) and urokinase-type plasminogen activator (uPA) in human astrocytes. Using the PAI-1 reporter constructs we show that the −58 to −51 proximal element mediates activation by both cytokines. This element is already bound by c-fos/c-jun heterodimers in unstimulated astrocytes, and treatment with cytokine strongly stimulates both expression of c-fos and binding of c-fos/c-jun heterodimers. In addition, IL-1 activates an inhibitory mechanism that downregulates PAI-1 expression after longer exposure to this cytokine. Overexpression of dominant-negative signal transducer and activator of transcription-1 (STAT1), STAT3, STAT5 and inhibitor of nuclear factor kB (IkB) suppressed OSM/IL-1-induced expression of the PAI-1 reporter construct. We conclude that OSM and IL-1 regulate the PAI-1 gene expression via up-regulating c-fos levels and subsequent binding of c-fos/c-jun heterodimers to the proximal element of the PAI-1 gene. PMID:12390531

  15. Src kinase regulation by phosphorylation and dephosphorylation

    SciTech Connect

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

    2005-05-27

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

  16. Selective up-regulation of GLT-1 in cultured astrocytes exposed to soluble mediators released by activated microglia.

    PubMed

    Tilleux, Sébastien; Goursaud, Stéphanie; Hermans, Emmanuel

    2009-01-01

    Impaired glial glutamate uptake is commonly involved in neuronal damages observed in acute and chronic nervous disorders. As nervous insults are frequently associated with local inflammation involving microglia, this study aims at exploring the link between activated microglia and altered glutamate uptake in astrocytes. The regulation of the expression and activity of type 1 glutamate transporter (GLT-1) was examined after exposing cultures of rat astrocytes to conditioned medium from lipopolysaccharide-activated microglia cultures. Significant increases in GLT-1 mRNA expression and dihydrokainate sensitive uptake of aspartate were observed after 72h of treatment. These effects were reproduced by direct exposure of the astrocyte cultures to tumor necrosis factor alpha, a major cytokine released by activated microglia. The regulation of GLT-1 activity in response to inflammatory stimuli was also evidenced in cells exposed to dibutyryl cAMP, recognised as a model of reactive astrocytes in which the expression of this glutamate transporter is constitutively enhanced. Taken together, these results suggest that the GLT-1-dependent control of glutamate neurotransmission by either naive or chemically activated astrocytes is influenced by microglia-mediated inflammation. PMID:19428805

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

    PubMed

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

    2015-11-15

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

  18. Up-regulation of gamma-glutamyl transpeptidase (GGT) activity in growth perturbed C6 astrocytes.

    PubMed

    Mares, V; Malík, R; Lisá, V; Sedo, A

    2005-05-20

    Activity of gamma-glutamyl transpeptidase (GGT) was studied in astrocyte-like C6 glial cells modulated in growth and maturation by different concentration of serum and dibutyryl cyclic AMP (Db-cAMP) supplement in culture medium. After reduction of serum concentration from 10% to 0.1%, the number of GGT positive cells determined histochemically increased 3.1 times and the GGT activity/mg protein in whole cell lysates was 5.1 times higher. In cultures with 0.1% serum + Db-cAMP, the histochemically and biochemically assayed GGT activity exceeded 5.1 and 7.9 times the values measured in control 10% serum cultures, respectively. The up-regulation of GGT was accompanied by an inhibition of proliferation, enhanced differentiation and hypertrophy of cells. In addition, the process of metabolic perturbation and/or cellular stress was revealed in these cultures by the (i) growth-support release followed by shrinkage and death of a small number of cells and (ii) higher oxidation of 2'7'dichlorofluorescein diacetate to its fluorescent form in the adherent/viable cells. The observed up-regulation of GGT is considered to primarily reflect increased metabolism of glutathione and/or the maintenance of the redox potential in cells stressed by sub-optimal concentration of serum and Db-cAMP supplement. The concomitant cellular hypertrophy and differentiation and their relationship to increased activity of GGT await further investigation. The study suggests that up-regulation of GGT can contribute to adaptation of astrocytic cells to metabolic and/or oxidative perturbances occurring under various pathological conditions, including radiation- and drug-induced toxicity. PMID:15893589

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

    Vlasova-St Louis, Irina; Bohjanen, Paul R

    2016-01-01

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

  1. Neuronal activity mediated regulation of glutamate transporter GLT-1 surface diffusion in rat astrocytes in dissociated and slice cultures.

    PubMed

    Al Awabdh, Sana; Gupta-Agarwal, Swati; Sheehan, David F; Muir, James; Norkett, Rosalind; Twelvetrees, Alison E; Griffin, Lewis D; Kittler, Josef T

    2016-07-01

    The astrocytic GLT-1 (or EAAT2) is the major glutamate transporter for clearing synaptic glutamate. While the diffusion dynamics of neurotransmitter receptors at the neuronal surface are well understood, far less is known regarding the surface trafficking of transporters in subcellular domains of the astrocyte membrane. Here, we have used live-cell imaging to study the mechanisms regulating GLT-1 surface diffusion in astrocytes in dissociated and brain slice cultures. Using GFP-time lapse imaging, we show that GLT-1 forms stable clusters that are dispersed rapidly and reversibly upon glutamate treatment in a transporter activity-dependent manner. Fluorescence recovery after photobleaching and single particle tracking using quantum dots revealed that clustered GLT-1 is more stable than diffuse GLT-1 and that glutamate increases GLT-1 surface diffusion in the astrocyte membrane. Interestingly, the two main GLT-1 isoforms expressed in the brain, GLT-1a and GLT-1b, are both found to be stabilized opposed to synapses under basal conditions, with GLT-1b more so. GLT-1 surface mobility is increased in proximity to activated synapses and alterations of neuronal activity can bidirectionally modulate the dynamics of both GLT-1 isoforms. Altogether, these data reveal that astrocytic GLT-1 surface mobility, via its transport activity, is modulated during neuronal firing, which may be a key process for shaping glutamate clearance and glutamatergic synaptic transmission. GLIA 2016;64:1252-1264. PMID:27189737

  2. Neuronal activity mediated regulation of glutamate transporter GLT‐1 surface diffusion in rat astrocytes in dissociated and slice cultures

    PubMed Central

    Al Awabdh, Sana; Gupta‐Agarwal, Swati; Sheehan, David F.; Muir, James; Norkett, Rosalind; Twelvetrees, Alison E.; Griffin, Lewis D.

    2016-01-01

    The astrocytic GLT‐1 (or EAAT2) is the major glutamate transporter for clearing synaptic glutamate. While the diffusion dynamics of neurotransmitter receptors at the neuronal surface are well understood, far less is known regarding the surface trafficking of transporters in subcellular domains of the astrocyte membrane. Here, we have used live‐cell imaging to study the mechanisms regulating GLT‐1 surface diffusion in astrocytes in dissociated and brain slice cultures. Using GFP‐time lapse imaging, we show that GLT‐1 forms stable clusters that are dispersed rapidly and reversibly upon glutamate treatment in a transporter activity‐dependent manner. Fluorescence recovery after photobleaching and single particle tracking using quantum dots revealed that clustered GLT‐1 is more stable than diffuse GLT‐1 and that glutamate increases GLT‐1 surface diffusion in the astrocyte membrane. Interestingly, the two main GLT‐1 isoforms expressed in the brain, GLT‐1a and GLT‐1b, are both found to be stabilized opposed to synapses under basal conditions, with GLT‐1b more so. GLT‐1 surface mobility is increased in proximity to activated synapses and alterations of neuronal activity can bidirectionally modulate the dynamics of both GLT‐1 isoforms. Altogether, these data reveal that astrocytic GLT‐1 surface mobility, via its transport activity, is modulated during neuronal firing, which may be a key process for shaping glutamate clearance and glutamatergic synaptic transmission. GLIA 2016;64:1252–1264 PMID:27189737

  3. Regulation of Macropinocytosis by Diacylglycerol Kinase ζ

    PubMed Central

    Pomoransky, Julia L.; Parks, Robin J.; Trinkle-Mulcahy, Laura; Bell, John C.; Gee, Stephen H.

    2015-01-01

    Macropinosomes arise from the closure of plasma membrane ruffles to bring about the non-selective uptake of nutrients and solutes into cells. The morphological changes underlying ruffle formation and macropinosome biogenesis are driven by actin cytoskeleton rearrangements under the control of the Rho GTPase Rac1. We showed previously that Rac1 is activated by diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show DGKζ is required for optimal macropinocytosis induced by growth factor stimulation of mouse embryonic fibroblasts. Time-lapse imaging of live cells and quantitative analysis revealed DGKζ was associated with membrane ruffles and nascent macropinosomes. Macropinocytosis was attenuated in DGKζ-null cells, as determined by live imaging and vaccinia virus uptake experiments. Moreover, macropinosomes that did form in DGKζ-null cells were smaller than those found in wild type cells. Rescue of this defect required DGKζ catalytic activity, consistent with it also being required for Rac1 activation. A constitutively membrane bound DGKζ mutant substantially increased the size of macropinosomes and potentiated the effect of a constitutively active Rac1 mutant on macropinocytosis. Collectively, our results suggest DGKζ functions in concert with Rac1 to regulate macropinocytosis. PMID:26701304

  4. 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.

  5. Extracellular signal-regulated kinase phosphorylation in forebrain neurones contributes to osmoregulatory mechanisms

    PubMed Central

    Dine, Julien; Ducourneau, Vincent R R; Fénelon, Valérie S; Fossat, Pascal; Amadio, Aurélie; Eder, Matthias; Israel, Jean-Marc; Oliet, Stéphane H R; Voisin, Daniel L

    2014-01-01

    Vasopressin secretion from the magnocellular neurosecretory cells (MNCs) is crucial for body fluid homeostasis. Osmotic regulation of MNC activity involves the concerted modulation of intrinsic mechanosensitive ion channels, taurine release from local astrocytes as well as excitatory inputs derived from osmosensitive forebrain regions. Extracellular signal-regulated protein kinases (ERK) are mitogen-activated protein kinases that transduce extracellular stimuli into intracellular post-translational and transcriptional responses, leading to changes in intrinsic neuronal properties and synaptic function. Here, we investigated whether ERK activation (i.e. phosphorylation) plays a role in the functioning of forebrain osmoregulatory networks. We found that within 10 min after intraperitoneal injections of hypertonic saline (3 m, 6 m) in rats, many phosphoERK-immunopositive neurones were observed in osmosensitive forebrain regions, including the MNC containing supraoptic nuclei. The intensity of ERK labelling was dose-dependent. Reciprocally, slow intragastric infusions of water that lower osmolality reduced basal ERK phosphorylation. In the supraoptic nucleus, ERK phosphorylation predominated in vasopressin neurones vs. oxytocin neurones and was absent from astrocytes. Western blot experiments confirmed that phosphoERK expression in the supraoptic nucleus was dose dependent. Intracerebroventricular administration of the ERK phosphorylation inhibitor U 0126 before a hyperosmotic challenge reduced the number of both phosphoERK-immunopositive neurones and Fos expressing neurones in osmosensitive forebrain regions. Blockade of ERK phosphorylation also reduced hypertonically induced depolarization and an increase in firing of the supraoptic MNCs recorded in vitro. It finally reduced hypertonically induced vasopressin release in the bloodstream. Altogether, these findings identify ERK phosphorylation as a new element contributing to the osmoregulatory mechanisms of

  6. Eicosanoid receptor subtype-mediated opposing regulation of TLR-stimulated expression of astrocyte glial-derived neurotrophic factor

    PubMed Central

    Li, Xianwu; Cudaback, Eiron; Breyer, Richard M.; Montine, Kathleen S.; Keene, C. Dirk; Montine, Thomas J.

    2012-01-01

    A major therapeutic target for Parkinson's disease (PD) is providing increased glial-derived neurotrophic factor (GDNF) to dopaminergic neurons. We tested the hypothesis that innate immune activation increases astrocyte GDNF production and that this is regulated by specific eicosanoid receptors. Innate immune-activated primary murine astrocytes were assayed for GDNF expression and secretion. Controls were agent vehicle exposure and wild-type mice. Rank order for up to 10-fold selectively increased GDNF expression was activators of TLR3 > TLR2 or TLR4 > TLR9. TLR3 activator-stimulated GDNF expression was selectively JNK-dependent, followed cyclooxygenase (COX)-2, was coincident with membranous PGE2 synthase, and was not significantly altered by a nonspecific COX- or a COX-2-selective inhibitor. Specific eicosanoid receptors had opposing effects on TLR3 activator-induced GDNF expression: ∼60% enhancement by blocking or ablating of PGE2 receptor subtype 1 (EP1), ∼30% enhancement by activating PGF2α receptor or thromboxane receptor, or ∼15% enhancement by activating EP4. These results demonstrate functionally antagonistic eicosanoid receptor subtype regulation of innate immunity-induced astrocyte GDNF expression and suggest that selective inhibition of EP1 signaling might be a means to augment astrocyte GDNF secretion in the context of innate immune activation in diseased regions of brain in PD.—Li, X., Cudaback, E., Breyer, R. M., Montine, K. S., Keene, C. D., Montine, T. J. Eicosanoid receptor subtype-mediated opposing regulation of Toll-like receptor-stimulated expression of astrocyte glial-derived neurotrophic factor. PMID:22499581

  7. Diphenylarsinic Acid Induced Activation of Cultured Rat Cerebellar Astrocytes: Phosphorylation of Mitogen-Activated Protein Kinases, Upregulation of Transcription Factors, and Release of Brain-Active Cytokines.

    PubMed

    Negishi, Takayuki; Matsumoto, Mami; Kojima, Mikiya; Asai, Ryota; Kanehira, Tomoko; Sakaguchi, Fumika; Takahata, Kazuaki; Arakaki, Rina; Aoyama, Yohei; Yoshida, Hikari; Yoshida, Kenji; Yukawa, Kazunori; Tashiro, Tomoko; Hirano, Seishiro

    2016-03-01

    Diphenylarsinic acid (DPAA) was detected as the primary compound responsible for the arsenic poisoning that occurred in Kamisu, Ibaraki, Japan, where people using water from a well that was contaminated with a high level of arsenic developed neurological (mostly cerebellar) symptoms and dysregulation of regional cerebral blood flow. To understand the underlying molecular mechanism of DPAA-induced cerebellar symptoms, we focused on astrocytes, which have a brain-protective function. Incubation with 10 µM DPAA for 96 h promoted cell proliferation, increased the expression of antioxidative stress proteins (heme oxygenase-1 and heat shock protein 70), and induced the release of cytokines (MCP-1, adrenomedullin, FGF2, CXCL1, and IL-6). Furthermore, DPAA overpoweringly increased the phosphorylation of three major mitogen-activated protein kinases (MAPKs) (ERK1/2, p38MAPK, and SAPK/JNK), which indicated MAPK activation, and subsequently induced expression and/or phosphorylation of transcription factors (Nrf2, CREB, c-Jun, and c-Fos) in cultured rat cerebellar astrocytes. Structure-activity relationship analyses of DPAA and other related pentavalent organic arsenicals revealed that DPAA at 10 µM activated astrocytes most effective among organic arsenicals tested at the same dose. These results suggest that in a cerebellum exposed to DPAA, abnormal activation of the MAPK-transcription factor pathway and irregular secretion of these neuroactive, glioactive, and/or vasoactive cytokines in astrocytes can be the direct/indirect cause of functional abnormalities in surrounding neurons, glial cells, and vascular cells: This in turn might lead to the onset of cerebellar symptoms and disruption of cerebral blood flow. PMID:26645585

  8. Regulation of NF-{kappa}B activity in astrocytes: effects of flavonoids at dietary-relevant concentrations

    SciTech Connect

    Spilsbury, Alison; Vauzour, David; Spencer, Jeremy P.E.; Rattray, Marcus

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer We tested the hypothesis that low concentrations of flavonoids inhibit NF-{kappa}B in astrocytes. Black-Right-Pointing-Pointer Primary cultured astrocytes possess a functional {kappa}B-system, measured using luciferase assays. Black-Right-Pointing-Pointer Seven flavonoids (100 nM-1 {mu}M) failed to reduce NF-{kappa}B activity in astrocytes. Black-Right-Pointing-Pointer Four flavonoids (100 nM-1 {mu}M) failed to reduce TNFa-stimulated NF-{kappa}B activity in astrocytes. Black-Right-Pointing-Pointer (-)-Epicatechin did not regulate nuclear translocation of the NF-{kappa}B subunit, p65. -- Abstract: Neuroinflammation plays an important role in the progression of neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Sustained activation of nuclear transcription factor {kappa}B (NF-{kappa}B) is thought to play an important role in the pathogenesis of neurodegenerative disorders. Flavonoids have been shown to possess antioxidant and anti-inflammatory properties and we investigated whether flavonoids, at submicromolar concentrations relevant to their bioavailability from the diet, were able to modulate NF-{kappa}B signalling in astrocytes. Using luciferase reporter assays, we found that tumour necrosis factor (TNF{alpha}, 150 ng/ml) increased NF-{kappa}B-mediated transcription in primary cultures of mouse cortical astrocytes, which was abolished on co-transfection of a dominant-negative I{kappa}B{alpha} construct. In addition, TNF{alpha} increased nuclear localisation of p65 as shown by immunocytochemistry. To investigate potential flavonoid modulation of NF-{kappa}B activity, astrocytes were treated with flavonoids from different classes; flavan-3-ols ((-)-epicatechin and (+)-catechin), flavones (luteolin and chrysin), a flavonol (kaempferol) or the flavanones (naringenin and hesperetin) at dietary-relevant concentrations (0.1-1 {mu}M) for 18 h. None of the flavonoids modulated constitutive or TNF

  9. Pyruvate kinase: function, regulation and role in cancer

    PubMed Central

    Israelsen, William J.; Vander Heiden, Matthew G.

    2015-01-01

    Pyruvate kinase is an enzyme that catalyzes the conversion of phosphoenolpyruvate and ADP to pyruvate and ATP in glycolysis and plays a role in regulating cell metabolism. There are four mammalian pyruvate kinase isoforms with unique tissue expression patterns and regulatory properties. The M2 isoform of pyruvate kinase (PKM2) supports anabolic metabolism and is expressed both in cancer and normal tissue. The enzymatic activity of PKM2 is allosterically regulated by both intracellular signaling pathways and metabolites; PKM2 thus integrates signaling and metabolic inputs to modulate glucose metabolism according to the needs of the cell. Recent advances have increased our understanding of metabolic regulation by pyruvate kinase, raised new questions, and suggested the possibility of non-canonical PKM2 functions to regulate gene expression and cell cycle progression via protein-protein interactions and protein kinase activity. Here we review the structure, function, and regulation of pyruvate kinase and discuss how these properties enable regulation of PKM2 for cell proliferation and tumor growth. PMID:26277545

  10. The SH2 domain of Abl kinases regulates kinase autophosphorylation by controlling activation loop accessibility

    NASA Astrophysics Data System (ADS)

    Lamontanara, Allan Joaquim; Georgeon, Sandrine; Tria, Giancarlo; Svergun, Dmitri I.; Hantschel, Oliver

    2014-11-01

    The activity of protein kinases is regulated by multiple molecular mechanisms, and their disruption is a common driver of oncogenesis. A central and almost universal control element of protein kinase activity is the activation loop that utilizes both conformation and phosphorylation status to determine substrate access. In this study, we use recombinant Abl tyrosine kinases and conformation-specific kinase inhibitors to quantitatively analyse structural changes that occur after Abl activation. Allosteric SH2-kinase domain interactions were previously shown to be essential for the leukemogenesis caused by the Bcr-Abl oncoprotein. We find that these allosteric interactions switch the Abl activation loop from a closed to a fully open conformation. This enables the trans-autophosphorylation of the activation loop and requires prior phosphorylation of the SH2-kinase linker. Disruption of the SH2-kinase interaction abolishes activation loop phosphorylation. Our analysis provides a molecular mechanism for the SH2 domain-dependent activation of Abl that may also regulate other tyrosine kinases.

  11. A Tale of Two Stories: Astrocyte Regulation of Synaptic Depression and Facilitation

    PubMed Central

    De Pittà, Maurizio; Volman, Vladislav; Berry, Hugues; Ben-Jacob, Eshel

    2011-01-01

    Short-term presynaptic plasticity designates variations of the amplitude of synaptic information transfer whereby the amount of neurotransmitter released upon presynaptic stimulation changes over seconds as a function of the neuronal firing activity. While a consensus has emerged that the resulting decrease (depression) and/or increase (facilitation) of the synapse strength are crucial to neuronal computations, their modes of expression in vivo remain unclear. Recent experimental studies have reported that glial cells, particularly astrocytes in the hippocampus, are able to modulate short-term plasticity but the mechanism of such a modulation is poorly understood. Here, we investigate the characteristics of short-term plasticity modulation by astrocytes using a biophysically realistic computational model. Mean-field analysis of the model, supported by intensive numerical simulations, unravels that astrocytes may mediate counterintuitive effects. Depending on the expressed presynaptic signaling pathways, astrocytes may globally inhibit or potentiate the synapse: the amount of released neurotransmitter in the presence of the astrocyte is transiently smaller or larger than in its absence. But this global effect usually coexists with the opposite local effect on paired pulses: with release-decreasing astrocytes most paired pulses become facilitated, namely the amount of neurotransmitter released upon spike i+1 is larger than that at spike i, while paired-pulse depression becomes prominent under release-increasing astrocytes. Moreover, we show that the frequency of astrocytic intracellular Ca2+ oscillations controls the effects of the astrocyte on short-term synaptic plasticity. Our model explains several experimental observations yet unsolved, and uncovers astrocytic gliotransmission as a possible transient switch between short-term paired-pulse depression and facilitation. This possibility has deep implications on the processing of neuronal spikes and resulting

  12. Regulation of astrocyte glutamate transporter-1 (GLT1) and aquaporin-4 (AQP4) expression in a model of epilepsy.

    PubMed

    Hubbard, Jacqueline A; Szu, Jenny I; Yonan, Jennifer M; Binder, Devin K

    2016-09-01

    Astrocytes regulate extracellular glutamate and water homeostasis through the astrocyte-specific membrane proteins glutamate transporter-1 (GLT1) and aquaporin-4 (AQP4), respectively. The role of astrocytes and the regulation of GLT1 and AQP4 in epilepsy are not fully understood. In this study, we investigated the expression of GLT1 and AQP4 in the intrahippocampal kainic acid (IHKA) model of temporal lobe epilepsy (TLE). We used real-time polymerase chain reaction (RT-PCR), Western blot, and immunohistochemical analysis at 1, 4, 7, and 30days after kainic acid-induced status epilepticus (SE) to determine hippocampal glial fibrillary acidic protein (GFAP, a marker for reactive astrocytes), GLT1, and AQP4 expression changes during the development of epilepsy (epileptogenesis). Following IHKA, all mice had SE and progressive increases in GFAP immunoreactivity and GFAP protein expression out to 30days post-SE. A significant initial increase in dorsal hippocampal GLT1 immunoreactivity and protein levels were observed 1day post SE and followed by a marked downregulation at 4 and 7days post SE with a return to near control levels by 30days post SE. AQP4 dorsal hippocampal protein expression was significantly downregulated at 1day post SE and was followed by a gradual return to baseline levels with a significant increase in ipsilateral protein levels by 30days post SE. Transient increases in GFAP and AQP4 mRNA were also observed. Our findings suggest that specific molecular changes in astrocyte glutamate transporters and water channels occur during epileptogenesis in this model, and suggest the novel therapeutic strategy of restoring glutamate and water homeostasis. PMID:27155358

  13. Testosterone Protects Mitochondrial Function and Regulates Neuroglobin Expression in Astrocytic Cells Exposed to Glucose Deprivation.

    PubMed

    Toro-Urrego, Nicolas; Garcia-Segura, Luis M; Echeverria, Valentina; Barreto, George E

    2016-01-01

    Testosterone is a hormone that has been shown to confer neuroprotection from different insults affecting the central nervous system (CNS). Testosterone induces this protection by different mechanisms that include the activation of anti-apoptotic pathways that are directly implicated in neuronal survival. However, little attention has been devoted to its actions on glial cells. In the present study, we have assessed whether testosterone exerts protection in a human astrocyte cell model, the T98G cells. Our results indicate that testosterone improves cell survival and mitochondrial membrane potential and reduces nuclear fragmentation and reactive oxygen species (ROS) generation. These effects were accompanied by a positive regulation of neuroglobin, an oxygen-binding and sensor protein, which may serve as a regulator of ROS and nitrogen reactive species (NOS), and these protective effects of testosterone may be at least in part mediated by estradiol and DHT. In conclusion, these findings suggest that astroglia may mediate some of the protective actions of testosterone in the brain upon pathological conditions. PMID:27445795

  14. Testosterone Protects Mitochondrial Function and Regulates Neuroglobin Expression in Astrocytic Cells Exposed to Glucose Deprivation

    PubMed Central

    Toro-Urrego, Nicolas; Garcia-Segura, Luis M.; Echeverria, Valentina; Barreto, George E.

    2016-01-01

    Testosterone is a hormone that has been shown to confer neuroprotection from different insults affecting the central nervous system (CNS). Testosterone induces this protection by different mechanisms that include the activation of anti-apoptotic pathways that are directly implicated in neuronal survival. However, little attention has been devoted to its actions on glial cells. In the present study, we have assessed whether testosterone exerts protection in a human astrocyte cell model, the T98G cells. Our results indicate that testosterone improves cell survival and mitochondrial membrane potential and reduces nuclear fragmentation and reactive oxygen species (ROS) generation. These effects were accompanied by a positive regulation of neuroglobin, an oxygen-binding and sensor protein, which may serve as a regulator of ROS and nitrogen reactive species (NOS), and these protective effects of testosterone may be at least in part mediated by estradiol and DHT. In conclusion, these findings suggest that astroglia may mediate some of the protective actions of testosterone in the brain upon pathological conditions. PMID:27445795

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

    SciTech Connect

    Roskoski, Robert

    2010-08-27

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

  16. Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory

    PubMed Central

    Orr, Anna G.; Hsiao, Edward C.; Wang, Max M.; Ho, Kaitlyn; Kim, Daniel H.; Wang, Xin; Guo, Weikun; Kang, Jing; Yu, Gui-Qiu; Adame, Anthony; Devidze, Nino; Dubal, Dena B.; Masliah, Eliezer; Conklin, Bruce R.; Mucke, Lennart

    2014-01-01

    Astrocytes express a variety of G protein-coupled receptors and might influence cognitive functions, such as learning and memory. However, the roles of astrocytic Gs-coupled receptors in cognitive function are not known. We found that humans with Alzheimer’s disease (AD) had increased levels of the Gs-coupled adenosine receptor A2A in astrocytes. Conditional genetic removal of these receptors enhanced long-term memory in young and aging mice, and increased the levels of Arc/Arg3.1, an immediate-early gene required for long-term memory. Chemogenetic activation of astrocytic Gs-coupled signaling reduced long-term memory in mice without affecting learning. Similar to humans with AD, aging mice expressing human amyloid precursor protein (hAPP) showed increased levels of astrocytic A2A receptors. Conditional genetic removal of these receptors enhanced memory in aging hAPP mice. Together, these findings establish a regulatory role for astrocytic Gs-coupled receptors in memory and suggest that AD-linked increases in astrocytic A2A receptor levels contribute to memory loss. PMID:25622143

  17. Probenecid protects against oxygen-glucose deprivation injury in primary astrocytes by regulating inflammasome activity.

    PubMed

    Jian, Zhihong; Ding, Shuai; Deng, Hongping; Wang, Jun; Yi, Wei; Wang, Lei; Zhu, Shengmei; Gu, Lijuan; Xiong, Xiaoxing

    2016-07-15

    Inflammation is extremely important in the development of cerebral ischemia/reperfusion injury. Pannexin 1 (Panx1) channel has been reported to activate inflammasome in astrocytes and be involved in ischemic injury, but this damage effect is reversed by a Panx1 inhibitor-probenecid. However, the mechanism of probenecid protects against cerebral ischemia/reperfusion injury remains unclear. In present study, we hypothesized that probenecid protected astrocytes from ischemia/reperfusion injury in vitro by modulating the inflammasome. Primary cultured neocortical astrocytes were exposed to oxygen-glucose deprivation/reoxygenation (OGD/RX) and probenecid was added in this model. Viability and nuclear morphology of astrocytes, production of reactive oxygen species (ROS), protein expressions of NLRP3 (NOD-like receptor protein 3), caspase-1, and AQP4 (Aquaporins 4), as well as release of cellular HMGB1 and IL-1β were observed to evaluate the effect and mechanisms of probenecid on OGD/reoxygenated astrocytes. Probenecid did not affect cell viability at concentrations of 1, 5, 10, and 100μM but induced significant astrocytes death at 500μM. Probenecid inhibited cell death and ROS generation in astrocytes subjected to 6h of OGD and 24h of reoxygenation. The expression levels of NLRP3, caspase-1, and AQP4 increased after 6h of OGD, but probenecid treatment attenuated this increase. Moreover, the extracellular release of IL-1β and HMGB1 from OGD/reoxygenated astrocytes increased significantly. However, treatment by probenecid resulted in substantial reduction of these proteins levels in extracellular space. In conclusion, The Panx1 inhibitor, probenecid, which was administered before OGD, provided protective effects on the OGD/reoxygenation model of cultured astrocytes by modulating inflammasome activity and downregulating AQP4 expression. PMID:27154322

  18. P2Y2 receptor up-regulation induced by guanosine or UTP in rat brain cultured astrocytes.

    PubMed

    Ballerini, P; Di Iorio, P; Caciagli, F; Rathbone, M P; Jiang, S; Nargi, E; Buccella, S; Giuliani, P; D'Alimonte, I; Fischione, G; Masciulli, A; Romano, S; Ciccarelli, R

    2006-01-01

    Among P2 metabotropic ATP receptors, P2Y2 subtype seems to be peculiar as its upregulation triggers important biological events in different cells types. In non-stimulated cells including astrocytes, P2Y2 receptors are usually expressed at levels lower than P2Y1 sites, however the promoter region of the P2Y2 receptors has not yet been studied and little is known about the mechanisms underlying the regulation of the expression of this ATP receptor. We showed that not only UTP and ATP are the most potent and naturally occurring agonist for P2Y2 sites, but also guanosine induced an up-regulation of astrocyte P2Y2 receptor mRNA evaluated by Northern blot analysis. We also focused our attention on this nucleoside since in our previous studies it was reported to be released by cultured astrocytes and to exert different neuroprotective effects. UTP and guanosine-evoked P2Y2 receptor up-regulation in rat brain cultured astrocytes was linked to an increased P2Y2-mediated intracellular calcium response, thus suggesting an increased P2Y2 activity. Actinomycin D, a RNA polymerase inhibitor, abrogated both UTP and guanosine-mediated P2Y2 up-regulation, thus indicating that de novo transcription was required. The effect of UTP and guanosine was also evaluated in astrocytes pretreated with different inhibitors of signal transduction pathways including ERK, PKC and PKA reported to be involved in the regulation of other cell surface receptor mRNAs. The results show that ERK1-2/MAPK pathway play a key role in the P2Y2 receptor up-regulation mediated by either UTP or guanosine. Moreover, our data suggest that PKA is also involved in guanosine-induced transcriptional activation of P2Y2 mRNA and that increased intracellular calcium levels and PKC activation may also mediate P2Y2 receptor up-regulation triggered by UTP. The extracellular release of ATP under physiological and pathological conditions has been widely studied. On the contrary, little is known about the release of

  19. Regulation of mitochondrial protein import by cytosolic kinases.

    PubMed

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

    2011-01-21

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

  20. Cell cycle regulation of a Xenopus Wee1-like kinase.

    PubMed Central

    Mueller, P R; Coleman, T R; Dunphy, W G

    1995-01-01

    Using a polymerase chain reaction-based strategy, we have isolated a gene encoding a Wee1-like kinase from Xenopus eggs. The recombinant Xenopus Wee1 protein efficiently phosphorylates Cdc2 exclusively on Tyr-15 in a cyclin-dependent manner. The addition of exogenous Wee1 protein to Xenopus cell cycle extracts results in a dose-dependent delay of mitotic initiation that is accompanied by enhanced tyrosine phosphorylation of Cdc2. The activity of the Wee1 protein is highly regulated during the cell cycle: the interphase, underphosphorylated form of Wee1 (68 kDa) phosphorylates Cdc2 very efficiently, whereas the mitotic, hyperphosphorylated version (75 kDa) is weakly active as a Cdc2-specific tyrosine kinase. The down-modulation of Wee1 at mitosis is directly attributable to phosphorylation, since dephosphorylation with protein phosphatase 2A restores its kinase activity. During interphase, the activity of this Wee1 homolog does not vary in response to the presence of unreplicated DNA. The mitosis-specific phosphorylation of Wee1 is due to at least two distinct kinases: the Cdc2 protein and another activity (kinase X) that may correspond to an MPM-2 epitope kinase. These studies indicate that the down-regulation of Wee1-like kinase activity at mitosis is a multistep process that occurs after other biochemical reactions have signaled the successful completion of S phase. Images PMID:7749193

  1. Regulation of the syncytin-1 promoter in human astrocytes by multiple sclerosis-related cytokines

    SciTech Connect

    Mameli, Giuseppe . E-mail: viross@uniss.it; Astone, Vito; Khalili, Kamel; Serra, Caterina; Sawaya, Bassel E.; Dolei, Antonina

    2007-05-25

    Syncytin-1 has a physiological role during early pregnancy, as mediator of trophoblast fusion into the syncytiotrophoblast layer, hence allowing embryo implantation. In addition, its expression in nerve tissue has been proposed to contribute to the pathogenesis of multiple sclerosis (MS). Syncytin-1 is the env glycoprotein of the ERVWE1 component of the W family of human endogenous retroviruses (HERV), located on chromosome 7q21-22, in a candidate region for genetic susceptibility to MS. The mechanisms of ERVWE1 regulation in nerve tissue remain to be identified. Since there are correlations between some cytokines and MS outcome, we examined the regulation of the syncytin-1 promoter by MS-related cytokines in human U-87MG astrocytic cells. Using transient transfection assays, we observed that the MS-detrimental cytokines TNF{alpha}, interferon-{gamma}, interleukin-6, and interleukin-1 activate the ERVWE1 promoter, while the MS-protective interferon-{beta} is inhibitory. The effects of cytokines are reduced by the deletion of the cellular enhancer domain of the promoter that contains binding sites for several transcription factors. In particular, we found that TNF{alpha} had the ability to activate the ERVWE1 promoter through an NF-{kappa}B-responsive element located within the enhancer domain of the promoter. Electrophoretic mobility shift and ChIP assays showed that TNF{alpha} enhances the binding of the p65 subunit of NF-{kappa}B, to its cognate site within the promoter. The effect of TNF{alpha} is abolished by siRNA directed against p65. Taken together, these results illustrate a role for p65 in regulating the ERVWE1 promoter and in TNF{alpha}-mediated induction of syncytin-1 in multiple sclerosis.

  2. Phytochemicals and botanical extracts regulate NF-κB and Nrf2/ARE reporter activities in DI TNC1 astrocytes.

    PubMed

    Ajit, Deepa; Simonyi, Agnes; Li, Runting; Chen, Zihong; Hannink, Mark; Fritsche, Kevin L; Mossine, Valeri V; Smith, Robert E; Dobbs, Thomas K; Luo, Rensheng; Folk, William R; Gu, Zezong; Lubahn, Dennis B; Weisman, Gary A; Sun, Grace Y

    2016-07-01

    The increase in oxidative stress and inflammatory responses associated with neurodegenerative diseases has drawn considerable attention towards understanding the transcriptional signaling pathways involving NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and Nrf2 (Nuclear Factor Erythroid 2-like 2). Our recent studies with immortalized murine microglial cells (BV-2) demonstrated effects of botanical polyphenols to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) and enhance Nrf2-mediated antioxidant responses (Sun et al., 2015). In this study, an immortalized rat astrocyte (DI TNC1) cell line expressing a luciferase reporter driven by the NF-κB or the Nrf2/Antioxidant Response Element (ARE) promoter was used to assess regulation of these two pathways by phytochemicals such as quercetin, rutin, cyanidin, cyanidin-3-O-glucoside, as well as botanical extracts from Withania somnifera (Ashwagandha), Sutherlandia frutescens (Sutherlandia) and Euterpe oleracea (Açaí). Quercetin effectively inhibited LPS-induced NF-κB reporter activity and stimulated Nrf2/ARE reporter activity in DI TNC1 astrocytes. Cyanidin and the glycosides showed similar effects but only at much higher concentrations. All three botanical extracts effectively inhibited LPS-induced NF-κB reporter activity. These extracts were capable of enhancing ARE activity by themselves and further enhanced ARE activity in the presence of LPS. Quercetin and botanical extracts induced Nrf2 and HO-1 protein expression. Interestingly, Ashwagandha extract was more active in inducing Nrf2 and HO-1 expression in DI TNC1 astrocytes as compared to Sutherlandia and Açaí extracts. In summary, this study demonstrated NF-kB and Nrf2/ARE promoter activities in DI TNC1 astrocytes, and further showed differences in ability for specific botanical polyphenols and extracts to down-regulate LPS-induced NF-kB and up-regulate the NRF2/ARE activities in these cells. PMID:27166148

  3. Astrocytic dynamin-like protein 1 regulates neuronal protection against excitotoxicity in Parkinson disease.

    PubMed

    Hoekstra, Jake G; Cook, Travis J; Stewart, Tessandra; Mattison, Hayley; Dreisbach, Max T; Hoffer, Zachary S; Zhang, Jing

    2015-02-01

    Mitochondrial dynamics has recently become an area of piqued interest in neurodegenerative disorders, including Parkinson disease (PD); however, the contribution of astrocytes to these disorders remains unclear. Here, we show that the level of dynamin-like protein 1 (Dlp1; official name DNM1L), which promotes mitochondrial fission, is lower in astrocytes from the brains of PD patients, and that decreased astrocytic Dlp1 likely represents a relatively early event in PD pathogenesis. In support of this conclusion, we show that Dlp1 knockdown dramatically affects mitochondrial morphological characteristics and localization in astrocytes, impairs the ability of astrocytes to adequately protect neurons from the excitotoxic effects of glutamate, and increases intracellular Ca(2+) in response to extracellular glutamate, resulting from compromised intracellular Ca(2+) buffering. Taken together, our results suggest that astrocytic mitochondrial Dlp1 is a key protein in mitochondrial dynamics and decreased Dlp1 may interfere with neuron survival in PD by disrupting Ca(2+)-coupled glutamate uptake. PMID:25482923

  4. Astrocytic Dynamin-Like Protein 1 Regulates Neuronal Protection against Excitotoxicity in Parkinson Disease

    PubMed Central

    Hoekstra, Jake G.; Cook, Travis J.; Stewart, Tessandra; Mattison, Hayley; Dreisbach, Max T.; Hoffer, Zachary S.; Zhang, Jing

    2016-01-01

    Mitochondrial dynamics has recently become an area of piqued interest in neurodegenerative disorders, including Parkinson disease (PD); however, the contribution of astrocytes to these disorders remains unclear. Here, we show that the level of dynamin-like protein 1 (Dlp1; official name DNM1L), which promotes mitochondrial fission, is lower in astrocytes from the brains of PD patients, and that decreased astrocytic Dlp1 likely represents a relatively early event in PD pathogenesis. In support of this conclusion, we show that Dlp1 knockdown dramatically affects mitochondrial morphological characteristics and localization in astrocytes, impairs the ability of astrocytes to adequately protect neurons from the excitotoxic effects of glutamate, and increases intracellular Ca2+ in response to extracellular glutamate, resulting from compromised intracellular Ca2+ buffering. Taken together, our results suggest that astrocytic mitochondrial Dlp1 is a key protein in mitochondrial dynamics and decreased Dlp1 may interfere with neuron survival in PD by disrupting Ca2+-coupled glutamate uptake. PMID:25482923

  5. Protein kinaseregulates vaccinia-related kinase 1 in DNA damage–induced apoptosis

    PubMed Central

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

    2011-01-01

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

  6. DJ-1 deficiency impairs glutamate uptake into astrocytes via the regulation of flotillin-1 and caveolin-1 expression

    PubMed Central

    Kim, Jin-Mo; Cha, Seon-Heui; Choi, Yu Ree; Jou, Ilo; Joe, Eun-Hye; Park, Sang Myun

    2016-01-01

    Parkinson’s disease (PD) is a common chronic and progressive neurodegenerative disorder. Although the cause of PD is still poorly understood, mutations in many genes including SNCA, parkin, PINK1, LRRK2, and DJ-1 have been identified in the familial forms of PD. It was recently proposed that alterations in lipid rafts may cause the neurodegeneration shown in PD. Here, we observe that DJ-1 deficiency decreased the expression of flotillin-1 (flot-1) and caveolin-1 (cav-1), the main protein components of lipid rafts, in primary astrocytes and MEF cells. As a mechanism, DJ-1 regulated flot-1 stability by direct interaction, however, decreased cav-1 expression may not be a direct effect of DJ-1, but rather as a result of decreased flot-1 expression. Dysregulation of flot-1 and cav-1 by DJ-1 deficiency caused an alteration in the cellular cholesterol level, membrane fluidity, and alteration in lipid rafts-dependent endocytosis. Moreover, DJ-1 deficiency impaired glutamate uptake into astrocytes, a major function of astrocytes in the maintenance of CNS homeostasis, by altering EAAT2 expression. This study will be helpful to understand the role of DJ-1 in the pathogenesis of PD, and the modulation of lipid rafts through the regulation of flot-1 or cav-1 may be a novel therapeutic target for PD. PMID:27346864

  7. Mitogen-Activated Protein Kinase Kinase 3 Is Required for Regulation during Dark-Light Transition.

    PubMed

    Lee, Horim

    2015-07-01

    Plant growth and development are coordinately orchestrated by environmental cues and phytohormones. Light acts as a key environmental factor for fundamental plant growth and physiology through photosensory phytochromes and underlying molecular mechanisms. Although phytochromes are known to possess serine/threonine protein kinase activities, whether they trigger a signal transduction pathway via an intracellular protein kinase network remains unknown. In analyses of mitogen-activated protein kinase kinase (MAPKK, also called MKK) mutants, the mkk3 mutant has shown both a hypersensitive response in plant hormone gibberellin (GA) and a less sensitive response in red light signaling. Surprisingly, light-induced MAPK activation in wild-type (WT) seedlings and constitutive MAPK phosphorylation in dark-grown mkk3 mutant seedlings have also been found, respectively. Therefore, this study suggests that MKK3 acts in negative regulation in darkness and in light-induced MAPK activation during dark-light transition. PMID:26082029

  8. Protopanaxatriol Ginsenoside Rh1 Upregulates Phase II Antioxidant Enzyme Gene Expression in Rat Primary Astrocytes: Involvement of MAP Kinases and Nrf2/ARE Signaling

    PubMed Central

    Jung, Ji-Sun; Lee, Sang-Yoon; Kim, Dong-Hyun; Kim, Hee-Sun

    2016-01-01

    Oxidative stress activates several intracellular signaling cascades that may have deleterious effects on neuronal cell survival. Thus, controlling oxidative stress has been suggested as an important strategy for prevention and/or treatment of neurodegenerative diseases. In this study, we found that ginsenoside Rh1 inhibited hydrogen peroxide-induced reactive oxygen species generation and subsequent cell death in rat primary astrocytes. Rh1 increased the expression of phase II antioxidant enzymes, such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreductase 1, superoxide dismutase-2, and catalase, that are under the control of Nrf2/ARE signaling pathways. Further mechanistic studies showed that Rh1 increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to the antioxidant response element (ARE), and increased the ARE-mediated transcription activities in rat primary astrocytes. Analysis of signaling pathways revealed that MAP kinases are important in HO-1 expression, and act by modulating ARE-mediated transcriptional activity. Therefore, the upregulation of antioxidant enzymes by Rh1 may provide preventive therapeutic potential for various neurodegenerative diseases that are associated with oxidative stress. PMID:26759699

  9. Adenosine Receptors Differentially Regulate the Expression of Regulators of G-Protein Signalling (RGS) 2, 3 and 4 in Astrocyte-Like Cells

    PubMed Central

    Eusemann, Till Nicolas; Willmroth, Frank; Fiebich, Bernd; Biber, Knut; van Calker, Dietrich

    2015-01-01

    The “regulators of g-protein signalling” (RGS) comprise a large family of proteins that limit by virtue of their GTPase accelerating protein domain the signal transduction of G-protein coupled receptors. RGS proteins have been implicated in various neuropsychiatric diseases such as schizophrenia, drug abuse, depression and anxiety and aggressive behaviour. Since conditions associated with a large increase of adenosine in the brain such as seizures or ischemia were reported to modify the expression of some RGS proteins we hypothesized that adenosine might regulate RGS expression in neural cells. We measured the expression of RGS-2,-3, and -4 in both transformed glia cells (human U373 MG astrocytoma cells) and in primary rat astrocyte cultures stimulated with adenosine agonists. Expression of RGS-2 mRNA as well as RGS2 protein was increased up to 30-fold by adenosine agonists in astrocytes. The order of potency of agonists and the blockade by the adenosine A2B-antagonist MRS1706 indicated that this effect was largely mediated by adenosine A2B receptors. However, a smaller effect was observed due to activation of adenosine A2A receptors. In astrocytoma cells adenosine agonists elicited an increase in RGS-2 expression solely mediated by A2B receptors. Expression of RGS-3 was inhibited by adenosine agonists in both astrocytoma cells and astrocytes. However while this effect was mediated by A2B receptors in astrocytoma cells it was mediated by A2A receptors in astrocytes as assessed by the order of potency of agonists and selective blockade by the specific antagonists MRS1706 and ZM241385 respectively. RGS-4 expression was inhibited in astrocytoma cells but enhanced in astrocytes by adenosine agonists. PMID:26263491

  10. Sox2 Promotes Malignancy in Glioblastoma by Regulating Plasticity and Astrocytic Differentiation12

    PubMed Central

    Berezovsky, Artem D.; Poisson, Laila M.; Cherba, David; Webb, Craig P.; Transou, Andrea D.; Lemke, Nancy W.; Hong, Xin; Hasselbach, Laura A.; Irtenkauf, Susan M.; Mikkelsen, Tom; deCarvalho, Ana C.

    2014-01-01

    The high-mobility group–box transcription factor sex-determining region Y–box 2 (Sox2) is essential for the maintenance of stem cells from early development to adult tissues. Sox2 can reprogram differentiated cells into pluripotent cells in concert with other factors and is overexpressed in various cancers. In glioblastoma (GBM), Sox2 is a marker of cancer stemlike cells (CSCs) in neurosphere cultures and is associated with the proneural molecular subtype. Here, we report that Sox2 expression pattern in GBM tumors and patient-derived mouse xenografts is not restricted to a small percentage of cells and is coexpressed with various lineage markers, suggesting that its expression extends beyond CSCs to encompass more differentiated neoplastic cells across molecular subtypes. Employing a CSC derived from a patient with GBM and isogenic differentiated cell model, we show that Sox2 knockdown in the differentiated state abolished dedifferentiation and acquisition of CSC phenotype. Furthermore, Sox2 deficiency specifically impaired the astrocytic component of a biphasic gliosarcoma xenograft model while allowing the formation of tumors with sarcomatous phenotype. The expression of genes associated with stem cells and malignancy were commonly downregulated in both CSCs and serum-differentiated cells on Sox2 knockdown. Genes previously shown to be associated with pluripontency and CSCs were only affected in the CSC state, whereas embryonic stem cell self-renewal genes and cytokine signaling were downregulated, and the Wnt pathway activated in differentiated Sox2-deficient cells. Our results indicate that Sox2 regulates the expression of key genes and pathways involved in GBM malignancy, in both cancer stemlike and differentiated cells, and maintains plasticity for bidirectional conversion between the two states, with significant clinical implications. PMID:24726753

  11. Palmitate induces transcriptional regulation of BACE1 and presenilin by STAT3 in neurons mediated by astrocytes

    PubMed Central

    Liu, Li; Martin, Rebecca; Kohler, Garrett; Chan, Christina

    2013-01-01

    Deregulation of calcium has been implicated in neurodegenerative diseases, including Alzheimer’s disease (AD). Previously, we showed that saturated free-fatty acid, palmitate, causes AD-like changes in primary cortical neurons mediated by astrocytes. However, the molecular mechanisms by which conditioned media from astrocytes cultured in palmitate induces AD-like changes in neurons are unknown. This study demonstrates that this condition media from astrocytes elevates calcium level in the neurons, which subsequently increases calpain activity, a calcium-dependent protease, leading to enhance p25/Cdk5 activity and phosphorylation and activation of the STAT3 (signal transducer and activator of transcription) transcription factor. Inhibiting calpain or Cdk5 significantly reduces the upregulation in nuclear level of pSTAT3, which we found to transcriptionally regulate both BACE1 and presenilin-1, the latter is a catalytic subunit of γ-secretase. Decreasing pSTAT3 levels reduced the mRNA levels of both BACE1 and presenilin-1 to near control levels. These data demonstrate a signal pathway leading to the activation of STAT3, and the generation of the amyloid peptide. Thus, our results suggest that STAT3 is an important potential therapeutic target of AD pathogenesis. PMID:23968646

  12. A secretory kinase complex regulates extracellular protein phosphorylation.

    PubMed

    Cui, Jixin; Xiao, Junyu; Tagliabracci, Vincent S; Wen, Jianzhong; Rahdar, Meghdad; Dixon, Jack E

    2015-01-01

    Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation. PMID:25789606

  13. Astrocyte Stellation, a Process Dependent on Rac1 Is Sustained by the Regulated Exocytosis of Enlargeosomes

    PubMed Central

    Racchetti, Gabriella; D'Alessandro, Rosalba; Meldolesi, Jacopo

    2012-01-01

    Cultured astrocytes exhibit a flat/epitelioid phenotype much different from the star-like phenotype of tissue astrocytes. Upon exposure to treatments that affect the small GTPase Rho and/or its effector ROCK, however, flat astrocytes undergo stellation, with restructuring of cytoskeleton and outgrowth of processes with lamellipodia, assuming a phenotype closer to that exhibited in situ. The mechanisms of this change are known only in part. Using the ROCK blocker drug Y27632, which induces rapid (tens of min), dose-dependent and reversible stellations, we focused on two specific aspects of the process: its dependence on small GTPases and the large surface expansion of the cells. Contrary to previous reports, we found stellation to be governed by the small G protein Rac1, up to disappearance of the process when Rac1 was downregulated or blocked by a specific drug. In contrast cdc42, the other G-protein often involved in phenotype changes, appeared not involved. The surface expansion concomitant to cytoskeleton restructuring, also dependent on Rac1, was found to be at least partially sustained by the exocytosis of enlargeosomes, small vesicles distinct from classical cell organelles, which are abundant in astrocytes. Exhaustion of stellation induced by repeated administrations of Y27632 correlated with the decrease of the enlargeosome pool. A whole-cell process like stellation of cultured astrocytes might be irrelevant in the brain tissue. However, local restructuring of the cytoskeleton coordinate with surface expansion, occurring at critical cell sites and sustained by mechanisms analogous to those of stellation, might be of importance in both astrocyte physiology and pathology. © 2011 Wiley Periodicals, Inc. PMID:22144092

  14. Fluoxetin Upregulates Connexin 43 Expression in Astrocyte

    PubMed Central

    Mostafavi, Hossein; Khaksarian, Mojtaba; Joghataei, Mohammad Taghi; Hassanzadeh, Gholamreza; Soleimani, Masoud; Eftekhari, Sanaz; Soleimani, Mansooreh; Mousavizadeh, Kazem; Hadjighassem, Mahmoud Reza

    2014-01-01

    Introduction Recent studies have shown that astrocytes play major roles in normal and disease condition of the central nervous system including multiple sclerosis (MS). Molecular target therapy studies in MS have revealed that connexin-43 (Cx43) and Aquaporin-4 (AQP4) contents of astrocytes undergo expression alteration. Fluoxetine had some effects in MS patients unrelated to its known antidepressant effects. Some of fluoxetine effects were attributed to its capability of cAMP signaling pathway stimulation. This study aimed to investigate possible acute effects of fluoxetine on Cx43 and AQP4 expression in astrocyte. Methods Astrocytoma cells were treated for 24 hours with fluoxetine (10 and 20 µg/ml) with or without adenyl cyclase (AC) and protein kinase A (PKA) inhibition. Cx43 expression at both mRNA and protein levels and AQP4 expression at mRNA level were evaluated. Results Acquired results showed that fluoxetine with and without AC and PKA inhibition resulted in Cx43 up-regulation both in mRNA and protein levels, whereas AQP4 expression have not changed. Discussion In conclusion, data showed that fluoxetine alone and in the absence of serotonin acutely up-regulated Cx43 expression in astrocytes that can be assumed in molecular target therapy of MS patients. It seems that cAMP involvement in fluoxetine effects need more researches. PMID:25436087

  15. The molecular regulation of Janus kinase (JAK) activation.

    PubMed

    Babon, Jeffrey J; Lucet, Isabelle S; Murphy, James M; Nicola, Nicos A; Varghese, Leila N

    2014-08-15

    The JAK (Janus kinase) family members serve essential roles as the intracellular signalling effectors of cytokine receptors. This family, comprising JAK1, JAK2, JAK3 and TYK2 (tyrosine kinase 2), was first described more than 20 years ago, but the complexities underlying their activation, regulation and pleiotropic signalling functions are still being explored. Here, we review the current knowledge of their physiological functions and the causative role of activating and inactivating JAK mutations in human diseases, including haemopoietic malignancies, immunodeficiency and inflammatory diseases. At the molecular level, recent studies have greatly advanced our knowledge of the structures and organization of the component FERM (4.1/ezrin/radixin/moesin)-SH2 (Src homology 2), pseudokinase and kinase domains within the JAKs, the mechanism of JAK activation and, in particular, the role of the pseudokinase domain as a suppressor of the adjacent tyrosine kinase domain's catalytic activity. We also review recent advances in our understanding of the mechanisms of negative regulation exerted by the SH2 domain-containing proteins, SOCS (suppressors of cytokine signalling) proteins and LNK. These recent studies highlight the diversity of regulatory mechanisms utilized by the JAK family to maintain signalling fidelity, and suggest alternative therapeutic strategies to complement existing ATP-competitive kinase inhibitors. PMID:25057888

  16. A molecular ruler regulates cytoskeletal remodelling by the Rho kinases

    PubMed Central

    Truebestein, Linda; Elsner, Daniel J.; Fuchs, Elisabeth; Leonard, Thomas A.

    2015-01-01

    The Rho-associated coiled-coil kinases (ROCK) are essential regulators of the actin cytoskeleton; however, the structure of a full-length ROCK is unknown and the mechanisms by which its kinase activity is controlled are not well understood. Here we determine the low-resolution structure of human ROCK2 using electron microscopy, revealing it to be a constitutive dimer, 120 nm in length, with a long coiled-coil tether linking the kinase and membrane-binding domains. We find, in contrast to previous reports, that ROCK2 activity does not appear to be directly regulated by binding to membranes, RhoA, or by phosphorylation. Instead, we show that changing the length of the tether modulates ROCK2 function in cells, suggesting that it acts as a molecular ruler. We present a model in which ROCK activity is restricted to a discrete region of the actin cytoskeleton, governed by the length of its coiled-coil. This represents a new type of spatial control, and hence a new paradigm for kinase regulation. PMID:26620183

  17. Allosteric regulation of focal adhesion kinase by PIP₂ and ATP.

    PubMed

    Zhou, Jing; Bronowska, Agnieszka; Le Coq, Johanne; Lietha, Daniel; Gräter, Frauke

    2015-02-01

    Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that regulates cell signaling, proliferation, migration, and development. A major mechanism of regulation of FAK activity is an intramolecular autoinhibitory interaction between two of its domains--the catalytic and FERM domains. Upon cell adhesion to the extracellular matrix, FAK is being translocated toward focal adhesion sites and activated. Interactions of FAK with phosphoinositide phosphatidylinsositol-4,5-bis-phosphate (PIP₂) are required to activate FAK. However, the molecular mechanism of the activation remains poorly understood. Recent fluorescence resonance energy transfer experiments revealed a closure of the FERM-kinase interface upon ATP binding, which is reversed upon additional binding of PIP₂. Here, we addressed the allosteric regulation of FAK by performing all-atom molecular-dynamics simulations of a FAK fragment containing the catalytic and FERM domains, and comparing the dynamics in the absence or presence of ATP and PIP₂. As a major conformational change, we observe a closing and opening motion upon ATP and additional PIP₂ binding, respectively, in good agreement with the fluorescence resonance energy transfer experiments. To reveal how the binding of the regulatory PIP₂ to the FERM F2 lobe is transduced to the very distant F1/N-lobe interface, we employed force distribution analysis. We identified a network of mainly charged residue-residue interactions spanning from the PIP₂ binding site to the distant interface between the kinase and FERM domains, comprising candidate residues for mutagenesis to validate the predicted mechanism of FAK activation. PMID:25650936

  18. Regulation of Multicellular Spheroids by MAPK and FYN Kinase.

    PubMed

    Lee, Casey; Ramos, Daniel M

    2016-08-01

    Understanding of the biology of oral squamous cell carcinoma (SCC) has not progressed significantly in the past 60 years, with 5-year survival remaining at approximately 50%. The epidemic of Human Papilloma Virus and its associated SCC warrants a renewed emphasis on fully understanding this disease. We previously used the 3-dimensional multicellular spheroid (MCS) model system to evaluate SCC behavior more accurately. In this study, we determined that SCC growth in MCS approximates epithelial to mesenchymal transition. Organization of an MCS requires the full-length β6 integrin subunit and its maintenance requires mitogen-activated protein kinase (MAPK). Limiting FYN kinase activation results in the down-regulation of E-cadherin, β-catenin and an increase in expression of N-cadherin and SNAIL. These results indicate that the microenvironment and growth patterns in an MCS are complex and require MAPK and FYN kinase. PMID:27466485

  19. pH regulation of an egg cortex tyrosine kinase.

    PubMed

    Jiang, W P; Veno, P A; Wood, R W; Peaucellier, G; Kinsey, W H

    1991-07-01

    Fertilization of the echinoderm egg is known to result in the phosphorylation, on tyrosine, of a high-molecular-weight cortical protein (HMWCP) localized in the egg cortex. Studies using various parthenogenic agents indicate that this phosphorylation event occurs in response to the alkaline shift in cytoplasmic pHi which normally occurs 1 to 2 min after fertilization. In the present study, the purified egg cell surface complex was used as in vitro system to determine whether a small alkaline shift in pH, such as occurs upon fertilization, could stimulate the activity of the egg cortex-associated tyrosine kinase toward endogenous protein substrates. The results demonstrated that the cell surface complex is highly enriched in a tyrosine kinase activity which accounts for the majority of the protein kinase activity in this preparation. The activity of this tyrosine kinase toward the HMWCP and other cortical proteins was highly dependent on pH over the range pH 6.8 to 7.3. This indicates that the fertilization-associated change in cytoplasmic pH would be sufficient to trigger increased tyrosine phosphorylation of the high-molecular-weight cortical protein in vivo. The regulation of tyrosine phosphorylation by small changes in pH represents a novel control mechanism in which a tyrosine protein kinase may act as a pH-sensitive transducer. PMID:2060713

  20. The Modulation of Neurotrophin and Epigenetic Regulators: Implication for Astrocyte Proliferation and Neuronal Cell Apoptosis After Spinal Cord Injury

    PubMed Central

    2016-01-01

    Objective To investigate alterations in the expression of the main regulators of neuronal survival and death related to astrocytes and neuronal cells in the brain in a mouse model of spinal cord injury (SCI). Methods Eight-week-old male imprinting control region mice (n=36; 30–35 g) were used in this study and randomly assigned to two groups: the naïve control group (n=18) and SCI group (n=18). The mice in both groups were randomly allocated to the following three time points: 3 days, 1 week, and 2 weeks (n=6 each). The expression levels of regulators such as brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), histone deacetylase 1 (HDAC1), and methyl-CpG-binding protein 2 (MeCP 2) in the brain were evaluated following thoracic contusive SCI. In addition, the number of neuronal cells in the motor cortex (M1 and M2 areas) and the number of astrocytes in the hippocampus were determined by immunohistochemistry. Results BDNF expression was significantly elevated at 2 weeks after injury (p=0.024). The GDNF level was significantly elevated at 3 days (p=0.042). The expression of HDAC1 was significantly elevated at 1 week (p=0.026). Following SCI, compared with the control the number of NeuN-positive cells in the M1 and M2 areas gradually and consistently decreased at 2 weeks after injury. In contrast, the number of astrocytes was significantly increased at 1 week (p=0.029). Conclusion These results demonstrate that the upregulation of BDNF, GDNF and HDAC1 might play on important role in brain reorganization after SCI. PMID:27606261

  1. 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.

  2. 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

  3. The molecular regulation of Janus kinase (JAK) activation

    PubMed Central

    2014-01-01

    The Janus Kinase (JAK) family members serve essential roles as the intracellular signalling effectors of cytokine receptors. This family, comprising JAK1, JAK2, JAK3 and TYK2, was first described more than 20 years ago, but the complexities underlying their activation, regulation and pleiotropic signalling functions are still being explored. Here, we review the current knowledge of their physiological functions and the causative role of activating and inactivating JAK mutations in human diseases, including haematopoietic malignancies, immunodeficiency and inflammatory diseases. At the molecular level, recent studies have greatly advanced our knowledge of the structures and organisation of the component FERM-SH2, pseudokinase and kinase domains within the JAKs, the mechanism of JAK activation and, in particular, the role of the pseudokinase domain as a suppressor of the adjacent tyrosine kinase domain's catalytic activity. We also review recent advances in our understanding of the mechanisms of negative regulation exerted by the SH2 domain containing proteins, SOCS (Suppressors of Cytokine Signalling) proteins and Lnk. These recent advances highlight the diversity of regulatory mechanisms utilised by the JAK family to maintain signalling fidelity, and suggest alternative therapeutic strategies to complement existing ATP-competitive kinase inhibitors. PMID:25057888

  4. 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

  5. Kinase-dependent Regulation of Monoamine Neurotransmitter Transporters.

    PubMed

    Bermingham, Daniel P; Blakely, Randy D

    2016-10-01

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

  6. Elucidating the Role of Injury-Induced Electric Fields (EFs) in Regulating the Astrocytic Response to Injury in the Mammalian Central Nervous System

    PubMed Central

    Baer, Matthew L.; Henderson, Scott C.; Colello, Raymond J.

    2015-01-01

    Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These astrocytic responses to injury occur in a clear temporal sequence and, by their intensity and duration, can have both beneficial and detrimental effects on the repair of damaged CNS tissue. Studies on highly regenerative tissues in non-mammalian vertebrates have demonstrated that the intensity of direct-current extracellular electric fields (EFs) at the injury site, which are 50–100 fold greater than in uninjured tissue, represent a potent signal to drive tissue repair. In contrast, a 10-fold EF increase has been measured in many injured mammalian tissues where limited regeneration occurs. As the astrocytic response to CNS injury is crucial to the reparative outcome, we exposed purified rat cortical astrocytes to EF intensities associated with intact and injured mammalian tissues, as well as to those EF intensities measured in regenerating non-mammalian vertebrate tissues, to determine whether EFs may contribute to the astrocytic injury response. Astrocytes exposed to EF intensities associated with uninjured tissue showed little change in their cellular behavior. However, astrocytes exposed to EF intensities associated with injured tissue showed a dramatic increase in migration and proliferation. At EF intensities associated with regenerating non-mammalian vertebrate tissues, these cellular responses were even more robust and included morphological changes consistent with a regenerative phenotype. These findings suggest that endogenous EFs may be a crucial signal for regulating the astrocytic response to injury and that their manipulation may be a novel target for facilitating CNS repair. PMID:26562295

  7. Elucidating the Role of Injury-Induced Electric Fields (EFs) in Regulating the Astrocytic Response to Injury in the Mammalian Central Nervous System.

    PubMed

    Baer, Matthew L; Henderson, Scott C; Colello, Raymond J

    2015-01-01

    Injury to the vertebrate central nervous system (CNS) induces astrocytes to change their morphology, to increase their rate of proliferation, and to display directional migration to the injury site, all to facilitate repair. These astrocytic responses to injury occur in a clear temporal sequence and, by their intensity and duration, can have both beneficial and detrimental effects on the repair of damaged CNS tissue. Studies on highly regenerative tissues in non-mammalian vertebrates have demonstrated that the intensity of direct-current extracellular electric fields (EFs) at the injury site, which are 50-100 fold greater than in uninjured tissue, represent a potent signal to drive tissue repair. In contrast, a 10-fold EF increase has been measured in many injured mammalian tissues where limited regeneration occurs. As the astrocytic response to CNS injury is crucial to the reparative outcome, we exposed purified rat cortical astrocytes to EF intensities associated with intact and injured mammalian tissues, as well as to those EF intensities measured in regenerating non-mammalian vertebrate tissues, to determine whether EFs may contribute to the astrocytic injury response. Astrocytes exposed to EF intensities associated with uninjured tissue showed little change in their cellular behavior. However, astrocytes exposed to EF intensities associated with injured tissue showed a dramatic increase in migration and proliferation. At EF intensities associated with regenerating non-mammalian vertebrate tissues, these cellular responses were even more robust and included morphological changes consistent with a regenerative phenotype. These findings suggest that endogenous EFs may be a crucial signal for regulating the astrocytic response to injury and that their manipulation may be a novel target for facilitating CNS repair. PMID:26562295

  8. Cell fate regulation governed by a repurposed bacterial histidine kinase

    SciTech Connect

    Childers, W. Seth; Xu, Qingping; Mann, Thomas H.; Mathews, Irimpan I.; Blair, Jimmy A.; Deacon, Ashley M.; Shapiro, Lucy; Stock, Ann M.

    2014-10-28

    One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK~P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.

  9. Cell Fate Regulation Governed by a Repurposed Bacterial Histidine Kinase

    PubMed Central

    Mann, Thomas H.; Mathews, Irimpan I.; Blair, Jimmy A.; Deacon, Ashley M.; Shapiro, Lucy

    2014-01-01

    One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK∼P over DivK, which is modulated by an allosteric intramolecular interaction between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation. PMID:25349992

  10. Cell fate regulation governed by a repurposed bacterial histidine kinase

    DOE PAGESBeta

    Childers, W. Seth; Xu, Qingping; Mann, Thomas H.; Mathews, Irimpan I.; Blair, Jimmy A.; Deacon, Ashley M.; Shapiro, Lucy; Stock, Ann M.

    2014-10-28

    One of the simplest organisms to divide asymmetrically is the bacterium Caulobacter crescentus. The DivL pseudo-histidine kinase, positioned at one cell pole, regulates cell-fate by controlling the activation of the global transcription factor CtrA via an interaction with the response regulator (RR) DivK. DivL uniquely contains a tyrosine at the histidine phosphorylation site, and can achieve these regulatory functions in vivo without kinase activity. Determination of the DivL crystal structure and biochemical analysis of wild-type and site-specific DivL mutants revealed that the DivL PAS domains regulate binding specificity for DivK~P over DivK, which is modulated by an allosteric intramolecular interactionmore » between adjacent domains. We discovered that DivL's catalytic domains have been repurposed as a phosphospecific RR input sensor, thereby reversing the flow of information observed in conventional histidine kinase (HK)-RR systems and coupling a complex network of signaling proteins for cell-fate regulation.« less

  11. Globular adiponectin induces a pro-inflammatory response in human astrocytic cells

    SciTech Connect

    Wan, Zhongxiao; Mah, Dorrian; Simtchouk, Svetlana; Klegeris, Andis; Little, Jonathan P.

    2014-03-28

    Highlights: • Adiponectin receptors are expressed in human astrocytes. • Globular adiponectin induces secretion of IL-6 and MCP-1 from cultured astrocytes. • Adiponectin may play a pro-inflammatory role in astrocytes. - Abstract: Neuroinflammation, mediated in part by activated brain astrocytes, plays a critical role in the development of neurodegenerative disorders, including Alzheimer’s disease (AD). Adiponectin is the most abundant adipokine secreted from adipose tissue and has been reported to exert both anti- and pro-inflammatory effects in peripheral tissues; however, the effects of adiponectin on astrocytes remain unknown. Shifts in peripheral concentrations of adipokines, including adiponectin, could contribute to the observed link between midlife adiposity and increased AD risk. The aim of the present study was to characterize the effects of globular adiponectin (gAd) on pro-inflammatory cytokine mRNA expression and secretion in human U373 MG astrocytic cells and to explore the potential involvement of nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and phosphatidylinositide 3-kinases (PI3 K) signaling pathways in these processes. We demonstrated expression of adiponectin receptor 1 (adipoR1) and adipoR2 in U373 MG cells and primary human astrocytes. gAd induced secretion of interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1, and gene expression of IL-6, MCP-1, IL-1β and IL-8 in U373 MG cells. Using specific inhibitors, we found that NF-κB, p38MAPK and ERK1/2 pathways are involved in gAd-induced induction of cytokines with ERK1/2 contributing the most. These findings provide evidence that gAd may induce a pro-inflammatory phenotype in human astrocytes.

  12. Diacylglycerol kinase regulation of protein kinase D during oxidative stress-induced intestinal cell injury

    SciTech Connect

    Song Jun; Li Jing; Mourot, Joshua M.; Mark Evers, B.; Chung, Dai H.

    2008-10-17

    We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury; however, the exact role of DAG kinase (DGK){zeta}, an isoform expressed in intestine, during this process is unknown. We sought to determine the role of DGK during oxidative stress-induced intestinal cell injury and whether DGK acts as an upstream regulator of PKD. Inhibition of DGK with R59022 compound or DGK{zeta} siRNA transfection decreased H{sub 2}O{sub 2}-induced RIE-1 cell apoptosis as measured by DNA fragmentation and increased PKD phosphorylation. Overexpression of kinase-dead DGK{zeta} also significantly increased PKD phosphorylation. Additionally, endogenous nuclear DGK{zeta} rapidly translocated to the cytoplasm following H{sub 2}O{sub 2} treatment. Our findings demonstrate that DGK is involved in the regulation of oxidative stress-induced intestinal cell injury. PKD activation is induced by DGK{zeta}, suggesting DGK is an upstream regulator of oxidative stress-induced activation of the PKD signaling pathway in intestinal epithelial cells.

  13. Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels.

    PubMed

    Lee, Jaekwang; Han, Young-Eun; Favorov, Oleg; Tommerdahl, Mark; Whitsel, Barry; Lee, C Justin

    2016-04-01

    Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC. PMID:27122993

  14. Fluoride Induces a Volume Reduction in CA1 Hippocampal Slices Via MAP Kinase Pathway Through Volume Regulated Anion Channels

    PubMed Central

    Lee, Jaekwang; Han, Young-Eun; Favorov, Oleg; Tommerdahl, Mark; Whitsel, Barry

    2016-01-01

    Regulation of cell volume is an important aspect of cellular homeostasis during neural activity. This volume regulation is thought to be mediated by activation of specific transporters, aquaporin, and volume regulated anion channels (VRAC). In cultured astrocytes, it was reported that swelling-induced mitogen-activated protein (MAP) kinase activation is required to open VRAC, which are thought to be important in regulatory volume decrease and in the response of CNS to trauma and excitotoxicity. It has been also described that sodium fluoride (NaF), a recognized G-protein activator and protein phosphatase inhibitor, leads to a significant MAP kinase activation in endothelial cells. However, NaF's effect in volume regulation in the brain is not known yet. Here, we investigated the mechanism of NaF-induced volume change in rat and mouse hippocampal slices using intrinsic optical signal (IOS) recording, in which we measured relative changes in intracellular and extracellular volume as changes in light transmittance through brain slices. We found that NaF (1~5 mM) application induced a reduction in light transmittance (decreased volume) in CA1 hippocampus, which was completely reversed by MAP kinase inhibitor U0126 (10 µM). We also observed that NaF-induced volume reduction was blocked by anion channel blockers, suggesting that NaF-induced volume reduction could be mediated by VRAC. Overall, our results propose a novel molecular mechanism of NaF-induced volume reduction via MAP kinase signaling pathway by activation of VRAC. PMID:27122993

  15. Nitric oxide: a regulator of eukaryotic initiation factor 2 kinases.

    PubMed

    Tong, Lingying; Heim, Rachel A; Wu, Shiyong

    2011-06-15

    Generation of nitric oxide (NO(•)) can upstream induce and downstream mediate the kinases that phosphorylate the α subunit of eukaryotic initiation factor 2 (eIF2α), which plays a critical role in regulating gene expression. There are four known eIF2α kinases (EIF2AKs), and NO(•) affects each one uniquely. Whereas NO(•) directly activates EIF2AK1 (HRI), it indirectly activates EIF2AK3 (PERK). EIF2AK4 (GCN2) is activated by depletion of l-arginine, which is used by nitric oxide synthase (NOS) during the production of NO(•). Finally EIF2AK2 (PKR), which can mediate inducible NOS expression and therefore NO(•) production, can also be activated by NO(•). The production of NO(•) and activation of EIF2AKs coordinately regulate physiological and pathological events such as innate immune response and cell apoptosis. PMID:21463677

  16. Phosphatidylinositol-3-kinase regulates mast cell ion channel activity.

    PubMed

    Lam, Rebecca S; Shumilina, Ekaterina; Matzner, Nicole; Zemtsova, Irina M; Sobiesiak, Malgorzata; Lang, Camelia; Felder, Edward; Dietl, Paul; Huber, Stephan M; Lang, Florian

    2008-01-01

    Stimulation of the mast cell IgE-receptor (FcepsilonRI) by antigen leads to stimulation of Ca(2+) entry with subsequent mast cell degranulation and release of inflammatory mediators. Ca(2+) further activates Ca(2+)-activated K(+) channels, which in turn provide the electrical driving force for Ca(2+) entry. Since phosphatidylinositol (PI)-3-kinase has previously been shown to be required for mast cell activation and degranulation, we explored, whether mast cell Ca(2+) and Ca(2+)-activated K(+) channels may be sensitive to PI3-kinase activity. Whole-cell patch clamp experiments and Fura-2 fluorescence measurements for determination of cytosolic Ca(2+) concentration were performed in mouse bone marrow-derived mast cells either treated or untreated with the PI3-kinase inhibitors LY-294002 (10 muM) and wortmannin (100 nM). Antigen-stimulated Ca(2+) entry but not Ca(2+) release from the intracellular stores was dramatically reduced upon PI3-kinase inhibition. Ca(2+) entry was further inhibited by TRPV blocker ruthenium red (10 muM). Ca(2+) entry following readdition after Ca(+)-store depletion with thapsigargin was again decreased by LY-294002, pointing to inhibition of store-operated channels (SOCs). Moreover, inhibition of PI3-kinase abrogated IgE-stimulated, but not ionomycin-induced stimulation of Ca(2+)-activated K(+) channels. These observations disclose PI3-kinase-dependent regulation of Ca(2+) entry and Ca(2+)-activated K(+)-channels, which in turn participate in triggering mast cell degranulation. PMID:18769043

  17. Regulation of ABC Transporter Function Via Phosphorylation by Protein Kinases

    PubMed Central

    Stolarczyk, Elzbieta I.; Reiling, Cassandra J.; Paumi, Christian M.

    2011-01-01

    ATP-binding cassette (ABC) transporters are multispanning membrane proteins that utilize ATP to move a broad range of substrates across cellular membranes. ABC transporters are involved in a number of human disorders and diseases [1]. Overexpression of a subset of the transporters has been closely linked to multidrug resistance in both bacteria and viruses and in cancer. A poorly understood and important aspect of ABC transporter biology is the role of phosphorylation as a mechanism to regulate transporter function. In this review, we summarize the current literature addressing the role of phosphorylation in regulating ABC transporter function. A comprehensive list of all the phosphorylation sites that have been identified for the human ABC transporters is presented, and we discuss the role of individual kinases in regulating transporter function. We address the potential pitfalls and difficulties associated with identifying phosphorylation sites and the corresponding kinase(s), and we discuss novel techniques that may circumvent these problems. We conclude by providing a brief perspective on studying ABC transporter phosphorylation. PMID:21118091

  18. Protein-tyrosine Phosphatase and Kinase Specificity in Regulation of SRC and Breast Tumor Kinase* ♦

    PubMed Central

    Fan, Gaofeng; Aleem, Saadat; Yang, Ming; Miller, W. Todd; Tonks, Nicholas K.

    2015-01-01

    Despite significant evidence to the contrary, the view that phosphatases are “nonspecific” still pervades the field. Systems biology approaches to defining how signal transduction pathways are integrated at the level of whole organisms also often downplay the contribution of phosphatases, defining them as “erasers” that serve merely to restore the system to its basal state. Here, we present a study that counteracts the idea of “nonspecific phosphatases.” We have characterized two structurally similar and functionally related kinases, BRK and SRC, which are regulated by combinations of activating autophosphorylation and inhibitory C-terminal sites of tyrosine phosphorylation. We demonstrated specificity at the level of the kinases in that SRMS phosphorylated the C terminus of BRK, but not SRC; in contrast, CSK is the kinase responsible for C-terminal phosphorylation of SRC, but not BRK. For the phosphatases, we observed that RNAi-mediated suppression of PTP1B resulted in opposing effects on the activity of BRK and SRC and have defined the mechanisms underlying this specificity. PTP1B inhibited BRK by directly dephosphorylating the Tyr-342 autophosphorylation site. In contrast, PTP1B potentiated SRC activity, but not by dephosphorylating SRC itself directly; instead, PTP1B regulated the interaction between CBP/PAG and CSK. SRC associated with, and phosphorylated, the transmembrane protein CBP/PAG at Tyr-317, resulting in CSK recruitment. We identified PAG as a substrate of PTP1B, and dephosphorylation abolished recruitment of the inhibitory kinase CSK. Overall, these findings illustrate how the combinatorial effects of PTKs and PTPs may be integrated to regulate signaling, with both classes of enzymes displaying exquisite specificity. PMID:25897081

  19. Modulation of Factors Affecting Optic Nerve Head Astrocyte Migration

    PubMed Central

    Miao, Haixi; Crabb, Andrea W.; Hernandez, M. Rosario

    2010-01-01

    Purpose. The authors investigated the role of myosin light chain kinase (MYLK) and transforming growth factor beta (TGFβ) receptor pathways in optic nerve head (ONH) astrocyte migration. They further investigated how the expression of these genes is altered by elevated hydrostatic pressure (HP). Methods. PCR was used to determine the isoforms of MYLK expressed in ONH astrocytes. siRNAs against MYLK (all isoforms) and TGFβ receptor 2 (TGFBR2) were prepared and tested for effects on the migration of cultured ONH astrocytes. Finally, the effects of elevated HP (24–96 hours) on the expression of MYLK isoforms and selected TGFβ pathway components were measured. Results. Multiple isoforms of MYLK are present in ONH astrocytes from Caucasian (CA) and African American (AA) donors. Both populations express the short form (MYLK-130) and the long form (MYLK-210) of MYLK and a splicing variant within MYLK-210. MYLK-directed siRNA decreased MYLK expression and cell migration compared with control siRNA. siRNA directed against TGFβ receptor 2 also decreased cell migration compared with control and decreased extracellular matrix genes regulated by TGFβ signaling. Elevated HP increased the expression of MYLK-130 and MYLK-210 in both populations of astrocytes. However, TGFβ2 was uniquely upregulated by exposure to elevated HP in CA compared with AA astrocytes. Conclusions. Differential expression of TGFβ pathway genes and MYLK isoforms observed in populations of glaucomatous astrocytes applies to the elevated HP model system. MYLK may be a new target for intervention in glaucoma to alter reactive astrocyte migration in the ONH. PMID:20375339

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

    PubMed

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

    2008-12-01

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

  1. Regulation of bradykinin-induced activation of volume-sensitive outwardly rectifying anion channels by Ca2+ nanodomains in mouse astrocytes

    PubMed Central

    Akita, Tenpei; Okada, Yasunobu

    2011-01-01

    Abstract Volume-sensitive outwardly rectifying (VSOR) anion channels play a key role in a variety of essential cell functions including cell volume regulation, cell death induction and intercellular communications. We previously demonstrated that, in cultured mouse cortical astrocytes, VSOR channels are activated in response to an inflammatory mediator, bradykinin, even without an increase in cell volume. Here we report that this VSOR channel activation must be mediated firstly by ‘nanodomains’ of high [Ca2+]i generated at the sites of both Ca2+ release from intracellular Ca2+ stores and Ca2+ entry at the plasma membrane. Bradykinin elicited a [Ca2+]i rise, initially caused by Ca2+ release and then by Ca2+ entry. Suppression of the [Ca2+]i rise by removal of extracellular Ca2+ and by depletion of Ca2+ stores suppressed the VSOR channel activation in a graded manner. Quantitative RT-PCR and suppression of gene expression with small interfering RNAs indicated that Orai1, TRPC1 and TRPC3 channels are involved in the Ca2+ entry and especially the entry through TRPC1 channels is strongly involved in the bradykinin-induced activation of VSOR channels. Moreover, Ca2+-dependent protein kinases Cα and β were found to mediate the activation after the [Ca2+]i rise through inducing generation of reactive oxygen species. Intracellular application of a slow Ca2+ chelator, EGTA, at 10 mm or a fast chelator, BAPTA, at 1 mm, however, had little effect on the VSOR channel activation. Application of BAPTA at 10 mm suppressed significantly the activation to one-third. These suggest that the VSOR channel activation induced by bradykinin is regulated by Ca2+ in the vicinity of individual Ca2+ release and entry channels, providing a basis for local control of cell volume regulation and intercellular communications. PMID:21690189

  2. FGF and stress regulate CREB and ATF-1 via a pathway involving p38 MAP kinase and MAPKAP kinase-2.

    PubMed Central

    Tan, Y; Rouse, J; Zhang, A; Cariati, S; Cohen, P; Comb, M J

    1996-01-01

    Fibroblast growth factor (FGF) activates a protein kinase cascade in SK-N-MC cells that regulates gene expression at a cyclic-AMP response element (CRE) by stimulating the transcriptional activity of CREB. The activation of CREB is prevented by a dominant negative mutant of Ras and triggered via the same site (Ser133) that becomes phosphorylated in response to cyclic AMP and Ca2+. However, the effect of FGF is not mediated by cyclic AMP-dependent protein kinase, TPA-sensitive isoforms of protein kinase-C, p70S6K or p90rsk (all of which phosphorylate CREB at Ser133 in vitro). Instead, we identify the FGF-stimulated CREB kinase as MAP kinase-activated protein (MAPKAP) kinase-2, an enzyme that lies immediately downstream of p38 MAP kinase, in a pathway that is also stimulated by cellular stresses. We show that MAPKAP kinase-2 phosphorylates CREB at Ser133 in vitro, that the FGF- or stress-induced activation of MAPKAP kinase-2 and phosphorylation of CREB and ATF-1 are prevented by similar concentrations of the specific p38 MAP kinase inhibitor SB 203580, and that MAPKAP kinase-2 is the only detectable SB 203580-sensitive CREB kinase in SK-N-MC cell extracts. We also show that transfection of RK/p38 MAP kinase in SK-N-MC cells, but not transfection of p44 MAP kinase, activates Gal4-CREB-dependent transcription via Ser133. These findings identify a new growth factor and stress-activated signaling pathway that regulates gene expression at the CRE. Images PMID:8887554

  3. Tyrosine Kinase Inhibition Regulates Early Systemic Immune Changes and Modulates the Neuroimmune Response in α-Synucleinopathy

    PubMed Central

    Hebron, Michaeline L.; Lonskaya, Irina; Olopade, Paul; Selby, Sandra T.; Pagan, Fernando; Moussa, Charbel E-H

    2015-01-01

    Objectives Neuro-inflammation is common in α-Synucleinopathies and Tauopathies; and evidence suggests a link between the tyrosine kinase Abl and neurodegeneration. Abl upregulates α-Synuclein and promotes Tau hyper-phosphorylation (p-Tau), while Abl inhibitors facilitate autophagic clearance. Methods A model of α-Synucleinopathy harboring human mutant A53T α-Synuclein and exhibits concomitant increase in murine p-Tau was used to determine the immunological response to Abl inhibition. Results Age-dependent alterations of brain immunity, including loss of IL-10 and decreased levels of IL-2 and IL-3 were observed in old A53T mice. Brain CCL2 and CCL5 were decreased, but CX3CL1 remained constantly elevated. Young A53T mice exhibited differential systemic and central immune profiles in parallel with increased blood markers of adaptive immunity, suggesting an early systemic immune response. Tyrosine kinase inhibitors (TKIs), including nilotinib and bosutinib reduced brain and peripheral α-Synuclein and p-Tau and modulated blood immunological responses. TKIs did not affect brain IL-10, but they changed the levels of all measured blood immune markers, except CX3CL1. TKIs altered microglia morphology and reduced the number of astrocyte and dendritic cells, suggesting beneficial regulation of microglia. Conclusions These data indicate that tyrosine kinase inhibition affects neuro-inflammation via early changes of the peripheral immune profile, leading to modulation of the neuro-immune response to α-Synuclein and p-Tau. PMID:25635231

  4. Oscillatory Dynamics of the Extracellular Signal-regulated Kinase Pathway

    SciTech Connect

    Shankaran, Harish; Wiley, H. S.

    2010-12-01

    The extracellular signal-regulated kinase (ERK) pathway is a central signaling pathway in development and disease and is regulated by multiple negative and positive feedback loops. Recent studies have shown negative feedback from ERK to upstream regulators can give rise to biochemical oscillations with a periodicity of between 15-30 minutes. Feedback due to the stimulated transcription of negative regulators of the ERK pathway can also give rise to transcriptional oscillations with a periodicity of 1-2h. The biological significance of these oscillations is not clear, but recent evidence suggests that transcriptional oscillations participate in developmental processes, such as somite formation. Biochemical oscillations are more enigmatic, but could provide a mechanism for encoding different types of inputs into a common signaling pathway.

  5. A secretory kinase complex regulates extracellular protein phosphorylation

    PubMed Central

    Cui, Jixin; Xiao, Junyu; Tagliabracci, Vincent S; Wen, Jianzhong; Rahdar, Meghdad; Dixon, Jack E

    2015-01-01

    Although numerous extracellular phosphoproteins have been identified, the protein kinases within the secretory pathway have only recently been discovered, and their regulation is virtually unexplored. Fam20C is the physiological Golgi casein kinase, which phosphorylates many secreted proteins and is critical for proper biomineralization. Fam20A, a Fam20C paralog, is essential for enamel formation, but the biochemical function of Fam20A is unknown. Here we show that Fam20A potentiates Fam20C kinase activity and promotes the phosphorylation of enamel matrix proteins in vitro and in cells. Mechanistically, Fam20A is a pseudokinase that forms a functional complex with Fam20C, and this complex enhances extracellular protein phosphorylation within the secretory pathway. Our findings shed light on the molecular mechanism by which Fam20C and Fam20A collaborate to control enamel formation, and provide the first insight into the regulation of secretory pathway phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.06120.001 PMID:25789606

  6. Mechanosensitive Kinases Regulate Stiffness-Induced Cardiomyocyte Maturation

    PubMed Central

    Young, Jennifer L.; Kretchmer, Kyle; Ondeck, Matthew G.; Zambon, Alexander C.; Engler, Adam J.

    2014-01-01

    Cells secrete and assemble extracellular matrix throughout development, giving rise to time-dependent, tissue-specific stiffness. Mimicking myocardial matrix stiffening, i.e. ~10-fold increase over 1 week, with a hydrogel system enhances myofibrillar organization of embryonic cardiomyocytes compared to static hydrogels, and thus we sought to identify specific mechanosensitive proteins involved. Expression and/or phosphorylation state of 309 unique protein kinases were examined in embryonic cardiomyocytes plated on either dynamically stiffening or static mature myocardial stiffness hydrogels. Gene ontology analysis of these kinases identified cardiogenic pathways that exhibited time-dependent up-regulation on dynamic versus static matrices, including PI3K/AKT and p38 MAPK, while GSK3β, a known antagonist of cardiomyocyte maturation, was down-regulated. Additionally, inhibiting GSK3β on static matrices improved spontaneous contraction and myofibril organization, while inhibiting agonist AKT on dynamic matrices reduced myofibril organization and spontaneous contraction, confirming its role in mechanically-driven maturation. Together, these data indicate that mechanically-driven maturation is at least partially achieved via active mechanosensing at focal adhesions, affecting expression and phosphorylation of a variety of protein kinases important to cardiomyogenesis. PMID:25236849

  7. Interacting Protein Kinases Involved in the Regulation of Flagellar Length

    PubMed Central

    Erdmann, Maja; Scholz, Anne; Melzer, Inga M.; Schmetz, Christel; Wiese, Martin

    2006-01-01

    A striking difference of the life stages of the protozoan parasite Leishmania is a long flagellum in the insect stage promastigotes and a rudimentary organelle in the mammalian amastigotes. LmxMKK, a mitogen-activated protein (MAP) kinase kinase from Leishmania mexicana, is required for growth of a full-length flagellum. We identified LmxMPK3, a MAP kinase homologue, with a similar expression pattern as LmxMKK being not detectable in amastigotes, up-regulated during the differentiation to promastigotes, constantly expressed in promastigotes, and shut down during the differentiation to amastigotes. LmxMPK3 null mutants resemble the LmxMKK knockouts with flagella reduced to one-fifth of the wild-type length, stumpy cell bodies, and vesicles and membrane fragments in the flagellar pocket. A constitutively activated recombinant LmxMKK activates LmxMPK3 in vitro. Moreover, LmxMKK is likely to be directly involved in the phosphorylation of LmxMPK3 in vivo. Finally, LmxMPK3 is able to phosphorylate LmxMKK, indicating a possible feedback regulation. This is the first time that two interacting components of a signaling cascade have been described in the genus Leishmania. Moreover, we set the stage for the analysis of reversible phosphorylation in flagellar morphogenesis. PMID:16467378

  8. Generation of reactive astrocytes from NG2 cells is regulated by sonic hedgehog.

    PubMed

    Honsa, Pavel; Valny, Martin; Kriska, Jan; Matuskova, Hana; Harantova, Lenka; Kirdajova, Denisa; Valihrach, Lukas; Androvic, Peter; Kubista, Mikael; Anderova, Miroslava

    2016-09-01

    NG2 cells, a fourth glial cell type in the adult mammalian central nervous system, produce oligodendrocytes in the healthy nervous tissue, and display wide differentiation potential under pathological conditions, where they could give rise to reactive astrocytes. The factors that control the differentiation of NG2 cells after focal cerebral ischemia (FCI) are largely unknown. Here, we used transgenic Cspg4-cre/Esr1/ROSA26Sortm14(CAG-tdTomato) mice, in which tamoxifen administration triggers the expression of red fluorescent protein (tomato) specifically in NG2 cells and cells derived therefrom. Differentiation potential (in vitro and in vivo) of tomato-positive NG2 cells from control or postischemic brains was determined using the immunohistochemistry, single cell RT-qPCR and patch-clamp method. The ischemic injury was induced by middle cerebral artery occlusion, a model of FCI. Using genetic fate-mapping method, we identified sonic hedgehog (Shh) as an important factor that influences differentiation of NG2 cells into astrocytes in vitro. We also manipulated Shh signaling in the adult mouse brain after FCI. Shh signaling activation significantly increased the number of astrocytes derived from NG2 cells in the glial scar around the ischemic lesion, while Shh signaling inhibition caused the opposite effect. Since Shh signaling modifications did not change the proliferation rate of NG2 cells, we can conclude that Shh has a direct influence on the differentiation of NG2 cells and therefore, on the formation and composition of a glial scar, which consequently affects the degree of the brain damage. GLIA 2016;64:1518-1531. PMID:27340757

  9. Ribosomal protein S6 kinase 1 signaling regulates mammalian lifespan

    PubMed Central

    Selman, Colin; Tullet, Jennifer M.A.; Wieser, Daniela; Irvine, Elaine; Lingard, Steven J.; Choudhury, Agharul I.; Claret, Marc; Al-Qassab, Hind; Carmignac, Danielle; Ramadani, Faruk; Woods, Angela; Robinson, Iain C.A.; Schuster, Eugene; Batterham, Rachel L.; Kozma, Sara C.; Thomas, George; Carling, David; Okkenhaug, Klaus; Thornton, Janet M.; Partridge, Linda; Gems, David; Withers, Dominic J.

    2016-01-01

    Caloric restriction (CR) protects against aging and disease but the mechanisms by which this affects mammalian lifespan are unclear. We show in mice that deletion of the nutrient-responsive mTOR (mammalian target of rapamycin) signaling pathway component ribosomal S6 protein kinase 1 (S6K1) led to increased lifespan and resistance to age-related pathologies such as bone, immune and motor dysfunction and loss of insulin sensitivity. Deletion of S6K1 induced gene expression patterns similar to those seen in CR or with pharmacological activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), a conserved regulator of the metabolic response to CR. Our results demonstrate that S6K1 influences healthy mammalian lifespan, and suggest therapeutic manipulation of S6K1 and AMPK might mimic CR and provide broad protection against diseases of aging. PMID:19797661

  10. Protein Kinase C and Extracellular Signal-Regulated Kinase Regulate Movement, Attachment, Pairing and Egg Release in Schistosoma mansoni

    PubMed Central

    Ressurreição, Margarida; De Saram, Paulu; Kirk, Ruth S.; Rollinson, David; Emery, Aidan M.; Page, Nigel M.; Davies, Angela J.; Walker, Anthony J.

    2014-01-01

    Protein kinases C (PKCs) and extracellular signal-regulated kinases (ERKs) are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using ‘smart’ antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated) throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance. PMID:24921927

  11. Brassinosteroid regulated kinases (BRKs) that mediate brassinosteroid signal transduction and uses thereof

    DOEpatents

    Wang, Zhi-Yong; Tang, Wenqiang

    2013-09-24

    The present invention identifies a novel family of kinases regulated by brassinosteroids, referred to as BRKs (brassinosteroid regulated kinases) or BSKs (brassinosteroid signaling kinases). The present invention provides methods for modulating the response of a plant cell to a brassinosteroid using BRKs.

  12. Sonic hedgehog (Shh) regulates the expression of angiogenic growth factors in oxygen-glucose-deprived astrocytes by mediating the nuclear receptor NR2F2.

    PubMed

    Li, Yanan; Xia, Yuanpeng; Wang, Yong; Mao, Ling; Gao, Yuan; He, Quanwei; Huang, Ming; Chen, Shengcai; Hu, Bo

    2013-06-01

    Sonic hedgehog (Shh) has been found to regulate the angiogenic growth factor such as VEGF, Ang-1, and Ang-2 during ischemic insults, but the underlying mechanism is not fully understood. In this study, we employed oxygen-glucose deprivation (OGD) in astrocytes to mimic the ischemia in vitro. We found that OGD could induce the expressions of VEGF, Ang-1, and Ang-2, with the expression of Shh signaling components increased. Moreover, inhibiting the Shh signaling pathway with 5EI, a specific antibody, could decrease the expressions of VEGF, Ang-1, and Ang-2. Furthermore, the administration of exogenous Shh could induce the expressions of VEGF, Ang-1, and Ang-2 in astrocytes. The results of silencing Gli-1, or NR2F2, exhibited that exogenous Shh could regulate the expressions of VEGF, Ang-1, and Ang-2 in astrocytes by activating the NR2F2, but not the Gli-1. These results suggested that Shh could regulate the angiogenic growth factor after ischemic insults in astrocytes, and the regulation was partially mediated by the NR2F2. PMID:23378030

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

    PubMed

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

    2016-06-01

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

  14. Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases

    PubMed Central

    Beurel, Eleonore; Grieco, Steven F.; Jope, Richard S.

    2014-01-01

    Glycogen synthase kinase-3 (GSK3) may be the busiest kinase in most cells, with over 100 known substrates to deal with. How does GSK3 maintain control to selectively phosphorylate each substrate, and why was it evolutionarily favorable for GSK3 to assume such a large responsibility? GSK3 must be particularly adaptable for incorporating new substrates into its repertoire, and we discuss the distinct properties of GSK3 that may contribute to its capacity to fulfill its roles in multiple signaling pathways. The mechanisms regulating GSK3 (predominantly post-translational modifications, substrate priming, cellular trafficking, protein complexes) have been reviewed previously, so here we focus on newly identified complexities in these mechanisms, how each of these regulatory mechanism contributes to the ability of GSK3 to select which substrates to phosphorylate, and how these mechanisms may have contributed to its adaptability as new substrates evolved. The current understanding of the mechanisms regulating GSK3 is reviewed, as are emerging topics in the actions of GSK3, particularly its interactions with receptors and receptor-coupled signal transduction events, and differential actions and regulation of the two GSK3 isoforms, GSK3α and GSK3β. Another remarkable characteristic of GSK3 is its involvement in many prevalent disorders, including psychiatric and neurological diseases, inflammatory diseases, cancer, and others. We address the feasibility of targeting GSK3 therapeutically, and provide an update of its involvement in the etiology and treatment of several disorders. PMID:25435019

  15. Phosphoinositide 3-kinase and Bruton's tyrosine kinase regulate overlapping sets of genes in B lymphocytes

    PubMed Central

    Fruman, David A.; Ferl, Gregory Z.; An, Sam S.; Donahue, Amber C.; Satterthwaite, Anne B.; Witte, Owen N.

    2002-01-01

    Bruton's tyrosine kinase (Btk) acts downstream of phosphoinositide 3-kinase (PI3K) in a pathway required for B cell receptor (BCR)-dependent proliferation. We used DNA microarrays to determine what fraction of genes this pathway influences and to investigate whether PI3K and Btk mediate distinct gene regulation events. As complete loss-of-function mutations in PI3K and Btk alter B cell subpopulations and may cause compensatory changes in gene expression, we used B cells with partial loss of function in either PI3K or Btk. Only about 5% of the BCR-dependent gene expression changes were significantly affected by reduced PI3K or Btk. The results indicate that PI3K and Btk share target genes, and that PI3K influences additional genes independently of Btk. These data are consistent with PI3K acting through Btk and other effectors to regulate expression of a critical subset of BCR target genes that determine effective entry into the cell cycle. PMID:11756681

  16. Myosin light chain kinase regulates cell polarization independently of membrane tension or Rho kinase

    PubMed Central

    Lou, Sunny S.; Diz-Muñoz, Alba; Weiner, Orion D.; Fletcher, Daniel A.

    2015-01-01

    Cells polarize to a single front and rear to achieve rapid actin-based motility, but the mechanisms preventing the formation of multiple fronts are unclear. We developed embryonic zebrafish keratocytes as a model system for investigating establishment of a single axis. We observed that, although keratocytes from 2 d postfertilization (dpf) embryos resembled canonical fan-shaped keratocytes, keratocytes from 4 dpf embryos often formed multiple protrusions despite unchanged membrane tension. Using genomic, genetic, and pharmacological approaches, we determined that the multiple-protrusion phenotype was primarily due to increased myosin light chain kinase (MLCK) expression. MLCK activity influences cell polarity by increasing myosin accumulation in lamellipodia, which locally decreases protrusion lifetime, limiting lamellipodial size and allowing for multiple protrusions to coexist within the context of membrane tension limiting protrusion globally. In contrast, Rho kinase (ROCK) regulates myosin accumulation at the cell rear and does not determine protrusion size. These results suggest a novel MLCK-specific mechanism for controlling cell polarity via regulation of myosin activity in protrusions. PMID:25918227

  17. Complement regulatory protein expression by a human oligodendrocyte cell line: cytokine regulation and comparison with astrocytes.

    PubMed Central

    Gasque, P; Morgan, B P

    1996-01-01

    Rat oligodendrocytes spontaneously activate complement (C) and lack the C inhibitor CD59. As a consequence, rat oligodendrocytes are susceptible to lysis by autologous C in vitro. Expression of C inhibitors on human oligodendrocytes in vitro and other human glia has yet to be well characterized. We have previously shown expression at the mRNA level of the membrane inhibitors CD59, decay-accelerating factor (DAF; CD55) and membrane cofactor protein (MCP; CD46) in human astrocytes. We here examine the expression of membrane and secreted C inhibitors by the oligodendrocyte cell line, HOG. HOG cells abundantly expressed CD59, assessed at protein and mRNA level, and expressed DAF and MCP, albeit at a lower level. Expression of all three inhibitors was enhanced by incubation with interferon-gamma or with phorbol ester (PMA). Complement receptor type 1 (CR1; CD35) was neither expressed constitutively nor induced by cytokines. HOG also constitutively secreted C1-inhibitor, S-protein and clusterin. Factor H was secreted only after stimulation with cytokines. C4b binding protein was expressed at a very low level and was detected only at the mRNA level by reverse transcriptase-polymerase chain reaction (RT-PCR). For comparison, astrocyte expression of CD59, DAF, MCP and CR1 was confirmed at the mRNA and protein levels. HOG did not activate C spontaneously, as judged by the lack of deposition of C fragments, and were not lysed by C even after inhibition of CD59 and DAF using specific monoclonal antibodies. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8958045

  18. The Rheb-mTOR pathway is upregulated in reactive astrocytes of the injured spinal cord

    PubMed Central

    Codeluppi, Simone; Svensson, Camilla I.; Hefferan, Michael P.; Valencia, Fatima; Silldorff, Morgan D.; Marsala, Martin; Pasquale, Elena B.

    2009-01-01

    Astrocytes in the central nervous system respond to tissue damage by becoming reactive. They migrate, undergo hypertrophy, and form a glial scar that inhibits axon regeneration. Therefore, limiting astrocytic responses represents a potential therapeutic strategy to improve functional recovery. It was recently shown that the epidermal growth factor (EGF) receptor is upregulated in astrocytes after injury and promotes their transformation into reactive astrocytes. Furthermore, EGF receptor inhibitors were shown to enhance axon regeneration in the injured optic nerve and promote recovery after spinal cord injury. However, the signaling pathways involved were not elucidated. Here we show that in cultures of adult spinal cord astrocytes EGF activates the mTOR pathway, a key regulator of astrocyte physiology. This occurs through Akt-mediated phosphorylation of the GTPase-activating protein Tuberin, which inhibits Tuberin’s ability to inactivate the small GTPase Rheb. Indeed, we found that Rheb is required for EGF-dependent mTOR activation in spinal cord astrocytes, whereas the Ras-MAP kinase pathway does not appear to be involved. Moreover, astrocyte growth and EGF-dependent chemoattraction were inhibited by the mTOR-selective drug rapamycin. We also detected elevated levels of activated EGF receptor and mTOR signaling in reactive astrocytes in vivo in an ischemic model of spinal cord injury. Furthermore, increased Rheb expression likely contributes to mTOR activation in the injured spinal cord. Interestingly, injured rats treated with rapamycin showed reduced signs of reactive gliosis, suggesting that rapamycin could be used to harness astrocytic responses in the damaged nervous system to promote an environment more permissive to axon regeneration. PMID:19176818

  19. The Rheb-mTOR pathway is upregulated in reactive astrocytes of the injured spinal cord.

    PubMed

    Codeluppi, Simone; Svensson, Camilla I; Hefferan, Michael P; Valencia, Fatima; Silldorff, Morgan D; Oshiro, Masakatsu; Marsala, Martin; Pasquale, Elena B

    2009-01-28

    Astrocytes in the CNS respond to tissue damage by becoming reactive. They migrate, undergo hypertrophy, and form a glial scar that inhibits axon regeneration. Therefore, limiting astrocytic responses represents a potential therapeutic strategy to improve functional recovery. It was recently shown that the epidermal growth factor (EGF) receptor is upregulated in astrocytes after injury and promotes their transformation into reactive astrocytes. Furthermore, EGF receptor inhibitors were shown to enhance axon regeneration in the injured optic nerve and promote recovery after spinal cord injury. However, the signaling pathways involved were not elucidated. Here we show that in cultures of adult spinal cord astrocytes EGF activates the mTOR pathway, a key regulator of astrocyte physiology. This occurs through Akt-mediated phosphorylation of the GTPase-activating protein Tuberin, which inhibits Tuberin's ability to inactivate the small GTPase Rheb. Indeed, we found that Rheb is required for EGF-dependent mTOR activation in spinal cord astrocytes, whereas the Ras-MAP kinase pathway does not appear to be involved. Moreover, astrocyte growth and EGF-dependent chemoattraction were inhibited by the mTOR-selective drug rapamycin. We also detected elevated levels of activated EGF receptor and mTOR signaling in reactive astrocytes in vivo in an ischemic model of spinal cord injury. Furthermore, increased Rheb expression likely contributes to mTOR activation in the injured spinal cord. Interestingly, injured rats treated with rapamycin showed reduced signs of reactive gliosis, suggesting that rapamycin could be used to harness astrocytic responses in the damaged nervous system to promote an environment more permissive to axon regeneration. PMID:19176818

  20. Evolutionary Adaptations of Plant AGC Kinases: From Light Signaling to Cell Polarity Regulation

    PubMed Central

    Rademacher, Eike H.; Offringa, Remko

    2012-01-01

    Signaling and trafficking over membranes involves a plethora of transmembrane proteins that control the flow of compounds or relay specific signaling events. Next to external cues, internal stimuli can modify the activity or abundance of these proteins at the plasma membrane (PM). One such regulatory mechanism is protein phosphorylation by membrane-associated kinases, several of which are AGC kinases. The AGC kinase family is one of seven kinase families that are conserved in all eukaryotic genomes. In plants evolutionary adaptations introduced specific structural changes within the AGC kinases that most likely allow modulation of kinase activity by external stimuli (e.g., light). Starting from the well-defined structural basis common to all AGC kinases we review the current knowledge on the structure-function relationship in plant AGC kinases. Nine of the 39 Arabidopsis AGC kinases have now been shown to be involved in the regulation of auxin transport. In particular, AGC kinase-mediated phosphorylation of the auxin transporters ABCB1 and ABCB19 has been shown to regulate their activity, while auxin transporters of the PIN family are located to different positions at the PM depending on their phosphorylation status, which is a result of counteracting AGC kinase and PP6 phosphatase activities. We therefore focus on regulation of AGC kinase activity in this context. Identified structural adaptations of the involved AGC kinases may provide new insight into AGC kinase functionality and demonstrate their position as central hubs in the cellular network controlling plant development and growth. PMID:23162562

  1. Creatine kinase in cell cycle regulation and cancer.

    PubMed

    Yan, Yong-Bin

    2016-08-01

    The phosphocreatine-creatine kinase (CK) shuttle system is increasingly recognized as a fundamental mechanism for ATP homeostasis in both excitable and non-excitable cells. Many intracellular processes are ATP dependent. Cell division is a process requiring a rapid rate of energy turnover. Cell cycle regulation is also a key point to understanding the mechanisms underlying cancer progression. It has been known for about 40 years that aberrant CK levels are associated with various cancers and for over 30 years that CK is involved in mitosis regulation. However, the underlying molecular mechanisms have not been investigated sufficiently until recently. By maintaining ATP at sites of high-energy demand, CK can regulate cell cycle progression by affecting the intracellular energy status as well as by influencing signaling pathways that are essential to activate cell division and cytoskeleton reorganization. Aberrant CK levels may impair cell viability under normal or stressed conditions and induce cell death. The involvement of CK in cell cycle regulation and cellular energy metabolism makes it a potential diagnostic biomarker and therapeutic target in cancer. To understand the multiple physiological/pathological functions of CK, it is necessary to identify CK-binding partners and regulators including proteins, non-coding RNAs and participating endogenous small molecular weight chemical compounds. This review will focus on molecular mechanisms of CK in cell cycle regulation and cancer progression. It will also discuss the implications of recent mechanistic studies, the emerging problems and future challenges of the multifunctional enzyme CK. PMID:27020776

  2. Silencing TRPM7 in Mouse Cortical Astrocytes Impairs Cell Proliferation and Migration via ERK and JNK Signaling Pathways

    PubMed Central

    Zeng, Zhao; Leng, Tiandong; Feng, Xuechao; Sun, Huawei; Inoue, Koichi; Zhu, Li; Xiong, Zhi-Gang

    2015-01-01

    Transient receptor potential melastatin 7 (TRPM7), a non-selective cation channel, is highly expressed expressed in the brain and plays a critical role in ischemic neuronal death. Astrocyte, the most abundant cell type in central nervous system (CNS), exerts many essential functions in the physiological and pathological conditions. Here we investigated the expression and functions of the TRPM7 channel in mouse cortical astrocytes. Using reverse transcription (RT)-PCR, immunostaining, western blot and patch clamp recording, we showed that functional TRPM7 channel is expressed in cultured mouse cortical astrocytes. Knocking down TRPM7 with specific siRNA impairs the proliferation and migration of astrocytes by 40.2% ± 3.9% and 40.1% ± 11.5%, respectively. Consistently, inhibition of TRPM7 with 2-aminoethoxydiphenyl borate (2-APB) also decreases astrocyte proliferation and migration by 46.1% ± 2.5% and 64.2% ± 2.4%. MAPKs and Akt signaling pathways have been shown to be implicated in TRPM7-mediated responses including cell proliferation and migration. Our data show that suppression of TRPM7 in astrocytes reduces the phosphorylation of extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK), but not p38 mitogen-activated protein kinase and Akt. In addition, TRPM7, as a cation channel, has been involved in the Ca2+ and Mg2+ homeostasis in several types of cells. In our study, we found that silencing TRPM7 decreases the intracellular basal Mg2+ concentration without affecting Ca2+ concentration in astrocytes. However, an addition of Mg2+ to the growth medium could not rescue the impaired proliferation of astrocytes. Together, our data suggest that TRPM7 channel may play a critical role in the proliferation and migration of astrocytes via the ERK and JNK pathways. PMID:25799367

  3. Structural and functional diversity in the activity and regulation of DAPK-related protein kinases.

    PubMed

    Temmerman, Koen; Simon, Bertrand; Wilmanns, Matthias

    2013-11-01

    Within the large group of calcium/calmodulin-dependent protein kinases (CAMKs) of the human kinome, there is a distinct branch of highly related kinases that includes three families: death-associated protein-related kinases, myosin light-chain-related kinases and triple functional domain protein-related kinases. In this review, we refer to these collectively as DMT kinases. There are several functional features that span the three families, such as a broad involvement in apoptotic processes, cytoskeletal association and cellular plasticity. Other CAMKs contain a highly conserved HRD motif, which is a prerequisite for kinase regulation through activation-loop phosphorylation, but in all 16 members of the DMT branch, this is replaced by an HF/LD motif. This DMT kinase signature motif substitutes phosphorylation-dependent active-site interactions with a local hydrophobic core that maintains an active kinase conformation. Only about half of the DMT kinases have an additional autoregulatory domain, C-terminal to the kinase domain that binds calcium/calmodulin in order to regulate kinase activity. Protein substrates have been identified for some of the DMT kinases, but little is known about the mechanism of recognition. Substrate conformation could be an equally important parameter in substrate recognition as specific preferences in sequence position. Taking the data together, this kinase branch encapsulates a treasure trove of features that renders it distinct from many other protein kinases and calls for future research activities in this field. PMID:23745726

  4. Extracellular signal-regulated kinases modulate capacitation of human spermatozoa.

    PubMed

    Luconi, M; Barni, T; Vannelli, G B; Krausz, C; Marra, F; Benedetti, P A; Evangelista, V; Francavilla, S; Properzi, G; Forti, G; Baldi, E

    1998-06-01

    Recent evidence indicates the presence of p21 Ras and of a protein with characteristics similar to mitogen-activated protein kinases (MAPKs), also known as extracellular signal-regulated kinases (ERKs), in mammalian spermatozoa, suggesting the occurrence of the Ras/ERK cascade in these cells. In the present study we investigated the subcellular localization of ERKs and their biological functions in human spermatozoa. Immunohistochemistry, immunofluorescence, confocal microscopy, and immunoelectron microscopy demonstrated localization of ERKs in the postacrosomal region of spermatozoa. After stimulation of acrosome reaction with the calcium ionophore A23187 and progesterone, ERKs were mostly localized at the level of the equatorial region, indicating redistribution of these proteins in acrosome-reacted spermatozoa. Two proteins of 42 and 44 kDa that are tyrosine phosphorylated in a time-dependent manner during in vitro capacitation were identified as p42 (ERK-2) and p44 (ERK-1) by means of specific antibodies. The increase in tyrosine phosphorylation of these proteins during capacitation was accompanied by increased kinase activity, as determined by the ability of ERK-1 and ERK-2 to phosphorylate the substrate myelin basic protein. The role of this activity in the occurrence of sperm capacitation was also investigated by using PD098059, an inhibitor of the MAPK cascade. The presence of this compound during in vitro capacitation inhibits ERK activation and significantly reduces the ability of spermatozoa to undergo the acrosome reaction in response to progesterone. Since only capacitated spermatozoa are able to respond to progesterone, these data strongly indicate that ERKs are involved in the regulation of capacitation. In summary, our data demonstrate the presence of functional ERKs in human spermatozoa and indicate that these enzymes are involved in activation of these cells during capacitation, providing new insight in clarifying the molecular mechanisms and the

  5. Hypothermia protects against oxygen-glucose deprivation-induced neuronal injury by down-regulating the reverse transport of glutamate by astrocytes as mediated by neurons.

    PubMed

    Wang, D; Zhao, Y; Zhang, Y; Zhang, T; Shang, X; Wang, J; Liu, Y; Kong, Q; Sun, B; Mu, L; Liu, X; Wang, G; Li, H

    2013-05-01

    Glutamate is the major mediator of excitotoxic neuronal death following cerebral ischemia. Under severe ischemic conditions, glutamate transporters can functionally reverse to release glutamate, thereby inducing further neuronal injury. Hypothermia has been shown to protect neurons from brain ischemia. However, the mechanism(s) involved remain unclear. Therefore, the aim of this study was to investigate the mechanism(s) mediating glutamate release during brain ischemia-reperfusion injury under hypothermic conditions. Neuron/astrocyte co-cultures were exposed to oxygen-glucose deprivation (OGD) at various temperatures for 2h, and cell viability was assayed 12h after reoxygenation. PI and MAP-2 staining demonstrated that hypothermia significantly decreased neuronal injury. Furthermore, [(3)H]-glutamate uptake assays showed that hypothermia protected rat primary cortical cultures against OGD reoxygenation-induced injury. Protein levels of the astrocytic glutamate transporter, GLT-1, which is primarily responsible for the clearance of extracellular glutamate, were also found to be reduced in a temperature-dependent manner. In contrast, expression of GLT-1 in astrocyte-enriched cultures was found to significantly increase following the addition of neuron-conditioned medium maintained at 37 °C, and to a lesser extent with neuron-conditioned medium at 33 °C. In conclusion, the neuroprotective effects of hypothermia against brain ischemia-reperfusion injury involve down-regulation of astrocytic GLT-1, which mediates the reverse transport of glutamate. Moreover, this process may be regulated by molecules secreted by stressed neurons. PMID:23402854

  6. Mitogen-Activated Protein Kinase Kinase 3 Regulates Seed Dormancy in Barley.

    PubMed

    Nakamura, Shingo; Pourkheirandish, Mohammad; Morishige, Hiromi; Kubo, Yuta; Nakamura, Masako; Ichimura, Kazuya; Seo, Shigemi; Kanamori, Hiroyuki; Wu, Jianzhong; Ando, Tsuyu; Hensel, Goetz; Sameri, Mohammad; Stein, Nils; Sato, Kazuhiro; Matsumoto, Takashi; Yano, Masahiro; Komatsuda, Takao

    2016-03-21

    Seed dormancy has fundamental importance in plant survival and crop production; however, the mechanisms regulating dormancy remain unclear [1-3]. Seed dormancy levels generally decrease during domestication to ensure that crops successfully germinate in the field. However, reduction of seed dormancy can cause devastating losses in cereals like wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) due to pre-harvest sprouting, the germination of mature seed (grain) on the mother plant when rain occurs before harvest. Understanding the mechanisms of dormancy can facilitate breeding of crop varieties with the appropriate levels of seed dormancy [4-8]. Barley is a model crop [9, 10] and has two major seed dormancy quantitative trait loci (QTLs), SD1 and SD2, on chromosome 5H [11-19]. We detected a QTL designated Qsd2-AK at SD2 as the single major determinant explaining the difference in seed dormancy between the dormant cultivar "Azumamugi" (Az) and the non-dormant cultivar "Kanto Nakate Gold" (KNG). Using map-based cloning, we identified the causal gene for Qsd2-AK as Mitogen-activated Protein Kinase Kinase 3 (MKK3). The dormant Az allele of MKK3 is recessive; the N260T substitution in this allele decreases MKK3 kinase activity and appears to be causal for Qsd2-AK. The N260T substitution occurred in the immediate ancestor allele of the dormant allele, and the established dormant allele became prevalent in barley cultivars grown in East Asia, where the rainy season and harvest season often overlap. Our findings show fine-tuning of seed dormancy during domestication and provide key information for improving pre-harvest sprouting tolerance in barley and wheat. PMID:26948880

  7. NEMO-LIKE KINASE REGULATES POSTNATAL SKELETAL HOMEOSTASIS

    PubMed Central

    Canalis, Ernesto; Kranz, Lauren; Zanotti, Stefano

    2014-01-01

    Nemo-like kinase (Nlk) is related to the mitogen-activated protein (MAP) kinases and known to regulate signaling pathways involved in osteoblastogenesis. In vitro Nlk suppresses osteoblastogenesis, but the consequences of the Nlk inactivation in the skeleton in vivo are unknown. To study the function of Nlk, NlkloxP/loxP mice, where the Nlk exon2 is flanked by loxP sequences, were mated with mice expressing the Cre recombinase under the control of the paired-related homeobox gene 1 (Prx1) enhancer (Prx1-Cre), the Osterix (Osx-Cre) or the osteocalcin/bone gamma carboxyglutamate protein (Bglap-Cre) promoter. Prx1-Cre;NlkΔ/Δ mice did not exhibit a skeletal phenotype except for a modest increase in trabecular number and connectivity observed only in 3 month old male mice. Osx-Cre;NlkΔ/Δ male and female mice exhibited an increase in trabecular bone volume secondary to an increased trabecular number at 3 months of age. Bone histomorphometry revealed a decrease in osteoclast number and eroded surface in male mice, and decreased osteoblast number and function in female mice. Expression of osteoprotegerin mRNA was increased in calvarial extracts, explaining the decreased osteoclast and osteoblast number. The conditional deletion of Nlk in mature osteoblasts (Bglap-Cre;NlkΔ/Δ) resulted in no skeletal phenotype in 1 to 6 month old male or female mice. In conclusion, when expressed in undifferentiated osteoblasts, Nlk is a negative regulator of skeletal homeostasis possibly by targeting signals that regulate osteoclastogenesis and bone resorption. PMID:24664870

  8. p38 MAP kinase regulates circadian rhythms in Drosophila.

    PubMed

    Vrailas-Mortimer, Alysia D; Ryan, Sarah M; Avey, Matthew J; Mortimer, Nathan T; Dowse, Harold; Sanyal, Subhabrata

    2014-12-01

    The large repertoire of circadian rhythms in diverse organisms depends on oscillating central clock genes, input pathways for entrainment, and output pathways for controlling rhythmic behaviors. Stress-activated p38 MAP Kinases (p38K), although sparsely investigated in this context, show circadian rhythmicity in mammalian brains and are considered part of the circadian output machinery in Neurospora. We find that Drosophila p38Kb is expressed in clock neurons, and mutants in p38Kb either are arrhythmic or have a longer free-running periodicity, especially as they age. Paradoxically, similar phenotypes are observed through either transgenic inhibition or activation of p38Kb in clock neurons, suggesting a requirement for optimal p38Kb function for normal free-running circadian rhythms. We also find that p38Kb genetically interacts with multiple downstream targets to regulate circadian locomotor rhythms. More specifically, p38Kb interacts with the period gene to regulate period length and the strength of rhythmicity. In addition, we show that p38Kb suppresses the arrhythmic behavior associated with inhibition of a second p38Kb target, the transcription factor Mef2. Finally, we find that manipulating p38K signaling in free-running conditions alters the expression of another downstream target, MNK/Lk6, which has been shown to cycle with the clock and to play a role in regulating circadian rhythms. These data suggest that p38Kb may affect circadian locomotor rhythms through the regulation of multiple downstream pathways. PMID:25403440

  9. Pyruvate Kinase M2: A Potential Target for Regulating Inflammation

    PubMed Central

    Alves-Filho, Jose C.; Pålsson-McDermott, Eva M.

    2016-01-01

    Pyruvate kinase (PK) is the enzyme responsible for catalyzing the last step of glycolysis. Of the four PK isoforms expressed in mammalian cells, PKM2 has generated the most interest due to its impact on changes in cellular metabolism observed in cancer as well as in activated immune cells. As our understanding of dysregulated metabolism in cancer develops, and in light of the growing field of immunometabolism, intense efforts are in place to define the mechanism by which PKM2 regulates the metabolic profile of cancer as well as of immune cells. The enzymatic activity of PKM2 is heavily regulated by endogenous allosteric effectors as well as by intracellular signaling pathways, affecting both the enzymatic activity of PKM2 as a PK and the regulation of the recently described non-canonical nuclear functions of PKM2. We here review the current literature on PKM2 and its regulation, and discuss the potential for this protein as a therapeutic target in inflammatory disorders. PMID:27148264

  10. Regulation of ERK Kinase by MEK1 Kinase Inhibition in the Brain*

    PubMed Central

    Tassin, Tara C.; Benavides, David R.; Plattner, Florian; Nishi, Akinori; Bibb, James A.

    2015-01-01

    Metabotropic (slow) and ionotropic (fast) neurotransmission are integrated by intracellular signal transduction mechanisms involving protein phosphorylation/dephosphorylation to achieve experience-dependent alterations in brain circuitry. ERK is an important effector of both slow and fast forms of neurotransmission and has been implicated in normal brain function and CNS diseases. Here we characterize phosphorylation of the ERK-activating protein kinase MEK1 by Cdk5, ERK, and Cdk1 in vitro in intact mouse brain tissue and in the context of an animal behavioral paradigm of stress. Cdk5 only phosphorylates Thr-292, whereas ERK and Cdk1 phosphorylate both Thr-292 and Thr-286 MEK1. These sites interact in a kinase-specific manner and inhibit the ability of MEK1 to activate ERK. Thr-292 and Thr-286 MEK1 are phosphorylated in most mouse brain regions to stoichiometries of ∼5% or less. Phosphorylation of Thr-292 MEK1 is regulated by cAMP-dependent signaling in mouse striatum in a manner consistent with negative feedback inhibition in response to ERK activation. Protein phosphatase 1 and 2A contribute to the maintenance of the basal phosphorylation state of both Thr-292 and Thr-286 MEK1 and that of ERK. Activation of the NMDA class of ionotropic glutamate receptors reduces inhibitory MEK1 phosphorylation, whereas forced swim, a paradigm of acute stress, attenuates Thr-292 MEK1 phosphorylation. Together, the data indicate that these inhibitory MEK1 sites phosphorylated by Cdk5 and ERK1 serve as mechanistic points of convergence for the regulation of ERK signaling by both slow and fast neurotransmission. PMID:25971971

  11. Regulation of striatal astrocytic receptor for advanced glycation end-products variants in an early stage of experimental Parkinson's disease.

    PubMed

    Viana, Sofia D; Valero, Jorge; Rodrigues-Santos, Paulo; Couceiro, Patrícia; Silva, Andréa M; Carvalho, Félix; Ali, Syed F; Fontes-Ribeiro, Carlos A; Pereira, Frederico C

    2016-08-01

    Convincing evidence indicates that advanced glycation end-products and danger-associated protein S100B play a role in Parkinson's disease (PD). These agents operate through the receptor for advanced glycation end-products (RAGE), which displays distinct isoforms playing protective/deleterious effects. However, the nature of RAGE variants has been overlooked in PD studies. Hence, we attempted to characterize RAGE regulation in early stages of PD striatal pathology. A neurotoxin-based rodent model of PD was used in this study, through administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to C57BL/6 mice. Animals were killed 6 h post-MPTP to assess S100B/RAGE contents (RT-qPCR, ELISA) and RAGE isoform density (WB) and cellular distribution (immunohistochemistry). Dopaminergic and gliotic status were also mapped (HPLC-ED, WB, immunohistochemistry). At this preliminary stage of MPTP-induced PD in mice, RAGE inhibitory isoforms were increased whereas full-length RAGE was not affected. This putative cytoprotective RAGE phenotype paired an inflammatory and pro-oxidant setting fueling DAergic denervation. Increased RAGE inhibitory variants occur in astrocytes showing higher S100B density but no overt signs of hypertrophy or NF-κB activation, a canonical effector of RAGE. These findings expand our understanding of the toxic effect of MPTP on striatum and offer first in vivo evidence of RAGE being a responder in early stages of astrogliosis dynamics, supporting a protective rather tissue-destructive phenotype of RAGE in the initial phase of PD degeneration. These data lay the groundwork for future studies on the relevance of astrocytic RAGE in DAergic neuroprotection strategies. We report increased antagonistic RAGE variants paralleling S100B up-regulation in early stages of MPTP-induced astrogliosis dynamics . We propose that selective RAGE regulation reflects a self-protective mechanism to maintain low levels of RAGE ligands , preventing long

  12. A kinome wide screen identifies novel kinases involved in regulation of monoamine transporter function.

    PubMed

    Vuorenpää, Anne; Ammendrup-Johnsen, Ina; Jørgensen, Trine N; Gether, Ulrik

    2016-09-01

    The high affinity transporters for the monoamine neurotransmitters, dopamine, norepinephrine, and serotonin, play a key role in controlling monoaminergic neurotransmission. It is believed that the transporters (DAT, NET and SERT, respectively) are subject to tight regulation by the cellular signaling machinery to maintain monoaminergic homeostasis. Kinases constitute a pivotal role in cellular signaling, however, the regulation of monoamine transporters by the entire ensemble of kinases is unknown. Here, we perform a whole human kinome RNA interference screen to identify novel kinases involved in regulation of monoamine transporter function and surface expression. A primary screen in HEK 293 cells stably expressing DAT or SERT with siRNAs against 573 human kinases revealed 93 kinases putatively regulating transporter function. All 93 hits, which also included kinases previously implicated in monoamine transporter regulation, such as Protein kinase B (Akt) and mitogen-activated protein kinases (MAPK), were validated with a new set of siRNAs in a secondary screen. In this screen we assessed both changes in uptake and surface expression leading to selection of 11 kinases for further evaluation in HEK 293 cells transiently expressing DAT, SERT or NET. Subsequently, three kinases; salt inducible kinase 3 (SIK3), cAMP-dependent protein kinase catalytic subunit alpha (PKA C-α) and protein kinase X-linked (PrKX); were selected for additional exploration in catecholaminergic CATH.a differentiated cells (CAD) and rat chromocytoma (PC12) cells. Whereas SIK3 likely transcriptionally regulated expression of the three transfected transporters, depletion of PKA C-α was shown to decrease SERT function. Depletion of PrKX caused decreased surface expression and function of DAT without changing protein levels, suggesting that PrKX stabilizes the transporter at the cell surface. Summarized, our data provide novel insight into kinome regulation of the monoamine transporters and

  13. Estrogen protects against dopamine neuron toxicity in primary mesencephalic cultures through an indirect P13K/Akt mediated astrocyte pathway.

    PubMed

    Bains, Mona; Roberts, James L

    2016-01-01

    Astrocytes regulate neuronal homeostasis and have been implicated in affecting the viability and functioning of surrounding neurons under stressed and injured conditions. Previous data from our lab suggests indirect actions of estrogen through ERα in neighboring astroglia to protect dopamine neurons against 1-methyl-4-phenylpyridinium (MPP(+)) toxicity in mouse mesencephalic cultures. We further evaluate estrogen signaling in astrocytes and the mechanism of estrogen's indirect neuroprotective effects on dopamine neurons. Primary mesencephalic cultures pre-treated with 17β-estradiol and the membrane impermeable estrogen, E2-BSA, were both neuroprotective against MPP(+) -induced dopamine neuron toxicity, suggesting membrane-initiated neuroprotection. ERα was found in the plasma membrane of astrocyte cultures and colocalized with the lipid raft marker, flotillin-1. A 17β-estradiol time course revealed a significant increase in Akt, which was inhibited by the PI3 kinase inhibitor, LY294004. Estrogen conditioned media collected from pure astrocyte cultures rescued glial deficient mesencephalic cultures from MPP(+). This indirect estrogen-mediated neuroprotective effect in mesencephalic cultures was significantly reduced when PI3 kinase signaling in astrocytes was blocked prior to collecting estrogen-conditioned media using the irreversible PI3 kinase inhibitor, Wortmannin. Estrogen signaling via astrocytes is rapidly initiated at the membrane level and requires PI3 kinase signaling in order to protect primary mesencephalic dopamine neurons from MPP(+) neurotoxicity. PMID:26520464

  14. Receptor Tyrosine Kinases: Molecular Switches Regulating CNS Axon Regeneration

    PubMed Central

    Vigneswara, Vasanthy; Kundi, Sarina; Ahmed, Zubair

    2012-01-01

    The poor or lack of injured adult central nervous system (CNS) axon regeneration results in devastating consequences and poor functional recovery. The interplay between the intrinsic and extrinsic factors contributes to robust inhibition of axon regeneration of injured CNS neurons. The insufficient or lack of trophic support for injured neurons is considered as one of the major obstacles contributing to their failure to survive and regrow their axons after injury. In the CNS, many of the signalling pathways associated with neuronal survival and axon regeneration are regulated by several classes of receptor tyrosine kinases (RTK) that respond to a variety of ligands. This paper highlights and summarises the most relevant recent findings pertinent to different classes of the RTK family of molecules, with a particular focus on elucidating their role in CNS axon regeneration. PMID:22848811

  15. Parkin Regulates the Activity of Pyruvate Kinase M2*

    PubMed Central

    Liu, Kun; Li, Fanzhou; Han, Haichao; Chen, Yue; Mao, Zebin; Luo, Jianyuan; Zhao, Yingming; Zheng, Bin; Gu, Wei; Zhao, Wenhui

    2016-01-01

    Parkin, a ubiquitin E3 ligase, is mutated in most cases of autosomal recessive early onset Parkinson disease. It was discovered that Parkin is also mutated in glioblastoma and other human malignancies and that it inhibits tumor cell growth. Here, we identified pyruvate kinase M2 (PKM2) as a unique substrate for parkin through biochemical purification. We found that parkin interacts with PKM2 both in vitro and in vivo, and this interaction dramatically increases during glucose starvation. Ubiquitylation of PKM2 by parkin does not affect its stability but decreases its enzymatic activity. Parkin regulates the glycolysis pathway and affects the cell metabolism. Our studies revealed the novel important roles of parkin in tumor cell metabolism and provided new insight for therapy of Parkinson disease. PMID:26975375

  16. Parkin Regulates the Activity of Pyruvate Kinase M2.

    PubMed

    Liu, Kun; Li, Fanzhou; Han, Haichao; Chen, Yue; Mao, Zebin; Luo, Jianyuan; Zhao, Yingming; Zheng, Bin; Gu, Wei; Zhao, Wenhui

    2016-05-01

    Parkin, a ubiquitin E3 ligase, is mutated in most cases of autosomal recessive early onset Parkinson disease. It was discovered that Parkin is also mutated in glioblastoma and other human malignancies and that it inhibits tumor cell growth. Here, we identified pyruvate kinase M2 (PKM2) as a unique substrate for parkin through biochemical purification. We found that parkin interacts with PKM2 both in vitro and in vivo, and this interaction dramatically increases during glucose starvation. Ubiquitylation of PKM2 by parkin does not affect its stability but decreases its enzymatic activity. Parkin regulates the glycolysis pathway and affects the cell metabolism. Our studies revealed the novel important roles of parkin in tumor cell metabolism and provided new insight for therapy of Parkinson disease. PMID:26975375

  17. Cyclin-dependent kinase 5 regulates degranulation in human eosinophils.

    PubMed

    Odemuyiwa, Solomon O; Ilarraza, Ramses; Davoine, Francis; Logan, Michael R; Shayeganpour, Anooshirvan; Wu, Yingqi; Majaesic, Carina; Adamko, Darryl J; Moqbel, Redwan; Lacy, Paige

    2015-04-01

    Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation. PMID:25346443

  18. Follicle-stimulating Hormone Activates Extracellular Signal-regulated Kinase but Not Extracellular Signal-regulated Kinase Kinase through a 100-kDa Phosphotyrosine Phosphatase*

    PubMed Central

    Cottom, Joshua; Salvador, Lisa M.; Maizels, Evelyn T.; Reierstad, Scott; Park, Youngkyu; Carr, Daniel W.; Davare, Monika A.; Hell, Johannes W.; Palmer, Stephen S.; Dent, Paul; Kawakatsu, Hisaaki; Ogata, Masato; Hunzicker-Dunn, Mary

    2006-01-01

    In this report we sought to elucidate the mechanism by which the follicle-stimulating hormone (FSH) receptor signals to promote activation of the p42/p44 extracellular signal-regulated protein kinases (ERKs) in granulosa cells. Results show that the ERK kinase MEK and upstream intermediates Raf-1, Ras, Src, and L-type Ca2+ channels are already partially activated in vehicle-treated cells and that FSH does not further activate them. This tonic stimulatory pathway appears to be restrained at the level of ERK by a 100-kDa phosphotyrosine phosphatase that associates with ERK in vehicle-treated cells and promotes dephosphorylation of its regulatory Tyr residue, resulting in ERK inactivation. FSH promotes the phosphorylation of this phosphotyrosine phosphatase and its dissociation from ERK, relieving ERK from inhibition and resulting in its activation by the tonic stimulatory pathway and consequent translocation to the nucleus. Consistent with this premise, FSH-stimulated ERK activation is inhibited by the cell-permeable protein kinase A-specific inhibitor peptide Myr-PKI as well as by inhibitors of MEK, Src, a Ca2+ channel blocker, and chelation of extracellular Ca2+. These results suggest that FSH stimulates ERK activity in immature granulosa cells by relieving an inhibition imposed by a 100-kDa phosphotyrosine phosphatase. PMID:12493768

  19. Inositol Hexakisphosphate Kinase 3 Regulates Metabolism and Lifespan in Mice.

    PubMed

    Moritoh, Yusuke; Oka, Masahiro; Yasuhara, Yoshitaka; Hozumi, Hiroyuki; Iwachidow, Kimihiko; Fuse, Hiromitsu; Tozawa, Ryuichi

    2016-01-01

    Inositol hexakisphosphate kinase 3 (IP6K3) generates inositol pyrophosphates, which regulate diverse cellular functions. However, little is known about its own physiological role. Here, we show the roles of IP6K3 in metabolic regulation. We detected high levels of both mouse and human IP6K3 mRNA in myotubes and muscle tissues. In human myotubes, IP6K3 was upregulated by dexamethasone treatment, which is known to inhibit glucose metabolism. Furthermore, Ip6k3 expression was elevated under diabetic, fasting, and disuse conditions in mouse skeletal muscles. Ip6k3(-/-) mice demonstrated lower blood glucose, reduced circulating insulin, deceased fat mass, lower body weight, increased plasma lactate, enhanced glucose tolerance, lower glucose during an insulin tolerance test, and reduced muscle Pdk4 expression under normal diet conditions. Notably, Ip6k3 deletion extended animal lifespan with concomitant reduced phosphorylation of S6 ribosomal protein in the heart. In contrast, Ip6k3(-/-) mice showed unchanged skeletal muscle mass and no resistance to the effects of high fat diet. The current observations suggest novel roles of IP6K3 in cellular regulation, which impact metabolic control and lifespan. PMID:27577108

  20. Serum and Glucocorticoid Regulated Kinase 1 in Sodium Homeostasis

    PubMed Central

    Lou, Yiyun; Zhang, Fan; Luo, Yuqin; Wang, Liya; Huang, Shisi; Jin, Fan

    2016-01-01

    The ubiquitously expressed serum and glucocorticoid regulated kinase 1 (SGK1) is tightly regulated by osmotic and hormonal signals, including glucocorticoids and mineralocorticoids. Recently, SGK1 has been implicated as a signal hub for the regulation of sodium transport. SGK1 modulates the activities of multiple ion channels and carriers, such as epithelial sodium channel (ENaC), voltage-gated sodium channel (Nav1.5), sodium hydrogen exchangers 1 and 3 (NHE1 and NHE3), sodium-chloride symporter (NCC), and sodium-potassium-chloride cotransporter 2 (NKCC2); as well as the sodium-potassium adenosine triphosphatase (Na+/K+-ATPase) and type A natriuretic peptide receptor (NPR-A). Accordingly, SGK1 is implicated in the physiology and pathophysiology of Na+ homeostasis. Here, we focus particularly on recent findings of SGK1’s involvement in Na+ transport in renal sodium reabsorption, hormone-stimulated salt appetite and fluid balance and discuss the abnormal SGK1-mediated Na+ reabsorption in hypertension, heart disease, edema with diabetes, and embryo implantation failure. PMID:27517916

  1. Serum and Glucocorticoid Regulated Kinase 1 in Sodium Homeostasis.

    PubMed

    Lou, Yiyun; Zhang, Fan; Luo, Yuqin; Wang, Liya; Huang, Shisi; Jin, Fan

    2016-01-01

    The ubiquitously expressed serum and glucocorticoid regulated kinase 1 (SGK1) is tightly regulated by osmotic and hormonal signals, including glucocorticoids and mineralocorticoids. Recently, SGK1 has been implicated as a signal hub for the regulation of sodium transport. SGK1 modulates the activities of multiple ion channels and carriers, such as epithelial sodium channel (ENaC), voltage-gated sodium channel (Nav1.5), sodium hydrogen exchangers 1 and 3 (NHE1 and NHE3), sodium-chloride symporter (NCC), and sodium-potassium-chloride cotransporter 2 (NKCC2); as well as the sodium-potassium adenosine triphosphatase (Na⁺/K⁺-ATPase) and type A natriuretic peptide receptor (NPR-A). Accordingly, SGK1 is implicated in the physiology and pathophysiology of Na⁺ homeostasis. Here, we focus particularly on recent findings of SGK1's involvement in Na⁺ transport in renal sodium reabsorption, hormone-stimulated salt appetite and fluid balance and discuss the abnormal SGK1-mediated Na⁺ reabsorption in hypertension, heart disease, edema with diabetes, and embryo implantation failure. PMID:27517916

  2. Regulation of Endothelial Permeability by Src Kinase Signaling

    PubMed Central

    Hu, Guochang; Place, Aaron T.; Minshall, Richard D.

    2010-01-01

    An important function of the endothelium is to regulate the transport of liquid and solutes across the semi-permeable vascular endothelial barrier. Two cellular pathways have been identified controlling endothelial barrier function. The normally restrictive paracellular pathway, which can become “leaky” during inflammation when gaps are induced between endothelial cells at the level of adherens and tight junctional complexes, and the transcellular pathway, which transports plasma proteins the size of albumin via transcytosis in vesicle carriers originating from cell surface caveolae. During non-inflammatory conditions, caveolae-mediated transport may be the primary mechanism of vascular permeability regulation of fluid phase molecules as well as lipids, hormones, and peptides that bind avidly to albumin. Src family protein tyrosine kinases have been implicated in the upstream signaling pathways that lead to endothelial hyperpermeability through both the paracellular and transcellular pathways. Endothelial barrier dysfunction not only affects vascular homeostasis and cell metabolism, but also governs drug delivery to underlying cells and tissues. In this review of the field, we discuss the current understanding of Src signaling in regulating paracellular and transcellular endothelial permeability pathways and effects on endogenous macromolecule and drug delivery. PMID:17897637

  3. Inositol Hexakisphosphate Kinase 3 Regulates Metabolism and Lifespan in Mice

    PubMed Central

    Moritoh, Yusuke; Oka, Masahiro; Yasuhara, Yoshitaka; Hozumi, Hiroyuki; Iwachidow, Kimihiko; Fuse, Hiromitsu; Tozawa, Ryuichi

    2016-01-01

    Inositol hexakisphosphate kinase 3 (IP6K3) generates inositol pyrophosphates, which regulate diverse cellular functions. However, little is known about its own physiological role. Here, we show the roles of IP6K3 in metabolic regulation. We detected high levels of both mouse and human IP6K3 mRNA in myotubes and muscle tissues. In human myotubes, IP6K3 was upregulated by dexamethasone treatment, which is known to inhibit glucose metabolism. Furthermore, Ip6k3 expression was elevated under diabetic, fasting, and disuse conditions in mouse skeletal muscles. Ip6k3−/− mice demonstrated lower blood glucose, reduced circulating insulin, deceased fat mass, lower body weight, increased plasma lactate, enhanced glucose tolerance, lower glucose during an insulin tolerance test, and reduced muscle Pdk4 expression under normal diet conditions. Notably, Ip6k3 deletion extended animal lifespan with concomitant reduced phosphorylation of S6 ribosomal protein in the heart. In contrast, Ip6k3−/− mice showed unchanged skeletal muscle mass and no resistance to the effects of high fat diet. The current observations suggest novel roles of IP6K3 in cellular regulation, which impact metabolic control and lifespan. PMID:27577108

  4. Casein kinase 2 dependent phosphorylation of neprilysin regulates receptor tyrosine kinase signaling to Akt.

    PubMed

    Siepmann, Martin; Kumar, Sathish; Mayer, Günter; Walter, Jochen

    2010-01-01

    Neprilysin (NEP) is a type II membrane metalloproteinase that cleaves physiologically active peptides at the cell surface thus regulating the local concentration of these peptides available for receptor binding and signal transduction. In addition, the cytoplasmic N-terminal domain of NEP interacts with the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) thereby regulating intracellular signaling via Akt. Thus, NEP serves dual functions in extracellular and intracellular signal transduction. Here, we show that NEP undergoes phosphorylation at serine residue 6 within the N-terminal cytoplasmic domain. In vitro and cell culture experiments demonstrate that Ser 6 is efficiently phosphorylated by protein kinase CK2. The phosphorylation of the cytoplasmic domain of NEP inhibits its interaction with PTEN. Interestingly, expression of a pseudophosphorylated NEP variant (Ser6Asp) abrogates the inhibitory effect of NEP on insulin/insulin-like growth factor-1 (IGF-1) stimulated activation of Akt. Thus, our data demonstrate a regulatory role of CK2 in the interaction of NEP with PTEN and insulin/IGF-1 signaling. PMID:20957047

  5. ENaC γ-expressing astrocytes in the circumventricular organs, white matter, and ventral medullary surface: sites for Na+ regulation by glial cells

    PubMed Central

    Miller, Rebecca L.; Loewy, Arthur D.

    2014-01-01

    Using a double immunofluorescence procedure, we report the discovery of a novel group of fibrous astrocytes that co-express epithelial sodium channel (ENaC) γ-subunit protein along with glial acidic fibrillary protein (GFAP). These cells are concentrated along the borders of the sensory circumventricular organs (CVOs), embedded in the white matter (e.g., optic nerve/chiasm, anterior commissure, corpus callosum, pyramidal tract) and are components of the pia mater. In the CVOs, a compact collection of ENaC γ-immunoreactive glial fibers form the lamina terminalis immediately rostral to the organum vasculosum of the lamina terminalis (OVLT). Astrocyte processes can be traced into the median preoptic nucleus – a region implicated in regulation of sodium homeostasis. In the subfornical organ (SFO), ENaC γ-GFAP astrocytes lie in its lateral border, but not in the ventromedial core. In the AP, a dense ENaC γ-GFAP glial fibers form the interface between the AP and nucleus tractus solitarius; this area is termed the subpostremal region. Antibodies against the ENaC α- or β-subunit proteins do not immunostain these regions. In contrast, the antibodies against the ENaC γ-subunit protein react weakly with neuronal cell bodies in the CVOs. Besides affecting glial-neural functions in the CVOs, the astrocytes found in the white matter may affect saltatory nerve conduction, serving as a sodium buffer. The ENaC γ-expressing astrocytes of the ventral medulla send processes into the raphe pallidus which intermingle with the serotoninergic (5-HT) neurons found in this region as well as with the other nearby 5-HT neurons distributed along ventral medullary surface. PMID:24145067

  6. ENaC γ-expressing astrocytes in the circumventricular organs, white matter, and ventral medullary surface: sites for Na+ regulation by glial cells.

    PubMed

    Miller, Rebecca L; Loewy, Arthur D

    2013-11-01

    Using a double immunofluorescence procedure, we report the discovery of a novel group of fibrous astrocytes that co-express epithelial sodium channel (ENaC) γ-subunit protein along with glial acidic fibrillary protein (GFAP). These cells are concentrated along the borders of the sensory circumventricular organs (CVOs), embedded in the white matter (e.g., optic nerve/chiasm, anterior commissure, corpus callosum, pyramidal tract) and are components of the pia mater. In the CVOs, a compact collection of ENaC γ-immunoreactive glial fibers form the lamina terminalis immediately rostral to the organum vasculosum of the lamina terminalis (OVLT). Astrocyte processes can be traced into the median preoptic nucleus - a region implicated in regulation of sodium homeostasis. In the subfornical organ (SFO), ENaC γ-GFAP astrocytes lie in its lateral border, but not in the ventromedial core. In the area postrema (AP), a dense ENaC γ-GFAP glial fibers form the interface between the AP and nucleus tractus solitarius; this area is termed the subpostremal region. Antibodies against the ENaC α- or β-subunit proteins do not immunostain these regions. In contrast, the antibodies against the ENaC γ-subunit protein react weakly with neuronal cell bodies in the CVOs. Besides affecting glial-neural functions in the CVOs, the astrocytes found in the white matter may affect saltatory nerve conduction, serving as a sodium buffer. The ENaC γ-expressing astrocytes of the ventral medulla send processes into the raphe pallidus which intermingle with the serotoninergic (5-HT) neurons found in this region as well as with the other nearby 5-HT neurons distributed along ventral medullary surface. PMID:24145067

  7. Effects of agrin on the expression and distribution of the water channel protein aquaporin-4 and volume regulation in cultured astrocytes.

    PubMed

    Noell, Susan; Fallier-Becker, Petra; Beyer, Cordian; Kröger, Stephan; Mack, Andreas F; Wolburg, Hartwig

    2007-10-01

    Agrin is a heparan sulfate proteoglycan of the extracellular matrix and is known for organizing the postsynaptic differentiation of the neuromuscular junction. Increasing evidence also suggests roles for agrin in the developing CNS, including the formation and maintenance of the blood-brain barrier. Here we describe effects of agrin on the expression and distribution of the water channel protein aquaporin-4 (AQP4) and on the swelling capacity of cultured astrocytes of newborn mice. If astrocytes were cultured on a substrate containing poly DL-ornithine, anti-AQP4 immunoreactivity was evenly and diffusely distributed. If, however, astrocytes were cultured in the presence of agrin-conditioned medium, we observed an increase in the intensity of AQP4-specific membrane-associated staining. Freeze-fracture studies revealed a clustering of orthogonal arrays of particles, representing a structural equivalent of AQP4, when exogenous agrin was present in the astrocyte cultures. Neuronal and non-neuronal agrin isoforms (agrin A0B0 and agrin A4B8, respectively) were able to induce membrane-associated AQP4 staining. Water transport capacity as well as the density of orthogonal arrays of intramembranous particles was increased in astrocytes cultured with the neuronal agrin isoform A4B8, but not with the endothelial and meningeal isoform A0B0. RT-PCR demonstrated that agrin A4B8 increased the level of the M23 splice variant of AQP4 and decreased the level of the M1 splice variant of AQP4. Implications for the regulation and maintenance of the blood-brain barrier including oedema formation under pathological conditions are discussed. PMID:17927773

  8. Regulation and functions of sphingosine kinases in the brain

    PubMed Central

    Bryan, Lauren; Kordula, Tomasz; Spiegel, Sarah; Milstien, Sheldon

    2009-01-01

    It has long been known that sphingolipids, especially sphingomyelin, a principal component of myelin, are highly enriched in the central nervous system and are structural components of all eukaryotic cell membranes. In the last few years, substantial evidence has accumulated from studies of many types of cells demonstrating that in addition to their structural roles, their breakdown products form a new class of signaling molecules with potent and myriad regulatory effects on essentially every cell in the body. While the sphingolipid metabolites sphingosine and its precursor ceramide have been associated with cell growth arrest and apoptosis, sphingosine-1-phosphate (S1P) enhances proliferation, differentiation, and cell survival as well as regulates many physiological and pathological processes. The relative levels of these three interconvertible sphingolipid metabolites, and thus cell fate, are strongly influenced by the activity of sphingosine kinases, of which there are two isoforms, designated SphK1 and SphK2, the enzymes that phosphorylate sphingosine to produce S1P. Not much is yet known of the importance of S1P in the central nervous system. Therefore, this review is focused on current knowledge of regulation of SphK1 and SphK2 on both transcriptional and post-translational levels and the functions of these isozymes and their product S1P and its receptors in the central nervous system. PMID:18485923

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

    PubMed Central

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

    2013-01-01

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

  10. The role of Tec family kinases in the regulation of T-helper-cell differentiation.

    PubMed

    Boucheron, Nicole; Ellmeier, Wilfried

    2012-04-01

    ABSTRACT Members of the Tec kinase family (Tec, Btk, Itk, Rlk, and Bmx) play an important role during innate and adaptive immune responses, and mutations in Tec family kinases are linked with immunodeficiencies in humans and mice. Three members of the Tec kinase family are expressed in T cells (Tec, Itk, and Rlk), and biochemical and genetic studies have revealed important roles for Tec family kinases during T-cell development and in the control of T-cell function. Here the authors review the role of Tec family kinases in the regulation of T-helper-cell differentiation. PMID:22449074

  11. Cdc42 Regulation of Kinase Activity and Signaling by the Yeast p21-Activated Kinase Ste20

    PubMed Central

    Lamson, Rachel E.; Winters, Matthew J.; Pryciak, Peter M.

    2002-01-01

    The Saccharomyces cerevisiae kinase Ste20 is a member of the p21-activated kinase (PAK) family with several functions, including pheromone-responsive signal transduction. While PAKs are usually activated by small G proteins and Ste20 binds Cdc42, the role of Cdc42-Ste20 binding has been controversial, largely because Ste20 lacking its entire Cdc42-binding (CRIB) domain retains kinase activity and pheromone response. Here we show that, unlike CRIB deletion, point mutations in the Ste20 CRIB domain that disrupt Cdc42 binding also disrupt pheromone signaling. We also found that Ste20 kinase activity is stimulated by GTP-bound Cdc42 in vivo and this effect is blocked by the CRIB point mutations. Moreover, the Ste20 CRIB and kinase domains bind each other, and mutations that disrupt this interaction cause hyperactive kinase activity and bypass the requirement for Cdc42 binding. These observations demonstrate that the Ste20 CRIB domain is autoinhibitory and that this negative effect is antagonized by Cdc42 to promote Ste20 kinase activity and signaling. Parallel results were observed for filamentation pathway signaling, suggesting that the requirement for Cdc42-Ste20 interaction is not qualitatively different between the mating and filamentation pathways. While necessary for pheromone signaling, the role of the Cdc42-Ste20 interaction does not require regulation by pheromone or the pheromone-activated Gβγ complex, because the CRIB point mutations also disrupt signaling by activated forms of the kinase cascade scaffold protein Ste5. In total, our observations indicate that Cdc42 converts Ste20 to an active form, while pathway stimuli regulate the ability of this active Ste20 to trigger signaling through a particular pathway. PMID:11940652

  12. S6 Kinase Reflects and Regulates Ethanol-Induced Sedation

    PubMed Central

    Acevedo, Summer F.; Peru y Colón de Portugal, Raniero L.; Gonzalez, Dante A.; Rodan, Aylin R.

    2015-01-01

    Alcohol use disorders (AUDs) affect people at great individual and societal cost. Individuals at risk for AUDs are sensitive to alcohol's rewarding effects and/or resistant to its aversive and sedating effects. The molecular basis for these traits is poorly understood. Here, we show that p70 S6 kinase (S6k), acting downstream of the insulin receptor (InR) and the small GTPase Arf6, is a key mediator of ethanol-induced sedation in Drosophila. S6k signaling in the adult nervous system determines flies' sensitivity to sedation. Furthermore, S6k activity, measured via levels of phosphorylation (P-S6k), is a molecular marker for sedation and overall neuronal activity: P-S6k levels are decreased when neurons are silenced, as well as after acute ethanol sedation. Conversely, P-S6k levels rebound upon recovery from sedation and are increased when neuronal activity is enhanced. Reducing neural activity increases sensitivity to ethanol-induced sedation, whereas neuronal activation decreases ethanol sensitivity. These data suggest that ethanol has acute silencing effects on adult neuronal activity, which suppresses InR/Arf6/S6k signaling and results in behavioral sedation. In addition, we show that activity of InR/Arf6/S6k signaling determines flies' behavioral sensitivity to ethanol-induced sedation, highlighting this pathway in acute responses to ethanol. SIGNIFICANCE STATEMENT Genetic factors play a major role in the development of addiction. Identifying these genes and understanding their molecular mechanisms is a necessary first step in the development of targeted therapeutic intervention. Here, we show that signaling from the insulin receptor in Drosophila neurons determines flies' sensitivity to ethanol-induced sedation. We show that this signaling cascade includes the small GTPase Arf6 and S6 kinase (S6k). In addition, activity of S6k is regulated by acute ethanol exposure and by neuronal activity. S6k activity is therefore both an acute target of ethanol exposure and

  13. p38 and Extracellular Signal-Regulated Kinases Regulate the Myogenic Program at Multiple Steps

    PubMed Central

    Wu, Zhenguo; Woodring, Pamela J.; Bhakta, Kunjan S.; Tamura, Kumiko; Wen, Fang; Feramisco, James R.; Karin, Michael; Wang, Jean Y. J.; Puri, Pier Lorenzo

    2000-01-01

    The extracellular signals which regulate the myogenic program are transduced to the nucleus by mitogen-activated protein kinases (MAPKs). We have investigated the role of two MAPKs, p38 and extracellular signal-regulated kinase (ERK), whose activities undergo significant changes during muscle differentiation. p38 is rapidly activated in myocytes induced to differentiate. This activation differs from those triggered by stress and cytokines, because it is not linked to Jun–N-terminal kinase stimulation and is maintained during the whole process of myotube formation. Moreover, p38 activation is independent of a parallel promyogenic pathway stimulated by insulin-like growth factor 1. Inhibition of p38 prevents the differentiation program in myogenic cell lines and human primary myocytes. Conversely, deliberate activation of endogenous p38 stimulates muscle differentiation even in the presence of antimyogenic cues. Much evidence indicates that p38 is an activator of MyoD: (i) p38 kinase activity is required for the expression of MyoD-responsive genes, (ii) enforced induction of p38 stimulates the transcriptional activity of a Gal4-MyoD fusion protein and allows efficient activation of chromatin-integrated reporters by MyoD, and (iii) MyoD-dependent myogenic conversion is reduced in mouse embryonic fibroblasts derived from p38α−/− embryos. Activation of p38 also enhances the transcriptional activities of myocyte enhancer binding factor 2A (MEF2A) and MEF2C by direct phosphorylation. With MEF2C, selective phosphorylation of one residue (Thr293) is a tissue-specific activating signal in differentiating myocytes. Finally, ERK shows a biphasic activation profile, with peaks of activity in undifferentiated myoblasts and postmitotic myotubes. Importantly, activation of ERK is inhibitory toward myogenic transcription in myoblasts but contributes to the activation of myogenic transcription and regulates postmitotic responses (i.e., hypertrophic growth) in myotubes. PMID

  14. Pre-LTP requires extracellular signal-regulated kinase in the ACC

    PubMed Central

    Yamanaka, Manabu; Tian, Zhen; Darvish-Ghane, Soroush

    2016-01-01

    The extracellular signal-regulated kinase is an important protein kinase for cortical plasticity. Long-term potentiation in the anterior cingulate cortex is believed to play important roles in chronic pain, fear, and anxiety. Previous studies of extracellular signal-regulated kinase are mainly focused on postsynaptic form of long-term potentiation (post-long-term potentiation). Little is known about the relationship between extracellular signal-regulated kinase and presynaptic long-term potentiation (pre-long-term potentiation) in cortical synapses. In this study, we examined whether pre-long-term potentiation in the anterior cingulate cortex requires the activation of presynaptic extracellular signal-regulated kinase. We found that p42/p44 mitogen-activated protein kinase inhibitors, PD98059 and U0126, suppressed the induction of pre-long-term potentiation. By contrast, these inhibitors did not affect the maintenance of pre-long-term potentiation. Using pharmacological inhibitors, we found that pre-long-term potentiation recorded for 1 h did not require transcriptional or translational processes. Our results strongly indicate that the activation of presynaptic extracellular signal-regulated kinase is required for the induction of pre-long-term potentiation, and this involvement may explain the contribution of extracellular signal-regulated kinase to mood disorders. PMID:27178245

  15. Pre-LTP requires extracellular signal-regulated kinase in the ACC.

    PubMed

    Yamanaka, Manabu; Tian, Zhen; Darvish-Ghane, Soroush; Zhuo, Min

    2016-02-01

    The extracellular signal-regulated kinase is an important protein kinase for cortical plasticity. Long-term potentiation in the anterior cingulate cortex is believed to play important roles in chronic pain, fear, and anxiety. Previous studies of extracellular signal-regulated kinase are mainly focused on postsynaptic form of long-term potentiation (post-long-term potentiation). Little is known about the relationship between extracellular signal-regulated kinase and presynaptic long-term potentiation (pre-long-term potentiation) in cortical synapses. In this study, we examined whether pre-long-term potentiation in the anterior cingulate cortex requires the activation of presynaptic extracellular signal-regulated kinase. We found that p42/p44 mitogen-activated protein kinase inhibitors, PD98059 and U0126, suppressed the induction of pre-long-term potentiation. By contrast, these inhibitors did not affect the maintenance of pre-long-term potentiation. Using pharmacological inhibitors, we found that pre-long-term potentiation recorded for 1 h did not require transcriptional or translational processes. Our results strongly indicate that the activation of presynaptic extracellular signal-regulated kinase is required for the induction of pre-long-term potentiation, and this involvement may explain the contribution of extracellular signal-regulated kinase to mood disorders. PMID:27178245

  16. Regulation of store-operated calcium entry by calcium-independent phospholipase A2 in rat cerebellar astrocytes.

    PubMed

    Singaravelu, Karthika; Lohr, Christian; Deitmer, Joachim W

    2006-09-13

    We have studied store-operated Ca2+ entry (SOCE) in Bergmann glia and granule cell layer astrocytes in acute brain slices of the rat cerebellum, using the Ca2+-sensitive fluorescent dye Fluo-4 and confocal laser scanning microscopy. Astrocytes were identified by their morphology, location, and their Ca2+ response in K+-free solution. Depletion of Ca2+ stores by cyclopiazonic acid (CPA) (20 microM) induced SOCE in both types of astrocyte. A similar Ca2+ influx was elicited by the calmodulin antagonist calmidazolium (CMZ) (1 microM). The SOCE channel blocker 2-aminoethoxy-diphenylborate (2-APB) (100 microM) and the Ca2+ release-activated channel blocker 3,5-bistrifluoromethyl pyrazole derivative (BTP2) (20 microM) suppressed the CPA- and the CMZ-induced Ca2+ influx. Pretreatment of acute slices with the specific Ca2+-independent phospholipase A2 (iPLA2) inhibitor bromoenol lactone (BEL) (25 microM) blocked the CPA- and the CMZ-induced Ca2+ influx. The lysophospholipid products of iPLA2, lysophosphatidylcholine (250 nM) and lysophosphatidylinositol (250 nM), but not lysophosphatidic acid (250 nM), induced a BTP2- and 2-APB-sensitive, but BEL-insensitive, Ca2+ influx. CPA or CMZ enhanced the BEL-sensitive enzymatic activity of iPLA2 in cerebellar astrocyte culture. Inhibition of iPLA2 expression by specific antisense oligodeoxynucleotide of iPLA2 reduced the SOCE and the Ca2+ store refilling in cultured astrocytes. Spontaneous Ca2+ oscillations in astrocytes in situ were reduced after inhibiting SOCE channels or iPLA2 activity. The results suggest that the depletion of Ca2+ stores activates iPLA2 to open Ca2+ channels in the plasma membrane by the formation of lysophospholipids in astrocytes, presumably to refill the stores and allow normal Ca2+ signaling. PMID:16971542

  17. MEK Kinase 2 and the Adaptor Protein Lad Regulate Extracellular Signal-Regulated Kinase 5 Activation by Epidermal Growth Factor via Src

    PubMed Central

    Sun, Weiyong; Wei, Xudong; Kesavan, Kamala; Garrington, Timothy P.; Fan, Ruihua; Mei, Junjie; Anderson, Steven M.; Gelfand, Erwin W.; Johnson, Gary L.

    2003-01-01

    Lad is an SH2 domain-containing adaptor protein that binds MEK kinase 2 (MEKK2), a mitogen-activated protein kinase (MAPK) kinase kinase for the extracellular signal-regulated kinase 5 (ERK5) and JNK pathways. Lad and MEKK2 are in a complex in resting cells. Antisense knockdown of Lad expression and targeted gene disruption of MEKK2 expression results in loss of epidermal growth factor (EGF) and stress stimuli-induced activation of ERK5. Activation of MEKK2 and the ERK5 pathway by EGF and stress stimuli is dependent on Src kinase activity. The Lad-binding motif is encoded within amino acids 228 to 282 in the N terminus of MEKK2, and expression of this motif blocks Lad-MEKK2 interaction, resulting in inhibition of Src-dependent activation of MEKK2 and ERK5. JNK activation by EGF is similarly inhibited by loss of Lad or MEKK2 expression and by blocking the interaction of MEKK2 and Lad. Our studies demonstrate that Src kinase activity is required for ERK5 activation in response to EGF, MEKK2 expression is required for ERK5 activation by Src, Lad and MEKK2 association is required for Src activation of ERK5, and EGF and Src stimulation of ERK5-regulated MEF2-dependent promoter activity requires a functional Lad-MEKK2 signaling complex. PMID:12640115

  18. Pyruvate dehydrogenase kinase regulates hepatitis C virus replication.

    PubMed

    Jung, Gwon-Soo; Jeon, Jae-Han; Choi, Yeon-Kyung; Jang, Se Young; Park, Soo Young; Kim, Sung-Woo; Byun, Jun-Kyu; Kim, Mi-Kyung; Lee, Sungwoo; Shin, Eui-Cheol; Lee, In-Kyu; Kang, Yu Na; Park, Keun-Gyu

    2016-01-01

    During replication, hepatitis C virus (HCV) utilizes macromolecules produced by its host cell. This process requires host cellular metabolic reprogramming to favor elevated levels of aerobic glycolysis. Therefore, we evaluated whether pyruvate dehydrogenase kinase (PDK), a mitochondrial enzyme that promotes aerobic glycolysis, can regulate HCV replication. Levels of c-Myc, hypoxia-inducible factor-1α (HIF-1α), PDK1, PDK3, glucokinase, and serine biosynthetic enzymes were compared between HCV-infected and uninfected human liver and Huh-7.5 cells infected with or without HCV. Protein and mRNA expression of c-Myc, HIF-1α, and glycolytic enzymes were significantly higher in HCV-infected human liver and hepatocytes than in uninfected controls. This increase was accompanied by upregulation of serine biosynthetic enzymes, suggesting cellular metabolism was altered toward facilitated nucleotide synthesis essential for HCV replication. JQ1, a c-Myc inhibitor, and dichloroacetate (DCA), a PDK inhibitor, decreased the expression of glycolytic and serine synthetic enzymes in HCV-infected hepatocytes, resulting in suppressed viral replication. Furthermore, when co-administered with IFN-α or ribavirin, DCA further inhibited viral replication. In summary, HCV reprograms host cell metabolism to favor glycolysis and serine biosynthesis; this is mediated, at least in part, by increased PDK activity, which provides a surplus of nucleotide precursors. Therefore, blocking PDK activity might have therapeutic benefits against HCV replication. PMID:27471054

  19. Extracellular signal-regulated kinases in pain of peripheral origin.

    PubMed

    White, John P M; Cibelli, Mario; Fidalgo, Antonio Rei; Nagy, Istvan

    2011-01-10

    Activation of members of the family of enzymes known as extracellular signal-regulated kinases (ERKs) is now known to be involved in the development and/or maintenance of the pain associated with many inflammatory conditions, such as herniated spinal disc pain, chronic inflammatory articular pain, and the pain associated with bladder inflammation. Moreover, ERKs are implicated in the development of neuropathic pain signs in animals which are subjected to the lumbar 5 spinal nerve ligation model and the chronic constriction injury model of neuropathic pain. The position has now been reached where all scientists working on pain subjects ought to be aware of the importance of ERKs, if only because certain of these enzymes are increasingly employed as experimental markers of nociceptive processing. Here, we introduce the reader, first, to the intracellular context in which these enzymes function. Thereafter, we consider the involvement of ERKs in mediating nociceptive signalling to the brain resulting from noxious stimuli at the periphery which will be interpreted by the brain as pain of peripheral origin. PMID:20950608

  20. Pyruvate dehydrogenase kinase regulates hepatitis C virus replication

    PubMed Central

    Jung, Gwon-Soo; Jeon, Jae-Han; Choi, Yeon-Kyung; Jang, Se Young; Park, Soo Young; Kim, Sung-Woo; Byun, Jun-Kyu; Kim, Mi-Kyung; Lee, Sungwoo; Shin, Eui-Cheol; Lee, In-Kyu; Kang, Yu Na; Park, Keun-Gyu

    2016-01-01

    During replication, hepatitis C virus (HCV) utilizes macromolecules produced by its host cell. This process requires host cellular metabolic reprogramming to favor elevated levels of aerobic glycolysis. Therefore, we evaluated whether pyruvate dehydrogenase kinase (PDK), a mitochondrial enzyme that promotes aerobic glycolysis, can regulate HCV replication. Levels of c-Myc, hypoxia-inducible factor-1α (HIF-1α), PDK1, PDK3, glucokinase, and serine biosynthetic enzymes were compared between HCV-infected and uninfected human liver and Huh-7.5 cells infected with or without HCV. Protein and mRNA expression of c-Myc, HIF-1α, and glycolytic enzymes were significantly higher in HCV-infected human liver and hepatocytes than in uninfected controls. This increase was accompanied by upregulation of serine biosynthetic enzymes, suggesting cellular metabolism was altered toward facilitated nucleotide synthesis essential for HCV replication. JQ1, a c-Myc inhibitor, and dichloroacetate (DCA), a PDK inhibitor, decreased the expression of glycolytic and serine synthetic enzymes in HCV-infected hepatocytes, resulting in suppressed viral replication. Furthermore, when co-administered with IFN-α or ribavirin, DCA further inhibited viral replication. In summary, HCV reprograms host cell metabolism to favor glycolysis and serine biosynthesis; this is mediated, at least in part, by increased PDK activity, which provides a surplus of nucleotide precursors. Therefore, blocking PDK activity might have therapeutic benefits against HCV replication. PMID:27471054

  1. Transcriptional Regulation by Protein Kinase A in Cryptococcus neoformans

    PubMed Central

    Hu, Guanggan; Steen, Barbara R; Lian, Tianshun; Sham, Anita P; Tam, Nicola; Tangen, Kristin L; Kronstad, James W

    2007-01-01

    A defect in the PKA1 gene encoding the catalytic subunit of cyclic adenosine 5′-monophosphate (cAMP)–dependent protein kinase A (PKA) is known to reduce capsule size and attenuate virulence in the fungal pathogen Cryptococcus neoformans. Conversely, loss of the PKA regulatory subunit encoded by pkr1 results in overproduction of capsule and hypervirulence. We compared the transcriptomes between the pka1 and pkr1 mutants and a wild-type strain, and found that PKA influences transcript levels for genes involved in cell wall synthesis, transport functions such as iron uptake, the tricarboxylic acid cycle, and glycolysis. Among the myriad of transcriptional changes in the mutants, we also identified differential expression of ribosomal protein genes, genes encoding stress and chaperone functions, and genes for secretory pathway components and phospholipid synthesis. The transcriptional influence of PKA on these functions was reminiscent of the linkage between transcription, endoplasmic reticulum stress, and the unfolded protein response in Saccharomyces cerevisiae. Functional analyses confirmed that the PKA mutants have a differential response to temperature stress, caffeine, and lithium, and that secretion inhibitors block capsule production. Importantly, we also found that lithium treatment limits capsule size, thus reinforcing potential connections between this virulence trait and inositol and phospholipid metabolism. In addition, deletion of a PKA-regulated gene, OVA1, revealed an epistatic relationship with pka1 in the control of capsule size and melanin formation. OVA1 encodes a putative phosphatidylethanolamine-binding protein that appears to negatively influence capsule production and melanin accumulation. Overall, these findings support a role for PKA in regulating the delivery of virulence factors such as the capsular polysaccharide to the cell surface and serve to highlight the importance of secretion and phospholipid metabolism as potential targets for

  2. Focal adhesion kinases and calcium/calmodulin-dependent protein kinases regulate protein tyrosine phosphorylation in stallion sperm.

    PubMed

    González-Fernández, Lauro; Macías-García, Beatriz; Loux, Shavahn C; Varner, Dickson D; Hinrichs, Katrin

    2013-06-01

    Protein tyrosine phosphorylation (PY) is a hallmark of sperm capacitation. In stallion sperm, calcium inhibits PY at pH <7.8, mediated by calmodulin. To explore the mechanism of that inhibition, we incubated stallion sperm in media without added calcium, with calcium, or with calcium plus the calmodulin inhibitor W-7 (Ca/W-7 treatment). Treatment with inhibitors of calcium/calmodulin-dependent kinases, protein kinase A (PRKA), or Src family kinases suppressed the PY induced by the absence of added calcium, but not that induced by the Ca/W-7 treatment, indicating that PY in the absence of added calcium occurred via the canonical PRKA pathway, but that PY in the Ca/W-7 treatment did not. This suggested that when calmodulin was inhibited, calcium stimulated PY via a noncanonical pathway. Incubation with PF-431396, an inhibitor of focal adhesion kinases (FAKs), a family of calcium-induced protein tyrosine kinases, inhibited the PY induced both by the absence of added calcium and by the Ca/W-7 treatment. Western blotting demonstrated that both FAK family members, protein tyrosine kinases 2 and 2B, were phosphorylated in the absence of added calcium and in the Ca/W-7 treatment, but not in the presence of calcium without calmodulin inhibitors. Inhibition of FAK proteins inhibited PY in stallion sperm incubated under capacitating conditions (in the presence of calcium, bovine serum albumin, and bicarbonate at pH >7.8). These results show for the first time a role for calcium/calmodulin-dependent kinases in PRKA-dependent sperm PY; a non-PRKA-dependent pathway regulating sperm PY; and the apparent involvement of the FAK family of protein tyrosine kinases downstream in both pathways. PMID:23595906

  3. Extracellular signal-regulated kinase-2 within the ventral tegmental area regulates responses to stress.

    PubMed

    Iñiguez, Sergio D; Vialou, Vincent; Warren, Brandon L; Cao, Jun-Li; Alcantara, Lyonna F; Davis, Lindsey C; Manojlovic, Zarko; Neve, Rachael L; Russo, Scott J; Han, Ming-Hu; Nestler, Eric J; Bolaños-Guzmán, Carlos A

    2010-06-01

    Neurotrophic factors and their signaling pathways have been implicated in the neurobiological adaptations in response to stress and the regulation of mood-related behaviors. A candidate signaling molecule implicated in mediating these cellular responses is the extracellular signal-regulated kinase (ERK1/2), although its functional role in mood regulation remains to be fully elucidated. Here we show that acute (1 d) or chronic (4 weeks) exposure to unpredictable stress increases phosphorylation of ERK1/2 and of two downstream targets (ribosomal S6 kinase and mitogen- and stress-activated protein kinase 1) within the ventral tegmental area (VTA), an important substrate for motivated behavior and mood regulation. Using herpes simplex virus-mediated gene transfer to assess the functional significance of this ERK induction, we show that overexpressing ERK2 within the VTA increases susceptibility to stress as measured in the forced swim test, responses to unconditioned nociceptive stimuli, and elevated plus maze in Sprague Dawley male rats, and in the tail suspension test and chronic social defeat stress procedure in C57BL/6 male mice. In contrast, blocking ERK2 activity in the VTA produces stress-resistant behavioral responses in these same assays and also blocks a chronic stress-induced reduction in sucrose preference. The effects induced by ERK2 blockade were accompanied by decreases in the firing frequency of VTA dopamine neurons, an important electrophysiological hallmark of resilient-like behavior. Together, these results strongly implicate a role for ERK2 signaling in the VTA as a key modulator of responsiveness to stress and mood-related behaviors. PMID:20519540

  4. Astrocytic CCAAT/Enhancer Binding Protein δ Regulates Neuronal Viability and Spatial Learning Ability via miR-135a.

    PubMed

    Chu, Yu-Yi; Ko, Chiung-Yuan; Wang, Wei-Jan; Wang, Shao-Ming; Gean, Po-Wu; Kuo, Yu-Min; Wang, Ju-Ming

    2016-08-01

    The progression of Alzheimer's disease (AD) has been associated with astrocytes-induced neuroinflammation. However, the detailed mechanism of astrocytes associated with learning impairments and neuronal loss in AD is poorly defined. Here, we provide novel evidences that astrocytic miR-135a is critical for neuronal viability and spatial learning ability in vivo. The AppTg/Cebpd (-/-) mice showed a spatial learning improvement compared with the APPswe/PS1/E9 bigenic (AppTg) mice. miR-135a was found to be a CCAAT/enhancer binding protein δ (CEBPD) responsive miRNA and can repress the transcription of thrombospondin 1 (THBS1) / Thbs1 (mouse) via its 3'-untranslated region (3'UTR). We used different experimental approaches to attenuate the expression of CEBPD/Cebpd (mouse) or miR-135a in astrocytes and found the following results: increase in THBS1/Thbs1 expression, decrease in neuronal apoptosis, and increase in growth of neurites. Importantly, injection of miR-135a antagonist (AM135a) into the brain of AppTg mice was found to prevent neuronal apoptosis and improved the spatial learning ability. Together, our findings demonstrate a critical function for the astrocytic CEBPD, and point to miR-135a antagonist as an attractive therapeutic target for the treatment of Alzheimer's disease. PMID:26208701

  5. A Metabotropic-Like Flux-Independent NMDA Receptor Regulates Ca2+ Exit from Endoplasmic Reticulum and Mitochondrial Membrane Potential in Cultured Astrocytes

    PubMed Central

    Montes de Oca Balderas, Pavel; Aguilera, Penélope

    2015-01-01

    Astrocytes were long thought to be only structural cells in the CNS; however, their functional properties support their role in information processing and cognition. The ionotropic glutamate N-methyl D-aspartate (NMDA) receptor (NMDAR) is critical for CNS functions, but its expression and function in astrocytes is still a matter of research and debate. Here, we report immunofluorescence (IF) labeling in rat cultured cortical astrocytes (rCCA) of all NMDAR subunits, with phenotypes suggesting their intracellular transport, and their mRNA were detected by qRT-PCR. IF and Western Blot revealed GluN1 full-length synthesis, subunit critical for NMDAR assembly and transport, and its plasma membrane localization. Functionally, we found an iCa2+ rise after NMDA treatment in Fluo-4-AM labeled rCCA, an effect blocked by the NMDAR competitive inhibitors D(-)-2-amino-5-phosphonopentanoic acid (APV) and Kynurenic acid (KYNA) and dependent upon GluN1 expression as evidenced by siRNA knock down. Surprisingly, the iCa2+ rise was not blocked by MK-801, an NMDAR channel blocker, or by extracellular Ca2+ depletion, indicating flux-independent NMDAR function. In contrast, the IP3 receptor (IP3R) inhibitor XestosponginC did block this response, whereas a Ryanodine Receptor inhibitor did so only partially. Furthermore, tyrosine kinase inhibition with genistein enhanced the NMDA elicited iCa2+ rise to levels comparable to those reached by the gliotransmitter ATP, but with different population dynamics. Finally, NMDA depleted the rCCA mitochondrial membrane potential (mΔψ) measured with JC-1. Our results demonstrate that rCCA express NMDAR subunits which assemble into functional receptors that mediate a metabotropic-like, non-canonical, flux-independent iCa2+ increase. PMID:25954808

  6. Novel autophosphorylation sites of Src family kinases regulate kinase activity and SH2 domain-binding capacity.

    PubMed

    Weir, Marion E; Mann, Jacqueline E; Corwin, Thomas; Fulton, Zachary W; Hao, Jennifer M; Maniscalco, Jeanine F; Kenney, Marie C; Roman Roque, Kristal M; Chapdelaine, Elizabeth F; Stelzl, Ulrich; Deming, Paula B; Ballif, Bryan A; Hinkle, Karen L

    2016-04-01

    Src family tyrosine kinases (SFKs) are critical players in normal and aberrant biological processes. While phosphorylation importantly regulates SFKs at two known tyrosines, large-scale phosphoproteomics have revealed four additional tyrosines commonly phosphorylated in SFKs. We found these novel tyrosines to be autophosphorylation sites. Mimicking phosphorylation at the C-terminal site to the activation loop decreased Fyn activity. Phosphomimetics and direct phosphorylation at the three SH2 domain sites increased Fyn activity while reducing phosphotyrosine-dependent interactions. While 68% of human SH2 domains exhibit conservation of at least one of these tyrosines, few have been found phosphorylated except when found in cis to a kinase domain. PMID:27001024

  7. Manipulation of adenosine kinase affects sleep regulation in mice

    PubMed Central

    Palchykova, Svitlana; Winsky-Sommerer, Raphaelle; Shen, Hai-Ying; Boison, Detlev; Gerling, Andrea; Tobler, Irene

    2010-01-01

    Sleep and sleep intensity are enhanced by adenosine and its receptor agonists, while adenosine receptor antagonists induce wakefulness. Adenosine kinase (ADK) is the primary enzyme metabolizing adenosine in adult brain. To investigate whether adenosine metabolism or clearance affects sleep we recorded sleep in mice with engineered mutations in Adk. Adk-tg mice over-express a transgene encoding the cytoplasmic isoform of ADK in the brain, but lack the nuclear isoform of the enzyme. Wild-type mice and Adk+/− mice that have a 50% reduction of the cytoplasmic and the nuclear isoforms of ADK served as controls. Adk-tg mice showed a remarkable reduction of EEG power in low frequencies in all vigilance states and in theta activity (6.25–11 Hz) in REM sleep and waking. Adk-tg mice were awake 58 min more per day than wild-type mice and spent significantly less time in REM sleep (102±3 vs 128±3 min in wild-type). After sleep deprivation slow-wave activity (0.75–4 Hz), the intensity component of NREM sleep, increased significantly less in Adk-tg mice and their slow-wave energy was reduced. In contrast, the vigilance states and EEG spectra of Adk+/− and wild-type mice did not differ. Our data suggest that over-expression of the cytoplasmic isoform of ADK is sufficient to alter sleep physiology. ADK might orchestrate neurotransmitter pathways involved in the generation of EEG oscillations and regulation of sleep. PMID:20881134

  8. Mechanism of abnormal growth in astrocytes derived from a mouse model of GM2 gangliosidosis.

    PubMed

    Kawashima, Nagako; Tsuji, Daisuke; Okuda, Tetsuya; Itoh, Kohji; Nakayama, Ken-ichi

    2009-11-01

    Sandhoff disease is a progressive neurodegenerative disorder caused by mutations in the HEXB gene which encodes the beta-subunit of N-acetyl-beta-hexosaminidase A and B, resulting in the accumulation of the ganglioside GM2. We isolated astrocytes from the neonatal brain of Sandhoff disease model mice in which the N-acetyl-beta-hexosaminidase beta-subunit gene is genetically disrupted (ASD). Glycolipid profiles revealed that GM2/GA2 accumulated in the lysosomes and not on the cell surface of ASD astrocytes. In addition, GM3 was increased on the cell surface. We found remarkable differences in the cell proliferation of ASD astrocytes when compared with cells isolated from wild-type mice, with a faster growth rate of ASD cells. In addition, we observed increased extracellular, signal-regulated kinase (ERK) phosphorylation in ASD cells, but Akt phosphorylation was decreased. Furthermore, the phosphorylation of ERK in ASD cells was not dependent upon extracellular growth factors. Treatment of ASD astrocytes with recombinant N-acetyl-beta-hexosaminidase A resulted in a decrease of their growth rate and ERK phosphorylation. These results indicated that the up-regulation of ERK phosphorylation and the increase in proliferation of ASD astrocytes were dependent upon GM2/GA2 accumulation. These findings may represent a mechanism in linking the nerve cell death and reactive gliosis observed in Sandhoff disease. PMID:19765188

  9. Pyruvate Kinase M2 Regulates Gene Transcription by Acting as A Protein Kinase

    PubMed Central

    Gao, Xueliang; Wang, Haizhen; Jenny, J. Yang; Liu, Xiaowei; Liu, Zhi-Ren

    2012-01-01

    Summary Pyruvate kinase isoform M2 (PKM2) is a glycolysis enzyme catalyzing conversion of phosphoenolpyruvate (PEP) to pyruvate with transferring a phosphate from PEP to ADP. We report here that PKM2 localizes to the cell nucleus. The levels of nuclear PKM2 correlate with cell proliferation. PKM2 activates transcription of MEK5 by phosphorylating stat3 at Y705. In vitro phosphorylation assays show that PKM2 is a protein kinase using PEP as phosphate donor. ADP competes with the protein substrate binding, indicating that the substrate may bind to the ADP site of PKM2. Our experiments suggest that PKM2 dimer is an active protein kinase, while the tetramer is an active pyruvate kinase. Expression a PKM2 mutant that exists as a dimer promotes cell proliferation, indicating that protein kinase activity of PKM2 plays a role in promoting cell proliferation. Our study reveals an important link between metabolism alteration and gene expression during tumor transformation and progression. PMID:22306293

  10. Regulation of the Target of Rapamycin and Other Phosphatidylinositol 3-Kinase-Related Kinases by Membrane Targeting

    PubMed Central

    De Cicco, Maristella; Abd Rahim, Munirah S.; Dames, Sonja A.

    2015-01-01

    Phosphatidylinositol 3-kinase-related kinases (PIKKs) play vital roles in the regulation of cell growth, proliferation, survival, and consequently metabolism, as well as in the cellular response to stresses such as ionizing radiation or redox changes. In humans six family members are known to date, namely mammalian/mechanistic target of rapamycin (mTOR), ataxia-telangiectasia mutated (ATM), ataxia- and Rad3-related (ATR), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), suppressor of morphogenesis in genitalia-1 (SMG-1), and transformation/transcription domain-associated protein (TRRAP). All fulfill rather diverse functions and most of them have been detected in different cellular compartments including various cellular membranes. It has been suggested that the regulation of the localization of signaling proteins allows for generating a locally specific output. Moreover, spatial partitioning is expected to improve the reliability of biochemical signaling. Since these assumptions may also be true for the regulation of PIKK function, the current knowledge about the regulation of the localization of PIKKs at different cellular (membrane) compartments by a network of interactions is reviewed. Membrane targeting can involve direct lipid-/membrane interactions as well as interactions with membrane-anchored regulatory proteins, such as, for example, small GTPases, or a combination of both. PMID:26426064

  11. Caveolin-1 regulates shear stress-dependent activation of extracellular signal-regulated kinase

    NASA Technical Reports Server (NTRS)

    Park, H.; Go, Y. M.; Darji, R.; Choi, J. W.; Lisanti, M. P.; Maland, M. C.; Jo, H.

    2000-01-01

    Fluid shear stress activates a member of the mitogen-activated protein (MAP) kinase family, extracellular signal-regulated kinase (ERK), by mechanisms dependent on cholesterol in the plasma membrane in bovine aortic endothelial cells (BAEC). Caveolae are microdomains of the plasma membrane that are enriched with cholesterol, caveolin, and signaling molecules. We hypothesized that caveolin-1 regulates shear activation of ERK. Because caveolin-1 is not exposed to the outside, cells were minimally permeabilized by Triton X-100 (0.01%) to deliver a neutralizing, polyclonal caveolin-1 antibody (pCav-1) inside the cells. pCav-1 then bound to caveolin-1 and inhibited shear activation of ERK but not c-Jun NH(2)-terminal kinase. Epitope mapping studies showed that pCav-1 binds to caveolin-1 at two regions (residues 1-21 and 61-101). When the recombinant proteins containing the epitopes fused to glutathione-S-transferase (GST-Cav(1-21) or GST-Cav(61-101)) were preincubated with pCav-1, only GST-Cav(61-101) reversed the inhibitory effect of the antibody on shear activation of ERK. Other antibodies, including m2234, which binds to caveolin-1 residues 1-21, had no effect on shear activation of ERK. Caveolin-1 residues 61-101 contain the scaffolding and oligomerization domains, suggesting that binding of pCav-1 to these regions likely disrupts the clustering of caveolin-1 or its interaction with signaling molecules involved in the shear-sensitive ERK pathway. We suggest that caveolae-like domains play a critical role in the mechanosensing and/or mechanosignal transduction of the ERK pathway.

  12. Scaffold Proteins Regulating Extracellular Regulated Kinase Function in Cardiac Hypertrophy and Disease

    PubMed Central

    Liang, Yan; Sheikh, Farah

    2016-01-01

    The mitogen activated protein kinase (MAPK)-extracellular regulated kinase 1/2 (ERK1/2) pathway is a central downstream signaling pathway that is activated in cardiac muscle cells during mechanical and agonist-mediated hypertrophy. Studies in genetic mouse models deficient in ERK-associated MAPK components pathway have further reinforced a direct role for this pathway in stress-induced cardiac hypertrophy and disease. However, more recent studies have highlighted that these signaling pathways may exert their regulatory functions in a more compartmentalized manner in cardiac muscle. Emerging data has uncovered specific MAPK scaffolding proteins that tether MAPK/ERK signaling specifically at the sarcomere and plasma membrane in cardiac muscle and show that deficiencies in these scaffolding proteins alter ERK activity and phosphorylation, which are then critical in altering the cardiac myocyte response to stress-induced hypertrophy and disease progression. In this review, we provide insights on ERK-associated scaffolding proteins regulating cardiac myofilament function and their impact on cardiac hypertrophy and disease. PMID:26973524

  13. Astrocyte calcium signaling: the third wave.

    PubMed

    Bazargani, Narges; Attwell, David

    2016-02-01

    The discovery that transient elevations of calcium concentration occur in astrocytes, and release 'gliotransmitters' which act on neurons and vascular smooth muscle, led to the idea that astrocytes are powerful regulators of neuronal spiking, synaptic plasticity and brain blood flow. These findings were challenged by a second wave of reports that astrocyte calcium transients did not mediate functions attributed to gliotransmitters and were too slow to generate blood flow increases. Remarkably, the tide has now turned again: the most important calcium transients occur in fine astrocyte processes not resolved in earlier studies, and new mechanisms have been discovered by which astrocyte [Ca(2+)]i is raised and exerts its effects. Here we review how this third wave of discoveries has changed our understanding of astrocyte calcium signaling and its consequences for neuronal function. PMID:26814587

  14. Creating Order from Chaos: Cellular Regulation by Kinase Anchoring

    PubMed Central

    Scott, John D.; Dessauer, Carmen W.; Tasken, Kjetil

    2012-01-01

    Second messenger responses rely on where and when the enzymes that propagate these signals become active. Spatial and temporal organization of certain signaling enzymes is controlled in part by A-kinase anchoring proteins (AKAPs). This family of regulatory proteins was originally classified on the basis of their ability to compartmentalize the cyclic adenosine monophosphate (cAMP)-dependent protein kinase (also known as protein kinase A, or PKA). However, it is now recognized that AKAPs position G protein–coupled receptors, adenylyl cyclases, G proteins, and their effector proteins in relation to protein kinases and signal termination enzymes such as phosphodiesterases and protein phosphatases. This arrangement offers a simple and efficient means to limit the scope, duration, and directional flow of information to sites deep within the cell. This review focuses on the pros and cons of reagents that define the biological role of kinase anchoring inside cells and discusses recent advances in our understanding of anchored second messenger signaling in the cardiovascular and immune systems. PMID:23043438

  15. Bioenergetic Mechanisms in Astrocytes May Contribute to Amyloid Plaque Deposition and Toxicity*

    PubMed Central

    Fu, Wen; Shi, Diya; Westaway, David; Jhamandas, Jack H.

    2015-01-01

    Alzheimer disease (AD) is characterized neuropathologically by synaptic disruption, neuronal loss, and deposition of amyloid β (Aβ) protein in brain structures that are critical for memory and cognition. There is increasing appreciation, however, that astrocytes, which are the major non-neuronal glial cells, may play an important role in AD pathogenesis. Unlike neurons, astrocytes are resistant to Aβ cytotoxicity, which may, in part, be related to their greater reliance on glycolytic metabolism. Here we show that, in cultures of human fetal astrocytes, pharmacological inhibition or molecular down-regulation of a main enzymatic regulator of glycolysis, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase (PFKFB3), results in increased accumulation of Aβ within and around astrocytes and greater vulnerability of these cells to Aβ toxicity. We further investigated age-dependent changes in PFKFB3 and astrocytes in AD transgenic mice (TgCRND8) that overexpress human Aβ. Using a combination of Western blotting and immunohistochemistry, we identified an increase in glial fibrillary acidic protein expression in astrocytes that paralleled the escalation of the Aβ plaque burden in TgCRND8 mice in an age-dependent manner. Furthermore, PFKFB3 expression also demonstrated an increase in these mice, although at a later age (9 months) than GFAP and Aβ. Immunohistochemical staining showed significant reactive astrogliosis surrounding Aβ plaques with increased PFKFB3 activity in 12-month-old TgCRND8 mice, an age when AD pathology and behavioral deficits are fully manifested. These studies shed light on the unique bioenergetic mechanisms within astrocytes that may contribute to the development of AD pathology. PMID:25814669

  16. Astrocytic Actions on Extrasynaptic Neuronal Currents

    PubMed Central

    Pál, Balázs

    2015-01-01

    In the last few decades, knowledge about astrocytic functions has significantly increased. It was demonstrated that astrocytes are not passive elements of the central nervous system (CNS), but active partners of neurons. There is a growing body of knowledge about the calcium excitability of astrocytes, the actions of different gliotransmitters and their release mechanisms, as well as the participation of astrocytes in the regulation of synaptic functions and their contribution to synaptic plasticity. However, astrocytic functions are even more complex than being a partner of the “tripartite synapse,” as they can influence extrasynaptic neuronal currents either by releasing substances or regulating ambient neurotransmitter levels. Several types of currents or changes of membrane potential with different kinetics and via different mechanisms can be elicited by astrocytic activity. Astrocyte-dependent phasic or tonic, inward or outward currents were described in several brain areas. Such currents, together with the synaptic actions of astrocytes, can contribute to neuromodulatory mechanisms, neurosensory and -secretory processes, cortical oscillatory activity, memory, and learning or overall neuronal excitability. This mini-review is an attempt to give a brief summary of astrocyte-dependent extrasynaptic neuronal currents and their possible functional significance. PMID:26696832

  17. Extracellular α-crystallin protects astrocytes from cell death through activation of MAPK, PI3K/Akt signaling pathway and blockade of ROS release from mitochondria.

    PubMed

    Zhu, Zhihui; Li, Rongyu; Stricker, Rolf; Reiser, Georg

    2015-09-16

    α-Crystallin with two isoforms, αA-crystallin (HSPB4) and αB-crystallin (HSPB5), is found in eye lens, spleen, lung, kidney, cornea, skin, but also in brain. Several studies revealed roles of αA/αB-crystallin in regulating cell viability and protection in the central nervous system. We previously demonstrated that α-crystallin serves as an intracellular protectant in astrocytes. Compared to well-studied intracellular functions of α-crystallin, there is limited proof for the role of α-crystallin as extracellular protectant. In order to clarify protective effects of extracellular αA/αB-crystallin, we exposed astrocytes to the toxic agents, staurosporine or C2-ceramide, or serum-starvation in the presence of αA/αB-crystallin. Extracellular αA/αB-crystallin protected astrocytes from staurosporine- and C2-ceramide-induced cell death. In addition, extracellular αB-crystallin/HSPB5 effectively promoted astrocytes viability through phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinases (p38) and c-Jun N-terminal kinases (JNK) signaling pathways under serum-deprivation. Furthermore, αB-crystallin/HSPB5 decreases the staurosporine-mediated cleavage of caspase 3 through PI3K/Akt signaling preventing apoptosis of astrocytes. Thus, the current study indicates that extracellular αA/αB-crystallin protects astrocytes exposed to various harmful stimuli. Furthermore, application of αB-crystallin/HSPB5 to isolated rat brain mitochondria inhibits ROS generation induced by complex III inhibition with Antimycin A. PMID:25998538

  18. An intrinsic adenylate kinase activity regulates gating of the ABC transporter CFTR.

    PubMed

    Randak, Christoph; Welsh, Michael J

    2003-12-26

    Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP binding cassette (ABC) transporter family. Like other ABC transporters, it can hydrolyze ATP. Yet while ATP hydrolysis influences channel gating, it has long seemed puzzling that CFTR would require this reaction because anions flow passively through CFTR. Moreover, no other ion channel is known to require the large energy of ATP hydrolysis to gate. We found that CFTR also has adenylate kinase activity (ATP + AMP <=> ADP + ADP) that regulates gating. When functioning as an adenylate kinase, CFTR showed positive cooperativity for ATP suggesting its two nucleotide binding domains may dimerize. Thus, channel activity could be regulated by two different enzymatic reactions, ATPase and adenylate kinase, that share a common ATP binding site in the second nucleotide binding domain. At physiologic nucleotide concentrations, adenylate kinase activity, rather than ATPase activity may control gating, and therefore involve little energy consumption. PMID:14697202

  19. Disentangling the Role of Astrocytes in Alcohol Use Disorder.

    PubMed

    Adermark, Louise; Bowers, M Scott

    2016-09-01

    Several laboratories recently identified that astrocytes are critical regulators of addiction machinery. It is now known that astrocyte pathology is a common feature of ethanol (EtOH) exposure in both humans and animal models, as even brief EtOH exposure is sufficient to elicit long-lasting perturbations in astrocyte gene expression, activity, and proliferation. Astrocytes were also recently shown to modulate the motivational properties of EtOH and other strongly reinforcing stimuli. Given the role of astrocytes in regulating glutamate homeostasis, a crucial component of alcohol use disorder (AUD), astrocytes might be an important target for the development of next-generation alcoholism treatments. This review will outline some of the more prominent features displayed by astrocytes, how these properties are influenced by acute and long-term EtOH exposure, and future directions that may help to disentangle astrocytic from neuronal functions in the etiology of AUD. PMID:27476876

  20. Abl Kinases Regulate HGF/Met Signaling Required for Epithelial Cell Scattering, Tubulogenesis and Motility

    PubMed Central

    Li, Ran; Knight, Jennifer F.; Park, Morag; Pendergast, Ann Marie

    2015-01-01

    Tight regulation of receptor tyrosine kinases (RTKs) is crucial for normal development and homeostasis. Dysregulation of RTKs signaling is associated with diverse pathological conditions including cancer. The Met RTK is the receptor for hepatocyte growth factor (HGF) and is dysregulated in numerous human tumors. Here we show that Abl family of non-receptor tyrosine kinases, comprised of Abl (ABL1) and Arg (ABL2), are activated downstream of the Met receptor, and that inhibition of Abl kinases dramatically suppresses HGF-induced cell scattering and tubulogenesis. We uncover a critical role for Abl kinases in the regulation of HGF/Met-dependent RhoA activation and RhoA-mediated actomyosin contractility and actin cytoskeleton remodeling in epithelial cells. Moreover, treatment of breast cancer cells with Abl inhibitors markedly decreases Met-driven cell migration and invasion. Notably, expression of a transforming mutant of the Met receptor in the mouse mammary epithelium results in hyper-activation of both Abl and Arg kinases. Together these data demonstrate that Abl kinases link Met activation to Rho signaling and Abl kinases are required for Met-dependent cell scattering, tubulogenesis, migration, and invasion. Thus, inhibition of Abl kinases might be exploited for the treatment of cancers driven by hyperactivation of HGF/Met signaling. PMID:25946048

  1. The Tec Kinase-Regulated Phosphoproteome Reveals a Mechanism for the Regulation of Inhibitory Signals in Murine Macrophages.

    PubMed

    Tampella, Giacomo; Kerns, Hannah M; Niu, Deqiang; Singh, Swati; Khim, Socheath; Bosch, Katherine A; Garrett, Meghan E; Moguche, Albanus; Evans, Erica; Browning, Beth; Jahan, Tahmina A; Nacht, Mariana; Wolf-Yadlin, Alejandro; Plebani, Alessandro; Hamerman, Jessica A; Rawlings, David J; James, Richard G

    2015-07-01

    Previous work has shown conflicting roles for Tec family kinases in regulation of TLR-dependent signaling in myeloid cells. In the present study, we performed a detailed investigation of the role of the Tec kinases Btk and Tec kinases in regulating TLR signaling in several types of primary murine macrophages. We demonstrate that primary resident peritoneal macrophages deficient for Btk and Tec secrete less proinflammatory cytokines in response to TLR stimulation than do wild-type cells. In contrast, we found that bone marrow-derived and thioglycollate-elicited peritoneal macrophages deficient for Btk and Tec secrete more proinflammatory cytokines than do wild-type cells. We then compared the phosphoproteome regulated by Tec kinases and LPS in primary peritoneal and bone marrow-derived macrophages. From this analysis we determined that Tec kinases regulate different signaling programs in these cell types. In additional studies using bone marrow-derived macrophages, we found that Tec and Btk promote phosphorylation events necessary for immunoreceptor-mediated inhibition of TLR signaling. Taken together, our results are consistent with a model where Tec kinases (Btk, Tec, Bmx) are required for TLR-dependent signaling in many types of myeloid cells. However, our data also support a cell type-specific TLR inhibitory role for Btk and Tec that is mediated by immunoreceptor activation and signaling via PI3K. PMID:26026062

  2. Regulation of hemeoxygenase-1 gene expression by Nrf2 and c-Jun in tertiary butylhydroquinone-stimulated rat primary astrocytes

    SciTech Connect

    Park, Jin-Sun; Kim, Hee-Sun

    2014-05-16

    Highlights: • tBHQ increased HO-1 mRNA and protein levels in rat primary astrocytes. • tBHQ enhanced HO-1 gene transcription in an ARE-dependent manner. • tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. • Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. • Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction. - Abstract: Hemeoxygenase-1 (HO-1) is a phase II antioxidant enzyme that is primarily involved in detoxification and cytoprotection in a variety of tissues. However, the mechanism underlying HO-1 gene expression remains unclear. In the present study, we investigated the regulation of HO-1 expression in primary cultured astrocytes by using the natural antioxidant compound tertiary butylhydroquinone (tBHQ). We found that tBHQ increased HO-1 mRNA and protein levels. Promoter analysis revealed that tBHQ enhanced HO-1 gene transcription in an antioxidant response element (ARE)-dependent manner. In addition, tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. Small interfering RNA (siRNA) experiments demonstrated that Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. Thus, Nrf2 knockdown reduced the basal level of HO-1 expression but did not affect the fold induction by tBHQ. On the other hand, knockdown of c-Jun diminished tBHQ-mediated induction of HO-1 without affecting basal expression. The data suggest that Nrf2 generally modulates the basal expression of HO-1, while c-Jun mediates HO-1 induction in response to tBHQ. The results of co-immunoprecipitation assays demonstrated a physical interaction between Nrf2 and c-Jun in tBHQ-treated astrocytes. The results suggest that Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction in tBHQ-treated rat primary astrocytes.

  3. Two distinct mechanisms for negative regulation of the Wee1 protein kinase.

    PubMed Central

    Tang, Z; Coleman, T R; Dunphy, W G

    1993-01-01

    The Wee1 protein kinase negatively regulates the entry into mitosis by catalyzing the inhibitory tyrosine phosphorylation of the Cdc2 protein. To examine the potential mechanisms for Wee1 regulation during the cell cycle, we have introduced a recombinant form of the fission yeast Wee1 protein kinase into Xenopus egg extracts. We find that the Wee1 protein undergoes dramatic changes in its phosphorylation state and kinase activity during the cell cycle. The Wee1 protein oscillates between an underphosphorylated 107 kDa form during interphase and a hyperphosphorylated 170 kDa version at mitosis. The mitosis-specific hyperphosphorylation of the Wee1 protein results in a substantial reduction in its activity as a Cdc2-specific tyrosine kinase. This phosphorylation occurs in the N-terminal region of the protein that lies outside the C-terminal catalytic domain, which was recently shown to be a substrate for the fission yeast Nim1 protein kinase. These experiments demonstrate the existence of a Wee1 regulatory system, consisting of both a Wee1-inhibitory kinase and a Wee1-stimulatory phosphatase, which controls the phosphorylation of the N-terminal region of the Wee1 protein. Moreover, these findings indicate that there are apparently two potential mechanisms for negative regulation of the Wee1 protein, one involving phosphorylation of its C-terminal domain by the Nim1 protein and the other involving phosphorylation of its N-terminal region by a different kinase. Images PMID:7504624

  4. Increasing freezing tolerance: kinase regulation of ICE1.

    PubMed

    Zhan, Xiangqiang; Zhu, Jian-Kang; Lang, Zhaobo

    2015-02-01

    Cold temperatures trigger the ICE1-CBF-COR transcriptional cascade in plants, which reprograms gene expression to increase freezing tolerance. In this issue of Developmental Cell, Ding et al. (2015) report that cold stress activates the protein kinase OST1 to phosphorylate and thereby stabilize and stimulate ICE1. This enhances plant tolerance to freezing temperatures. PMID:25669879

  5. Adenosine Monophosphate-activated Protein Kinase Regulates Interleukin-1β Expression and Glial Glutamate Transporter Function in Rodents with Neuropathic Pain

    PubMed Central

    Maixner, Dylan W.; Yan, Xisheng; Gao, Mei; Yadav, Ruchi; Weng, Han-Rong

    2015-01-01

    Background Neuroinflammation and dysfunctional glial glutamate transporters (GTs) in the spinal dorsal horn (SDH) are implicated in the genesis of neuropathic pain. We determined if adenosine monophosphate-activated protein kinase (AMPK) in the SDH regulates these processes in rodents with neuropathic pain. Methods Hind paw withdrawal responses to radiant heat and mechanical stimuli were used to assess nociceptive behaviors. Spinal markers related to neuroinflammation and glial GTs were determined by Western blotting. AMPK activities were manipulated pharmacologically and genetically. Regulation of glial GTs was determined by measuring protein expression and activities of glial GTs. Results AMPK activities were reduced in the SDH of rats (n = 5) with thermal hyperalgesia induced by nerve injury, which were accompanied with the activation of astrocytes, increased production of interleukin-1beta and activities of glycogen synthase kinase 3β, and suppressed protein expression of glial glutamate transporter-1. Thermal hyperalgesia was reversed by spinal activation of AMPK in neuropathic rats (n = 10), and induced by inhibiting spinal AMPK in naïve rats (n = 7 to 8). Spinal AMPKα knockdown (n = 6) and AMPKα1 conditional knockout (n = 6) induced thermal hyperalgesia and mechanical allodynia. These genetic alterations mimicked the changes of molecular markers induced by nerve injury. Pharmacological activation of AMPK enhanced glial GT activity in mice with neuropathic pain (n = 8) and attenuated glial glutamate transporter-1 internalization induced by interleukin-1β (n = 4). Conclusion These findings suggest enhancing spinal AMPK activities could be an effective approach for the treatment of neuropathic pain. PMID:25710409

  6. PERK pathway is involved in oxygen-glucose-serum deprivation-induced NF-kB activation via ROS generation in spinal cord astrocytes.

    PubMed

    Liu, Jinbo; Du, Lijian

    2015-11-13

    Mitochondrial dysfunction is a direct target of hypoxic/ischemic stress in astrocytes, which results in the increased production of reactive oxygen species (ROS). Previous reports showed that ROS can activate NF-kB in spinal cord astrocytes, which occurs as a secondary injury during the pathological process of spinal cord injury (SCI). Protein kinase RNA (PKR)-like ER kinase (PERK) plays an important role in mitochondrial dysfunction. To elucidate the specific role of PERK in hypoxic/ischemic-induced NF-kB activation in spinal astrocytes, we utilized an in vitro oxygen-glucose deprivation (OGD) model, which showed an enhanced formation of ROS and NF-kB activation. Knockdown of PERK resulted in reduced activation of PERK and ROS generation in astrocytes under OGD conditions. Notably, the knockdown of PERK also induced NF-kB activation in astrocytes. These data suggest that PERK is required for the hypoxic/ischemic-induced-dependent regulation of ROS and that it is involved in NF-kB activation in the astrocytes. PMID:26454173

  7. Lipopolysaccharide Induces Degradation of Connexin43 in Rat Astrocytes via the Ubiquitin-Proteasome Proteolytic Pathway

    PubMed Central

    Liao, Chih-Kai; Jeng, Chung-Jiuan; Wang, Hwai-Shi; Wang, Shu-Huei; Wu, Jiahn-Chun

    2013-01-01

    The astrocytic syncytium plays a critical role in maintaining the homeostasis of the brain through the regulation of gap junction intercellular communication (GJIC). Changes to GJIC in response to inflammatory stimuli in astrocytes may have serious effects on the brain. We have previously shown that lipopolysaccharide (LPS) reduces connexin43 (Cx43) expression and GJIC in cultured rat astrocytes via a toll-like receptor 4-mediated signaling pathway. In the present study, treatment of astrocytes with LPS resulted in a significant increase in levels of the phosphorylated forms of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) -1, -2, and -3 for up to 18 h. An increase in nuclear transcription factor NF-κB levels was also observed after 8 h of LPS treatment and was sustained for up to 18 h. The LPS-induced decrease in Cx43 protein levels and inhibition of GJIC were blocked by the SAPK/JNK inhibitor SP600125, but not by the NF-κB inhibitor BAY11-7082. Following blockade of de novo protein synthesis by cycloheximide, LPS accelerated Cx43 degradation. Moreover, the LPS-induced downregulation of Cx43 was blocked following inhibition of 26S proteasome activity using the reversible proteasome inhibitor MG132 or the irreversible proteasome inhibitor lactacystin. Immunoprecipitation analyses revealed an increased association of Cx43 with both ubiquitin and E3 ubiquitin ligase Nedd4 in astrocytes after LPS stimulation for 6 h and this effect was prevented by SP600125. Taken together, these results suggest that LPS stimulation leads to downregulation of Cx43 expression and GJIC in rat astrocytes by activation of SAPK/JNK and the ubiquitin-proteasome proteolytic pathway. PMID:24236122

  8. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    SciTech Connect

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M.H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

    2014-10-02

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.

  9. ATM kinase activity modulates Fas sensitivity through the regulation of FLIP in lymphoid cells.

    PubMed

    Stagni, Venturina; di Bari, Maria Giovanna; Cursi, Silvia; Condò, Ivano; Cencioni, Maria Teresa; Testi, Roberto; Lerenthal, Yaniv; Cundari, Enrico; Barilà, Daniela

    2008-01-15

    Ataxia telangiectasia (A-T) is a rare cancer-predisposing genetic disease, caused by the lack of functional ATM kinase, a major actor of the double strand brakes (DSB) DNA-damage response. A-T patients show a broad and diverse phenotype, which includes an increased rate of lymphoma and leukemia development. Fas-induced apoptosis plays a fundamental role in the homeostasis of the immune system and its defects have been associated with autoimmunity and lymphoma development. We therefore investigated the role of ATM kinase in Fas-induced apoptosis. Using A-T lymphoid cells, we could show that ATM deficiency causes resistance to Fas-induced apoptosis. A-T cells up-regulate FLIP protein levels, a well-known inhibitor of Fas-induced apoptosis. Reconstitution of ATM kinase activity was sufficient to decrease FLIP levels and to restore Fas sensitivity. Conversely, genetic and pharmacologic ATM kinase inactivation resulted in FLIP protein up-regulation and Fas resistance. Both ATM and FLIP are aberrantly regulated in Hodgkin lymphoma. Importantly, we found that reconstitution of ATM kinase activity decreases FLIP protein levels and restores Fas sensitivity in Hodgkin lymphoma-derived cells. Overall, these data identify a novel molecular mechanism through which ATM kinase may regulate the immune system homeostasis and impair lymphoma development. PMID:17932249

  10. Caveolin-1 is involved in reactive oxygen species-induced SHP-2 activation in astrocytes

    PubMed Central

    Yun, Ji Hee; Park, Soo Jung; Jo, Ara; Jou, Ilo; Park, Jung Soo

    2011-01-01

    Recent evidence supports a neuroprotective role of Src homology 2-containing protein tyrosine phosphatase 2 (SHP-2) against ischemic brain injury. However, the molecular mechanisms of SHP-2 activation and those governing how SHP-2 exerts its function under oxidative stress conditions are not well understood. Recently we have reported that reactive oxygen species (ROS)-mediated oxidative stress promotes the phosphorylation of endogenous SHP-2 through lipid rafts, and that this phosphorylation strongly occurs in astrocytes, but not in microglia. To investigate the molecules involved in events leading to phosphorylation of SHP-2, raft proteins were analyzed using astrocytes and microglia. Interestingly, caveolin-1 and -2 were detected only in astrocytes but not in microglia, whereas flotillin-1 was expressed in both cell types. To examine whether the H2O2-dependent phosphorylation of SHP-2 is mediated by caveolin-1, we used specific small interfering RNA (siRNA) to downregulate caveolin-1 expression. In the presence of caveolin-1 siRNA, the level of SHP-2 phosphorylation induced by H2O2 was significantly decreased, compared with in the presence of control siRNA. Overexpression of caveolin-1 effectively increased H2O2-induced SHP-2 phosphorylation in microglia. Lastly, H2O2 induced extracellular signal-regulated kinase (ERK) activation in astrocytes through caveolin-1. Our results suggest that caveolin-1 is involved in astrocyte-specific intracellular responses linked to the SHP-2-mediated signaling cascade following ROS-induced oxidative stress. PMID:21918362

  11. The Pim-1 Protein Kinase Is an Important Regulator of MET Receptor Tyrosine Kinase Levels and Signaling

    PubMed Central

    Xiong, Ying; Song, Jin H.; Mahajan, Sandeep; DuPont, Rachel; McEachern, Kristen; DeAngelo, Daniel J.; Cortes, Jorge E.; Minden, Mark D.; Ebens, Allen; Mims, Alice; LaRue, Amanda C.

    2014-01-01

    MET, the receptor for hepatocyte growth factor (HGF), plays an important role in signaling normal and tumor cell migration and invasion. Here, we describe a previously unrecognized mechanism that promotes MET expression in multiple tumor cell types. The levels of the Pim-1 protein kinase show a positive correlation with the levels of MET protein in human tumor cell lines and patient-derived tumor materials. Using small interfering RNA (siRNA), Pim knockout mice, small-molecule inhibitors, and overexpression of Pim-1, we confirmed this correlation and found that Pim-1 kinase activity regulates HGF-induced tumor cell migration, invasion, and cell scattering. The novel biochemical mechanism for these effects involves the ability of Pim-1 to control the translation of MET by regulating the phosphorylation of eukaryotic initiation factor 4B (eIF4B) on S406. This targeted phosphorylation is required for the binding of eIF4B to the eIF3 translation initiation complex. Importantly, Pim-1 action was validated by the evaluation of patient blood and bone marrow from a phase I clinical trial of a Pim kinase inhibitor, AZD1208. These results suggest that Pim inhibitors may have an important role in the treatment of patients where MET is driving tumor biology. PMID:24777602

  12. The Cotton Mitogen-Activated Protein Kinase Kinase 3 Functions in Drought Tolerance by Regulating Stomatal Responses and Root Growth.

    PubMed

    Wang, Chen; Lu, Wenjing; He, Xiaowen; Wang, Fang; Zhou, Yuli; Guo, Xulei; Guo, Xingqi

    2016-08-01

    Mitogen-activated protein kinase (MAPK) cascades play critical roles in signal transduction processes in eukaryotes. The MAPK kinases (MAPKKs) that link MAPKK kinases (MAPKKKs) and MAPKs are key components of MAPK cascades. However, the intricate regulatory mechanisms that control MAPKKs under drought stress conditions are not fully understood, especially in cotton (Gossypium hirsutum) Here, we isolated and characterized the cotton group B MAPKK gene GhMKK3 Overexpressing GhMKK3 in Nicotiana benthamiana enhanced tolerance to drought, and the results of RNA sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR) assays suggest that GhMKK3 plays an important role in responses to abiotic stresses by regulating stomatal responses and root hair growth. Further evidence demonstrated that overexpressing GhMKK3 promoted root growth and ABA-induced stomatal closure. In contrast, silencing GhMKK3 in cotton using virus-induced gene silencing (VIGS) resulted in the opposite phenotypes. More importantly, we identified an ABA- and drought-induced MAPK cascade that is composed of GhMKK3, GhMPK7 and GhPIP1 that compensates for deficiency in the MAPK cascade pathway in cotton under drought stress conditions. Together, these findings significantly improve our understanding of the mechanism by which GhMKK3 positively regulates drought stress responses. PMID:27335349

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

    PubMed Central

    Tavalin, Steven J.

    2008-01-01

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

  14. Trafficking of astrocytic vesicles in hippocampal slices

    SciTech Connect

    Potokar, Maja; Kreft, Marko; Celica Biomedical Center, Technology Park 24, 1000 Ljubljana ; Lee, So-Young; Takano, Hajime; Haydon, Philip G.; Zorec, Robert; Celica Biomedical Center, Technology Park 24, 1000 Ljubljana

    2009-12-25

    The increasingly appreciated role of astrocytes in neurophysiology dictates a thorough understanding of the mechanisms underlying the communication between astrocytes and neurons. In particular, the uptake and release of signaling substances into/from astrocytes is considered as crucial. The release of different gliotransmitters involves regulated exocytosis, consisting of the fusion between the vesicle and the plasma membranes. After fusion with the plasma membrane vesicles may be retrieved into the cytoplasm and may continue to recycle. To study the mobility implicated in the retrieval of secretory vesicles, these structures have been previously efficiently and specifically labeled in cultured astrocytes, by exposing live cells to primary and secondary antibodies. Since the vesicle labeling and the vesicle mobility properties may be an artifact of cell culture conditions, we here asked whether the retrieving exocytotic vesicles can be labeled in brain tissue slices and whether their mobility differs to that observed in cell cultures. We labeled astrocytic vesicles and recorded their mobility with two-photon microscopy in hippocampal slices from transgenic mice with fluorescently tagged astrocytes (GFP mice) and in wild-type mice with astrocytes labeled by Fluo4 fluorescence indicator. Glutamatergic vesicles and peptidergic granules were labeled by the anti-vesicular glutamate transporter 1 (vGlut1) and anti-atrial natriuretic peptide (ANP) antibodies, respectively. We report that the vesicle mobility parameters (velocity, maximal displacement and track length) recorded in astrocytes from tissue slices are similar to those reported previously in cultured astrocytes.

  15. Profilin Isoforms Modulate Astrocytic Morphology and the Motility of Astrocytic Processes

    PubMed Central

    Schweinhuber, Stefanie K.; Meßerschmidt, Tania; Hänsch, Robert; Korte, Martin; Rothkegel, Martin

    2015-01-01

    The morphology of astrocytic processes determines their close structural association with synapses referred to as the ‘tripartite synapse’. Concerted morphological plasticity processes at tripartite synapses are supposed to shape neuronal communication. Morphological changes in astrocytes as well as the motility of astrocytic processes require remodeling of the actin cytoskeleton. Among the regulators of fast timescale actin-based motility, the actin binding protein profilin 1 has recently been shown to control the activity-dependent outgrowth of astrocytic processes. Here, we demonstrate that cultured murine astrocytes in addition to the ubiquitous profilin 1 also express the neuronal isoform profilin 2a. To analyze the cellular function of both profilins in astrocytes, we took advantage of a shRNA mediated isoform-specific downregulation. Interestingly, consistent with earlier results in neurons, we found redundant as well as isoform-specific functions of both profilins in modulating cellular physiology. The knockdown of either profilin 1 or profilin 2a led to a significant decrease in cell spreading of astrocytes. In contrast, solely the knockdown of profilin 2a resulted in a significantly reduced morphological complexity of astrocytes in both dissociated and slice culture astrocytes. Moreover, both isoforms proved to be crucial for forskolin-induced astrocytic stellation. Furthermore, forskolin treatment resulted in isoform-specific changes in the phosphorylation level of profilin 1 and profilin 2a, leading to a PKA-dependent phosphorylation of profilin 2a. In addition, transwell assays revealed an involvement of both isoforms in the motility of astrocytic processes, while FRAP analysis displayed an isoform-specific role of profilin 1 in the regulation of actin dynamics in peripheral astrocytic processes. Taken together, we suggest profilin isoforms to be important modulators of astrocytic morphology and motility with overlapping as well as isoform

  16. Stimulatory and inhibitory protein kinase C consensus sequences regulate the cystic fibrosis transmembrane conductance regulator.

    PubMed

    Chappe, Valerie; Hinkson, Deborah A; Howell, L Daniel; Evagelidis, Alexandra; Liao, Jie; Chang, Xiu-Bao; Riordan, John R; Hanrahan, John W

    2004-01-01

    Protein kinase C (PKC) phosphorylation stimulates the cystic fibrosis transmembrane conductance regulator (CFTR) channel and enhances its activation by protein kinase A (PKA) through mechanisms that remain poorly understood. We have examined the effects of mutating consensus sequences for PKC phosphorylation and report here evidence for both stimulatory and inhibitory sites. Sequences were mutated in subsets and the mutants characterized by patch clamping. Activation of a 4CA mutant (S707A/S790A/T791A/S809A) by PKA was similar to that of wild-type CFTR and was enhanced by PKC, whereas responses of 3CA (T582A/T604A/S641A) and 2CA (T682A/S686A) channels to PKA were both drastically reduced (>90%). When each mutation in the 3CA and 2CA constructs was studied individually in a wild-type background, T582, T604, and S686 were found to be essential for PKA activation. Responses were restored when these three residues were reintroduced simultaneously into a 9CA mutant lacking all nine PKC consensus sequences (R6CA revertant); however, PKC phosphorylation was not required for this rescue. Nevertheless, two of the sites (T604 and S686) were phosphorylated in vitro, and PKC alone partially activated wild-type CFTR, the 4CA mutant, and the point mutants T582A and T604A, but not S686A channels, indicating that PKC does act at S686. The region encompassing S641 and T682 is inhibitory, because S641A enhanced activation by PKA, and T682A channels had 4-fold larger responses to PKC compared to wild-type channels. These results identify functionally important PKC consensus sequences on CFTR and will facilitate studies of its convergent regulation by PKC and PKA. PMID:14695900

  17. Stimulatory and inhibitory protein kinase C consensus sequences regulate the cystic fibrosis transmembrane conductance regulator

    PubMed Central

    Chappe, Valerie; Hinkson, Deborah A.; Howell, L. Daniel; Evagelidis, Alexandra; Liao, Jie; Chang, Xiu-Bao; Riordan, John R.; Hanrahan, John W.

    2004-01-01

    Protein kinase C (PKC) phosphorylation stimulates the cystic fibrosis transmembrane conductance regulator (CFTR) channel and enhances its activation by protein kinase A (PKA) through mechanisms that remain poorly understood. We have examined the effects of mutating consensus sequences for PKC phosphorylation and report here evidence for both stimulatory and inhibitory sites. Sequences were mutated in subsets and the mutants characterized by patch clamping. Activation of a 4CA mutant (S707A/S790A/T791A/S809A) by PKA was similar to that of wild-type CFTR and was enhanced by PKC, whereas responses of 3CA (T582A/T604A/S641A) and 2CA (T682A/S686A) channels to PKA were both drastically reduced (>90%). When each mutation in the 3CA and 2CA constructs was studied individually in a wild-type background, T582, T604, and S686 were found to be essential for PKA activation. Responses were restored when these three residues were reintroduced simultaneously into a 9CA mutant lacking all nine PKC consensus sequences (R6CA revertant); however, PKC phosphorylation was not required for this rescue. Nevertheless, two of the sites (T604 and S686) were phosphorylated in vitro, and PKC alone partially activated wild-type CFTR, the 4CA mutant, and the point mutants T582A and T604A, but not S686A channels, indicating that PKC does act at S686. The region encompassing S641 and T682 is inhibitory, because S641A enhanced activation by PKA, and T682A channels had 4-fold larger responses to PKC compared to wild-type channels. These results identify functionally important PKC consensus sequences on CFTR and will facilitate studies of its convergent regulation by PKC and PKA. PMID:14695900

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

    PubMed Central

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

    2016-01-01

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

  19. Ligand-Induced Asymmetry in Histidine Sensor Kinase Complex Regulates Quorum Sensing

    SciTech Connect

    Neiditch,M.; Federle, M.; Pompeani, A.; Kelly, R.; Swem, D.; Jeffrey, P.; Bassler, B.; Hughson, F.

    2006-01-01

    Bacteria sense their environment using receptors of the histidine sensor kinase family, but how kinase activity is regulated by ligand binding is not well understood. Autoinducer-2 (AI-2), a secreted signaling molecule originally identified in studies of the marine bacterium Vibrio harveyi, regulates quorum-sensing responses and allows communication between different bacterial species. AI-2 signal transduction in V. harveyi requires the integral membrane receptor LuxPQ, comprised of periplasmic binding protein (LuxP) and histidine sensor kinase (LuxQ) subunits. Combined X-ray crystallographic and functional studies show that AI-2 binding causes a major conformational change within LuxP, which in turn stabilizes a quaternary arrangement in which two LuxPQ monomers are asymmetrically associated. We propose that formation of this asymmetric quaternary structure is responsible for repressing the kinase activity of both LuxQ subunits and triggering the transition of V. harveyi into quorum-sensing mode.

  20. Per-Arnt-Sim Kinase (PASK): An Emerging Regulator of Mammalian Glucose and Lipid Metabolism

    PubMed Central

    Zhang, Dan-dan; Zhang, Ji-gang; Wang, Yu-zhu; Liu, Ying; Liu, Gao-lin; Li, Xiao-yu

    2015-01-01

    Per-Arnt-Sim Kinase (PASK) is an evolutionarily-conserved nutrient-responsive protein kinase that regulates lipid and glucose metabolism, mitochondrial respiration, phosphorylation, and gene expression. Recent data suggests that mammalian PAS kinase is involved in glucose metabolism and acts on pancreatic islet α/β cells and glycogen synthase (GS), affecting insulin secretion and blood glucose levels. In addition, PASK knockout mice (PASK-/-) are protected from obesity, liver triglyceride accumulation, and insulin resistance when fed a high-fat diet, implying that PASK may be a new target for metabolic syndrome (MetS) treatment as well as the cellular nutrients and energy sensors—adenosine monophosphate (AMP)-activated protein kinase (AMPK) and the targets of rapamycin (m-TOR). In this review, we will briefly summarize the regulation of PASK on mammalian glucose and lipid metabolism and its possible mechanism, and further explore the potential targets for MetS therapy. PMID:26371032

  1. Regulation of Sphingolipid Biosynthesis by the Morphogenesis Checkpoint Kinase Swe1.

    PubMed

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

    2016-01-29

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

  2. Regulation of therapeutic resistance in cancers by receptor tyrosine kinases

    PubMed Central

    Chen, Mei-Kuang; Hung, Mien-Chie

    2016-01-01

    In response to DNA damage lesions due to cellular stress, DNA damage response (DDR) pathways are activated to promote cell survival and genetic stability or unrepaired lesion-induced cell death. Current cancer treatments predominantly utilize DNA damaging agents, such as irradiation and chemotherapy drugs, to inhibit cancer cell proliferation and induce cell death through the activation of DDR. However, a portion of cancer patients is reported to develop therapeutic resistance to these DDR-inducing agents. One significant resistance mechanism in cancer cells is oncogenic kinase overexpression, which promotes cell survival by enhancing DNA damage repair pathways and evading cell cycle arrest. Among the oncogenic kinases, overexpression of receptor tyrosine kinases (RTKs) is reported in many of solid tumors, and numerous clinical trials targeting RTKs are currently in progress. As the emerging trend in cancer treatment combines DNA damaging agents and RTK inhibitors, it is important to understand the substrates of RTKs relative to the DDR pathways. In addition, alteration of RTK expression and their phosphorylated substrates can serve as biomarkers to stratify patients for combination therapies. In this review, we summarize the deleterious effects of RTKs on the DDR pathways and the emerging biomarkers for personalized therapy. PMID:27186434

  3. Serum and glucocorticoid-regulated kinase 1 regulates neutrophil clearance during inflammation resolution.

    PubMed

    Burgon, Joseph; Robertson, Anne L; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R; Walker, Paul; Hoggett, Emily E; Ward, Jonathan R; Farrow, Stuart N; Zuercher, William J; Jeffrey, Philip; Savage, Caroline O; Ingham, Philip W; Hurlstone, Adam F; Whyte, Moira K B; Renshaw, Stephen A

    2014-02-15

    The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid-regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease. PMID:24431232

  4. Serum and Glucocorticoid Regulated Kinase 1 (SGK1) Regulates Neutrophil Clearance During Inflammation Resolution

    PubMed Central

    Burgon, Joseph; Robertson, Anne L.; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R.; Walker, Paul; Hoggett, Emily E.; Ward, Jonathan R.; Farrow, Stuart N.; Zuercher, William J.; Jeffrey, Philip; Savage, Caroline O.; Ingham, Philip W.; Hurlstone, Adam F.; Whyte, Moira K. B.; Renshaw, Stephen A.

    2013-01-01

    The inflammatory response is integral to maintaining health, by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralise invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein Serum and Glucocorticoid Regulated Kinase 1 (SGK1). We have characterised the expression patterns and regulation of SGK family members in human neutrophils, and shown that inhibition of SGK activity completely abrogates the anti-apoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signalling, and thus may prove a valuable therapeutic target for the treatment of inflammatory disease. PMID:24431232

  5. The protein kinase A-regulated cardiac Cl- channel resembles the cystic fibrosis transmembrane conductance regulator.

    PubMed

    Nagel, G; Hwang, T C; Nastiuk, K L; Nairn, A C; Gadsby, D C

    1992-11-01

    Stimulation of beta-adrenoceptors in cardiac ventricular myocytes activates a strong chloride ion conductance as a result of phosphorylation by cyclic AMP-dependent protein kinase (PKA). This Cl- conductance, which is time- and voltage-independent, counters the tendency of the simultaneously enhanced Ca2+ channel current to prolong the ventricular action potential. Using inside-out giant patches excised from guinea-pig myocytes, we show here that phosphorylation by the PKA catalytic subunit plus Mg-ATP elicits discrete Cl- channel currents. In almost symmetrical Cl- solutions (approximately 150 mM), unitary current amplitude scales with membrane potential, and reverses sign near 0 mV, to yield a single channel conductance of approximately 12 pS. Opening of the phosphorylated channels requires hydrolysable nucleoside triphosphate, indicating that phosphorylation by PKA is necessary, but not sufficient, for channel activation. The properties of these PKA-regulated cardiac Cl- channels are very similar, if not identical, to those of the cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial cell Cl- channel whose regulation is defective in patients with cystic fibrosis. The full cardiological impact of these Cl- channels and of their possible malfunction in patients with cystic fibrosis remains to be determined. PMID:1279437

  6. Casein kinase iepsilon in the wnt pathway: regulation of beta-catenin function.

    PubMed

    Sakanaka, C; Leong, P; Xu, L; Harrison, S D; Williams, L T

    1999-10-26

    Wnt and its intracellular effector beta-catenin regulate developmental and oncogenic processes. Using expression cloning to identify novel components of the Wnt pathway, we isolated casein kinase Iepsilon (CKIepsilon). CKIepsilon mimicked Wnt in inducing a secondary axis in Xenopus, stabilizing beta-catenin, and stimulating gene transcription in cells. Inhibition of endogenous CKIepsilon by kinase-defective CKIepsilon or CKIepsilon antisense-oligonucleotides attenuated Wnt signaling. CKIepsilon was in a complex with axin and other downstream components of the Wnt pathway, including Dishevelled. CKIepsilon appears to be a positive regulator of the pathway and a link between upstream signals and the complexes that regulate beta-catenin. PMID:10535959

  7. Role of Intrinsic and Extrinsic Factors in the Regulation of the Mitotic Checkpoint Kinase Bub1

    PubMed Central

    Breit, Claudia; Bange, Tanja; Petrovic, Arsen; Weir, John R.; Müller, Franziska; Vogt, Doro; Musacchio, Andrea

    2015-01-01

    The spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores to ensure accurate sister chromatid segregation during mitosis. The SAC members Bub1 and BubR1 are paralogs that underwent significant functional specializations during evolution. We report an in-depth characterization of the kinase domains of Bub1 and BubR1. BubR1 kinase domain binds nucleotides but is unable to deliver catalytic activity in vitro. Conversely, Bub1 is an active kinase regulated by intra-molecular phosphorylation at the P+1 loop. The crystal structure of the phosphorylated Bub1 kinase domain illustrates a hitherto unknown conformation of the P+1 loop docked into the active site of the Bub1 kinase. Both Bub1 and BubR1 bind Bub3 constitutively. A hydrodynamic characterization of Bub1:Bub3 and BubR1:Bub3 demonstrates both complexes to have 1:1 stoichiometry, with no additional oligomerization. Conversely, Bub1:Bub3 and BubR1:Bub3 combine to form a heterotetramer. Neither BubR1:Bub3 nor Knl1, the kinetochore receptor of Bub1:Bub3, modulate the kinase activity of Bub1 in vitro, suggesting autonomous regulation of the Bub1 kinase domain. We complement our study with an analysis of the Bub1 substrates. Our results contribute to the mechanistic characterization of a crucial cell cycle checkpoint. PMID:26658523

  8. Mechanisms of regulation of SNF1/AMPK/SnRK1 protein kinases

    PubMed Central

    Crozet, Pierre; Margalha, Leonor; Confraria, Ana; Rodrigues, Américo; Martinho, Cláudia; Adamo, Mattia; Elias, Carlos A.; Baena-González, Elena

    2014-01-01

    The SNF1 (sucrose non-fermenting 1)-related protein kinases 1 (SnRKs1) are the plant orthologs of the budding yeast SNF1 and mammalian AMPK (AMP-activated protein kinase). These evolutionarily conserved kinases are metabolic sensors that undergo activation in response to declining energy levels. Upon activation, SNF1/AMPK/SnRK1 kinases trigger a vast transcriptional and metabolic reprograming that restores energy homeostasis and promotes tolerance to adverse conditions, partly through an induction of catabolic processes and a general repression of anabolism. These kinases typically function as a heterotrimeric complex composed of two regulatory subunits, β and γ, and an α-catalytic subunit, which requires phosphorylation of a conserved activation loop residue for activity. Additionally, SNF1/AMPK/SnRK1 kinases are controlled by multiple mechanisms that have an impact on kinase activity, stability, and/or subcellular localization. Here we will review current knowledge on the regulation of SNF1/AMPK/SnRK1 by upstream components, post-translational modifications, various metabolites, hormones, and others, in an attempt to highlight both the commonalities of these essential eukaryotic kinases and the divergences that have evolved to cope with the particularities of each one of these systems. PMID:24904600

  9. Mechanisms of regulation of SNF1/AMPK/SnRK1 protein kinases.

    PubMed

    Crozet, Pierre; Margalha, Leonor; Confraria, Ana; Rodrigues, Américo; Martinho, Cláudia; Adamo, Mattia; Elias, Carlos A; Baena-González, Elena

    2014-01-01

    The SNF1 (sucrose non-fermenting 1)-related protein kinases 1 (SnRKs1) are the plant orthologs of the budding yeast SNF1 and mammalian AMPK (AMP-activated protein kinase). These evolutionarily conserved kinases are metabolic sensors that undergo activation in response to declining energy levels. Upon activation, SNF1/AMPK/SnRK1 kinases trigger a vast transcriptional and metabolic reprograming that restores energy homeostasis and promotes tolerance to adverse conditions, partly through an induction of catabolic processes and a general repression of anabolism. These kinases typically function as a heterotrimeric complex composed of two regulatory subunits, β and γ, and an α-catalytic subunit, which requires phosphorylation of a conserved activation loop residue for activity. Additionally, SNF1/AMPK/SnRK1 kinases are controlled by multiple mechanisms that have an impact on kinase activity, stability, and/or subcellular localization. Here we will review current knowledge on the regulation of SNF1/AMPK/SnRK1 by upstream components, post-translational modifications, various metabolites, hormones, and others, in an attempt to highlight both the commonalities of these essential eukaryotic kinases and the divergences that have evolved to cope with the particularities of each one of these systems. PMID:24904600

  10. WNK2 kinase is a novel regulator of essential neuronal cation-chloride cotransporters.

    PubMed

    Rinehart, Jesse; Vázquez, Norma; Kahle, Kristopher T; Hodson, Caleb A; Ring, Aaron M; Gulcicek, Erol E; Louvi, Angeliki; Bobadilla, Norma A; Gamba, Gerardo; Lifton, Richard P

    2011-08-26

    NKCC1 and KCC2, related cation-chloride cotransporters (CCC), regulate cell volume and γ-aminobutyric acid (GABA)-ergic neurotranmission by modulating the intracellular concentration of chloride [Cl(-)]. These CCCs are oppositely regulated by serine-threonine phosphorylation, which activates NKCC1 but inhibits KCC2. The kinase(s) that performs this function in the nervous system are not known with certainty. WNK1 and WNK4, members of the WNK (with no lysine [K]) kinase family, either directly or via the downstream SPAK/OSR1 Ste20-type kinases, regulate the furosemide-sensitive NKCC2 and the thiazide-sensitive NCC, kidney-specific CCCs. What role the novel WNK2 kinase plays in this regulatory cascade, if any, is unknown. Here, we show that WNK2, unlike other WNKs, is not expressed in kidney; rather, it is a neuron-enriched kinase primarily expressed in neocortical pyramidal cells, thalamic relay cells, and cerebellar granule and Purkinje cells in both the developing and adult brain. Bumetanide-sensitive and Cl(-)-dependent (86)Rb(+) uptake assays in Xenopus laevis oocytes revealed that WNK2 promotes Cl(-) accumulation by reciprocally activating NKCC1 and inhibiting KCC2 in a kinase-dependent manner, effectively bypassing normal tonicity requirements for cotransporter regulation. TiO(2) enrichment and tandem mass spectrometry studies demonstrate WNK2 forms a protein complex in the mammalian brain with SPAK, a known phosphoregulator of NKCC1. In this complex, SPAK is phosphorylated at Ser-383, a consensus WNK recognition site. These findings suggest a role for WNK2 in the regulation of CCCs in the mammalian brain, with implications for both cell volume regulation and/or GABAergic signaling. PMID:21733846

  11. The intricate regulation and complex functions of the Class III phosphoinositide 3-kinase Vps34.

    PubMed

    Backer, Jonathan M

    2016-08-01

    The Class III phosphoinositide 3-kinase Vps34 (vacuolar protein sorting 34) plays important roles in endocytic trafficking, macroautophagy, phagocytosis, cytokinesis and nutrient sensing. Recent studies have provided exciting new insights into the structure and regulation of this lipid kinase, and new cellular functions for Vps34 have emerged. This review critically examines the wealth of new data on this important enzyme, and attempts to integrate these findings with current models of Vps34 signalling. PMID:27470591

  12. The Tec kinase-regulated phosphoproteome reveals a mechanism for the regulation of inhibitory signals in murine macrophages

    PubMed Central

    Tampella, Giacomo; Kerns, Hannah M.; Niu, Deqiang; Singh, Swati; Khim, Socheath; Bosch, Katherine A.; Garrett, Meghan E.; Moguche, Albanus; Evans, Erica; Browning, Beth; Jahan, Tahmina A.; Nacht, Mariana; Wolf-Yadlin, Alejandro; Plebani, Alessandro; Hamerman, Jessica A.; Rawlings, David J.; James, Richard G.

    2015-01-01

    Previous work has shown conflicting roles for Tec family kinases in regulation of Toll-like receptor (TLR)-dependent signalling in myeloid cells. In the present study, we performed a detailed investigation of the role of Btk and Tec kinases in regulating TLR signalling in several types of primary murine macrophages. We demonstrate that primary resident peritoneal macrophages deficient for Btk and Tec secrete less pro-inflammatory cytokines in response to TLR stimulation than wild type cells. In contrast, we found that bone marrow-derived and thioglycollate-elicited peritoneal macrophages deficient for Btk and Tec secrete more pro-inflammatory cytokines than wild type cells. We then compared the phosphoproteome regulated by Tec kinases and lipopolysaccharide in primary peritoneal and bone marrow derived macrophages. From this analysis we determined that Tec kinases regulate different signalling programs in these cell types. In additional studies using bone marrow-derived macrophages, we find that Tec and Btk promote phosphorylation events necessary for immunoreceptor-mediated inhibition of TLR signalling. Taken together, our results are consistent with a model where Tec kinases (Btk, Tec, Bmx) are required for TLR-dependent signalling in many types of myeloid cells. However, our data also support a cell type-specific TLR-inhibitory role for Btk and Tec that is mediated by immunoreceptor activation and signalling via PI3K. PMID:26026062

  13. Astrocytes: Orchestrating synaptic plasticity?

    PubMed

    De Pittà, M; Brunel, N; Volterra, A

    2016-05-26

    Synaptic plasticity is the capacity of a preexisting connection between two neurons to change in strength as a function of neural activity. Because synaptic plasticity is the major candidate mechanism for learning and memory, the elucidation of its constituting mechanisms is of crucial importance in many aspects of normal and pathological brain function. In particular, a prominent aspect that remains debated is how the plasticity mechanisms, that encompass a broad spectrum of temporal and spatial scales, come to play together in a concerted fashion. Here we review and discuss evidence that pinpoints to a possible non-neuronal, glial candidate for such orchestration: the regulation of synaptic plasticity by astrocytes. PMID:25862587

  14. Synbindin in Extracellular Signal-Regulated Protein Kinase Spatial Regulation and Gastric Cancer Aggressiveness

    PubMed Central

    2013-01-01

    Background The molecular mechanisms that control the aggressiveness of gastric cancer (GC) remain poorly defined. Here we show that synbindin contributes to the aggressiveness of GC by activating extracellular signal-regulated protein kinase (ERK) signaling on the Golgi apparatus. Methods Expression of synbindin was examined in normal gastric mucosa (n = 44), intestinal metaplastic gastric mucosa (n = 66), and GC tissues (n=52), and the biological effects of synbindin on tumor growth and ERK signaling were detected in cultured cells, nude mice, and human tissue samples. The interaction between synbindin and mitogen-activated protein kinase kinase (MEK1)/ERK was determined by immunofluorescence and fluorescence resonance energy transfer assays. The transactivation of synbindin by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) was detected using luciferase reporter assay and chromatin immunoprecipitation. Results High expression of synbindin was associated with larger tumor size (120.8 vs 44.8cm3; P = .01), advanced tumor node metastasis (TNM) stage (P = .003), and shorter patient survival (hazard ratio = 1.51; 95% confidence interval [CI] = 1.01 to 2.27; P = .046). Synbindin promotes cell proliferation and invasion by activating ERK2 on the Golgi apparatus, and synbindin is directly transactivated by NF-κB. Synbindin expression level was statistically significantly higher in human GCs with activated ERK2 than those with low ERK2 activity (intensity score of 11.5, 95% CI = 10.4 to 12.4 vs intensity score of 4.6, 95% CI 3.9 to 5.3; P < .001). Targeting synbindin in xenograft tumors decreased ERK2 phosphorylation and statistically significantly reduced tumor volume (451.2mm3, 95% CI = 328.3 to 574.1 vs 726.1mm3, 95% CI = 544.2 to 908.2; P = .01). Conclusions Synbindin contributes to malignant phenotypes of GC by activating ERK on the Golgi, and synbindin is a potential biomarker and therapeutic target for GC. PMID:24104608

  15. Phosphoinositide 3-kinase p85beta regulates invadopodium formation

    PubMed Central

    Cariaga-Martínez, Ariel E.; Cortés, Isabel; García, Esther; Pérez-García, Vicente; Pajares, María J.; Idoate, Miguel A.; Redondo-Muñóz, Javier; Antón, Inés M.; Carrera, Ana C.

    2014-01-01

    ABSTRACT The acquisition of invasiveness is characteristic of tumor progression. Numerous genetic changes are associated with metastasis, but the mechanism by which a cell becomes invasive remains unclear. Expression of p85β, a regulatory subunit of phosphoinositide-3-kinase, markedly increases in advanced carcinoma, but its mode of action is unknown. We postulated that p85β might facilitate cell invasion. We show that p85β localized at cell adhesions in complex with focal adhesion kinase and enhanced stability and maturation of cell adhesions. In addition, p85β induced development at cell adhesions of an F-actin core that extended several microns into the cell z-axis resembling the skeleton of invadopodia. p85β lead to F-actin polymerization at cell adhesions by recruiting active Cdc42/Rac at these structures. In accordance with p85β function in invadopodium-like formation, p85β levels increased in metastatic melanoma and p85β depletion reduced invadopodium formation and invasion. These results show that p85β enhances invasion by inducing cell adhesion development into invadopodia-like structures explaining the metastatic potential of tumors with increased p85β levels. PMID:25217619

  16. Cell cycle regulation by the NEK family of protein kinases.

    PubMed

    Fry, Andrew M; O'Regan, Laura; Sabir, Sarah R; Bayliss, Richard

    2012-10-01

    Genetic screens for cell division cycle mutants in the filamentous fungus Aspergillus nidulans led to the discovery of never-in-mitosis A (NIMA), a serine/threonine kinase that is required for mitotic entry. Since that discovery, NIMA-related kinases, or NEKs, have been identified in most eukaryotes, including humans where eleven genetically distinct proteins named NEK1 to NEK11 are expressed. Although there is no evidence that human NEKs are essential for mitotic entry, it is clear that several NEK family members have important roles in cell cycle control. In particular, NEK2, NEK6, NEK7 and NEK9 contribute to the establishment of the microtubule-based mitotic spindle, whereas NEK1, NEK10 and NEK11 have been implicated in the DNA damage response. Roles for NEKs in other aspects of mitotic progression, such as chromatin condensation, nuclear envelope breakdown, spindle assembly checkpoint signalling and cytokinesis have also been proposed. Interestingly, NEK1 and NEK8 also function within cilia, the microtubule-based structures that are nucleated from basal bodies. This has led to the current hypothesis that NEKs have evolved to coordinate microtubule-dependent processes in both dividing and non-dividing cells. Here, we review the functions of the human NEKs, with particular emphasis on those family members that are involved in cell cycle control, and consider their potential as therapeutic targets in cancer. PMID:23132929

  17. Protein kinase C is involved in the regulation of several calreticulin posttranslational modifications.

    PubMed

    Cristina Castañeda-Patlán, M; Razo-Paredes, Roberto; Carrisoza-Gaytán, Rolando; González-Mariscal, Lorenza; Robles-Flores, Martha

    2010-01-01

    Calreticulin (CRT) is a highly versatile lectin-like chaperone that affects many cellular functions both inside and outside the endoplasmic reticulum lumen. We previously reported that calreticulin interacts with several protein kinase C isozymes both in vitro and in vivo. The aim of this study was to elucidate the molecular determinants involved in the association between these proteins and the biochemical significance of their interaction. Using full-length or CRT-domain constructs expressed as GST-fusion proteins, we found that protein kinase C binds to the CRT N domain in overlay and pull-down assays. Phosphorylation experiments showed that only this CRT domain is phosphorylated by the kinase. Lectin blot analysis demonstrated that CRT is modified by N-glycosylation, but this modification did not affect its interaction with protein kinase C. We also demonstrated that although both domains of protein kinase C theta can bind to CRT, it is the catalytic one that binds with higher affinity to CRT. Immunofluorescence studies showed that CRT and PKC co-localize mainly at the ER (estimated in 35%). Activation of protein kinase C induced caused transient changes in CRT localization, and unexpectedly, also induced changes in posttranslational modifications found in the protein: CRT N-glycosylation is abolished, whereas tyrosine phosphorylation and O-linked beta-N-acetylglucosamine modification are increased. Together, these findings suggest that protein kinase C is involved in the regulation of CRT function. PMID:19800981

  18. Molecular mechanism for the regulation of rho-kinase by dimerization and its inhibition by fasudil.

    PubMed

    Yamaguchi, Hiroto; Kasa, Miyuki; Amano, Mutsuki; Kaibuchi, Kozo; Hakoshima, Toshio

    2006-03-01

    Rho-kinase is a key regulator of cytoskeletal events and a promising drug target in the treatment of vascular diseases and neurological disorders. Unlike other protein kinases, Rho-kinase requires both N- and C-terminal extension segments outside the kinase domain for activity, although the details of this requirement have been elusive. The crystal structure of an active Rho-kinase fragment containing the kinase domain and both the extensions revealed a head-to-head homodimer through the N-terminal extension forming a helix bundle that structurally integrates the C-terminal extension. This structural organization enables binding of the C-terminal hydrophobic motif to the N-terminal lobe, which defines the correct disposition of helix alphaC that is important for the catalytic activity. The bound inhibitor fasudil significantly alters the conformation and, consequently, the mode of interaction with the catalytic cleft that contains local structural changes. Thus, both kinase and drug conformational pliability and stability confer selectivity. PMID:16531242

  19. HIPK family kinases bind and regulate the function of the CCR4-NOT complex.

    PubMed

    Rodriguez-Gil, Alfonso; Ritter, Olesja; Hornung, Juliane; Stekman, Hilda; Krüger, Marcus; Braun, Thomas; Kremmer, Elisabeth; Kracht, Michael; Schmitz, M Lienhard

    2016-06-15

    The serine/threonine kinase HIPK2 functions as a regulator of developmental processes and as a signal integrator of a wide variety of stress signals, such as DNA damage, hypoxia, and reactive oxygen intermediates. Because the kinase is generated in a constitutively active form, its expression levels are restricted by a variety of different mechanisms. Here we identify the CCR4-NOT complex as a new regulator of HIPK2 abundance. Down-regulation or knockout of the CCR4-NOT complex member CNOT2 leads to reduced HIPK2 protein levels without affecting the expression level of HIPK1 or HIPK3. A fraction of all HIPK family members associates with the CCR4-NOT components CNOT2 and CNOT3. HIPKs also phosphorylate the CCR4-NOT complex, a feature that is shared with their yeast progenitor kinase, YAK1. Functional assays reveal that HIPK2 and HIPK1 restrict CNOT2-dependent mRNA decay. HIPKs are well known regulators of transcription, but the mutual regulation between CCR4-NOT and HIPKs extends the regulatory potential of these kinases by enabling posttranscriptional gene regulation. PMID:27122605

  20. An Integrative Approach for the Large-scale Identification of Human Genome Kinases Regulating Cancer Metastasis

    PubMed Central

    Zhang, Hanshuo; Wu, Pu-Yen; Ma, Ming; Ye, Yanzheng; Hao, Yang; Yang, Junyu; Yin, Shenyi; Sun, Changhong; Phan, John H.; Wang, May D.; Xi, Jianzhong Jeff

    2016-01-01

    Kinases regulate the majority of biological processes and become one of important groups of drug targets. To identify more kinases being potential for cancer therapy, we developed an integrative approach for the large-scale screen of functional genes capable of regulating the main traits of cancer metastasis, including cell migration as well as invasion. We first employed self-assembled cell microarray (SAMcell) to screen functional genes that regulate cancer cell migration using a siRNA library targeting 710 human genome kinase genes. We identified 81 genes capable of significantly regulating cancer cell migration. Following with invasion assays and bio-informatics analysis, we discovered that 16 genes with differentially expression in cancer samples can regulate both cell migration and invasion, among which 10 genes have been well known to play critical roles in the cancer development. The remaining 6 genes were experimentally validated to have the capacities of regulating the metastasis-related traits, including cell proliferation, apoptosis and anoikis activities besides cell motility. Together, these findings provide a new insight into the therapeutic use of human kinases. PMID:23751374

  1. Allosteric Activation of Bacterial Response Regulators: the Role of the Cognate Histidine Kinase Beyond Phosphorylation

    PubMed Central

    Trajtenberg, Felipe; Albanesi, Daniela; Ruétalo, Natalia; Botti, Horacio; Mechaly, Ariel E.; Nieves, Marcos; Aguilar, Pablo S.; Cybulski, Larisa; Larrieux, Nicole; de Mendoza, Diego

    2014-01-01

    ABSTRACT Response regulators are proteins that undergo transient phosphorylation, connecting specific signals to adaptive responses. Remarkably, the molecular mechanism of response regulator activation remains elusive, largely because of the scarcity of structural data on multidomain response regulators and histidine kinase/response regulator complexes. We now address this question by using a combination of crystallographic data and functional analyses in vitro and in vivo, studying DesR and its cognate sensor kinase DesK, a two-component system that controls membrane fluidity in Bacillus subtilis. We establish that phosphorylation of the receiver domain of DesR is allosterically coupled to two distinct exposed surfaces of the protein, controlling noncanonical dimerization/tetramerization, cooperative activation, and DesK binding. One of these surfaces is critical for both homodimerization- and kinase-triggered allosteric activations. Moreover, DesK induces a phosphorylation-independent activation of DesR in vivo, uncovering a novel and stringent level of specificity among kinases and regulators. Our results support a model that helps to explain how response regulators restrict phosphorylation by small-molecule phosphoryl donors, as well as cross talk with noncognate sensors. PMID:25406381

  2. HIPK family kinases bind and regulate the function of the CCR4-NOT complex

    PubMed Central

    Rodriguez-Gil, Alfonso; Ritter, Olesja; Hornung, Juliane; Stekman, Hilda; Krüger, Marcus; Braun, Thomas; Kremmer, Elisabeth; Kracht, Michael; Schmitz, M. Lienhard

    2016-01-01

    The serine/threonine kinase HIPK2 functions as a regulator of developmental processes and as a signal integrator of a wide variety of stress signals, such as DNA damage, hypoxia, and reactive oxygen intermediates. Because the kinase is generated in a constitutively active form, its expression levels are restricted by a variety of different mechanisms. Here we identify the CCR4-NOT complex as a new regulator of HIPK2 abundance. Down-regulation or knockout of the CCR4-NOT complex member CNOT2 leads to reduced HIPK2 protein levels without affecting the expression level of HIPK1 or HIPK3. A fraction of all HIPK family members associates with the CCR4-NOT components CNOT2 and CNOT3. HIPKs also phosphorylate the CCR4-NOT complex, a feature that is shared with their yeast progenitor kinase, YAK1. Functional assays reveal that HIPK2 and HIPK1 restrict CNOT2-dependent mRNA decay. HIPKs are well known regulators of transcription, but the mutual regulation between CCR4-NOT and HIPKs extends the regulatory potential of these kinases by enabling posttranscriptional gene regulation. PMID:27122605

  3. Spatial Regulation of Histidine Kinases Governing Biofilm Formation in Bacillus subtilis▿ †

    PubMed Central

    McLoon, Anna L.; Kolodkin-Gal, Ilana; Rubinstein, Shmuel M.; Kolter, Roberto; Losick, Richard

    2011-01-01

    Bacillus subtilis is able to form architecturally complex biofilms on solid medium due to the production of an extracellular matrix. A master regulator that controls the expression of the genes involved in matrix synthesis is Spo0A, which is activated by phosphorylation via a phosphorelay involving multiple histidine kinases. Here we report that four kinases, KinA, KinB, KinC, and KinD, help govern biofilm formation but that their contributions are partially masked by redundancy. We show that the kinases fall into two categories and that the members of each pair (one pair comprising KinA and KinB and the other comprising KinC and KinD) are partially redundant with each other. We also show that the kinases are spatially regulated: KinA and KinB are active principally in the older, inner regions of the colony, and KinC and KinD function chiefly in the younger, outer regions. These conclusions are based on the morphology of kinase mutants, real-time measurements of gene expression using luciferase as a reporter, and confocal microscopy using a fluorescent protein as a reporter. Our findings suggest that multiple signals from the older and younger regions of the colony are integrated by the kinases to determine the overall architecture of the biofilm community. PMID:21097618

  4. Brassinosteroid-regulated GSK3/Shaggy-like Kinases Phosphorylate Mitogen-activated Protein (MAP) Kinase Kinases, Which Control Stomata Development in Arabidopsis thaliana*

    PubMed Central

    Khan, Mamoona; Rozhon, Wilfried; Bigeard, Jean; Pflieger, Delphine; Husar, Sigrid; Pitzschke, Andrea; Teige, Markus; Jonak, Claudia; Hirt, Heribert; Poppenberger, Brigitte

    2013-01-01

    Brassinosteroids (BRs) are steroid hormones that coordinate fundamental developmental programs in plants. In this study we show that in addition to the well established roles of BRs in regulating cell elongation and cell division events, BRs also govern cell fate decisions during stomata development in Arabidopsis thaliana. In wild-type A. thaliana, stomatal distribution follows the one-cell spacing rule; that is, adjacent stomata are spaced by at least one intervening pavement cell. This rule is interrupted in BR-deficient and BR signaling-deficient A. thaliana mutants, resulting in clustered stomata. We demonstrate that BIN2 and its homologues, GSK3/Shaggy-like kinases involved in BR signaling, can phosphorylate the MAPK kinases MKK4 and MKK5, which are members of the MAPK module YODA-MKK4/5-MPK3/6 that controls stomata development and patterning. BIN2 phosphorylates a GSK3/Shaggy-like kinase recognition motif in MKK4, which reduces MKK4 activity against its substrate MPK6 in vitro. In vivo we show that MKK4 and MKK5 act downstream of BR signaling because their overexpression rescued stomata patterning defects in BR-deficient plants. A model is proposed in which GSK3-mediated phosphorylation of MKK4 and MKK5 enables for a dynamic integration of endogenous or environmental cues signaled by BRs into cell fate decisions governed by the YODA-MKK4/5-MPK3/6 module. PMID:23341468

  5. Protein Kinase C Regulates Ionic Conductance in Hippocampal Pyramidal Neurons: Electrophysiological Effects of Phorbol Esters

    NASA Astrophysics Data System (ADS)

    Baraban, Jay M.; Snyder, Solomon H.; Alger, Bradley E.

    1985-04-01

    The vertebrate central nervous system contains very high concentrations of protein kinase C, a calcium-and phospholipid-stimulated phosphorylating enzyme. Phorbol esters, compounds with inflammatory and tumor-promoting properties, bind to and activate this enzyme. To clarify the role of protein kinase C in neuronal function, we have localized phorbol ester receptors in the rat hippocampus by autoradiography and examined the electrophysiological effects of phorbol esters on hippocampal pyramidal neurons in vitro. Phorbol esters blocked a calcium-dependent potassium conductance. In addition, phorbol esters blocked the late hyperpolarization elicited by synaptic stimulation even though other synaptic potentials were not affected. The potencies of several phorbol esters in exerting these actions paralleled their affinities for protein kinase C, suggesting that protein kinase C regulates membrane ionic conductance.

  6. PI3 kinase regulation of skeletal muscle hypertrophy and atrophy.

    PubMed

    Glass, David J

    2010-01-01

    Activation of the PI3 kinase pathway can induce skeletal muscle hypertrophy, defined as an increase in skeletal muscle mass. In mammals, skeletal muscle hypertrophy occurs as a result of an increase in the size, as opposed to the number, of pre-existing skeletal muscle fibers. This pathway's effects on skeletal muscle have been implicated most prominently downstream of Insulin-like growth factor 1 signaling. IGF-1's pro-hypertrophy activity comes predominantly through its ability to activate the Phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Akt is a serine-threonine protein kinase that can induce protein synthesis and block the transcriptional upregulation of key mediators of skeletal muscle atrophy, the E3 ubiquitin ligases MuRF1 and MAFbx (also called Atrogin-1), by phosphorylating and thereby inhibiting the nuclear translocation of the FOXO (also called "forkhead") family of transcription factors. Once phosphorylated by Akt, the FOXOs are excluded from the nucleus, and upregulation of MuRF1 and MAFbx is blocked. MuRF1 and MAFbx mediate atrophy by ubiquitinating particular protein substrates, causing them to undergo degradation by the proteasome. MuRF1's substrates include several components of the sarcomeric thick filament, including Myosin Heavy Chain (MyHC). Thus, by blocking MuRF1 activation, IGF-1 helps prevent the breakdown of the thick filament under atrophy conditions.IGF1/PI3K/Akt signaling also can dominantly inhibit the effects of a secreted protein called "myostatin," which is a member of the TGFβ family of proteins. Deletion or inhibition of myostatin causes an increase in skeletal muscle size, because myostatin acts both to inhibit myoblast differentiation and to block the Akt pathway. Thus by blocking myostatin, PI3K/Akt activation stimulates differentiation and protein synthesis by this distinct mechanism. Myostatin induces the phosphorylation and activation of the transcription factors of Smad2 and Smad3, downstream of the Act

  7. Regulation of midbody formation and function by mitotic kinases.

    PubMed

    D'Avino, Pier Paolo; Capalbo, Luisa

    2016-05-01

    Cytokinesis is the final phase of cell division and safeguards the correct distribution of genomic and cytoplasmic materials between the two nascent daughter cells. The final separation, or abscission, of the daughter cells depends on the proper assembly of an organelle at the intercellular bridge, the midbody, which acts as a platform for the recruitment and organisation of various proteins involved in both the control and execution of the abscission process. Recent studies have led to the identification of the mechanisms, signalling pathways and molecules that control the two tightly linked processes of midbody formation and abscission. Here we review our current knowledge of the role that mitotic kinases play in these processes and offer our perspectives on the potential future challenges that await researchers in the field. PMID:26802517

  8. Tomato thymidine kinase is subject to inefficient TTP feedback regulation.

    PubMed

    Larsen, N B; Munch-Petersen, B; Piškur, J

    2014-01-01

    A promising suicide gene therapy system to treat gliomas has been reported: the thymidine kinase 1 from tomato (toTK1) combined with the nucleoside analog pro-drug zidovudine (azidothymidine, AZT), which is known to penetrate the blood-brain barrier. Transduction with toTK1 has been found to efficiently increase the sensitivity of human glioblastoma cells to AZT, and nude rats with intracranial glioblastoma grafts have shown significantly improved survival when treated with the toTK1/AZT system. We show in our paper that the strong suicidal effect of AZT together with toTK1 may be explained by reduced TTP-mediated feedback inhibition of the AZT phosphorylation. PMID:24940681

  9. The oncogenic PIM kinase family regulates drug resistance through multiple mechanisms.

    PubMed

    Isaac, Methvin; Siu, Allan; Jongstra, Jan

    2011-01-01

    Resistance to chemotherapeutic drugs is a significant clinical problem for the treatment of cancer patients and has been linked to the activation of survival pathways and expression of multidrug efflux transporters. Thus inhibition of these survival pathways or efflux transporter expression may increase the efficacy of drug treatment. Here we review the role of the oncogenic PIM kinase family in regulating important proliferation and survival pathways in cancer cells and the involvement of PIM kinases in the expression and activity of MDR-1 and BCRP, two of the most important drug efflux transporters. PIM kinases are over expressed in various types of tumors and regulate the activation of signaling pathways that are important for tumor cell proliferation, survival and expression of drug efflux proteins. This makes PIM kinases attractive targets for the development of anti-cancer chemotherapeutic drugs. Focussing mainly on solid tumors, we provide an update on the literature describing the tumorigenic functions of PIM kinases. Also we provide an overview of the development of selective small molecule PIM kinase inhibitors. Because of the intense effort by pharmaceutical companies and academia it is reasonable to expect that PIM kinase inhibitors will enter the clinic in the foreseeable future. We therefore finish this review with a discussion on the most efficient application of these PIM inhibitors. This includes a consideration of which tumor type is the most appropriate target for treatment, how to select the patient population that stands to gain the most from treatment with PIM inhibitors, which molecular markers are suitable to follow the course of treatment and whether PIM kinase inhibitors should be used as monotherapy or in combination with other cytotoxic agents. PMID:21601509

  10. Targeting Abl Kinases to Regulate Vascular Leak During Sepsis and ARDS

    PubMed Central

    Rizzo, Alicia N.; Aman, Jurjan; van Nieuw Amerongen, Geerten P.; Dudek, Steven M.

    2015-01-01

    The vascular endothelium separates circulating fluid and inflammatory cells from the surrounding tissues. Vascular leak occurs in response to wide-spread inflammatory processes, such as sepsis and Acute Respiratory Distress Syndrome (ARDS), due to the formation of gaps between endothelial cells (EC). Although these disorders are leading causes of mortality in the ICU, no medical therapies exist to restore EC barrier function. Recent evidence highlights a key role for the Abl family of non-receptor tyrosine kinases in regulating vascular barrier integrity. These kinases have well-described roles in cancer progression and neuronal morphogenesis, but their functions in the vasculature have remained enigmatic until recently. The Abl family kinases, c-Abl (Abl1) and Abl related gene (Arg, Abl2), phosphorylate several cytoskeletal effectors that mediate vascular permeability, including myosin light chain kinase, cortactin, vinculin, and β-catenin. They also regulate cell-cell and cell-matrix junction dynamics, and the formation of actin-based cellular protrusions in multiple cell types. Additionally, both c-Abl and Arg are activated by hyperoxia and contribute to oxidant-induced EC injury. These numerous roles of Abl kinases in EC and the current clinical usage of imatinib and other Abl kinase inhibitors have spurred recent interest in repurposing these drugs for the treatment of vascular barrier dysfunction. This review will describe the structure and function of Abl kinases with an emphasis on their roles in mediating vascular barrier integrity. We will also provide a critical evaluation of the potential for exploiting Abl kinase inhibition as a novel therapy for inflammatory vascular leak syndromes. PMID:25814671

  11. Protein kinase B and extracellular signal-regulated kinase contribute to the chondroprotective effect of morroniside on osteoarthritis chondrocytes

    PubMed Central

    Cheng, Liang; Zeng, Guoqing; Liu, Zejun; Zhang, Bing; Cui, Xu; Zhao, Honghai; Zheng, Xinpeng; Song, Gang; Kang, Jian; Xia, Chun

    2015-01-01

    Despite extensive studies on the multifaceted roles of morroniside, the main active constituent of iridoid glycoside from Corni Fructus, the effect of morroniside on osteoarthritis (OA) chondrocytes remains poorly understood. Here, we investigated the influence of morroniside on cultured human OA chondrocytes and a rat experimental model of OA. The results showed that morroniside enhanced the cell viability and the levels of proliferating cell nuclear antigen expression (PCNA), type II collagen and aggrecan in human OA chondrocytes, indicating that morroniside promoted chondrocyte survival and matrix synthesis. Furthermore, different doses of morroniside activated protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) in human OA chondrocytes, and in turn, triggered AKT/S6 and ERK/P70S6K/S6 pathway, respectively. The PI3K/AKT inhibitor LY294002 or the MEK/ERK inhibitor U0126 attenuated the effect of morroniside on human OA chondrocytes, indicating that the activation of AKT and ERK contributed to the regulation of morroniside in human OA chondrocytes. In addition, the intra-articular injection of morroniside elevated the level of proteoglycans in cartilage matrix and the thickness of articular cartilage in a rat experimental model of OA, with the increase of AKT and ERK activation. As a consequence, morroniside has chondroprotective effect on OA chondrocytes, and may have the therapeutic potential for OA treatment. PMID:25754021

  12. Cocaine regulates protein kinase B and glycogen synthase kinase-3 activity in selective regions of rat brain

    PubMed Central

    SA, Perrine; JS, Miller; EM, Unterwald

    2008-01-01

    Protein kinase B (Akt) signaling regulates dopamine-mediated locomotor behaviors. Here the ability of cocaine to regulate Akt and glycogen synthase kinase-3 (GSK3) was studied. Rats were injected with cocaine or saline in a binge-pattern, which consisted of 3 daily injections of 15 mg/kg cocaine or 1 ml/kg saline spaced one hour apart for 1, 3 or 14 days. Amygdala, nucleus accumbens, caudate putamen and hippocampus tissues were dissected 30 minutes following the last injection and analyzed for phosphorylated and total Akt and GSK3(α & β) protein levels using Western blot analysis. Phosphorylation of Akt on the threonine-308 residue was significantly reduced in the nucleus accumbens and increased in the amygdala after 1 day of cocaine treatment; however, these effects were not accompanied by a significant decrease in GSK3 phosphorylation. Phosphorylation of Akt and GSK3 were significantly reduced after 14 days of cocaine administration, an effect that was only observed in the amygdala. Cocaine did not alter Akt or GSK3 phosphorylation in the caudate putamen or hippocampus. The findings in nucleus accumbens may reflect dopaminergic motor-stimulant activity caused by acute cocaine, whereas the effects in amygdala may be associated with changes in emotional state that occur after acute and chronic cocaine exposure. PMID:18717814

  13. Protein kinase B and extracellular signal-regulated kinase contribute to the chondroprotective effect of morroniside on osteoarthritis chondrocytes.

    PubMed

    Cheng, Liang; Zeng, Guoqing; Liu, Zejun; Zhang, Bing; Cui, Xu; Zhao, Honghai; Zheng, Xinpeng; Song, Gang; Kang, Jian; Xia, Chun

    2015-08-01

    Despite extensive studies on the multifaceted roles of morroniside, the main active constituent of iridoid glycoside from Corni Fructus, the effect of morroniside on osteoarthritis (OA) chondrocytes remains poorly understood. Here, we investigated the influence of morroniside on cultured human OA chondrocytes and a rat experimental model of OA. The results showed that morroniside enhanced the cell viability and the levels of proliferating cell nuclear antigen expression (PCNA), type II collagen and aggrecan in human OA chondrocytes, indicating that morroniside promoted chondrocyte survival and matrix synthesis. Furthermore, different doses of morroniside activated protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) in human OA chondrocytes, and in turn, triggered AKT/S6 and ERK/P70S6K/S6 pathway, respectively. The PI3K/AKT inhibitor LY294002 or the MEK/ERK inhibitor U0126 attenuated the effect of morroniside on human OA chondrocytes, indicating that the activation of AKT and ERK contributed to the regulation of morroniside in human OA chondrocytes. In addition, the intra-articular injection of morroniside elevated the level of proteoglycans in cartilage matrix and the thickness of articular cartilage in a rat experimental model of OA, with the increase of AKT and ERK activation. As a consequence, morroniside has chondroprotective effect on OA chondrocytes, and may have the therapeutic potential for OA treatment. PMID:25754021

  14. Glycogen Synthase Kinase 3β Is Positively Regulated by Protein Kinase Cζ-Mediated Phosphorylation Induced by Wnt Agonists

    PubMed Central

    Tejeda-Muñoz, Nydia; González-Aguilar, Héctor; Santoyo-Ramos, Paula; Castañeda-Patlán, M. Cristina

    2015-01-01

    The molecular events that drive Wnt-induced regulation of glycogen synthase kinase 3β (GSK-3β) activity are poorly defined. In this study, we found that protein kinase Cζ (PKCζ) and GSK-3β interact mainly in colon cancer cells. Wnt stimulation induced a rapid GSK-3β redistribution from the cytoplasm to the nuclei in malignant cells and a transient PKC-mediated phosphorylation of GSK-3β at a different site from serine 9. In addition, while Wnt treatment induced a decrease in PKC-mediated phosphorylation of GSK-3β in nonmalignant cells, in malignant cells, this phosphorylation was increased. Pharmacological inhibition and small interfering RNA (siRNA)-mediated silencing of PKCζ abolished all of these effects, but unexpectedly, it also abolished the constitutive basal activity of GSK-3β. In vitro activity assays demonstrated that GSK-3β phosphorylation mediated by PKCζ enhanced GSK-3β activity. We mapped Ser147 of GSK-3β as the site phosphorylated by PKCζ, i.e., its mutation into alanine abolished GSK-3β activity, resulting in β-catenin stabilization and increased transcriptional activity, whereas phosphomimetic replacement of Ser147 by glutamic acid maintained GSK-3β basal activity. Thus, we found that PKCζ phosphorylates GSK-3β at Ser147 to maintain its constitutive activity in resting cells and that Wnt stimulation modifies the phosphorylation of Ser147 to regulate GSK-3β activity in opposite manners in normal and malignant colon cells. PMID:26711256

  15. G Protein-coupled Receptor Kinase 5 Phosphorylates Nucleophosmin and Regulates Cell Sensitivity to Polo-like Kinase 1 Inhibition*

    PubMed Central

    So, Christopher H.; Michal, Allison M.; Mashayekhi, Rouzbeh; Benovic, Jeffrey L.

    2012-01-01

    G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, leading to their desensitization and endocytosis. GRKs have also been implicated in phosphorylating other classes of proteins and can localize in a variety of cellular compartments, including the nucleus. Here, we attempted to identify potential nuclear substrates for GRK5. Our studies reveal that GRK5 is able to interact with and phosphorylate nucleophosmin (NPM1) both in vitro and in intact cells. NPM1 is a nuclear protein that regulates a variety of cell functions including centrosomal duplication, cell cycle control, and apoptosis. GRK5 interaction with NPM1 is mediated by the N-terminal domain of each protein, and GRK5 primarily phosphorylates NPM1 at Ser-4, a site shared with polo-like kinase 1 (PLK1). NPM1 phosphorylation by GRK5 and PLK1 correlates with the sensitivity of cells to undergo apoptosis with cells having higher GRK5 levels being less sensitive and cells with lower GRK5 being more sensitive to PLK1 inhibitor-induced apoptosis. Taken together, our results demonstrate that GRK5 phosphorylates Ser-4 in nucleophosmin and regulates the sensitivity of cells to PLK1 inhibition. PMID:22467873

  16. How do kinases contribute to tonicity-dependent regulation of the transcription factor NFAT5?

    PubMed Central

    Zhou, Xiaoming

    2016-01-01

    NFAT5 plays a critical role in maintaining the renal functions. Its dis-regulation in the kidney leads to or is associated with certain renal diseases or disorders, most notably the urinary concentration defect. Hypertonicity, which the kidney medulla is normally exposed to, activates NFAT5 through phosphorylation of a signaling molecule or NFAT5 itself. Hypotonicity inhibits NFAT5 through a similar mechanism. More than a dozen of protein and lipid kinases have been identified to contribute to tonicity-dependent regulation of NFAT5. Hypertonicity activates NFAT5 by increasing its nuclear localization and transactivating activity in the early phase and protein abundance in the late phase. The known mechanism for inhibition of NFAT5 by hypotonicity is a decrease of nuclear NFAT5. The present article reviews the effect of each kinase on NFAT5 nuclear localization, transactivation and protein abundance, and the relationship among these kinases, if known. Cyclosporine A and tacrolimus suppress immune reactions by inhibiting the phosphatase calcineurin-dependent activation of NFAT1. It is hoped that this review would stimulate the interest to seek explanations from the NFAT5 regulatory pathways for certain clinical presentations and to explore novel therapeutic approaches based on the pathways. On the basic science front, this review raises two interesting questions. The first one is how these kinases can specifically signal to NFAT5 in the context of hypertonicity or hypotonicity, because they also regulate other cellular activities and even opposite activities in some cases. The second one is why these many kinases, some of which might have redundant functions, are needed to regulate NFAT5 activity. This review reiterates the concept of signaling through cooperation. Cells need these kinases working in a coordinated way to provide the signaling specificity that is lacking in the individual one. Redundancy in regulation of NFAT5 is a critical strategy for cells to

  17. Mitogen-Activated Protein Kinase Phosphatase 2 Regulates the Inflammatory Response in Sepsis▿

    PubMed Central

    Cornell, Timothy T.; Rodenhouse, Paul; Cai, Qing; Sun, Lei; Shanley, Thomas P.

    2010-01-01

    Sepsis results from a dysregulation of the regulatory mechanisms of the pro- and anti-inflammatory response to invading pathogens. The mitogen-activated protein (MAP) kinase cascades are key signal transduction pathways involved in the cellular production of cytokines. The dual-specific phosphatase 1 (DUSP 1), mitogen-activated protein kinase phosphatase-1 (MKP-1), has been shown to be an important negative regulator of the inflammatory response by regulating the p38 and Jun N-terminal protein kinase (JNK) MAP kinase pathways to influence pro- and anti-inflammatory cytokine production. MKP-2, also a dual-specific phosphatase (DUSP 4), is a phosphatase highly homologous with MKP-1 and is known to regulate MAP kinase signaling; however, its role in regulating the inflammatory response is not known. We hypothesized a regulatory role for MKP-2 in the setting of sepsis. Mice lacking the MKP-2 gene had a survival advantage over wild-type mice when challenged with intraperitoneal lipopolysaccharide (LPS) or a polymicrobial infection via cecal ligation and puncture. The MKP-2−/− mice also exhibited decreased serum levels of both pro-inflammatory cytokines (tumor necrosis factor alpha [TNF-α], interleukin-1β [IL-1β], IL-6) and anti-inflammatory cytokines (IL-10) following endotoxin challenge. Isolated bone marrow-derived macrophages (BMDMs) from MKP-2−/− mice showed increased phosphorylation of the extracellular signal-regulated kinase (ERK), decreased phosphorylation of JNK and p38, and increased induction of MKP-1 following LPS stimulation. The capacity for cytokine production increased in MKP-2−/− BMDMs following MKP-1 knockdown. These data support a mechanism by which MKP-2 targets ERK deactivation, thereby decreasing MKP-1 and thus removing the negative inhibition of MKP-1 on cytokine production. PMID:20351138

  18. Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases

    NASA Technical Reports Server (NTRS)

    Boo, Yong Chool; Jo, Hanjoong

    2003-01-01

    Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca2+/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases-protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases-are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation.

  19. Neuron-astrocyte signaling and epilepsy.

    PubMed

    Seifert, Gerald; Steinhäuser, Christian

    2013-06-01

    Astrocytes express a plethora of ion channels, neurotransmitter receptors and transporters and thus are endowed with the machinery to sense and respond to neuronal activity. Recent studies have implicated astrocytes in important physiological roles in the CNS, such as synchronization of neuronal firing, ion homeostasis, neurotransmitter uptake, glucose metabolism and regulation of the vascular tone. Astrocytes are abundantly coupled through gap junctions allowing them to redistribute elevated K(+) concentration from sites of excessive neuronal activity. Growing evidence now suggests that dysfunctional astrocytes are crucial players in epilepsy. Investigation of specimens from patients with pharmacoresistant temporal lobe epilepsy and epilepsy models revealed alterations in expression, localization and function of astroglial K(+) and water channels, entailing impaired K(+) buffering. Moreover, malfunction of glutamate transporters and the astrocytic glutamate-converting enzyme, glutamine synthetase, as observed in epileptic tissue suggested that astrocyte dysfunction is causative of hyperexcitation, seizure spread and neurotoxicity. Accordingly, dysfunctional astrocytes should be considered as promising targets for new therapeutic strategies. In this review, we will summarize current knowledge of astrocyte dysfunction in temporal lobe epilepsy and discuss putative mechanisms underlying these alterations. PMID:21925173

  20. Retinoic acid isomers up-regulate ATP binding cassette A1 and G1 and cholesterol efflux in rat astrocytes: implications for their therapeutic and teratogenic effects.

    PubMed

    Chen, Jing; Costa, Lucio G; Guizzetti, Marina

    2011-09-01

    Recent studies suggest that retinoids may be effective in the treatment of Alzheimer's disease, although exposure to an excess of retinoids during gestation causes teratogenesis. Cholesterol is essential for brain development, but high levels of cholesterol have been associated with Alzheimer's disease. We hypothesized that retinoic acid may affect cholesterol homeostasis in rat astrocytes, which regulate cholesterol distribution in the brain, through the up-regulation of cholesterol transporters ATP binding cassette (Abc)a1 and Abcg1. Tretinoin, 13-cis retinoic acid (13-cis-RA), 9-cis-RA, and the selective retinoid X receptor (RXR) agonist methoprene significantly increased cholesterol efflux induced by cholesterol acceptors and protein levels of Abca1 by 2.3- (± 0.25), 3.6- (± 0.42), 4.1- (± 0.5), and 1.75- (± 0.43) fold, respectively, and Abcg1 by 2.1- (± 0.26), 2.2- (± 0.33), 2.5- (± 0.23), and 2.2- (± 0.21) fold, respectively. 13-cis-RA and 9-cis-RA also significantly increased mRNA levels of Abca1 (maximal induction 7.3 ± 0.42 and 2.7 ± 0.17, respectively) and Abcg1 (maximal induction 2.0 ± 0.18 and 1.8 ± 0.09, respectively), and the levels of membrane-bound Abca1 (2.5 ± 0.3 and 2.5 ± 0.40-fold increase, respectively), whereas they significantly decreased intracellular cholesterol content without affecting cholesterol synthesis. The effect of 9-cis-RA on cholesterol homeostasis in astrocytes can be ascribed to the activation of RXR, whereas the effects of 13-cis-RA and tretinoin were independent of either RXRs or retinoic acid receptors. These findings suggest that retinoids affect cholesterol homeostasis in astrocytes and that this effect may be involved in both their therapeutic and teratogenic actions. PMID:21628419

  1. Insulin Receptor Substrate 2-mediated Phosphatidylinositol 3-kinase Signaling Selectively Inhibits Glycogen Synthase Kinase 3β to Regulate Aerobic Glycolysis*

    PubMed Central

    Landis, Justine; Shaw, Leslie M.

    2014-01-01

    Insulin receptor substrate 1 (IRS-1) and IRS-2 are cytoplasmic adaptor proteins that mediate the activation of signaling pathways in response to ligand stimulation of upstream cell surface receptors. Despite sharing a high level of homology and the ability to activate PI3K, only Irs-2 positively regulates aerobic glycolysis in mammary tumor cells. To determine the contribution of Irs-2-dependent PI3K signaling to this selective regulation, we generated an Irs-2 mutant deficient in the recruitment of PI3K. We identified four tyrosine residues (Tyr-649, Tyr-671, Tyr-734, and Tyr-814) that are essential for the association of PI3K with Irs-2 and demonstrate that combined mutation of these tyrosines inhibits glucose uptake and lactate production, two measures of aerobic glycolysis. Irs-2-dependent activation of PI3K regulates the phosphorylation of specific Akt substrates, most notably glycogen synthase kinase 3β (Gsk-3β). Inhibition of Gsk-3β by Irs-2-dependent PI3K signaling promotes glucose uptake and aerobic glycolysis. The regulation of unique subsets of Akt substrates by Irs-1 and Irs-2 may explain their non-redundant roles in mammary tumor biology. Taken together, our study reveals a novel mechanism by which Irs-2 signaling preferentially regulates tumor cell metabolism and adds to our understanding of how this adaptor protein contributes to breast cancer progression. PMID:24811175

  2. Phosphorylation of FEZ1 by Microtubule Affinity Regulating Kinases regulates its function in presynaptic protein trafficking

    PubMed Central

    Butkevich, Eugenia; Härtig, Wolfgang; Nikolov, Miroslav; Erck, Christian; Grosche, Jens; Urlaub, Henning; Schmidt, Christoph F.; Klopfenstein, Dieter R.; Chua, John Jia En

    2016-01-01

    Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated intracellular transport. The regulatory mechanisms governing adapter functions and the spectrum of cargoes recognized by individual adapters remain poorly defined. Here, we show that cargoes transported by the Kinesin-1 adapter FEZ1 are enriched for presynaptic components and identify that specific phosphorylation of FEZ1 at its serine 58 regulatory site is mediated by microtubule affinity-regulating kinases (MARK/PAR-1). Loss of MARK/PAR-1 impairs axonal transport, with adapter and cargo abnormally co-aggregating in neuronal cell bodies and axons. Presynaptic specializations are markedly reduced and distorted in FEZ1 and MARK/PAR-1 mutants. Strikingly, abnormal co-aggregates of unphosphorylated FEZ1, Kinesin-1 and its putative cargoes are present in brains of transgenic mice modelling aspects of Alzheimer’s disease, a neurodegenerative disorder exhibiting impaired axonal transport and altered MARK activity. Our findings suggest that perturbed FEZ1-mediated synaptic delivery of proteins arising from abnormal signalling potentially contributes to the process of neurodegeneration. PMID:27247180

  3. Phosphorylation of FEZ1 by Microtubule Affinity Regulating Kinases regulates its function in presynaptic protein trafficking.

    PubMed

    Butkevich, Eugenia; Härtig, Wolfgang; Nikolov, Miroslav; Erck, Christian; Grosche, Jens; Urlaub, Henning; Schmidt, Christoph F; Klopfenstein, Dieter R; Chua, John Jia En

    2016-01-01

    Adapters bind motor proteins to cargoes and therefore play essential roles in Kinesin-1 mediated intracellular transport. The regulatory mechanisms governing adapter functions and the spectrum of cargoes recognized by individual adapters remain poorly defined. Here, we show that cargoes transported by the Kinesin-1 adapter FEZ1 are enriched for presynaptic components and identify that specific phosphorylation of FEZ1 at its serine 58 regulatory site is mediated by microtubule affinity-regulating kinases (MARK/PAR-1). Loss of MARK/PAR-1 impairs axonal transport, with adapter and cargo abnormally co-aggregating in neuronal cell bodies and axons. Presynaptic specializations are markedly reduced and distorted in FEZ1 and MARK/PAR-1 mutants. Strikingly, abnormal co-aggregates of unphosphorylated FEZ1, Kinesin-1 and its putative cargoes are present in brains of transgenic mice modelling aspects of Alzheimer's disease, a neurodegenerative disorder exhibiting impaired axonal transport and altered MARK activity. Our findings suggest that perturbed FEZ1-mediated synaptic delivery of proteins arising from abnormal signalling potentially contributes to the process of neurodegeneration. PMID:27247180

  4. Production of recombinant human apoptosis signal-regulating kinase 1 (ASK1) in Escherichia coli.

    PubMed

    Volynets, Galyna P; Gorbatiuk, Oksana B; Kukharenko, Oleksandr P; Usenko, Mariya O; Yarmoluk, Sergiy M

    2016-10-01

    Apoptosis signal-regulating kinase 1 (ASK1) is a mediator of the MAPK signaling cascade, which regulates different cellular processes including apoptosis, cell survival, and differentiation. The increased activity of ASK1 is associated with a number of human diseases and this protein kinase is considered as promising therapeutic target. In the present study, the kinase domain of human ASK1 was expressed in Escherichia coli (E. coli) in soluble form. The expression level of ASK1 was around 0.3-0.47 g per 1 L after using auto-induction protocol or IPTG induction. A one-step on column method for the efficient purification of recombinant ASK1 was performed. Our approach yields sufficient amount of recombinant ASK1, which can be used for inhibitor screening assays and different crystallographic studies. PMID:27245507

  5. Freshly dissociated mature hippocampal astrocytes exhibit passive membrane conductance and low membrane resistance similarly to syncytial coupled astrocytes

    PubMed Central

    Du, Yixing; Ma, Baofeng; Kiyoshi, Conrad M.; Alford, Catherine C.; Wang, Wei

    2015-01-01

    Mature astrocytes exhibit a linear current-to-voltage K+ membrane conductance (passive conductance) and an extremely low membrane resistance (Rm) in situ. The combination of these electrophysiological characteristics establishes a highly negative and stable membrane potential that is essential for basic functions, such as K+ spatial buffering and neurotransmitter uptake. However, astrocytes are coupled extensively in situ. It remains to be determined whether the observed passive behavior and low Rm are attributable to the intrinsic properties of membrane ion channels or to gap junction coupling in functionally mature astrocytes. In the present study, freshly dissociated hippocampal tissues were used as a new model to examine this basic question in young adult animals. The morphologically intact single astrocytes could be reliably dissociated from animals postnatal day 21 and older. At this animal age, dissociated single astrocytes exhibit passive conductance and resting membrane potential similar to those exhibited by astrocytes in situ. To precisely measure the Rm from single astrocytes, dual-patch single-astrocyte recording was performed. We show that dissociated single astrocytes exhibit a low Rm similarly to syncytial coupled astrocytes. Functionally, the symmetric expression of high-K+ conductance enabled rapid change in the intracellular K+ concentrations in response to changing K+ drive force. Altogether, we demonstrate that freshly dissociated tissue preparation is a highly useful model for study of the functional expression and regulation of ion channels, receptors, and transporters in astrocytes and that passive behavior and low Rm are the intrinsic properties of mature astrocytes. PMID:25810481

  6. Phosphoinositide 3-kinase dependent regulation of Kv channels in dendritic cells.

    PubMed

    Shumilina, Ekaterina; Zahir, Naima; Xuan, Nguyen Thi; Lang, Florian

    2007-01-01

    The phosphoinositide 3 (PI3) kinase plays a pivotal role in the regulation of dendritic cells (DCs), antigen-presenting cells that are able to initiate primary immune responses and to establish immunological memory. PI3 kinase is an endogenous suppressor of interleukin 12 (IL-12) production in DCs that is triggered by Toll-like receptor signaling. Inhibition of IL-12 production limits T helper 1 (Th1) polarization. On the other hand, PI3 kinase is an important regulator of various ion channels. The present study aimed to explore whether ion channels in DCs are regulated by PI3 kinase and whether they are important for DC function. To this end, DCs were isolated from murine bone marrow and ion channel activity was determined by patch clamp. As a result, DCs express voltage-gated K(+) channels (Kv), which are blocked by Stichodactyla helianthus toxin (ShK, 2.5 nM). A significant upregulation of Kv currents was observed upon maturation of DCs as induced by stimulation of the cells with lipopolysaccharide (LPS, 0.1 microg/ml, 48 h). A dramatic increase of Kv current amplitude was observed following preincubation of the cells with LY294002 (100 nM), a specific inhibitor of PI3 kinase. PI3 kinase inhibitor wortmannin (100 nM) similarly increased Kv current. LY294002 treatment was further followed by a significant increase of IL-12 production. ShK (100 nM) significantly blunted the stimulation of IL-12 release by LPS but not when the cells were first pretreated with LY294002. The observations point to Kv channel sensitive and Kv channel insensitive regulation of DC function. PMID:17982262

  7. Regulation of Mammalian Cone Phototransduction by Recoverin and Rhodopsin Kinase*

    PubMed Central

    Sakurai, Keisuke; Chen, Jeannie; Khani, Shahrokh C.; Kefalov, Vladimir J.

    2015-01-01

    Cone photoreceptors function under daylight conditions and are essential for color perception and vision with high temporal and spatial resolution. A remarkable feature of cones is that, unlike rods, they remain responsive in bright light. In rods, light triggers a decline in intracellular calcium, which exerts a well studied negative feedback on phototransduction that includes calcium-dependent inhibition of rhodopsin kinase (GRK1) by recoverin. Rods and cones share the same isoforms of recoverin and GRK1, and photoactivation also triggers a calcium decline in cones. However, the molecular mechanisms by which calcium exerts negative feedback on cone phototransduction through recoverin and GRK1 are not well understood. Here, we examined this question using mice expressing various levels of GRK1 or lacking recoverin. We show that although GRK1 is required for the timely inactivation of mouse cone photoresponse, gradually increasing its expression progressively delays the cone response recovery. This surprising result is in contrast with the known effect of increasing GRK1 expression in rods. Notably, the kinetics of cone responses converge and become independent of GRK1 levels for flashes activating more than ∼1% of cone pigment. Thus, mouse cone response recovery in bright light is independent of pigment phosphorylation and likely reflects the spontaneous decay of photoactivated visual pigment. We also find that recoverin potentiates the sensitivity of cones in dim light conditions but does not contribute to their capacity to function in bright light. PMID:25673692

  8. Identification of Extracellular Signal-regulated Kinase 1 (ERK1) Direct Substrates using Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics*

    PubMed Central

    Xue, Liang; Wang, Pengcheng; Cao, Pianpian; Zhu, Jian-kang; Tao, W. Andy

    2014-01-01

    Kinase mediated phosphorylation signaling is extensively involved in cellular functions and human diseases, and unraveling phosphorylation networks requires the identification of substrates targeted by kinases, which has remained challenging. We report here a novel proteomic strategy to identify the specificity and direct substrates of kinases by coupling phosphoproteomics with a sensitive stable isotope labeled kinase reaction. A whole cell extract was moderately dephosphorylated and subjected to in vitro kinase reaction under the condition in which 18O-ATP is the phosphate donor. The phosphorylated proteins are then isolated and identified by mass spectrometry, in which the heavy phosphate (+85.979 Da) labeled phosphopeptides reveal the kinase specificity. The in vitro phosphorylated proteins with heavy phosphates are further overlapped with in vivo kinase-dependent phosphoproteins for the identification of direct substrates with high confidence. The strategy allowed us to identify 46 phosphorylation sites on 38 direct substrates of extracellular signal-regulated kinase 1, including multiple known substrates and novel substrates, highlighting the ability of this high throughput method for direct kinase substrate screening. PMID:25022875

  9. Homeodomain-interacting protein kinase 2 is the ionizing radiation-activated p53 serine 46 kinase and is regulated by ATM.

    PubMed

    Dauth, Ilka; Krüger, Jana; Hofmann, Thomas G

    2007-03-01

    Phosphorylation of p53 at Ser(46) is important to activate the apoptotic program. The protein kinase that phosphorylates p53 Ser(46) in response to DNA double-strand breaks is currently unknown. The identification of this kinase is of particular interest because it may contribute to the outcome of cancer therapy. Here, we report that ionizing radiation (IR) provokes homeodomain-interacting protein kinase 2 (HIPK2) accumulation, activation, and complex formation with p53. IR-induced HIPK2 up-regulation strictly correlates with p53 Ser(46) phosphorylation. Down-regulation of HIPK2 by RNA interference specifically inhibits IR-induced phosphorylation of p53 at Ser(46). Moreover, we show that HIPK2 activation after IR is regulated by the DNA damage checkpoint kinase ataxia telangiectasia mutated (ATM). Cells from ataxia telangiectasia patients show defects in HIPK2 accumulation. Concordantly, IR-induced HIPK2 accumulation is blocked by pharmacologic inhibition of ATM. Furthermore, ATM down-regulation by RNA interference inhibited IR-induced HIPK2 accumulation, whereas checkpoint kinase 2 deficiency showed no effect. Taken together, our findings indicate that HIPK2 is the IR-activated p53 Ser(46) kinase and is regulated by ATM. PMID:17332358

  10. Structure of DNA-dependent protein kinase: implications for its regulation by DNA.

    PubMed

    Leuther, K K; Hammarsten, O; Kornberg, R D; Chu, G

    1999-03-01

    DNA double-strand breaks are created by ionizing radiation or during V(D)J recombination, the process that generates immunological diversity. Breaks are repaired by an end-joining reaction that requires DNA-PKCS, the catalytic subunit of DNA-dependent protein kinase. DNA-PKCS is a 460 kDa serine-threonine kinase that is activated by direct interaction with DNA. Here we report its structure at 22 A resolution, as determined by electron crystallography. The structure contains an open channel, similar to those seen in other double-stranded DNA-binding proteins, and an enclosed cavity with three openings large enough to accommodate single-stranded DNA, with one opening adjacent to the open channel. Based on these structural features, we performed biochemical experiments to examine the interactions of DNA-PKCS with different DNA molecules. Efficient kinase activation required DNA longer than 12 bp, the minimal length of the open channel. Competition experiments demonstrated that DNA-PKCS binds to double- and single-stranded DNA via separate but interacting sites. Addition of unpaired single strands to a double-stranded DNA fragment stimulated kinase activation. These results suggest that activation of the kinase involves interactions with both double- and single-stranded DNA, as suggested by the structure. A model for how the kinase is regulated by DNA is described. PMID:10064579

  11. Structural Basis of Human p70 Ribosomal S6 Kinase-1 Regulation by Activation Loop Phosphorylation

    SciTech Connect

    Sunami, Tomoko; Byrne, Noel; Diehl, Ronald E.; Funabashi, Kaoru; Hall, Dawn L.; Ikuta, Mari; Patel, Sangita B.; Shipman, Jennifer M.; Smith, Robert F.; Takahashi, Ikuko; Zugay-Murphy, Joan; Iwasawa, Yoshikazu; Lumb, Kevin J.; Munshi, Sanjeev K.; Sharma, Sujata

    2010-03-04

    p70 ribosomal S6 kinase (p70S6K) is a downstream effector of the mTOR signaling pathway involved in cell proliferation, cell growth, cell-cycle progression, and glucose homeostasis. Multiple phosphorylation events within the catalytic, autoinhibitory, and hydrophobic motif domains contribute to the regulation of p70S6K. We report the crystal structures of the kinase domain of p70S6K1 bound to staurosporine in both the unphosphorylated state and in the 3{prime}-phosphoinositide-dependent kinase-1-phosphorylated state in which Thr-252 of the activation loop is phosphorylated. Unphosphorylated p70S6K1 exists in two crystal forms, one in which the p70S6K1 kinase domain exists as a monomer and the other as a domain-swapped dimer. The crystal structure of the partially activated kinase domain that is phosphorylated within the activation loop reveals conformational ordering of the activation loop that is consistent with a role in activation. The structures offer insights into the structural basis of the 3{prime}-phosphoinositide-dependent kinase-1-induced activation of p70S6K and provide a platform for the rational structure-guided design of specific p70S6K inhibitors.

  12. Phosphorylation of Src by phosphoinositide 3-kinase regulates beta-adrenergic receptor-mediated EGFR transactivation.

    PubMed

    Watson, Lewis J; Alexander, Kevin M; Mohan, Maradumane L; Bowman, Amber L; Mangmool, Supachoke; Xiao, Kunhong; Naga Prasad, Sathyamangla V; Rockman, Howard A

    2016-10-01

    β2-Adrenergic receptors (β2AR) transactivate epidermal growth factor receptors (EGFR) through formation of a β2AR-EGFR complex that requires activation of Src to mediate signaling. Here, we show that both lipid and protein kinase activities of the bifunctional phosphoinositide 3-kinase (PI3K) enzyme are required for β2AR-stimulated EGFR transactivation. Mechanistically, the generation of phosphatidylinositol (3,4,5)-tris-phosphate (PIP3) by the lipid kinase function stabilizes β2AR-EGFR complexes while the protein kinase activity of PI3K regulates Src activation by direct phosphorylation. The protein kinase activity of PI3K phosphorylates serine residue 70 on Src to enhance its activity and induce EGFR transactivation following βAR stimulation. This newly identified function for PI3K, whereby Src is a substrate for the protein kinase activity of PI3K, is of importance since Src plays a key role in pathological and physiological signaling. PMID:27169346

  13. Nercc1, a mammalian NIMA-family kinase, binds the Ran GTPase and regulates mitotic progression

    PubMed Central

    Roig, Joan; Mikhailov, Alexei; Belham, Christopher; Avruch, Joseph

    2002-01-01

    The protein kinase NIMA is an indispensable pleiotropic regulator of mitotic progression in Aspergillus. Although several mammalian NIMA-like kinases (Neks) are known, none appears to have the broad importance for mitotic regulation attributed to NIMA. Nercc1 is a new NIMA-like kinase that regulates chromosome alignment and segregation in mitosis. Its NIMA-like catalytic domain is followed by a noncatalytic tail containing seven repeats homologous to those of the Ran GEF, RCC1, a Ser/Thr/Pro-rich segment, and a coiled-coil domain. Nercc1 binds to another NIMA-like kinase, Nek6, and also binds specifically to the Ran GTPase through both its catalytic and its RCC1-like domains, preferring RanGDP in vivo. Nercc1 exists as a homooligomer and can autoactivate in vitro by autophosphorylation. Nercc1 is a cytoplasmic protein that is activated during mitosis and is avidly phosphorylated by active p34Cdc2. Microinjection of anti-Nercc1 antibodies in prophase results in spindle abnormalities and/or chromosomal misalignment. In Ptk2 cells the outcome is prometaphase arrest or aberrant chromosome segregation and aneuploidy, whereas in CFPAC-1 cells prolonged arrest in prometaphase is the usual response. Nercc1 and its partner Nek6 represent a new signaling pathway that regulates mitotic progression. PMID:12101123

  14. Cellular context–mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis

    PubMed Central

    Hellesøy, Monica; Lorens, James B.

    2015-01-01

    The formation of new blood vessels by sprouting angiogenesis is tightly regulated by contextual cues that affect angiogeneic growth factor signaling. Both constitutive activation and loss of Akt kinase activity in endothelial cells impair angiogenesis, suggesting that Akt dynamics mediates contextual microenvironmental regulation. We explored the temporal regulation of Akt in endothelial cells during formation of capillary-like networks induced by cell–cell contact with vascular smooth muscle cells (vSMCs) and vSMC-associated VEGF. Expression of constitutively active Akt1 strongly inhibited network formation, whereas hemiphosphorylated Akt1 epi-alleles with reduced kinase activity had an intermediate inhibitory effect. Conversely, inhibition of Akt signaling did not affect endothelial cell migration or morphogenesis in vSMC cocultures that generate capillary-like structures. We found that endothelial Akt activity is transiently blocked by proteasomal degradation in the presence of SMCs during the initial phase of capillary-like structure formation. Suppressed Akt activity corresponded to the increased endothelial MAP kinase signaling that was required for angiogenic endothelial morphogenesis. These results reveal a regulatory principle by which cellular context regulates Akt protein dynamics, which determines MAP kinase signaling thresholds necessary drive a morphogenetic program during angiogenesis. PMID:26023089

  15. Impact of Serine/Threonine Protein Kinases on the Regulation of Sporulation in Bacillus subtilis

    PubMed Central

    Pompeo, Frédérique; Foulquier, Elodie; Galinier, Anne

    2016-01-01

    Bacteria possess many kinases that catalyze phosphorylation of proteins on diverse amino acids including arginine, cysteine, histidine, aspartate, serine, threonine, and tyrosine. These protein kinases regulate different physiological processes in response to environmental modifications. For example, in response to nutritional stresses, the Gram-positive bacterium Bacillus subtilis can differentiate into an endospore; the initiation of sporulation is controlled by the master regulator Spo0A, which is activated by phosphorylation. Spo0A phosphorylation is carried out by a multi-component phosphorelay system. These phosphorylation events on histidine and aspartate residues are labile, highly dynamic and permit a temporal control of the sporulation initiation decision. More recently, another kind of phosphorylation, more stable yet still dynamic, on serine or threonine residues, was proposed to play a role in spore maintenance and spore revival. Kinases that perform these phosphorylation events mainly belong to the Hanks family and could regulate spore dormancy and spore germination. The aim of this mini review is to focus on the regulation of sporulation in B. subtilis by these serine and threonine phosphorylation events and the kinases catalyzing them. PMID:27148245

  16. Regulation of mixed-lineage kinase activation in JNK-dependent morphogenesis

    PubMed Central

    Garlena, Rebecca A.; Gonda, Rebecca L.; Green, Alyssa B.; Pileggi, Rachel M.; Stronach, Beth

    2010-01-01

    Normal cells respond appropriately to various signals, while sustaining proper developmental programs and tissue homeostasis. Inappropriate signal reception, response or attenuation, can upset the normal balance of signaling within cells, leading to dysfunction or tissue malformation. To understand the molecular mechanisms that regulate protein-kinase-based signaling in the context of tissue morphogenesis, we analyzed the domain requirements of Drosophila Slpr, a mixed-lineage kinase (MLK), for Jun N-terminal kinase (JNK) signaling. The N-terminal half of Slpr is involved in regulated signaling whereas the C-terminal half promotes cortical protein localization. The SH3 domain negatively regulates Slpr activity consistent with autoinhibition via a conserved proline motif. Also, like many kinases, conserved residues in the activation segment of the catalytic domain regulate Slpr. Threonine 295, in particular, is essential for function. Slpr activation requires dual input from the MAP4K Misshapen (Msn), through its C-terminal regulatory domain, and the GTPase Rac, which both bind to the LZ–CRIB region of Slpr in vitro. Although Rac is sufficient to activate JNK signaling, our results indicate that there are Slpr-independent functions for Rac in dorsal closure. Finally, expression of various Slpr constructs alone or with upstream activators reveals a wide-ranging response at the cell and tissue level. PMID:20736302

  17. Making the Auroras glow: regulation of Aurora A and B kinase function by interacting proteins

    PubMed Central

    Carmena, Mar; Ruchaud, Sandrine; Earnshaw, William C

    2009-01-01

    The conserved Aurora family of protein kinases have emerged as crucial regulators of mitosis and cytokinesis. Despite their high degree of homology, Aurora A and B have very distinctive localisations and functions: Aurora A associates with the spindle poles to regulate entry into mitosis, centrosome maturation and spindle assembly; Aurora B is a member of the Chromosomal Passenger Complex (CPC) that transfers from the inner centromere in early mitosis to the spindle midzone, equatorial cortex and midbody in late mitosis and cytokinesis. Aurora B functions include regulation of chromosome–microtubule interactions, cohesion, spindle stability and cytokinesis. This review will focus on how interacting proteins make this functional diversity possible by targeting the kinases to different subcellular locations and regulating their activity. PMID:19836940

  18. Making the Auroras glow: regulation of Aurora A and B kinase function by interacting proteins.

    PubMed

    Carmena, Mar; Ruchaud, Sandrine; Earnshaw, William C

    2009-12-01

    The conserved Aurora family of protein kinases have emerged as crucial regulators of mitosis and cytokinesis. Despite their high degree of homology, Aurora A and B have very distinctive localisations and functions: Aurora A associates with the spindle poles to regulate entry into mitosis, centrosome maturation and spindle assembly; Aurora B is a member of the Chromosomal Passenger Complex (CPC) that transfers from the inner centromere in early mitosis to the spindle midzone, equatorial cortex and midbody in late mitosis and cytokinesis. Aurora B functions include regulation of chromosome-microtubule interactions, cohesion, spindle stability and cytokinesis. This review will focus on how interacting proteins make this functional diversity possible by targeting the kinases to different subcellular locations and regulating their activity. PMID:19836940

  19. Regulation of yeast pyruvate kinase by ultrasensitive allostery independent of phosphorylation

    PubMed Central

    Xu, Yi-Fan; Zhao, Xin; Glass, David S.; Absalan, Farnaz; Perlman, David H.; Broach, James R.; Rabinowitz, Joshua D.

    2012-01-01

    Summary Allostery and covalent modification are major means of fast-acting metabolic regulation. Their relative roles in responding to environmental changes remain, however, unclear. Here we examine this issue, using as a case study the rapid decrease in pyruvate kinase flux in yeast upon glucose removal. The main pyruvate kinase isozyme (Cdc19) is phosphorylated in response to environmental cues. It also exhibits positively-cooperative (ultrasensitive) allosteric activation by fructose-1,6-bisphosphate (FBP). Glucose removal causes accumulation of Cdc19’s substrate, phosphoenolpyruvate. This response is retained in strains with altered protein-kinase-A or AMP-activated-protein-kinase activity or with CDC19 carrying mutated phosphorylation sites. In contrast, yeast engineered with a CDC19 point mutation that ablates FBP-based regulation fail to accumulate phosphoenolpyruvate. They also fail to grow on ethanol and slowly resume growth upon glucose upshift. Thus, while yeast pyruvate kinase is covalently modified in response to glucose availability, its activity is controlled almost exclusively by ultrasensitive allostery. PMID:22902555

  20. TRPM6 kinase activity regulates TRPM7 trafficking and inhibits cellular growth under hypomagnesic conditions

    PubMed Central

    Brandao, Katherine; Deason-Towne, Francina; Zhao, Xiaoyun; Perraud, Anne-Laure; Schmitz, Carsten

    2014-01-01

    The channel kinases TRPM6 and TRPM7 are both members of the melastatin related transient receptor potential (TRPM) subfamily of ion channels and the only known fusions of an ion channel pore with a kinase domain. TRPM6 and TRPM7 form functional, tetrameric channel complexes at the plasma membrane by heteromerization. TRPM6 was previously shown to cross-phosphorylate TRPM7 on threonine residues, but not vice versa. Genetic studies demonstrated that TRPM6 and TRPM7 fulfill non-redundant functions, and that each channel contributes uniquely to the regulation of Mg2+ homeostasis. Although there are indications that TRPM6 and TRPM7 can influence each other’s cellular distribution and activity, little is known about the functional relationship between these two channel-kinases. In the present study, we examined how TRPM6 kinase activity influences TRPM7 serine phosphorylation, intracellular trafficking, and cell surface expression of TRPM7, as well as Mg2+-dependent cellular growth. We found TRPM7 serine phosphorylation via the TRPM6 kinase, but no TRPM6 serine phosphorylation via the TRPM7 kinase. Intracellular trafficking of TRPM7 was altered in HEK-293 epithelial kidney cells and DT40 B cells in the presence of TRPM6 with intact kinase activity, independently of the availability of extracellular Mg2+, but TRPM6/7 surface labeling experiments indicate comparable levels of the TRPM6/7 channels at the plasma membrane. Furthermore, using a complementation approach in TRPM7-deficient DT40 B-cells, we demonstrated that wildtype TRPM6 inhibited cell growth under hypomagnesic cell culture conditions in cells co-expressing TRPM6 and TRPM7, however co-expression of a TRPM6 kinase dead mutant had no effect – a similar phenotype was also observed in TRPM6/7 co-expressing HEK-293 cells. Our results provide first clues about how heteromer formation between TRPM6 and TRPM7 influences the biological activity of these ion channels. We show that TRPM6 regulates TRPM7 intracellular

  1. The rice CK2 kinase regulates trafficking of phosphate transporters in response to phosphate levels.

    PubMed

    Chen, Jieyu; Wang, Yifeng; Wang, Fei; Yang, Jian; Gao, Mingxing; Li, Changying; Liu, Yingyao; Liu, Yu; Yamaji, Naoki; Ma, Jian Feng; Paz-Ares, Javier; Nussaume, Laurent; Zhang, Shuqun; Yi, Keke; Wu, Zhongchang; Wu, Ping

    2015-03-01

    Phosphate transporters (PTs) mediate phosphorus uptake and are regulated at the transcriptional and posttranslational levels. In one key mechanism of posttranslational regulation, phosphorylation of PTs affects their trafficking from the endoplasmic reticulum (ER) to the plasma membrane. However, the kinase(s) mediating PT phosphorylation and the mechanism leading to ER retention of phosphorylated PTs remain unclear. In this study, we identified a rice (Oryza sativa) kinase subunit, CK2β3, which interacts with PT2 and PT8 in a yeast two-hybrid screen. Also, the CK2α3/β3 holoenzyme phosphorylates PT8 under phosphate-sufficient conditions. This phosphorylation inhibited the interaction of PT8 with PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1, a key cofactor regulating the exit of PTs from the ER to the plasma membrane. Additionally, phosphorus starvation promoted CK2β3 degradation, relieving the negative regulation of PT phosphorus-insufficient conditions. In accordance, transgenic expression of a nonphosphorylatable version of OsPT8 resulted in elevated levels of that protein at the plasma membrane and enhanced phosphorus accumulation and plant growth under various phosphorus regimes. Taken together, these results indicate that CK2α3/β3 negatively regulates PTs and phosphorus status regulates CK2α3/β3. PMID:25724641

  2. GIT1/βPIX signaling proteins and PAK1 kinase regulate microtubule nucleation.

    PubMed

    Černohorská, Markéta; Sulimenko, Vadym; Hájková, Zuzana; Sulimenko, Tetyana; Sládková, Vladimíra; Vinopal, Stanislav; Dráberová, Eduarda; Dráber, Pavel

    2016-06-01

    Microtubule nucleation from γ-tubulin complexes, located at the centrosome, is an essential step in the formation of the microtubule cytoskeleton. However, the signaling mechanisms that regulate microtubule nucleation in interphase cells are largely unknown. In this study, we report that γ-tubulin is in complexes containing G protein-coupled receptor kinase-interacting protein 1 (GIT1), p21-activated kinase interacting exchange factor (βPIX), and p21 protein (Cdc42/Rac)-activated kinase 1 (PAK1) in various cell lines. Immunofluorescence microscopy revealed association of GIT1, βPIX and activated PAK1 with centrosomes. Microtubule regrowth experiments showed that depletion of βPIX stimulated microtubule nucleation, while depletion of GIT1 or PAK1 resulted in decreased nucleation in the interphase cells. These data were confirmed for GIT1 and βPIX by phenotypic rescue experiments, and counting of new microtubules emanating from centrosomes during the microtubule regrowth. The importance of PAK1 for microtubule nucleation was corroborated by the inhibition of its kinase activity with IPA-3 inhibitor. GIT1 with PAK1 thus represent positive regulators, and βPIX is a negative regulator of microtubule nucleation from the interphase centrosomes. The regulatory roles of GIT1, βPIX and PAK1 in microtubule nucleation correlated with recruitment of γ-tubulin to the centrosome. Furthermore, in vitro kinase assays showed that GIT1 and βPIX, but not γ-tubulin, serve as substrates for PAK1. Finally, direct interaction of γ-tubulin with the C-terminal domain of βPIX and the N-terminal domain of GIT1, which targets this protein to the centrosome, was determined by pull-down experiments. We propose that GIT1/βPIX signaling proteins with PAK1 kinase represent a novel regulatory mechanism of microtubule nucleation in interphase cells. PMID:27012601

  3. Identification of Nuclear Protein Targets for Six Leukemogenic Tyrosine Kinases Governed by Post-Translational Regulation

    PubMed Central

    Pierce, Andrew; Williamson, Andrew; Jaworska, Ewa; Griffiths, John R.; Taylor, Sam; Walker, Michael; O’Dea, Mark Aspinall; Spooncer, Elaine; Unwin, Richard D.; Poolman, Toryn; Ray, David; Whetton, Anthony D.

    2012-01-01

    Mutated tyrosine kinases are associated with a number of different haematological malignancies including myeloproliferative disorders, lymphoma and acute myeloid leukaemia. The potential commonalities in the action of six of these leukemogenic proteins on nuclear proteins were investigated using systematic proteomic analysis. The effects on over 3600 nuclear proteins and 1500 phosphopeptide sites were relatively quantified in seven isogenic cell lines. The effects of the kinases were diverse although some commonalities were found. Comparison of the nuclear proteomic data with transcriptome data and cytoplasmic proteomic data indicated that the major changes are due to post-translational mechanisms rather than changes in mRNA or protein distribution. Analysis of the promoter regions of genes whose protein levels changed in response to the kinases showed the most common binding site found was that for NFκB whilst other sites such as those for the glucocorticoid receptor were also found. Glucocorticoid receptor levels and phosphorylation were decreased by all 6 PTKs. Whilst Glucocorticoid receptor action can potentiate NFκB action those proteins where genes have NFκB binding sites were in often regulated post-translationally. However all 6 PTKs showed evidence of NFkB pathway modulation via activation via altered IkB and NFKB levels. Validation of a common change was also undertaken with PMS2, a DNA mismatch repair protein. PMS2 nuclear levels were decreased in response to the expression of all 6 kinases, with no concomitant change in mRNA level or cytosolic protein level. Response to thioguanine, that requires the mismatch repair pathway, was modulated by all 6 oncogenic kinases. In summary common targets for 6 oncogenic PTKs have been found that are regulated by post-translational mechanisms. They represent potential new avenues for therapies but also demonstrate the post-translational regulation is a key target of leukaemogenic kinases. PMID:22745689

  4. Digital implementation of a biological astrocyte model and its application.

    PubMed

    Soleimani, Hamid; Bavandpour, Mohammad; Ahmadi, Arash; Abbott, Derek

    2015-01-01

    This paper presents a modified astrocyte model that allows a convenient digital implementation. This model is aimed at reproducing relevant biological astrocyte behaviors, which provide appropriate feedback control in regulating neuronal activities in the central nervous system. Accordingly, we investigate the feasibility of a digital implementation for a single astrocyte and a biological neuronal network model constructed by connecting two limit-cycle Hopf oscillators to an implementation of the proposed astrocyte model using oscillator-astrocyte interactions with weak coupling. Hardware synthesis, physical implementation on field-programmable gate array, and theoretical analysis confirm that the proposed astrocyte model, with considerably low hardware overhead, can mimic biological astrocyte model behaviors, resulting in desynchronization of the two coupled limit-cycle oscillators. PMID:25532161

  5. Kinetic studies on the regulation of rabbit liver pyruvate kinase

    PubMed Central

    Irving, M. G.; Williams, J. F.

    1973-01-01

    Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K+ and optimum activity was recorded with 30mm-K+, 4mm-MgADP−, with a MgADP−/ADP2− ratio of 50:1, but inhibition occurred with K+ concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg2+ was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (nH=2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent Km for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis–Menten response was obtained when phosphoenolpyruvate was the variable substrate (Km=0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg2+. PMID:4722439

  6. Actin cytoskeleton organization regulated by the PAK family of protein kinases.

    PubMed

    Eby, J J; Holly, S P; van Drogen, F; Grishin, A V; Peter, M; Drubin, D G; Blumer, K J

    1998-08-27

    Cdc42, Rac1 and other Rho-type GTPases regulate gene expression, cell proliferation and cytoskeletal architecture [1,2]. A challenge is to identify the effectors of Cdc42 and Rac1 that mediate these biological responses. Protein kinases of the p21-activated kinase (PAK) family bind activated Rac1 and Cdc42, and switch on mitogen-activated protein (MAP) kinase pathways; however, their roles in regulating actin cytoskeleton organization have not been clearly established [3-5]. Here, we show that mutants of the budding yeast Saccharomyces cerevisiae lacking the PAK homologs Ste20 and Cla4 exhibit actin cytoskeletal defects, in vivo and in vitro, that resemble those of cdc42-1 mutants. Moreover, STE20 overexpression suppresses cdc42-1 growth defects and cytoskeletal defects in vivo, and Ste20 kinase corrects the actin-assembly defects of permeabilized cdc42-1 cells in vitro. Thus, PAKs are effectors of Cdc42 in pathways that regulate the organization of the cortical actin cytoskeleton. PMID:9742399

  7. Regulation of the death-associated protein kinase 1 expression and autophagy via ATF6 requires apoptosis signal-regulating kinase 1.

    PubMed

    Gade, Padmaja; Manjegowda, Srikanta B; Nallar, Shreeram C; Maachani, Uday B; Cross, Alan S; Kalvakolanu, Dhananjaya V

    2014-11-01

    The death-associated protein kinase 1 (DAPK1) is an important regulator of cell death and autophagy. Recently, we have identified that ATF6, an endoplasmic reticulum-resident transcription factor, in association with the transcription factor CEBP-β, regulates the gamma interferon (IFN-γ)-induced expression of Dapk1 (P. Gade et al., Proc. Natl. Acad. Sci. U. S. A. 109:10316-10321, 2012, doi.org/10.1073/pnas.1119273109). IFN-γ-induced proteolytic processing of ATF6 and phosphorylation of C/EBP-β were essential for the formation of a novel transcriptional complex that regulates DAPK1. Here, we report that IFN-γ activates the ASK1-MKK3/MKK6-p38 mitogen-activated protein kinase (MAPK) pathway for controlling the activity of ATF6. The terminal enzyme in this pathway, p38 MAPK, phosphorylates a critical threonine residue in ATF6 upstream of its DNA binding domain. ATF6 mutants defective for p38 MAPK phosphorylation fail to undergo proteolytic processing in the Golgi apparatus and drive IFN-γ-induced gene expression and autophagy. We also show that mice lacking Ask1 are highly susceptible to lethal bacterial infection owing to defective autophagy. Together, these results identify a novel host defense pathway controlled by IFN-γ signaling. PMID:25135476

  8. Integration of Apoptosis Signal-Regulating Kinase 1-Mediated Stress Signaling with the Akt/Protein Kinase B-IκB Kinase Cascade

    PubMed Central

    Puckett, Mary C.; Goldman, Erinn H.; Cockrell, Lisa M.; Huang, Bei; Kasinski, Andrea L.; Du, Yuhong; Wang, Cun-Yu; Lin, Anning; Ichijo, Hidenori; Khuri, Fadlo

    2013-01-01

    Cellular processes are tightly controlled through well-coordinated signaling networks that respond to conflicting cues, such as reactive oxygen species (ROS), endoplasmic reticulum (ER) stress signals, and survival factors to ensure proper cell function. We report here a direct interaction between inhibitor of κB kinase (IKK) and apoptosis signal-regulating kinase 1 (ASK1), unveiling a critical node at the junction of survival, inflammation, and stress signaling networks. IKK can be activated by growth factor stimulation or tumor necrosis factor alpha engagement. IKK forms a complex with and phosphorylates ASK1 at a sensor site, Ser967, leading to the recruitment of 14-3-3, counteracts stress signal-triggered ASK1 activation, and suppresses ASK1-mediated functions. An inhibitory role of IKK in JNK signaling has been previously reported to depend on NF-κB-mediated gene expression. Our data suggest that IKK has a dual role: a transcription-dependent and a transcription-independent action in controlling the ASK1-JNK axis, coupling IKK to ROS and ER stress response. Direct phosphorylation of ASK1 by IKK also defines a novel IKK phosphorylation motif. Because of the intimate involvement of ASK1 in diverse diseases, the IKK/ASK1 interface offers a promising target for therapeutic development. PMID:23530055

  9. Distinct roles for protein kinase C isoforms in regulating platelet purinergic receptor function.

    PubMed

    Mundell, Stuart J; Jones, Matthew L; Hardy, Adam R; Barton, Johanna F; Beaucourt, Stephanie M; Conley, Pamela B; Poole, Alastair W

    2006-09-01

    ADP is a critical regulator of platelet activation, mediating its actions through two G protein-coupled receptors (GPCRs), P2Y1 and P2Y12. We have shown previously that the receptors are functionally desensitized, in a homologous manner, by distinct kinase-dependent mechanisms in which P2Y1 is regulated by protein kinase C (PKC) and P2Y12 by G protein-coupled receptor kinases. In this study, we addressed whether different PKC isoforms play different roles in regulating the trafficking and activity of these two GPCRs. Expression of PKCalpha and PKCdelta dominant-negative mutants in 1321N1 cells revealed that both isoforms regulated P2Y1 receptor signaling and trafficking, although only PKCdelta was capable of regulating P2Y12, in experiments in which PKC was directly activated by the phorbol ester phorbol 12-myristate 13-acetate (PMA). These results were paralleled in human platelets, in which PMA reduced subsequent ADP-induced P2Y1 and P2Y12 receptor signaling. PKC isoform-selective inhibitors revealed that novel, but not conventional, isoforms of PKC regulate P2Y12 function, whereas both novel and classic isoforms regulate P2Y1 activity. It is also noteworthy that we studied receptor internalization in platelets by a radioligand binding approach showing that both receptors internalize rapidly in these cells. ADP-induced P2Y1 receptor internalization is attenuated by PKC inhibitors, whereas that of the P2Y12 receptor is unaffected. Both P2Y1 and P2Y12 receptors can also undergo PMA-stimulated internalization, and here again, novel but not classic PKCs regulate P2Y12, whereas both novel and classic isoforms regulate P2Y1 internalization. This study therefore is the first to reveal distinct roles for PKC isoforms in the regulation of platelet P2Y receptor function and trafficking. PMID:16804093

  10. Reciprocal Regulation of AKT and MAP Kinase Dictates Virus-Host Cell Fusion ▿

    PubMed Central

    Sharma, Nishi R.; Mani, Prashant; Nandwani, Neha; Mishra, Rajakishore; Rana, Ajay; Sarkar, Debi P.

    2010-01-01

    Viruses of the Paramyxoviridae family bind to their host cells by using hemagglutinin-neuraminidase (HN), which enhances fusion protein (F)-mediated membrane fusion. Although respiratory syncytial virus and parainfluenza virus 5 of this family are suggested to trigger host cell signaling during infection, the virus-induced intracellular signals dictating virus-cell fusion await elucidation. Using an F- or HN-F-containing reconstituted envelope of Sendai virus, another paramyxovirus, we revealed the role and regulation of AKT1 and Raf/MEK/ERK cascades during viral fusion with liver cells. Our observation that extracellular signal-regulated kinase (ERK) activation promotes viral fusion via ezrin-mediated cytoskeletal rearrangements, whereas AKT1 attenuates fusion by promoting phosphorylation of F protein, indicates a counteractive regulation of viral fusion by reciprocal activation of AKT1 and mitogen-activated protein kinase (MAPK) cascades, establishing a novel conceptual framework for a therapeutic strategy. PMID:20164223

  11. Mitogen-activated protein kinase activation down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in G2-arrested oocytes.

    PubMed Central

    Abrieu, A; Dorée, M; Picard, A

    1997-01-01

    The G2 arrest of oocytes from frogs, clams, and starfish requires that preformed cyclin B-cdc2 complexes [prematuration-promoting factor (MPF)] be kept in an inactive form that is largely due to inhibitory phosphorylation of this pre-MPF. We have investigated the role of mitogen-activated protein (MAP) kinase in the activation of this pre-MPF. The cytoplasm of both frog and starfish oocytes contains an activity that can rapidly inactivate injected MPF. When the MAP kinase of G2-arrested starfish or Xenopus oocytes was prematurely activated by microinjection of c-mos or Ste-11 delta N fusion proteins, the rate and extent of MPF inactivation was much reduced. Both effects were suppressed by expression of the specific MAP kinase phosphatase Pyst 1. These results show that MAP kinase down-regulates a mechanism that inactivates cyclin B-cdc2 kinase in Xenopus oocytes. In starfish oocytes, however, MAP kinase activation occurs only after germinal vesicle breakdown, much after MPF activation. In this case, down-regulation of the cyclin B-cdc2 inhibiting pathway is a sensitive response to hormonal stimulation that does not require MAP kinase activation. Images PMID:9190205

  12. EBNA3C regulates p53 through induction of Aurora kinase B.

    PubMed

    Jha, Hem C; Yang, Karren; El-Naccache, Darine W; Sun, Zhiguo; Robertson, Erle S

    2015-03-20

    In multicellular organisms p53 maintains genomic integrity through activation of DNA repair, and apoptosis. EBNA3C can down regulate p53 transcriptional activity. Aurora kinase (AK) B phosphorylates p53, which leads to degradation of p53. Aberrant expression of AK-B is a hallmark of numerous human cancers. Therefore changes in the activities of p53 due to AK-B and EBNA3C expression is important for understanding EBV-mediated cell transformation. Here we show that the activities of p53 and its homolog p73 are dysregulated in EBV infected primary cells which can contribute to increased cell transformation. Further, we showed that the ETS-1 binding site is crucial for EBNA3C-mediated up-regulation of AK-B transcription. Further, we determined the Ser 215 residue of p53 is critical for functional regulation by AK-B and EBNA3C and that the kinase domain of AK-B which includes amino acid residues 106, 111 and 205 was important for p53 regulation. AK-B with a mutation at residue 207 was functionally similar to wild type AK-B in terms of its kinase activities and knockdown of AK-B led to enhanced p73 expression independent of p53. This study explores an additional mechanism by which p53 is regulated by AK-B and EBNA3C contributing to EBV-induced B-cell transformation. PMID:25691063

  13. EBNA3C regulates p53 through induction of Aurora kinase B

    PubMed Central

    Jha, Hem C.; Yang, Karren; El-Naccache, Darine W.; Sun, Zhiguo; Robertson, Erle S.

    2015-01-01

    In multicellular organisms p53 maintains genomic integrity through activation of DNA repair, and apoptosis. EBNA3C can down regulate p53 transcriptional activity. Aurora kinase (AK) B phosphorylates p53, which leads to degradation of p53. Aberrant expression of AK-B is a hallmark of numerous human cancers. Therefore changes in the activities of p53 due to AK-B and EBNA3C expression is important for understanding EBV-mediated cell transformation. Here we show that the activities of p53 and its homolog p73 are dysregulated in EBV infected primary cells which can contribute to increased cell transformation. Further, we showed that the ETS-1 binding site is crucial for EBNA3C-mediated up-regulation of AK-B transcription. Further, we determined the Ser 215 residue of p53 is critical for functional regulation by AK-B and EBNA3C and that the kinase domain of AK-B which includes amino acid residues 106, 111 and 205 was important for p53 regulation. AK-B with a mutation at residue 207 was functionally similar to wild type AK-B in terms of its kinase activities and knockdown of AK-B led to enhanced p73 expression independent of p53. This study explores an additional mechanism by which p53 is regulated by AK-B and EBNA3C contributing to EBV-induced B-cell transformation. PMID:25691063

  14. Ca2+/Calmodulin-Dependent Protein Kinase Kinases (CaMKKs) Effects on AMP-Activated Protein Kinase (AMPK) Regulation of Chicken Sperm Functions

    PubMed Central

    Nguyen, Thi Mong Diep; Combarnous, Yves; Praud, Christophe; Duittoz, Anne; Blesbois, Elisabeth

    2016-01-01

    Sperm require high levels of energy to ensure motility and acrosome reaction (AR) accomplishment. The AMP-activated protein kinase (AMPK) has been demonstrated to be strongly involved in the control of these properties. We address here the question of the potential role of calcium mobilization on AMPK activation and function in chicken sperm through the Ca2+/calmodulin-dependent protein kinase kinases (CaMKKs) mediated pathway. The presence of CaMKKs and their substrates CaMKI and CaMKIV was evaluated by western-blotting and indirect immunofluorescence. Sperm were incubated in presence or absence of extracellular Ca2+, or of CaMKKs inhibitor (STO-609). Phosphorylations of AMPK, CaMKI, and CaMKIV, as well as sperm functions were evaluated. We demonstrate the presence of both CaMKKs (α and β), CaMKI and CaMKIV in chicken sperm. CaMKKα and CaMKI were localized in the acrosome, the midpiece, and at much lower fluorescence in the flagellum, whereas CaMKKβ was mostly localized in the flagellum and much less in the midpiece and the acrosome. CaMKIV was only present in the flagellum. The presence of extracellular calcium induced an increase in kinases phosphorylation and sperm activity. STO-609 reduced AMPK phosphorylation in the presence of extracellular Ca2+ but not in its absence. STO-609 did not affect CaMKIV phosphorylation but decreased CaMKI phosphorylation and this inhibition was quicker in the presence of extracellular Ca2+ than in its absence. STO-609 efficiently inhibited sperm motility and AR, both in the presence and absence of extracellular Ca2+. Our results show for the first time the presence of CaMKKs (α and β) and one of its substrate, CaMKI in different subcellular compartments in germ cells, as well as the changes in the AMPK regulation pathway, sperm motility and AR related to Ca2+ entry in sperm through the Ca2+/CaM/CaMKKs/CaMKI pathway. The Ca2+/CaMKKs/AMPK pathway is activated only under conditions of extracellular Ca2+ entry in the cells

  15. TRAF6 upregulation in spinal astrocytes maintains neuropathic pain by integrating TNF-α and IL-1β signaling

    PubMed Central

    Lu, Ying; Jiang, Bao-Chun; Cao, De-Li; Zhang, Zhi-Jun; Zhang, Xin; Ji, Ru-Rong; Gao, Yong-Jing

    2014-01-01

    The proinflammatory cytokines TNF-α and IL-1β have been strongly implicated in the pathogenesis of neuropathic pain, but the intracellular signaling of these cytokines in glial cells are not fully understood. Tumor necrosis factor receptor associated factor 6 (TRAF6) plays a key role in signal transduction in the TNF receptor superfamily and the interleukin-1 receptor superfamily. In this study, we investigated the role of TRAF6 in neuropathic pain in mice following spinal nerve ligation (SNL). SNL induced persistent TRAF6 upregulation in the spinal cord. Interestingly, TRAF6 was mainly colocalized with the astrocytic marker GFAP on SNL day 10 and partially expressed in microglia on SNL day 3. In cultured astrocytes, TRAF6 was up-regulated after exposure to TNF-α or IL-1β. TNF-α or IL-1β also increased CCL2 expression, which was suppressed by both siRNA and shRNA targeting TRAF6. TRAF6 siRNA treatment also inhibited the phosphorylation of c-Jun N-terminal kinase (JNK) in astrocytes induced by TNF-α or IL-1β. JNK inhibitor D-NKI-1 dose-dependently decreased IL-1β-induced CCL2 expression. Moreover, spinal injection of TRAF6 siRNA decreased intrathecal TNF-α-or IL-1β-induced allodynia and hyperalgesia. Spinal TRAF6 inhibition via TRAF6 siRNA, shRNA lentivirus, or antisense oligodeoxynucleotides partially reversed SNL-induced neuropathic pain and spinal CCL2 expression. Finally, intrathecal injection of TNF-α-activated astrocytes induced mechanical allodynia, which was attenuated by pretreatment of astrocytes with TRAF6 siRNA. Taken together, the results suggest that TRAF6, upregulated in spinal cord astrocytes in the late phase after nerve injury, maintains neuropathic pain by integrating TNF-α and IL-1β signaling and activating the JNK/CCL2 pathway in astrocytes. PMID:25267210

  16. Extracellular signal-regulated kinases 1 and 2 activation in endothelial cells exposed to cyclic strain

    NASA Technical Reports Server (NTRS)

    Ikeda, M.; Takei, T.; Mills, I.; Kito, H.; Sumpio, B. E.

    1999-01-01

    The aim of this study was to determine whether extracellular signal-regulated kinases 1/2 (ERK1/ERK2) are activated and might play a role in enhanced proliferation and morphological change induced by strain. Bovine aortic endothelial cells (BAEC) were subjected to an average of 6 or 10% strain at a rate of 60 cycles/min for up to 4 h. Cyclic strain caused strain- and time-dependent phosphorylation and activation of ERK1/ERK2. Peak phosphorylation and activation of ERK1/ERK2 induced by 10% strain were at 10 min. A specific ERK1/ERK2 kinase inhibitor, PD-98059, inhibited phosphorylation and activation of ERK1/ERK2 but did not inhibit the increased cell proliferation and cell alignment induced by strain. Treatment of BAEC with 2,5-di-tert-butyl-1, 4-benzohydroquinone, to deplete inositol trisphosphate-sensitive calcium storage, and gadolinium chloride, a Ca2+ channel blocker, did not inhibit the activation of ERK1/ERK2. Strain-induced ERK1/ERK2 activation was partly inhibited by the protein kinase C inhibitor calphostin C and completely inhibited by the tyrosine kinase inhibitor genistein. These data suggest that 1) ERK1/ERK2 are not critically involved in the strain-induced cell proliferation and orientation, 2) strain-dependent activation of ERK1/ERK2 is independent of intracellular and extracellular calcium mobilization, and 3) protein kinase C activation and tyrosine kinase regulate strain-induced activation of ERK1/ERK2.

  17. Protein kinase Cζ exhibits constitutive phosphorylation and phosphatidylinositol-3,4,5-triphosphate-independent regulation

    PubMed Central

    Tobias, Irene S.; Kaulich, Manuel; Kim, Peter K.; Simon, Nitya; Jacinto, Estela; Dowdy, Steven F.; King, Charles C.; Newton, Alexandra C.

    2016-01-01

    Atypical protein kinase C (aPKC) isoenzymes are key modulators of insulin signalling, and their dysfunction correlates with insulin-resistant states in both mice and humans. Despite the engaged interest in the importance of aPKCs to type 2 diabetes, much less is known about the molecular mechanisms that govern their cellular functions than for the conventional and novel PKC isoenzymes and the functionally-related protein kinase B (Akt) family of kinases. Here we show that aPKC is constitutively phosphorylated and, using a genetically-encoded reporter for PKC activity, basally active in cells. Specifically, we show that phosphorylation at two key regulatory sites, the activation loop and turn motif, of the aPKC PKCζ in multiple cultured cell types is constitutive and independently regulated by separate kinases: ribosome-associated mammalian target of rapamycin complex 2 (mTORC2) mediates co-translational phosphorylation of the turn motif, followed by phosphorylation at the activation loop by phosphoinositide-dependent kinase-1 (PDK1). Live cell imaging reveals that global aPKC activity is constitutive and insulin unresponsive, in marked contrast to the insulin-dependent activation of Akt monitored by an Akt-specific reporter. Nor does forced recruitment to phosphoinositides by fusing the pleckstrin homology (PH) domain of Akt to the kinase domain of PKCζ alter either the phosphorylation or activity of PKCζ. Thus, insulin stimulation does not activate PKCζ through the canonical phosphatidylinositol-3,4,5-triphosphate-mediated pathway that activates Akt, contrasting with previous literature on PKCζ activation. These studies support a model wherein an alternative mechanism regulates PKCζ-mediated insulin signalling that does not utilize conventional activation via agonist-evoked phosphorylation at the activation loop. Rather, we propose that scaffolding near substrates drives the function of PKCζ. PMID:26635352

  18. Disrupting astrocyte-neuron lactate transfer persistently reduces conditioned responses to cocaine.

    PubMed

    Boury-Jamot, B; Carrard, A; Martin, J L; Halfon, O; Magistretti, P J; Boutrel, B

    2016-08-01

    A central problem in the treatment of drug addiction is the high risk of relapse often precipitated by drug-associated cues. The transfer of glycogen-derived lactate from astrocytes to neurons is required for long-term memory. Whereas blockade of drug memory reconsolidation represents a potential therapeutic strategy, the role of astrocyte-neuron lactate transport in long-term conditioning has received little attention. By infusing an inhibitor of glycogen phosphorylase into the basolateral amygdala of rats, we report that disruption of astrocyte-derived lactate not only transiently impaired the acquisition of a cocaine-induced conditioned place preference but also persistently disrupted an established conditioning. The drug memory was rescued by L-Lactate co-administration through a mechanism requiring the synaptic plasticity-related transcription factor Zif268 and extracellular signal-regulated kinase (ERK) signalling pathway but not the brain-derived neurotrophic factor (Bdnf). The long-term amnesia induced by glycogenolysis inhibition and the concomitant decreased expression of phospho-ERK were both restored with L-Lactate co-administration. These findings reveal a critical role for astrocyte-derived lactate in positive memory formation and highlight a novel amygdala-dependent reconsolidation process, whose disruption may offer a novel therapeutic target to reduce the long-lasting conditioned responses to cocaine. PMID:26503760

  19. Astrocytes as potential targets to suppress inflammatory demyelinating lesions in multiple sclerosis.

    PubMed

    De Keyser, Jacques; Laureys, Guy; Demol, Frauke; Wilczak, Nadine; Mostert, Jop; Clinckers, Ralph

    2010-11-01

    A hallmark of multiple sclerosis (MS) is the occurrence of focal inflammatory demyelinating lesions in the central nervous system. The prevailing view that activated anti-myelin T cells inherently mediate these lesions has been challenged after observations that these T cells, which are part of the normal immune repertoire, can also intermittently become activated in healthy people and subjects with other diseases. Astrocytes in the white matter of subjects with MS are deficient in beta(2) adrenergic receptors. Stimulation of beta(2) adrenergic receptors increases cAMP, leading to activation of protein kinase A (PKA). beta(2) adrenergic receptor deficiency will reduce the suppressive action of PKA on coactivator class II transactivator (CIITA), which is a key regulator of interferon gamma-induced major histocompatibility (MHC) class II molecule transcription. The expression of MHC class II may deviate astrocytes to function as facultative antigen presenting cells, which can then initiate the inflammatory cascade. In a proof of concept study in MS subjects it was shown that fluoxetine, which activates PKA in astrocytes, reduced the development of focal inflammatory lesions. If confirmed and extended by additional studies, suppressing the antigen presenting capacity of astrocytes could be a novel therapeutic option for the treatment of MS. PMID:20178822

  20. Sucrose-induced Receptor Kinase SIRK1 Regulates a Plasma Membrane Aquaporin in Arabidopsis*

    PubMed Central

    Wu, Xu Na; Sanchez Rodriguez, Clara; Pertl-Obermeyer, Heidi; Obermeyer, Gerhard; Schulze, Waltraud X.

    2013-01-01

    The transmembrane receptor kinase family is the largest protein kinase family in Arabidopsis, and it contains the highest fraction of proteins with yet uncharacterized functions. Here, we present functions of SIRK1, a receptor kinase that was previously identified with rapid transient phosphorylation after sucrose resupply to sucrose-starved seedlings. SIRK1 was found to be an active kinase with increasing activity in the presence of an external sucrose supply. In sirk1 T-DNA insertional mutants, the sucrose-induced phosphorylation patterns of several membrane proteins were strongly reduced; in particular, pore-gating phosphorylation sites in aquaporins were affected. SIRK1-GFP fusions were found to directly interact with aquaporins in affinity pull-down experiments on microsomal membrane vesicles. Furthermore, protoplast swelling assays of sirk1 mutants and SIRK1-GFP expressing lines confirmed a direct functional interaction of receptor kinase SIRK1 and aquaporins as substrates for phosphorylation. A lack of SIRK1 expression resulted in the failure of mutant protoplasts to control water channel activity upon changes in external sucrose concentrations. We propose that SIRK1 is involved in the regulation of sucrose-specific osmotic responses through direct interaction with and activation of an aquaporin via phosphorylation and that the duration of this response is controlled by phosphorylation-dependent receptor internalization. PMID:23820729

  1. Protein kinase NII and the regulation of rDNA transcription in mammalian cells.

    PubMed Central

    Belenguer, P; Baldin, V; Mathieu, C; Prats, H; Bensaid, M; Bouche, G; Amalric, F

    1989-01-01

    Transcription of ribosomal RNA genes is generally accepted to correlate with cell growth. Using primary cultures of adult bovine aortic endothelial (ABAE) cells, we have shown that transcription of rDNA in confluent cells falls to 5% of the transcription level in growing cells. Protein kinase NII appears to be a limiting factor to promote rDNA transcription in isolated nuclei of confluent cells. Protein kinase NII was detected by immunocytochemistry in the cytoplasm, nuclei and nucleoli of growing cells while it was no longer present in nucleoli of confluent cells. The kinase activity, in isolated nuclei, was estimated by endogenous phosphorylation of a specific substrate, nucleolin. A 10% residual activity was present in confluent cell nuclei compared to growing cell nuclei. Concomitantly, the transcription 'in vitro' of rDNA in the corresponding nuclei was also highly reduced (by 85%). Addition of exogenous protein kinase NII to confluent cell nuclei induced a strong increase in the phosphorylation of specific proteins including nucleolin. In parallel, the transcription of rDNA was increased by a factor of 5, to nearly the level observed in nuclei prepared from growing cells. These data suggest that, in confluent cells, factors necessary for rDNA transcription machinery are present but inactive in the nucleolus and that the phosphorylation of one or several of these factors (nucleolin, topoisomerase I,...) by protein kinase NII is a key event in the regulation of rDNA transcription. Images PMID:2780290

  2. A lipid-regulated docking site on vinculin for protein kinase C.

    PubMed

    Ziegler, Wolfgang H; Tigges, Ulrich; Zieseniss, Anke; Jockusch, Brigitte M

    2002-03-01

    During cell spreading, binding of actin-organizing proteins to acidic phospholipids and phosphorylation are important for localization and activity of these proteins at nascent cell-matrix adhesion sites. Here, we report on a transient interaction between the lipid-dependent protein kinase Calpha and vinculin, an early component of these sites, during spreading of HeLa cells on collagen. In vitro binding of protein kinase Calpha to vinculin tail was found dependent on free calcium and acidic phospholipids but independent of a functional kinase domain. The interaction was enhanced by conditions that favor the oligomerization of vinculin. Phosphorylation by protein kinase Calpha reached 1.5 mol of phosphate/mol of vinculin tail and required the C-terminal hydrophobic hairpin, a putative phosphatidylinositol 4,5-bisphosphate-binding site. Mass spectroscopy of peptides derived from in vitro phosphorylated vinculin tail identified phosphorylation of serines 1033 and 1045. Inhibition of C-terminal phospholipid binding at the vinculin tail by mutagenesis or deletion reduced the rate of phosphorylation to < or =50%. We suggest a possible mechanism whereby phospholipid-regulated conformational changes in vinculin may lead to exposure of a docking site for protein kinase Calpha and subsequent phosphorylation of vinculin and/or vinculin interaction partners, thereby affecting the formation of cell adhesion complexes. PMID:11741957

  3. Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

    NASA Technical Reports Server (NTRS)

    Liu, Z.; Xia, M.; Poovaiah, B. W.

    1998-01-01

    cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.

  4. Endocannabinoids regulate the activity of astrocytic hemichannels and the microglial response against an injury: In vivo studies.

    PubMed

    Vázquez, Carmen; Tolón, Rosa María; Pazos, María Ruth; Moreno, Marta; Koester, Erin C; Cravatt, Benjamin F; Hillard, Cecilia J; Romero, Julián

    2015-07-01

    Anandamide (AEA) is an endocannabinoid (EC) that modulates multiple functions in the CNS and that is released in areas of injury, exerting putative neuroprotective actions. In the present study, we have used intravital microscopy to analyze the role of the EC system in the glial response against an acute insult. Our data show that AEA modulates astroglial function in vivo by increasing connexin-43 hemichannel (HC) activity. Furthermore, the genetic inactivation of the AEA-degrading enzyme, fatty acid amide hydrolase (FAAH), also increased HC activity and enhanced the microglial response against an acute injury to the brain parenchyma, effects that were mediated by cannabinoid CB1 receptors. The contribution of ATP released through an astrocytic HC was critical for the microglial response, as this was prevented by the use of the HC blocker flufenamic acid and by apyrase. As could be expected, brain concentrations of AEA, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) were elevated in FAAH-null mice, while 2-arachidonoylglycerol (2-AG) concentrations remained unaltered. In summary, these findings demonstrate that AEA modifies glial functions by promoting an enhanced pro-inflammatory glial response in the brain. PMID:25917763

  5. ASTROCYTES: EMERGING STARS IN LEUKODYSTROPHY PATHOGENESIS

    PubMed Central

    Lanciotti, Angela; Brignone, Maria Stefania; Bertini, Enrico; Petrucci, Tamara C.; Aloisi, Francesca; Ambrosini, Elena

    2013-01-01

    Astrocytes are the predominant glial cell population in the central nervous system (CNS). Once considered only passive scaffolding elements, astrocytes are now recognised as cells playing essential roles in CNS development and function. They control extracellular water and ion homeostasis, provide substrates for energy metabolism, and regulate neurogenesis, myelination and synaptic transmission. Due to these multiple activities astrocytes have been implicated in almost all brain pathologies, contributing to various aspects of disease initiation, progression and resolution. Evidence is emerging that astrocyte dysfunction can be the direct cause of neurodegeneration, as shown in Alexander’s disease where myelin degeneration is caused by mutations in the gene encoding the astrocyte-specific cytoskeleton protein glial fibrillary acidic protein. Recent studies point to a primary role for astrocytes in the pathogenesis of other genetic leukodystrophies such as megalencephalic leukoencephalopathy with subcortical cysts and vanishing white matter disease. The aim of this review is to summarize current knowledge of the pathophysiological role of astrocytes focusing on their contribution to the development of the above mentioned leukodystrophies and on new perspectives for the treatment of neurological disorders. PMID:24340223

  6. Loose excitation-secretion coupling in astrocytes.

    PubMed

    Vardjan, Nina; Parpura, Vladimir; Zorec, Robert

    2016-05-01

    Astrocytes play an important housekeeping role in the central nervous system. Additionally, as secretory cells, they actively participate in cell-to-cell communication, which can be mediated by membrane-bound vesicles. The gliosignaling molecules stored in these vesicles are discharged into the extracellular space after the vesicle membrane fuses with the plasma membrane. This process is termed exocytosis, regulated by SNARE proteins, and triggered by elevations in cytosolic calcium levels, which are necessary and sufficient for exocytosis in astrocytes. For astrocytic exocytosis, calcium is sourced from the intracellular endoplasmic reticulum store, although its entry from the extracellular space contributes to cytosolic calcium dynamics in astrocytes. Here, we discuss calcium management in astrocytic exocytosis and the properties of the membrane-bound vesicles that store gliosignaling molecules, including the vesicle fusion machinery and kinetics of vesicle content discharge. In astrocytes, the delay between the increase in cytosolic calcium activity and the discharge of secretions from the vesicular lumen is orders of magnitude longer than that in neurons. This relatively loose excitation-secretion coupling is likely tailored to the participation of astrocytes in modulating neural network processing. PMID:26358496

  7. Artificial Astrocytes Improve Neural Network Performance

    PubMed Central

    Porto-Pazos, Ana B.; Veiguela, Noha; Mesejo, Pablo; Navarrete, Marta; Alvarellos, Alberto; Ibáñez, Oscar; Pazos, Alejandro; Araque, Alfonso

    2011-01-01

    Compelling evidence indicates the existence of bidirectional communication between astrocytes and neurons. Astrocytes, a type of glial cells classically considered to be passive supportive cells, have been recently demonstrated to be actively involved in the processing and regulation of synaptic information, suggesting that brain function arises from the activity of neuron-glia networks. However, the actual impact of astrocytes in neural network function is largely unknown and its application in artificial intelligence remains untested. We have investigated the consequences of including artificial astrocytes, which present the biologically defined properties involved in astrocyte-neuron communication, on artificial neural network performance. Using connectionist systems and evolutionary algorithms, we have compared the performance of artificial neural networks (NN) and artificial neuron-glia networks (NGN) to solve classification problems. We show that the degree of success of NGN is superior to NN. Analysis of performances of NN with different number of neurons or different architectures indicate that the effects of NGN cannot be accounted for an increased number of network elements, but rather they are specifically due to astrocytes. Furthermore, the relative efficacy of NGN vs. NN increases as the complexity of the network increases. These results indicate that artificial astrocytes improve neural network performance, and established the concept of Artificial Neuron-Glia Networks, which represents a novel concept in Artificial Intelligence with implications in computational science as well as in the understanding of brain function. PMID:21526157

  8. Artificial astrocytes improve neural network performance.

    PubMed

    Porto-Pazos, Ana B; Veiguela, Noha; Mesejo, Pablo; Navarrete, Marta; Alvarellos, Alberto; Ibáñez, Oscar; Pazos, Alejandro; Araque, Alfonso

    2011-01-01

    Compelling evidence indicates the existence of bidirectional communication between astrocytes and neurons. Astrocytes, a type of glial cells classically considered to be passive supportive cells, have been recently demonstrated to be actively involved in the processing and regulation of synaptic information, suggesting that brain function arises from the activity of neuron-glia networks. However, the actual impact of astrocytes in neural network function is largely unknown and its application in artificial intelligence remains untested. We have investigated the consequences of including artificial astrocytes, which present the biologically defined properties involved in astrocyte-neuron communication, on artificial neural network performance. Using connectionist systems and evolutionary algorithms, we have compared the performance of artificial neural networks (NN) and artificial neuron-glia networks (NGN) to solve classification problems. We show that the degree of success of NGN is superior to NN. Analysis of performances of NN with different number of neurons or different architectures indicate that the effects of NGN cannot be accounted for an increased number of network elements, but rather they are specifically due to astrocytes. Furthermore, the relative efficacy of NGN vs. NN increases as the complexity of the network increases. These results indicate that artificial astrocytes improve neural network performance, and established the concept of Artificial Neuron-Glia Networks, which represents a novel concept in Artificial Intelligence with implications in computational science as well as in the understanding of brain function. PMID:21526157

  9. Modified Wu-Zi-Yan-Zong prescription, a traditional Chinese polyherbal formula, suppresses lipopolysaccharide-induced neuroinflammatory processes in rat astrocytes via NF-κB and JNK/p38 MAPK signaling pathways.

    PubMed

    Zeng, Ke-Wu; Zhang, Tai; Fu, Hong; Liu, Geng-Xin; Wang, Xue-Mei

    2012-01-15

    Neuroinflammation plays an important role in several neurodegenerative diseases. In this study, we investigated the anti-inflammatory properties of modified Wu-Zi-Yan-Zong prescription (MWP), a traditional Chinese polyherbal formula, in primary cultured rat astrocytes treated with lipopolysaccharide (LPS). The results showed that MWP significantly inhibited release of nitric oxide (NO) and prostaglandin E (PGE), as well as expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 in LPS-induced rat astrocytes. Mechanism study indicated that MWP significantly inhibited nuclear factor-kappa B (NF-κB) inflammatory signaling pathway through attenuating inhibitor of nuclear factor-κB (IκB) degradation and down-regulating IκB kinases (IKKs) phosphorylation level. Moreover, MWP also decreased c-Jun NH(2)-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) phosphorylation, which play an important role in the induction of proinflammatory gene expressions. At last, MWP protected neurons from LPS-activated astrocytes in neuron-astrocyte co-culture system. Taken together, our results suggest that MWP may act to suppress neuroinflammatory response in LPS-stimulated rat astrocytes via NF-κB and JNK/p38 MAPK signaling cascades, and MWP may be a useful agent for prevention and treatment of neuroinflammatory disease. PMID:21893401

  10. Astrocyte Aquaporin Dynamics in Health and Disease

    PubMed Central

    Potokar, Maja; Jorgačevski, Jernej; Zorec, Robert

    2016-01-01

    The family of aquaporins (AQPs), membrane water channels, consists of diverse types of proteins that are mainly permeable to water; some are also permeable to small solutes, such as glycerol and urea. They have been identified in a wide range of organisms, from microbes to vertebrates and plants, and are expressed in various tissues. Here, we focus on AQP types and their isoforms in astrocytes, a major glial cell type in the central nervous system (CNS). Astrocytes have anatomical contact with the microvasculature, pia, and neurons. Of the many roles that astrocytes have in the CNS, they are key in maintaining water homeostasis. The processes involved in this regulation have been investigated intensively, in particular regulation of the permeability and expression patterns of different AQP types in astrocytes. Three aquaporin types have been described in astrocytes: aquaporins AQP1 and AQP4 and aquaglyceroporin AQP9. The aim here is to review their isoforms, subcellular localization, permeability regulation, and expression patterns in the CNS. In the human CNS, AQP4 is expressed in normal physiological and pathological conditions, but astrocytic expression of AQP1 and AQP9 is mainly associated with a pathological state. PMID:27420057

  11. Astrocyte Aquaporin Dynamics in Health and Disease.

    PubMed

    Potokar, Maja; Jorgačevski, Jernej; Zorec, Robert

    2016-01-01

    The family of aquaporins (AQPs), membrane water channels, consists of diverse types of proteins that are mainly permeable to water; some are also permeable to small solutes, such as glycerol and urea. They have been identified in a wide range of organisms, from microbes to vertebrates and plants, and are expressed in various tissues. Here, we focus on AQP types and their isoforms in astrocytes, a major glial cell type in the central nervous system (CNS). Astrocytes have anatomical contact with the microvasculature, pia, and neurons. Of the many roles that astrocytes have in the CNS, they are key in maintaining water homeostasis. The processes involved in this regulation have been investigated intensively, in particular regulation of the permeability and expression patterns of different AQP types in astrocytes. Three aquaporin types have been described in astrocytes: aquaporins AQP1 and AQP4 and aquaglyceroporin AQP9. The aim here is to review their isoforms, subcellular localization, permeability regulation, and expression patterns in the CNS. In the human CNS, AQP4 is expressed in normal physiological and pathological conditions, but astrocytic expression of AQP1 and AQP9 is mainly associated with a pathological state. PMID:27420057

  12. Regulation of osteoclast structure and function by FAK family kinases

    PubMed Central

    Ray, Brianne J.; Thomas, Keena; Huang, Cynthia S.; Gutknecht, Michael F.; Botchwey, Edward A.; Bouton, Amy H.

    2012-01-01

    Osteoclasts are highly specialized cells that resorb bone and contribute to bone remodeling. Diseases such as osteoporosis and osteolytic bone metastasis occur when osteoclast-mediated bone resorption takes place in the absence of concurrent bone synthesis. Considerable effort has been placed on identifying molecules that regulate the bone resorption activity of osteoclasts. To this end, we investigated unique and overlapping functions of members of the FAK family (FAK and Pyk2) in osteoclast functions. With the use of a conditional knockout mouse model, in which FAK is selectively targeted for deletion in osteoclast precursors (FAKΔmyeloid), we found that loss of FAK resulted in reduced bone resorption by osteoclasts in vitro, coincident with impaired signaling through the CSF-1R. However, bone architecture appeared normal in FAKΔmyeloid mice, suggesting that Pyk2 might functionally compensate for reduced FAK levels in vivo. This was supported by data showing that podosome adhesion structures, which are essential for bone degradation, were significantly more impaired in osteoclasts when FAK and Pyk2 were reduced than when either molecule was depleted individually. We conclude that FAK contributes to cytokine signaling and bone resorption in osteoclasts and partially compensates for the absence of Pyk2 to maintain proper adhesion structures in these cells. PMID:22941736

  13. Ribosomal S6 Kinase 2 Is a Key Regulator in Tumor Promoter–Induced Cell Transformation

    PubMed Central

    Cho, Yong-Yeon; Yao, Ke; Kim, Hong-Gyum; Kang, Bong Seok; Zheng, Duo; Bode, Ann M.; Dong, Zigang

    2010-01-01

    The ribosomal S6 kinase 2 (RSK2), a member of the p90RSK (RSK) family of proteins, is a widely expressed serine/threonine kinase that is activated by extracellular signal-regulated kinase 1/2 and phosphoinositide-dependent kinase 1 in response to many growth factors and peptide hormones. Its activation signaling enhances cell survival. However, the roles of RSK2 in cell transformation have not yet been elucidated. Here, we found that RSK2 is a critical serine/threonine kinase for the regulation of cell transformation. When cells were stimulated with tumor promoters, such as epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA), phosphorylation of RSK was increased within 5 min. Cell proliferation was suppressed in RSK2–/– mouse embryonic fibroblasts (MEFs) compared with RSK2+/+ MEFs. Moreover, RSK2–/– MEFs accumulated at the G1 phase of the cell cycle under normal cell culture conditions as well as after stimulation with EGF or TPA. In the anchorage-independent cell transformation assay (soft agar), stable expression of RSK2 in JB6 cells significantly enhanced colony formation in either the presence or absence of tumor promoters. Furthermore, knockdown of RSK2 with small interfering RNA-RSK2 suppressed constitutively active Ras (RasG12V)-induced foci formation in NIH3T3 cells. In addition, kaempferol, an inhibitor of RSK2, suppressed EGF-induced colony formation of JB6 Cl41 cells in soft agar, which was associated with inhibition of histone H3 phosphorylation (Ser10). These results showed that RSK2 is a key regulator for cell transformation induced by tumor promoters such as EGF and TPA. PMID:17804722

  14. MEK1/2 dual-specificity protein kinases: Structure and regulation

    SciTech Connect

    Roskoski, Robert

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer MEK1 is activated by phosphorylation of S218 and S222 in its activation segment. Black-Right-Pointing-Pointer MEK1 activation requires KSR, which functions as a scaffold and a protein kinase. Black-Right-Pointing-Pointer S212 phosphorylation in the MEK1 activation segment is inhibitory. Black-Right-Pointing-Pointer MEK1 and MEK2 contain a catalytic and a regulatory spine. -- Abstract: MEK1 and MEK2 are related protein kinases that participate in the RAS-RAF-MEK-ERK signal transduction cascade. This cascade participates in the regulation of a large variety of processes including apoptosis, cell cycle progression, cell migration, differentiation, metabolism, and proliferation. Moreover, oncogenic mutations in RAS or B-RAF are responsible for a large proportion of human cancers. MEK1 is activated by phosphorylation of S218 and S222 in its activation segment as catalyzed by RAF kinases in an intricate process that involves a KSR scaffold. Besides functioning as a scaffold, the kinase activity of KSR is also required for MEK activation. MEK1 regulation is unusual in that S212 phosphorylation in its activation segment is inhibitory. Moreover, active ERK catalyzes a feedback inhibitory phosphorylation of MEK1 T292 that serves to downregulate the pathway.

  15. Serine/Threonine/Tyrosine Protein Kinase Phosphorylates Oleosin, a Regulator of Lipid Metabolic Functions1[OA

    PubMed Central

    Parthibane, Velayoudame; Iyappan, Ramachandiran; Vijayakumar, Anitha; Venkateshwari, Varadarajan; Rajasekharan, Ram

    2012-01-01

    Plant oils are stored in oleosomes or oil bodies, which are surrounded by a monolayer of phospholipids embedded with oleosin proteins that stabilize the structure. Recently, a structural protein, Oleosin3 (OLE3), was shown to exhibit both monoacylglycerol acyltransferase and phospholipase A2 activities. The regulation of these distinct dual activities in a single protein is unclear. Here, we report that a serine/threonine/tyrosine protein kinase phosphorylates oleosin. Using bimolecular fluorescence complementation analysis, we demonstrate that this kinase interacts with OLE3 and that the fluorescence was associated with chloroplasts. Oleosin-green fluorescent protein fusion protein was exclusively associated with the chloroplasts. Phosphorylated OLE3 exhibited reduced monoacylglycerol acyltransferase and increased phospholipase A2 activities. Moreover, phosphatidylcholine and diacylglycerol activated oleosin phosphorylation, whereas lysophosphatidylcholine, oleic acid, and Ca2+ inhibited phosphorylation. In addition, recombinant peanut (Arachis hypogaea) kinase was determined to predominantly phosphorylate serine residues, specifically serine-18 in OLE3. Phosphorylation levels of OLE3 during seed germination were determined to be higher than in developing peanut seeds. These findings provide direct evidence for the in vivo substrate selectivity of the dual-specificity kinase and demonstrate that the bifunctional activities of oleosin are regulated by phosphorylation. PMID:22434039

  16. A fungicide-responsive kinase as a tool for synthetic cell fate regulation.

    PubMed

    Furukawa, Kentaro; Hohmann, Stefan

    2015-08-18

    Engineered biological systems that precisely execute defined tasks have major potential for medicine and biotechnology. For instance, gene- or cell-based therapies targeting pathogenic cells may replace time- and resource-intensive drug development. Engineering signal transduction systems is a promising, yet presently underexplored approach. Here, we exploit a fungicide-responsive heterologous histidine kinase for pathway engineering and synthetic cell fate regulation in the budding yeast Saccharomyces cerevisiae. Rewiring the osmoregulatory Hog1 MAPK signalling system generates yeast cells programmed to execute three different tasks. First, a synthetic negative feedback loop implemented by employing the fungicide-responsive kinase and a fungicide-resistant derivative reshapes the Hog1 activation profile, demonstrating how signalling dynamics can be engineered. Second, combinatorial integration of different genetic parts including the histidine kinases, a pathway activator and chemically regulated promoters enables control of yeast growth and/or gene expression in a two-input Boolean logic manner. Finally, we implemented a genetic 'suicide attack' system, in which engineered cells eliminate target cells and themselves in a specific and controllable manner. Taken together, fungicide-responsive kinases can be applied in different constellations to engineer signalling behaviour. Sensitizing engineered cells to existing chemicals may be generally useful for future medical and biotechnological applications. PMID:26138483

  17. A fungicide-responsive kinase as a tool for synthetic cell fate regulation

    PubMed Central

    Furukawa, Kentaro; Hohmann, Stefan

    2015-01-01

    Engineered biological systems that precisely execute defined tasks have major potential for medicine and biotechnology. For instance, gene- or cell-based therapies targeting pathogenic cells may replace time- and resource-intensive drug development. Engineering signal transduction systems is a promising, yet presently underexplored approach. Here, we exploit a fungicide-responsive heterologous histidine kinase for pathway engineering and synthetic cell fate regulation in the budding yeast Saccharomyces cerevisiae. Rewiring the osmoregulatory Hog1 MAPK signalling system generates yeast cells programmed to execute three different tasks. First, a synthetic negative feedback loop implemented by employing the fungicide-responsive kinase and a fungicide-resistant derivative reshapes the Hog1 activation profile, demonstrating how signalling dynamics can be engineered. Second, combinatorial integration of different genetic parts including the histidine kinases, a pathway activator and chemically regulated promoters enables control of yeast growth and/or gene expression in a two-input Boolean logic manner. Finally, we implemented a genetic ‘suicide attack’ system, in which engineered cells eliminate target cells and themselves in a specific and controllable manner. Taken together, fungicide-responsive kinases can be applied in different constellations to engineer signalling behaviour. Sensitizing engineered cells to existing chemicals may be generally useful for future medical and biotechnological applications. PMID:26138483

  18. Cyclin E-dependent protein kinase activity regulates niche retention of Drosophila ovarian follicle stem cells

    PubMed Central

    Wang, Zhu A.; Kalderon, Daniel

    2009-01-01

    Whether stem cells have unique cell cycle machineries and how they integrate with niche interactions remains largely unknown. We identified a hypomorphic cyclin E allele WX that strongly impairs the maintenance of follicle stem cells (FSCs) in the Drosophila ovary but does not reduce follicle cell proliferation or germline stem cell maintenance. CycEWX protein can still bind to the cyclin-dependent kinase catalytic subunit Cdk2, but forms complexes with reduced protein kinase activity measured in vitro. By creating additional CycE variants with different degrees of kinase dysfunction and expressing these and CycEWX at different levels, we found that higher CycE-Cdk2 kinase activity is required for FSC maintenance than to support follicle cell proliferation. Surprisingly, cycEWX FSCs were lost from their niches rather than arresting proliferation. Furthermore, FSC function was substantially restored by expressing either excess DE-cadherin or excess E2F1/DP, the transcription factor normally activated by CycE-Cdk2 phosphorylation of retinoblastoma proteins. These results suggest that FSC maintenance through niche adhesion is regulated by inputs that normally control S phase entry, possibly as a quality control mechanism to ensure adequate stem cell proliferation. We speculate that a positive connection between central regulators of the cell cycle and niche retention may be a common feature of highly proliferative stem cells. PMID:19966222

  19. Extracellular signal-regulated kinase and phosphoinositol-3 kinase mediate IGF-1 induced proliferation of fetal sheep cardiomyocytes.

    PubMed

    Sundgren, Nathan C; Giraud, George D; Schultz, Jess M; Lasarev, Michael R; Stork, Philip J S; Thornburg, Kent L

    2003-12-01

    Growth of the fetal heart involves cardiomyocyte enlargement, division, and maturation. Insulin-like growth factor-1 (IGF-1) is implicated in many aspects of growth and is likely to be important in developmental heart growth. IGF-1 stimulates the IGF-1 receptor (IGF1R) and downstream signaling pathways, including extracellular signal-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3K). We hypothesized that IGF-1 stimulates cardiomyocyte proliferation and enlargement through stimulation of the ERK cascade and stimulates cardiomyocyte differentiation through the PI3K cascade. In vivo administration of Long R3 IGF-1 (LR3 IGF-1) did not stimulate cardiomyocyte hypertrophy but led to a decreased percentage of cells that were binucleated in vivo. In culture, LR3 IGF-1 increased myocyte bromodeoxyuridine (BrdU) uptake by three- to five-fold. The blockade of either ERK or PI3K signaling (by UO-126 or LY-294002, respectively) completely abolished BrdU uptake stimulated by LR3 IGF-1. LR3 IGF-1 did not increase footprint area, but as expected, phenylephrine stimulated an increase in binucleated cardiomyocyte size. We conclude that 1) IGF-1 through IGF1R stimulates cardiomyocyte division in vivo; hyperplastic growth is the most likely explanation of IGF-1 stimulated heart growth in vivo; 2) IGF-1 through IGF1R does not stimulate binucleation in vitro or in vivo; 3) IGF-1 through IGF1R does not stimulate hypertrophy either in vivo or in vitro; and 4) IGF-1 through IGF1R requires both ERK and PI3K signaling for proliferation of near-term fetal sheep cardiomyocytes in vitro. PMID:12947030

  20. Gliotransmission: Exocytotic release from astrocytes

    PubMed Central

    Parpura, Vladimir; Zorec, Robert

    2009-01-01

    Gliotransmitters are chemicals released from glial cells fulfilling a following set of criteria: i) they are synthesized by and/or stored in glia; ii) their regulated release is triggered by physiological and/or pathological stimuli; iii) they activate rapid (milliseconds to seconds) responses in neighboring cells; and iv) they play a role in (patho)physiological processes. Astrocytes can release a variety of gliotransmitters into the extracellular space using several different mechanisms. In this review, we focus on exocytotic mechanism(s) underlying the release of three classes of gliotransmitters: (i) amino acids, such as, glutamate and D-serine; (ii) nucleotides, like adenosine 5'-triphosphate; and (iii) peptides, such as, atrial natriuretic peptide and brain-derived neurotrophic factor. It is becoming clear that astrocytes are endowed with elements that qualify them as cells communicating with neurons and other cells within the central nervous system by employing regulated exocytosis. PMID:19948188

  1. Inhibition of TREK-2 K(+) channels by PI(4,5)P2: an intrinsic mode of regulation by intracellular ATP via phosphatidylinositol kinase.

    PubMed

    Woo, Joohan; Shin, Dong Hoon; Kim, Hyun Jong; Yoo, Hae Young; Zhang, Yin-Hua; Nam, Joo Hyun; Kim, Woo Kyung; Kim, Sung Joon

    2016-08-01

    TWIK-related two-pore domain K(+) channels 1 and 2 (TREKs) are activated under various physicochemical conditions. However, the directions in which they are regulated by PI(4,5)P2 and intracellular ATP are not clearly presented yet. In this study, we investigated the effects of ATP and PI(4,5)P2 on overexpressed TREKs (HEK293T and COS-7) and endogenously expressed TREK-2 (mouse astrocytes and WEHI-231 B cells). In all of these cells, both TREK-1 and TREK-2 currents were spontaneously increased by dialysis with ATP-free pipette solution for whole-cell recording (ITREK-1,w-c and ITREK-2w-c) or by membrane excision for inside-out patch clamping without ATP (ITREK-1,i-o and ITREK-2,i-o). Steady state ITREK-2,i-o was reversibly decreased by 3 mM ATP applied to the cytoplasmic side, and this reduction was prevented by wortmannin, a PI-kinase inhibitor. An exogenous application of PI(4,5)P2 inhibited the spontaneously increased ITREKs,i-o, suggesting that intrinsic PI(4,5)P2 maintained by intracellular ATP and PI kinase may set the basal activity of TREKs in the intact cells. The inhibition of intrinsic TREK-2 by ATP was more prominent in WEHI-231 cells than astrocytes. Interestingly, unspecific screening of negative charges by poly-L-lysine also inhibited ITREK-2,i-o. Application of PI(4,5)P2 after the poly-L-lysine treatment showed dose-dependent dual effects, initial activation and subsequent inhibition of ITREK-2,i-o at low and high concentrations, respectively. In HEK293T cells coexpressing TREK-2 and a voltage-sensitive PI(4,5)P2 phosphatase, sustained depolarization increased ITREK-2,w-c initially (<5 s) but then decreased the current below the control level. In HEK293T cells coexpressing TREK-2 and type 3 muscarinic receptor, application of carbachol induced transient activation and sustained suppression of ITREK-2,w-c and cell-attached ITREK-2. The inhibition of TREK-2 by unspecific electrostatic quenching, extensive dephosphorylation, or sustained hydrolysis

  2. Leptin suppresses adenosine triphosphate-induced impairment of spinal cord astrocytes.

    PubMed

    Li, Baoman; Qi, Shuang; Sun, Guangfeng; Yang, Li; Han, Jidong; Zhu, Yue; Xia, Maosheng

    2016-10-01

    Spinal cord injury (SCI) causes long-term disability and has no clinically effective treatment. After SCI, adenosine triphosphate (ATP) may be released from neuronal cells and astrocytes in large amounts. Our previous studies have shown that the extracellular release of ATP increases the phosphorylation of cytosolic phospholipase A2 (cPLA2 ) and triggers the rapid release of arachidonic acid (AA) and prostaglandin E2 (PGE2) via the stimulation of epidermal growth factor receptor (EGFR) and the downstream phosphorylation of extracellular-regulated protein kinases 1 and 2. Leptin, a glycoprotein, induces the activation of the Janus kinase (JAK2)/signal transducers and activators of transcription-3 (Stat3) pathway via the leptin receptor. In this study, we found that 1) prolonged leptin treatment suppressed the ATP-stimulated release of AA and PGE2 from cultured spinal cord astrocytes; 2) leptin elevated the expression of caveolin-1 (Cav-1) via the JAK2/Stat3 signaling pathway; 3) Cav-1 blocked the interaction between Src and EGFR, thereby inhibiting the phosphorylation of EGFR and cPLA2 and attenuating the release of AA or PGE2; 4) pretreatment with leptin decreased ;he level of apoptosis and the release of interleukin-6 from cocultured neurons and astrocytes; and 5) leptin improved the recovery of locomotion in mice after SCI. Our results highlight leptin as a promising therapeutic agent for SCI. © 2016 Wiley Periodicals, Inc. PMID:27316329

  3. Guanosine effect on cholesterol efflux and apolipoprotein E expression in astrocytes.

    PubMed

    Ballerini, Patrizia; Ciccarelli, Renata; Di Iorio, Patrizia; Buccella, Silvana; D'Alimonte, Iolanda; Giuliani, Patricia; Masciulli, Arianna; Nargi, Eleonora; Beraudi, Alina; Rathbone, Michel P; Caciagli, Francesco

    2006-11-01

    The main source of cholesterol in the central nervous system (CNS) is represented by glial cells, mainly astrocytes, which also synthesise and secrete apolipoproteins, in particular apolipoprotein E (ApoE), the major apolipoprotein in the brain, thus generating cholesterol-rich high density lipoproteins (HDLs). This cholesterol trafficking, even though still poorly known, is considered to play a key role in different aspects of neuronal plasticity and in the stabilisation of synaptic transmission. Moreover, cell cholesterol depletion has recently been linked to a reduction in amyloid beta formation. Here we demonstrate that guanosine, which we previously reported to exert several neuroprotective effects, was able to increase cholesterol efflux from astrocytes and C6 rat glioma cells in the absence of exogenously added acceptors. In this effect the phosphoinositide 3 kinase/extracellular signal-regulated kinase 1/2 (PI3K/ERK1/2) pathway seems to play a pivotal role. Guanosine was also able to increase the expression of ApoE in astrocytes, whereas it did not modify the levels of ATP-binding cassette protein A1 (ABCA1), considered the main cholesterol transporter in the CNS. Given the emerging role of cholesterol balance in neuronal repair, these effects provide evidence for a role of guanosine as a potential pharmacological tool in the modulation of cholesterol homeostasis in the brain. PMID:18404467

  4. Extracellular-signal-regulated kinase 5 modulates the antioxidant response by transcriptionally controlling Sirtuin 1 expression in leukemic cells.

    PubMed

    Lopez-Royuela, Nuria; Rathore, Moeez G; Allende-Vega, Nerea; Annicotte, Jean-Sébastien; Fajas, Lluis; Ramachandran, Bindu; Gulick, Tod; Villalba, Martin

    2014-08-01

    Cancer cell metabolism differs from that of non-transformed cells in the same tissue. This specific metabolism gives tumor cells growing advantages besides the effect in increasing anabolism. One of these advantages is immune evasion mediated by a lower expression of the mayor histocompatibility complex class I molecules. The extracellular-signal-regulated kinase-5 regulates both mayor histocompatibility complex class I expression and metabolic activity. However, the mechanisms underlying are largely unknown. We show here that extracellular-signal-regulated kinase-5 regulates the transcription of the NADH(+)-dependent histone deacetylase silent mating type information regulation 2 homolog 1 (Sirtuin 1) in leukemic Jurkat T cells. This involves the activation of the transcription factor myocyte enhancer factor-2 and its binding to the sirt1 promoter. In addition, extracellular-signal-regulated kinase-5 is required for T cell receptor-induced and oxidative stress-induced full Sirtuin 1 expression. Extracellular-signal-regulated kinase-5 induces the expression of promoters containing the antioxidant response elements through a Sirtuin 1-dependent pathway. On the other hand, down modulation of extracellular-signal-regulated kinase-5 expression impairs the anti-oxidant response. Notably, the extracellular-signal-regulated kinase-5 inhibitor BIX02189 induces apoptosis in acute myeloid leukemia tumor cells without affecting T cells from healthy donors. Our results unveil a new pathway that modulates metabolism in tumor cells. This pathway represents a promising therapeutic target in cancers with deep metabolic layouts such as acute myeloid leukemia. PMID:24880091

  5. Different Regulation of p53 Expression by Cadmium Exposure in Kidney, Liver, Intestine, Vasculature, and Brain Astrocytes

    PubMed Central

    Lee, Jin-Yong; Tokumoto, Maki; Hattori, Yuta; Fujiwara, Yasuyuki; Shimada, Akinori; Satoh, Masahiko

    2016-01-01

    Chronic exposure to cadmium (Cd) is known to adversely affect renal function. Our previous studies indicated that Cd induces p53-dependent apoptosis by inhibiting gene expression of the ubiquitin-conjugating enzyme (Ube) 2d family in both human and rat proximal tubular cells. In this study, the effects of Cd on protein expression of p53 and apoptotic signals in the kidney and liver of mice exposed to Cd for 12 months were examined, as well as the effects of Cd on p53 protein levels and gene expression of the Ube2d family in various cell lines. Results showed that in the kidney of mice exposed to 300 ppm Cd for 12 months, there was overaccumulation of p53 proteins in addition to the induction of apoptosis, which was triggered specifically in the proximal tubules. Interestingly, the site of apoptosis was the same as that of p53 accumulation in the proximal tubules. In the liver of mice chronically exposed to Cd, gene expression of the Ube2d family tended to be slightly decreased, together with slight apoptosis without the accumulation of p53 protein. In rat small intestine epithelial (IEC-6) cells, Cd decreased not only the p53 protein level but also gene expression of Ube2d1, Ube2d2 and Ube2d4. In human brain microvascular endothelial cells (HBMECs), Cd did not suppress gene expression of the Ube2d family, but increased the p53 protein level. In human brain astrocytes (HBASTs), Cd only increased gene expression of UBE2D3. These results suggest that Cd-induced apoptosis through p53 protein is associated with renal toxicity but not hepatic toxicity, and the modification of p53 protein by Cd may vary depending on cell type. PMID:26977261

  6. Protein Kinase D Enzymes as Regulators of EMT and Cancer Cell Invasion.

    PubMed

    Durand, Nisha; Borges, Sahra; Storz, Peter

    2016-01-01

    The Protein Kinase D (PKD) isoforms PKD1, PKD2, and PKD3 are effectors of the novel Protein Kinase Cs (nPKCs) and diacylglycerol (DAG). PKDs impact diverse biological processes like protein transport, cell migration, proliferation, epithelial to mesenchymal transition (EMT) and apoptosis. PKDs however, have distinct effects on these functions. While PKD1 blocks EMT and cell migration, PKD2 and PKD3 tend to drive both processes. Given the importance of EMT and cell migration to the initiation and progression of various malignancies, abnormal expression of PKDs has been reported in multiple types of cancers, including breast, pancreatic and prostate cancer. In this review, we discuss how EMT and cell migration are regulated by PKD isoforms and the significance of this regulation in the context of cancer development. PMID:26848698

  7. 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

  8. A novel calmodulin-β-PIX interaction and its implication in receptor tyrosine kinase regulation.

    PubMed

    Singh, Vinay K; Munro, Kim; Jia, Zongchao

    2012-09-01

    Calmodulin (CaM), a ubiquitous calcium-binding protein, regulates numerous cellular processes, primarily in response to calcium flux. We have identified and characterized a novel interaction between CaM and β-p21-activated kinase interacting exchange factor (β-PIX), a putative guanine exchange factor implicated in cell signaling, using affinity pull-down assays, co-immunoprecipitation, co-localization and circular dichroism studies. Fluorescence-based titration and isothermal titration calorimetry experiments revealed a Ca(2+)-dependent binding mechanism (K(D)≤10μM). Further, we show that CaM participates in a multi-protein complex involving β-PIX and E3 ubiquitin ligase c-Cbl (casitas B-cell lymphoma), which may play a critical role in receptor tyrosine kinase regulation and downstream signaling. PMID:22588125

  9. Protein Kinase D Enzymes as Regulators of EMT and Cancer Cell Invasion

    PubMed Central

    Durand, Nisha; Borges, Sahra; Storz, Peter

    2016-01-01

    The Protein Kinase D (PKD) isoforms PKD1, PKD2, and PKD3 are effectors of the novel Protein Kinase Cs (nPKCs) and diacylglycerol (DAG). PKDs impact diverse biological processes like protein transport, cell migration, proliferation, epithelial to mesenchymal transition (EMT) and apoptosis. PKDs however, have distinct effects on these functions. While PKD1 blocks EMT and cell migration, PKD2 and PKD3 tend to drive both processes. Given the importance of EMT and cell migration to the initiation and progression of various malignancies, abnormal expression of PKDs has been reported in multiple types of cancers, including breast, pancreatic and prostate cancer. In this review, we discuss how EMT and cell migration are regulated by PKD isoforms and the significance of this regulation in the context of cancer development. PMID:26848698

  10. Anaplastic Lymphoma Kinase Acts in the Drosophila Mushroom Body to Negatively Regulate Sleep

    PubMed Central

    Bai, Lei; Sehgal, Amita

    2015-01-01

    Though evidence is mounting that a major function of sleep is to maintain brain plasticity and consolidate memory, little is known about the molecular pathways by which learning and sleep processes intercept. Anaplastic lymphoma kinase (Alk), the gene encoding a tyrosine receptor kinase whose inadvertent activation is the cause of many cancers, is implicated in synapse formation and cognitive functions. In particular, Alk genetically interacts with Neurofibromatosis 1 (Nf1) to regulate growth and associative learning in flies. We show that Alk mutants have increased sleep. Using a targeted RNAi screen we localized the negative effects of Alk on sleep to the mushroom body, a structure important for both sleep and memory. We also report that mutations in Nf1 produce a sexually dimorphic short sleep phenotype, and suppress the long sleep phenotype of Alk. Thus Alk and Nf1 interact in both learning and sleep regulation, highlighting a common pathway in these two processes. PMID:26536237

  11. Slow Inhibition and Conformation Selective Properties of Extracellular Signal-Regulated Kinase 1 and 2 Inhibitors

    PubMed Central

    Rudolph, Johannes; Xiao, Yao; Pardi, Arthur; Ahn, Natalie G.

    2016-01-01

    The mitogen-activated protein (MAP) kinase pathway is a target for anticancer therapy, validated using inhibitors of B-Raf and MAP kinase kinase (MKK) 1 and 2. Clinical outcomes show a high frequency of acquired resistance in patient tumors, involving upregulation of activity of the MAP kinase, extracellular signal-regulated kinase (ERK) 1 and 2. Thus, inhibitors for ERK1/2 are potentially important for targeted therapeutics against cancer. The structures and potencies of different ERK inhibitors have been published, but their kinetic mechanisms have not been characterized. Here we perform enzyme kinetic studies on six representative ERK inhibitors, with potencies varying from 100 pM to 20 μM. Compounds with significant biological activity (IC50 < 100 nM) that inhibit in the subnanomolar range (Vertex-11e and SCH772984) display slow-onset inhibition and represent the first inhibitors of ERK2 known to demonstrate slow dissociation rate constants (values of 0.2 and 1.1 h−1, respectively). Furthermore, we demonstrate using kinetic competition assays that Vertex-11e binds with differing affinities to ERK2 in its inactive, unphosphorylated and active, phosphorylated forms. Finally, two-dimensional heteronuclear multiple-quantum correlation nuclear magnetic resonance experiments reveal that distinct conformational states are formed in complexes of Vertex-11e with inactive and active ERK2. Importantly, two conformers interconvert in equilibrium in the active ERK2 apoenzyme, but Vertex-11e strongly shifts the equilibrium completely to one conformer. Thus, a high-affinity, slow dissociation inhibitor stabilizes different enzyme conformations depending on the activity state of ERK2 and reveals properties of conformational selection toward the active kinase. PMID:25350931

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

    PubMed Central

    Sundararaman, Ananthalakshmy; Amirtham, Usha; Rangarajan, Annapoorni

    2016-01-01

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

  13. Optimization of microtubule affinity regulating kinase (MARK) inhibitors with improved physical properties.

    PubMed

    Sloman, David L; Noucti, Njamkou; Altman, Michael D; Chen, Dapeng; Mislak, Andrea C; Szewczak, Alexander; Hayashi, Mansuo; Warren, Lee; Dellovade, Tammy; Wu, Zhenhua; Marcus, Jacob; Walker, Deborah; Su, Hua-Poo; Edavettal, Suzanne C; Munshi, Sanjeev; Hutton, Michael; Nuthall, Hugh; Stanton, Matthew G

    2016-09-01

    Inhibition of microtubule affinity regulating kinase (MARK) represents a potentially attractive means of arresting neurofibrillary tangle pathology in Alzheimer's disease. This manuscript outlines efforts to optimize a pyrazolopyrimidine series of MARK inhibitors by focusing on improvements in potency, physical properties and attributes amenable to CNS penetration. A unique cylcyclohexyldiamine scaffold was identified that led to remarkable improvements in potency, opening up opportunities to reduce MW, Pgp efflux and improve pharmacokinetic properties while also conferring improved solubility. PMID:27491711

  14. Critical role of glycogen synthase kinase-3ß in regulating the avian heterophil response to Salmonella enterica serovar Enteritidis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A microarray-assisted gene expression screen of chicken heterophils revealed glycogen synthase kinase-3ß (GSK-3ß), a multifunctional Ser/Thr kinase, to be consistently up-regulated 30-180 min following stimulation with Salmonella enterica serovar Enteritidis (S. Enteritidis). The present study was ...

  15. Protein kinase C-mediated phosphorylation and functional regulation of dopamine transporters in striatal synaptosomes.

    PubMed

    Vaughan, R A; Huff, R A; Uhl, G R; Kuhar, M J

    1997-06-13

    Dopamine transporters (DATs) are members of a family of Na+- and Cl--dependent neurotransmitter transporters responsible for the rapid clearance of dopamine from synaptic clefts. The predicted primary sequence of DAT contains numerous consensus phosphorylation sites. In this report we demonstrate that DATs undergo endogenous phosphorylation in striatal synaptosomes that is regulated by activators of protein kinase C. Rat striatal synaptosomes were metabolically labeled with [32P]orthophosphate, and solubilized homogenates were subjected to immunoprecipitation with an antiserum specific for DAT. Basal phosphorylation occurred in the absence of exogenous treatments, and the phosphorylation level was rapidly increased when synaptosomes were treated with the phosphatase inhibitors okadaic acid or calyculin. Treatment of synaptosomes with the protein kinase C activator phorbol 12-myristate 13-acetate (PMA) also increased the level of phosphate incorporation. This occurred within 10 min and was dosedependent between 0.1 and 1 microM PMA. DAT phosphorylation was also significantly increased by two other protein kinase C activators, (-)-indolactam V and 1-oleoyl-2-acetyl-sn-glycerol. The inactive phorbol ester 4alpha-phorbol 12,13-didecanoate at 10 microM was without effect, and PMA-induced phosphorylation was blocked by treatment of synaptosomes with the protein kinase C inhibitors staurosporine and bisindoylmaleimide. These results indicate that DATs undergo rapid in vivo phosphorylation in response to protein kinase C activation and that a robust mechanism exists in synaptosomes for DAT dephosphorylation. Dopamine transport activity in synaptosomes was reduced by all treatments that promoted DAT phosphorylation, with comparable dose, time, and inhibitor characteristics. The change in transport activity was produced by a reduction in Vmax with no significant effect on the Km for dopamine. These results suggest that synaptosomal dopamine transport activity is regulated by

  16. Regulation of Microtubule Dynamics through Phosphorylation on Stathmin by Epstein-Barr Virus Kinase BGLF4*

    PubMed Central

    Chen, Po-Wen; Lin, Sue-Jane; Tsai, Shu-Chun; Lin, Jiun-Han; Chen, Mei-Ru; Wang, Jiin-Tarng; Lee, Chung-Pei; Tsai, Ching-Hwa

    2010-01-01

    Stathmin is an important microtubule (MT)-destabilizing protein, and its activity is differently attenuated by phosphorylation at one or more of its four phosphorylatable serine residues (Ser-16, Ser-25, Ser-38, and Ser-63). This phosphorylation of stathmin plays important roles in mitotic spindle formation. We observed increasing levels of phosphorylated stathmin in Epstein-Barr virus (EBV)-harboring lymphoblastoid cell lines (LCLs) and nasopharyngeal carcinoma (NPC) cell lines during the EBV lytic cycle. These suggest that EBV lytic products may be involved in the regulation of stathmin phosphorylation. BGLF4 is an EBV-encoded kinase and has similar kinase activity to cdc2, an important kinase that phosphorylates serine residues 25 and 38 of stathmin during mitosis. Using an siRNA approach, we demonstrated that BGLF4 contributes to the phosphorylation of stathmin in EBV-harboring NPC. Moreover, we confirmed that BGLF4 interacts with and phosphorylates stathmin using an in vitro kinase assay and an in vivo two-dimensional electrophoresis assay. Interestingly, unlike cdc2, BGLF4 was shown to phosphorylate non-proline directed serine residues of stathmin (Ser-16) and it mediated phosphorylation of stathmin predominantly at serines 16, 25, and 38, indicating that BGLF4 can down-regulate the activity of stathmin. Finally, we demonstrated that the pattern of MT organization was changed in BGLF4-expressing cells, possibly through phosphorylation of stathmin. In conclusion, we have shown that a viral Ser/Thr kinase can directly modulate the activity of stathmin and this contributes to alteration of cellular MT dynamics and then may modulate the associated cellular processes. PMID:20110360

  17. Regulation of a plant SNF1-related protein kinase by glucose-6-phosphate

    SciTech Connect

    Toroser, D.; Plaut, Z.; Huber, S.C.

    2000-05-01

    One of the major protein kinases (PK{sub III}) that phosphorylates serine-158 of spinach sucrose-phosphate synthase (SPS), which is responsible for light/dark modulation of activity, is known to be a member of the SNF1-related family of protein kinases. In the present study, the authors have developed a fluorescence-based continuous assay for measurement of PK{sub III} activity. Using the continuous assay, along with the fixed-time-point {sup 32}P-incorporation assay, they demonstrate that PK{sub III} activity is inhibited by glucose-6-phosphate (Glc-6-P). Relative inhibition by Glc-6-P was increased by decreasing pH from 8.5 to 5.5 and by reducing the concentration of Mg{sup 2+} in the assay from 10 to 2 nM. Under likely physiological conditions (PH 7.0 and 2 mM Mg{sup 2+}), 10 nM Glc-6-P inhibited kinase activity approximately 70%. Inhibition by Glc-6-P could not be ascribed to contaminants in the commercial preparations. Other metabolites inhibited PK{sub III} in the following order: Glc-6-P > mannose-6-P, fructose-1,6P{sub 2} > ribose-5-P, 3-PGA, fructose-6-P. Inorganic phosphate, Glc, and AMP were not inhibitory, and free Glc did not reverse the inhibition by Glc-6-P. Because SNF1-related protein kinases are thought to function broadly in the regulation of enzyme activity and gene expression, Glc-6-P inhibition of PK{sub III} activity potentially provides a mechanism for metabolic regulation of the reactions catalyzed by these important protein kinases.

  18. Phosphorylation of the RNA-binding protein Dazl by MAPKAP kinase 2 regulates spermatogenesis.

    PubMed

    Williams, Patrick A; Krug, Michael S; McMillan, Emily A; Peake, Jasmine D; Davis, Tara L; Cocklin, Simon; Strochlic, Todd I

    2016-08-01

    Developing male germ cells are exquisitely sensitive to environmental insults such as heat and oxidative stress. An additional characteristic of these cells is their unique dependence on RNA-binding proteins for regulating posttranscriptional gene expression and translational control. Here we provide a mechanistic link unifying these two features. We show that the germ cell-specific RNA-binding protein deleted in azoospermia-like (Dazl) is phosphorylated by MAPKAP kinase 2 (MK2), a stress-induced protein kinase activated downstream of p38 MAPK. We demonstrate that phosphorylation of Dazl by MK2 on an evolutionarily conserved serine residue inhibits its interaction with poly(A)-binding protein, resulting in reduced translation of Dazl-regulated target RNAs. We further show that transgenic expression of wild-type human Dazl but not a phosphomimetic form in the Drosophila male germline can restore fertility to flies deficient in boule, the Drosophila orthologue of human Dazl. These results illuminate a novel role for MK2 in spermatogenesis, expand the repertoire of RNA-binding proteins phosphorylated by this kinase, and suggest that signaling by the p38-MK2 pathway is a negative regulator of spermatogenesis via phosphorylation of Dazl. PMID:27280388

  19. Cell cycle regulation of Greatwall kinase nuclear localization facilitates mitotic progression

    PubMed Central

    Wang, Peng; Galan, Jacob A.; Normandin, Karine; Bonneil, Éric; Hickson, Gilles R.; Roux, Philippe P.; Thibault, Pierre

    2013-01-01

    Cell division requires the coordination of critical protein kinases and phosphatases. Greatwall (Gwl) kinase activity inactivates PP2A-B55 at mitotic entry to promote the phosphorylation of cyclin B–Cdk1 substrates, but how Gwl is regulated is poorly understood. We found that the subcellular localization of Gwl changed dramatically during the cell cycle in Drosophila. Gwl translocated from the nucleus to the cytoplasm in prophase. We identified two critical nuclear localization signals in the central, poorly characterized region of Gwl, which are required for its function. The Polo kinase associated with and phosphorylated Gwl in this region, promoting its binding to 14-3-3ε and its localization to the cytoplasm in prophase. Our results suggest that cyclin B–Cdk1 phosphorylation of Gwl is also required for its nuclear exclusion by a distinct mechanism. We show that the nucleo-cytoplasmic regulation of Gwl is essential for its functions in vivo and propose that the spatial regulation of Gwl at mitotic entry contributes to the mitotic switch. PMID:23857770

  20. The mucolipidosis IV Ca2+ channel TRPML1 (MCOLN1) is regulated by the TOR kinase

    PubMed Central

    Onyenwoke, Rob U.; Sexton, Jonathan Z.; Yan, Feng; Díaz, María Cristina Huertas; Forsberg, Lawrence J.; Major, Michael B.; Brenman, Jay E.

    2015-01-01

    Autophagy is a complex pathway regulated by numerous signalling events that recycles macromolecules and may be perturbed in lysosomal storage disorders (LSDs). During autophagy, aberrant regulation of the lysosomal Ca2+ efflux channel TRPML1 [transient receptor potential mucolipin 1 (MCOLN1)], also known as MCOLN1, is solely responsible for the human LSD mucolipidosis type IV (MLIV); however, the exact mechanisms involved in the development of the pathology of this LSD are unknown. In the present study, we provide evidence that the target of rapamycin (TOR), a nutrient-sensitive protein kinase that negatively regulates autophagy, directly targets and inactivates the TRPML1 channel and thereby functional autophagy, through phosphorylation. Further, mutating these phosphorylation sites to unphosphorylatable residues proved to block TOR regulation of the TRPML1 channel. These findings suggest a mechanism for how TOR activity may regulate the TRPML1 channel. PMID:26195823

  1. A Digital Realization of Astrocyte and Neural Glial Interactions.

    PubMed

    Hayati, Mohsen; Nouri, Moslem; Haghiri, Saeed; Abbott, Derek

    2016-04-01

    The implementation of biological neural networks is a key objective of the neuromorphic research field. Astrocytes are the largest cell population in the brain. With the discovery of calcium wave propagation through astrocyte networks, now it is more evident that neuronal networks alone may not explain functionality of the strongest natural computer, the brain. Models of cortical function must now account for astrocyte activities as well as their relationships with neurons in encoding and manipulation of sensory information. From an engineering viewpoint, astrocytes provide feedback to both presynaptic and postsynaptic neurons to regulate their signaling behaviors. This paper presents a modified neural glial interaction model that allows a convenient digital implementation. This model can reproduce relevant biological astrocyte behaviors, which provide appropriate feedback control in regulating neuronal activities in the central nervous system (CNS). Accordingly, we investigate the feasibility of a digital implementation for a single astrocyte constructed by connecting a two coupled FitzHugh Nagumo (FHN) neuron model to an implementation of the proposed astrocyte model using neuron-astrocyte interactions. Hardware synthesis, physical implementation on FPGA, and theoretical analysis confirm that the proposed neuron astrocyte model, with significantly low hardware cost, can mimic biological behavior such as the regulation of postsynaptic neuron activity and the synaptic transmission mechanisms. PMID:26390499

  2. p21-activated kinase 4 regulates mitotic spindle positioning and orientation.

    PubMed

    Bompard, Guillaume; Morin, Nathalie

    2012-01-01

    During mitosis, microtubules (MTs) are massively rearranged into three sets of highly dynamic MTs that are nucleated from the centrosomes to form the mitotic spindle. Tight regulation of spindle positioning in the dividing cell and chromosome alignment at the center of the metaphase spindle are required to ensure perfect chromosome segregation and to position the cytokinetic furrow that will specify the two daughter cells. Spindle positioning requires regulation of MT dynamics, involving depolymerase activities together with cortical and kinetochore-mediated pushing and pulling forces acting on astral MTs and kinetochore fibres. These forces rely on MT motor activities. Cortical pulling forces exerted on astral MTs depend upon dynein/dynactin complexes and are essential in both symmetric and asymmetric cell division. A well-established spindle positioning pathway regulating the cortical targeting of dynein/dynactin involves the conserved LGN (Leu-Gly-Asn repeat-enriched-protein) and NuMA (microtubule binding nuclear mitotic apparatus protein) complex. Spindle orientation is also regulated by integrin-mediated cell adhesion and actin retraction fibres that respond to mechanical stress and are influenced by the microenvironment of the dividing cell. Altering the capture of astral MTs or modulating pulling forces affects spindle position, which can impair cell division, differentiation and embryogenesis. In this general scheme, the activity of mitotic kinases such as Auroras and Plk1 (Polo-like kinase 1) is crucial. Recently, the p21-activated kinases (PAKs) emerged as novel important players in mitotic progression. In our recent article, we demonstrated that PAK4 regulates spindle positioning in symmetric cell division. In this commentary, and in light of recent published studies, we discuss how PAK4 could participate in the regulation of mechanisms involved in spindle positioning and orientation. PMID:22960742

  3. Modulation of extracellular signal-related kinase, cyclin D1, glial fibrillary acidic protein, and vimentin expression in estradiol-pretreated astrocyte cultures treated with competence and progression growth factors.

    PubMed

    Bramanti, Vincenzo; Grasso, Sonia; Tibullo, Daniele; Giallongo, Cesarina; Raciti, Giuseppina; Viola, Maria; Avola, Roberto

    2015-09-01

    The present study seeks to elucidate the interactions between the "competence" growth factor basic fibroblast growth factor (bFGF) and/or estrogen 17β-estradiol and the "progression" growth factors epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and insulin (INS) on DNA labeling and also cyclin D1, extracellular signal-related kinase 1/2 (ERK1/2), glial fibrillary acidic protein (GFAP), and vimentin expression in astroglial cultures under different experimental conditions. Pretreatment for 24 hr with bFGF and subsequent exposure for 36 hr to estradiol (E2 ) and EGF, IGF-I, or INS stimulated DNA labeling in the last 12 hr, especially when the cultures were treated with progression growth factors. bFGF pretreatment and subsequent treatment with E2 for 36 hr stimulated DNA labeling. The 36-hr E2 treatment alone did not significantly decrease DNA labeling, but contemporary addition of E2 with two or three growth factors stimulated DNA labeling remarkably. When E2 was coadded with growth factors, a significantly increased DNA labeling was observed, demonstrating an astroglial synergistic mitogenic effect evoked by contemporary treatment with growth factors in the presence of estrogens. Cyclin D1 expression was markedly increased when astrocyte cultures were pretreated for 36 hr with E2 and subsequently treated with two or three competence and progression growth factors. A highly significant increase of ERK1/2 expression was observed after all the treatments (EGF, bFGF, INS, IGF-I alone or in combination with two or three growth factors). GFAP and vimentin expression was markedly increased when the cultures were treated with two or three growth factors. In conclusion, our data demonstrate estradiol-growth factor cross-talk during astroglial cell proliferation and differentiation in culture. PMID:26053243

  4. Spleen tyrosine kinase regulates mammary epithelial cell proliferation in mammary glands of dairy cows.

    PubMed

    Hou, Xiaoming; Lin, Lin; Xing, Weinan; Yang, Yang; Duan, Xiaoyu; Li, Qingzhang; Gao, Xuejun; Lin, Ye

    2016-05-01

    Spleen tyrosine kinase (SYK) is a nonreceptor tyrosine kinase that has been considered a hematopoietic cell-specific signal transducer involved in cell proliferation and differentiation. However, the role of SYK in normal mammary gland is still poorly understood. Here we show that SYK is expressed in mammary glands of dairy cows. Expression of SYK was higher in dry period mammary tissues than in lactating mammary tissues. Knockdown and overexpression of SYK affected dairy cow mammary epithelial cell proliferation as well as the expression of signal molecules involved in proliferation, including protein kinase B (PKB, also known as AKT1), p42/44 mitogen-activated protein kinase (MAPK), and signal transducer and activator of transcription 5 (STAT5). Dual-luciferase reporter assay showed that SYK increased the transcriptional activity of the AKT1 promoter, and cis-elements within the AKT1 promoter region from -439 to -84 bp mediated this regulation. These results suggest that SYK affects mammary epithelial cell proliferation by activating AKT1 at the transcriptional level in mammary glands of dairy cows, which is important for the mammary remodeling process in dry cows as well as for increasing persistency of lactation in lactating cows. PMID:26947307

  5. Myricetin inhibits UVB-induced angiogenesis by regulating PI-3 kinase in vivo.

    PubMed

    Jung, Sung Keun; Lee, Ki Won; Byun, Sanguine; Lee, Eun Jung; Kim, Jong-Eun; Bode, Ann M; Dong, Zigang; Lee, Hyong Joo

    2010-05-01

    Myricetin is one of the principal phytochemicals in onions, berries and red wine. Previous studies showed that myricetin exhibits potent anticancer and chemopreventive effects. The present study examined the effect of myricetin on ultraviolet (UV) B-induced angiogenesis in an SKH-1 hairless mouse skin tumorigenesis model. Topical treatment with myricetin inhibited repetitive UVB-induced neovascularization in SKH-1 hairless mouse skin. The induction of vascular endothelial growth factor, matrix metalloproteinase (MMP)-9 and MMP-13 expression by chronic UVB irradiation was significantly suppressed by myricetin treatment. Immunohistochemical and western blot analyses revealed that myricetin inhibited UVB-induced hypoxia inducible factor-1alpha expression in mouse skin. Western blot analysis and kinase assay data revealed that myricetin suppressed UVB-induced phosphatidylinositol-3 (PI-3) kinase activity and subsequently attenuated the UVB-induced phosphorylation of Akt/p70(S6K) in mouse skin lysates. A pull-down assay revealed the direct binding of PI-3 kinase and myricetin in mouse skin lysates. Our results indicate that myricetin suppresses UVB-induced angiogenesis by regulating PI-3 kinase activity in vivo in mouse skin. PMID:20008033

  6. Spm1, a stress-activated MAP kinase that regulates morphogenesis in S.pombe.

    PubMed Central

    Zaitsevskaya-Carter, T; Cooper, J A

    1997-01-01

    A gene encoding a novel MAP kinase family member, Spm1, was isolated from the fission yeast Schizosaccharomyces pombe. Overproduction of Spm1 inhibits proliferation. Disruption of the spm1+ gene interferes with cell separation and morphogenesis. Under conditions of nutrient limitation, hypertonic stress or elevated temperature, spm1 delta cells grow as short branched filaments in which the cell walls and septa are thickened, suggesting defects in polarized growth and cell wall remodeling. At high osmolarity, spm1 delta cells fail to form colonies. The Spm1 protein is tyrosine phosphorylated and activated in response to osmotic and heat stress, consistent with a role for Spm1 in adaptation to these conditions. Two other S.pombe MAP kinases are known, Spk1, required for sexual differentiation and sporulation, and Spc1/Sty1/Phh1, which is activated in hypertonic conditions. However, the distinctive features of the spm1 delta mutant phenotype and direct biochemical assays suggest that Spm1 does not lie on other known MAP kinase pathways. Our results demonstrate the existence of a new MAP kinase pathway that regulates cell wall remodeling and cytokinesis in response to environmental stresses. PMID:9135147

  7. Cross-regulation between Aurora B and Citron kinase controls midbody architecture in cytokinesis

    PubMed Central

    McKenzie, Callum; Bassi, Zuni I.; Debski, Janusz; Gottardo, Marco; Callaini, Giuliano; Dadlez, Michal; D'Avino, Pier Paolo

    2016-01-01

    Cytokinesis culminates in the final separation, or abscission, of the two daughter cells at the end of cell division. Abscission relies on an organelle, the midbody, which forms at the intercellular bridge and is composed of various proteins arranged in a precise stereotypic pattern. The molecular mechanisms controlling midbody organization and function, however, are obscure. Here we show that proper midbody architecture requires cross-regulation between two cell division kinases, Citron kinase (CIT-K) and Aurora B, the kinase component of the chromosomal passenger complex (CPC). CIT-K interacts directly with three CPC components and is required for proper midbody architecture and the orderly arrangement of midbody proteins, including the CPC. In addition, we show that CIT-K promotes Aurora B activity through phosphorylation of the INCENP CPC subunit at the TSS motif. In turn, Aurora B controls CIT-K localization and association with its central spindle partners through phosphorylation of CIT-K's coiled coil domain. Our results identify, for the first time, a cross-regulatory mechanism between two kinases during cytokinesis, which is crucial for establishing the stereotyped organization of midbody proteins. PMID:27009191

  8. Acute toxicity of doxorubicin on isolated perfused heart: response of kinases regulating energy supply.

    PubMed

    Tokarska-Schlattner, Malgorzata; Zaugg, Michael; da Silva, Rafaela; Lucchinetti, Eliana; Schaub, Marcus C; Wallimann, Theo; Schlattner, Uwe

    2005-07-01

    Doxorubicin (DXR) is a widely used and efficient anticancer drug. However, its application is limited by the risk of severe cardiotoxicity. Impairment of cardiac high-energy phosphate homeostasis is an important manifestation of both acute and chronic DXR cardiotoxic action. Using the Langendorff model of the perfused rat heart, we characterized the acute effects of 1-h perfusion with 2 or 20 microM DXR on two key kinases in cardiac energy metabolism, creatine kinase (CK) and AMP-activated protein kinase (AMPK), and related them to functional responses of the perfused heart and structural integrity of the contractile apparatus as well as drug accumulation in cardiomyocytes. DXR-induced changes in CK were dependent on the isoenzyme, with a shift in protein levels of cytosolic isoenzymes from muscle-type CK to brain-type CK, and a destabilization of octamers of the mitochondrial isoenzyme (sarcometric mitochondrial CK) accompanied by drug accumulation in mitochondria. Interestingly, DXR rapidly reduced the protein level and phosphorylation of AMPK as well as phosphorylation of its target, acetyl-CoA-carboxylase. AMPK was strongly affected already at 2 microM DXR, even before substantial cardiac dysfunction occurred. Impairment of CK isoenzymes was mostly moderate but became significant at 20 microM DXR. Only at 2 microM DXR did upregulation of brain-type CK compensate for inactivation of other isoenzymes. These results suggest that an impairment of kinase systems regulating cellular energy homeostasis is involved in the development of DXR cardiotoxicity. PMID:15764680

  9. Protein Kinase C Overactivity Impairs Prefrontal Cortical Regulation of Working Memory

    NASA Astrophysics Data System (ADS)

    Birnbaum, S. G.; Yuan, P. X.; Wang, M.; Vijayraghavan, S.; Bloom, A. K.; Davis, D. J.; Gobeske, K. T.; Sweatt, J. D.; Manji, H. K.; Arnsten, A. F. T.

    2004-10-01

    The prefrontal cortex is a higher brain region that regulates thought, behavior, and emotion using representational knowledge, operations often referred to as working memory. We tested the influence of protein kinase C (PKC) intracellular signaling on prefrontal cortical cognitive function and showed that high levels of PKC activity in prefrontal cortex, as seen for example during stress exposure, markedly impair behavioral and electrophysiological measures of working memory. These data suggest that excessive PKC activation can disrupt prefrontal cortical regulation of behavior and thought, possibly contributing to signs of prefrontal cortical dysfunction such as distractibility, impaired judgment, impulsivity, and thought disorder.

  10. Influence of thyroid hormones on maturation of rat cerebellar astrocytes.

    PubMed

    Manzano, Jimena; Bernal, Juan; Morte, Beatriz

    2007-05-01

    Thyroid hormone influences brain maturation through interaction with nuclear receptors and regulation of gene expression. Their role on astrocyte maturation remains unclear. We have analyzed the role of thyroid hormone in rat cerebellar astrocyte maturation by comparing the sequential patterns of intermediate filament expression in normal and hypothyroid animals. During normal development astroglial cells sequentially express nestin, vimentin, and glial fibrillary acidic protein. Differentiated astrocytes appeared in the superior medullary vellum by postnatal day 2 and reached the white mater and internal granular layer by postnatal day 4. Intermediate filament marker expression was transiently lost from postnatal days 6 to 8 in anterior lobes, without an increased apoptosis. Vimentin expression was replaced by glial fibrillary acidic protein between postnatal days 10 and 32. The differentiated astrocytes were evenly distributed throughout the cerebellar slices, including the internal granular layer. Differences between normal and hypothyroid rats were observed starting from postnatal day 4, with lack of differentiated astrocytes in the internal granular layer. The transient decrease of astrocyte markers immunoreactivity in the anterior lobe did not take place in hypothyroid rats. The vimentin-glial fibrillary acidic protein transition was delayed and most differentiated astrocytes remained confined to the white matter. The results indicate that thyroid hormone deficiency induces a delay and a partial arrest of astrocyte differentiation. Astrocytes express thyroid hormone receptor alpha and beta subtypes suggesting that astrocytes are direct target cells of thyroid hormones. PMID:17408906

  11. 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

  12. Induction of proinflammatory mediators requires activation of the TRAF, NIK, IKK and NF-κB signal transduction pathway in astrocytes infected with Escherichia coli

    PubMed Central

    Kim, J M; Oh, Y-K; Lee, J H; Im, D Y; Kim, Y-J; Youn, J; Lee, C-H; Son, H; Lee, Y-S; Park, J Y; Choi, I-H

    2005-01-01

    Escherichia coli is associated with inflammation in the brain. To investigate whether astrocytes are involved in E. coil-induced inflammation, we assessed the levels of expression of proinflammatory mediators produced by E. coli-infected astrocytes. E. coli infection in primary human astrocytes and cell lines increased expression of the CXC chemokine IL-8/GRO-α, the CC chemokine MCP-1, TNF-α, and iNOS. E. coli infection activated p65/p50 heterodimeric NF-κB and concurrently decreased the signals of IκBα. Blocking the NF-κB signals by IκBα-superrepressor-containing retrovirus or antisense p50 oligonucleotide transfection resulted in down-regulation of expression of the proinflammatory mediators. Furthermore, superrepressors of IκBα, IκB kinase (IKK) or NF-κB inducing kinase (NIK) inhibited the up-regulated expression of the downstream target genes of NF-κB such as IL-8 and MCP-1, and superrepressors of TNF receptor-associated factor (TRAF)2 and TRAF5 also inhibited expression of the E. coli-induced target genes of NF-κB. These results indicate that proinflammatory mediators such as the CXC chemokine IL-8/GRO-α, the CC chemokine MCP-1, TNF-α, and iNOS can be expressed in E. coli-infected astrocytes via an NF-κB pathway, suggesting that these mediators may contribute to inflammation in the brain, including infiltration of inflammatory cells. PMID:15932506

  13. Focal Adhesion Kinase regulates cell-cell contact formation in epithelial cells via modulation of Rho

    SciTech Connect

    Playford, Martin P.; Vadali, Kavita; Cai Xinming; Burridge, Keith; Schaller, Michael D.

    2008-10-15

    Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase that plays a key role in cellular processes such as cell adhesion, migration, proliferation and survival. Recent studies have also implicated FAK in the regulation of cell-cell adhesion. Here, evidence is presented showing that siRNA-mediated suppression of FAK levels in NBT-II cells and expression of dominant negative mutants of FAK caused loss of epithelial cell morphology and inhibited the formation of cell-cell adhesions. Rac and Rho have been implicated in the regulation of cell-cell adhesions and can be regulated by FAK signaling. Expression of active Rac or Rho in NBT-II cells disrupted formation of cell-cell contacts, thus promoting a phenotype similar to FAK-depleted cells. The loss of intercellular contacts in FAK-depleted cells is prevented upon expression of a dominant negative Rho mutant, but not a dominant negative Rac mutant. Inhibition of FAK decreased tyrosine phosphorylation of p190RhoGAP and elevated the level of GTP-bound Rho. This suggests that FAK regulates cell-cell contact formation by regulation of Rho.

  14. SIRT1 phosphorylation by AMP-activated protein kinase regulates p53 acetylation

    PubMed Central

    Lau, Alan W; Liu, Pengda; Inuzuka, Hiroyuki; Gao, Daming

    2014-01-01

    The deacetylase SIRT1 regulates multiple biological processes including cellular metabolism and aging. Importantly, SIRT1 can also inactivate the p53 tumor suppressor via deacetylation, suggesting a role in oncogenesis. Recently, SIRT1 was shown to be released from its endogenous inhibitor DBC1 by a process requiring AMPK and the phosphorylation of SIRT1 by yet undefined kinase(s). Here we provide further evidence that AMPK directly phosphorylates SIRT1 on T344, releasing it from DBC1. Furthermore, a phospho-mimetic SIRT1 (T334E) showed decreased binding to DBC1, supporting the importance of this phosphorylation in AMPK-mediated regulation of SIRT1 activity. In addition, inhibition of AMPK by Compound C led to increased p53 acetylation, suggesting a role for the AMPK/SIRT1 pathway in regulating p53 signaling. Together, our results support a hypothesis that AMPK negatively regulates p53 acetylation via phosphorylation of SIRT1 on T344. Furthermore, our findings also define the AMPK/SIRT1 axis as a possible targetable pathway to regulate p53 function. PMID:24959379

  15. Protein Kinase C Beta Regulates the D2-Like Dopamine Autoreceptor

    PubMed Central

    Luderman, Kathryn D.; Chen, Rong; Ferris, Mark J.; Jones, Sara R.; Gnegy, Margaret E.

    2014-01-01

    The focus of this study was the regulation of the D2-like dopamine autoreceptor (D2 autoreceptor) by protein kinase Cβ, a member of the protein kinase C (PKC) family. Together with the dopamine transporter, the D2 autoreceptor regulates the level of extracellular dopamine and thus dopaminergic signaling. PKC regulates neuronal signaling via several mechanisms, including desensitizing autoreceptors to increase the release of several different neurotransmitters. Here, using both PKCβ−/− mice and specific PKCβ inhibitors, we demonstrated that a lack of PKCβ activity enhanced the D2 autoreceptor-stimulated decrease in dopamine release following both chemical and electrical stimulations. Inhibition of PKCβ increased surface localization of D2R in mouse striatal synaptosomes, which could underlie the greater sensitivity to quinpirole following inhibition of PKCβ. PKCβ−/− mice displayed greater sensitivity to the quinpirole-induced suppression of locomotor activity, demonstrating that the regulation of the D2 autoreceptor by PKCβ is physiologically significant. Overall, we have found that PKCβ downregulates the D2 autoreceptor, providing an additional layer of regulation for dopaminergic signaling. We propose that in the absence of PKCβ activity, surface D2 autoreceptor localization and thus D2 autoreceptor signaling is increased, leading to less dopamine in the extracellular space and attenuated dopaminergic signaling. PMID:25446677

  16. Phosphorylation and inactivation of yeast 6-phosphofructo-2-kinase contribute to the regulation of glycolysis under hypotonic stress.

    PubMed

    Dihazi, H; Kessler, R; Eschrich, K

    2001-12-01

    Phosphorylation of yeast 6-phosphofructo-2-kinase and its role for the regulation of glycolysis under hypoosmotic conditions were investigated. 6-Phosphofructo-2-kinase was found to be phosphorylated in vitro by protein kinase C at serine 652 and thereby inactivated. Protein phosphatase 2A reversed the phosphorylative inhibition of the enzyme. When yeast cells were shifted to hypotonic media, 6-phosphofructo-2-kinase was found to be phosphorylated and inactivated. Under in vivo conditions, two phosphate residues were incorporated into the enzyme. One of them is bound to serine 652, indicating that this modification was probably caused by yeast protein kinase C1. The second phosphate is bound to Ser8 within the N-terminal peptide T(1-41) which contains several serine residues but no protein kinase C recognition sequence. Site-directed mutagenesis confirmed that the phosphorylation of serine 652 but not the N-terminal modification is responsible for the in vivo inactivation of 6-phosphofructo-2-kinase. The obtained results suggest that the phosphorylation of 6-phosphofructo-2-kinase mediates a response of the cells to an activation of the hypoosmolarity MAP kinase pathway. Via a suppression of glycolysis, the inactivation of 6-phosphofructo-2-kinase is expected to be responsible for the observed accumulation of glucose 6-phosphate, an essential precursor of the cell wall glucans, and the decrease of glycerol, an important osmolyte. PMID:11724581

  17. Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase.

    PubMed

    Brunskill, N J; Stuart, J; Tobin, A B; Walls, J; Nahorski, S

    1998-05-15

    Receptor-mediated endocytosis of albumin is an important function of the kidney proximal tubule epithelium. We have measured endocytosis of [125I]-albumin in opossum kidney cells and examined the regulation of this process by phosphatidylinositide 3-kinase (PI 3-kinase). Albumin endocytosis was inhibited by both wortmannin (IC50 6.9 nM) and LY294002 (IC50 6.5 microM) at concentrations that suggested the involvement of PI 3-kinase in its regulation. Recycling rates were unaffected. We transfected OK cells with either a wild-type p85 subunit of PI 3-kinase, or a dominant negative form of the p85 subunit (Deltap85) using the LacSwitch expression system. Transfects were screened by immunoblotting with anti-PI 3-kinase antibodies. Under basal conditions, transfects demonstrated no expression of p85 or Deltap85, but expression was briskly induced by treatment of the cells with IPTG (EC50 13.7 microM). Inhibition of PI 3-kinase activity by Deltap85 was confirmed by in vitro kinase assay of anti-phosphotyrosine immunoprecipitates from transfected cells stimulated with insulin. Expression of Deltap85 resulted in marked inhibition of albumin endocytosis, predominantly as a result of reduction of the Vmax of the transport process. Expression of p85 had no significant effect on albumin uptake. The results demonstrate that PI 3-kinase regulates an early step in the receptor-mediated endocytosis of albumin by kidney proximal tubular cells. PMID:9593770

  18. ERECTA and BAK1 Receptor Like Kinases Interact to Regulate Immune Responses in Arabidopsis.

    PubMed

    Jordá, Lucía; Sopeña-Torres, Sara; Escudero, Viviana; Nuñez-Corcuera, Beatriz; Delgado-Cerezo, Magdalena; Torii, Keiko U; Molina, Antonio

    2016-01-01

    ERECTA (ER) receptor-like kinase (RLK) regulates Arabidopsis thaliana organ growth, and inflorescence and stomatal development by interacting with the ERECTA-family genes (ERf) paralogs, ER-like 1 (ERL1) and ERL2, and the receptor-like protein (RLP) TOO MANY MOUTHS (TMM). ER also controls immune responses and resistance to pathogens such as the bacterium Pseudomonas syringae pv. tomato DC3000 (Pto) and the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM). We found that er null-mutant plants overexpressing an ER dominant-negative version lacking the cytoplasmic kinase domain (ERΔK) showed an enhanced susceptibility to PcBMM, suggesting that ERΔK associates and forms inactive complexes with additional RLKs/RLPs required for PcBMM resistance. Genetic analyses demonstrated that ER acts in a combinatorial specific manner with ERL1, ERL2, and TMM to control PcBMM resistance. Moreover, BAK1 (BRASSINOSTEROID INSENSITIVE 1-associated kinase 1) RLK, which together with ERf/TMM regulates stomatal patterning and resistance to Pto, was also found to have an unequal contribution with ER in regulating immune responses and resistance to PcBMM. Co-immunoprecipitation experiments in Nicotiana benthamiana further demonstrated BAK1-ER protein interaction. The secreted epidermal pattern factor peptides (EPF1 and EPF2), which are perceived by ERf members to specify stomatal patterning, do not seem to regulate ER-mediated immunity to PcBMM, since their inducible overexpression in A. thaliana did not impact on PcBMM resistance. Our results indicate that the multiproteic receptorsome formed by ERf, TMM and BAK1 modulates A. thaliana resistance to PcBMM, and suggest that the cues underlying ERf/TMM/BAK1-mediated immune responses are distinct from those regulating stomatal pattering. PMID:27446127

  19. Capsaicinoids regulate airway anion transporters through Rho kinase- and cyclic AMP-dependent mechanisms.

    PubMed

    Hibino, Yoshitaka; Morise, Masahiro; Ito, Yasushi; Mizutani, Takefumi; Matsuno, Tadakatsu; Ito, Satoru; Hashimoto, Naozumi; Sato, Mitsuo; Kondo, Masashi; Imaizumi, Kazuyoshi; Hasegawa, Yoshinori

    2011-10-01

    To investigate the effects of capsaicinoids on airway anion transporters, we recorded and analyzed transepithelial currents in human airway epithelial Calu-3 cells. Application of capsaicin (100 μM) attenuated vectorial anion transport, estimated as short-circuit currents (I(SC)), before and after stimulation by forskolin (10 μM) with concomitant reduction of cytosolic cyclic AMP (cAMP) levels. The capsaicin-induced inhibition of I(SC) was also observed in the response to 8-bromo-cAMP (1 mM, a cell-permeable cAMP analog) and 3-isobutyl-1-methylxanthine (1 mM, an inhibitor of phosphodiesterases). The capsaicin-induced inhibition of I(SC) was attributed to suppression of bumetanide (an inhibitor of the basolateral Na(+)-K(+)-2 Cl(-) cotransporter 1)- and 4,4'-dinitrostilbene-2,2'-disulfonic acid (an inhibitor of basolateral HCO(3)(-)-dependent anion transporters)-sensitive components, which reflect anion uptake via basolateral cAMP-dependent anion transporters. In contrast, capsaicin potentiated apical Cl(-) conductance, which reflects conductivity through the cystic fibrosis transmembrane conductance regulator, a cAMP-regulated Cl(-) channel. All these paradoxical effects of capsaicin were mimicked by capsazepine. Forskolin application also increased phosphorylated myosin phosphatase target subunit 1, and the phosphorylation was prevented by capsaicin and capsazepine, suggesting that these capsaicinoids assume aspects of Rho kinase inhibitors. We also found that the increments in apical Cl(-) conductance were caused by conventional Rho kinase inhibitors, Y-27632 (20 μM) and HA-1077 (20 μM), with selective inhibition of basolateral Na(+)-K(+)-2 Cl(-) cotransporter 1. Collectively, capsaicinoids inhibit cAMP-mediated anion transport through down-regulation of basolateral anion uptake, paradoxically accompanied by up-regulation of apical cystic fibrosis transmembrane conductance regulator-mediated anion conductance. The latter is mediated by inhibition of Rho-kinase

  20. ERECTA and BAK1 Receptor Like Kinases Interact to Regulate Immune Responses in Arabidopsis

    PubMed Central

    Jordá, Lucía; Sopeña-Torres, Sara; Escudero, Viviana; Nuñez-Corcuera, Beatriz; Delgado-Cerezo, Magdalena; Torii, Keiko U.; Molina, Antonio

    2016-01-01

    ERECTA (ER) receptor-like kinase (RLK) regulates Arabidopsis thaliana organ growth, and inflorescence and stomatal development by interacting with the ERECTA-family genes (ERf) paralogs, ER-like 1 (ERL1) and ERL2, and the receptor-like protein (RLP) TOO MANY MOUTHS (TMM). ER also controls immune responses and resistance to pathogens such as the bacterium Pseudomonas syringae pv. tomato DC3000 (Pto) and the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM). We found that er null-mutant plants overexpressing an ER dominant-negative version lacking the cytoplasmic kinase domain (ERΔK) showed an enhanced susceptibility to PcBMM, suggesting that ERΔK associates and forms inactive complexes with additional RLKs/RLPs required for PcBMM resistance. Genetic analyses demonstrated that ER acts in a combinatorial specific manner with ERL1, ERL2, and TMM to control PcBMM resistance. Moreover, BAK1 (BRASSINOSTEROID INSENSITIVE 1-associated kinase 1) RLK, which together with ERf/TMM regulates stomatal patterning and resistance to Pto, was also found to have an unequal contribution with ER in regulating immune responses and resistance to PcBMM. Co-immunoprecipitation experiments in Nicotiana benthamiana further demonstrated BAK1-ER protein interaction. The secreted epidermal pattern factor peptides (EPF1 and EPF2), which are perceived by ERf members to specify stomatal patterning, do not seem to regulate ER-mediated immunity to PcBMM, since their inducible overexpression in A. thaliana did not impact on PcBMM resistance. Our results indicate that the multiproteic receptorsome formed by ERf, TMM and BAK1 modulates A. thaliana resistance to PcBMM, and suggest that the cues underlying ERf/TMM/BAK1-mediated immune responses are distinct from those regulating stomatal pattering. PMID:27446127

  1. ErbB receptor signaling in astrocytes: a mediator of neuron-glia communication in the mature central nervous system.

    PubMed

    Sharif, Ariane; Prevot, Vincent

    2010-11-01

    Astrocytes are now recognized as active players in the developing and mature central nervous system. Each astrocyte contacts vascular structures and thousands of synapses within discrete territories. These cells receive a myriad of inputs and generate appropriate responses to regulate the function of brain microdomains. Emerging evidence has implicated receptors of the ErbB tyrosine kinase family in the integration and processing of neuronal inputs by astrocytes: ErbB receptors can be activated by a wide range of neuronal stimuli; they control critical steps of glutamate-glutamine metabolism; and they regulate the biosynthesis and release of various glial-derived neurotrophic factors, gliomediators and gliotransmitters. These key properties of astrocytic ErbB signaling in neuron-glia interactions have significance for the physiology of the mature central nervous system, as exemplified by the central control of reproduction within the hypothalamus, and are also likely to contribute to pathological situations, since both dysregulation of ErbB signaling and glial dysfunction occur in many neurological disorders. PMID:20685225

  2. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A.

    PubMed

    Borthwick, Lee A; Kerbiriou, Mathieu; Taylor, Christopher J; Cozza, Giorgio; Lascu, Ioan; Postel, Edith H; Cassidy, Diane; Trouvé, Pascal; Mehta, Anil; Robson, Louise; Muimo, Richmond

    2016-01-01

    Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36-54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351-727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia. PMID:26950439

  3. Mitogen-activated protein kinase/extracellular signal-regulated kinase 2 regulates cytoskeletal organization and chemotaxis via catalytic and microtubule-specific interactions.

    PubMed Central

    Reszka, A A; Bulinski, J C; Krebs, E G; Fischer, E H

    1997-01-01

    The extracellular signal-regulated kinases (ERKs) 1 and 2 are mitogen-activated protein kinases that act as key components in a signaling cascade linking growth factor receptors to the cytoskeleton and the nucleus. ERK2 mutants have been used to alter cytoskeletal regulation in Chinese hamster ovary cells without affecting cell growth or feedback signaling. Mutation of the unique loop L6 (residues 91-95), which is in a portion of the molecule that is cryptic upon the binding of ERK2 to the microtubules (MTs), generated significant morphological alterations. Most notable phenotypes were observed after expression of a combined mutant incorporating changes to both L6 and the TEY phosphorylation lip, including a 70% increase in cell spreading. Actin stress fibers in these cells, which normally formed a single broad parallel array, were arranged in three or more orientations or in fan-like arrays. MTs, which ordinarily extend longitudinally from the centrosome, spread radially, covering a larger surface area. Single, but not the double, mutations of the Thr and Tyr residues of the TEY phosphorylation lip caused a ca. 25% increase in cell spreading, accompanied by a threefold increase in chemotactic cell migration. Mutation of Lys-52 triggered a 48% increase in cell spreading but no alteration to chemotaxis. These findings suggest that wild-type ERK2 inhibits the organization of the cytoskeleton, the spreading of the cell, and chemotactic migration. This involves control of the orientation of actin and MTs and the positioning of focal adhesions via regulatory interactions that may occur on the MTs. Images PMID:9243503

  4. Role of Interaction and Nucleoside Diphosphate Kinase B in Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator Function by cAMP-Dependent Protein Kinase A

    PubMed Central

    Borthwick, Lee A.; Kerbiriou, Mathieu; Taylor, Christopher J.; Cozza, Giorgio; Lascu, Ioan; Postel, Edith H.; Cassidy, Diane; Trouvé, Pascal; Mehta, Anil; Robson, Louise; Muimo, Richmond

    2016-01-01

    Cystic fibrosis results from mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-dependent protein kinase A (PKA) and ATP-regulated chloride channel. Here, we demonstrate that nucleoside diphosphate kinase B (NDPK-B, NM23-H2) forms a functional complex with CFTR. In airway epithelia forskolin/IBMX significantly increases NDPK-B co-localisation with CFTR whereas PKA inhibitors attenuate complex formation. Furthermore, an NDPK-B derived peptide (but not its NDPK-A equivalent) disrupts the NDPK-B/CFTR complex in vitro (19-mers comprising amino acids 36–54 from NDPK-B or NDPK-A). Overlay (Far-Western) and Surface Plasmon Resonance (SPR) analysis both demonstrate that NDPK-B binds CFTR within its first nucleotide binding domain (NBD1, CFTR amino acids 351–727). Analysis of chloride currents reflective of CFTR or outwardly rectifying chloride channels (ORCC, DIDS-sensitive) showed that the 19-mer NDPK-B peptide (but not its NDPK-A equivalent) reduced both chloride conductances. Additionally, the NDPK-B (but not NDPK-A) peptide also attenuated acetylcholine-induced intestinal short circuit currents. In silico analysis of the NBD1/NDPK-B complex reveals an extended interaction surface between the two proteins. This binding zone is also target of the 19-mer NDPK-B peptide, thus confirming its capability to disrupt NDPK-B/CFTR complex. We propose that NDPK-B forms part of the complex that controls chloride currents in epithelia. PMID:26950439

  5. Kinase-Dependent and -Independent Roles of EphA2 in the Regulation of Prostate Cancer Invasion and Metastasis

    PubMed Central

    Taddei, Maria Letizia; Parri, Matteo; Angelucci, Adriano; Onnis, Barbara; Bianchini, Francesca; Giannoni, Elisa; Raugei, Giovanni; Calorini, Lido; Rucci, Nadia; Teti, Anna; Bologna, Mauro; Chiarugi, Paola

    2009-01-01

    Ligand-activated Eph tyrosine kinases regulate cellular repulsion, morphology, adhesion, and motility. EphA2 kinase is frequently up-regulated in several different types of cancers, including prostate, breast, colon, and lung carcinomas, as well as in melanoma. The existing data do not clarify whether EphA2 receptor phosphorylation or its simple overexpression, which likely leads to Eph kinase-independent responses, plays a role in the progression of malignant prostate cancer. In this study, we address the role of EphA2 tyrosine phosphorylation in prostate carcinoma cell adhesion, motility, invasion, and formation of metastases. Tumor cells expressing kinase-deficient EphA2 mutants, as well as an EphA2 variant lacking the cytoplasmic domain, are defective in ephrinA1-mediated cell rounding, retraction fiber formation, de-adhesion from the extracellular matrix, RhoA and Rac1 GTPase regulation, three-dimensional matrix invasion, and in vivo metastasis, suggesting a key role for EphA2 kinase activity. Nevertheless, EphA2 regulation of cell motility and invasion, as well as the formation of bone and visceral tumor colonies, reveals a component of both EphA2 kinase-dependent and -independent features. These results uncover a differential requirement for EphA2 kinase activity in the regulation of prostate carcinoma metastasis outcome, suggesting that although the kinase activity of EphA2 is required for the regulation of cell adhesion and cytoskeletal rearrangement, some distinct kinase-dependent and -independent pathways likely cooperate to drive cancer cell migration, invasion, and metastasis outcome. PMID:19264906

  6. Targeting astrocytes in bipolar disorder.

    PubMed

    Peng, Liang; Li, Baoman; Verkhratsky, Alexei

    2016-06-01

    Astrocytes are homeostatic cells of the central nervous system, which are critical for development and maintenance of synaptic transmission and hence of synaptically connected neuronal ensembles. Astrocytic densities are reduced in bipolar disorder, and therefore deficient astroglial function may contribute to overall disbalance in neurotransmission and to pathological evolution. Classical anti-bipolar drugs (lithium salts, valproic acid and carbamazepine) affect expression of astroglial genes and modify astroglial signalling and homeostatic cascades. Many effects of both antidepressant and anti-bipolar drugs are exerted through regulation of glutamate homeostasis and glutamatergic transmission, through K(+) buffering, through regulation of calcium-dependent phospholipase A2 (that controls metabolism of arachidonic acid) or through Ca(2+) homeostatic and signalling pathways. Sometimes anti-depressant and anti-bipolar drugs exert opposite effects, and some effects on gene expression in drug treated animals are opposite in neurones vs. astrocytes. Changes in the intracellular pH induced by anti-bipolar drugs affect uptake of myo-inositol and thereby signalling via inositoltrisphosphate (InsP3), this being in accord with one of the main theories of mechanism of action for these drugs. PMID:27015045

  7. The ubiquitin-associated domain of AMPK-related kinases regulates conformation and LKB1-mediated phosphorylation and activation

    PubMed Central

    Jaleel, Mahaboobi; Villa, Fabrizio; Deak, Maria; Toth, Rachel; Prescott, Alan R.; van Aalten, Daan M. F.; Alessi, Dario R.

    2006-01-01

    Recent work indicates that the LKB1 tumour suppressor protein kinase, which is mutated in Peutz–Jeghers cancer syndrome, phosphorylates and activates a group of protein kinases that are related to AMPK (AMP-activated protein kinase). Ten of the 14 AMPK-related protein kinases activated by LKB1, including SIK (salt-induced kinase), MARK (microtubule-affinity-regulating kinase) and BRSK (brain-specific kinase) isoforms, possess a ubiquitin-associated (UBA) domain immediately C-terminal to the kinase catalytic domain. These are the only protein kinases in the human genome known to possess a UBA domain, but their roles in regulating AMPK-related kinases are unknown. We have investigated the roles that the UBA domain may play in regulating these enzymes. Limited proteolysis of MARK2 revealed that the kinase and UBA domains were contained within a fragment that was resistant to trypsin proteolysis. SAXS (small-angle X-ray scattering) analysis of inactive and active LKB1-phosphorylated MARK2 revealed that activation of MARK2 is accompanied by a significant conformational change that alters the orientation of the UBA domain with respect to the catalytic domain. Our results indicate that none of the UBA domains found in AMPK-related kinases interact with polyubiquitin or other ubiquitin-like molecules. Instead, the UBA domains appear to play an essential conformational role and are required for the LKB1-mediated phosphorylation and activation of AMPK-related kinases. This is based on the findings that mutation or removal of the UBA domains of several AMPK-related kinases, including isoforms of MARK, SIK and BRSK, markedly impaired the catalytic activity and LKB1-mediated phosphorylation of these enzymes. We also provide evidence that the UBA domains do not function as LKB1–STRAD (STE20-related adaptor)–MO25 (mouse protein 25) docking/interacting sites and that mutations in the UBA domain of SIK suppressed the ability of SIK to localize within punctate regions of the

  8. TRPA1 Channels Are Regulators of Astrocyte Basal Calcium Levels and Long-Term Potentiation via Constitutive d-Serine Release

    PubMed Central

    Shigetomi, Eiji; Jackson-Weaver, Olan; Huckstepp, Robert T.

    2013-01-01

    Astrocytes are found throughout the brain where they make extensive contacts with neurons and synapses. Astrocytes are known to display intracellular Ca2+ signals and release signaling molecules such as d-serine into the extracellular space. However, the role(s) of astrocyte Ca2+ signals in hippocampal long-term potentiation (LTP), a form of synaptic plasticity involved in learning and memory, remains unclear. Here, we explored a recently discovered novel TRPA1 channel-mediated transmembrane Ca2+ flux pathway in astrocytes. Specifically, we determined whether block or genetic deletion of TRPA1 channels affected LTP of Schaffer collateral to CA1 pyramidal neuron synapses. Using pharmacology, TRPA1−/− mice, imaging, electrophysiology, and d-serine biosensors, our data indicate that astrocyte TRPA1 channels contribute to basal Ca2+ levels and are required for constitutive d-serine release into the extracellular space, which contributes to NMDA receptor-dependent LTP. The findings have broad relevance for the study of astrocyte–neuron interactions by demonstrating how TRPA1 channel-mediated fluxes contribute to astrocyte basal Ca2+ levels and neuronal function via constitutive d-serine release. PMID:23761909

  9. Integrin-linked kinase regulates the niche of quiescent epidermal stem cells

    PubMed Central

    Morgner, Jessica; Ghatak, Sushmita; Jakobi, Tobias; Dieterich, Christoph; Aumailley, Monique; Wickström, Sara A.

    2015-01-01

    Stem cells reside in specialized niches that are critical for their function. Quiescent hair follicle stem cells (HFSCs) are confined within the bulge niche, but how the molecular composition of the niche regulates stem cell behaviour is poorly understood. Here we show that integrin-linked kinase (ILK) is a key regulator of the bulge extracellular matrix microenvironment, thereby governing the activation and maintenance of HFSCs. ILK mediates deposition of inverse laminin (LN)-332 and LN-511 gradients within the basement membrane (BM) wrapping the hair follicles. The precise BM composition tunes activities of Wnt and transforming growth factor-β pathways and subsequently regulates HFSC activation. Notably, reconstituting an optimal LN microenvironment restores the altered signalling in ILK-deficient cells. Aberrant stem cell activation in ILK-deficient epidermis leads to increased replicative stress, predisposing the tissue to carcinogenesis. Overall, our findings uncover a critical role for the BM niche in regulating stem cell activation and thereby skin homeostasis. PMID:26349061

  10. Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development

    PubMed Central

    Loponte, Sara; Segré, Chiara V.; Senese, Silvia; Miccolo, Claudia; Santaguida, Stefano; Deflorian, Gianluca; Citro, Simona; Mattoscio, Domenico; Pisati, Federica; Moser, Mirjam A.; Visintin, Rosella; Seiser, Christian; Chiocca, Susanna

    2016-01-01

    Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology. PMID:27458029

  11. Protein kinase Darkener of apricot and its substrate EF1γ regulate organelle transport along microtubules.

    PubMed

    Serpinskaya, Anna S; Tuphile, Karine; Rabinow, Leonard; Gelfand, Vladimir I

    2014-01-01

    Regulation of organelle transport along microtubules is important for proper distribution of membrane organelles and protein complexes in the cytoplasm. RNAi-mediated knockdown in cultured Drosophila S2 cells demonstrates that two microtubule-binding proteins, a unique isoform of Darkener of apricot (DOA) protein kinase, and its substrate, translational elongation factor EF1γ, negatively regulate transport of several classes of membrane organelles along microtubules. Inhibition of transport by EF1γ requires its phosphorylation by DOA on serine 294. Together, our results indicate a new role for two proteins that have not previously been implicated in regulation of the cytoskeleton. These results further suggest that the biological role of some of the proteins binding to the microtubule track is to regulate cargo transport along these tracks. PMID:24163433

  12. Negative regulation of Caenorhabditis elegans epidermal damage responses by death-associated protein kinase

    PubMed Central

    Tong, Amy; Lynn, Grace; Ngo, Vy; Wong, Daniel; Moseley, Sarah L.; Ewbank, Jonathan J.; Goncharov, Alexandr; Wu, Yi-Chun; Pujol, Nathalie; Chisholm, Andrew D.

    2009-01-01

    Wounding of epidermal layers triggers multiple coordinated responses to damage. We show here that the Caenorhabditis elegans ortholog of the tumor suppressor death-associated protein kinase, dapk-1, acts as a previously undescribed negative regulator of barrier repair and innate immune responses to wounding. Loss of DAPK-1 function results in constitutive formation of scar-like structures in the cuticle, and up-regulation of innate immune responses to damage. Overexpression of DAPK-1 represses innate immune responses to needle wounding. Up-regulation of innate immune responses in dapk-1 requires the TIR-1/p38 signal transduction pathway; loss of function in this pathway synergizes with dapk-1 to drastically reduce adult lifespan. Our results reveal a previously undescribed function for the DAPK tumor suppressor family in regulation of epithelial damage responses. PMID:19164535

  13. Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development.

    PubMed

    Loponte, Sara; Segré, Chiara V; Senese, Silvia; Miccolo, Claudia; Santaguida, Stefano; Deflorian, Gianluca; Citro, Simona; Mattoscio, Domenico; Pisati, Federica; Moser, Mirjam A; Visintin, Rosella; Seiser, Christian; Chiocca, Susanna

    2016-01-01

    Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology. PMID:27458029

  14. Differences in kinase-mediated regulation of cell cycle progression in normal and transformed cells

    SciTech Connect

    Crissman, H.A.; Gadbois, D.M.; Tobey, R.A.; Stevenson, A.P.; Kraemer, P.M.; Bustos, L.D.; Dickson, J.A.; Bradbury, E.M. )

    1993-01-01

    Staurosporine (Stsp), a general protein kinase inhibitor, was used to investigate the role of kinase-mediated mechanisms in regulating mammalian cell proliferation. Low levels of Stsp (1-2nM) prevented nontransformed cells from entering S phase, indicating that protein phosphorylation processes are essential for commitment of DNA replication in normal cells. Cells resumed cycling when Stsp was removed. The period of sensitivity of nontransformed human diploid fibroblasts to low levels of the drug commenced 3 h later than the G0/G1 boundary and extended through the G1/S boundary. The initial block point at 3 h corresponds neither to the serum nor the amino acid restriction point. In contrast, neither low nor high concentrations (100nm) of Stsp affected G1 progression of transformed cells. High drug concentrations blocked normal cells in G1 and G2 but affected only G2-progression in transformed cells. These results indicate that kinase-mediated regulation of DNA replication is lost as a result of neoplastic transformation, but the G2-arrest mechanism remains intact.

  15. Changes in Dynamics upon Oligomerization Regulate Substrate Binding and Allostery in Amino Acid Kinase Family Members

    PubMed Central

    Marcos, Enrique; Crehuet, Ramon; Bahar, Ivet

    2011-01-01

    Oligomerization is a functional requirement for many proteins. The interfacial interactions and the overall packing geometry of the individual monomers are viewed as important determinants of the thermodynamic stability and allosteric regulation of oligomers. The present study focuses on the role of the interfacial interactions and overall contact topology in the dynamic features acquired in the oligomeric state. To this aim, the collective dynamics of enzymes belonging to the amino acid kinase family both in dimeric and hexameric forms are examined by means of an elastic network model, and the softest collective motions (i.e., lowest frequency or global modes of motions) favored by the overall architecture are analyzed. Notably, the lowest-frequency modes accessible to the individual subunits in the absence of multimerization are conserved to a large extent in the oligomer, suggesting that the oligomer takes advantage of the intrinsic dynamics of the individual monomers. At the same time, oligomerization stiffens the interfacial regions of the monomers and confers new cooperative modes that exploit the rigid-body translational and rotational degrees of freedom of the intact monomers. The present study sheds light on the mechanism of cooperative inhibition of hexameric N-acetyl-L-glutamate kinase by arginine and on the allosteric regulation of UMP kinases. It also highlights the significance of the particular quaternary design in selectively determining the oligomer dynamics congruent with required ligand-binding and allosteric activities. PMID:21980279

  16. TCR-induced Akt serine 473 phosphorylation is regulated by protein kinase C-alpha

    SciTech Connect

    Yang, Lifen; Qiao, Guilin; Ying, Haiyan; Zhang, Jian; Yin, Fei

    2010-09-10

    Research highlights: {yields} Conventional PKC positively regulates TCR-induced phosphorylation of Akt. {yields} PKC-alpha is the PDK-2 responsible for phosphorylating Akt at Ser{sup 473} upon TCR stimulation. {yields} Knockdown of PKC-alpha decreases TCR-induced Akt phosphorylation. -- Abstract: Akt signaling plays a central role in T cell functions, such as proliferation, apoptosis, and regulatory T cell development. Phosphorylation at Ser{sup 473} in the hydrophobic motif, along with Thr{sup 308} in its activation loop, is considered necessary for Akt function. It is widely accepted that phosphoinositide-dependent kinase 1 (PDK-1) phosphorylates Akt at Thr{sup 308}, but the kinase(s) responsible for phosphorylating Akt at Ser{sup 473} (PDK-2) remains elusive. The existence of PDK-2 is considered to be specific to cell type and stimulus. PDK-2 in T cells in response to TCR stimulation has not been clearly defined. In this study, we found that conventional PKC positively regulated TCR-induced Akt Ser{sup 473} phosphorylation. PKC-alpha purified from T cells can phosphorylate Akt at Ser{sup 473} in vitro upon TCR stimulation. Knockdown of PKC-alpha in T-cell-line Jurkat cells reduced TCR-induced phosphorylation of Akt as well as its downstream targets. Thus our results suggest that PKC-alpha is a candidate for PDK-2 in T cells upon TCR stimulation.

  17. The lipid kinase PIP5K1C regulates pain signaling and sensitization

    PubMed Central

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

    2014-01-01

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

  18. JAK2 Tyrosine Kinase Phosphorylates and Is Negatively Regulated by Centrosomal Protein Ninein

    PubMed Central

    Jay, Jennifer; Hammer, Alan; Nestor-Kalinoski, Andrea

    2014-01-01

    JAK2 is a cytoplasmic tyrosine kinase critical for cytokine signaling. In this study, we have identified a novel centrosome-associated complex containing ninein and JAK2. We have found that active JAK2 localizes around the mother centrioles, where it partly colocalizes with ninein, a protein involved in microtubule (MT) nucleation and anchoring. We demonstrated that JAK2 is an important regulator of centrosome function. Depletion of JAK2 or use of JAK2-null cells causes defects in MT anchoring and increased numbers of cells with mitotic defects; however, MT nucleation is unaffected. We showed that JAK2 directly phosphorylates the N terminus of ninein while the C terminus of ninein inhibits JAK2 kinase activity in vitro. Overexpressed wild-type (WT) or C-terminal (amino acids 1179 to 1931) ninein inhibits JAK2. This ninein-dependent inhibition of JAK2 significantly decreases prolactin- and interferon gamma (IFN-γ)-induced tyrosyl phosphorylation of STAT1 and STAT5. Downregulation of ninein enhances JAK2 activation. These results indicate that JAK2 is a novel member of centrosome-associated complex and that this localization regulates both centrosomal function and JAK2 kinase activity, thus controlling cytokine-activated molecular pathways. PMID:25332239

  19. c-Abl kinase regulates the protein binding activity of c-Crk.

    PubMed Central

    Feller, S M; Knudsen, B; Hanafusa, H

    1994-01-01

    c-Crk is a proto-oncogene product composed largely of Src homology (SH) 2 and 3 domains. We have identified a kinase activity, which binds to the first Crk SH3 domain and phosphorylates c-Crk on tyrosine 221 (Y221), as c-Abl. c-Abl has a strong preference for c-Crk, when compared with common tyrosine kinase substrates. The phosphorylation of c-Crk Y221 creates a binding site for the Crk SH2 domain. Bacterially expressed c-Crk protein lacks phosphorylation on Y221 and can bind specifically to several proteins, while mammalian c-Crk, which is phosphorylated on tyrosine, remains uncomplexed. The protein binding activity of c-Crk is therefore likely regulated by a mechanism similar to that of the Src family kinases. v-Crk is truncated before c-Crk Y221 and forms constitutive complexes with c-Abl and other proteins. Our results suggest that c-Abl regulates c-Crk function and that it could be involved in v-Crk transformation. Images PMID:8194526

  20. Differential regulation and role of Interleukin-1 Receptor Associated Kinase-M in innate immunity signaling

    PubMed Central

    Su, Jianmin; Xie, Qifa; Wilson, Ingred; Li, Liwu

    2007-01-01

    Toll-like-receptor mediated signaling is finely regulated by a complex intracellular protein network including the interleukin-1 receptor associate kinases (IRAKs). IRAK-4, 1, and 2 may positively regulate innate immunity signaling through the activation of various downstream kinases such as MAPKs. In contrast, IRAK-M plays an inhibitory role through unknown mechanism. In this report, we show that IRAK-M is ubiquitously present in the cell, and becomes exclusively cytoplasmic upon bacterial lipoprotein Pam3CSK4 challenge. Furthermore, using bone marrow derived macrophages (BMDM) from wild type, IRAK1−/−, and IRAK-M−/− mice, we have herein demonstrated that IRAK-M selectively attenuates bacterial lipopeptide Pam3CSK4-induced p38 activation, but not ERK or JNK. IRAK1−/− and IRAK-M−/− BMDM display distinct activation profile of various MAP kinases upon Pam3CSK4 challenge, indicating that IRAK-M exerts its inhibitory effect through an IRAK1 independent pathway. Pam3CSK4 challenge leads to rapid decrease of MKP-1 protein level in IRAK-M−/− BMDM as well as THP-1 cells with decreased IRAK-M expression through siRNA interference. Our findings indicate that IRAK-M selectively attenuates p38 activation and inhibits innate immunity through stabilizing MKP-1. PMID:17379480

  1. Algal dual-specificity tyrosine phosphorylation-regulated kinase, triacylglycerol accumulation regulator1, regulates accumulation of triacylglycerol in nitrogen or sulfur deficiency.

    PubMed

    Kajikawa, Masataka; Sawaragi, Yuri; Shinkawa, Haruka; Yamano, Takashi; Ando, Akira; Kato, Misako; Hirono, Masafumi; Sato, Naoki; Fukuzawa, Hideya

    2015-06-01

    Although microalgae accumulate triacylglycerol (TAG) and starch in response to nutrient-deficient conditions, the regulatory mechanisms are poorly understood. We report here the identification and characterization of a kinase, triacylglycerol accumulation regulator1 (TAR1), that is a member of the yeast (Saccharomyces cerevisiae) Yet another kinase1 (Yak1) subfamily in the dual-specificity tyrosine phosphorylation-regulated kinase family in a green alga (Chlamydomonas reinhardtii). The kinase domain of TAR1 showed auto- and transphosphorylation activities. A TAR1-defective mutant, tar1-1, accumulated TAG to levels 0.5- and 0.1-fold of those of a wild-type strain in sulfur (S)- and nitrogen (N)-deficient conditions, respectively. In N-deficient conditions, tar1-1 showed more pronounced arrest of cell division than the wild type, had increased cell size and cell dry weight, and maintained chlorophyll and photosynthetic activity, which were not observed in S-deficient conditions. In N-deficient conditions, global changes in expression levels of N deficiency-responsive genes in N assimilation and tetrapyrrole metabolism were noted between tar1-1 and wild-type cells. These results indicated that TAR1 is a regulator of TAG accumulation in S- and N-deficient conditions, and it functions in cell growth and repression of photosynthesis in conditions of N deficiency. PMID:25922058

  2. Algal Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase, Triacylglycerol Accumulation Regulator1, Regulates Accumulation of Triacylglycerol in Nitrogen or Sulfur Deficiency1[OPEN

    PubMed Central

    Kajikawa, Masataka; Sawaragi, Yuri; Shinkawa, Haruka; Yamano, Takashi; Ando, Akira; Kato, Misako; Hirono, Masafumi; Sato, Naoki

    2015-01-01

    Although microalgae accumulate triacylglycerol (TAG) and starch in response to nutrient-deficient conditions, the regulatory mechanisms are poorly understood. We report here the identification and characterization of a kinase, triacylglycerol accumulation regulator1 (TAR1), that is a member of the yeast (Saccharomyces cerevisiae) Yet another kinase1 (Yak1) subfamily in the dual-specificity tyrosine phosphorylation-regulated kinase family in a green alga (Chlamydomonas reinhardtii). The kinase domain of TAR1 showed auto- and transphosphorylation activities. A TAR1-defective mutant, tar1-1, accumulated TAG to levels 0.5- and 0.1-fold of those of a wild-type strain in sulfur (S)- and nitrogen (N)-deficient conditions, respectively. In N-deficient conditions, tar1-1 showed more pronounced arrest of cell division than the wild type, had increased cell size and cell dry weight, and maintained chlorophyll and photosynthetic activity, which were not observed in S-deficient conditions. In N-deficient conditions, global changes in expression levels of N deficiency-responsive genes in N assimilation and tetrapyrrole metabolism were noted between tar1-1 and wild-type cells. These results indicated that TAR1 is a regulator of TAG accumulation in S- and N-deficient conditions, and it functions in cell growth and repression of photosynthesis in conditions of N deficiency. PMID:25922058

  3. Proteomic analysis reveals GIT1 as a novel mTOR complex component critical for mediating astrocyte survival.

    PubMed

    Smithson, Laura J; Gutmann, David H

    2016-06-15

    As a critical regulator of cell growth, the mechanistic target of rapamycin (mTOR) protein operates as part of two molecularly and functionally distinct complexes. Herein, we demonstrate that mTOR complex molecular composition varies in different somatic tissues. In astrocytes and neural stem cells, we identified G-protein-coupled receptor kinase-interacting protein 1 (GIT1) as a novel mTOR-binding protein, creating a unique mTOR complex lacking Raptor and Rictor. Moreover, GIT1 binding to mTOR is regulated by AKT activation and is essential for mTOR-mediated astrocyte survival. Together, these data reveal that mTOR complex function is partly dictated by its molecuflar composition in different cell types. PMID:27340174

  4. Mechanisms underlying the protein-kinase mediated regulation of the HERG potassium channel synthesis

    PubMed Central

    Krishnan, Yamini; Li, Yan; Zheng, Renjian; Kanda, Vikram; McDonald, Thomas V.

    2012-01-01

    The HERG (human ether-a-go-go related gene) potassium channel aids in repolarization of the cardiomyocyte membrane at the end of each action potential. We have previously shown that sustained protein kinase A or C (PKA and PKC) activity specifically enhances channel synthesis over the course of hours to days in heterologous expression and cardiac myocytes. The kinase-mediated augmentation of the channel is post-transcriptional and occurs near or at the endoplasmic reticulum. Here we report our further investigations into the mechanisms of kinase-mediated augmentation of HERG channel protein. We show that HERG channel phosphorylation alone is not sufficient for the PKA-dependent increase to occur. In vitro translation studies indicate that an additional factor is required for the process. Pharmacologic inhibitors suggest that the channel augmentation is not due to kinase-mediated alteration in proteasome or lysosome activity. PKA activation had no effect on stability of HERG mRNA and polyribosomal profiling showed that kinase activity did not elevate translation from low to high rates. Transcriptional inhibition results suggest that the additional cellular factor is a PKA-regulated protein. Together, these findings suggest that PKA-mediated augmentation of HERG abundance is more complex than previously appreciated involving enhancement of already active translation rates, phosphorylation of the channel protein and at least one other cAMP/PKA-responsive protein. Further exploration of molecular components of this regulatory pathway will be necessary to determine exact mechanism and the biomedical impact of this process in vivo. PMID:22613764

  5. Identification of genes associated with the astrocyte-specific gene Gfap during astrocyte differentiation

    PubMed Central

    Ito, Kenji; Sanosaka, Tsukasa; Igarashi, Katsuhide; Ideta-Otsuka, Maky; Aizawa, Akira; Uosaki, Yuichi; Noguchi, Azumi; Arakawa, Hirokazu; Nakashima, Kinichi; Takizawa, Takumi

    2016-01-01

    Chromosomes and genes are non-randomly arranged within the mammalian cell nucleus, and gene clustering is of great significance in transcriptional regulation. However, the relevance of gene clustering and their expression during the differentiation of neural precursor cells (NPCs) into astrocytes remains unclear. We performed a genome-wide enhanced circular chromosomal conformation capture (e4C) to screen for genes associated with the astrocyte-specific gene glial fibrillary acidic protein (Gfap) during astrocyte differentiation. We identified 18 genes that were specifically associated with Gfap and expressed in NPC-derived astrocytes. Our results provide additional evidence for the functional significance of gene clustering in transcriptional regulation during NPC differentiation. PMID:27041678

  6. PfIRR Interacts with HrIGF-I and Activates the MAP-kinase and PI3-kinase Signaling Pathways to Regulate Glycogen Metabolism in Pinctada fucata

    PubMed Central

    Shi, Yu; He, Mao-xian

    2016-01-01

    The insulin-induced mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways are major intracellular signaling modules and conserved among eukaryotes that are known to regulate diverse cellular processes. However, they have not been investigated in the mollusk species Pinctada fucata. Here, we demonstrate that insulin-related peptide receptor of P. fucata (pfIRR) interacts with human recombinant insulin-like growth factor I (hrIGF-I), and stimulates the MAPK and PI3K signaling pathways in P. fucata oocytes. We also show that inhibition of pfIRR by the inhibitor PQ401 significantly attenuates the basal and hrIGF-I-induced phosphorylation of MAPK and PI3K/Akt at amino acid residues threonine 308 and serine 473. Furthermore, our experiments show that there is cross-talk between the MAPK and PI3K/Akt pathways, in which MAPK kinase positively regulates the PI3K pathway, and PI3K positively regulates the MAPK cascade. Intramuscular injection of hrIGF-I stimulates the PI3K and MAPK pathways to increase the expression of pfirr, protein phosphatase 1, glucokinase, and the phosphorylation of glycogen synthase, decreases the mRNA expression of glycogen synthase kinase-3 beta, decreases glucose levels in hemocytes, and increases glycogen levels in digestive glands. These results suggest that the MAPK and PI3K pathways in P. fucata transmit the hrIGF-I signal to regulate glycogen metabolism. PMID:26911653

  7. Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis.

    PubMed

    Weigel, Christoph; Veldwijk, Marlon R; Oakes, Christopher C; Seibold, Petra; Slynko, Alla; Liesenfeld, David B; Rabionet, Mariona; Hanke, Sabrina A; Wenz, Frederik; Sperk, Elena; Benner, Axel; Rösli, Christoph; Sandhoff, Roger; Assenov, Yassen; Plass, Christoph; Herskind, Carsten; Chang-Claude, Jenny; Schmezer, Peter; Popanda, Odilia

    2016-01-01

    Radiotherapy is a fundamental part of cancer treatment but its use is limited by the onset of late adverse effects in the normal tissue, especially radiation-induced fibrosis. Since the molecular causes for fibrosis are largely unknown, we analyse if epigenetic regulation might explain inter-individual differences in fibrosis risk. DNA methylation profiling of dermal fibroblasts obtained from breast cancer patients prior to irradiation identifies differences associated with fibrosis. One region is characterized as a differentially methylated enhancer of diacylglycerol kinase alpha (DGKA). Decreased DNA methylation at this enhancer enables recruitment of the profibrotic transcription factor early growth response 1 (EGR1) and facilitates radiation-induced DGKA transcription in cells from patients later developing fibrosis. Conversely, inhibition of DGKA has pronounced effects on diacylglycerol-mediated lipid homeostasis and reduces profibrotic fibroblast activation. Collectively, DGKA is an epigenetically deregulated kinase involved in radiation response and may serve as a marker and therapeutic target for personalized radiotherapy. PMID:26964756

  8. Role of diacylglycerol kinase in cellular regulatory processes: a new regulator for cardiomyocyte hypertrophy.

    PubMed

    Takeishi, Yasuchika; Goto, Kaoru; Kubota, Isao

    2007-09-01

    Diacylglycerol (DAG) kinase (DGK) phosphorylates and converts DAG to phosphatidic acid. DGK regulates cellular DAG levels and attenuates DAG signaling. The 10 mammalian DGK isoforms have been identified to date. In cardiac myocytes, DGKalpha, epsilon, and zeta are expressed, and DGKzeta is the predominant isoform. DGKzeta inhibits protein kinase C (PKC) activation and subsequent hypertrophic programs in response to endothelin-1 (ET-1) in neonatal rat cardiomyocytes. DGKzeta blocks cardiac hypertrophy induced by G protein-coupled receptor agonists and pressure overload in vivo. DGKzeta attenuates ventricular remodeling and improves survival after myocardial infarction. These data provide a novel insight for subcellular mechanisms of cardiac hypertrophy and heart failure, and DGKzeta may be a new therapeutic target to prevent cardiac hypertrophy and progression to heart failure. PMID:17659347

  9. Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis

    PubMed Central

    Weigel, Christoph; Veldwijk, Marlon R.; Oakes, Christopher C.; Seibold, Petra; Slynko, Alla; Liesenfeld, David B.; Rabionet, Mariona; Hanke, Sabrina A.; Wenz, Frederik; Sperk, Elena; Benner, Axel; Rösli, Christoph; Sandhoff, Roger; Assenov, Yassen; Plass, Christoph; Herskind, Carsten; Chang-Claude, Jenny; Schmezer, Peter; Popanda, Odilia

    2016-01-01

    Radiotherapy is a fundamental part of cancer treatment but its use is limited by the onset of late adverse effects in the normal tissue, especially radiation-induced fibrosis. Since the molecular causes for fibrosis are largely unknown, we analyse if epigenetic regulation might explain inter-individual differences in fibrosis risk. DNA methylation profiling of dermal fibroblasts obtained from breast cancer patients prior to irradiation identifies differences associated with fibrosis. One region is characterized as a differentially methylated enhancer of diacylglycerol kinase alpha (DGKA). Decreased DNA methylation at this enhancer enables recruitment of the profibrotic transcription factor early growth response 1 (EGR1) and facilitates radiation-induced DGKA transcription in cells from patients later developing fibrosis. Conversely, inhibition of DGKA has pronounced effects on diacylglycerol-mediated lipid homeostasis and reduces profibrotic fibroblast activation. Collectively, DGKA is an epigenetically deregulated kinase involved in radiation response and may serve as a marker and therapeutic target for personalized radiotherapy. PMID:26964756

  10. Metallothionein gene expression is regulated by serum factors and activators of protein kinase C.

    PubMed Central

    Imbra, R J; Karin, M

    1987-01-01

    The exact physiological role of metallothionein (MT) is not clear. It has been suggested that these low-molecular-weight, highly inducible, heavy-metal-binding proteins serve in the regulation of intracellular Zn metabolism. Among the Zn-requiring systems are several enzymes involved in DNA replication and repair. Therefore, during periods of active DNA synthesis there is likely to be an increased demand for Zn, which could be met by elevated MT synthesis. For that reason, we examined whether stimulation of cellular proliferation leads to increased expression of MT. We report here that treatment of cultured mammalian cells with serum growth factors and activators of protein kinase C, all of which are known to have growth stimulatory activity, led to induction of MT mRNA. One of the required steps in the signal transduction pathways triggered by these agents, ending in MT induction, appears to be the activation of protein kinase C. Images PMID:3600629

  11. Regulation of Beta-Cell Function and Mass by the Dual Leucine Zipper Kinase.

    PubMed

    Oetjen, Elke

    2016-06-01

    Diabetes mellitus is one of the most rapidly increasing diseases worldwide, whereby approximately 90-95% of patients suffer from type 2 diabetes. Considering its micro- and macrovascular complications like blindness and myocardial infarction, a reliable anti-diabetic treatment is needed. Maintaining the function and the mass of the insulin producing beta-cells despite elevated levels of beta-cell-toxic prediabetic signals represents a desirable mechanism of action of anti-diabetic drugs. The dual leucine zipper kinase (DLK) inhibits the action of two transcription factors within the beta-cell, thereby interfering with insulin secretion and production and the conservation of beta-cell mass. Furthermore, DLK action is regulated by prediabetic signals. Hence, the inhibition of this kinase might protect beta-cells against beta-cell-toxic prediabetic signals and prevent the development of diabetes. DLK might thus present a novel drug target for the treatment of diabetes mellitus type 2. PMID:27100796

  12. Dynamic conformational ensembles regulate casein kinase-1 isoforms: Insights from molecular dynamics and molecular docking studies.

    PubMed

    Singh, Surya Pratap; Gupta, Dwijendra K

    2016-04-01

    Casein kinase-1 (CK1) isoforms actively participate in the down-regulation of canonical Wnt signaling pathway; however recent studies have shown their active roles in oncogenesis of various tissues through this pathway. Functional loss of two isoforms (CK1-α/ε) has been shown to activate the carcinogenic pathway which involves the stabilization of of cytoplasmic β-catenin. Development of anticancer therapeutics is very laborious task and depends upon the structural and conformational details of the target. This study focuses on, how the structural dynamics and conformational changes of two CK1 isoforms are synchronized in carcinogenic pathway. The conformational dynamics in kinases is the responsible for their action as has been supported by the molecular docking experiments. PMID:26788877

  13. A-kinase anchoring proteins: molecular regulators of the cardiac stress response.

    PubMed

    Diviani, Dario; Maric, Darko; Pérez López, Irene; Cavin, Sabrina; Del Vescovo, Cosmo D

    2013-04-01

    In response to stress or injury the heart undergoes a pathological remodeling process, associated with hypertrophy, cardiomyocyte death and fibrosis, that ultimately causes cardiac dysfunction and heart failure. It has become increasingly clear that signaling events associated with these pathological cardiac remodeling events are regulated by scaffolding and anchoring proteins, which allow coordination of pathological signals in space and time. A-kinase anchoring proteins (AKAPs) constitute a family of functionally related proteins that organize multiprotein signaling complexes that tether the cAMP-dependent protein kinase (PKA) as well as other signaling enzymes to ensure integration and processing of multiple signaling pathways. This review will discuss the role of AKAPs in the cardiac response to stress. Particular emphasis will be given to the adaptative process associated with cardiac hypoxia as well as the remodeling events linked to cardiac hypertrophy and heart failure. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction. PMID:22889610

  14. Regulation of Protein Phosphatase 1I by Cdc25C-associated Kinase 1 (C-TAK1) and PFTAIRE Protein Kinase*

    PubMed Central

    Platholi, Jimcy; Federman, Anna; Detert, Julia A.; Heerdt, Paul; Hemmings, Hugh C.

    2014-01-01

    Protein phosphatase 1I (PP-1I) is a major endogenous form of protein phosphatase 1 (PP-1) that consists of the core catalytic subunit PP-1c and the regulatory subunit inhibitor 2 (I-2). Phosphorylation of the Thr-72 residue of I-2 is required for activation of PP-1I. We studied the effects of two protein kinases identified previously in purified brain PP-1I by mass spectrometry, Cdc25C-associated kinase 1 (C-TAK1) and PFTAIRE (PFTK1) kinase, for their ability to regulate PP-1I. Purified C-TAK1 phosphorylated I-2 in reconstituted PP-1I (PP-1c·I-2) on Ser-71, which resulted in partial inhibition of its ATP-dependent phosphatase activity and inhibited subsequent phosphorylation of Thr-72 by the exogenous activating kinase GSK-3. In contrast, purified PFTK1 phosphorylated I-2 at Ser-86, a site known to potentiate Thr-72 phosphorylation and activation of PP-1I phosphatase activity by GSK-3. These findings indicate that brain PP-1I associates with and is regulated by the associated protein kinases C-TAK1 and PFTK1. Multisite phosphorylation of the I-2 regulatory subunit of PP-1I leads to activation or inactivation of PP-1I through bidirectional modulation of Thr-72 phosphorylation, the critical activating residue of I-2. PMID:25028520

  15. Protein kinase C and tyrosine kinase pathways regulate lipopolysaccharide-induced nitric oxide synthase activity in RAW 264.7 murine macrophages.

    PubMed Central

    Paul, A; Pendreigh, R H; Plevin, R

    1995-01-01

    1. In RAW 264.7 macrophages, lipopolysaccharide (LPS) and gamma-interferon (IFN gamma) alone or in combination stimulated the induction of nitric oxide synthase (iNOS) activity and increased the expression of the 130 kDa isoform of NOS. 2. LPS-induced NOS activity was reduced by incubation with CD14 neutralising antibodies and abolished in macrophages deprived of serum. 3. LPS stimulated a small increase in protein kinase C (PKC) activity in RAW 264.7 macrophages which was dependent on the presence of serum. However, IFN gamma did not potentiate LPS-stimulated PKC activity. 4. The protein kinase C inhibitor, Ro-318220, abolished both LPS- and IFN gamma-stimulated protein kinase C activity and the induction of NOS activity. 5. LPS- and IFN gamma-induced NOS activity was reduced by the tyrosine kinase inhibitor genestein. Genestein also reduced LPS-stimulated protein kinase C activity but did not affect the response to the protein kinase C activator, tetradecanoylphorbol acetate (TPA). 6. Nicotinamide, an inhibitor of poly-ADP ribosylation, abolished LPS- and IFN gamma-induced NOS activity. 7. Brefeldin A, an inhibitor of a factor which stimulates nucleotide exchange activity on the 21 kDa ADP-ribosylation factor, ARF, reduced LPS- and IFN gamma-induced NOS activity by approximately 80%. 8. These results suggest the involvement of protein kinase C, tyrosine kinase and poly-ADP ribosylation pathways in the regulation of the induction of nitric oxide synthase in RAW 264.7 macrophages by LPS and IFN gamma. Images Figure 2 PMID:7533621

  16. Stargazing: Monitoring subcellular dynamics of brain astrocytes.

    PubMed

    Benjamin Kacerovsky, J; Murai, K K

    2016-05-26

    Astrocytes are major non-neuronal cell types in the central nervous system that regulate a variety of processes in the brain including synaptic transmission, neurometabolism, and cerebrovasculature tone. Recent discoveries have revealed that astrocytes perform very specialized and heterogeneous roles in brain homeostasis and function. Exactly how astrocytes fulfill such diverse roles in the brain remains to be fully understood and is an active area of research. In this review, we focus on the complex subcellular anatomical features of protoplasmic gray matter astrocytes in the mature, healthy brain that likely empower these cells with the ability to detect and respond to changes in neuronal and synaptic activity. In particular, we discuss how intricate processes on astrocytes allow these cells to communicate with neurons and their synapses and strategically deliver specific cellular organelles such as mitochondria and ribosomes to active compartments within the neuropil. Understanding the properties of these structural elements will lead to a better understanding of how astrocytes function in the healthy and diseased brain. PMID:26162237

  17. Protein kinase C -dependent regulation of synaptosomal glutamate uptake under conditions of hypergravity

    NASA Astrophysics Data System (ADS)

    Borisova, Tatiana; Krisanova, Natalia; Borisov, Arseniy; Sivko, Roman

    Glutamate is not only the main excitatory neurotransmitter in the mammalian CNS, but also a potent neurotoxin. Excessive concentration of ambient glutamate over activates glutamate receptors and causes neurotoxicity. Uptake of glutamate from the extracellular space into nerve cells was mediated by sodium-dependent glutamate transporters located in the plasma membrane. It was shown that the activity of glutamate transporters in rat brain nerve terminals was decreased after hypergravity (centrifugation of rats in special containers at 10 G for 1 hour). This decrease may result from the reduction in the number of glutamate transporters expressed in the plasma membrane of nerve terminals after hypergravity that was regulated by protein kinase C. The possibility of the involvement of protein kinase C in the regulation of the activity of glutamate transporters was assessed under conditions of hypergravity. The effect of protein kinase C inhibitor GF 109 203X on synaptosomal L-[14C]glutamate uptake was analysed. It was shown that the inhibitor decreased L-[14C]glutamate uptake by 15 % in control but did not influence it after hypergravity. In control, the initial velocity of L-[14C]glutamate uptake in the presence of the inhibitor decreased from 2.5 ± 0.2 nmol x min-1 x mg-1 of proteins to 2.17 ± 0.1 nmol x min-1 x mg-1 of proteins, whereas after hypergravity this value lowered from 2.05 ± 0.1 nmol x min-1 x mg-1 of proteins to 2.04 ± 0.1 nmol x min-1 x mg-1 of proteins. Thus, protein kinase C -dependent alteration in the cell surface expression of glutamate transporters may be one of the causes of a decrease in the activity of glutamate transporters after hypergravity.

  18. Regulation of the human WEE1Hu CDK tyrosine 15-kinase during the cell cycle.

    PubMed Central

    Watanabe, N; Broome, M; Hunter, T

    1995-01-01

    In higher eukaryotes, the cyclin-dependent kinases (CDKs) are negatively regulated by phosphorylation on threonine 14 (T14) and tyrosine 15 (Y15). In fission yeast, the Wee1 and mitosis inhibitory kinase 1 (Mik1) protein kinases phosphorylate Y15 in Cdc2. WEE1Hu is the only known protein kinase that can carry out this inhibitory phosphorylation on Y15 in higher eukaryotes. In the present study, we examined the endogenous products of WEE1Hu in human cells and found that the original WEE1Hu cDNA lacked 214 amino acids at the N-terminus. The predicted full-length protein has weak, but significant, similarity over its entire length with Mik1. Thus, we suggest that 'WEE1Hu' is a Mik1-related protein rather than a Wee1 homologue. When isolated in immunoprecipitates, the endogenous WEE1Hu phosphorylated several cyclin-associated CDKs on Y15. WEE1Hu activity increased during S and G2 phases in parallel with the level of protein. Its activity decreased at M phase when WEE1Hu became transiently hyperphosphorylated. In addition, a decrease in WEE1Hu protein level was observed at M/G1 phase. Apparently, the hyperphosphorylation and degradation in combination caused inactivation of WEE1Hu at M phase and the following G1 phase. These results suggest that the activity of WEE1Hu is regulated by phosphorylation and proteolytic degradation, and that WEE1Hu plays a role in inhibiting mitosis before M phase by phosphorylating cyclin B1-Cdc2. Images PMID:7743995

  19. Diacylglycerol kinase-zeta localization in skeletal muscle is regulated by phosphorylation and interaction with syntrophins.

    PubMed

    Abramovici, Hanan; Hogan, Angela B; Obagi, Christopher; Topham, Matthew K; Gee, Stephen H

    2003-11-01

    Syntrophins are scaffolding proteins that link signaling molecules to dystrophin and the cytoskeleton. We previously reported that syntrophins interact with diacylglycerol kinase-zeta (DGK-zeta), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show syntrophins and DGK-zeta form a complex in skeletal muscle whose translocation from the cytosol to the plasma membrane is regulated by protein kinase C-dependent phosphorylation of the DGK-zeta MARCKS domain. DGK-zeta mutants that do not bind syntrophins were mislocalized, and an activated mutant of this sort induced atypical changes in the actin cytoskeleton, indicating syntrophins are important for localizing DGK-zeta and regulating its activity. Consistent with a role in actin organization, DGK-zeta and syntrophins were colocalized with filamentous (F)-actin and Rac in lamellipodia and ruffles. Moreover, extracellular signal-related kinase-dependent phosphorylation of DGK-zeta regulated its association with the cytoskeleton. In adult muscle, DGK-zeta was colocalized with syntrophins on the sarcolemma and was concentrated at neuromuscular junctions (NMJs), whereas in type IIB fibers it was found exclusively at NMJs. DGK-zeta was reduced at the sarcolemma of dystrophin-deficient mdx mouse myofibers but was specifically retained at NMJs, indicating that dystrophin is important for the sarcolemmal but not synaptic localization of DGK-zeta. Together, our findings suggest syntrophins localize DGK-zeta signaling complexes at specialized domains of muscle cells, which may be critical for the proper control of lipid-signaling pathways regulating actin organization. In dystrophic muscle, mislocalized DGK-zeta may cause abnormal cytoskeletal changes that contribute to disease pathogenesis. PMID:14551255

  20. The Golgi apparatus regulates cGMP-dependent protein kinase I compartmentation and proteolysis.

    PubMed

    Kato, Shin; Chen, Jingsi; Cornog, Katherine H; Zhang, Huili; Roberts, Jesse D

    2015-06-01

    cGMP-dependent protein kinase I (PKGI) is an important effector of cGMP signaling that regulates vascular smooth muscle cell (SMC) phenotype and proliferation. PKGI has been detected in the perinuclear region of cells, and recent data indicate that proprotein convertases (PCs) typically resident in the Golgi apparatus (GA) can stimulate PKGI proteolysis and generate a kinase fragment that localizes to the nucleus and regulates gene expression. However, the role of the endomembrane system in PKGI compartmentation and processing is unknown. Here, we demonstrate that PKGI colocalizes with endoplasmic reticulum (ER), ER-Golgi intermediate compartment, GA cisterna, and trans-Golgi network proteins in pulmonary artery SMC and cell lines. Moreover, PKGI localizes with furin, a trans-Golgi network-resident PC known to cleave PKGI. ER protein transport influences PKGI localization because overexpression of a constitutively inactive Sar1 transgene caused PKGI retention in the ER. Additionally, PKGI appears to reside within the GA because PKGI immunoreactivity was determined to be resistant to cytosolic proteinase K treatment in live cells. The GA appears to play a role in PKGI proteolysis because overexpression of inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate, not only tethered heterologous PKGI-β to the ER and decreased its localization to the GA, but also diminished PKGI proteolysis and nuclear translocation. Also, inhibiting intra-GA protein transport with monensin was observed to decrease PKGI cleavage. These studies detail a role for the endomembrane system in regulating PKGI compartmentation and proteolysis. Moreover, they support the investigation of mechanisms regulating PKGI-dependent nuclear cGMP signaling in the pulmonary vasculature with Golgi dysfunction. PMID:25855081

  1. The Golgi apparatus regulates cGMP-dependent protein kinase I compartmentation and proteolysis

    PubMed Central

    Kato, Shin; Chen, Jingsi; Cornog, Katherine H.; Zhang, Huili

    2015-01-01

    cGMP-dependent protein kinase I (PKGI) is an important effector of cGMP signaling that regulates vascular smooth muscle cell (SMC) phenotype and proliferation. PKGI has been detected in the perinuclear region of cells, and recent data indicate that proprotein convertases (PCs) typically resident in the Golgi apparatus (GA) can stimulate PKGI proteolysis and generate a kinase fragment that localizes to the nucleus and regulates gene expression. However, the role of the endomembrane system in PKGI compartmentation and processing is unknown. Here, we demonstrate that PKGI colocalizes with endoplasmic reticulum (ER), ER-Golgi intermediate compartment, GA cisterna, and trans-Golgi network proteins in pulmonary artery SMC and cell lines. Moreover, PKGI localizes with furin, a trans-Golgi network-resident PC known to cleave PKGI. ER protein transport influences PKGI localization because overexpression of a constitutively inactive Sar1 transgene caused PKGI retention in the ER. Additionally, PKGI appears to reside within the GA because PKGI immunoreactivity was determined to be resistant to cytosolic proteinase K treatment in live cells. The GA appears to play a role in PKGI proteolysis because overexpression of inositol 1,4,5-trisphosphate receptor-associated cGMP kinase substrate, not only tethered heterologous PKGI-β to the ER and decreased its localization to the GA, but also diminished PKGI proteolysis and nuclear translocation. Also, inhibiting intra-GA protein transport with monensin was observed to decrease PKGI cleavage. These studies detail a role for the endomembrane system in regulating PKGI compartmentation and proteolysis. Moreover, they support the investigation of mechanisms regulating PKGI-dependent nuclear cGMP signaling in the pulmonary vasculature with Golgi dysfunction. PMID:25855081

  2. Jelly Belly Trans-Synaptic Signaling to Anaplastic Lymphoma Kinase Regulates Neurotransmission Strength and Synapse Architecture

    PubMed Central

    Rohrbough, Jeffrey; Kent, Karla S.; Broadie, Kendal; Weiss, Joseph B.

    2012-01-01

    In Drosophila the secreted signaling molecule Jelly Belly (Jeb) activates Anaplastic Lymphoma Kinase (Alk), a receptor tyrosine kinase, in multiple developmental and adult contexts. We have shown previously that Jeb and Alk are highly enriched at Drosophila synapses within the CNS neuropil and neuromuscular junction (NMJ) and postulated a conserved intercellular signaling function. At the embryonic and larval NMJ Jeb is localized in the motor neuron presynaptic terminal whereas Alk is concentrated in the muscle postsynaptic domain surrounding boutons, consistent with anterograde trans-synaptic signaling. Here, we show by functional inhibition of Jeb-Alk signaling that neurotransmission is regulated by Jeb secretion. Jeb is a novel negative regulator of neuromuscular transmission. Reduction or inhibtion of Alk function results in enhanced synaptic transmission. Activation of Alk conversely inhibits synaptic transmission. Restoration of wildtype postsynaptic Alk expression in Alk partial loss-of-function mutants rescues NMJ transmission phenotypes and confirms that postsynaptic Alk regulates NMJ transmission. The effects of impaired Alk signaling on neurotransmission are observed in the absence of associated changes in NMJ structure. Complete removal of Jeb in motor neurons, however, disrupts both presynaptic bouton architecture and postsynaptic differentiation. Non-physiologic activation of Alk signaling also negatively regulates NMJ growth. Activation of Jeb-Alk signaling triggers the Ras-MAP kinase cascade in both pre- and postsynaptic compartments. These novel roles for Jeb-Alk signaling in the modulation of synaptic function and structure have potential implications for recently reported Alk functions in human addiction, retention of spatial memory, cognitive dysfunction in neurofibromatosis and the pathogenesis of amyotrophic lateral sclerosis. PMID:22949158

  3. Platelet-derived growth factor (PDGF)-BB-mediated induction of monocyte chemoattractant protein 1 in human astrocytes: implications for HIV-associated neuroinflammation

    PubMed Central

    2012-01-01

    Chemokine (C-C motif) ligand 2, also known as monocyte chemoattractant protein 1 (MCP-1) is an important factor for the pathogenesis of HIV-associated neurocognitive disorders (HAND). The mechanisms of MCP-1-mediated neuropathogenesis, in part, revolve around its neuroinflammatory role and the recruitment of monocytes into the central nervous system (CNS) via the disrupted blood-brain barrier (BBB). We have previously demonstrated that HIV-1/HIV-1 Tat upregulate platelet-derived growth factor (PDGF)-BB, a known cerebrovascular permeant; subsequently, the present study was aimed at exploring the regulation of MCP-1 by PDGF-BB in astrocytes with implications in HAND. Specifically, the data herein demonstrate that exposure of human astrocytes to HIV-1 LAI elevated PDGF-B and MCP-1 levels. Furthermore, treating astrocytes with the human recombinant PDGF-BB protein significantly increased the production and release of MCP-1 at both the RNA and protein levels. MCP-1 induction was regulated by activation of extracellular-signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinas