Ca2+ -stimulated adenylyl cyclases regulate ERK-dependent activation of MSK1 during fear conditioning.

PubMed

Sindreu, Carlos Balet; Scheiner, Zachary S; Storm, Daniel R

2007-01-04

The cAMP and ERK/MAP kinase (MAPK) signal transduction pathways are critical for hippocampus-dependent memory, a process that depends on CREB-mediated transcription. However, the extent of crosstalk between these pathways and the downstream CREB kinase activated during memory formation has not been elucidated. Here we report that PKA, MAPK, and MSK1, a CREB kinase, are coactivated in a subset of hippocampal CA1 pyramidal neurons following contextual fear conditioning. Activation of PKA, MAPK, MSK1, and CREB is absolutely dependent on Ca(2+)-stimulated adenylyl cyclase activity. We conclude that adenylyl cyclase activity supports the activation of MAPK, and that MSK1 is the major CREB kinase activated during training for contextual memory.

Ca2+-Stimulated Adenylyl Cyclases Regulate ERK-Dependent Activation of MSK1 During Fear Conditioning

PubMed Central

Sindreu, Carlos Balet; Scheiner, Zachary S.; Storm, Daniel R.

2007-01-01

The cAMP and ERK/MAP kinase (MAPK) signal transduction pathways are critical for hippocampus-dependent memory, a process that depends on CREB-mediated transcription. However, the extent of crosstalk between these pathways and the downstream CREB kinase activated during memory formation have not been elucidated. Here we report that PKA, MAPK, and MSK1, a CREB kinase, are co-activated in a subset of hippocampal CA1 pyramidal neurons following contextual fear conditioning. Activation of PKA, MAPK, MSK1, and CREB is absolutely dependent on Ca2+-stimulated adenylyl cyclase activity. We conclude that adenylyl cyclase activity supports the activation of MAPK, and that MSK1 is the major CREB kinase activated during training for contextual memory. PMID:17196532

c-Jun controls the efficiency of MAP kinase signaling by transcriptional repression of MAP kinase phosphatases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sprowles, Amy; Robinson, Dan; Wu Yimi

2005-08-15

The mammalian JNK signaling pathway regulates the transcriptional response of cells to environmental stress, including UV irradiation. This signaling pathway is composed of a classical MAP kinase cascade; activation results in phosphorylation of the transcription factor substrates c-Jun and ATF2, and leads to changes in gene expression. The defining components of this pathway are conserved in the fission yeast S. pombe, where the genetic studies have shown that the ability of the JNK homolog Spc1 to be activated in response to UV irradiation is dependent on the presence of the transcription factor substrate Atf1. We have used genetic analysis tomore » define the role of c-Jun in activation of the mammalian JNK signaling pathway. Our results show that optimal activation of JNK requires the presence of its transcription factor substrate c-Jun. Mutational analysis shows that the ability of c-Jun to support efficient activation of JNK requires the ability of Jun to bind DNA, suggesting a transcriptional mechanism. Consistent with this, we show that c-Jun represses the expression of several MAP kinase phosphatases. In the absence of c-Jun, the increased expression of MAP kinase phosphatases leads to impaired activation of the ERK, JNK, and p38 MAP kinases after pathway activation. The results show that one function of c-Jun is to regulate the efficiency of signaling by the ERK, p38, and JNK MAP kinases, a function that is likely to affect cellular responses to many different stimuli.« less

Activation of MAP kinase kinase (MEK) and Ras by cholecystokinin in rat pancreatic acini.

PubMed

Duan, R D; Zheng, C F; Guan, K L; Williams, J A

1995-06-01

Cholecystokinin (CCK) has recently been shown to activate mitogen-activated protein (MAP) kinase in rat pancreatic acini [Duan and Williams, Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G401-G408, 1994]. To evaluate the mechanism of MAP kinase activation, we studied the effects of CCK on MAP kinase kinase (MEK) in rat pancreatic acini. Two forms of MEK were identified by immunoblotting, using antibodies specific to MEK1 and MEK2. MEK activity in acinar extracts and after immunoprecipitation with anti-MEK was detected using a recombinant fusion protein, glutathione S-transferase-MAP kinase, as a substrate. MEK activity rapidly increased after stimulation of acini by CCK, with significant stimulation at 1 min and a maximal effect at 5 min, followed by a slow decline to slightly above control levels after 30 min. The threshold concentration of CCK was approximately 10 pM, and the maximal effect was induced by 1 nM CCK, which increased MEK activity by 120%. In addition to CCK, bombesin and carbachol, but not secretin or vasoactive intestinal peptide, enhanced MEK activity. Phorbol ester mimicked the effect of CCK, whereas ionomycin and thapsigargin failed to activate MEK. We further studied the activation of Ras, an important component leading to activation of MEK by growth factors. Ras in acini was immunoprecipitated and identified by Western blotting. CCK and 12-O-tetradecanoylphorbol-13-acetate stimulated the incorporation of GTP into Ras, a requirement for its activation, reaching a maximum at 10 min of approximately 120% over control. In conclusion, the activation of MAP kinase by CCK can be explained by activation of MEK and may involve the activation of Ras by a protein kinase C-dependent mechanism.

SB202190 affects cell response to hydroxyurea-induced genotoxic stress in root meristems of Vicia faba.

PubMed

Winnicki, Konrad; Maszewski, Janusz

2012-11-01

Genotoxic stress caused by a variety of chemical and physical agents may lead to DNA breaks and genome instability. Response to DNA damage depends on ATM/ATR sensor kinases and their downstream proteins, which arrange cell cycle checkpoints. Activation of ATM (ataxia-telangiectasia-mutated)/ATR (ATM and Rad 3-related) signaling pathway triggers cell cycle arrest (by keeping cyclin-Cdk complexes inactive), combined with gamma-phosphorylation of histone H2A.X and induction of DNA repair processes. However, genotoxic stress activates also mitogen-activated protein kinases (MAPKs) which may control the functions of checkpoint proteins both directly, by post-translational modifications, or indirectly, by regulation of their expression. Our results indicate that in root meristem cells of Vicia faba, MAP kinase signaling pathway takes part in response to hydroxyurea-induced genotoxic stress. It is shown that SB202190, an inhibitor of p38 MAP kinase, triggers PCC (premature chromosome condensation) more rapidly, but only if cell cycle checkpoints are alleviated by caffeine. Since SB202190 and, independently, caffeine reduces HU-mediated histone H4 Lys5 acetylation, it may be that there is a cooperation of MAP kinase signaling pathways and ATM/ATR-dependent checkpoints during response to genotoxic stress. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Activation of the Cph1-Dependent MAP Kinase Signaling Pathway Induces White-Opaque Switching in Candida albicans

PubMed Central

Ramírez-Zavala, Bernardo; Weyler, Michael; Gildor, Tsvia; Schmauch, Christian; Kornitzer, Daniel; Arkowitz, Robert; Morschhäuser, Joachim

2013-01-01

Depending on the environmental conditions, the pathogenic yeast Candida albicans can undergo different developmental programs, which are controlled by dedicated transcription factors and upstream signaling pathways. C. albicans strains that are homozygous at the mating type locus can switch from the normal yeast form (white) to an elongated cell type (opaque), which is the mating-competent form of this fungus. Both white and opaque cells use the Ste11-Hst7-Cek1/Cek2 MAP kinase signaling pathway to react to the presence of mating pheromone. However, while opaque cells employ the transcription factor Cph1 to induce the mating response, white cells recruit a different downstream transcription factor, Tec1, to promote the formation of a biofilm that facilitates mating of opaque cells in the population. The switch from the white to the opaque cell form is itself induced by environmental signals that result in the upregulation of the transcription factor Wor1, the master regulator of white-opaque switching. To get insight into the upstream signaling pathways controlling the switch, we expressed all C. albicans protein kinases from a tetracycline-inducible promoter in a switching-competent strain. Screening of this library of strains showed that a hyperactive form of Ste11 lacking its N-terminal domain (Ste11ΔN467) efficiently stimulated white cells to switch to the opaque phase, a behavior that did not occur in response to pheromone. Ste11ΔN467-induced switching specifically required the downstream MAP kinase Cek1 and its target transcription factor Cph1, but not Cek2 and Tec1, and forced expression of Cph1 also promoted white-opaque switching in a Wor1-dependent manner. Therefore, depending on the activation mechanism, components of the pheromone-responsive MAP kinase pathway can be reconnected to stimulate an alternative developmental program, switching of white cells to the mating-competent opaque phase. PMID:24130492

Investigating small molecules to inhibit germinal center kinase-like kinase (GLK/MAP4K3) upstream of PKCθ phosphorylation: Potential therapy to modulate T cell dependent immunity.

PubMed

May-Dracka, Tricia L; Arduini, Robert; Bertolotti-Ciarlet, Andrea; Bhisetti, Govinda; Brickelmaier, Margot; Cahir-McFarland, Ellen; Enyedy, Istvan; Fontenot, Jason D; Hesson, Thomas; Little, Kevin; Lyssikatos, Joe; Marcotte, Douglas; McKee, Timothy; Murugan, Paramasivam; Patterson, Thomas; Peng, Hairuo; Rushe, Mia; Silvian, Laura; Spilker, Kerri; Wu, Ping; Xin, Zhili; Burkly, Linda C

2018-06-01

Germinal center kinase-like kinase (GLK, also known as MAP4K3) has been hypothesized to have an effect on key cellular activities, including inflammatory responses. GLK is required for activation of protein kinase C-θ (PKCθ) in T cells. Controlling the activity of T helper cell responses could be valuable for the treatment of autoimmune diseases. This approach circumvents previous unsuccessful approaches to target PKCθ directly. The use of structure based drug design, aided by the first crystal structure of GLK, led to the discovery of several inhibitors that demonstrate potent inhibition of GLK biochemically and in relevant cell lines. Copyright © 2018 Elsevier Ltd. All rights reserved.

Inhibiting Src family tyrosine kinase activity blocks glutamate signalling to ERK1/2 and Akt/PKB but not JNK in cultured striatal neurones.

PubMed

Crossthwaite, Andrew J; Valli, Haseeb; Williams, Robert J

2004-03-01

Glutamate receptor activation of mitogen-activated protein (MAP) kinase signalling cascades has been implicated in diverse neuronal functions such as synaptic plasticity, development and excitotoxicity. We have previously shown that Ca2+-influx through NMDA receptors in cultured striatal neurones mediates the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt/protein kinase B (PKB) through a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent pathway. Exposing neurones to the Src family tyrosine kinase inhibitor PP2, but not the inactive analogue PP3, inhibited NMDA receptor-induced phosphorylation of ERK1/2 and Akt/PKB in a concentration-dependent manner, and reduced cAMP response element-binding protein (CREB) phosphorylation. To establish a link between Src family tyrosine kinase-mediated phosphorylation and PI 3-kinase signalling, affinity precipitation experiments were performed with the SH2 domains of the PI 3-kinase regulatory subunit p85. This revealed a Src-dependent phosphorylation of a focal adhesion kinase (FAK)-p85 complex on glutamate stimulation. Demonstrating that PI3-kinase is not ubiquitously involved in NMDA receptor signal transduction, the PI 3-kinase inhibitors wortmannin and LY294002 did not prevent NMDA receptor Ca2+-dependent phosphorylation of c-Jun N-terminal kinase 1/2 (JNK1/2). Further, inhibiting Src family kinases increased NMDA receptor-dependent JNK1/2 phosphorylation, suggesting that Src family kinase-dependent cascades may physiologically limit signalling to JNK. These results demonstrate that Src family tyrosine kinases and PI3-kinase are pivotal regulators of NMDA receptor signalling to ERK/Akt and JNK in striatal neurones.

Metalloproteinase-dependent transforming growth factor-alpha release mediates neurotensin-stimulated MAP kinase activation in human colonic epithelial cells.

PubMed

Zhao, Dezheng; Zhan, Yanai; Koon, Hon Wai; Zeng, Huiyan; Keates, Sarah; Moyer, Mary P; Pothoulakis, Charalabos

2004-10-15

Expression of the neuropeptide neurotensin (NT) and its high affinity receptor (NTR1) is increased during the course of Clostridium difficile toxin A-induced acute colitis, and NTR1 antagonism attenuates the severity of toxin A-induced inflammation. We recently demonstrated in non-transformed human colonic epithelial NCM460 cells that NT treatment caused activation of a Ras-mediated MAP kinase pathway that significantly contributes to NT-induced interleukin-8 (IL-8) secretion. Here we used NCM460 cells, which normally express low levels of NTR1, and NCM460 cells stably transfected with NTR1 to identify the upstream signaling molecules involved in NT-NTR1-mediated MAP kinase activation. We found that inhibition of the epidermal growth factor receptor (EGFR) by either an EGFR neutralizing antibody or by its specific inhibitor AG1478 (0.2 microm) blocked NT-induced MAP kinase activation. Moreover, NT stimulated tyrosine phosphorylation of the EGFR, and pretreatment with a broad spectrum metalloproteinase inhibitor batimastat reduced NT-induced MAP kinase activation. Using neutralizing antibodies against the EGFR ligands EGF, heparin-binding-EGF, transforming growth factor-alpha (TGFalpha), or amphiregulin we have shown that only the anti-TGFalpha antibody significantly decreases NT-induced phosphorylation of EGFR and MAP kinases. Furthermore, inhibition of the EGF receptor by AG1478 significantly reduced NT-induced IL-8 promoter activity and IL-8 secretion. This is the first report demonstrating that NT binding to NTR1 transactivates the EGFR and that this response is linked to NT-mediated proinflammatory signaling. Our findings indicate that matrix metalloproteinase-mediated release of TGFalpha and subsequent EGFR transactivation triggers a NT-mediated MAP kinase pathway that leads to IL-8 gene expression in human colonic epithelial cells.

Phosphorylated and Nonphosphorylated PfMAP2 Are Localized in the Nucleus, Dependent on the Stage of Plasmodium falciparum Asexual Maturation

PubMed Central

Dahalan, Farah Aida; Sidek, Hasidah Mohd; Murtey, Mogana Das; Embi, Mohammed Noor; Ibrahim, Jamaiah; Fei Tieng, Lim; Zakaria, Nurul Aiezzah

2016-01-01

Plasmodium falciparum mitogen-activated protein (MAP) kinases, a family of enzymes central to signal transduction processes including inflammatory responses, are a promising target for antimalarial drug development. Our study shows for the first time that the P. falciparum specific MAP kinase 2 (PfMAP2) is colocalized in the nucleus of all of the asexual erythrocytic stages of P. falciparum and is particularly elevated in its phosphorylated form. It was also discovered that PfMAP2 is expressed in its highest quantity during the early trophozoite (ring form) stage and significantly reduced in the mature trophozoite and schizont stages. Although the phosphorylated form of the kinase is always more prevalent, its ratio relative to the nonphosphorylated form remained constant irrespective of the parasites' developmental stage. We have also shown that the TSH motif specifically renders PfMAP2 genetically divergent from the other plasmodial MAP kinase activation sites using Neighbour Joining analysis. Furthermore, TSH motif-specific designed antibody is crucial in determining the location of the expression of the PfMAP2 protein. However, by using immunoelectron microscopy, PPfMAP2 were detected ubiquitously in the parasitized erythrocytes. In summary, PfMAP2 may play a far more important role than previously thought and is a worthy candidate for research as an antimalarial. PMID:27525262

The TAM-family receptor Mer mediates production of HGF through the RhoA-dependent pathway in response to apoptotic cells.

PubMed

Park, Hyun-Jung; Baen, Ji-Yeon; Lee, Ye-Ji; Choi, Youn-Hee; Kang, Jihee Lee

2012-08-01

The TAM receptor protein tyrosine kinases Tyro3, Axl, and Mer play important roles in macrophage function. We investigated the roles of the TAM receptors in mediating the induction of hepatocyte growth factor (HGF) during the interaction of macrophages with apoptotic cells. Mer-specific neutralizing antibody, small interfering RNA (siRNA), and a recombinant Mer protein (Mer/Fc) inhibited HGF mRNA and protein expression, as well as activation of RhoA, Akt, and specific mitogen-activated protein (MAP) kinases in response to apoptotic cells. Inhibition of Axl or Tyro3 with specific antibodies, siRNA, or Fc-fusion proteins did not prevent apoptotic cell-induced HGF mRNA and protein expression and did not inhibit activation of the postreceptor signaling molecules RhoA and certain MAP kinases, including extracellular signal-regulated protein kinase and c-Jun NH(2)-terminal kinase. However, Axl- and Tyro3-specific blockers did inhibit the activation of Akt and p38 MAP kinase in response to apoptotic cells. In addition, none of the TAM receptors mediated the effects of apoptotic cells on transforming growth factor-β or epidermal growth factor mRNA expression. However, they were involved in the induction of vascular endothelial growth factor mRNA expression. Our data provide evidence that when macrophages interact with apoptotic cells, only Mer of the TAM-family receptors is responsible for mediating transcriptional HGF production through a RhoA-dependent pathway.

PTEN-mediated ERK1/2 inhibition and paradoxical cellular proliferation following Pnck overexpression

PubMed Central

Deb, Tushar B; Barndt, Robert J; Zuo, Annie H; Sengupta, Surojeet; Coticchia, Christine M; Johnson, Michael D

2014-01-01

Pregnancy upregulated non-ubiquitous calmodulin kinase (Pnck), a novel calmodulin kinase, is significantly overexpressed in breast and renal cancers. We present evidence that at high cell density, overexpression of Pnck in HEK 293 cells inhibits serum-induced extracellular signal-regulated kinase (ERK1/ERK2) activation. ERK1/2 inhibition is calcium-dependent and Pnck kinase activity is required for ERK1/2 inhibition, since expression of a kinase-dead (K44A) and a catalytic loop phosphorylation mutant (T171A) Pnck protein is unable to inhibit ERK 1/2 activity. Ras is constitutively active at high cell density, and Pnck does not alter Ras activation, suggesting that Pnck inhibition of ERK1/2 activity is independent of Ras activity. Pnck inhibition of serum-induced ERK1/2 activity is lost in cells in which phosphatase and tensin homolog (PTEN) is suppressed, suggesting that Pnck inhibition of ERK1/2 activity is mediated by PTEN. Overexpression of protein phosphatase-active but lipid phosphatase-dead PTEN protein inhibits ERK1/2 activity in control cells and enhances Pnck-mediated ERK1/2 inhibition, suggesting that Pnck increases availability of protein phosphatase active PTEN for ERK1/2 inhibition. Pnck is a stress-responsive kinase; however, serum-induced p38 MAP kinase activity is also downregulated by Pnck in a Pnck kinase- and PTEN-dependent manner, similar to ERK1/2 inhibition. Pnck overexpression increases proliferation, which is inhibited by PTEN knockdown, implying that PTEN acts as a paradoxical promoter of proliferation in ERK1/2 and p38 MAP kinase phosphorylation-inhibited, Pnck-overexpressing cells. Overall, these data reveal a novel function of Pnck in the regulation of ERK1/2 and p38 MAP kinase activity and cell proliferation, which is mediated by paradoxical PTEN functions. The possible biological implications of these data are discussed. PMID:24552815

TRAF6 and Src kinase activity regulates Cot activation by IL-1.

PubMed

Rodríguez, Cristina; Pozo, Maite; Nieto, Elvira; Fernández, Margarita; Alemany, Susana

2006-09-01

Cot is one of the MAP kinase kinase kinases that regulates the ERK1/ERK2 pathway under physiological conditions. Cot is activated by LPS, by inducing its dissociation from the inactive p105 NFkappaB-Cot complex in macrophages. Here, we show that IL-1 promotes a 10-fold increase in endogenous Cot activity and that Cot is the only MAP kinase kinase kinase that activates ERK1/ERK2 in response to this cytokine. Moreover, in cells where the expression of Cot is blocked, IL-1 fails to induce an increase in IL-8 and MIP-1betamRNA levels. The activation of Cot-MKK1-ERK1/ERK2 signalling pathway by IL-1 is dependent on the activity of the transducer protein TRAF6. Most important, IL-1-induced ERK1/ERK2 activation is inhibited by PP1, a known inhibitor of Src tyrosine kinases, but this tyrosine kinase activity is not required for IL-1 to activate other MAP kinases such as p38 and JNK. This Src kinases inhibitor does not block the dissociation and subsequently degradation of Cot in response to IL-1, indicating that other events besides Cot dissociation are required to activate Cot. All these data highlight the specific requirements for activation of the Cot-MKK1-ERK1/ERK2 pathway and provide evidence that Cot controls the functions of IL-1 that are mediated by ERK1/ERK2.

The MAP kinase pathway coordinates crossover designation with disassembly of synaptonemal complex proteins during meiosis

PubMed Central

Nadarajan, Saravanapriah; Mohideen, Firaz; Tzur, Yonatan B; Ferrandiz, Nuria; Crawley, Oliver; Montoya, Alex; Faull, Peter; Snijders, Ambrosius P; Cutillas, Pedro R; Jambhekar, Ashwini; Blower, Michael D; Martinez-Perez, Enrique; Harper, J Wade; Colaiacovo, Monica P

2016-01-01

Asymmetric disassembly of the synaptonemal complex (SC) is crucial for proper meiotic chromosome segregation. However, the signaling mechanisms that directly regulate this process are poorly understood. Here we show that the mammalian Rho GEF homolog, ECT-2, functions through the conserved RAS/ERK MAP kinase signaling pathway in the C. elegans germline to regulate the disassembly of SC proteins. We find that SYP-2, a SC central region component, is a potential target for MPK-1-mediated phosphorylation and that constitutively phosphorylated SYP-2 impairs the disassembly of SC proteins from chromosomal domains referred to as the long arms of the bivalents. Inactivation of MAP kinase at late pachytene is critical for timely disassembly of the SC proteins from the long arms, and is dependent on the crossover (CO) promoting factors ZHP-3/RNF212/Zip3 and COSA-1/CNTD1. We propose that the conserved MAP kinase pathway coordinates CO designation with the disassembly of SC proteins to ensure accurate chromosome segregation. DOI: http://dx.doi.org/10.7554/eLife.12039.001 PMID:26920220

Pyroglutamic acid stimulates DNA synthesis in rat primary hepatocytes through the mitogen-activated protein kinase pathway.

PubMed

Inoue, Shinjiro; Okita, Yoichi; de Toledo, Andreia; Miyazaki, Hiroyuki; Hirano, Eiichi; Morinaga, Tetsuo

2015-01-01

We purified pyroglutamic acid from human placental extract and identified it as a potent stimulator of rat primary hepatocyte DNA synthesis. Pyroglutamic acid dose-dependently stimulated DNA synthesis, and this effect was inhibited by PD98059, a dual specificity mitogen-activated protein kinase kinase 1 (MAP2K1) inhibitor. Therefore, pyroglutamic acid stimulated DNA synthesis in rat primary hepatocytes via MAPK signaling.

Whole-brain activity mapping onto a zebrafish brain atlas.

PubMed

Randlett, Owen; Wee, Caroline L; Naumann, Eva A; Nnaemeka, Onyeka; Schoppik, David; Fitzgerald, James E; Portugues, Ruben; Lacoste, Alix M B; Riegler, Clemens; Engert, Florian; Schier, Alexander F

2015-11-01

In order to localize the neural circuits involved in generating behaviors, it is necessary to assign activity onto anatomical maps of the nervous system. Using brain registration across hundreds of larval zebrafish, we have built an expandable open-source atlas containing molecular labels and definitions of anatomical regions, the Z-Brain. Using this platform and immunohistochemical detection of phosphorylated extracellular signal–regulated kinase (ERK) as a readout of neural activity, we have developed a system to create and contextualize whole-brain maps of stimulus- and behavior-dependent neural activity. This mitogen-activated protein kinase (MAP)-mapping assay is technically simple, and data analysis is completely automated. Because MAP-mapping is performed on freely swimming fish, it is applicable to studies of nearly any stimulus or behavior. Here we demonstrate our high-throughput approach using pharmacological, visual and noxious stimuli, as well as hunting and feeding. The resultant maps outline hundreds of areas associated with behaviors.

Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

PubMed Central

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

2005-01-01

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

Nitrosative/oxidative stress conditions regulate thioredoxin-interacting protein (TXNIP) expression and thioredoxin-1 (TRX-1) nuclear localization.

PubMed

Ogata, Fernando Toshio; Batista, Wagner Luiz; Sartori, Adriano; Gesteira, Tarsis Ferreira; Masutani, Hiroshi; Arai, Roberto Jun; Yodoi, Junji; Stern, Arnold; Monteiro, Hugo Pequeno

2013-01-01

Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras-ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H₂O₂, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases.

The MAP kinase substrate MKS1 is a regulator of plant defense responses

PubMed Central

Andreasson, Erik; Jenkins, Thomas; Brodersen, Peter; Thorgrimsen, Stephan; Petersen, Nikolaj H T; Zhu, Shijiang; Qiu, Jin-Long; Micheelsen, Pernille; Rocher, Anne; Petersen, Morten; Newman, Mari-Anne; Bjørn Nielsen, Henrik; Hirt, Heribert; Somssich, Imre; Mattsson, Ole; Mundy, John

2005-01-01

Arabidopsis MAP kinase 4 (MPK4) functions as a regulator of pathogen defense responses, because it is required for both repression of salicylic acid (SA)-dependent resistance and for activation of jasmonate (JA)-dependent defense gene expression. To understand MPK4 signaling mechanisms, we used yeast two-hybrid screening to identify the MPK4 substrate MKS1. Analyses of transgenic plants and genome-wide transcript profiling indicated that MKS1 is required for full SA-dependent resistance in mpk4 mutants, and that overexpression of MKS1 in wild-type plants is sufficient to activate SA-dependent resistance, but does not interfere with induction of a defense gene by JA. Further yeast two-hybrid screening revealed that MKS1 interacts with the WRKY transcription factors WRKY25 and WRKY33. WRKY25 and WRKY33 were shown to be in vitro substrates of MPK4, and a wrky33 knockout mutant was found to exhibit increased expression of the SA-related defense gene PR1. MKS1 may therefore contribute to MPK4-regulated defense activation by coupling the kinase to specific WRKY transcription factors. PMID:15990873

Phosphorylation of WAVE2 by MAP kinases regulates persistent cell migration and polarity

PubMed Central

Danson, Christopher M.; Pocha, Shirin M.; Bloomberg, Graham B.; Cory, Giles O.

2009-01-01

Summary The WAVE family of proteins has long been implicated in the stimulus-dependent generation of lamellipodia at the leading edge of migrating cells, with WAVE2 in particular implicated in the formation of peripheral ruffles and chemotactic migration. However, the lack of direct visualisation of cell migration in WAVE2 mutants or knockdowns has made defining the mechanisms of WAVE2 regulation during cell migration difficult. We have characterised three MAP kinase phosphorylation sites within WAVE2 and analysed fibroblast behaviour in a scratch-wound model following introduction of transgenes encoding phospho-defective WAVE2. The cells exhibited an increase in migration speed, a decrease in the persistence of migration, and disruption of polarisation of the Golgi apparatus. All these effects could be mimicked by acute knockdown of endogenous WAVE2 expression with RNAi, indicating that phosphorylation of WAVE2 by MAP kinases regulates cell polarity during migration. PMID:18032787

Phosphorylation of WAVE2 by MAP kinases regulates persistent cell migration and polarity.

PubMed

Danson, Christopher M; Pocha, Shirin M; Bloomberg, Graham B; Cory, Giles O

2007-12-01

The WAVE family of proteins has long been implicated in the stimulus-dependent generation of lamellipodia at the leading edge of migrating cells, with WAVE2 in particular implicated in the formation of peripheral ruffles and chemotactic migration. However, the lack of direct visualisation of cell migration in WAVE2 mutants or knockdowns has made defining the mechanisms of WAVE2 regulation during cell migration difficult. We have characterised three MAP kinase phosphorylation sites within WAVE2 and analysed fibroblast behaviour in a scratch-wound model following introduction of transgenes encoding phospho-defective WAVE2. The cells exhibited an increase in migration speed, a decrease in the persistence of migration, and disruption of polarisation of the Golgi apparatus. All these effects could be mimicked by acute knockdown of endogenous WAVE2 expression with RNAi, indicating that phosphorylation of WAVE2 by MAP kinases regulates cell polarity during migration.

Curcumin Stimulates Proliferation of Spinal Cord Neural Progenitor Cells via a Mitogen-Activated Protein Kinase Signaling Pathway

PubMed Central

Son, Sihoon; Cho, Dae-Chul; Kim, Hye-Jeong; Sung, Joo-Kyung; Bae, Jae-Sung

2014-01-01

Objective The aims of our study are to evaluate the effect of curcumin on spinal cord neural progenitor cell (SC-NPC) proliferation and to clarify the mechanisms of mitogen-activated protein (MAP) kinase signaling pathways in SC-NPCs. Methods We established cultures of SC-NPCs, extracted from the spinal cord of Sprague-Dawley rats weighing 250 g to 350 g. We measured proliferation rates of SC-NPCs after curcumin treatment at different dosage. The immuno-blotting method was used to evaluate the MAP kinase signaling protein that contains extracellular signal-regulated kinases (ERKs), p38, c-Jun NH2-terminal kinases (JNKs) and β-actin as the control group. Results Curcumin has a biphasic effect on SC-NPC proliferation. Lower dosage (0.1, 0.5, 1 µM) of curcumin increased SC-NPC proliferation. However, higher dosage decreased SC-NPC proliferation. Also, curcumin stimulates proliferation of SC-NPCs via the MAP kinase signaling pathway, especially involving the p-ERK and p-38 protein. The p-ERK protein and p38 protein levels varied depending on curcumin dosage (0.5 and 1 µM, p<0.05). Conclusion Curcumin can stimulate proliferation of SC-NPCs via ERKs and the p38 signaling pathway in low concentrations. PMID:25289117

Whole-brain activity mapping onto a zebrafish brain atlas

PubMed Central

Randlett, Owen; Wee, Caroline L.; Naumann, Eva A.; Nnaemeka, Onyeka; Schoppik, David; Fitzgerald, James E.; Portugues, Ruben; Lacoste, Alix M.B.; Riegler, Clemens; Engert, Florian; Schier, Alexander F.

2015-01-01

In order to localize the neural circuits involved in generating behaviors, it is necessary to assign activity onto anatomical maps of the nervous system. Using brain registration across hundreds of larval zebrafish, we have built an expandable open source atlas containing molecular labels and anatomical region definitions, the Z-Brain. Using this platform and immunohistochemical detection of phosphorylated-Extracellular signal-regulated kinase (ERK/MAPK) as a readout of neural activity, we have developed a system to create and contextualize whole brain maps of stimulus- and behavior-dependent neural activity. This MAP-Mapping (Mitogen Activated Protein kinase – Mapping) assay is technically simple, fast, inexpensive, and data analysis is completely automated. Since MAP-Mapping is performed on fish that are freely swimming, it is applicable to nearly any stimulus or behavior. We demonstrate the utility of our high-throughput approach using hunting/feeding, pharmacological, visual and noxious stimuli. The resultant maps outline hundreds of areas associated with behaviors. PMID:26778924

Suppression of proliferation and neurite extension of human neuroblastoma SH-SY5Y cells on immobilized Psathyrella velutina lectin.

PubMed

Kitamura, Noriaki; Ikekita, Masahiko; Hayakawa, Satoru; Funahashi, Hisayuki; Furukawa, Kiyoshi

2004-02-01

Glycoproteins from mammalian brain tissues contain unique N-linked oligosaccharides terminating with beta-N-acetylglucosamine residues. Lectin blot analysis of membrane glycoprotein samples from human neuroblastoma SH-SY5Y cells showed that several protein bands bind to Psathylera velutina lectin (PVL), which interacts with beta-N-acetylglucosamine-terminating oligosaccharides. No lectin positive bands were detected by digestion with jack bean beta-N-acetyl-hexosaminidase or N-glycanase before incubation with the lectin, indicating that the cells contain beta-N-acetylglucosamine-terminating N-linked oligosaccharides. When cells were cultured in dishes with different concentrations of PVL, the cell proliferation was inhibited in a dose-dependent manner. Similarly, the neurite extension, which was stimulated with nerve growth factor, was also inhibited in a manner dependent on the lectin dose. Cell proliferation and neurite extension were recovered by the addition of 10 mM N-acetylglucosamine into the medium. Immunoblot analysis of the activation of mitogen-activated protein (MAP) kinases and protein kinase C revealed that phosphorylation of 42-kDa and 44-kDa MAP kinases and 80-kDa protein kinase C are inhibited when SH-SY5Y cells are cultured in PVL-coated dishes, but are restored by the addition of the haptenic sugar into the medium, indicating that MAP kinase and protein kinase C pathways are inhibited by interaction with immobilized PVL. These results indicate that beta-N-acetylglucosamine-terminating N-linked oligosaccharides expressed on neural cells can induce intracellular signals upon binding to extracellular receptors, and are important for growth regulation of neural cells. Copyright 2003 Wiley-Liss, Inc.

Interactive roles of Ras, insulin receptor substrate-1, and proteins with Src homology-2 domains in insulin signaling in Xenopus oocytes.

PubMed

Chuang, L M; Hausdorff, S F; Myers, M G; White, M F; Birnbaum, M J; Kahn, C R

1994-11-04

Insulin receptor substrate-1 (IRS-1) serves as the major immediate substrate of insulin/insulin-like growth factor (IGF)-1 receptors and following tyrosine phosphorylation binds to specific Src homology-2 (SH2) domain-containing proteins including the p85 subunit of phosphatidylinositol (PI) 3-kinase and GRB2, a molecule believed to link IRS-1 to the Ras pathway. To investigate how these SH2-containing signaling molecules interact to regulate insulin/IGF-1 action, IRS-1, glutathione S-transferase (GST)-SH2 domain fusion proteins and Ras proteins were microinjected into Xenopus oocytes. We found that pleiotropic insulin actions are mediated by IRS-1 through two independent, but convergent, pathways involving PI 3-kinase and GRB2. Thus, microinjection of GST-fusion proteins of either p85 or GRB2 inhibited IRS-1-dependent activation of mitogen-activated protein (MAP) and S6 kinases and oocyte maturation, although only the GST-SH2 of p85 reduced insulin-stimulated PI 3-kinase activation. Co-injection of a dominant negative Ras (S17N) with IRS-1 inhibited insulin-stimulated MAP and S6 kinase activation. Micro-injection of activated [Arg12,Thr59]Ras increased basal MAP and S6 kinase activities and sensitized the oocytes to insulin-stimulated maturation without altering insulin-stimulated PI 3-kinase. The Ras-enhanced oocyte maturation response, but not the elevated basal level of MAP and S6 kinase, was partially blocked by the SH2-p85, but not SH2-GRB2. These data strongly suggest that IRS-1 can mediate many of insulin's actions on cellular enzyme activation and cell cycle progression requires binding and activation of multiple different SH2-domain proteins.

Roles of cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1.

PubMed

Shinohara, M; Kodama, A; Matozaki, T; Fukuhara, A; Tachibana, K; Nakanishi, H; Takai, Y

2001-06-01

Gab-1 is a multiple docking protein that is tyrosine phosphorylated by receptor tyrosine kinases such as c-Met, hepatocyte growth factor/scatter factor receptor, and epidermal growth factor receptor. We have now demonstrated that cell-cell adhesion also induces marked tyrosine phosphorylation of Gab-1 and that disruption of cell-cell adhesion results in its dephosphorylation. An anti-E-cadherin antibody decreased cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas the expression of E-cadherin specifically induced tyrosine phosphorylation of Gab-1. A relatively selective inhibitor of Src family kinases reduced cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas expression of a dominant-negative mutant of Csk increased it. Disruption of cell-cell adhesion, which reduced tyrosine phosphorylation of Gab-1, also reduced the activation of mitogen-activated protein kinase and Akt in response to cell-cell adhesion. These results indicate that E-cadherin-mediated cell-cell adhesion induces tyrosine phosphorylation by a Src family kinase of Gab-1, thereby regulating the activation of Ras/MAP kinase and phosphatidylinositol 3-kinase/Akt cascades.

Endothelial NOS-dependent activation of c-Jun NH(2)- terminal kinase by oxidized low-density lipoprotein

NASA Technical Reports Server (NTRS)

Go, Y. M.; Levonen, A. L.; Moellering, D.; Ramachandran, A.; Patel, R. P.; Jo, H.; Darley-Usmar, V. M.

2001-01-01

Oxidized low-density lipoprotein (oxLDL) is known to activate a number of signal transduction pathways in endothelial cells. Among these are the c-Jun NH(2)-terminal kinase (JNK), also known as stress-activated protein kinase, and extracellular signal-regulated kinase (ERK). These mitogen-activated protein kinases (MAP kinase) determine cell survival in response to environmental stress. Interestingly, JNK signaling involves redox-sensitive mechanisms and is activated by reactive oxygen and nitrogen species derived from both NADPH oxidases, nitric oxide synthases (NOS), peroxides, and oxidized low-density lipoprotein (oxLDL). The role of endothelial NOS (eNOS) in the activation of JNK in response to oxLDL has not been examined. Herein, we show that on exposure of endothelial cells to oxLDL, both ERK and JNK are activated through independent signal transduction pathways. A key role of eNOS activation through a phosphatidylinositol-3-kinase-dependent mechanism leading to phosphorylation of eNOS is demonstrated for oxLDL-dependent activation of JNK. Moreover, we show that activation of ERK by oxLDL is critical in protection against the cytotoxicity of oxLDL.

Expression and purification of functional JNK2beta2: perspectives on high-level production of recombinant MAP kinases.

PubMed

Savopoulos, John W; Dowd, Stephen; Armour, Carolyn; Carter, Paul S; Greenwood, Catherine J; Mills, David; Powell, David; Pettman, Gary R; Jenkins, Owen; Walsh, Frank S; Philpott, Karen L

2002-02-01

The mitogen-activated protein (MAP) kinases are a group of serine/threonine kinases that mediate intracellular signal transduction in response to environmental stimuli including stress, growth factors, and various cytokines. Of this family, the c-Jun N-terminal kinases (JNKs) are members which, depending on cell type, have been shown to activate the transcription of genes involved in the inflammatory response, apoptosis, and hypertrophy. Here we report the use Baculovirus/Sf9 cells to produce milligram quantities of recombinant JNK2beta2 substrate which could be purified to >90% as judged by SDS-PAGE. In addition, we report a novel method for the site-specific biotinylation for this enzyme and demonstrate that the biotinylated product is an authentic substrate of the upstream kinases MKK4 and 7 and can phosphorylate a downstream target, ATF-2. We also show that the phosphorylated product can be captured efficiently on streptavidin-coated beads for use in scintillation proximity assays. Copyright 2002 Elsevier Science (USA).

NFκB-Associated Pathways in Progression of Chemoresistance to 5-Fluorouracil in an In Vitro Model of Colonic Carcinoma.

PubMed

Körber, Maria Isabel; Staribacher, Anna; Ratzenböck, Ina; Steger, Günther; Mader, Robert M

2016-04-01

Drug resistance to 5-fluorouracil (5-FU) is a major obstacle in colonic cancer treatment. Activation of nuclear factor-kappa B (NFκB), mitogen-activated protein kinase kinase kinase 8 (MAP3K8) and protein kinase B (AKT) is thought to protect cancer cells against therapy-induced cytotoxicity. Using cytotoxicity assays and immunoblotting, the impact of inhibitory strategies addressing NFκB, AKT and MAP3K8 in chemoresistance was evaluated in a colonic cancer model in vitro. This model consisted of the cell lines SW480 and SW620, and three subclones with increasing degrees of chemoresistance in order to mimic the development of secondary resistance. NFκB protein p65 was selectively activated in all resistant cell lines. Consequently, several inhibitors of NFκB, MAP3K8 and AKT effectively circumvented this chemoresistance. As a cellular reaction, NFκB inhibition may trigger a feedback loop resulting in activation of extracellular signal-regulated kinase. The results suggest that chemoresistance to 5-FU in this colonic carcinoma model (cell lines SW480 and SW620) is strongly dependent on NFκB activation. The efficacy of MAP3K8 inhibition in our model potentially uncovers a new mechanism to circumvent 5-FU resistance. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

N-acetylcysteine attenuates TNF-α-induced p38 MAP kinase activation and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells

PubMed Central

Hashimoto, Shu; Gon, Yasuhiro; Matsumoto, Ken; Takeshita, Ikuko; Horie, Takashi

2001-01-01

We have previously shown that tumour necrosis factor-α (TNF-α) activates p38 mitogen-activated protein (MAP) kinase to produce interleukin-8 (IL-8) by human pulmonary vascular endothelial cells. Reactive oxygen species (ROS) including H2O2 generated by TNF-α can act as signalling intermediates for cytokine induction; therefore, scavenging ROS by anti-oxidants is important for the regulation of cytokine production. However, the effect of N-acetylcysteine (NAC), which acts as a precursor of glutathione (GSH) synthesis, on TNF-α-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells has not been determined. To clarify these issues, we examined the effect of NAC on TNF-α-induced activation of p38 MAP kinase, MAP kinase kinase (MKK) 3 and MKK6 which are upstream regulators of p38 MAP kinase, and p38 MAP kinase-mediated IL-8 production. Human pulmonary vascular endothelial cells that had been preincubated with NAC were stimulated with TNF-α and then the activation of p38 MAP kinase and MKK3/MKK6 in the cells and IL-8 concentrations in the culture supernatants were determined. Intracellular GSH levels increased in NAC-treated cells. NAC attenuated TNF-α-induced activation of p38 MAP kinase and MKK3/MKK6. NAC attenuated p38 MAP kinase-mediated IL-8 production by TNF-α-stimulated cells. These results indicate that the cellular reduction and oxidation (redox) regulated by intracellular GSH is critical for TNF-α-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells, and we emphasize that anti-oxidant therapy is an important strategy for the treatment of acute lung injury. PMID:11156586

N-acetylcysteine attenuates TNF-alpha-induced p38 MAP kinase activation and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells.

PubMed

Hashimoto, S; Gon, Y; Matsumoto, K; Takeshita, I; Horie, T

2001-01-01

1. We have previously shown that tumour necrosis factor-alpha (TNF-alpha) activates p38 mitogen-activated protein (MAP) kinase to produce interleukin-8 (IL-8) by human pulmonary vascular endothelial cells. Reactive oxygen species (ROS) including H(2)O(2) generated by TNF-alpha can act as signalling intermediates for cytokine induction; therefore, scavenging ROS by anti-oxidants is important for the regulation of cytokine production. However, the effect of N-acetylcysteine (NAC), which acts as a precursor of glutathione (GSH) synthesis, on TNF-alpha-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells has not been determined. To clarify these issues, we examined the effect of NAC on TNF-alpha-induced activation of p38 MAP kinase, MAP kinase kinase (MKK) 3 and MKK6 which are upstream regulators of p38 MAP kinase, and p38 MAP kinase-mediated IL-8 production. 2. Human pulmonary vascular endothelial cells that had been preincubated with NAC were stimulated with TNF-alpha and then the activation of p38 MAP kinase and MKK3/MKK6 in the cells and IL-8 concentrations in the culture supernatants were determined. 3. Intracellular GSH levels increased in NAC-treated cells. 4. NAC attenuated TNF-alpha-induced activation of p38 MAP kinase and MKK3/MKK6. 5. NAC attenuated p38 MAP kinase-mediated IL-8 production by TNF-alpha-stimulated cells. 6. These results indicate that the cellular reduction and oxidation (redox) regulated by intracellular GSH is critical for TNF-alpha-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells, and we emphasize that anti-oxidant therapy is an important strategy for the treatment of acute lung injury.

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

PubMed Central

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

2013-01-01

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

Effects of Cot expression on the nuclear translocation of NF-kappaB in RBL-2H3 cells.

PubMed

Chikamatsu, Satomi; Furuno, Tadahide; Kinoshita, Yosuke; Inoh, Yoshikazu; Hirashima, Naohide; Teshima, Reiko; Nakanishi, Mamoru

2007-03-01

Cot is a serine/threonine protein kinase and is classified as a mitogen-activated protein (MAP) kinase kinase kinase. Overexpression of this protein has been shown to activate the extracellular signal-regulated kinase, the c-Jun N-terminal kinase, and the p38 MAP kinase pathways and to stimulate NF-AT and NF-kappaB-dependent transcription. Here we have shown that Cot kinase activity is intimately involved in the high affinity receptor for IgE (FcvarepsilonRI)-mediated nuclear translocation of NF-kappaB1 independent of NF-kappaB-inducing kinase (NIK) in rat basophilic leukemia (RBL-2H3) cells. A transfected green fluorescent protein-tagged NF-kappaB1 (GFP-NF-kappaB1) resided in the cytoplasm in RBL-2H3 cells and it remained in the cytoplasm even when Cot tagged with red fluorescent protein (Cot-RFP) was co-expressed. Western blotting analysis showed that IkappaB kinases (IKKs) were expressed in RBL-2H3 cells but NIK was not. GFP-NF-kappaB1 translocated from the cytoplasm to the nucleus after the aggregation of FcvarepsilonRI in Cot-transfected cells but not in kinase-deficient Cot-transfected cells. This finding gives a new insight into the role of Cot in the FcvarepsilonRI-mediated NF-kappaB activation in mast cells.

Herpes simplex virus 2 VP22 phosphorylation induced by cellular and viral kinases does not influence intracellular localization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Geiss, Brian J.; Cano, Gina L.; Tavis, John E.

2004-12-05

Phosphorylation of the herpes simplex virus (HSV) VP22 protein is regulated by cellular kinases and the UL13 viral kinase, but the sites at which these enzymes induce phosphorylation of HSV-2 VP22 are not known. Using serine-to-alanine mutants to map phosphorylation sites on HSV-2 VP22 in cells, we made three major observations. First, phosphorylation by a cellular kinase mapped to serines 70, 71, and/or 72 within CKII consensus sites analogous to previously identified phosphorylation sites in HSV-1 VP22. Second, we mapped UL13-mediated phosphorylation of HSV-2 VP22 to serines 28 and 34, describing for the first time UL13-dependent phosphorylation sites on VP22.more » Third, previously identified VP22-associated cellular kinase sites in HSV-1 VP22 (serines 292 and 294) were not phosphorylated in HSV-2 VP22 (serines 291 and 293). VP22 expressed alone accumulated in the cytoplasm and to a lesser extent in the nucleus. Phosphorylation by endogenous cellular kinase(s) did not alter the localization of VP22. Co-expression of HSV-2 VP22 with active UL13, but not with enzymatically inactive UL13, resulted in nuclear accumulation of VP22 and altered nuclear morphology. Surprisingly, redistribution of VP22 to the nucleus occurred independently of UL13-induced phosphorylation of VP22. The altered nuclear morphology of UL13-expressing cells was not due to apoptosis. These results demonstrate that phosphorylation of HSV-2 VP22 at multiple serine residues is induced by UL13 and cellular kinase(s), and that the nuclear/cytoplasmic distribution of VP22 is independent of its phosphorylation status but is controlled indirectly by UL13 kinase activity.« less

TAK1 (MAP3K7) inhibition promotes apoptosis in KRAS-dependent colon cancers

PubMed Central

Singh, Anurag; Sweeney, Michael F.; Yu, Min; Burger, Alexa; Greninger, Patricia; Benes, Cyril; Haber, Daniel A.; Settleman, Jeff

2012-01-01

Summary Colon cancers frequently harbor KRAS mutations, yet only a subset of KRAS-mutant colon cancer cell lines are dependent upon KRAS signaling for survival. In a screen for kinases that promote survival of KRAS-dependent colon cancer cells, we found that the TAK1 kinase (MAP3K7) is required for tumor cell viability. The induction of apoptosis by RNAi-mediated depletion or pharmacologic inhibition of TAK1 is linked to its suppression of hyperactivated Wnt signaling, evident in both endogenous and genetically reconstituted cells. In APC-mutant/KRAS-dependent cells, KRAS stimulates BMP-7 secretion and BMP signaling, leading to TAK1 activation and enhancement of Wnt-dependent transcription. An in vitro-derived “TAK1-dependency signature” is enriched in primary human colon cancers with mutations in both APC and KRAS, suggesting potential clinical utility in stratifying patient populations. Together, these findings identify TAK1 inhibition as a potential therapeutic strategy for a treatment-refractory subset of colon cancers exhibiting aberrant KRAS and Wnt pathway activation. PMID:22341439

Proteolytic Inhibition of Salmonella enterica Serovar Typhimurium-Induced Activation of the Mitogen-Activated Protein Kinases ERK and JNK in Cultured Human Intestinal Cells

PubMed Central

Mynott, Tracey L.; Crossett, Ben; Prathalingam, S. Radhika

2002-01-01

Bromelain, a mixture of cysteine proteases from pineapple stems, blocks signaling by the mitogen-activated protein (MAP) kinases extracellular regulated kinase 1 (ERK-1) and ERK-2, inhibits inflammation, and protects against enterotoxigenic Escherichia coli infection. In this study, we examined the effect of bromelain on Salmonella enterica serovar Typhimurium infection, since an important feature of its pathogenesis is its ability to induce activation of ERK-1 and ERK-2, which leads to internalization of bacteria and induction of inflammatory responses. Our results show that bromelain dose dependently blocks serovar Typhimurium-induced ERK-1, ERK-2, and c-Jun NH2-terminal kinase (JNK) activation in Caco-2 cells. Bromelain also blocked signaling induced by carbachol and anisomycin, pharmacological MAP kinase agonists. Despite bromelain inhibition of serovar Typhimurium-induced MAP kinase signaling, it did not prevent subsequent invasion of the Caco-2 cells by serovar Typhimurium or alter serovar Typhimurium -induced decreases in resistance across Caco-2 monolayers. Surprisingly, bromelain also did not block serovar Typhimurium-induced interleukin-8 (IL-8) secretion but synergized with serovar Typhimurium to enhance IL-8 production. We also found that serovar Typhimurium does not induce ERK phosphorylation in Caco-2 cells in the absence of serum but that serovar Typhimurium-induced invasion and decreases in monolayer resistance are unaffected. Collectively, these data indicate that serovar Typhimurium-induced invasion of Caco-2 cells, changes in the resistance of epithelial cell monolayers, and IL-8 production can occur independently of the ERK and JNK signaling pathways. Data also confirm that bromelain is a novel inhibitor of MAP kinase signaling pathways and suggest a novel role for proteases as inhibitors of signal transduction pathways in intestinal epithelial cells. PMID:11748167

The c-mos proto-oncogene protein kinase turns on and maintains the activity of MAP kinase, but not MPF, in cell-free extracts of Xenopus oocytes and eggs.

PubMed Central

Nebreda, A R; Hunt, T

1993-01-01

During studies of the activation and inactivation of the cyclin B-p34cdc2 protein kinase (MPF) in cell-free extracts of Xenopus oocytes and eggs, we found that a bacterially expressed fusion protein between the Escherichia coli maltose-binding protein and the Xenopus c-mos protein kinase (malE-mos) activated a 42 kDa MAP kinase. The activation of MAP kinase on addition of malE-mos was consistent, whereas the activation of MPF was variable and failed to occur in some oocyte extracts in which cyclin A or okadaic acid activated both MPF and MAP kinase. In cases when MPF activation was transient, MAP kinase activity declined after MPF activity was lost, and MAP kinase, but not MPF, could be maintained at a high level by the presence of malE-mos. When intact oocytes were treated with progesterone, however, the activation of MPF and MAP kinase occurred simultaneously, in contrast to the behaviour of extracts. These observations suggest that one role of c-mos may be to maintain high MAP kinase activity in meiosis. They also imply that the activation of MPF and MAP kinase in vivo are synchronous events that normally rely on an agent that has still to be identified. Images PMID:8387916

COT drives resistance to RAF inhibition through MAP kinase pathway reactivation.

PubMed

Johannessen, Cory M; Boehm, Jesse S; Kim, So Young; Thomas, Sapana R; Wardwell, Leslie; Johnson, Laura A; Emery, Caroline M; Stransky, Nicolas; Cogdill, Alexandria P; Barretina, Jordi; Caponigro, Giordano; Hieronymus, Haley; Murray, Ryan R; Salehi-Ashtiani, Kourosh; Hill, David E; Vidal, Marc; Zhao, Jean J; Yang, Xiaoping; Alkan, Ozan; Kim, Sungjoon; Harris, Jennifer L; Wilson, Christopher J; Myer, Vic E; Finan, Peter M; Root, David E; Roberts, Thomas M; Golub, Todd; Flaherty, Keith T; Dummer, Reinhard; Weber, Barbara L; Sellers, William R; Schlegel, Robert; Wargo, Jennifer A; Hahn, William C; Garraway, Levi A

2010-12-16

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.

Nitrosative/Oxidative Stress Conditions Regulate Thioredoxin-Interacting Protein (TXNIP) Expression and Thioredoxin-1 (TRX-1) Nuclear Localization

PubMed Central

Ogata, Fernando Toshio; Batista, Wagner Luiz; Sartori, Adriano; Gesteira, Tarsis Ferreira; Masutani, Hiroshi; Arai, Roberto Jun; Yodoi, Junji; Stern, Arnold; Monteiro, Hugo Pequeno

2013-01-01

Thioredoxin (TRX-1) is a multifunctional protein that controls the redox status of other proteins. TRX-1 can be found in the extracellular milieu, cytoplasm and nucleus, and it has distinct functions in each environment. Previously, we studied the intracellular localization of TRX-1 and its relationship with the activation of the p21Ras - ERK1/2 MAP Kinases signaling pathway. In situations where this pathway was activated by stress conditions evoked by a nitrosothiol, S-nitroso-N-acetylpenicillamine (SNAP), TRX-1 accumulated in the nuclear compartment due to nitrosylation of p21Ras and activation of downstream ERK1/2 MAP kinases. Presently, we demonstrate that ERK1/2 MAP Kinases activation and spatial distribution within cells trigger TRX-1 nuclear translocation through down-regulation of the physiological inhibitor of TRX-1, Thioredoxin Interacting Protein (TXNIP). Once activated by the oxidants, SNAP and H2O2, the ERK1/2 MAP kinases migrate to the nucleus. This is correlated with down-regulation of TXNIP. In the presence of the MEK inhibitors (PD98059 or UO126), or in cells transfected with the Protein Enriched in Astrocytes (PEA-15), a cytoplasmic anchor of ERK1/2 MAP kinases, TRX-1 nuclear migration and TXNIP down-regulation are no longer observed in cells exposed to oxidants. On the other hand, over-expression of TXNIP abolishes nuclear migration of TRX-1 under nitrosative/oxidative stress conditions, whereas gene silencing of TXNIP facilitates nuclear migration even in the absence of stress conditions. Studies based on the TXNIP promoter support this regulation. In conclusion, changes in TRX-1 compartmentalization under nitrosative/oxidative stress conditions are dependent on the expression levels of TXNIP, which are regulated by cellular compartmentalization and activation of the ERK1/2 MAP kinases. PMID:24376827

Calcium-dependent protein kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates.

PubMed

Curran, Amy; Chang, Ing-Feng; Chang, Chia-Lun; Garg, Shilpi; Miguel, Rodriguez Milla; Barron, Yoshimi D; Li, Ying; Romanowsky, Shawn; Cushman, John C; Gribskov, Michael; Harmon, Alice C; Harper, Jeffrey F

2011-01-01

The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca(2+)-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with K(M) ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies.

Calcium-Dependent Protein Kinases from Arabidopsis Show Substrate Specificity Differences in an Analysis of 103 Substrates

PubMed Central

Curran, Amy; Chang, Ing-Feng; Chang, Chia-Lun; Garg, Shilpi; Miguel, Rodriguez Milla; Barron, Yoshimi D.; Li, Ying; Romanowsky, Shawn; Cushman, John C.; Gribskov, Michael; Harmon, Alice C.; Harper, Jeffrey F.

2011-01-01

The identification of substrates represents a critical challenge for understanding any protein kinase-based signal transduction pathway. In Arabidopsis, there are more than 1000 different protein kinases, 34 of which belong to a family of Ca2+-dependent protein kinases (CPKs). While CPKs are implicated in regulating diverse aspects of plant biology, from ion transport to transcription, relatively little is known about isoform-specific differences in substrate specificity, or the number of phosphorylation targets. Here, in vitro kinase assays were used to compare phosphorylation targets of four CPKs from Arabidopsis (CPK1, 10, 16, and 34). Significant differences in substrate specificity for each kinase were revealed by assays using 103 different substrates. For example CPK16 phosphorylated Serine 109 in a peptide from the stress-regulated protein, Di19-2 with KM ∼70 μM, but this site was not phosphorylated significantly by CPKs 1, 10, or 34. In contrast, CPKs 1, 10, and 34 phosphorylated 93 other peptide substrates not recognized by CPK16. Examples of substrate specificity differences among all four CPKs were verified by kinetic analyses. To test the correlation between in vivo phosphorylation events and in vitro kinase activities, assays were performed with 274 synthetic peptides that contained phosphorylation sites previously mapped in proteins isolated from plants (in vivo-mapped sites). Of these, 74 (27%) were found to be phosphorylated by at least one of the four CPKs tested. This 27% success rate validates a robust strategy for linking the activities of specific kinases, such as CPKs, to the thousands of in planta phosphorylation sites that are being uncovered by emerging technologies. PMID:22645532

Cooperation between STAT5 and phosphatidylinositol 3-kinase in the IL-3-dependent survival of a bone marrow derived cell line.

PubMed

Rosa Santos, S C; Dumon, S; Mayeux, P; Gisselbrecht, S; Gouilleux, F

2000-02-24

Cytokine-dependent activation of distinct signaling pathways is a common scheme thought to be required for the subsequent programmation into cell proliferation and survival. The PI 3-kinase/Akt, Ras/MAP kinase, Ras/NFIL3 and JAK/STAT pathways have been shown to participate in cytokine mediated suppression of apoptosis in various cell types. However the relative importance of these signaling pathways seems to depend on the cellular context. In several cases, individual inhibition of each pathway is not sufficient to completely abrogate cytokine mediated cell survival suggesting that cooperation between these pathways is required. Here we showed that individual inhibition of STAT5, PI 3-kinase or MEK activities did not or weakly affected the IL-3 dependent survival of the bone marrow derived Ba/F3 cell line. However, the simultaneous inhibition of STAT5 and PI 3-kinase activities but not that of STAT5 and MEK reduced the IL-3 dependent survival of Ba/F3. Analysis of the expression of the Bcl-2 members indicated that phosphorylation of Bad and Bcl-x expression which are respectively regulated by the PI 3-kinase/Akt pathway and STAT5 probably explain this cooperation. Furthermore, we showed by co-immunoprecipitation studies and pull down experiments with fusion proteins encoding the GST-SH2 domains of p85 that STAT5 in its phosphorylated form interacts with the p85 subunit of the PI 3-kinase. These results indicate that the activations of STAT5 and the PI 3-kinase by IL-3 in Ba/F3 cells are tightly connected and cooperate to mediate IL-3-dependent suppression of apoptosis by modulating Bad phosphorylation and Bcl-x expression.

Angiotensin II stimulates calcium-dependent activation of c-Jun N-terminal kinase.

PubMed Central

Zohn, I E; Yu, H; Li, X; Cox, A D; Earp, H S

1995-01-01

In GN4 rat liver epithelial cells, angiotensin II (Ang II) and other agonists which activate phospholipase C stimulate tyrosine kinase activity in a calcium-dependent, protein kinase C (PKC)-independent manner. Since Ang II also produces a proliferative response in these cells, we investigated downstream signaling elements traditionally linked to growth control by tyrosine kinases. First, Ang II, like epidermal growth factor (EGF), stimulated AP-1 binding activity in a PKC-independent manner. Because increases in AP-1 can reflect induction of c-Jun and c-Fos, we examined the activity of the mitogen-activated protein (MAP) kinase family members Erk-1 and -2 and the c-Jun N-terminal kinase (JNK), which are known to influence c-Jun and c-Fos transcription. Ang II stimulated MAP kinase (MAPK) activity but only approximately 50% as effectively as EGF; again, these effects were independent of PKC. Ang II also produced a 50- to 200-fold activation of JNK in a PKC-independent manner. Unlike its smaller effect on MAPK, Ang II was approximately four- to sixfold more potent in activating JNK than EGF was. Although others had reported a lack of calcium ionophore-stimulated JNK activity in lymphocytes and several other cell lines, we examined the role of calcium in GN4 cells. The following results suggest that JNK activation in rat liver epithelial cells is at least partially Ca(2+) dependent: (i) norepinephrine and vasopressin hormones that increase inositol 1,4,5-triphosphate stimulated JNK; (ii) both thapsigargin, a compound that produces an intracellular Ca(2+) signal, and Ca(2+) ionophores stimulated a dramatic increase in JNK activity (up to 200-fold); (iii) extracellular Ca(2+) chelation with ethylene glycol tetraacetic acid (EGTA) inhibited JNK activation by ionophore and intracellular chelation with 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl-ester (BAPTA-AM) partially inhibited JNK activation by Ang II or thapsigargin; and (iv) JNK activation by Ang II was inhibited by pretreatment of cells with thapsigargin and EGTA, a procedure which depletes intracellular Ca(2+) stores. JNK activation following Ang II stimulation did not involve calmodulin; either W-7 nor calmidizolium, in concentrations sufficient to inhibit Ca(2+)/calmodulin-dependent kinase II, blocked JNK activation by Ang II. In contrast, genistein, in concentrations sufficient to inhibit Ca(2+)-dependent tyrosine phosphorylation, prevented Ang II and thapsigargin-induced JNK activation. In summary, in GN4 rat liver epithelial cells, Ang II stimulates JNK via a novel Ca(2+)-dependent pathway. The inhibition by genistein suggest that Ca(2+)-dependent tyrosine phosphorylation may modulate the JNK pathway in a cell type-specific manner, particularly in cells with a readily detectable Ca(2+)-regulated tyrosine kinase. PMID:7565768

Proteolytic Degradation of SCOP in the Hippocampus Contributes to Activation of MAP Kinase and Memory

PubMed Central

Shimizu, Kimiko; Phan, Trongha; Mansuy, Isabelle; Storm, Daniel R.

2007-01-01

Summary Because activation of Erk1/2 MAP kinase (MAPK) is critical for hippocampus-dependent memory, there is considerable interest in mechanisms for regulation of MAPK during memory formation. Here we report that MAPK and CREB-mediated transcription are negatively regulated by SCOP (SCN Circadian Oscillatory Protein) and that SCOP is proteolyzed by calpain when hippocampal neurons are stimulated by BDNF, KCl depolarization, or NMDA. Moreover, training for novel object memory decreases SCOP in the hippocampus. To determine if hippocampus-dependent memory is influenced by SCOP in vivo, we generated a transgenic mouse strain for the inducible overexpression of SCOP in the forebrain. Overexpression of SCOP completely blocked memory for novel objects. We conclude that degradation of SCOP by calpain contributes to activation of MAPK during memory formation. PMID:17382888

Use of LC-MS/MS and Bayes' theorem to identify protein kinases that phosphorylate aquaporin-2 at Ser256.

PubMed

Bradford, Davis; Raghuram, Viswanathan; Wilson, Justin L L; Chou, Chung-Lin; Hoffert, Jason D; Knepper, Mark A; Pisitkun, Trairak

2014-07-15

In the renal collecting duct, binding of AVP to the V2 receptor triggers signaling changes that regulate osmotic water transport. Short-term regulation of water transport is dependent on vasopressin-induced phosphorylation of aquaporin-2 (AQP2) at Ser256. The protein kinase that phosphorylates this site is not known. We use Bayes' theorem to rank all 521 rat protein kinases with regard to the likelihood of a role in Ser256 phosphorylation on the basis of prior data and new experimental data. First, prior probabilities were estimated from previous transcriptomic and proteomic profiling data, kinase substrate specificity data, and evidence for kinase regulation by vasopressin. This ranking was updated using new experimental data describing the effects of several small-molecule kinase inhibitors with known inhibitory spectra (H-89, KN-62, KN-93, and GSK-650394) on AQP2 phosphorylation at Ser256 in inner medullary collecting duct suspensions. The top-ranked kinase was Ca2+/calmodulin-dependent protein kinase II (CAMK2), followed by protein kinase A (PKA) and protein kinase B (AKT). Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based in vitro phosphorylation studies compared the ability of three highly ranked kinases to phosphorylate AQP2 and other inner medullary collecting duct proteins, PKA, CAMK2, and serum/glucocorticoid-regulated kinase (SGK). All three proved capable of phosphorylating AQP2 at Ser256, although CAMK2 and PKA were more potent than SGK. The in vitro phosphorylation experiments also identified candidate protein kinases for several additional phosphoproteins with likely roles in collecting duct regulation, including Nedd4-2, Map4k4, and 3-phosphoinositide-dependent protein kinase 1. We conclude that Bayes' theorem is an effective means of integrating data from multiple data sets in physiology.

Substituted N-aryl-6-pyrimidinones: A new class of potent, selective, and orally active p38 MAP kinase inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Devadas, Balekudru; Selness, Shaun R.; Xing, Li

2012-02-28

A novel series of highly potent and selective p38 MAP kinase inhibitors was developed originating from a substituted N-aryl-6-pyrimidinone scaffold. SAR studies coupled with in vivo evaluations in rat arthritis model culminated in the identification of 10 with excellent oral efficacy. Compound 10 exhibited a significantly enhanced dissolution rate compared to 1, translating to a high oral bioavailability (>90%) in rat. In animal studies 10 inhibited LPS-stimulated production of tumor necrosis factor-{alpha} in a dose-dependent manner and demonstrated robust efficacy comparable to dexamethasone in a rat streptococcal cell wall-induced arthritis model.

Complementation of Myelodysplastic Syndrome Clones with Lentivirus Expression Libraries

DTIC Science & Technology

2012-07-01

Description HRAS Homo sapiens v-Ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS), transcript 1 CDC25C Homo sapiens cell division cycle 25...homolog C (CDC25C), transcript variant 1 MYC Homo sapiens v-myc myeloctomatosis viral oncogene homolog (avian) (MYC) MAP3K7 Homo sapiens mitogen...activated protein kinase kinase kinase 7 (MAP3K7) MAP3K8 Homo sapiens mitogen-activated protein kinase kinase kinase 8 (MAP3K8) SF3B1 Homo sapiens splicing

Complementation of Myelodysplastic Syndrome Clones with Lentivirus Expression Libraries

DTIC Science & Technology

2013-01-01

Description HRAS Homo sapiens v-Ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS), transcript 1 CDC25C Homo sapiens cell division cycle 25...homolog C (CDC25C), transcript variant 1 MYC Homo sapiens v-myc myeloctomatosis viral oncogene homolog (avian) (MYC) MAP3K7 Homo sapiens mitogen...activated protein kinase kinase kinase 7 (MAP3K7) MAP3K8 Homo sapiens mitogen-activated protein kinase kinase kinase 8 (MAP3K8) SF3B1 Homo sapiens

Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6.

PubMed

Ichimura, K; Mizoguchi, T; Yoshida, R; Yuasa, T; Shinozaki, K

2000-12-01

Mitogen-activated protein kinase (MAP kinase, MAPK) cascades play pivotal roles in signal transduction of extracellular stimuli, such as environmental stresses and growth regulators, in various organisms. Arabidopsis thaliana MAP kinases constitute a gene family, but stimulatory signals for each MAP kinase have not been elucidated. Here we show that environmental stresses such as low temperature, low humidity, hyper-osmolarity, touch and wounding induce rapid and transient activation of the Arabidopsis MAP kinases ATMPK4 and ATMPK6. Activation of ATMPK4 and ATMPK6 was associated with tyrosine phosphorylation but not with the amounts of mRNA or protein. Kinetics during activation differ between these two MAP kinases. These results suggest that ATMPK4 and ATMPK6 are involved in distinct signal transduction pathways responding to these environmental stresses.

The NO-cGMP-PKG Signaling Pathway Regulates Synaptic Plasticity and Fear Memory Consolidation in the Lateral Amygdala via Activation of ERK/MAP Kinase

ERIC Educational Resources Information Center

Ota, Kristie T.; Pierre, Vicki J.; Ploski, Jonathan E.; Queen, Kaila; Schafe, Glenn E.

2008-01-01

Recent studies have shown that nitric oxide (NO) signaling plays a crucial role in memory consolidation of Pavlovian fear conditioning and in synaptic plasticity in the lateral amygdala (LA). In the present experiments, we examined the role of the cGMP-dependent protein kinase (PKG), a downstream effector of NO, in fear memory consolidation and…

Dichotomal effect of space flight-associated microgravity on stress-activated protein kinases in innate immunity

PubMed Central

Verhaar, Auke P.; Hoekstra, Elmer; Tjon, Angela S. W.; Utomo, Wesley K.; Deuring, J. Jasper; Bakker, Elvira R. M.; Muncan, Vanesa; Peppelenbosch, Maikel P.

2014-01-01

Space flight strongly moderates human immunity but is in general well tolerated. Elucidation of the mechanisms by which zero gravity interacts with human immunity may provide clues for developing rational avenues to deal with exaggerated immune responses, e.g. as in autoimmune disease. Using two sounding rockets and one manned Soyuz launch, the influence of space flight on immunological signal transduction provoked by lipopolysaccharide (LPS) stimulation was investigated in freshly isolated peripheral blood monocytes and was compared to samples obtained from on-board centrifuge-loaded 1 g controls. The effect of microgravity on immunological signal transduction is highly specific, since LPS dependent Jun-N-terminal kinase activation is impaired in the 0 g condition, while the corresponding LPS dependent activation of p38 MAP kinase remains unaffected. Thus our results identify Jun-N-terminal kinase as a relevant target in immunity for microgravity and support using Jun-N-terminal kinase specific inhibitors for combating autoimmune disease. PMID:24968806

Dichotomal effect of space flight-associated microgravity on stress-activated protein kinases in innate immunity.

PubMed

Verhaar, Auke P; Hoekstra, Elmer; Tjon, Angela S W; Utomo, Wesley K; Deuring, J Jasper; Bakker, Elvira R M; Muncan, Vanesa; Peppelenbosch, Maikel P

2014-06-27

Space flight strongly moderates human immunity but is in general well tolerated. Elucidation of the mechanisms by which zero gravity interacts with human immunity may provide clues for developing rational avenues to deal with exaggerated immune responses, e.g. as in autoimmune disease. Using two sounding rockets and one manned Soyuz launch, the influence of space flight on immunological signal transduction provoked by lipopolysaccharide (LPS) stimulation was investigated in freshly isolated peripheral blood monocytes and was compared to samples obtained from on-board centrifuge-loaded 1 g controls. The effect of microgravity on immunological signal transduction is highly specific, since LPS dependent Jun-N-terminal kinase activation is impaired in the 0 g condition, while the corresponding LPS dependent activation of p38 MAP kinase remains unaffected. Thus our results identify Jun-N-terminal kinase as a relevant target in immunity for microgravity and support using Jun-N-terminal kinase specific inhibitors for combating autoimmune disease.

Systems-level identification of PKA-dependent signaling in epithelial cells.

PubMed

Isobe, Kiyoshi; Jung, Hyun Jun; Yang, Chin-Rang; Claxton, J'Neka; Sandoval, Pablo; Burg, Maurice B; Raghuram, Viswanathan; Knepper, Mark A

2017-10-17

G protein stimulatory α-subunit (G αs )-coupled heptahelical receptors regulate cell processes largely through activation of protein kinase A (PKA). To identify signaling processes downstream of PKA, we deleted both PKA catalytic subunits using CRISPR-Cas9, followed by a "multiomic" analysis in mouse kidney epithelial cells expressing the G αs -coupled V2 vasopressin receptor. RNA-seq (sequencing)-based transcriptomics and SILAC (stable isotope labeling of amino acids in cell culture)-based quantitative proteomics revealed a complete loss of expression of the water-channel gene Aqp2 in PKA knockout cells. SILAC-based quantitative phosphoproteomics identified 229 PKA phosphorylation sites. Most of these PKA targets are thus far unannotated in public databases. Surprisingly, 1,915 phosphorylation sites with the motif x-(S/T)-P showed increased phosphooccupancy, pointing to increased activity of one or more MAP kinases in PKA knockout cells. Indeed, phosphorylation changes associated with activation of ERK2 were seen in PKA knockout cells. The ERK2 site is downstream of a direct PKA site in the Rap1GAP, Sipa1l1, that indirectly inhibits Raf1. In addition, a direct PKA site that inhibits the MAP kinase kinase kinase Map3k5 (ASK1) is upstream of JNK1 activation. The datasets were integrated to identify a causal network describing PKA signaling that explains vasopressin-mediated regulation of membrane trafficking and gene transcription. The model predicts that, through PKA activation, vasopressin stimulates AQP2 exocytosis by inhibiting MAP kinase signaling. The model also predicts that, through PKA activation, vasopressin stimulates Aqp2 transcription through induction of nuclear translocation of the acetyltransferase EP300, which increases histone H3K27 acetylation of vasopressin-responsive genes (confirmed by ChIP-seq).

Resveratrol Inhibits the Epidermal Growth Factor-Induced Migration of Osteoblasts: the Suppression of SAPK/JNK and Akt.

PubMed

Kawabata, Tetsu; Tokuda, Haruhiko; Fujita, Kazuhiko; Kainuma, Shingo; Sakai, Go; Matsushima-Nishiwaki, Rie; Kozawa, Osamu; Otsuka, Takanobu

2017-01-01

Resveratrol is a polyphenol enriched in the skins of grapes and berries, that shows various beneficial effects for human health. In the present study, we investigated the mechanism behind the epidermal growth factor (EGF)-induced migration of osteoblast-like MC3T3-E1 cells, and the effect of resveratrol on this cell migration. The cell migration was examined using Boyden chamber, and phosphorylation of each kinase was analyzed by Western blotting. The EGF-induced migration was suppressed by PD98059, an inhibitor of MEK1/2, as well as SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of SAPK/JNK, and deguelin, an inhibitor of Akt. In contrast, rapamycin, an inhibitor of upstream kinase of p70 S6 kinase, and fasudil, an inhibitor of Rho-kinase, hardly affected the migration. Resveratrol significantly reduced the EGF-induced migration in a dose-dependent manner. SRT1720, an SIRT1 activator, suppressed the migration by EGF. In addition, resveratrol markedly attenuated the EGF-induced phosphorylation of SAPK/JNK and Akt without affecting the phosphorylation of p44/p42 MAP kinase or p38 MAP kinase. The phosphorylation of SAPK/JNK and Akt induced by EGF was down-regulated by SRT1720. Our results strongly suggest that resveratrol reduces the EGF-stimulated migration of osteoblasts via suppression of SAPK and Akt, and that the inhibitory effect of resveratrol is mediated in part via SIRT1. © 2017 The Author(s). Published by S. Karger AG, Basel.

Raf Kinase Inhibitory Protein protects cells against locostatin-mediated inhibition of migration.

PubMed

Shemon, Anne N; Eves, Eva M; Clark, Matthew C; Heil, Gary; Granovsky, Alexey; Zeng, Lingchun; Imamoto, Akira; Koide, Shohei; Rosner, Marsha Rich

2009-06-24

Raf Kinase Inhibitory Protein (RKIP, also PEBP1), a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function. We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP-depleted HeLa cells or RKIP-deficient (RKIP(-/-)) mouse embryonic fibroblasts (MEFs) to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP(-/-) MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle. These results suggest that locostatin's effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells.

Acquisition of contextual discrimination involves the appearance of a RAS-GRF1/p38 mitogen-activated protein (MAP) kinase-mediated signaling pathway that promotes long term potentiation (LTP).

PubMed

Jin, Shan-Xue; Arai, Junko; Tian, Xuejun; Kumar-Singh, Rajendra; Feig, Larry A

2013-07-26

RAS-GRF1 is a guanine nucleotide exchange factor with the ability to activate RAS and RAC GTPases in response to elevated calcium levels. We previously showed that beginning at 1 month of age, RAS-GRF1 mediates NMDA-type glutamate receptor (NMDAR)-induction of long term depression in the CA1 region of the hippocampus of mice. Here we show that beginning at 2 months of age, when mice first acquire the ability to discriminate between closely related contexts, RAS-GRF1 begins to contribute to the induction of long term potentiation (LTP) in the CA1 hippocampus by mediating the action of calcium-permeable, AMPA-type glutamate receptors (CP-AMPARs). Surprisingly, LTP induction by CP-AMPARs through RAS-GRF1 occurs via activation of p38 MAP kinase rather than ERK MAP kinase, which has more frequently been linked to LTP. Moreover, contextual discrimination is blocked by knockdown of Ras-Grf1 expression specifically in the CA1 hippocampus, infusion of a p38 MAP kinase inhibitor into the CA1 hippocampus, or the injection of an inhibitor of CP-AMPARs. These findings implicate the CA1 hippocampus in the developmentally dependent capacity to distinguish closely related contexts through the appearance of a novel LTP-supporting signaling pathway.

Acquisition of Contextual Discrimination Involves the Appearance of a RAS-GRF1/p38 Mitogen-activated Protein (MAP) Kinase-mediated Signaling Pathway That Promotes Long Term Potentiation (LTP)*

PubMed Central

Jin, Shan-Xue; Arai, Junko; Tian, Xuejun; Kumar-Singh, Rajendra; Feig, Larry A.

2013-01-01

RAS-GRF1 is a guanine nucleotide exchange factor with the ability to activate RAS and RAC GTPases in response to elevated calcium levels. We previously showed that beginning at 1 month of age, RAS-GRF1 mediates NMDA-type glutamate receptor (NMDAR)-induction of long term depression in the CA1 region of the hippocampus of mice. Here we show that beginning at 2 months of age, when mice first acquire the ability to discriminate between closely related contexts, RAS-GRF1 begins to contribute to the induction of long term potentiation (LTP) in the CA1 hippocampus by mediating the action of calcium-permeable, AMPA-type glutamate receptors (CP-AMPARs). Surprisingly, LTP induction by CP-AMPARs through RAS-GRF1 occurs via activation of p38 MAP kinase rather than ERK MAP kinase, which has more frequently been linked to LTP. Moreover, contextual discrimination is blocked by knockdown of Ras-Grf1 expression specifically in the CA1 hippocampus, infusion of a p38 MAP kinase inhibitor into the CA1 hippocampus, or the injection of an inhibitor of CP-AMPARs. These findings implicate the CA1 hippocampus in the developmentally dependent capacity to distinguish closely related contexts through the appearance of a novel LTP-supporting signaling pathway. PMID:23766509

PDK1-dependent activation of atypical PKC leads to degradation of the p21 tumour modifier protein

PubMed Central

Scott, Mary T.; Ingram, Angela; Ball, Kathryn L.

2002-01-01

p21WAF1/CIP1 contributes to positive and negative growth control on multiple levels. We previously mapped phosphorylation sites within the C-terminal domain of p21 that regulate proliferating cell nucear antigen binding. In the current study, a kinase has been fractionated from mammalian cells that stoichiometrically phosphorylates p21 at the Ser146 site, and the enzyme has been identified as an insulin-responsive atypical protein kinase C (aPKC). Expression of PKCζ or activation of the endogenous kinase by 3-phosphoinositide dependent protein kinase-1 (PDK1) decreased the half-life of p21. Conversely, dnPKCζ or dnPDK1 increased p21 protein half-life, and a PDK1-dependent increase in the rate of p21 degradation was mediated by aPKC. Insulin stimulation gave a biphasic response with a rapid transient decrease in p21 protein levels during the initial signalling phase that was dependent on phosphatidylinositol 3- kinase, PKC and proteasome activity. Thus, aPKC provides a physiological signal for the degradation of p21. The rapid degradation of p21 protein during the signalling phase of insulin stimulation identifies a novel link between energy metabolism and a key modulator of cell cycle progression. PMID:12485998

Procarcinogenic effects of cyclosporine A are mediated through the activation of TAK1/TAB1 signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Jianmin; Walsh, Stephanie B.; Verney, Zoe M.

Research highlights: {yields} Organ transplant recipients are highly susceptible to early skin cancer development. {yields} CsA-mediated TGFB1-dependent TAK1/TAB1 signaling augments invasive tumor growth. {yields} CsA enhances accumulation of upstream kinases, ZMP, AMPK and IRAK to activate TAK1. {yields} TAK1 mediates enhanced proliferation and reduced apoptosis via CsA-dependent NF{kappa}B. -- Abstract: Cyclosporine A (CsA) is an immunosuppressive drug commonly used for maintaining chronic immune suppression in organ transplant recipients. It is known that patients receiving CsA manifest increased growth of aggressive non-melanoma skin cancers. However, the underlying mechanism by which CsA augments tumor growth is not fully understood. Here, we showmore » that CsA augments the growth of A431 epidermoid carcinoma xenograft tumors by activating tumor growth factor {beta}-activated kinase1 (TAK1). The activation of TAK1 by CsA occurs at multiple levels by kinases ZMP, AMPK and IRAK. TAK1 forms heterodimeric complexes with TAK binding protein 1 and 2 (TAB1/TAB2) which in term activate nuclear factor {kappa}B (NF{kappa}B) and p38 MAP kinase. Transcriptional activation of NF{kappa}B is evidenced by IKK{beta}-mediated phosphorylation-dependent degradation of I{kappa}B and consequent nuclear translocation of p65. This also leads to enhancement in the expression of its transcriptional target genes cyclin D1, Bcl2 and COX-2. Similarly, activation of p38 leads to enhanced inflammation-related signaling shown by increased phosphorylation of MAPKAPK2 and which in turn phosphorylates its substrate HSP27. Activation of both NF{kappa}B and p38 MAP kinase provide mitogenic stimuli to augment the growth of SCCs.« less

Role of phosphatidylinositol 3-kinase in angiotensin II regulation of norepinephrine neuromodulation in brain neurons of the spontaneously hypertensive rat.

PubMed

Yang, H; Raizada, M K

1999-04-01

Chronic stimulation of norepinephrine (NE) neuromodulation by angiotensin II (Ang II) involves activation of the Ras-Raf-MAP kinase signal transduction pathway in Wistar Kyoto (WKY) rat brain neurons. This pathway is only partially responsible for this heightened action of Ang II in the spontaneously hypertensive rat (SHR) brain neurons. In this study, we demonstrate that the MAP kinase-independent signaling pathway in the SHR neuron involves activation of PI3-kinase and protein kinase B (PKB/Akt). Ang II stimulated PI3-kinase activity in both WKY and SHR brain neurons and was accompanied by its translocation from the cytoplasmic to the nuclear compartment. Although the magnitude of stimulation by Ang II was comparable, the stimulation was more persistent in the SHR neuron compared with the WKY rat neuron. Inhibition of PI3-kinase had no significant effect in the WKY rat neuron. However, it caused a 40-50% attenuation of the Ang II-induced increase in norepinephrine transporter (NET) and tyrosine hydroxylase (TH) mRNAs and [3H]-NE uptake in the SHR neuron. In contrast, inhibition of MAP kinase completely attenuated Ang II stimulation of NET and TH mRNA levels in the WKY rat neuron, whereas it caused only a 45% decrease in the SHR neuron. However, an additive attenuation was observed when both kinases of the SHR neurons were inhibited. Ang II also stimulated PKB/Akt activity in both WKY and SHR neurons. This stimulation was 30% higher and lasted longer in the SHR neuron compared with the WKY rat neuron. In conclusion, these observations demonstrate an exclusive involvement of PI3-kinase-PKB-dependent signaling pathway in a heightened NE neuromodulatory action of Ang II in the SHR neuron. Thus, this study offers an excellent potential for the development of new therapies for the treatment of centrally mediated hypertension.

A role for Pyk2 and Src in linking G-protein-coupled receptors with MAP kinase activation.

PubMed

Dikic, I; Tokiwa, G; Lev, S; Courtneidge, S A; Schlessinger, J

1996-10-10

The mechanisms by which mitogenic G-protein-coupled receptors activate the MAP kinase signalling pathway are poorly understood. Candidate protein tyrosine kinases that link G-protein-coupled receptors with MAP kinase include Src family kinases, the epidermal growth factor receptor, Lyn and Syk. Here we show that lysophosphatidic acid (LPA) and bradykinin induce tyrosine phosphorylation of Pyk2 and complex formation between Pyk2 and activated Src. Moreover, tyrosine phosphorylation of Pyk2 leads to binding of the SH2 domain of Src to tyrosine 402 of Pyk2 and activation of Src. Transient overexpression of a dominant interfering mutant of Pyk2 or the protein tyrosine kinase Csk reduces LPA- or bradykinin-induced activation of MAP kinase. LPA- or bradykinin-induced MAP kinase activation was also inhibited by overexpression of dominant interfering mutants of Grb2 and Sos. We propose that Pyk2 acts with Src to link Gi- and Gq-coupled receptors with Grb2 and Sos to activate the MAP kinase signalling pathway in PC12 cells.

Loss of Mitogen-Activated Protein Kinase Kinase Kinase 4 (MAP3K4) Reveals a Requirement for MAPK Signalling in Mouse Sex Determination

PubMed Central

Bogani, Debora; Siggers, Pam; Brixey, Rachel; Warr, Nick; Beddow, Sarah; Edwards, Jessica; Williams, Debbie; Wilhelm, Dagmar; Koopman, Peter; Flavell, Richard A.; Chi, Hongbo; Ostrer, Harry; Wells, Sara; Cheeseman, Michael; Greenfield, Andy

2009-01-01

Sex determination in mammals is controlled by the presence or absence of the Y-linked gene SRY. In the developing male (XY) gonad, sex-determining region of the Y (SRY) protein acts to up-regulate expression of the related gene, SOX9, a transcriptional regulator that in turn initiates a downstream pathway of testis development, whilst also suppressing ovary development. Despite the requirement for a number of transcription factors and secreted signalling molecules in sex determination, intracellular signalling components functioning in this process have not been defined. Here we report a role for the phylogenetically ancient mitogen-activated protein kinase (MAPK) signalling pathway in mouse sex determination. Using a forward genetic screen, we identified the recessive boygirl (byg) mutation. On the C57BL/6J background, embryos homozygous for byg exhibit consistent XY gonadal sex reversal. The byg mutation is an A to T transversion causing a premature stop codon in the gene encoding MAP3K4 (also known as MEKK4), a mitogen-activated protein kinase kinase kinase. Analysis of XY byg/byg gonads at 11.5 d post coitum reveals a growth deficit and a failure to support mesonephric cell migration, both early cellular processes normally associated with testis development. Expression analysis of mutant XY gonads at the same stage also reveals a dramatic reduction in Sox9 and, crucially, Sry at the transcript and protein levels. Moreover, we describe experiments showing the presence of activated MKK4, a direct target of MAP3K4, and activated p38 in the coelomic region of the XY gonad at 11.5 d post coitum, establishing a link between MAPK signalling in proliferating gonadal somatic cells and regulation of Sry expression. Finally, we provide evidence that haploinsufficiency for Map3k4 accounts for T-associated sex reversal (Tas). These data demonstrate that MAP3K4-dependent signalling events are required for normal expression of Sry during testis development, and create a novel entry point into the molecular and cellular mechanisms underlying sex determination in mice and disorders of sexual development in humans. PMID:19753101

A Conserved p38 Mitogen-Activated Protein Kinase Pathway Regulates Drosophila Immunity Gene Expression

PubMed Central

Han, Zhiqiang Stanley; Enslen, Hervé; Hu, Xiaodi; Meng, Xiangjun; Wu, I-Huan; Barrett, Tamera; Davis, Roger J.; Ip, Y. Tony

1998-01-01

Accumulating evidence suggests that the insect and mammalian innate immune response is mediated by homologous regulatory components. Proinflammatory cytokines and bacterial lipopolysaccharide stimulate mammalian immunity by activating transcription factors such as NF-κB and AP-1. One of the responses evoked by these stimuli is the initiation of a kinase cascade that leads to the phosphorylation of p38 mitogen-activated protein (MAP) kinase on Thr and Tyr within the motif Thr-Gly-Tyr, which is located within subdomain VIII. We have investigated the possible involvement of the p38 MAP kinase pathway in the Drosophila immune response. Two genes that are highly homologous to the mammalian p38 MAP kinase were molecularly cloned and characterized. Furthermore, genes that encode two novel Drosophila MAP kinase kinases, D-MKK3 and D-MKK4, were identified. D-MKK3 is an efficient activator of both Drosophila p38 MAP kinases, while D-MKK4 is an activator of D-JNK but not D-p38. These data establish that Drosophila indeed possesses a conserved p38 MAP kinase signaling pathway. We have examined the role of the D-p38 MAP kinases in the regulation of insect immunity. The results revealed that one of the functions of D-p38 is to attenuate antimicrobial peptide gene expression following exposure to lipopolysaccharide. PMID:9584193

AT1 receptor-mediated uptake of angiotensin II and NHE-3 expression in proximal tubule cells through a microtubule-dependent endocytic pathway.

PubMed

Li, Xiao C; Hopfer, Ulrich; Zhuo, Jia L

2009-11-01

Angiotensin II (ANG II) is taken up by proximal tubule (PT) cells via AT1 (AT1a) receptor-mediated endocytosis, but the underlying cellular mechanisms remain poorly understood. The present study tested the hypothesis that the microtubule- rather than the clathrin-dependent endocytic pathway regulates AT1-mediated uptake of ANG II and ANG II-induced sodium and hydrogen exchanger-3 (NHE-3) expression in PT cells. The expression of AT1 receptors, clathrin light (LC) and heavy chain (HC) proteins, and type 1 microtubule-associated proteins (MAPs; MAP-1A and MAP-1B) in PT cells were knocked down by their respective small interfering (si) RNAs before AT1-mediated FITC-ANG II uptake and ANG II-induced NHE-3 expression were studied. AT1 siRNAs inhibited AT1 expression and blocked ANG II-induced NHE-3 expression in PT cells, as expected (P < 0.01). Clathrin LC or HC siRNAs knocked down their respective proteins by approximately 90% with a peak response at 24 h, and blocked the clathrin-dependent uptake of Alexa Fluor 594-transferrin (P < 0.01). However, neither LC nor HC siRNAs inhibited AT1-mediated uptake of FITC-ANG II or affected ANG II-induced NHE-3 expression. MAP-1A or MAP-1B siRNAs markedly knocked down MAP-1A or MAP-1B proteins in a time-dependent manner with peak inhibitions at 48 h (>76.8%, P < 0.01). MAP protein knockdown resulted in approximately 52% decreases in AT1-mediated FITC-ANG II uptake and approximately 66% decreases in ANG II-induced NHE-3 expression (P < 0.01). These effects were associated with threefold decreases in ANG II-induced MAP kinases ERK 1/2 activation (P < 0.01), but not with altered AT1 expression or clathrin-dependent transferrin uptake. Both losartan and AT1a receptor deletion in mouse PT cells completely abolished the effects of MAP-1A knockdown on ANG II-induced NHE-3 expression and activation of MAP kinases ERK1/2. Our findings suggest that the alternative microtubule-dependent endocytic pathway, rather than the canonical clathrin-dependent pathway, plays an important role in AT1 (AT1a)-mediated uptake of extracellular ANG II and ANG II-induced NHE-3 expression in PT cells.

Raf Kinase Inhibitory Protein Protects Cells against Locostatin-Mediated Inhibition of Migration

PubMed Central

Shemon, Anne N.; Eves, Eva M.; Clark, Matthew C.; Heil, Gary; Granovsky, Alexey; Zeng, Lingchun; Imamoto, Akira

2009-01-01

Background Raf Kinase Inhibitory Protein (RKIP, also PEBP1), a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function. Methods/Findings We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP-depleted HeLa cells or RKIP-deficient (RKIP−/−) mouse embryonic fibroblasts (MEFs) to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP−/− MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle. Conclusions/Significance These results suggest that locostatin's effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells. PMID:19551145

The Aspergillus fumigatus SchASCH9 kinase modulates SakAHOG1 MAP kinase activity and it is essential for virulence

PubMed Central

Alves de Castro, Patrícia; dos Reis, Thaila Fernanda; Dolan, Stephen K.; Manfiolli, Adriana Oliveira; Brown, Neil Andrew; Jones, Gary W.; Doyle, Sean; Riaño-Pachón, Diego M.; Squina, Fábio Márcio; Caldana, Camila; Singh, Ashutosh; Del Poeta, Maurizio; Hagiwara, Daisuke; Silva-Rocha, Rafael; Goldman, Gustavo H.

2016-01-01

Summary The serine-threonine kinase TOR, the Target of Rapamycin, is an important regulator of nutrient, energy and stress signaling in eukaryotes. Sch9, a Ser/Thr kinase of AGC family (the cAMP-dependent PKA, cGMP- dependent protein kinase G and phospholipid-dependent protein kinase C family), is a substrate of TOR. Here, we characterized the fungal opportunistic pathogen Aspergillus fumigatus Sch9 homologue (SchA). The schA null mutant was sensitive to rapamycin, high concentrations of calcium, hyperosmotic stress and SchA was involved in iron metabolism. The ΔschA null mutant showed increased phosphorylation of SakA, the A. fumigatus Hog1 homologue. The schA null mutant has increased and decreased trehalose and glycerol accumulation, respectively, suggesting SchA performs different roles for glycerol and trehalose accumulation during osmotic stress. The schA was transcriptionally regulated by osmotic stress and this response was dependent on SakA and MpkC. The double ΔschA ΔsakA and ΔschA ΔmpkC mutants were more sensitive to osmotic stress than the corresponding parental strains. Transcriptomics and proteomics identified direct and indirect targets of SchA post-exposure to hyperosmotic stress. Finally, ΔschA was avirulent in a low dose murine infection model. Our results suggest there is a complex network of interactions amongst the A. fumigatus TOR, SakA and SchA pathways. PMID:27538790

The Aspergillus fumigatus SchASCH9 kinase modulates SakAHOG1 MAP kinase activity and it is essential for virulence.

PubMed

Alves de Castro, Patrícia; Dos Reis, Thaila Fernanda; Dolan, Stephen K; Oliveira Manfiolli, Adriana; Brown, Neil Andrew; Jones, Gary W; Doyle, Sean; Riaño-Pachón, Diego M; Squina, Fábio Márcio; Caldana, Camila; Singh, Ashutosh; Del Poeta, Maurizio; Hagiwara, Daisuke; Silva-Rocha, Rafael; Goldman, Gustavo H

2016-11-01

The serine-threonine kinase TOR, the Target of Rapamycin, is an important regulator of nutrient, energy and stress signaling in eukaryotes. Sch9, a Ser/Thr kinase of AGC family (the cAMP-dependent PKA, cGMP- dependent protein kinase G and phospholipid-dependent protein kinase C family), is a substrate of TOR. Here, we characterized the fungal opportunistic pathogen Aspergillus fumigatus Sch9 homologue (SchA). The schA null mutant was sensitive to rapamycin, high concentrations of calcium, hyperosmotic stress and SchA was involved in iron metabolism. The ΔschA null mutant showed increased phosphorylation of SakA, the A. fumigatus Hog1 homologue. The schA null mutant has increased and decreased trehalose and glycerol accumulation, respectively, suggesting SchA performs different roles for glycerol and trehalose accumulation during osmotic stress. The schA was transcriptionally regulated by osmotic stress and this response was dependent on SakA and MpkC. The double ΔschA ΔsakA and ΔschA ΔmpkC mutants were more sensitive to osmotic stress than the corresponding parental strains. Transcriptomics and proteomics identified direct and indirect targets of SchA post-exposure to hyperosmotic stress. Finally, ΔschA was avirulent in a low dose murine infection model. Our results suggest there is a complex network of interactions amongst the A. fumigatus TOR, SakA and SchA pathways. © 2016 John Wiley & Sons Ltd.

Hydrogen peroxide inhibits Ca2+-dependent chloride secretion across colonic epithelial cells via distinct kinase signaling pathways and ion transport proteins

PubMed Central

Chappell, Alfred E.; Bunz, Michael; Smoll, Eric; Dong, Hui; Lytle, Christian; Barrett, Kim E.; McCole, Declan F.

2018-01-01

Reactive oxygen species (ROS) are key mediators in a number of inflammatory conditions, including inflammatory bowel disease (IBD). ROS, including hydrogen peroxide (H2O2), modulate intestinal epithelial ion transport and are believed to contribute to IBD-associated diarrhea. Intestinal crypt fluid secretion, driven by electrogenic Cl− secretion, hydrates and sterilizes the crypt, thus reducing bacterial adherence. Here, we show that pathophysiological concentrations of H2O2 inhibit Ca2+-dependent Cl− secretion across T84 colonic epithelial cells by elevating cytosolic Ca2+, which contributes to activation of two distinct signaling pathways. One involves recruitment of the Ca2+-responsive kinases, Src and Pyk-2, as well as extracellular signal-regulated kinase (ERK). A separate pathway recruits p38 MAP kinase and phosphoinositide 3-kinase (PI3-K) signaling. The ion transport response to Ca2+-dependent stimuli is mediated in part by K+ efflux through basolateral K+ channels and Cl− uptake by the Na+-K+-2Cl− cotransporter, NKCC1. We demonstrate that H2O2 inhibits Ca2+-dependent basolateral K+ efflux and also inhibits NKCC1 activity independently of inhibitory effects on apical Cl− conductance. Thus, we have demonstrated that H2O2 inhibits Ca2+-dependent Cl− secretion through multiple negative regulatory signaling pathways and inhibition of specific ion transporters. These findings increase our understanding of mechanisms by which inflammation disturbs intestinal epithelial function and contributes to intestinal pathophysiology.—Chappell, A. E., Bunz, M., Smoll, E., Dong, H., Lytle, C., Barrett, K. E., McCole, D. F. Hydrogen peroxide inhibits Ca2+-dependent chloride secretion across colonic epithelial cells via distinct kinase signaling pathways and ion transport proteins. FASEB J. 22, 000–000 (2008) PMID:18211955

Dual leucine zipper kinase-dependent PERK activation contributes to neuronal degeneration following insult

PubMed Central

Larhammar, Martin; Huntwork-Rodriguez, Sarah; Jiang, Zhiyu; Solanoy, Hilda; Sengupta Ghosh, Arundhati; Wang, Bei; Kaminker, Joshua S; Huang, Kevin; Eastham-Anderson, Jeffrey; Siu, Michael; Modrusan, Zora; Farley, Madeline M; Tessier-Lavigne, Marc; Lewcock, Joseph W; Watkins, Trent A

2017-01-01

The PKR-like endoplasmic reticulum kinase (PERK) arm of the Integrated Stress Response (ISR) is implicated in neurodegenerative disease, although the regulators and consequences of PERK activation following neuronal injury are poorly understood. Here we show that PERK signaling is a component of the mouse MAP kinase neuronal stress response controlled by the Dual Leucine Zipper Kinase (DLK) and contributes to DLK-mediated neurodegeneration. We find that DLK-activating insults ranging from nerve injury to neurotrophin deprivation result in both c-Jun N-terminal Kinase (JNK) signaling and the PERK- and ISR-dependent upregulation of the Activating Transcription Factor 4 (ATF4). Disruption of PERK signaling delays neurodegeneration without reducing JNK signaling. Furthermore, DLK is both sufficient for PERK activation and necessary for engaging the ISR subsequent to JNK-mediated retrograde injury signaling. These findings identify DLK as a central regulator of not only JNK but also PERK stress signaling in neurons, with both pathways contributing to neurodegeneration. DOI: http://dx.doi.org/10.7554/eLife.20725.001 PMID:28440222

Suppression of transforming growth factor-beta-induced apoptosis through a phosphatidylinositol 3-kinase/Akt-dependent pathway.

PubMed

Chen, R H; Su, Y H; Chuang, R L; Chang, T Y

1998-10-15

Insulin and insulin receptor substrate 1 (IRS-1) are capable of protecting liver cells from apoptosis induced by transforming growth factor-beta1 (TGF-beta). The Ras/mitogen-activated protein kinase (MAP kinase) and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways are both activated upon insulin stimulation and can protect against apoptosis under certain circumstances. We investigated which of these pathways is responsible for the protective effect of insulin on TGF-beta-induced apoptosis. An activated Ras, although elicited a strong mitogenic effect, could not protect Hep3B cells from TGF-beta-induced apoptosis. Furthermore, PD98059, a selective inhibitor of MEK, did not suppress the antiapoptotic effect of insulin. In contrast, the PI 3-kinase inhibitor, LY294002, efficiently blocked the effect of insulin. Protection against TGF-beta-induced apoptosis conferred by PI 3-kinase was further verified by stable transfection of an activated PI 3-kinase. Downstream targets of PI 3-kinase involved in this protection was further investigated. An activated Akt mimicked the antiapoptotic effect of insulin, whereas a dominant-negative Akt inhibited such effect. However, rapamycin, the p70S6 kinase inhibitor, had no effect on the protectivity of insulin against TGF-beta-induced apoptosis, suggesting that the antiapoptotic target of PI 3-kinase/Akt pathway is independent or lies upstream of the p70S6 kinase. The mechanism by which PI 3-kinase/Akt pathway interferes with the apoptotic signaling of TGF-beta was explored. Activation of PI 3-kinase did not lead to a suppression of Smad hetero-oligomerization or nuclear translocation but blocked TGF-beta-induced caspase-3-like activity. In summary, the PI 3-kinase/Akt pathway, but not the Ras/MAP kinase pathway, protects against TGF-beta-induced apoptosis by inhibiting a step downstream of Smad but upstream of caspase-3.

High humidity suppresses ssi4-mediated cell death and disease resistance upstream of MAP kinase activation, H2O2 production and defense gene expression.

PubMed

Zhou, Fasong; Menke, Frank L H; Yoshioka, Keiko; Moder, Wolfgang; Shirano, Yumiko; Klessig, Daniel F

2004-09-01

The Arabidopsis ssi4 mutant, which exhibits spontaneous lesion formation, constitutive expression of pathogenesis-related (PR) genes and enhanced resistance to virulent bacterial and oomycete pathogens, contains a gain-of-function mutation in a TIR-NBS-LRR type R gene. Epistatic analyses revealed that both PR gene expression and disease resistance are activated via a salicylic acid (SA)- and EDS1-dependent, but NPR1- and NDR1-independent signaling pathway. In this study, we demonstrate that in moderate relative humidity (RH; 60%), the ssi4 mutant accumulates H(2)O(2) and SA prior to lesion formation and displays constitutive activation of the MAP kinases AtMPK6 and AtMPK3. It also constitutively expresses a variety of defense-associated genes, including those encoding the WRKY transcription factors AtWRKY29 and AtWRKY6, the MAP kinases AtMPK6 and AtMPK3, the powdery mildew R proteins RPW8.1 and RPW8.2, EDS1 and PR proteins. All of these ssi4-induced responses, as well as the chlorotic, stunted morphology and enhanced disease resistance phenotype, are suppressed by high RH (95%) growth conditions. Thus, a humidity sensitive factor (HSF) appears to function at an early point in the ssi4 signaling pathway. All ssi4 phenotypes, except for MAP kinase activation, also were suppressed by the eds1-1 mutation. Thus, ssi4-induced MAP kinase activation occurs downstream of the HSF but either upstream of EDS1 or on a separate branch of the ssi4 signaling pathway. SA is a critical signaling component in ssi4-mediated defense responses. However, exogenously supplied SA failed to restore lesion formation in high RH-grown ssi4 plants, although it induced defense gene expression. Thus, additional signals also are involved.

SCO2 induces p53-mediated apoptosis by Thr845 phosphorylation of ASK-1 and dissociation of the ASK-1-Trx complex.

PubMed

Madan, Esha; Gogna, Rajan; Kuppusamy, Periannan; Bhatt, Madan; Mahdi, Abbas Ali; Pati, Uttam

2013-04-01

p53 prevents cancer via cell cycle arrest, apoptosis, and the maintenance of genome stability. p53 also regulates energy-generating metabolic pathways such as oxidative phosphorylation (OXPHOS) and glycolysis via transcriptional regulation of SCO2 and TIGAR. SCO2, a cytochrome c oxidase assembly factor, is a metallochaperone which is involved in the biogenesis of cytochrome c oxidase subunit II. Here we have shown that SCO2 functions as an apoptotic protein in tumor xenografts, thus providing an alternative pathway for p53-mediated apoptosis. SCO2 increases the generation of reactive oxygen species (ROS) and induces dissociation of the protein complex between apoptosis signal-regulating kinase 1 (ASK-1) (mitogen-activated protein kinase kinase kinase [MAPKKK]) and its cellular inhibitor, the redox-active protein thioredoxin (Trx). Furthermore, SCO2 induces phosphorylation of ASK-1 at the Thr(845) residue, resulting in the activation of the ASK-1 kinase pathway. The phosphorylation of ASK-1 induces the activation of mitogen-activated protein kinase kinases 4 and 7 (MAP2K4/7) and MAP2K3/6, which switches the c-Jun N-terminal protein kinase (JNK)/p38-dependent apoptotic cascades in cancer cells. Exogenous addition of the SCO2 gene to hypoxic cancer cells and hypoxic tumors induces apoptosis and causes significant regression of tumor xenografts. We have thus discovered a novel apoptotic function of SCO2, which activates the ASK-1 kinase pathway in switching "on" an alternate mode of p53-mediated apoptosis. We propose that SCO2 might possess a novel tumor suppressor function via the ROS-ASK-1 kinase pathway and thus could be an important candidate for anticancer gene therapy.

Terbinafine stimulates the pro-inflammatory responses in human monocytic THP-1 cells through an ERK signaling pathway.

PubMed

Mizuno, Katsuhiko; Fukami, Tatsuki; Toyoda, Yasuyuki; Nakajima, Miki; Yokoi, Tsuyoshi

2010-10-23

Oral antifungal terbinafine has been reported to cause liver injury with inflammatory responses in a small percentage of patients. However the underlying mechanism remains unknown. To examine the inflammatory reactions, we investigated whether terbinafine and other antifungal drugs increase the release of pro-inflammatory cytokines using human monocytic cells. Dose- and time-dependent changes in the mRNA expression levels and the release of interleukin (IL)-8 and tumor necrosis factor (TNF)α from human monocytic THP-1 and HL-60 cells with antifungal drugs were measured. Effects of terbinafine on the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK)1/2 were investigated. The release of IL-8 and TNFα from THP-1 and HL-60 cells was significantly increased by treatment with terbinafine but not by fluconazole, suggesting that terbinafine can stimulate monocytes and increase the pro-inflammatory cytokine release. Terbinafine also significantly increased the phosphorylation of ERK1/2 and p38 MAP kinase in THP-1 cells. Pretreatment with a MAP kinase/ERK kinase (MEK)1/2 inhibitor U0126 significantly suppressed the increase of IL-8 and TNFα levels by terbinafine treatment in THP-1 cells, but p38 MAPK inhibitor SB203580 did not. These results suggested that an ERK1/2 pathway plays an important role in the release of IL-8 and TNFα in THP-1 cells treated with terbinafine. The release of inflammatory mediators by terbinafine might be one of the mechanisms underlying immune-mediated liver injury. This in vitro method may be useful to predict adverse inflammatory reactions that lead to drug-induced liver injury. Copyright © 2010 Elsevier Inc. All rights reserved.

Phosphorylation-Dependent Regulation of Ryanodine Receptors

PubMed Central

Marx, Steven O.; Reiken, Steven; Hisamatsu, Yuji; Gaburjakova, Marta; Gaburjakova, Jana; Yang, Yi-Ming; Rosemblit, Nora; Marks, Andrew R.

2001-01-01

Ryanodine receptors (RyRs), intracellular calcium release channels required for cardiac and skeletal muscle contraction, are macromolecular complexes that include kinases and phosphatases. Phosphorylation/dephosphorylation plays a key role in regulating the function of many ion channels, including RyRs. However, the mechanism by which kinases and phosphatases are targeted to ion channels is not well understood. We have identified a novel mechanism involved in the formation of ion channel macromolecular complexes: kinase and phosphatase targeting proteins binding to ion channels via leucine/isoleucine zipper (LZ) motifs. Activation of kinases and phosphatases bound to RyR2 via LZs regulates phosphorylation of the channel, and disruption of kinase binding via LZ motifs prevents phosphorylation of RyR2. Elucidation of this new role for LZs in ion channel macromolecular complexes now permits: (a) rapid mapping of kinase and phosphatase targeting protein binding sites on ion channels; (b) predicting which kinases and phosphatases are likely to regulate a given ion channel; (c) rapid identification of novel kinase and phosphatase targeting proteins; and (d) tools for dissecting the role of kinases and phosphatases as modulators of ion channel function. PMID:11352932

KinMap: a web-based tool for interactive navigation through human kinome data.

PubMed

Eid, Sameh; Turk, Samo; Volkamer, Andrea; Rippmann, Friedrich; Fulle, Simone

2017-01-05

Annotations of the phylogenetic tree of the human kinome is an intuitive way to visualize compound profiling data, structural features of kinases or functional relationships within this important class of proteins. The increasing volume and complexity of kinase-related data underlines the need for a tool that enables complex queries pertaining to kinase disease involvement and potential therapeutic uses of kinase inhibitors. Here, we present KinMap, a user-friendly online tool that facilitates the interactive navigation through kinase knowledge by linking biochemical, structural, and disease association data to the human kinome tree. To this end, preprocessed data from freely-available sources, such as ChEMBL, the Protein Data Bank, and the Center for Therapeutic Target Validation platform are integrated into KinMap and can easily be complemented by proprietary data. The value of KinMap will be exemplarily demonstrated for uncovering new therapeutic indications of known kinase inhibitors and for prioritizing kinases for drug development efforts. KinMap represents a new generation of kinome tree viewers which facilitates interactive exploration of the human kinome. KinMap enables generation of high-quality annotated images of the human kinome tree as well as exchange of kinome-related data in scientific communications. Furthermore, KinMap supports multiple input and output formats and recognizes alternative kinase names and links them to a unified naming scheme, which makes it a useful tool across different disciplines and applications. A web-service of KinMap is freely available at http://www.kinhub.org/kinmap/ .

MAP kinase-independent signaling in angiotensin II regulation of neuromodulation in SHR neurons.

PubMed

Yang, H; Raizada, M K

1998-09-01

Angiotensin II (Ang II), via its interaction with the angiotensin type 1 (AT1) receptor subtype, causes enhanced stimulation of norepinephrine (NE) neuromodulation. This involves increased transcription of NE transporter, tyrosine hydroxylase, and dopamine ss-hydroxylase genes in Wistar-Kyoto rat (WKY) brain neurons. AT1 receptor-mediated regulation of certain signaling events (such as activation of the Ras-Raf-1-mitogen activated protein (MAP) kinase signaling pathway, nuclear translocation of transcription factors such as Fos and Jun, and the interactions of these factors with AP-1 binding sites) is involved in this NE neuromodulation (Lu et al. J Cell Biol. 1996;135:1609-1617). The aim of this study was to compare the signal transduction mechanism of Ang II regulation of NE neuromodulation in WKY and spontaneously hypertensive rat (SHR) brain neurons, in view of the fact that AT1 receptor expression and Ang II stimulation of NE neuromodulation are higher in SHR neurons compared with WKY neurons. Despite this hyperactivity, Ang II stimulation of Ras, Raf-1, and MAP kinase activities was comparable between the neurons from WKY and SHR. Similarly, central injections of Ang II caused a comparable stimulation of MAP kinase in the hypothalamic and brain stem areas of adult WKY and SHR. Inhibition of MAP kinase by either an MAP kinase kinase inhibitor (PD98059) or an MAP kinase antisense oligonucleotide completely attenuated the stimulatory effects of Ang II on [3H]-NE uptake, NE transporter mRNA, and tyrosine hydroxylase mRNA levels in WKY neurons. These treatments resulted in only 43% to 50% inhibition of [3H]-NE uptake and NE transporter and tyrosine hydroxylase mRNAs in SHR neurons. Thus, Ang II stimulation of NE neuromodulation was completely blocked by MAP kinase inhibition in WKY neurons and only partially blocked in the SHR neurons. These observations suggest the presence of an additional signal transduction pathway involved in NE neuromodulation in SHR neurons that is independent of the MAP kinase pathway.

Involvement of the mitogen activated protein kinase Hog1p in the response of Candida albicans to iron availability

PubMed Central

2013-01-01

Background Iron is an essential nutrient for almost all organisms, and generating iron limiting conditions for pathogens is one of the host defense strategies against microbial infections. Excess of iron can be toxic; therefore, iron uptake is tightly controlled. The high affinity iron uptake system of the opportunistic pathogenic yeast Candida albicans has been shown to be essential for virulence. Several transcription factors and regulators of iron uptake genes were identified, but the knowledge of signaling pathways is still limited. Gene expression profiling of the Δhog1 deletion mutant indicated an involvement of the mitogen activated protein (MAP) kinase Hog1p. However, the function of Hog1p in the response of C. albicans to iron availability was not studied in detail. Thus, we analyzed phenotypic and molecular responses of C. albicans to different iron concentrations particularly with respect to the activity of the Hog1p MAP kinase module. Results We observed flocculation of yeast cells, when the iron ion concentration was equal to or higher than 5 μM. This phenotype was dependent on the MAP kinase Hog1p and the corresponding MAP kinase kinase Pbs2p. Moreover, high extracellular iron ion concentrations led to hyper-phosphorylation of Hog1p. We determined lower amounts of multicopper ferroxidase (MCFO) proteins and lower ferric reductase activity, when the iron ion concentration in the medium was increased. This effect was also observed for the Δhog1 mutant. However, the amounts of MCFO proteins and the cell surface ferric reductase activity were increased in the Δhog1 in comparison to wild type cells. This effect was independent of iron availability in growth media. Conclusions In C. albicans, the MAP kinase Hog1p is part of the network regulating the response of the organism to iron availability. Hog1p was transiently phosphorylated under high iron concentrations and was essential for a flocculent phenotype. Furthermore, deletion of HOG1 led to increased levels of components of the reductive iron uptake system in comparison to the wild-type, independent of iron concentrations in the media. However, the additional induction of this system by low iron concentrations was independent of HOG1. PMID:23347662

Extracellular and intracellular cleavages of proBDNF required at two distinct stages of late-phase LTP

NASA Astrophysics Data System (ADS)

Pang, Petti T.; Nagappan, Guhan; Guo, Wei; Lu, Bai

2016-05-01

Although late-phase long-term potentiation (L-LTP) is implicated in long-term memory, its molecular mechanisms are largely unknown. Here we provide evidence that L-LTP can be divided into two stages: an induction stage (I) and a maintenance stage (II). Both stages require mature brain-derived neurotrophic factor (mBDNF), but involve distinct underlying mechanisms. Stage I requires secretion of existing proBDNF followed by extracellular cleavage by tPA/plasmin. Stage II depends on newly synthesized BDNF. Surprisingly, mBDNF at stage II is derived from intracellular cleavage of proBDNF by furin/PC1. Moreover, stage I involves BDNF-TrkB signaling mainly through MAP kinase, whereas all three signaling pathways (phospholipase C-γ, PI3 kinase, and MAP kinase) are required for the maintenance of L-LTP at stage II. These results reveal the molecular basis for two temporally distinct stages in L-LTP, and provide insights on how BDNF modulates this long-lasting synaptic alternation at two critical time windows.

MAP KINASE ERK 1/2 INHIBITORS INDUCE DYSMORPHOLOGY IN MOUSE WHOLE EMBRYO CULTURE

EPA Science Inventory

ROSEN, M.B. and E. S. HUNTER. Reproductive Toxicology Division, NHEERL, ORD, U.S. EPA, Research Triangle Park, North Carolina. MAP kinase Erk1/2 inhibitors induce dysmorphology in mouse whole embryo culture.

MAP Kinase signal transduction is associated with a variety ...

Glutamate-dependent transcriptional regulation in bergmann glia cells: involvement of p38 MAP kinase.

PubMed

Zepeda, Rossana C; Barrera, Iliana; Castelán, Francisco; Soto-Cid, Abraham; Hernández-Kelly, Luisa C; López-Bayghen, Esther; Ortega, Arturo

2008-07-01

Glutamate (Glu) is the major excitatory neurotransmitter in the Central Nervous System (CNS). Ionotropic and metabotropic glutamate receptors (GluRs) are present in neurons and glial cells and are involved in gene expression regulation. Mitogen-activated proteins kinases (MAPK) are critical for all the membrane to nuclei signaling pathways described so far. In cerebellar Bergmann glial cells, glutamate-dependent transcriptional regulation is partially dependent on p42/44 MAPK activity. Another member of this kinase family, p38 MAPK is activated by non-mitogenic stimuli through its Thr180/Tyr182 phosphorylation and phosphorylates cytoplasmic and nuclear protein targets involved in translational and transcriptional events. Taking into consideration that the role of p38MAPK in glial cells is not well understood, we demonstrate here that glutamate increases p38 MAPK phosphorylation in a time and dose dependent manner in cultured chick cerebellar Bergmann glial cells (BGC). Moreover, p38 MAPK is involved in the glutamate-induced transcriptional activation in these cells. Ionotropic as well as metabotropic glutamate receptors participate in p38 MAPK activation. The present findings demonstrate the involvement of p38 MAPK in glutamate-dependent gene expression regulation in glial cells.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Li; Nomanbhoy, Tyzoon; Gurbani, Deepak

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

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

PubMed Central

2015-01-01

We developed a pharmacophore model for type II inhibitors that was used to guide the construction of a library of kinase inhibitors. Kinome-wide selectivity profiling of the library resulted in the identification of a series of 4-substituted 1H-pyrrolo[2,3-b]pyridines that exhibited potent inhibitory activity against two mitogen-activated protein kinases (MAPKs), TAK1 (MAP3K7) and MAP4K2, as well as pharmacologically well interrogated kinases such as p38α (MAPK14) and ABL. Further investigation of the structure–activity relationship (SAR) resulted in the identification of potent dual TAK1 and MAP4K2 inhibitors such as 1 (NG25) and 2 as well as MAP4K2 selective inhibitors such as 16 and 17. Some of these inhibitors possess good pharmacokinetic properties that will enable their use in pharmacological studies in vivo. A 2.4 Å cocrystal structure of TAK1 in complex with 1 confirms that the activation loop of TAK1 assumes the DFG-out conformation characteristic of type II inhibitors. PMID:25075558

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

DOE PAGES

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

2014-07-17

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

Normal p21Ras/MAP kinase pathway expression and function in PBMC from patients with polycystic ovary disease.

PubMed

Buchs, A; Chagag, P; Weiss, M; Kish, E; Levinson, R; Aharoni, D; Rapoport, M J

2004-04-01

Polycystic ovary disease (PCOD) is associated with insulin resistance and increased prevalence of type II diabetes mellitus (T2DM). The p21Ras/MAP kinase is a major intracellular signaling pathway mediating insulin signaling in insulin responsive tissues. The expression, regulation and function of the p21Ras/MAP kinase pathway in PCOD patients were examined. Peripheral blood mononuclear cells (PBMC) were isolated from ten patients with PCOD and ten controls. The expression of p21Ras and its regulatory proteins; hSOS1 and p120GAP were studied. The basal and phytohemaglutinin (PHA) or insulin stimulated phosphorylation of MAP kinase was determined. Expression of p21Ras, and its regulatory proteins hSOS1 and p120GAP were similar in PCOD patients and controls. Basal, PHA and insulin stimulated phosphorylation of MAP kinase, were also comparable in the two groups as well as their PBMC proliferative response. These data indicate that the expression and overall function of the p21Ras/MAP kinase pathway remain intact in non-diabetic patients with PCOD.

Structure-based design, synthesis, and biological evaluation of imidazo[1,2-b]pyridazine-based p38 MAP kinase inhibitors.

PubMed

Kaieda, Akira; Takahashi, Masashi; Takai, Takafumi; Goto, Masayuki; Miyazaki, Takahiro; Hori, Yuri; Unno, Satoko; Kawamoto, Tomohiro; Tanaka, Toshimasa; Itono, Sachiko; Takagi, Terufumi; Hamada, Teruki; Shirasaki, Mikio; Okada, Kengo; Snell, Gyorgy; Bragstad, Ken; Sang, Bi-Ching; Uchikawa, Osamu; Miwatashi, Seiji

2018-02-01

We identified novel potent inhibitors of p38 MAP kinase using structure-based design strategy. X-ray crystallography showed that when p38 MAP kinase is complexed with TAK-715 (1) in a co-crystal structure, Phe169 adopts two conformations, where one interacts with 1 and the other shows no interaction with 1. Our structure-based design strategy shows that these two conformations converge into one via enhanced protein-ligand hydrophobic interactions. According to the strategy, we focused on scaffold transformation to identify imidazo[1,2-b]pyridazine derivatives as potent inhibitors of p38 MAP kinase. Among the herein described and evaluated compounds, N-oxide 16 exhibited potent inhibition of p38 MAP kinase and LPS-induced TNF-α production in human monocytic THP-1 cells, and significant in vivo efficacy in rat collagen-induced arthritis models. In this article, we report the discovery of potent, selective and orally bioavailable imidazo[1,2-b]pyridazine-based p38 MAP kinase inhibitors with pyridine N-oxide group. Copyright © 2018 Elsevier Ltd. All rights reserved.

Effects of Butyltins (BTs) on Mitogen-Activated-Protein Kinase Kinase Kinase (MAP3K) and Ras Activity in Human Natural Killer Cells

PubMed Central

Celada, Lindsay J.; Whalen, Margaret M.

2013-01-01

Butyltins (BTs) contaminate the environment and are found in human blood. BTs, tributyltin (TBT) and dibutyltin (DBT), diminish the cytotoxic function and levels of key proteins of human natural killer (NK) cells. NK cells are an initial immune defense against tumors, virally-infected cells and antibody-coated cells and thus critical to human health. The signaling pathways that regulate NK cell functions include mitogen-activated protein kinases (MAPKs). Studies have shown that exposure to BTs leads to the activation of specific MAPKs and MAPK kinases (MAP2Ks) in human NK cells. MAP2K kinases (MAP3Ks) are upstream activators of MAP2Ks, which then activate MAPKs. The current study examined if BT-induced activation of MAP3Ks was responsible for MAP2K and thus, MAPK activation. This study examines the effects of TBT and DBT on the total levels of two MAP3Ks, c-Raf and ASK1, as well as activating and inhibitory phosphorylation sites on these MAP3Ks. In addition, the immediate upstream activator of c-Raf, Ras, was examined for BT-induced alterations. Our results show significant activation of the MAP3K, c-Raf, in human NK cells within 10 minutes of TBT exposure and the MAP3K, ASK1, after one hour exposures to TBT. In addition, our results suggest that both TBT and DBT are impacting the regulation of c-Raf. PMID:24038145

Exposure to a specific time-varying electromagnetic field inhibits cell proliferation via cAMP and ERK signaling in cancer cells.

PubMed

Buckner, Carly A; Buckner, Alison L; Koren, Stan A; Persinger, Michael A; Lafrenie, Robert M

2018-04-01

Exposure to specific electromagnetic field (EMF) patterns can affect a variety of biological systems. We have shown that exposure to Thomas-EMF, a low-intensity, frequency-modulated (25-6 Hz) EMF pattern, inhibited growth and altered cell signaling in malignant cells. Exposure to Thomas-EMF for 1 h/day inhibited the growth of malignant cells including B16-BL6 mouse melanoma cells, MDA-MB-231, MDA-MB-468, BT-20, and MCF-7 human breast cancer and HeLa cervical cancer cells but did not affect non-malignant cells. The Thomas-EMF-dependent changes in cell proliferation were mediated by adenosine 3',5'-cyclic monophosphate (cAMP) and extracellular-signal-regulated kinase (ERK) signaling pathways. Exposure of malignant cells to Thomas-EMF transiently changed the level of cellular cAMP and promoted ERK phosphorylation. Pharmacologic inhibitors (SQ22536) and activators (forskolin) of cAMP production both blocked the ability of Thomas-EMF to inhibit cell proliferation, and an inhibitor of the MAP kinase pathway (PD98059) was able to partially block Thomas-EMF-dependent inhibition of cell proliferation. Genetic modulation of protein kinase A (PKA) in B16-BL6 cells also altered the effect of Thomas-EMF on cell proliferation. Cells transfected with the constitutively active form of PKA (PKA-CA), which interfered with ERK phosphorylation, also interfered with the Thomas-EMF effect on cell proliferation. The non-malignant cells did not show any EMF-dependent changes in cAMP levels, ERK phosphorylation, or cell growth. These data indicate that exposure to the specific Thomas-EMF pattern can inhibit the growth of malignant cells in a manner dependent on contributions from the cAMP and MAP kinase pathways. Bioelectromagnetics. 39;217-230, 2018. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Enteroaggregative Escherichia coli flagellin-induced interleukin-8 secretion requires Toll-like receptor 5-dependent p38 MAP kinase activation

PubMed Central

Khan, Mohammed A S; Kang, Jian; Steiner, Theodore S

2004-01-01

Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen that causes acute and chronic diarrhoea in a number of clinical settings. EAEC diarrhoea involves bacterial aggregation, adherence to intestinal epithelial cells and elaboration of several toxigenic bacterial mediators. Flagellin (FliC-EAEC), a major bacterial surface protein of EAEC, causes interleukin (IL)-8 release from several epithelial cell lines. The host response to flagellins from E. coli and several other bacteria is mediated by Toll-like receptor 5 (TLR5), which signals through nuclear factor kappa B (NF-κB) to induce transcription of pro-inflammatory cytokines. p38 mitogen-activating protein (MAP) kinase (MAPK) is a member of a family of stress-related kinases that influences a diverse range of cellular functions including host inflammatory responses to microbial products. We studied the role of p38 MAPK in FliC-EAEC-induced IL-8 secretion from Caco-2 human intestinal epithelial cells and THP-1 human monocytic cells. We found that IL-8 secretion from both cell types is dependent on p38 MAPK, which is phospho-activated in response to FliC-EAEC. The role of TLR5 in p38 MAPK-dependent IL-8 secretion was verified in HEp-2 cells transiently transfected with a TLR5 expression construct. Activation of interleukin-1 receptor-associated kinase (IRAK) was also observed in Caco-2 and TLR5-transfected HEp-2 cells after exposure to FliC-EAEC. Finally, we demonstrated that pharmacological inhibition of p38 MAPK reduced IL-8 transcription and mRNA levels, but did not affect NF-κB activation. Collectively, our results suggest that TLR5 mediates p38 MAPK-dependent IL-8 secretion from epithelial and monocytic cells incubated with FliC-EAEC, and that this effect requires IL-8 promoter activation independent of NF-κB nuclear migration. PMID:15270737

Functional wiring of the yeast kinome revealed by global analysis of genetic network motifs

PubMed Central

Sharifpoor, Sara; van Dyk, Dewald; Costanzo, Michael; Baryshnikova, Anastasia; Friesen, Helena; Douglas, Alison C.; Youn, Ji-Young; VanderSluis, Benjamin; Myers, Chad L.; Papp, Balázs; Boone, Charles; Andrews, Brenda J.

2012-01-01

A combinatorial genetic perturbation strategy was applied to interrogate the yeast kinome on a genome-wide scale. We assessed the global effects of gene overexpression or gene deletion to map an integrated genetic interaction network of synthetic dosage lethal (SDL) and loss-of-function genetic interactions (GIs) for 92 kinases, producing a meta-network of 8700 GIs enriched for pathways known to be regulated by cognate kinases. Kinases most sensitive to dosage perturbations had constitutive cell cycle or cell polarity functions under standard growth conditions. Condition-specific screens confirmed that the spectrum of kinase dosage interactions can be expanded substantially in activating conditions. An integrated network composed of systematic SDL, negative and positive loss-of-function GIs, and literature-curated kinase–substrate interactions revealed kinase-dependent regulatory motifs predictive of novel gene-specific phenotypes. Our study provides a valuable resource to unravel novel functional relationships and pathways regulated by kinases and outlines a general strategy for deciphering mutant phenotypes from large-scale GI networks. PMID:22282571

Activation of AMPKα2 in adipocytes is essential for nicotine-induced insulin resistance in vivo

PubMed Central

Wu, Yue; Song, Ping; Zhang, Wencheng; Liu, Junhui; Dai, Xiaoyan; Liu, Zhaoyu; Lu, Qiulun; Ouyang, Changhan; Xie, Zhonglin; Zhao, Zhengxing; Zhuo, Xiaozhen; Viollet, Benoit; Foretz, Marc; Wu, Jiliang; Yuan, Zuyi; Zou, Ming-Hui

2015-01-01

Cigarette smoking promotes body weight reduction in humans while paradoxically also promoting insulin resistance (IR) and hyperinsulinemia. The mechanisms behind these effects of smoking are unclear. Here, we show that nicotine, a major constitute of cigarette smoke, selectively activates AMP-activated protein kinase α2 (AMPKα2) in adipocytes, which, in turn, phosphorylates MAP kinase phosphatase-1 (MKP1) at serine 334, initiating a proteasome-dependent degradation of this latter protein. The nicotine-dependent reduction in MKP1 induces the aberrant activation of p38 mitogen-activated protein kinase and c-Jun amino-terminal kinase leading to increased phosphorylation of insulin receptor substrate 1 (IRS1) at serine 307. This phosphorylation of IRS1 leads to its degradation, Akt inhibition, and the loss of insulin-mediated inhibition of lipolysis. Consequently, nicotine increases lipolysis, which results in body weight reduction, but this increase also elevates the levels of circulating free fatty acids and thus causes IR in insulin-sensitive tissues. These results newly place AMPKα2 as an essential mediator of nicotine-induced whole-body IR in spite of reductions in adiposity. PMID:25799226

Diadenosine polyphosphates Ap3A and Ap4A, but not Ap5A or Ap6A, induce proliferation of vascular smooth muscle cells.

PubMed

Bobbert, Peter; Schlüter, Hartmut; Schultheiss, Heinz Peter; Reusch, Hans Peter

2008-05-15

Depending on the number of phosphate groups, diadenosine polyphosphates (ApnA, Ap3A, Ap4A, Ap5A and Ap6A) differ in properties such as proliferation, apoptosis, vasoconstriction and vasodilatation of vascular smooth muscle cells (VSMCs). Possible signaling pathways leading to effects such as proliferation are still unknown. This study examined the proliferative effects of diadenosine polyphosphates on VSMCs and their intracellular pathways. Proliferation of VSMCs was measured by the cell count and [(3)H] thymidine incorporation. Phosphorylation of the MAP kinases ERK1/2 was determined by Western blotting. Single-cell [Ca(2+)](i) measurements were done to determine the influence of [Ca(2+)](i) on intracellular signaling. Stress fiber formation was assessed by fluorescence microscopy to detect an influence of G alpha(12). Ap3A and Ap4A, but not Ap5A or Ap6A, were shown to increase proliferation of VSMCs by activating P2Y receptors, which leads to stimulation of the Ras-Raf-MEK-ERK1/2 cascade. Ap3A- and Ap4A-induced activation of the MAP kinases ERK1/2 was dependent on a signaling pathway that included the EGF receptor, PKC, PLCbeta and the increase of [Ca(2+)](i). In conclusion, Ap3A and Ap4A, but not Ap5A or Ap6A, induce proliferation of VSMCs by a signaling pathway that begins with activation of P2Y receptors and leads to stimulation of the MAP kinases ERK1/2.

Gab1 Acts as an Adapter Molecule Linking the Cytokine Receptor gp130 to ERK Mitogen-Activated Protein Kinase

PubMed Central

Takahashi-Tezuka, Mariko; Yoshida, Yuichi; Fukada, Toshiyuki; Ohtani, Takuya; Yamanaka, Yojiro; Nishida, Keigo; Nakajima, Koichi; Hibi, Masahiko; Hirano, Toshio

1998-01-01

Gab1 has structural similarities with Drosophila DOS (daughter of sevenless), which is a substrate of the protein tyrosine phosphatase Corkscrew. Both Gab1 and DOS have a pleckstrin homology domain and tyrosine residues, potential binding sites for various SH2 domain-containing adapter molecules when they are phosphorylated. We found that Gab1 was tyrosine phosphorylated in response to various cytokines, such as interleukin-6 (IL-6), IL-3, alpha interferon (IFN-α), and IFN-γ. Upon the stimulation of IL-6 or IL-3, Gab1 was found to form a complex with phosphatidylinositol (PI)-3 kinase and SHP-2, a homolog of Corkscrew. Mutational analysis of gp130, the common subunit of IL-6 family cytokine receptors, revealed that neither tyrosine residues of gp130 nor its carboxy terminus was required for tyrosine phosphorylation of Gab1. Expression of Gab1 enhanced gp130-dependent mitogen-activated protein (MAP) kinase ERK2 activation. A mutation of tyrosine 759, the SHP-2 binding site of gp130, abrogated the interactions of Gab1 with SHP-2 and PI-3 kinase as well as ERK2 activation. Furthermore, ERK2 activation was inhibited by a dominant negative p85 PI-3 kinase, wortmannin, or a dominant negative Ras. These observations suggest that Gab1 acts as an adapter molecule in transmitting signals to ERK MAP kinase for the cytokine receptor gp130 and that SHP-2, PI-3 kinase, and Ras are involved in Gab1-mediated ERK activation. PMID:9632795

Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

PubMed Central

Roth Flach, Rachel J.; Skoura, Athanasia; Matevossian, Anouch; Danai, Laura V.; Zheng, Wei; Cortes, Christian; Bhattacharya, Samit K.; Aouadi, Myriam; Hagan, Nana; Yawe, Joseph C.; Vangala, Pranitha; Menendez, Lorena Garcia; Cooper, Marcus P.; Fitzgibbons, Timothy P.; Buckbinder, Leonard; Czech, Michael P.

2015-01-01

Signalling pathways that control endothelial cell (EC) permeability, leukocyte adhesion and inflammation are pivotal for atherosclerosis initiation and progression. Here we demonstrate that the Sterile-20-like mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4), which has been implicated in inflammation, is abundantly expressed in ECs and in atherosclerotic plaques from mice and humans. On the basis of endothelial-specific MAP4K4 gene silencing and gene ablation experiments in Apoe−/− mice, we show that MAP4K4 in ECs markedly promotes Western diet-induced aortic macrophage accumulation and atherosclerotic plaque development. Treatment of Apoe−/− and Ldlr−/− mice with a selective small-molecule MAP4K4 inhibitor also markedly reduces atherosclerotic lesion area. MAP4K4 silencing in cultured ECs attenuates cell surface adhesion molecule expression while reducing nuclear localization and activity of NFκB, which is critical for promoting EC activation and atherosclerosis. Taken together, these results reveal that MAP4K4 is a key signalling node that promotes immune cell recruitment in atherosclerosis. PMID:26688060

Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ahn, Yu Mi; Clare, Michael; Ensinger, Carol L.

Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or themore » doubly phosphorylated form of p38-alpha kinase.« less

Membrane Transfer from Mononuclear Cells to Polymorphonuclear Neutrophils Transduces Cell Survival and Activation Signals in the Recipient Cells via Anti-Extrinsic Apoptotic and MAP Kinase Signaling Pathways.

PubMed

Li, Ko-Jen; Wu, Cheng-Han; Shen, Chieh-Yu; Kuo, Yu-Min; Yu, Chia-Li; Hsieh, Song-Chou

2016-01-01

The biological significance of membrane transfer (trogocytosis) between polymorphonuclear neutrophils (PMNs) and mononuclear cells (MNCs) remains unclear. We investigated the biological/immunological effects and molecular basis of trogocytosis among various immune cells in healthy individuals and patients with active systemic lupus erythematosus (SLE). By flow cytometry, we determined that molecules in the immunological synapse, including HLA class-I and-II, CD11b and LFA-1, along with CXCR1, are exchanged among autologous PMNs, CD4+ T cells, and U937 cells (monocytes) after cell-cell contact. Small interfering RNA knockdown of the integrin adhesion molecule CD11a in U937 unexpectedly enhanced the level of total membrane transfer from U937 to PMN cells. Functionally, phagocytosis and IL-8 production by PMNs were enhanced after co-culture with T cells. Total membrane transfer from CD4+ T to PMNs delayed PMN apoptosis by suppressing the extrinsic apoptotic molecules, BAX, MYC and caspase 8. This enhancement of activities of PMNs by T cells was found to be mediated via p38- and P44/42-Akt-MAP kinase pathways and inhibited by the actin-polymerization inhibitor, latrunculin B, the clathrin inhibitor, Pitstop-2, and human immunoglobulin G, but not by the caveolin inhibitor, methyl-β-cyclodextrin. In addition, membrane transfer from PMNs enhanced IL-2 production by recipient anti-CD3/anti-CD28 activated MNCs, and this was suppressed by inhibitors of mitogen-activated protein kinase (PD98059) and protein kinase C (Rottlerin). Of clinical significance, decreased total membrane transfer from PMNs to MNCs in patients with active SLE suppressed mononuclear IL-2 production. In conclusion, membrane transfer from MNCs to PMNs, mainly at the immunological synapse, transduces survival and activation signals to enhance PMN functions and is dependent on actin polymerization, clathrin activation, and Fcγ receptors, while membrane transfer from PMNs to MNCs depends on MAP kinase and PKC signaling. Defective membrane transfer from PMNs to MNCs in patients with active systemic lupus erythematous suppressed activated mononuclear IL-2 production.

Protein interactome analysis of 12 mitogen-activated protein kinase kinase kinase in rice using a yeast two-hybrid system.

PubMed

Singh, Raksha; Lee, Jae-Eun; Dangol, Sarmina; Choi, Jihyun; Yoo, Ran Hee; Moon, Jae Sun; Shim, Jae-Kyung; Rakwal, Randeep; Agrawal, Ganesh Kumar; Jwa, Nam-Soo

2014-01-01

The mitogen-activated protein kinase (MAPK) cascade is composed at least of MAP3K (for MAPK kinase kinase), MAP2K, and MAPK family modules. These components together play a central role in mediating extracellular signals to the cell and vice versa by interacting with their partner proteins. However, the MAP3K-interacting proteins remain poorly investigated in plants. Here, we utilized a yeast two-hybrid system and bimolecular fluorescence complementation in the model crop rice (Oryza sativa) to map MAP3K-interacting proteins. We identified 12 novel nonredundant interacting protein pairs (IPPs) representing 11 nonredundant interactors using 12 rice MAP3Ks (available as full-length cDNA in the rice KOME (http://cdna01.dna.affrc.go.jp/cDNA/) at the time of experimental design and execution) as bait and a rice seedling cDNA library as prey. Of the 12 MAP3Ks, only six had interacting protein partners. The established MAP3K interactome consisted of two kinases, three proteases, two forkhead-associated domain-containing proteins, two expressed proteins, one E3 ligase, one regulatory protein, and one retrotransposon protein. Notably, no MAP3K showed physical interaction with either MAP2K or MAPK. Seven IPPs (58.3%) were confirmed in vivo by bimolecular fluorescence complementation. Subcellular localization of 14 interactors, together involved in nine IPPs (75%) further provide prerequisite for biological significance of the IPPs. Furthermore, GO of identified interactors predicted their involvement in diverse physiological responses, which were supported by a literature survey. These findings increase our knowledge of the MAP3K-interacting proteins, help in proposing a model of MAPK modules, provide a valuable resource for developing a complete map of the rice MAPK interactome, and allow discussion for translating the interactome knowledge to rice crop improvement against environmental factors. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Transcription Factor Ste12 Mediates the Regulatory Role of the Tmk1 MAP Kinase in Mycoparasitism and Vegetative Hyphal Fusion in the Filamentous Fungus Trichoderma atroviride

PubMed Central

Gruber, Sabine; Zeilinger, Susanne

2014-01-01

Mycoparasitic species of the fungal genus Trichoderma are potent antagonists able to combat plant pathogenic fungi by direct parasitism. An essential step in this mycoparasitic fungus-fungus interaction is the detection of the fungal host followed by activation of molecular weapons in the mycoparasite by host-derived signals. The Trichoderma atroviride MAP kinase Tmk1, a homolog of yeast Fus3/Kss1, plays an essential role in regulating the mycoparasitic host attack, aerial hyphae formation and conidiation. However, the transcription factors acting downstream of Tmk1 are hitherto unknown. Here we analyzed the functions of the T. atroviride Ste12 transcription factor whose orthologue in yeast is targeted by the Fus3 and Kss1 MAP kinases. Deletion of the ste12 gene in T. atroviride not only resulted in reduced mycoparasitic overgrowth and lysis of host fungi but also led to loss of hyphal avoidance in the colony periphery and a severe reduction in conidial anastomosis tube formation and vegetative hyphal fusion events. The transcription of several orthologues of Neurospora crassa hyphal fusion genes was reduced upon ste12 deletion; however, the Δste12 mutant showed enhanced expression of mycoparasitism-relevant chitinolytic and proteolytic enzymes and of the cell wall integrity MAP kinase Tmk2. Based on the comparative analyses of Δste12 and Δtmk1 mutants, an essential role of the Ste12 transcriptional regulator in mediating outcomes of the Tmk1 MAPK pathway such as regulation of the mycoparasitic activity, hyphal fusion and carbon source-dependent vegetative growth is suggested. Aerial hyphae formation and conidiation, in contrast, were found to be independent of Ste12. PMID:25356841

Thioredoxin-1 promotes survival in cells exposed to S-nitrosoglutathione: Correlation with reduction of intracellular levels of nitrosothiols and up-regulation of the ERK1/2 MAP Kinases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arai, Roberto J.; Ogata, Fernando T.; Batista, Wagner L.

2008-12-01

Accumulating evidence indicates that post-translational protein modifications by nitric oxide and its derived species are critical effectors of redox signaling in cells. These protein modifications are most likely controlled by intracellular reductants. Among them, the importance of the 12 kDa dithiol protein thioredoxin-1 (TRX-1) has been increasingly recognized. However, the effects of TRX-1 in cells exposed to exogenous nitrosothiols remain little understood. We investigated the levels of intracellular nitrosothiols and survival signaling in HeLa cells over-expressing TRX-1 and exposed to S-nitrosoglutahione (GSNO). A role for TRX-1 expression on GSNO catabolism and cell viability was demonstrated by the concentration-dependent effects ofmore » GSNO on decreasing TRX-1 expression, activation of caspase-3, and increasing cell death. The over-expression of TRX-1 in HeLa cells partially attenuated caspase-3 activation and enhanced cell viability upon GSNO treatment. This was correlated with reduction of intracellular levels of nitrosothiols and increasing levels of nitrite and nitrotyrosine. The involvement of ERK, p38 and JNK pathways were investigated in parental cells treated with GSNO. Activation of ERK1/2 MAP kinases was shown to be critical for survival signaling. In cells over-expressing TRX-1, basal phosphorylation levels of ERK1/2 MAP kinases were higher and further increased after GSNO treatment. These results indicate that the enhanced cell viability promoted by TRX-1 correlates with its capacity to regulate the levels of intracellular nitrosothiols and to up-regulate the survival signaling pathway mediated by the ERK1/2 MAP kinases.« less

The transcription factor Ste12 mediates the regulatory role of the Tmk1 MAP kinase in mycoparasitism and vegetative hyphal fusion in the filamentous fungus Trichoderma atroviride.

PubMed

Gruber, Sabine; Zeilinger, Susanne

2014-01-01

Mycoparasitic species of the fungal genus Trichoderma are potent antagonists able to combat plant pathogenic fungi by direct parasitism. An essential step in this mycoparasitic fungus-fungus interaction is the detection of the fungal host followed by activation of molecular weapons in the mycoparasite by host-derived signals. The Trichoderma atroviride MAP kinase Tmk1, a homolog of yeast Fus3/Kss1, plays an essential role in regulating the mycoparasitic host attack, aerial hyphae formation and conidiation. However, the transcription factors acting downstream of Tmk1 are hitherto unknown. Here we analyzed the functions of the T. atroviride Ste12 transcription factor whose orthologue in yeast is targeted by the Fus3 and Kss1 MAP kinases. Deletion of the ste12 gene in T. atroviride not only resulted in reduced mycoparasitic overgrowth and lysis of host fungi but also led to loss of hyphal avoidance in the colony periphery and a severe reduction in conidial anastomosis tube formation and vegetative hyphal fusion events. The transcription of several orthologues of Neurospora crassa hyphal fusion genes was reduced upon ste12 deletion; however, the Δste12 mutant showed enhanced expression of mycoparasitism-relevant chitinolytic and proteolytic enzymes and of the cell wall integrity MAP kinase Tmk2. Based on the comparative analyses of Δste12 and Δtmk1 mutants, an essential role of the Ste12 transcriptional regulator in mediating outcomes of the Tmk1 MAPK pathway such as regulation of the mycoparasitic activity, hyphal fusion and carbon source-dependent vegetative growth is suggested. Aerial hyphae formation and conidiation, in contrast, were found to be independent of Ste12.

Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase

PubMed Central

Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

2016-01-01

Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase. PMID:26867010

Factor Xa Inhibitor Suppresses the Release of Phosphorylated HSP27 from Collagen-Stimulated Human Platelets: Inhibition of HSP27 Phosphorylation via p44/p42 MAP Kinase.

PubMed

Tsujimoto, Masanori; Kuroyanagi, Gen; Matsushima-Nishiwaki, Rie; Kito, Yuko; Enomoto, Yukiko; Iida, Hiroki; Ogura, Shinji; Otsuka, Takanobu; Tokuda, Haruhiko; Kozawa, Osamu; Iwama, Toru

2016-01-01

Selective inhibitors of factor Xa (FXa) are widely recognized as useful therapeutic tools for stroke prevention in non-valvular atrial fibrillation or venous thrombosis. Thrombin, which is rapidly generated from pro-thrombin through the activation of factor X to FXa, acts as a potent activator of human platelets. Thus, the reduction of thrombin generation by FXa inhibitor eventually causes a suppressive effect on platelet aggregation. However, little is known whether FXa inhibitors directly affect the function of human platelets. We have previously reported that collagen induces the phosphorylation of heat shock protein 27 (HSP27), a low-molecular weight heat shock protein via Rac-dependent activation of p44/p42 mitogen-activated protein (MAP) kinase in human platelets, eventually resulting in the release of HSP27. In the present study, we investigated the direct effect of FXa inhibitor on the collagen-induced human platelet activation. Rivaroxaban as well as edoxaban significantly reduced the collagen-induced phosphorylation of both HSP27 and p44/p42 MAP kinase without affecting the platelet aggregation. Rivaroxaban significantly inhibited the release of phosphorylated HSP27 from collagen-stimulated platelets but not the secretion of platelet derived growth factor-AB. In patients administrated with rivaroxaban, the collagen-induced levels of phosphorylated HSP27 were markedly diminished after 2 days of administration, which failed to affect the platelet aggregation. These results strongly suggest that FXa inhibitor reduces the collagen-stimulated release of phosphorylated HSP27 from human platelets due to the inhibition of HSP27 phosphorylation via p44/p42 MAP kinase.

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.

Acrylamide inhibits cellular differentiation of human neuroblastoma and glioblastoma cells.

PubMed

Chen, Jong-Hang; Chou, Chin-Cheng

2015-08-01

This study explores human neuroblastoma (SH-SY5Y) and human glioblastoma (U-1240 MG) cellular differentiation changes under exposure to acrylamide (ACR). Differentiation of SH-SY5Y and U-1240 MG cells were induced by retinoic acid (RA) and butyric acid (BA), respectively. Morphological observations and MTT assay showed that the induced cellular differentiation and cell proliferation were inhibited by ACR in a time- and dose-dependent manner. ACR co-treatment with RA attenuated SH-SY5Y expressions of neurofilament protein-L (NF-L), microtubule-associated protein 1b (MAP1b; 1.2 to 0.7, p < 0.001), MAP2c (2.2 to 0.8, p < 0.05), and Janus kinase1 (JAK1; 1.9 to 0.6, p < 0.001), while ACR co-treatment with BA attenuated U-1240 MG expressions of glial fibrillary acidic protein (GFAP), MAP1b (1.2 to 0.6, p < 0.001), MAP2c (1.5 to 0.7, p < 0.01), and JAK1 (2.1 to 0.5, p < 0.001), respectively. ACR also decreased the phosphorylation of extracellular-signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) in U-1240 MG cells, while caffeine reversed this suppression of ERK and JNK phosphorylation caused by ACR treatment. These results showed that RA-induced neurogenesis of SH-SY5Y and BA-induced astrogliogenesis of U-1240 MG cells were attenuated by ACR and were associated with down-regulation of MAPs expression and JAK-STAT signaling. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mitogen-Activated Protein Kinases Are Associated with the Regulation of Physiological Traits and Virulence in Fusarium oxysporum f. sp. cubense

PubMed Central

Ding, Zhaojian; Li, Minhui; Sun, Fei; Xi, Pinggen; Sun, Longhua; Zhang, Lianhui; Jiang, Zide

2015-01-01

Fusarium oxysporum f. sp. cubense (FOC) is an important soil-borne fungal pathogen causing devastating vascular wilt disease of banana plants and has become a great concern threatening banana production worldwide. However, little information is known about the molecular mechanisms that govern the expression of virulence determinants of this important fungal pathogen. In this study, we showed that null mutation of three mitogen-activated protein (MAP) kinase genes, designated as FoSlt2, FoMkk2 and FoBck1, respectively, led to substantial attenuation in fungal virulence on banana plants. Transcriptional analysis revealed that the MAP kinase signaling pathway plays a key role in regulation of the genes encoding production of chitin, peroxidase, beauvericin and fusaric acid. Biochemical analysis further confirmed the essential role of MAP kinases in modulating the production of fusaric acid, which was a crucial phytotoxin in accelerating development of Fusarium wilt symptoms in banana plants. Additionally, we found that the MAP kinase FoSlt2 was required for siderophore biosynthesis under iron-depletion conditions. Moreover, disruption of the MAP kinase genes resulted in abnormal hypha and increased sensitivity to Congo Red, Calcofluor White and H2O2. Taken together, these results depict the critical roles of MAP kinases in regulation of FOC physiology and virulence. PMID:25849862

Phospho-mimicking Atf1 mutants bypass the transcription activating function of the MAP kinase Sty1 of fission yeast.

PubMed

Sánchez-Mir, Laura; Salat-Canela, Clàudia; Paulo, Esther; Carmona, Mercè; Ayté, José; Oliva, Baldo; Hidalgo, Elena

2018-02-01

Stress-dependent activation of signaling cascades is often mediated by phosphorylation events, but the exact nature and role of these phosphorelays are frequently poorly understood. Here, we review which are the consequences of the stress-dependent phosphorylation of a transcription factor on gene activation. In fission yeast, the MAP kinase Sty1 is activated upon several environmental hazards and promotes cell adaptation and survival, greatly through activation of a gene program mediated by the transcription factor Atf1. Although described decades ago, the role of the phosphorylation of Atf1 by Sty1 is still a matter of debate. We present here a brief review of recent data, obtained through the characterization of several phosphorylation mutant derivatives of Atf1, demonstrating that Atf1 phosphorylation does not stabilize the factor nor stimulates its binding to DNA. Rather, it provides a structural platform of interaction with the transcriptional machinery. Based on these findings, future work will establish how this phosphorylated trans-activation domain promotes the massive gene expression shift allowing cellular adaptation to stress.

Ezrin is a cyclic AMP-dependent protein kinase anchoring protein.

PubMed Central

Dransfield, D T; Bradford, A J; Smith, J; Martin, M; Roy, C; Mangeat, P H; Goldenring, J R

1997-01-01

cAMP-dependent protein kinase (A-kinase) anchoring proteins (AKAPs) are responsible for the subcellular sequestration of the type II A-kinase. Previously, we identified a 78 kDa AKAP which was enriched in gastric parietal cells. We have now purified the 78 kDa AKAP to homogeneity from gastric fundic mucosal supernates using type II A-kinase regulatory subunit (RII) affinity chromatography. The purified 78 kDa AKAP was recognized by monoclonal antibodies against ezrin, the canalicular actin-associated protein. Recombinant ezrin produced in either Sf9 cells or bacteria also bound RII. Recombinant radixin and moesin, ezrin-related proteins, also bound RII in blot overlay. Analysis of recombinant truncations of ezrin mapped the RII binding site to a region between amino acids 373 and 439. This region contained a 14-amino-acid amphipathic alpha-helical putative RII binding region. A synthetic peptide containing the amphipathic helical region (ezrin409-438) blocked RII binding to ezrin, but a peptide with a leucine to proline substitution at amino acid 421 failed to inhibit RII binding. In mouse fundic mucosa, RII immunoreactivity redistributed from a predominantly cytosolic location in resting parietal cells, to a canalicular pattern in mucosa from animals stimulated with gastrin. These results demonstrate that ezrin is a major AKAP in gastric parietal cells and may function to tether type II A-kinase to a region near the secretory canaliculus. PMID:9009265

Amitriptyline induces early growth response-1 gene expression via ERK and JNK mitogen-activated protein kinase pathways in rat C6 glial cells.

PubMed

Chung, Eun Young; Shin, Soon Young; Lee, Young Han

2007-07-05

Astrocytes play important roles in guiding the construction of the nervous system, controlling extracellular ions and neurotransmitters, and regulating CNS synaptogenesis. Egr-1 is a transcription factor involved in neuronal differentiation and astrocyte cell proliferation. In this study, we investigated whether the tricyclic antidepressant (TCA) amitriptyline induces Egr-1 expression in astrocytes using rat C6 glioma cells as a model. We found that amitriptyline increased the expression of Egr-1 in a dose- and time-dependent manner. The amitriptyline-induced Egr-1 expression was mediated through serum response elements (SREs) in the Egr-1 promoter. SREs were activated by the Ets-domain transcription factor Elk-1 through the ERK and JNK mitogen-activated protein (MAP) kinase pathways. The inhibition of the ERK and JNK MAP kinase signals attenuated amitriptyline-induced transactivation of Gal4-Elk-1 and Egr-1 promoter activity. Our findings suggest that the induction of Egr-1 expression in astrocytes may be required to attain the therapeutic effects of antidepressant drugs.

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

PubMed

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

2016-07-29

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

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

PubMed

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

1998-08-15

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

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

PubMed Central

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

1998-01-01

Autophosphorylation-dependent protein kinase (auto-kinase) was identified from pig brain and liver on the basis of its unique autophosphorylation/activation property [Yang, Fong, Yu and Liu (1987) J. Biol. Chem. 262, 7034-7040; Yang, Chang and Soderling (1987) J. Biol. Chem. 262, 9421-9427]. Its substrate consensus sequence motif was determined as being -R-X-(X)-S*/T*-X3-S/T-. To characterize auto-kinase further, we partly sequenced the kinase purified from pig liver. The N-terminal sequence (VDGGAKTSDKQKKKAXMTDE) and two internal peptide sequences (EKLRTIV and LQNPEK/ILTP/FI) of auto-kinase were obtained. These sequences identify auto-kinase as a C-terminal catalytic fragment of p21-activated protein kinase 2 (PAK2 or gamma-PAK) lacking its N-terminal regulatory region. Auto-kinase can be recognized by an antibody raised against the C-terminal peptide of human PAK2 by immunoblotting. Furthermore the autophosphorylation site sequence of auto-kinase was successfully predicted on the basis of its substrate consensus sequence motif and the known PAK2 sequence, and was further demonstrated to be RST(P)MVGTPYWMAPEVVTR by phosphoamino acid analysis, manual Edman degradation and phosphopeptide mapping via the help of phosphorylation site analysis of a synthetic peptide corresponding to the sequence of PAK2 from residues 396 to 418. During the activation process, auto-kinase autophosphorylates mainly on a single threonine residue Thr402 (according to the sequence numbering of human PAK2). In addition, a phospho-specific antibody against a synthetic phosphopeptide containing this identified sequence was generated and shown to be able to differentially recognize the activated auto-kinase autophosphorylated at Thr402 but not the non-phosphorylated/inactive auto-kinase. Immunoblot analysis with this phospho-specific antibody further revealed that the change in phosphorylation level of Thr402 of auto-kinase was well correlated with the activity change of the kinase during both autophosphorylation/activation and protein phosphatase-mediated dephosphorylation/inactivation processes. Taken together, our results identify Thr402 as the regulatory autophosphorylation site of auto-kinase, which is a C-terminal catalytic fragment of PAK2. PMID:9693111

Mechanisms underlying modulation of monocarboxylate transporter 1 (MCT1) by somatostatin in human intestinal epithelial cells.

PubMed

Saksena, Seema; Theegala, Saritha; Bansal, Nikhil; Gill, Ravinder K; Tyagi, Sangeeta; Alrefai, Waddah A; Ramaswamy, Krishnamurthy; Dudeja, Pradeep K

2009-11-01

Somatostatin (SST), an important neuropeptide of the gastrointestinal tract has been shown to stimulate sodium chloride absorption and inhibit chloride secretion in the intestine. However, the effects of SST on luminal butyrate absorption in the human intestine have not been investigated. Earlier studies from our group and others have shown that monocarboxylate transporter (MCT1) plays an important role in the transport of butyrate in the human intestine. The present studies were undertaken to examine the effects of SST on butyrate uptake utilizing postconfluent human intestinal epithelial Caco2 cells. Apical SST treatment of Caco-2 cells for 30-60 min significantly increased butyrate uptake in a dose-dependent manner with maximal increase at 50 nM ( approximately 60%, P < 0.05). SST receptor 2 agonist, seglitide, mimicked the effects of SST on butyrate uptake. SST-mediated stimulation of butyrate uptake involved the p38 MAP kinase-dependent pathway. Kinetic studies demonstrated that SST increased the maximal velocity (V(max)) of the transporter by approximately twofold without any change in apparent Michaelis-Menten constant (K(m)). The higher butyrate uptake in response to SST was associated with an increase in the apical membrane levels of MCT1 protein parallel to a decrease in the intracellular MCT1 pool. MCT1 has been shown to interact specifically with CD147 glycoprotein/chaperone to facilitate proper expression and function of MCT1 at the cell surface. SST significantly enhanced the membrane levels of CD147 as well as its association with MCT1. This association was completely abolished by the specific p38 MAP kinase inhibitor, SB203580. Our findings demonstrate that increased MCT1 association with CD147 at the apical membrane in response to SST is p38 MAP kinase dependent and underlies the stimulatory effects of SST on butyrate uptake.

Raf kinase inhibitory protein: a signal transduction modulator and metastasis suppressor.

PubMed

Granovsky, Alexey E; Rosner, Marsha Rich

2008-04-01

Cells have a multitude of controls to maintain their integrity and prevent random switching from one biological state to another. Raf Kinase Inhibitory Protein (RKIP), a member of the phosphatidylethanolamine binding protein (PEBP) family, is representative of a new class of modulators of signaling cascades that function to maintain the "yin yang" or balance of biological systems. RKIP inhibits MAP kinase (Raf-MEK-ERK), G protein-coupled receptor (GPCR) kinase and NFkappaB signaling cascades. Because RKIP targets different kinases dependent upon its state of phosphorylation, RKIP also acts to integrate crosstalk initiated by multiple environmental stimuli. Loss or depletion of RKIP results in disruption of the normal cellular stasis and can lead to chromosomal abnormalities and disease states such as cancer. Since RKIP and the PEBP family have been reviewed previously, the goal of this analysis is to provide an update and highlight some of the unique features of RKIP that make it a critical player in the regulation of cellular signaling processes.

Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region.

PubMed

Ahn, Yu Mi; Clare, Michael; Ensinger, Carol L; Hood, Molly M; Lord, John W; Lu, Wei-Ping; Miller, David F; Patt, William C; Smith, Bryan D; Vogeti, Lakshminarayana; Kaufman, Michael D; Petillo, Peter A; Wise, Scott C; Abendroth, Jan; Chun, Lawrence; Clark, Robin; Feese, Michael; Kim, Hidong; Stewart, Lance; Flynn, Daniel L

2010-10-01

Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase. Copyright © 2010 Elsevier Ltd. All rights reserved.

Differential subcellular membrane recruitment of Src may specify its downstream signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diesbach, Philippe de; Medts, Thierry; Carpentier, Sarah

2008-04-15

Most Src family members are diacylated and constitutively associate with membrane 'lipid rafts' that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at 'rafts' remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to 'lipid rafts'; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 deg. C and floated into sucrose density gradients like caveolin-1 andmore » flotillin-2, i.e. 'lipid rafts'. By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe ({approx} 70%) cholesterol extraction with methyl-{beta}-cyclodextrin (M{beta}CD) did not abolish 'rafts' floatation, but strongly decreased Src association with floating 'rafts' and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to M{beta}CD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at 'non-raft' domains on endosomes, then via PI3-kinase-Akt on a distinct set of 'rafts' at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined.« less

Acrolein-induced activation of mitogen-activated protein kinase signaling is mediated by alkylation of thioredoxin reductase and thioredoxin 1.

PubMed

Randall, Matthew J; Spiess, Page C; Hristova, Milena; Hondal, Robert J; van der Vliet, Albert

2013-01-01

Cigarette smoking remains a major health concern worldwide, and many of the adverse effects of cigarette smoke (CS) can be attributed to its abundant electrophilic aldehydes, such as acrolein (2-propenal). Previous studies indicate that acrolein readily reacts with thioredoxin reductase 1 (TrxR1), a critical enzyme involved in regulation of thioredoxin (Trx)-mediated redox signaling, by alkylation at its selenocysteine (Sec) residue. Because alkylation of Sec within TrxR1 has significant implications for its enzymatic function, we explored the potential importance of TrxR1 alkylation in acrolein-induced activation or injury of bronchial epithelial cells. Exposure of human bronchial epithelial HBE1 cells to acrolein (1-30 μM) resulted in dose-dependent loss of TrxR thioredoxin reductase activity, which coincided with its alkylation, as determined by biotin hydrazide labeling, and was independent of initial GSH status. To test the involvement of TrxR1 in acrolein responses in HBE1 cells, we suppressed TrxR1 using siRNA silencing or augmented TrxR1 by cell supplementation with sodium selenite. Acrolein exposure of HBE1 cells induced dose-dependent activation of the MAP kinases, extracellular regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, and activation of JNK was markedly enhanced after selenite-mediated induction of TrxR1, and was associated with increased alkylation of TrxR1. Conversely, siRNA silencing of TrxR1 significantly suppressed the ability of acrolein to activate JNK, and also appeared to attenuate acrolein-dependent activation of ERK and p38. Alteration of initial TrxR1 levels by siRNA or selenite supplementation also affected initial Trx1 redox status and acrolein-mediated alkylation of Trx1, but did not significantly affect acrolein-mediated activation of HO-1 or cytotoxicity. Collectively, our findings indicate that alkylation of TrxR1 and/or Trx1 may contribute directly to acrolein-mediated activation of MAP kinases such as JNK, and may therefore be important in acrolein-induced alterations in airway epithelial function, as a contributing mechanism in tobacco-related respiratory disease.

Epigallocatechin activates haem oxygenase-1 expression via protein kinase Cδ and Nrf2

PubMed Central

Ogborne, Richard M.; Rushworth, Stuart A.; O’Connell, Maria A.

2008-01-01

The Nrf2/anti-oxidant response element (ARE) pathway plays an important role in regulating cellular anti-oxidants, including haem oxygenase-1 (HO-1). Various kinases have been implicated in the pathways leading to Nrf2 activation. Here, we investigated the effect of epigallocatechin (EGC) on ARE-mediated gene expression in human monocytic cells. EGC time and dose dependently increased HO-1 mRNA and protein expression but had minimal effect on expression of other ARE-regulated genes, including NAD(P)H:quinone oxidoreductase 1, glutathione cysteine ligase and ferritin. siRNA knock down of Nrf2 significantly inhibited EGC-induced HO-1 expression. Furthermore, inhibition of PKC by Ro-31-8220 dose dependently decreased EGC-induced HO-1 mRNA expression, whereas MAP kinase and phosphatidylinositol-3-kinase pathway inhibitors had no significant effect. EGC stimulated phosphorylation of PKCαβ and δ in THP-1 cells. PKCδ inhibition significantly decreased EGC-induced HO-1 mRNA expression, whereas PKCα- and β-specific inhibitors had no significant effect. These results demonstrate for the first time that EGC-induced HO-1 expression occurs via PKCδ and Nrf2. PMID:18586007

Thioredoxin Activates MKK4-NFκB Pathway in a Redox-dependent Manner to Control Manganese Superoxide Dismutase Gene Expression in Endothelial Cells*

PubMed Central

Kundumani-Sridharan, Venkatesh; Subramani, Jaganathan; Das, Kumuda C.

2015-01-01

The mitogen-activated protein kinase kinase 4 (MKK4) is activated via phosphorylation of Ser-257 and Thr-261 by upstream MAP3Ks and activates JNK and p38 MAPKs in response to cellular stress. We show that thioredoxin (Trx), a cellular redox protein, activates MKK4 via Cys-246 and Cys-266 residues as mutation of these residues renders MKK4 insensitive to phosphorylation by MAP3Ks, TNFα, or Trx. MKK4 is activated in vitro by reduced Trx but not oxidized Trx in the absence of an upstream kinase, suggesting that autophosphorylation of this protein occurs due to reduction of Cys-246 and Cys-266 by Trx. Additionally, mutation of Cys-246 and Cys-266 resulted in loss of kinase activity suggesting that the redox state of Cys-246 and Cys-266 is a critical determinant of MKK4 activation. Trx induces manganese superoxide dismutase (MnSOD) gene transcription by activating MKK4 via redox control of Cys-246 and Cys-266, as mutation of these residues abrogates MKK4 activation and MnSOD expression. We further show that MKK4 activates NFκB for its binding to the MnSOD promoter, which leads to AP-1 dissociation followed by MnSOD transcription. Taken together, our studies show that the redox status of Cys-246 and Cys-266 in MKK4 controls its activities independent of MAP3K, demonstrating integration of the endothelial redox environment to MAPK signaling. PMID:26028649

Phosphorylation of Dgk1 Diacylglycerol Kinase by Casein Kinase II Regulates Phosphatidic Acid Production in Saccharomyces cerevisiae.

PubMed

Qiu, Yixuan; Hassaninasab, Azam; Han, Gil-Soo; Carman, George M

2016-12-16

In the yeast Saccharomyces cerevisiae, Dgk1 diacylglycerol (DAG) kinase catalyzes the CTP-dependent phosphorylation of DAG to form phosphatidic acid (PA). The enzyme in conjunction with Pah1 PA phosphatase controls the levels of PA and DAG for the synthesis of triacylglycerol and membrane phospholipids, the growth of the nuclear/endoplasmic reticulum membrane, and the formation of lipid droplets. Little is known about how DAG kinase activity is regulated by posttranslational modification. In this work, we examined the phosphorylation of Dgk1 DAG kinase by casein kinase II (CKII). When phosphate groups were globally reduced using nonspecific alkaline phosphatase, Triton X-100-solubilized membranes from DGK1-overexpressing cells showed a 7.7-fold reduction in DAG kinase activity; the reduced enzyme activity could be increased 5.5-fold by treatment with CKII. Dgk1(1-77) expressed heterologously in Escherichia coli was phosphorylated by CKII on a serine residue, and its phosphorylation was dependent on time as well as on the concentrations of CKII, ATP, and Dgk1(1-77). We used site-specific mutagenesis, coupled with phosphorylation analysis and phosphopeptide mapping, to identify Ser-45 and Ser-46 of Dgk1 as the CKII target sites, with Ser-46 being the major phosphorylation site. In vivo, the S46A and S45A/S46A mutations of Dgk1 abolished the stationary phase-dependent stimulation of DAG kinase activity. In addition, the phosphorylation-deficient mutations decreased Dgk1 function in PA production and in eliciting pah1Δ phenotypes, such as the expansion of the nuclear/endoplasmic reticulum membrane, reduced lipid droplet formation, and temperature sensitivity. This work demonstrates that the CKII-mediated phosphorylation of Dgk1 regulates its function in the production of PA. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Signal-transducing protein phosphorylation cascades mediated by Ras/Rho proteins in the mammalian cell: the potential for multiplex signalling.

PubMed Central

Denhardt, D T

1996-01-01

The features of three distinct protein phosphorylation cascades in mammalian cells are becoming clear. These signalling pathways link receptor-mediated events at the cell surface or intracellular perturbations such as DNA damage to changes in cytoskeletal structure, vesicle transport and altered transcription factor activity. The best known pathway, the Ras-->Raf-->MEK-->ERK cascade [where ERK is extracellular-signal-regulated kinase and MEK is mitogen-activated protein (MAP) kinase/ERK kinase], is typically stimulated strongly by mitogens and growth factors. The other two pathways, stimulated primarily by assorted cytokines, hormones and various forms of stress, predominantly utilize p21 proteins of the Rho family (Rho, Rac and CDC42), although Ras can also participate. Diagnostic of each pathway is the MAP kinase component, which is phosphorylated by a unique dual-specificity kinase on both tyrosine and threonine in one of three motifs (Thr-Glu-Tyr, Thr-Phe-Tyr or Thr-Gly-Tyr), depending upon the pathway. In addition to activating one or more protein phosphorylation cascades, the initiating stimulus may also mobilize a variety of other signalling molecules (e.g. protein kinase C isoforms, phospholipid kinases, G-protein alpha and beta gamma subunits, phospholipases, intracellular Ca2+). These various signals impact to a greater or lesser extent on multiple downstream effectors. Important concepts are that signal transmission often entails the targeted relocation of specific proteins in the cell, and the reversible formation of protein complexes by means of regulated protein phosphorylation. The signalling circuits may be completed by the phosphorylation of upstream effectors by downstream kinases, resulting in a modulation of the signal. Signalling is terminated and the components returned to the ground state largely by dephosphorylation. There is an indeterminant amount of cross-talk among the pathways, and many of the proteins in the pathways belong to families of closely related proteins. The potential for more than one signal to be conveyed down a pathway simultaneously (multiplex signalling) is discussed. The net effect of a given stimulus on the cell is the result of a complex intracellular integration of the intensity and duration of activation of the individual pathways. The specific outcome depends on the particular signalling molecules expressed by the target cells and on the dynamic balance among the pathways. PMID:8836113

R-Ras Contributes to LTP and Contextual Discrimination

PubMed Central

Darcy, Michael J.; Jin, Shan-Xue; Feig, Larry A.

2014-01-01

The ability to discriminate between closely related contexts is a specific form of hippocampal-dependent learning that may be impaired in certain neurodegenerative disorders such as Alzheimer's and Down Syndrome. However, signaling pathways regulating this form of learning are poorly understood. Previous studies have shown that the calcium-dependent exchange factor Ras-GRF1, an activator of Rac, Ras and R-Ras GTPases, is important for this form of learning and memory. Moreover, the ability to discriminate contexts was linked to the ability of Ras-GRF1 to promote high-frequency stimulation (HFS)-LTP via the activation of p38 Map kinase. Here, we show that R-Ras is involved in this form of learning by using virally-delivered miRNAs targeting R-Ras into the CA1 region of dorsal hippocampus and observing impaired contextual discrimination. Like the loss of GRF1, knockdown of R-Ras in the CA1 also impairs the induction of HFS-LTP and p38 Map kinase. Nevertheless, experiments indicate that this involvement of R-Ras in HFS-LTP that is required for contextual discrimination is independent of Ras-GRF1. Thus, R-Ras is a novel regulator of a form of hippocampal-dependent LTP as well as learning and memory that is affected in certain forms of neurodegenerative diseases. PMID:25043327

A rice kinase-protein interaction map.

PubMed

Ding, Xiaodong; Richter, Todd; Chen, Mei; Fujii, Hiroaki; Seo, Young Su; Xie, Mingtang; Zheng, Xianwu; Kanrar, Siddhartha; Stevenson, Rebecca A; Dardick, Christopher; Li, Ying; Jiang, Hao; Zhang, Yan; Yu, Fahong; Bartley, Laura E; Chern, Mawsheng; Bart, Rebecca; Chen, Xiuhua; Zhu, Lihuang; Farmerie, William G; Gribskov, Michael; Zhu, Jian-Kang; Fromm, Michael E; Ronald, Pamela C; Song, Wen-Yuan

2009-03-01

Plants uniquely contain large numbers of protein kinases, and for the vast majority of the 1,429 kinases predicted in the rice (Oryza sativa) genome, little is known of their functions. Genetic approaches often fail to produce observable phenotypes; thus, new strategies are needed to delineate kinase function. We previously developed a cost-effective high-throughput yeast two-hybrid system. Using this system, we have generated a protein interaction map of 116 representative rice kinases and 254 of their interacting proteins. Overall, the resulting interaction map supports a large number of known or predicted kinase-protein interactions from both plants and animals and reveals many new functional insights. Notably, we found a potential widespread role for E3 ubiquitin ligases in pathogen defense signaling mediated by receptor-like kinases, particularly by the kinases that may have evolved from recently expanded kinase subfamilies in rice. We anticipate that the data provided here will serve as a foundation for targeted functional studies in rice and other plants. The application of yeast two-hybrid and TAPtag analyses for large-scale plant protein interaction studies is also discussed.

[MAP kinases--molecular transistors in animals and plants].

PubMed

Petersen, Morten; Brodersen, Peter; Mundy, John

2002-06-10

The survival of multicellular organisms depends on the ability of their cells to communicate with each other and to respond to environmental changes. A goal of modern biology is to uncover the processes by which these cellular signals are transduced. Recent studies have shown that MAP-kinases (MAPKs) are important constituents of such signal transduction pathways. MAPKs function as modules in phosphorelay cascades to activate or repress the activity of downstream target proteins. For example, recent research with knockout mice has shown that mammalian MAPKs are involved in the control of neuronal apoptosis and the activation of immune responses. These mammalian MAPKs exert their control by both promoting and inhibiting specific processes. Surprisingly, plants also use MAPKs to control their immune responses, and plant MAPKs also seem to play dual roles as positive and negative regulators. Such mechanistic similarities provide the basis for fruitful conceptual exchange between molecular research on animals and plants.

Pervanadate induces Mammalian Ste20 Kinase 3 (MST3) tyrosine phosphorylation but not activation.

PubMed

Kan, Wei-Chih; Lu, Te-Ling; Ling, Pin; Lee, Te-Hsiu; Cho, Chien-Yu; Huang, Chi-Ying F; Jeng, Wen-Yih; Weng, Yui-Ping; Chiang, Chun-Yen; Wu, Jin Bin; Lu, Te-Jung

2016-07-01

The yeast Ste20 (sterile) protein kinase, which is a serine/threonine kinase, responds to the stimulation of the G proteincoupled receptor (GPCR) pheromone receptor. Ste20 protein kinase serves as the critical component that links signaling from the GPCR/G proteins to the mitogen-activated protein kinase (MAPK) cascade in yeast. The yeast Ste20p functions as a MAP kinase kinase kinase kinase (MAP4K) in the pheromone response. Ste20-like kinases are structurally conserved from yeast to mammals. The mechanism by which MAP4K links GPCR to the MAPK pathway is less clearly defined in vertebrates. In addition to MAP4K, the tyrosine kinase cascade bridges G proteins and the MAPK pathway in vertebrate cells. Mammalian Ste20 Kinase 3 (MST3) has been categorized into the Ste20 family and has been reported to function in the regulation of cell polarity and migration. However, whether MST3 tyrosine phosphorylation regulates diverse signaling pathways is unknown. In this study, the tyrosine phosphatase inhibitor pervanadate was found to induce MST3 tyrosine phosphorylation in intact cells, and the activity of tyrosine-phosphorylated MST3 was measured. This tyrosine-directed phosphorylation was independent of MST3 activity. Parameters including protein conformation, Triton concentration and ionic concentration influenced the sensitivity of MST3 activity. Taken together, our data suggests that the serine/threonine kinase MST3 undergoes tyrosinedirected phosphorylation. The tyrosine-phosphorylated MST3 may create a docking site for the structurally conserved SH2/SH3 (Src Homology 2 and 3) domains within the Src oncoprotein. The unusual tyrosinephosphorylated MST3 may recruit MST3 to various signaling components. Copyright © 2016. Published by Elsevier Inc.

Importance of MAP Kinases during Protoperithecial Morphogenesis in Neurospora crassa

PubMed Central

Jeffree, Chris E.; Oborny, Radek; Boonyarungsrit, Patid; Read, Nick D.

2012-01-01

In order to produce multicellular structures filamentous fungi combine various morphogenetic programs that are fundamentally different from those used by plants and animals. The perithecium, the female sexual fruitbody of Neurospora crassa, differentiates from the vegetative mycelium in distinct morphological stages, and represents one of the more complex multicellular structures produced by fungi. In this study we defined the stages of protoperithecial morphogenesis in the N. crassa wild type in greater detail than has previously been described; compared protoperithecial morphogenesis in gene-deletion mutants of all nine mitogen-activated protein (MAP) kinases conserved in N. crassa; confirmed that all three MAP kinase cascades are required for sexual development; and showed that the three different cascades each have distinctly different functions during this process. However, only MAP kinases equivalent to the budding yeast pheromone response and cell wall integrity pathways, but not the osmoregulatory pathway, were essential for vegetative cell fusion. Evidence was obtained for MAP kinase signaling cascades performing roles in extracellular matrix deposition, hyphal adhesion, and envelopment during the construction of fertilizable protoperithecia. PMID:22900028

Cot kinase plays a critical role in Helicobacter pylori-induced IL-8 expression.

PubMed

Jang, Sungil; Kim, Jinmoon; Cha, Jeong-Heon

2017-04-01

Helicobacter pylori is a major pathogen causing various gastric diseases including gastric cancer. Infection of H. pylori induces pro-inflammatory cytokine IL-8 expression in gastric epithelial cells in the initial inflammatory process. It has been known that H. pylori can modulate Ras-Raf-Mek-Erk signal pathway for IL-8 induction. Recently, it has been shown that another signal molecule, cancer Osaka thyroid oncogene/tumor progression locus 2 (Cot/Tpl2) kinase, activates Mek and Erk and plays a role in the Erk pathway, similar to MAP3K signal molecule Raf kinase. Therefore, the objective of this study was to determine whether Cot kinase might be involved in IL-8 induction caused by H. pylori infection. AGS gastric epithelial cells were infected by H. pylori strain G27 or its isogenic mutants lacking cagA or type IV secretion system followed by treatment with Cot kinase inhibitor (KI) or siRNA specific for Cot kinase. Activation of Erk was assessed by Western blot analysis and expression of IL-8 was measured by ELISA. Treatment with Cot KI reduced both transient and sustained Erk activation. It also reduced early and late IL-8 secretion in the gastric epithelial cell line. Furthermore, siRNA knockdown of Cot inhibited early and late IL-8 secretion induced by H. pylori infection. Taken together, these results suggest that Cot kinase might play a critical role in H. pylori type IV secretion apparatus-dependent early IL-8 secretion and CagA-dependent late IL-8 secretion as an alternative signaling molecule in the Erk pathway.

Virtual screening filters for the design of type II p38 MAP kinase inhibitors: a fragment based library generation approach.

PubMed

Badrinarayan, Preethi; Sastry, G Narahari

2012-04-01

In this work, we introduce the development and application of a three-step scoring and filtering procedure for the design of type II p38 MAP kinase leads using allosteric fragments extracted from virtual screening hits. The design of the virtual screening filters is based on a thorough evaluation of docking methods, DFG-loop conformation, binding interactions and chemotype specificity of the 138 p38 MAP kinase inhibitors from Protein Data Bank bound to DFG-in and DFG-out conformations using Glide, GOLD and CDOCKER. A 40 ns molecular dynamics simulation with the apo, type I with DFG-in and type II with DFG-out forms was carried out to delineate the effects of structural variations on inhibitor binding. The designed docking-score and sub-structure filters were first tested on a dataset of 249 potent p38 MAP kinase inhibitors from seven diverse series and 18,842 kinase inhibitors from PDB, to gauge their capacity to discriminate between kinase and non-kinase inhibitors and likewise to selectively filter-in target-specific inhibitors. The designed filters were then applied in the virtual screening of a database of ten million (10⁷) compounds resulting in the identification of 100 hits. Based on their binding modes, 98 allosteric fragments were extracted from the hits and a fragment library was generated. New type II p38 MAP kinase leads were designed by tailoring the existing type I ATP site binders with allosteric fragments using a common urea linker. Target specific virtual screening filters can thus be easily developed for other kinases based on this strategy to retrieve target selective compounds. Copyright © 2012 Elsevier Inc. All rights reserved.

MAP4-regulated dynein-dependent trafficking of BTN3A1 controls the TBK1–IRF3 signaling axis

PubMed Central

Seo, Minji; Lee, Seong-Ok; Kim, Ji-Hoon; Hong, Yujin; Kim, Seongchan; Kim, Yeumin; Min, Dal-Hee; Kong, Young-Yun; Shin, Jinwook; Ahn, Kwangseog

2016-01-01

The innate immune system detects viral nucleic acids and induces type I interferon (IFN) responses. The RNA- and DNA-sensing pathways converge on the protein kinase TANK-binding kinase 1 (TBK1) and the transcription factor IFN-regulatory factor 3 (IRF3). Activation of the IFN signaling pathway is known to trigger the redistribution of key signaling molecules to punctate perinuclear structures, but the mediators of this spatiotemporal regulation have yet to be defined. Here we identify butyrophilin 3A1 (BTN3A1) as a positive regulator of nucleic acid-mediated type I IFN signaling. Depletion of BTN3A1 inhibits the cytoplasmic nucleic acid- or virus-triggered activation of IFN-β production. In the resting state, BTN3A1 is constitutively associated with TBK1. Stimulation with nucleic acids induces the redistribution of the BTN3A1–TBK1 complex to the perinuclear region, where BTN3A1 mediates the interaction between TBK1 and IRF3, leading to the phosphorylation of IRF3. Furthermore, we show that microtubule-associated protein 4 (MAP4) controls the dynein-dependent transport of BTN3A1 in response to nucleic acid stimulation, thereby identifying MAP4 as an upstream regulator of BTN3A1. Thus, the depletion of either MAP4 or BTN3A1 impairs cytosolic DNA- or RNA-mediated type I IFN responses. Our findings demonstrate a critical role for MAP4 and BTN3A1 in the spatiotemporal regulation of TBK1, a central player in the intracellular nucleic acid-sensing pathways involved in antiviral signaling. PMID:27911820

Tumor necrosis factor-alpha stimulation of calcitonin gene-related peptide expression and secretion from rat trigeminal ganglion neurons.

PubMed

Bowen, Elizabeth J; Schmidt, Thomas W; Firm, Christina S; Russo, Andrew F; Durham, Paul L

2006-01-01

Expression of the neuropeptide calcitonin gene-related peptide (CGRP) in trigeminal ganglion is implicated in neurovascular headaches and temporomandibular joint disorders. Elevation of cytokines contributes to the pathology of these diseases. However, a connection between cytokines and CGRP gene expression in trigeminal ganglion nerves has not been established. We have focused on the effects of the cytokine tumor necrosis factor-alpha (TNF-alpha). TNFR1 receptors were found on the majority of CGRP-containing rat trigeminal ganglion neurons. Treatment of cultures with TNF-alpha stimulated CGRP secretion. In addition, the intracellular signaling intermediate from the TNFR1 receptor, ceramide, caused a similar increase in CGRP release. TNF-alpha caused a coordinate increase in CGRP promoter activity. TNF-alpha treatment activated the transcription factor NF-kappaB, as well as the Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways. The importance of TNF-alpha induction of MAP kinase pathways was demonstrated by inhibiting MAP kinases with pharmacological reagents and gene transfer with an adenoviral vector encoding MAP kinase phosphatase-1 (MKP-1). We propose that selective and regulated inhibition of MAP kinases in trigeminal neurons may be therapeutically beneficial for inflammatory disorders involving elevated CGRP levels.

Tumor necrosis factor-α stimulation of calcitonin gene-related peptide expression and secretion from rat trigeminal ganglion neurons

PubMed Central

Bowen, Elizabeth J.; Schmidt, Thomas W.; Firm, Christina S.; Russo, Andrew F.; Durham, Paul L.

2006-01-01

Expression of the neuropeptide calcitonin gene-related peptide (CGRP) in trigeminal ganglion is implicated in neurovascular headaches and temporomandibular joint disorders. Elevation of cytokines contributes to the pathology of these diseases. However, a connection between cytokines and CGRP gene expression in trigeminal ganglion nerves has not been established. We have focused on the effects of the cytokine tumor necrosis factorα (TNFα). TNFR1 receptors were found on the majority of CGRP-containing rat trigeminal ganglion neurons. Treatment of cultures with TNFα stimulated CGRP secretion. In addition, the intracellular signaling intermediate from the TNFR1 receptor, ceramide, caused a similar increase in CGRP release. TNFα caused a coordinate increase in CGRP promoter activity. TNFα treatment activated the transcription factor NF-κB, as well as the Jun N-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase pathways. The importance of TNFα induction of MAP kinase pathways was demonstrated by inhibiting MAP kinases with pharmacological reagents and gene transfer with an adenoviral vector encoding MAP kinase phosphatase-1 (MKP-1). We propose that selective and regulated inhibition of MAP kinases in trigeminal neurons may be therapeutically beneficial for inflammatory disorders involving elevated CGRP levels. PMID:16277606

The MAP Kinase Cascade Is Activated prior to the Induction of Gliosis in the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) Model of Dopaminergic Neurotoxicity.

PubMed

Callaghan, James P; Martin, Parthena M; Mass, Marc J

1998-05-01

Injury to the central nervous system (CNS) provokes microglial activation and astrocytic hypertrophy at the site of damage. The signaling events that underlie these cellular responses remain unknown. Recent evidence has implicated tyrosine phosphorylation systems, in general, and the mitogen-activated protein kinase (MAP kinase) cascade, in particular, in the mediation of growth-associated events linked to neural degeneration, such as glial activation. 1 Moreover, an increase in the mRNA coding for the 14.3.3 protein, a known regulator of the MAP kinase pathway, 2 appears to be involved in methamphetamine neurotoxicity. 3 To examine the potential role of these protein kinase pathways in drug-induced damage to the CNS, we used the dopaminergic neurotoxicant, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), to damage nerve terminals in the mouse neostriatum and elicit a glial reaction. The onset of reactive gliosis then was verified by Northern blot analysis of glial fibrillary acidic protein (GFAP) mRNA and qualified by enzyme-linked immunosorbent assay (ELISA) of GFAP (protein). A single administration of MPTP (12.5 mg/kg, subcutaneously (s.c.)) to the C57B1/6J mouse resulted in a 10-fold increase in GFAP mRNA by 1 day and a 4-fold increase in GFAP (protein) by 2 days. To determine the potential role of protein tyrosine phosphorylation and MAP kinase activation in these events, blots of striatal homogenates were probed with antibodies directed against phospho-tyr 204 and phospho-thr 202, residues corresponding to the active sites of p42/44 MAP kinase. After mice were sacrificed by focused microwave irradiation to preserve steady-state phosphorylation, proteins from striatal homogenates were resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunoblots of these samples showed a number of phosphotyrosine-labeled bands, but there were no apparent differences between control and MPTP groups. In contrast, phospho-MAP kinase was elevated over 1.5-fold, 3-6hours post MPTP. These findings are suggestive of a role of the MAP kinase cascade in the early phase of injury-induced glial activation.

Polygalasaponin XXXII from Polygala tenuifolia root improves hippocampal-dependent learning and memory.

PubMed

Xue, Wei; Hu, Jin-feng; Yuan, Yu-he; Sun, Jian-dong; Li, Bo-yu; Zhang, Dong-ming; Li, Chuang-jun; Chen, Nai-hong

2009-09-01

The aim of this study was to investigate the cognition-enhancing activity and underlying mechanisms of a triterpenoid saponin (polygalasaponin XXXII, PGS32) isolated from the roots of Polygala tenuifolia Willd. The Morris water maze was used to evaluate the spatial learning and memory of mice. To detect the basic properties of synaptic transmission and long-term potentiation (LTP) in the dentate gyrus of rats, electrophysiological recordings were made of evoked potentials. Western blotting analysis and immunofluorescence assays were used to determine the phosphorylation of extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB), synapsin I and the expression of brain derived neurotrophic factor (BDNF). When administered at 0.125, 0.5, or 2 mg/kg, PGS32 could significantly prevent scopolamine-induced cognitive impairments in mice. Intracerebroventricular (icv) administration of PGS32 greatly enhanced basic synaptic transmission in the dentate gyrus of rats and induced LTP. In primary hippocampal neurons, as well as in the hippocampus of maze-trained mice, PGS32 activated the mitogen-activated protein (MAP) kinase cascade by promoting phosphorylation of ERK, CREB and synapsin I. The expression of BDNF was also greatly enhanced in the hippocampus. Our findings suggest that PGS32 can improve hippocampus-dependent learning and memory, possibly through improvement of synaptic transmission, activation of the MAP kinase cascade and enhancement of the level of BDNF. Therefore, PGS32 shows promise as a potential cognition-enhancing therapeutic drug.

Polygalasaponin XXXII from Polygala tenuifolia root improves hippocampal-dependent learning and memory

PubMed Central

Xue, Wei; Hu, Jin-feng; Yuan, Yu-he; Sun, Jian-dong; Li, Bo-yu; Zhang, Dong-ming; Li, Chuang-jun; Chen, Nai-hong

2009-01-01

Aim: The aim of this study was to investigate the cognition-enhancing activity and underlying mechanisms of a triterpenoid saponin (polygalasaponin XXXII, PGS32) isolated from the roots of Polygala tenuifolia Willd. Methods: The Morris water maze was used to evaluate the spatial learning and memory of mice. To detect the basic properties of synaptic transmission and long-term potentiation (LTP) in the dentate gyrus of rats, electrophysiological recordings were made of evoked potentials. Western blotting analysis and immunofluorescence assays were used to determine the phosphorylation of extracellular signal-regulated kinase (ERK), cAMP response element-binding protein (CREB), synapsin I and the expression of brain derived neurotrophic factor (BDNF). Results: When administered at 0.125, 0.5, or 2 mg/kg, PGS32 could significantly prevent scopolamine-induced cognitive impairments in mice. Intracerebroventricular (icv) administration of PGS32 greatly enhanced basic synaptic transmission in the dentate gyrus of rats and induced LTP. In primary hippocampal neurons, as well as in the hippocampus of maze-trained mice, PGS32 activated the mitogen-activated protein (MAP) kinase cascade by promoting phosphorylation of ERK, CREB and synapsin I. The expression of BDNF was also greatly enhanced in the hippocampus. Conclusion: Our findings suggest that PGS32 can improve hippocampus-dependent learning and memory, possibly through improvement of synaptic transmission, activation of the MAP kinase cascade and enhancement of the level of BDNF. Therefore, PGS32 shows promise as a potential cognition-enhancing therapeutic drug. PMID:19684611

Signaling through protein kinases and transcriptional regulators in Candida albicans.

PubMed

Dhillon, Navneet K; Sharma, Sadhna; Khuller, G K

2003-01-01

The human fungal pathogen Candida albicans switches from a budding yeast form to a polarized hyphal form in response to various external signals. This morphogenetic switching has been implicated in the development of pathogenicity. Several signaling pathways that regulate morphogenesis have been identified, including various transcription factors that either activate or repress hypha-specific genes. Two well-characterized pathways include the MAP kinase cascade and cAMP-dependent protein kinase pathway that regulate the transcription factors Cph1p and Efg1p, respectively. cAMP also appears to interplay with other second messengers: Ca2+, inositol tri-phosphates in regulating yeast-hyphal transition. Other, less-characterized pathways include two component histidine kinases, cyclin-dependent kinase pathway, and condition specific pathways such as pH and embedded growth conditions. Nrg1 and Rfg1 function as transcriptional repressors of hyphal genes via recruitment of Tup1 co-repressor complex. Different upstream signals converge into a common downstream output during hyphal switch. The levels of expression of several genes have been shown to be associated with hyphal morphogenesis rather than with a specific hypha-inducing condition. Hyphal development is also linked to the expression of a range of other virulence factors. This review explains the relative contribution of multiple pathways that could be used by Candida albican cells to sense subtle differences in the growth conditions of its native host environment.

Decursin inhibits growth of human bladder and colon cancer cells via apoptosis, G1-phase cell cycle arrest and extracellular signal-regulated kinase activation.

PubMed

Kim, Wun-Jae; Lee, Se-Jung; Choi, Young Deuk; Moon, Sung-Kwon

2010-04-01

Decursin, a pyranocoumarin isolated from the Korean Angelica gigas root, has demonstrated anti-cancer properties. In the present study, we found that decursin inhibited cell viability in cultured human urinary bladder cancer 235J cells and colon cancer HCT116 cells. The inhibited proliferation was due to apoptotic induction, because both cells treated with decursin dose-dependently showed a sub-G1 phase accumulation and an increased cytoplasmic DNA-histone complex. Cell death caused by decursin was also associated with the down-regulation of anti-apoptotic factor Bcl-2 and the up-regulation of pro-apoptotic molecules cytochrome c, caspase 3 and Bax. Treatment of both types of cancer cells with decursin resulted in G1-phase cell cycle arrest, as revealed by FACS analyses. In addition, decursin increased protein levels of p21WAF1 with a decrease in cyclins and cyclin dependent kinases (CDKs). Furthermore, decursin induced the activation of extracellular signal-regulated kinases (ERK) in both cancer cell lines, with the notable exceptions of c-Jun N-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase. Finally, pretreatment with ERK-specific inhibitor PD98059 reversed decursin-induced p21WAF1 expression and decursin-inhibited cell growth. Thus, these findings suggest that decursin has potential therapeutic efficacy for the treatment of bladder and colon cancer.

MicroRNA 27a-3p Regulates Antimicrobial Responses of Murine Macrophages Infected by Mycobacterium avium subspecies paratuberculosis by Targeting Interleukin-10 and TGF-β-Activated Protein Kinase 1 Binding Protein 2

PubMed Central

Hussain, Tariq; Zhao, Deming; Shah, Syed Zahid Ali; Wang, Jie; Yue, Ruichao; Liao, Yi; Sabir, Naveed; Yang, Lifeng; Zhou, Xiangmei

2018-01-01

Mycobacterium avium subspecies paratuberculosis (MAP) persistently survive and replicate in mononuclear phagocytic cells by adopting various strategies to subvert host immune response. Interleukin-10 (IL-10) upregulation via inhibition of macrophage bactericidal activity is a critical step for MAP survival and pathogenesis within the host cell. Mitogen-activated protein kinase p38 signaling cascade plays a crucial role in the elevation of IL-10 and progression of MAP pathogenesis. The contribution of microRNAs (miRNAs) and their influence on the activation of macrophages during MAP pathogenesis are still unclear. In the current study, we found that miRNA-27a-3p (miR-27a) expression is downregulated during MAP infection both in vivo and in vitro. Moreover, miR-27a is also downregulated in toll-like receptor 2 (TLR2)-stimulated murine macrophages (RAW264.7 and bone marrow-derived macrophage). ELISA and real-time qRT-PCR results confirm that overexpression of miR-27a inhibited MAP-induced IL-10 production in macrophages and upregulated pro-inflammatory cytokines, while miR-27a inhibitor counteracted these effects. Luciferase reporter assay results revealed that IL-10 and TGF-β-activated protein kinase 1 binding protein 2 (TAB 2) are potential targets of miR-27a. In addition, we demonstrated that miR-27a negatively regulates TAB 2 expression and diminishes TAB 2-dependent p38/JNK phosphorylation, ultimately downregulating IL-10 expression in MAP-infected macrophages. Furthermore, overexpression of miR-27a significantly inhibited the intracellular survival of MAP in infected macrophages. Our data show that miR-27a augments antimicrobial activities of macrophages and inhibits the expression of IL-10, demonstrating that miR-27a regulates protective innate immune responses during MAP infection and can be exploited as a novel therapeutic target in the control of intracellular pathogens, including paratuberculosis. PMID:29375563

The amoebal MAP kinase response to Legionella pneumophila is regulated by DupA.

PubMed

Li, Zhiru; Dugan, Aisling S; Bloomfield, Gareth; Skelton, Jason; Ivens, Alasdair; Losick, Vicki; Isberg, Ralph R

2009-09-17

The amoeba Dictyostelium discoideum can support replication of Legionella pneumophila. Here we identify the dupA gene, encoding a putative tyrosine kinase/dual-specificity phosphatase, in a screen for D. discoideum mutants altered in allowing L. pneumophila intracellular replication. Inactivation of dupA resulted in depressed L. pneumophila growth and sustained hyperphosphorylation of the amoebal MAP kinase ERK1, consistent with loss of a phosphatase activity. Bacterial challenge of wild-type amoebae induced dupA expression and resulted in transiently increased ERK1 phosphorylation, suggesting that dupA and ERK1 are part of a response to bacteria. Indeed, over 500 of the genes misregulated in the dupA(-) mutant were regulated in response to L. pneumophila infection, including some thought to have immune-like functions. MAP kinase phosphatases are known to be highly upregulated in macrophages challenged with L. pneumophila. Thus, DupA may regulate a MAP kinase response to bacteria that is conserved from amoebae to mammals.

3,5,6,7,8,3′,4′-Heptamethoxyflavone Ameliorates Depressive-Like Behavior and Hippocampal Neurochemical Changes in Chronic Unpredictable Mild Stressed Mice by Regulating the Brain-Derived Neurotrophic Factor: Requirement for ERK Activation

PubMed Central

Sawamoto, Atsushi; Okuyama, Satoshi; Amakura, Yoshiaki; Yoshimura, Morio; Yamada, Takashi; Yokogoshi, Hidehiko; Nakajima, Mitsunari

2017-01-01

We previously reported that the subcutaneous administration of 3,5,6,7,8,3′,4′-heptamethoxyflavone (HMF), a citrus polymethoxyflavone, attenuated depressive-like behavior and increased the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus of a corticosterone-induced depression-like mouse model. We herein demonstrated that (1) HMF was detectable in the brain 10 and 30 min after its oral administration, (2) orally administered HMF improved chronic unpredictable mild stress (CUMS)-induced pathological conditions, including body weight loss and depressive-like behavior, and CUMS-induced neurochemical changes, such as reduction in BDNF expression, decrease in neurogenesis, and decreased level of phosphorylated calcium-calmodulin-dependent protein kinase II in the hippocampus, and (3) these effects of HMF were inhibited by the pre-administration of U0126, a mitogen-activated protein (MAP) kinase inhibitor. These results suggest that orally administered HMF is beneficial for the upregulation of BDNF in the hippocampus via the extracellular signal-regulated kinase1/2 (ERK1/2)/MAP system, which may account for its antidepression effects. PMID:29023414

Chaperone Hsp27 Modulates AUF1 Proteolysis and AU-Rich Element-Mediated mRNA Degradation▿

PubMed Central

Knapinska, Anna M.; Gratacós, Frances M.; Krause, Christopher D.; Hernandez, Kristina; Jensen, Amber G.; Bradley, Jacquelyn J.; Wu, Xiangyue; Pestka, Sidney; Brewer, Gary

2011-01-01

AUF1 is an AU-rich element (ARE)-binding protein that recruits translation initiation factors, molecular chaperones, and mRNA degradation enzymes to the ARE for mRNA destruction. We recently found chaperone Hsp27 to be an AUF1-associated ARE-binding protein required for tumor necrosis factor alpha (TNF-α) mRNA degradation in monocytes. Hsp27 is a multifunctional protein that participates in ubiquitination of proteins for their degradation by proteasomes. A variety of extracellular stimuli promote Hsp27 phosphorylation on three serine residues—Ser15, Ser78, and Ser82—by a number of kinases, including the mitogen-activated protein (MAP) pathway kinases p38 and MK2. Activating either kinase stabilizes ARE mRNAs. Likewise, ectopic expression of phosphomimetic mutant forms of Hsp27 stabilizes reporter ARE mRNAs. Here, we continued to examine the contributions of Hsp27 to mRNA degradation. As AUF1 is ubiquitinated and degraded by proteasomes, we addressed the hypothesis that Hsp27 phosphorylation controls AUF1 levels to modulate ARE mRNA degradation. Indeed, selected phosphomimetic mutants of Hsp27 promote proteolysis of AUF1 in a proteasome-dependent fashion and render ARE mRNAs more stable. Our results suggest that the p38 MAP kinase (MAPK)-MK2–Hsp27 signaling axis may target AUF1 destruction by proteasomes, thereby promoting ARE mRNA stabilization. PMID:21245386

The BRAF{sup T1799A} mutation confers sensitivity of thyroid cancer cells to the BRAF{sup V600E} inhibitor PLX4032 (RG7204)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xing, Joanna; Liu, Ruixin; Xing, Mingzhao

2011-01-28

Research highlights: {yields} Exciting therapeutic potential has been recently reported for the BRAF{sup V600E} inhibitor PLX4032 in melanoma. {yields} We tested the effects of PLX4032 on the growth of thyroid cancer cells which often harbor the BRAF{sup V600E} mutation. {yields} We observed a potent BRAF{sup V600E}-dependent inhibition of thyroid cancer cells by PLX4032. {yields} We thus demonstrated an important therapeutic potential of PLX4032 for thyroid cancer. -- Abstract: Aberrant signaling of the Ras-Raf-MEK-ERK (MAP kinase) pathway driven by the mutant kinase BRAF{sup V600E}, as a result of the BRAF{sup T1799A} mutation, plays a fundamental role in thyroid tumorigenesis. This studymore » investigated the therapeutic potential of a BRAF{sup V600E}-selective inhibitor, PLX4032 (RG7204), for thyroid cancer by examining its effects on the MAP kinase signaling and proliferation of 10 thyroid cancer cell lines with wild-type BRAF or BRAF{sup T1799A} mutation. We found that PLX4032 could effectively inhibit the MAP kinase signaling, as reflected by the suppression of ERK phosphorylation, in cells harboring the BRAF{sup T1799A} mutation. PLX4032 also showed a potent and BRAF mutation-selective inhibition of cell proliferation in a concentration-dependent manner. PLX4032 displayed low IC{sub 50} values (0.115-1.156 {mu}M) in BRAF{sup V600E} mutant cells, in contrast with wild-type BRAF cells that showed resistance to the inhibitor with high IC{sub 50} values (56.674-1349.788 {mu}M). Interestingly, cells with Ras mutations were also sensitive to PLX4032, albeit moderately. Thus, this study has confirmed that the BRAF{sup T1799A} mutation confers cancer cells sensitivity to PLX4032 and demonstrated its specific potential as an effective and BRAF{sup T1799A} mutation-selective therapeutic agent for thyroid cancer.« less

Discovery of cellular substrates for protein kinase A using a peptide array screening protocol.

PubMed

Smith, F Donelson; Samelson, Bret K; Scott, John D

2011-08-15

Post-translational modification of proteins is a universal form of cellular regulation. Phosphorylation on serine, threonine, tyrosine or histidine residues by protein kinases is the most widespread and versatile form of covalent modification. Resultant changes in activity, localization or stability of phosphoproteins drives cellular events. MS and bioinformatic analyses estimate that ~30% of intracellular proteins are phosphorylated at any given time. Multiple approaches have been developed to systematically define targets of protein kinases; however, it is likely that we have yet to catalogue the full complement of the phosphoproteome. The amino acids that surround a phosphoacceptor site are substrate determinants for protein kinases. For example, basophilic enzymes such as PKA (protein kinase A), protein kinase C and calmodulin-dependent kinases recognize basic side chains preceding the target serine or threonine residues. In the present paper we describe a strategy using peptide arrays and motif-specific antibodies to identify and characterize previously unrecognized substrate sequences for protein kinase A. We found that the protein kinases PKD (protein kinase D) and MARK3 [MAP (microtubule-associated protein)-regulating kinase 3] can both be phosphorylated by PKA. Furthermore, we show that the adapter protein RIL [a product of PDLIM4 (PDZ and LIM domain protein 4)] is a PKA substrate that is phosphorylated on Ser(119) inside cells and that this mode of regulation may control its ability to affect cell growth. © The Authors Journal compilation © 2011 Biochemical Society

Independent localization of MAP2, CaMKIIα and β-actin RNAs in low copy numbers.

PubMed

Mikl, Martin; Vendra, Georgia; Kiebler, Michael A

2011-09-30

Messenger RNA localization involves the assembly of ribonucleoprotein particles (RNPs) and their subsequent transport along the cytoskeleton to their final destination. Here, we provide new evidence that microtubule-associated protein 2 (MAP2), calcium/calmodulin-dependent protein kinase II (CaMKIIα) and β-actin RNAs localize to dendrites in distinct RNPs, which contain--unexpectedly--very few RNA molecules. The number of MAP2 molecules per particle is affected by synaptic activity and Staufen 2, indicating that RNP composition is tightly controlled. Our data suggest that the independent localization of individual RNAs in low copy numbers could contribute to tighter temporal and spatial control of expression in neurons and synapse-specific plasticity.

Regulation of cotton (Gossypium hirsutum) drought responses by mitogen-activated protein (MAP) kinase cascade-mediated phosphorylation of GhWRKY59.

PubMed

Li, Fangjun; Li, Maoying; Wang, Ping; Cox, Kevin L; Duan, Liusheng; Dever, Jane K; Shan, Libo; Li, Zhaohu; He, Ping

2017-09-01

Drought is a key limiting factor for cotton (Gossypium spp.) production, as more than half of the global cotton supply is grown in regions with high water shortage. However, the underlying mechanism of the response of cotton to drought stress remains elusive. By combining genome-wide transcriptome profiling and a loss-of-function screen using virus-induced gene silencing, we identified Gossypium hirsutum GhWRKY59 as an important transcription factor that regulates the drought stress response in cotton. Biochemical and genetic analyses revealed a drought stress-activated mitogen-activated protein (MAP) kinase cascade consisting of GhMAP3K15-Mitogen-activated Protein Kinase Kinase 4 (GhMKK4)-Mitogen-activated Protein Kinase 6 (GhMPK6) that directly phosphorylates GhWRKY59 at residue serine 221. Interestingly, GhWRKY59 is required for dehydration-induced expression of GhMAPK3K15, constituting a positive feedback loop of GhWRKY59-regulated MAP kinase activation in response to drought stress. Moreover, GhWRKY59 directly binds to the W-boxes of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 2 (GhDREB2), which encodes a dehydration-inducible transcription factor regulating the plant hormone abscisic acid (ABA)-independent drought response. Our study identified a complete MAP kinase cascade that phosphorylates and activates a key WRKY transcription factor, and elucidated a regulatory module, consisting of GhMAP3K15-GhMKK4-GhMPK6-GhWRKY59-GhDREB2, that is involved in controlling the cotton drought response. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

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

Prostaglandin F(2alpha) stimulates tyrosine phosphorylation of phospholipase C-gamma1.

PubMed

Husain, Shahid; Jafri, Farahdiba

2002-10-11

In this study, we investigated the ability of prostaglandin F(2alpha) (PGF(2alpha)) to induce tyrosine phosphorylation of phospholipase C-gamma1 (PLC-gamma1) in cat iris sphincter smooth muscle (CISM) cells. PGF(2alpha)(1 microM) stimulated PLC-gamma1 tyrosine phosphorylation in a time- and dose-dependent manner with a maximum increase of 3-fold at 0.5min. The protein tyrosine kinase inhibitors, genistein, and tyrphostin A-25, blocked the stimulatory effects of PGF(2alpha), suggesting involvement of protein tyrosine kinase activity in the physiological actions of the PGF(2alpha). Furthermore, PGF(2alpha)-induced p42/p44 MAP kinase activation was also completely blocked by protein tyrosine kinase inhibitors. In summary, these findings show that PGF(2alpha) stimulates tyrosine phosphorylation of PLC-gamma1 in CISM cells and indicate that PGF(2alpha)-stimulated tyrosine phosphorylation is responsible for an early signal transduction event.

The kinases MEKK2 and MEKK3 regulate transforming growth factor-β-mediated helper T cell differentiation.

PubMed

Chang, Xing; Liu, Fang; Wang, Xiaofang; Lin, Aiping; Zhao, Hongyu; Su, Bing

2011-02-25

Mitogen-activated protein kinases (MAPKs) are key mediators of the T cell receptor (TCR) signals but their roles in T helper (Th) cell differentiation are unclear. Here we showed that the MAPK kinase kinases MEKK2 (encoded by Map3k2) and MEKK3 (encoded by Map3k3) negatively regulated transforming growth factor-β (TGF-β)-mediated Th cell differentiation. Map3k2(-/-)Map3k3(Lck-Cre/-) mice showed an abnormal accumulation of regulatory T (Treg) and Th17 cells in the periphery, consistent with Map3k2(-/-)Map3k3(Lck-Cre/-) naive CD4(+) T cells' differentiation into Treg and Th17 cells with a higher frequency than wild-type (WT) cells after TGF-β stimulation in vitro. In addition, Map3k2(-/-)Map3k3(Lck-Cre/-) mice developed more severe experimental autoimmune encephalomyelitis. Map3k2(-/-)Map3k3(Lck-Cre/-) T cells exhibited impaired phosphorylation of SMAD2 and SMAD3 proteins at their linker regions, which negatively regulated the TGF-β responses in T cells. Thus, the crosstalk between TCR-induced MAPK and the TGF-β signaling pathways is important in regulating Th cell differentiation. Copyright © 2011 Elsevier Inc. All rights reserved.

R-Ras contributes to LTP and contextual discrimination.

PubMed

Darcy, M J; Jin, S-X; Feig, L A

2014-09-26

The ability to discriminate between closely related contexts is a specific form of hippocampal-dependent learning that may be impaired in certain neurodegenerative disorders such as Alzheimer's and Down Syndrome. However, signaling pathways regulating this form of learning are poorly understood. Previous studies have shown that the calcium-dependent exchange factor Ras-GRF1, an activator of Rac, Ras and R-Ras GTPases, is important for this form of learning and memory. Moreover, the ability to discriminate contexts was linked to the ability of Ras-GRF1 to promote high-frequency stimulation long-term potentiation (HFS-LTP) via the activation of p38 Map kinase. Here, we show that R-Ras is involved in this form of learning by using virally-delivered miRNAs targeting R-Ras into the CA1 region of the dorsal hippocampus and observing impaired contextual discrimination. Like the loss of GRF1, knockdown of R-Ras in the CA1 also impairs the induction of HFS-LTP and p38 Map kinase. Nevertheless, experiments indicate that this involvement of R-Ras in HFS-LTP that is required for contextual discrimination is independent of Ras-GRF1. Thus, R-Ras is a novel regulator of a form of hippocampal-dependent LTP as well as learning and memory that is affected in certain forms of neurodegenerative diseases. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Protein Kinase G Induces an Immune Response in Cows Exposed to Mycobacterium avium Subsp. paratuberculosis

PubMed Central

Bach, Eviatar; Chaffer, Marcelo; Lai, Wanika; Keefe, Greg; Begg, Douglas J.

2018-01-01

To establish infection, pathogens secrete virulence factors, such as protein kinases and phosphatases, to modulate the signal transduction pathways used by host cells to initiate immune response. The protein MAP3893c is annotated in the genome sequence of Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease, as the serine/threonine protein kinase G (PknG). In this work, we report that PknG is a functional kinase that is secreted within macrophages at early stages of infection. The antigen is able to induce an immune response from cattle exposed to MAP in the form of interferon gamma production after stimulation of whole blood with PknG. These findings suggest that PknG may contribute to the pathogenesis of MAP by phosphorylating macrophage signalling and/or adaptor molecules as observed with other pathogenic mycobacterial species. PMID:29581962

Protein Kinase G Induces an Immune Response in Cows Exposed to Mycobacterium avium Subsp. paratuberculosis.

PubMed

Bach, Horacio; Richard-Greenblatt, Melissa; Bach, Eviatar; Chaffer, Marcelo; Lai, Wanika; Keefe, Greg; Begg, Douglas J

2018-01-01

To establish infection, pathogens secrete virulence factors, such as protein kinases and phosphatases, to modulate the signal transduction pathways used by host cells to initiate immune response. The protein MAP3893c is annotated in the genome sequence of Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease, as the serine/threonine protein kinase G (PknG). In this work, we report that PknG is a functional kinase that is secreted within macrophages at early stages of infection. The antigen is able to induce an immune response from cattle exposed to MAP in the form of interferon gamma production after stimulation of whole blood with PknG. These findings suggest that PknG may contribute to the pathogenesis of MAP by phosphorylating macrophage signalling and/or adaptor molecules as observed with other pathogenic mycobacterial species.

Calcium Hydroxide-induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells.

PubMed

Chen, Luoping; Zheng, Lisha; Jiang, Jingyi; Gui, Jinpeng; Zhang, Lingyu; Huang, Yan; Chen, Xiaofang; Ji, Jing; Fan, Yubo

2016-09-01

Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Biochemical Screening of Five Protein Kinases from Plasmodium falciparum against 14,000 Cell-Active Compounds

PubMed Central

Crowther, Gregory J.; Hillesland, Heidi K.; Keyloun, Katelyn R.; Reid, Molly C.; Lafuente-Monasterio, Maria Jose; Ghidelli-Disse, Sonja; Leonard, Stephen E.; He, Panqing; Jones, Jackson C.; Krahn, Mallory M.; Mo, Jack S.; Dasari, Kartheek S.; Fox, Anna M. W.; Boesche, Markus; El Bakkouri, Majida; Rivas, Kasey L.; Leroy, Didier; Hui, Raymond; Drewes, Gerard; Maly, Dustin J.; Van Voorhis, Wesley C.; Ojo, Kayode K.

2016-01-01

In 2010 the identities of thousands of anti-Plasmodium compounds were released publicly to facilitate malaria drug development. Understanding these compounds’ mechanisms of action—i.e., the specific molecular targets by which they kill the parasite—would further facilitate the drug development process. Given that kinases are promising anti-malaria targets, we screened ~14,000 cell-active compounds for activity against five different protein kinases. Collections of cell-active compounds from GlaxoSmithKline (the ~13,000-compound Tres Cantos Antimalarial Set, or TCAMS), St. Jude Children’s Research Hospital (260 compounds), and the Medicines for Malaria Venture (the 400-compound Malaria Box) were screened in biochemical assays of Plasmodium falciparum calcium-dependent protein kinases 1 and 4 (CDPK1 and CDPK4), mitogen-associated protein kinase 2 (MAPK2/MAP2), protein kinase 6 (PK6), and protein kinase 7 (PK7). Novel potent inhibitors (IC50 < 1 μM) were discovered for three of the kinases: CDPK1, CDPK4, and PK6. The PK6 inhibitors are the most potent yet discovered for this enzyme and deserve further scrutiny. Additionally, kinome-wide competition assays revealed a compound that inhibits CDPK4 with few effects on ~150 human kinases, and several related compounds that inhibit CDPK1 and CDPK4 yet have limited cytotoxicity to human (HepG2) cells. Our data suggest that inhibiting multiple Plasmodium kinase targets without harming human cells is challenging but feasible. PMID:26934697

Quantitative Phospho-proteomic Analysis of TNFα/NFκB Signaling Reveals a Role for RIPK1 Phosphorylation in Suppressing Necrotic Cell Death.

PubMed

Mohideen, Firaz; Paulo, Joao A; Ordureau, Alban; Gygi, Steve P; Harper, J Wade

2017-07-01

TNFα is a potent inducer of inflammation due to its ability to promote gene expression, in part via the NFκB pathway. Moreover, in some contexts, TNFα promotes Caspase-dependent apoptosis or RIPK1/RIPK3/MLKL-dependent necrosis. Engagement of the TNF Receptor Signaling Complex (TNF-RSC), which contains multiple kinase activities, promotes phosphorylation of several downstream components, including TAK1, IKKα/IKKβ, IκBα, and NFκB. However, immediate downstream phosphorylation events occurring in response to TNFα signaling are poorly understood at a proteome-wide level. Here we use Tandem Mass Tagging-based proteomics to quantitatively characterize acute TNFα-mediated alterations in the proteome and phosphoproteome with or without inhibition of the cIAP-dependent survival arm of the pathway with a SMAC mimetic. We identify and quantify over 8,000 phosphorylated peptides, among which are numerous known sites in the TNF-RSC, NFκB, and MAP kinase signaling systems, as well as numerous previously unrecognized phosphorylation events. Functional analysis of S320 phosphorylation in RIPK1 demonstrates a role for this event in suppressing its kinase activity, association with CASPASE-8 and FADD proteins, and subsequent necrotic cell death during inflammatory TNFα stimulation. This study provides a resource for further elucidation of TNFα-dependent signaling pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Dual modulation of ERK1/2 and p38 MAP kinase activities induced by minocycline reverses the neurotoxic effects of the prion protein fragment 90-231.

PubMed

Corsaro, Alessandro; Thellung, Stefano; Chiovitti, Katia; Villa, Valentina; Simi, Alessandro; Raggi, Federica; Paludi, Domenico; Russo, Claudio; Aceto, Antonio; Florio, Tullio

2009-02-01

Several in vitro and in vivo studies addressed the identification of molecular determinants of the neuronal death induced by PrP(Sc) or related peptides. We developed an experimental model to assess PrP(Sc) neurotoxicity using a recombinant polypeptide encompassing amino acids 90-231 of human PrP (hPrP90-231) that corresponds to the protease-resistant core of PrP(Sc) identified in prion-infected brains. By means of mild thermal denaturation, we can convert hPrP90-231 from a PrP(C)-like conformation into a PrP(Sc)-like structure. In virtue of these structural changes, hPrP90-231 powerfully affected the survival of SH-SY5Y cells, inducing caspase 3 and p38-dependent apoptosis, while in the native alpha-helix-rich conformation, hPrP90-231 did not induce cell toxicity. The aim of this study was to identify drugs able to block hPrP90-231 neurotoxic effects, focusing on minocycline, a tetracycline with known neuroprotective activity. hPrP90-231 caused a caspase 3-dependent apoptosis via the blockade of ERK1/2 activation and the subsequent activation of p38 MAP kinase. We propose that hPrP90-231-induced apoptosis is dependent on the inhibition of ERK1/2 responsiveness to neurotrophic factors, removing a tonic inhibition of p38 activity and resulting in caspase 3 activation. Minocycline prevented hPrP90-231-induced toxicity interfering with this mechanism: the pretreatment with this tetracycline restored ERK1/2 activity and reverted p38 and caspase 3 activities. The effects of minocycline were not mediated by the prevention of hPrP90-231 structural changes or cell internalization (differently from Congo Red). In conclusion, minocycline elicits anti-apoptotic effects against the neurotoxic activity of hPrP90-231 and these effects are mediated by opposite modulation of ERK1/2 and p38 MAP kinase activities.

Enhanced basal activation of mitogen-activated protein kinases in adipocytes from type 2 diabetes: potential role of p38 in the downregulation of GLUT4 expression.

PubMed

Carlson, Christian J; Koterski, Sandra; Sciotti, Richard J; Poccard, German Braillard; Rondinone, Cristina M

2003-03-01

Serine and threonine kinases may contribute to insulin resistance and the development of type 2 diabetes. To test the potential for members of the mitogen-activated protein (MAP) kinase family to contribute to type 2 diabetes, we examined basal and insulin-stimulated Erk 1/2, JNK, and p38 phosphorylation in adipocytes isolated from healthy and type 2 diabetic individuals. Maximal insulin stimulation increased the phosphorylation of Erk 1/2 and JNK in healthy control subjects but not type 2 diabetic patients. Insulin stimulation did not increase p38 phosphorylation in either healthy control subjects or type 2 diabetic patients. In type 2 diabetic adipocytes, the basal phosphorylation status of these MAP kinases was significantly elevated and was associated with decreased IRS-1 and GLUT4 in these fat cells. To determine whether MAP kinases were involved in the downregulation of IRS-1 and GLUT4 protein levels, selective inhibitors were used to inhibit these MAP kinases in 3T3-L1 adipocytes treated chronically with insulin. Inhibition of Erk 1/2, JNK, or p38 had no effect on insulin-stimulated reduction of IRS-1 protein levels. However, inhibition of the p38 pathway prevented the insulin-stimulated decrease in GLUT4 protein levels. In summary, type 2 diabetes is associated with an increased basal activation of the MAP kinase family. Furthermore, upregulation of the p38 pathway might contribute to the loss of GLUT4 expression observed in adipose tissue from type 2 diabetic patients.

[6]-Gingerol Induces Caspase-Dependent Apoptosis and Prevents PMA-Induced Proliferation in Colon Cancer Cells by Inhibiting MAPK/AP-1 Signaling

PubMed Central

Narayanan, Sai Shyam; Nath, Lekshmi R.; Thulasidasan, Arun Kumar T.; Soniya, Eppurathu Vasudevan; Anto, Ruby John

2014-01-01

We report mechanism-based evidence for the anticancer and chemopreventive efficacy of [6]-gingerol, the major active principle of the medicinal plant, Ginger (Zingiber officinale), in colon cancer cells. The compound was evaluated in two human colon cancer cell lines for its cytotoxic effect and the most sensitive cell line, SW-480, was selected for the mechanistic evaluation of its anticancer and chemopreventive efficacy. The non-toxic nature of [6]-gingerol was confirmed by viability assays on rapidly dividing normal mouse colon cells. [6]-gingerol inhibited cell proliferation and induced apoptosis as evidenced by externalization of phosphatidyl serine in SW-480, while the normal colon cells were unaffected. Sensitivity to [6]-gingerol in SW-480 cells was associated with activation of caspases 8, 9, 3 &7 and cleavage of PARP, which attests induction of apoptotic cell death. Mechanistically, [6]-gingerol down-regulated Phorbol Myristate Acetate (PMA) induced phosphorylation of ERK1/2 and JNK MAP kinases and activation of AP-1 transcription factor, but had only little effects on phosphorylation of p38 MAP kinase and activation of NF-kappa B. Additionally, it complemented the inhibitors of either ERK1/2 or JNK MAP kinase in bringing down the PMA-induced cell proliferation in SW-480 cells. We report the inhibition of ERK1/2/JNK/AP-1 pathway as a possible mechanism behind the anticancer as well as chemopreventive efficacy of [6]-gingerol against colon cancer. PMID:25157570

[6]-Gingerol induces caspase-dependent apoptosis and prevents PMA-induced proliferation in colon cancer cells by inhibiting MAPK/AP-1 signaling.

PubMed

Radhakrishnan, E K; Bava, Smitha V; Narayanan, Sai Shyam; Nath, Lekshmi R; Thulasidasan, Arun Kumar T; Soniya, Eppurathu Vasudevan; Anto, Ruby John

2014-01-01

We report mechanism-based evidence for the anticancer and chemopreventive efficacy of [6]-gingerol, the major active principle of the medicinal plant, Ginger (Zingiber officinale), in colon cancer cells. The compound was evaluated in two human colon cancer cell lines for its cytotoxic effect and the most sensitive cell line, SW-480, was selected for the mechanistic evaluation of its anticancer and chemopreventive efficacy. The non-toxic nature of [6]-gingerol was confirmed by viability assays on rapidly dividing normal mouse colon cells. [6]-gingerol inhibited cell proliferation and induced apoptosis as evidenced by externalization of phosphatidyl serine in SW-480, while the normal colon cells were unaffected. Sensitivity to [6]-gingerol in SW-480 cells was associated with activation of caspases 8, 9, 3 &7 and cleavage of PARP, which attests induction of apoptotic cell death. Mechanistically, [6]-gingerol down-regulated Phorbol Myristate Acetate (PMA) induced phosphorylation of ERK1/2 and JNK MAP kinases and activation of AP-1 transcription factor, but had only little effects on phosphorylation of p38 MAP kinase and activation of NF-kappa B. Additionally, it complemented the inhibitors of either ERK1/2 or JNK MAP kinase in bringing down the PMA-induced cell proliferation in SW-480 cells. We report the inhibition of ERK1/2/JNK/AP-1 pathway as a possible mechanism behind the anticancer as well as chemopreventive efficacy of [6]-gingerol against colon cancer.

WASH and Tsg101/ALIX-dependent diversion of stress-internalized EGFR from the canonical endocytic pathway

PubMed Central

Tomas, Alejandra; Vaughan, Simon O.; Burgoyne, Thomas; Sorkin, Alexander; Hartley, John A.; Hochhauser, Daniel; Futter, Clare E.

2015-01-01

Stress exposure triggers ligand-independent EGF receptor (EGFR) endocytosis, but its post-endocytic fate and role in regulating signalling are unclear. We show that the p38 MAP kinase-dependent, EGFR tyrosine kinase (TK)-independent EGFR internalization induced by ultraviolet light C (UVC) or the cancer therapeutic cisplatin, is followed by diversion from the canonical endocytic pathway. Instead of lysosomal degradation or plasma membrane recycling, EGFR accumulates in a subset of LBPA-rich perinuclear multivesicular bodies (MVBs) distinct from those carrying EGF-stimulated EGFR. Stress-internalized EGFR co-segregates with exogenously expressed pre-melanosomal markers OA1 and fibrillar PMEL, following early endosomal sorting by the actin polymerization-promoting WASH complex. Stress-internalized EGFR is retained intracellularly by continued p38 activity in a mechanism involving ubiquitin-independent, ESCRT/ALIX-dependent incorporation onto intraluminal vesicles (ILVs) of MVBs. In contrast to the internalization-independent EGF-stimulated activation, UVC/cisplatin-triggered EGFR activation depends on EGFR internalization and intracellular retention. EGFR signalling from this MVB subpopulation delays apoptosis and might contribute to chemoresistance. PMID:26066081

Endothelial atheroprotective and anti-inflammatory mechanisms.

PubMed

Berk, B C; Abe, J I; Min, W; Surapisitchat, J; Yan, C

2001-12-01

Atherosclerosis preferentially occurs in areas of turbulent flow and low fluid shear stress, whereas laminar flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF), have been shown to stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent data suggest that steady laminar flow decreases EC apoptosis and blocks TNF-mediated EC activation. EC apoptosis is likely important in the process termed "plaque erosion" that leads to platelet aggregation. Steady laminar flow inhibits EC apoptosis by preventing cell cycle entry, by increasing antioxidant mechanisms (e.g., superoxide dismutase), and by stimulating nitric oxide-dependent protective pathways that involve enzymes PI3-kinase and Akt. Conversely, our laboratory has identified nitric oxide-independent mechanisms that limit TNF signal transduction. TNF regulates gene expression in EC, in part, by stimulating mitogen-activated protein kinases (MAPK) which phosphorylate transcription factors. We hypothesized that fluid shear stress modulates TNF effects on EC by inhibiting TNF-mediated activation of MAP kinases. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm2) on TNF-stimulated activity of two MAP kinases: extracellular signal regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK). Flow alone stimulated ERK1/2 activity, but decreased JNK activity compared to static controls. TNF (10 ng/ml) alone activated both ERK1/2 and JNK maximally at 15 minutes in human umbilical vein EC (HUVEC). Pre-exposing HUVEC for 10 minutes to flow inhibited TNF activation of JNK by 46%, but it had no significant effect on ERK1/2 activation. Incubation of EC with PD98059, a specific mitogen-activated protein kinase kinase inhibitor, blocked the flow-mediated inhibition of TNF activation of JNK. Flow-mediated inhibition of JNK was unaffected by 0.1 mM L-nitroarginine, 100 pM 8-bromo-cyclic GMP, or 100 microM 8-bromo-cyclic AMP. Transfection studies with dominant negative constructs of the protein kinase MEK1 and MEK5 suggested an important role for BMK1 in flow-mediated regulation of TNF signals. In summary, the atheroprotective effects of steady laminar flow on the endothelium involve multiple synergistic mechanisms.

Stimulation of a Ca sup 2+ -dependent protein kinase by G sub M1 ganglioside in nerve growth factor-treated PC12 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hilbush, B.S.; Levine, J.M.

1991-07-01

The authors have investigated the ability of exogenous gangliosides to modulate nerve growth factor (NGF) signal transduction in PC12 cells. The effects of exogenous ganglioside G{sub M1} on multiple protein kinase activities were assayed by analyzing site-specific serine phosphorylation of tyrosine hydroxylase (TyrOHase) by two-dimensional phosphopeptide mapping. In the presence of NGF, exogenous G{sub M1} increased {sup 32}P incorporation into TyrOHase phosphopeptide T2, a Ca{sup 2+}/calmodulin-dependent protein kinase substrate whose phosphorylation is not normally affected by NGF treatment. In the absence of NGF, G{sub M1} treatment had no significant effects on TyrOHase phosphorylation. The removal of extracellular Ca{sup 2+} ormore » blockade of dihydropyridine-sensitive Ca{sup 2+} channels prevented the G{sub M1}-induced increases in {sup 32}P incorporation into phosphopeptide T2. Exogenous G{sub M1} also potentiated K{sup +} depolarization-induced increases in the phosphorylation of TyrOHase. These results suggest that the stimulatory effects of exogenous G{sub M1} ganglioside on NGF actions may be due to its ability to potentiate a Ca{sup 2+}-dependent signaling pathway.« less

MYB75 Phosphorylation by MPK4 Is Required for Light-Induced Anthocyanin Accumulation in Arabidopsis[OPEN

PubMed Central

Li, Shengnan; Wang, Wenyi; Gao, Jinlan; Yin, Kangquan; Wang, Rui; Wang, Chengcheng; Mundy, John

2016-01-01

Light is a major environmental cue affecting various physiological and metabolic processes in plants. Although plant photoreceptors are well characterized, the mechanisms by which light regulates downstream responses are less clear. In Arabidopsis thaliana, the accumulation of photoprotective anthocyanin pigments is light dependent, and the R2R3 MYB transcription factor MYB75/PAP1 regulates anthocyanin accumulation. Here, we report that MYB75 interacts with and is phosphorylated by MAP KINASE4 (MPK4). Their interaction is dependent on MPK4 kinase activity and is required for full function of MYB75. MPK4 can be activated in response to light and is involved in the light-induced accumulation of anthocyanins. We show that MPK4 phosphorylation of MYB75 increases its stability and is essential for light-induced anthocyanin accumulation. Our findings reveal an important role for a MAPK pathway in light signal transduction. PMID:27811015

ERK-dependent phosphorylation of the transcription initiation factor TIF-IA is required for RNA polymerase I transcription and cell growth.

PubMed

Zhao, Jian; Yuan, Xuejun; Frödin, Morten; Grummt, Ingrid

2003-02-01

Phosphorylation of transcription factors by mitogen-activated protein kinase (MAPK) cascades links cell signaling with the control of gene expression. Here we show that growth factors induce rRNA synthesis by activating MAPK-dependent signaling cascades that target the RNA polymerase I-specific transcription initiation factor TIF-IA. Activation of TIF-IA and ribosomal gene transcription is sensitive to PD98059, indicating that TIF-IA is targeted by MAPK in vivo. Phosphopeptide mapping and mutational analysis reveals two serine residues (S633 and S649) that are phosphorylated by ERK and RSK kinases. Replacement of S649 by alanine inactivates TIF-IA, inhibits pre-rRNA synthesis, and retards cell growth. The results provide a link between growth factor signaling, ribosome production, and cell growth, and may have a major impact on the mechanism of cell transformation.

Interdependence of Platelet-Derived Growth Factor and Estrogen-Signaling Pathways in Inducing Neonatal Rat Testicular Gonocytes Proliferation1

PubMed Central

Thuillier, Raphael; Mazer, Monty; Manku, Gurpreet; Boisvert, Annie; Wang, Yan; Culty, Martine

2010-01-01

We previously found that platelet-derived growth factor (PDGF) and 17beta-estradiol stimulate gonocyte proliferation in a dose-dependent, nonadditive manner. In the present study, we report that gonocytes express RAF1, MAP2K1, and MAPK1/3. Inhibition of RAF1 and MAP2K1/2, but not phosphoinositide-3-kinase, blocked PDGF-induced proliferation. AG-370, an inhibitor of PDGF receptor kinase activity, suppressed not only PDGF-induced proliferation but also that induced by 17beta-estradiol. In addition, RAF1 and MAP2K1/2 inhibitors blocked 17beta-estradiol-activated proliferation. The estrogen receptor antagonist ICI 182780 inhibited both the effects of 17beta-estradiol and PDGF. PDGF lost its stimulatory effect when steroid-depleted serum or no serum was used. Similarly, 17beta-estradiol did not induce gonocyte proliferation in the absence of PDGF. The xenoestrogens genistein, bisphenol A, and DES, but not coumestrol, stimulated gonocyte proliferation in a dose-dependent and PDGF-dependent manner similarly to 17beta-estradiol. Their effects were blocked by ICI 182780, suggesting that they act via the estrogen receptor. AG-370 blocked genistein and bisphenol A effects, demonstrating their requirement of PDGF receptor activation in a manner similar to 17beta-estradiol. These results demonstrate the interdependence of PDGF and estrogen pathways in stimulating in vitro gonocyte proliferation, suggesting that this critical step in gonocyte development might be regulated in vivo by the coordinated action of PDGF and estrogen. Thus, the inappropriate exposure of gonocytes to xenoestrogens might disrupt the crosstalk between the two pathways and potentially interfere with gonocyte development. PMID:20089883

Combinations of ERK and p38 MAPK inhibitors ablate tumor necrosis factor-alpha (TNF-alpha ) mRNA induction. Evidence for selective destabilization of TNF-alpha transcripts.

PubMed

Rutault, K; Hazzalin, C A; Mahadevan, L C

2001-03-02

Tumor necrosis factor-alpha (TNF-alpha) is a potent proinflammatory cytokine whose synthesis and secretion are implicated in diverse pathologies. Hence, inhibition of TNF-alpha transcription or translation and neutralization of its protein product represent major pharmaceutical strategies to control inflammation. We have studied the role of ERK and p38 mitogen-activated protein (MAP) kinase in controlling TNF-alpha mRNA levels in differentiated THP-1 cells and in freshly purified human monocytes. We show here that it is possible to produce virtually complete inhibition of lipopolysaccharide-stimulated TNF-alpha mRNA accumulation by using a combination of ERK and p38 MAP kinase inhibitors. Furthermore, substantial inhibition is achievable using combinations of 1 microm of each inhibitor, whereas inhibitors used individually are incapable of producing complete inhibition even at high concentrations. Finally, addressing mechanisms involved, we show that inhibition of p38 MAP kinase selectively destabilizes TNF-alpha transcripts but does not affect degradation of c-jun transcripts. These results impinge on the controversy in the literature surrounding the mode of action of MAP kinase inhibitors on TNF-alpha mRNA and suggest the use of combinations of MAP kinase inhibitors as an effective anti-inflammatory strategy.

The Bcr-Abl kinase regulates the actin cytoskeleton via a GADS/Slp-76/Nck1 adaptor protein pathway.

PubMed

Preisinger, Christian; Kolch, Walter

2010-05-01

Bcr-Abl is the transforming principle underlying chronic myelogenous leukaemia (CML). Here, we use a functional interaction proteomics approach to map pathways by which Bcr-Abl regulates defined cellular processes. The results show that Bcr-Abl regulates the actin cytoskeleton and non-apoptotic membrane blebbing via a GADS/Slp-76/Nck1 adaptor protein pathway. The binding of GADS to Bcr-Abl requires Bcr-Abl tyrosine kinase activity and is sensitive to the Bcr-Abl inhibitor imatinib, while the GADS/Slp-76 and Slp-76/Nck interactions are tyrosine phosphorylation independent. All three adaptor proteins co-localize with cortical actin in membrane blebs. Downregulation of each adaptor protein disrupts the actin cytoskeleton and membrane blebbing in a similar fashion and similar to imatinib. These findings highlight the importance of protein interaction dependent adaptor protein pathways in oncogenic kinase signaling. 2010 Elsevier Inc. All rights reserved.

Glucagon-like peptide-1 increases myocardial glucose uptake via p38alpha MAP kinase-mediated, nitric oxide-dependent mechanisms in conscious dogs with dilated cardiomyopathy.

PubMed

Bhashyam, Siva; Fields, Anjali V; Patterson, Brandy; Testani, Jeffrey M; Chen, Li; Shen, You-Tang; Shannon, Richard P

2010-07-01

We have shown that glucagon-like peptide-1 (GLP-1[7-36] amide) stimulates myocardial glucose uptake in dilated cardiomyopathy (DCM) independent of an insulinotropic effect. The cellular mechanisms of GLP-1-induced myocardial glucose uptake are unknown. Myocardial substrates and glucoregulatory hormones were measured in conscious, chronically instrumented dogs at control (n=6), DCM (n=9) and DCM after treatment with a 48-hour infusion of GLP-1 (7-36) amide (n=9) or vehicle (n=6). GLP-1 receptors and cellular pathways implicated in myocardial glucose uptake were measured in sarcolemmal membranes harvested from the 4 groups. GLP-1 stimulated myocardial glucose uptake (DCM: 20+/-7 nmol/min/g; DCM+GLP-1: 61+/-12 nmol/min/g; P=0.001) independent of increased plasma insulin levels. The GLP-1 receptors were upregulated in the sarcolemmal membranes (control: 98+/-2 density units; DCM: 256+/-58 density units; P=0.046) and were expressed in their activated (65 kDa) form in DCM. The GLP-1-induced increases in myocardial glucose uptake did not involve adenylyl cyclase or Akt activation but was associated with marked increases in p38alpha MAP kinase activity (DCM+vehicle: 97+/-22 pmol ATP/mg/min; DCM+GLP-1: 170+/-36 pmol ATP/mg/min; P=0.051), induction of nitric oxide synthase 2 (DCM+vehicle: 151+/-13 density units; DCM+GLP-1: 306+/-12 density units; P=0.001), and GLUT-1 translocation (DCM+vehicle: 21+/-3% membrane bound; DCM+GLP-1: 39+/-3% membrane bound; P=0.005). The effects of GLP-1 on myocardial glucose uptake were blocked by pretreatment with the p38alpha MAP kinase inhibitor or the nonspecific nitric oxide synthase inhibitor nitro-l-arginine. GLP-1 stimulates myocardial glucose uptake through a non-Akt-1-dependent mechanism by activating cellular pathways that have been identified in mediating chronic hibernation and the late phase of ischemic preconditioning.

Identification of a novel human kinase supporter of Ras (hKSR-2) that functions as a negative regulator of Cot (Tpl2) signaling.

PubMed

Channavajhala, Padma L; Wu, Leeying; Cuozzo, John W; Hall, J Perry; Liu, Wei; Lin, Lih-Ling; Zhang, Yuhua

2003-11-21

Kinase suppressor of Ras (KSR) is an integral and conserved component of the Ras signaling pathway. Although KSR is a positive regulator of the Ras/mitogen-activated protein (MAP) kinase pathway, the role of KSR in Cot-mediated MAPK activation has not been identified. The serine/threonine kinase Cot (also known as Tpl2) is a member of the MAP kinase kinase kinase (MAP3K) family that is known to regulate oncogenic and inflammatory pathways; however, the mechanism(s) of its regulation are not precisely known. In this report, we identify an 830-amino acid novel human KSR, designated hKSR-2, using predictions from genomic data base mining based on the structural profile of the KSR kinase domain. We show that, similar to the known human KSR, hKSR-2 co-immunoprecipitates with many signaling components of the Ras/MAPK pathway, including Ras, Raf, MEK-1, and ERK-1/2. In addition, we demonstrate that hKSR-2 co-immunoprecipitates with Cot and that co-expression of hKSR-2 with Cot significantly reduces Cot-mediated MAPK and NF-kappaB activation. This inhibition is specific to Cot, because Ras-induced ERK and IkappaB kinase-induced NF-kappaB activation are not significantly affected by hKSR-2 co-expression. Moreover, Cot-induced interleukin-8 production in HeLa cells is almost completely inhibited by the concurrent expression of hKSR-2, whereas transforming growth factor beta-activated kinase 1 (TAK1)/TAK1-binding protein 1 (TAB1)-induced interleukin-8 production is not affected by hKSR-2 co-expression. Taken together, these results indicate that hKSR-2, a new member of the KSR family, negatively regulates Cot-mediated MAP kinase and NF-kappaB pathway signaling.

PSM/SH2-B distributes selected mitogenic receptor signals to distinct components in the PI3-kinase and MAP kinase signaling pathways.

PubMed

Deng, Youping; Xu, Hu; Riedel, Heimo

2007-02-15

The Pro-rich, PH, and SH2 domain containing mitogenic signaling adapter PSM/SH2-B has been implicated as a cellular partner of various mitogenic receptor tyrosine kinases and related signaling mechanisms. Here, we report in a direct comparison of three peptide hormones, that PSM participates in the assembly of distinct mitogenic signaling complexes in response to insulin or IGF-I when compared to PDGF in cultured normal fibroblasts. The complex formed in response to insulin or IGF-I involves the respective peptide hormone receptor and presumably the established components leading to MAP kinase activation. However, our data suggest an alternative link from the PDGF receptor via PSM directly to MEK1/2 and consequently also to p44/42 activation, possibly through a scaffold protein. At least two PSM domains participate, the SH2 domain anticipated to link PSM to the respective receptor and the Pro-rich region in an association with an unidentified downstream component resulting in direct MEK1/2 and p44/42 regulation. The PDGF receptor signaling complex formed in response to PDGF involves PI 3-kinase in addition to the same components and interactions as described for insulin or IGF-I. PSM associates with PI 3-kinase via p85 and in addition the PSM PH domain participates in the regulation of PI 3-kinase activity, presumably through membrane interaction. In contrast, the PSM Pro-rich region appears to participate only in the MAP kinase signal. Both pathways contribute to the mitogenic response as shown by cell proliferation, survival, and focus formation. PSM regulates p38 MAP kinase activity in a pathway unrelated to the mitogenic response.

Independent localization of MAP2, CaMKIIα and β-actin RNAs in low copy numbers

PubMed Central

Mikl, Martin; Vendra, Georgia; Kiebler, Michael A

2011-01-01

Messenger RNA localization involves the assembly of ribonucleoprotein particles (RNPs) and their subsequent transport along the cytoskeleton to their final destination. Here, we provide new evidence that microtubule-associated protein 2 (MAP2), calcium/calmodulin-dependent protein kinase II (CaMKIIα) and β-actin RNAs localize to dendrites in distinct RNPs, which contain—unexpectedly—very few RNA molecules. The number of MAP2 molecules per particle is affected by synaptic activity and Staufen 2, indicating that RNP composition is tightly controlled. Our data suggest that the independent localization of individual RNAs in low copy numbers could contribute to tighter temporal and spatial control of expression in neurons and synapse-specific plasticity. PMID:21869818

Pharmacophore modeling of diverse classes of p38 MAP kinase inhibitors.

PubMed

Sarma, Rituparna; Sinha, Sharat; Ravikumar, Muttineni; Kishore Kumar, Madala; Mahmood, S K

2008-12-01

Mitogen-activated protein (MAP) p38 kinase is a serine-threonine protein kinase and its inhibitors are useful in the treatment of inflammatory diseases. Pharmacophore models were developed using HypoGen program of Catalyst with diverse classes of p38 MAP kinase inhibitors. The best pharmacophore hypothesis (Hypo1) with hydrogen-bond acceptor (HBA), hydrophobic (HY), hydrogen-bond donor (HBD), and ring aromatic (RA) as features has correlation coefficient of 0.959, root mean square deviation (RMSD) of 1.069 and configuration cost of 14.536. The model was validated using test set containing 119 compounds and had high correlation coefficient of 0.851. The results demonstrate that results obtained in this study can be considered to be useful and reliable tools in identifying structurally diverse compounds with desired biological activity.

Lead decreases cell survival, proliferation, and neuronal differentiation of primary cultured adult neural precursor cells through activation of the JNK and p38 MAP kinases

PubMed Central

Engstrom, Anna; Wang, Hao; Xia, Zhengui

2015-01-01

Adult hippocampal neurogenesis is the process whereby adult neural precursor cells (aNPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate adult-born, functional neurons in the hippocampus. This process is modulated by various extracellular and intracellular stimuli, and the adult-born neurons have been implicated in hippocampus-dependent learning and memory. However, studies on how neurotoxic agents affect this process and the underlying mechanisms are limited. The goal of this study was to determine whether lead, a heavy metal, directly impairs critical processes in adult neurogenesis and to characterize the underlying signaling pathways using primary cultured SGZ-aNPCs isolated from adult mice. We report here that lead significantly increases apoptosis and inhibits proliferation in SGZ-aNPCs. In addition, lead significantly impairs spontaneous neuronal differentiation and maturation. Furthermore, we found that activation of the c-Jun NH2-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase signaling pathways are important for lead cytotoxicity. Our data suggest that lead can directly act on adult neural stem cells and impair critical processes in adult hippocampal neurogenesis, which may contribute to its neurotoxicity and adverse effects on cognition in adults. PMID:25967738

Lead decreases cell survival, proliferation, and neuronal differentiation of primary cultured adult neural precursor cells through activation of the JNK and p38 MAP kinases.

PubMed

Engstrom, Anna; Wang, Hao; Xia, Zhengui

2015-08-01

Adult hippocampal neurogenesis is the process whereby adult neural precursor cells (aNPCs) in the subgranular zone (SGZ) of the dentate gyrus (DG) generate adult-born, functional neurons in the hippocampus. This process is modulated by various extracellular and intracellular stimuli, and the adult-born neurons have been implicated in hippocampus-dependent learning and memory. However, studies on how neurotoxic agents affect this process and the underlying mechanisms are limited. The goal of this study was to determine whether lead, a heavy metal, directly impairs critical processes in adult neurogenesis and to characterize the underlying signaling pathways using primary cultured SGZ-aNPCs isolated from adult mice. We report here that lead significantly increases apoptosis and inhibits proliferation in SGZ-aNPCs. In addition, lead significantly impairs spontaneous neuronal differentiation and maturation. Furthermore, we found that activation of the c-Jun NH2-terminal kinase (JNK) and p38 mitogen activated protein (MAP) kinase signaling pathways are important for lead cytotoxicity. Our data suggest that lead can directly act on adult neural stem cells and impair critical processes in adult hippocampal neurogenesis, which may contribute to its neurotoxicity and adverse effects on cognition in adults. Copyright © 2015 Elsevier Ltd. All rights reserved.

TGFβ1-mediated PI3K/Akt and p38 MAP kinase dependent alternative splicing of fibronectin extra domain A in human podocyte culture.

PubMed

Madne, Tarunkumar Hemraj; Dockrell, Mark Edward Carl

2018-04-30

Alternative splicing is an important gene regulation process to distribute proteins in health and diseases. Extra Domain A+ Fibronectin (EDA+Fn) is an alternatively spliced form of fibronectin (Fn) protein, present in the extra cellular matrix (ECM) and a recognised marker of various pathologies. TGFβ1 has been shown to induce alternative splicing of EDA+Fn in many cell types. Podocytes are spectacular cell type and play a key role in filtration and synthesise ECM proteins in renal physiology and pathology. In our previous study we have demonstrated expression and alternative splicing of EDA+Fn in basal condition in human podocytes culture. TGFβ1 further induced the basal expression and alternative splicing of EDA+Fn through Alk5 receptor and SR proteins. In this study, we have investigated TGFβ1 mediated signalling involved in alternative splicing of EDA+Fn in human podocytes. We have performed western blotting to characterise the expression of the EDA+Fn protein and other signalling proteins and RT-PCR to look for signalling pathways involved in regulation of alternative splicing of EDA+Fn in conditionally immortalised human podocytes culture.We have used TGFβ1 as a stimulator and SB431542, SB202190 and LY294002 for inhibitory studies. In this work, we have demonstrated in human podocytes culture TGFβ1 2.5ng/ml induced phosphorylation of Smad1/5/8, Smad2 and Smad3 via the ALK5 receptor. TGFβ1 significantly induced the PI3K/Akt pathway and the PI3K/Akt pathway inhibitor LY294002 significantly downregulated basal as well as TGFβ1 induced alternative splicing of EDA+Fn in human podocytes. In addition to this, TGFβ1 significantly induced the p38 MAP kinase signalling pathway and p38 MAP kinase signalling pathway inhibitor SB202190 downregulated the TGFβ1-mediated alternative splicing of EDA+Fn in human podocytes. The results with PI3K and p38 MAP kinase signalling pathway suggest that inhibiting PI3K signalling pathway downregulated the basal alternative splicing of EDA+Fn in human podocytes and its the inhibition of p38 Map Kinase signalling pathway which had specifically downregulated the TGFβ1 mediated alternative splicing of EDA+Fn in human podocytes culture. Activation of TGFβ1-mediated Smad1/5/8 via Alk5 receptor suggests that TGFβ1 signalling pathway involved Alk5/Alk1 receptor axis signalling in human podocytes.

Internalization of the human N-formyl peptide and C5a chemoattractant receptors occurs via clathrin-independent mechanisms.

PubMed

Gilbert, T L; Bennett, T A; Maestas, D C; Cimino, D F; Prossnitz, E R

2001-03-27

After stimulation by ligand, most G protein-coupled receptors (GPCRs) undergo rapid phosphorylation, followed by desensitization and internalization. In the case of the N-formyl peptide receptor (FPR), these latter two processing steps have been shown to be entirely dependent on phosphorylation of the receptor's carboxy terminus. We have previously demonstrated that FPR internalization can occur in the absence of receptor desensitization, indicating that FPR desensitization and internalization are regulated differentially. In this study, we have investigated whether human chemoattractant receptors internalize via clathrin-coated pits. Internalization of the FPR transiently expressed in HEK 293 cells was shown to be dependent upon receptor phosphorylation. Despite this, internalization of the FPR, as well as the C5a receptor, was demonstrated to be independent of the actions of arrestin, dynamin, and clathrin. In addition, we utilized fluorescence microscopy to visualize the FPR and beta(2)-adrenergic receptor as they internalized in the same cell, revealing distinct sites of internalization. Last, we found that a nonphosphorylatable mutant of the FPR, unable to internalize, was competent to activate p44/42 MAP kinase. Together, these results demonstrate not only that the FPR internalizes via an arrestin-, dynamin-, and clathrin-independent pathway but also that signal transduction to MAP kinases occurs in an internalization-independent manner.

Cardiotrophin-1 Induces Matrix Metalloproteinase-1 in Human Aortic Endothelial Cells

PubMed Central

Tokito, Akinori; Jougasaki, Michihisa; Ichiki, Tomoko; Hamasaki, Shuichi

2013-01-01

Rupture of an atherosclerotic plaque is a key event in the development of cardiovascular disorders, in which matrix metalloproteinase-1 (MMP-1) plays a crucial role by degradation of extracellular matrix resulting in plaque instability. Cardiotrophin-1 (CT-1), a member of interleukin-6-type proinflammatory cytokines, has potent cardiovascular actions and is highly expressed in vascular endothelium, however its role in atherosclerosis has not been fully elucidated to date. The present study was designed to investigate whether CT-1 induces MMP-1 in human aortic endothelial cells (HAECs). Ribonuclease protection assay demonstrated that MMP-1 gene level in HAECs was enhanced by the treatment of CT-1 in a dose- and time-dependent manner. Immunocytochemical staining, Western immunoblot analysis and enzyme-linked immunosorbent assay revealed that CT-1 augmented MMP-1 protein synthesis and secretion. MMP-1 activity assay revealed that MMP-1 present in the supernatant of HAECs was exclusively precursor form. Casein zymography disclosed proteolytic activity in the supernatant of HAECs, which was enhanced by CT-1 treatment. Furthermore, pharmacological inhibitor study indicated the important roles of extracellular signal-regulated kinase (ERK) 1/2, p38 mitogen-activated protein (MAP) kinase, c-Jun N-terminal kinase (JNK) and Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathways in mediating CT-1-induced MMP-1 gene and protein expression. These data reveal for the first time that CT-1 induces the proteolytic potential in HAECs by upregulating MMP-1 expression through ERK1/2, p38 MAP kinase, JNK and JAK/STAT pathways, and suggest that CT-1 may play an important role in the pathophysiology of atherosclerosis and plaque instability. PMID:23935888

Mitochondrial-derived hydrogen peroxide inhibits relaxation of bovine coronary arterial smooth muscle to hypoxia through stimulation of ERK MAP kinase.

PubMed

Gao, Qun; Zhao, Xiangmin; Ahmad, Mansoor; Wolin, Michael S

2009-12-01

Mitochondrial reactive oxygen species (ROS) are potentially important in vascular oxygen-sensing mechanisms because hypoxia appears to be a stimulus for mitochondrial ROS generation; however, scavenging of endogenous ROS does not alter relaxation of endothelium-denuded bovine coronary arteries (BCA) to hypoxia. The purpose of this study was to investigate the influence of increasing mitochondrial ROS on the relaxation of BCA to hypoxia. Increasing mitochondrial superoxide with inhibitors of electron transport (10 microM rotenone and antimycin) and by opening mitochondrial ATP-dependent K+ channels with 100 microM diazoxide were observed in this study to attenuate relaxation of BCA precontracted with 30 mM KCl to hypoxia by 68-76% and 38%, respectively. This effect of rotenone is not prevented by inhibiting NADPH oxidase (Nox) activation or scavenging superoxide with Peg-SOD; however, it is reversed 85% and 26% by increasing the consumption of intracellular peroxide by 0.1 mM ebselen and 32.5 U/ml Peg-catalase. Because inhibition of extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase (10 microM PD-98059), but not src kinase or rho kinase, also reverses the effects of rotenone by 69%, the peroxide-elicited force-enhancing effects of ERK appear to be attenuating the response to hypoxia. Rotenone increased the phosphorylation of ERK (by 163%). Activation of ERK in BCA with 0.1 mM peroxide or endogenous peroxide generated by stimulating Nox2 with a stretch treatment or contraction with 100 nM U-46619 also attenuated relaxation to hypoxia. Thus coronary arterial relaxation to hypoxia may be attenuated by pathophysiological conditions associated with increased peroxide generation by mitochondria or other sources that stimulate ERK.

Fungal Communication Requires the MAK-2 Pathway Elements STE-20 and RAS-2, the NRC-1 Adapter STE-50 and the MAP Kinase Scaffold HAM-5

PubMed Central

Dettmann, Anne; Heilig, Yvonne; Valerius, Oliver; Ludwig, Sarah; Seiler, Stephan

2014-01-01

Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell–cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell–cell communication in fungi and higher eukaryotes. PMID:25411845

Comparison of angiotensin II (Ang II) effects in the internal anal sphincter (IAS) and lower esophageal sphincter smooth muscles.

PubMed

Rattan, Satish; Fan, Ya-Ping; Puri, Rajinder N

2002-03-22

Studies were performed to compare the actions of Ang II in the internal anal sphincter (IAS) vs. lower esophageal sphincter (LES) smooth muscles in vitro, in opossum and rabbit. Studies also were carried out in isolated smooth muscle cells. In opossum, Ang II produced no discernible effects in the IAS, but did produce a concentration-dependent contraction in the LES. Conversely, in the rabbit, while Ang II caused a modest response in the LES, it caused a significant contraction in the IAS. The contractile responses of Ang II in the opossum LES were mostly resistant to different neurohumoral antagonists but were antagonized by AT1 antagonist losartan. AT2 antagonist PD 123,319, rather than inhibiting, prolonged the contractile action of Ang II. The contractile actions of Ang II in the opossum LES were not modified by the tyrosine kinase inhibitors (genistein and tyrphostin 1 x 10(-6) M) but were partially attenuated by the PKC inhibitor H-7 (1 x 10(-6) M), Ca2+ channel blocker nicardipine (1 x 10(-5) M), Rho kinase inhibitor HA-1077 (1 x 10(-7) M) or p(44/42) MAP kinase inhibitor PD 98059 (5 x 10(-5) M). The combination of HA-1077 and H-7 did not cause an additive attenuation of Ang II responses. Western blot analyses revealed the presence of both AT1 and AT2 receptors. We conclude that Ang lI-induced contraction of sphincteric smooth muscle occurs primarily by the activation of AT1 receptors at the smooth muscle cells and involves multiple pathways, influx of Ca2+, and PKC, Rho kinase and p(44/42) MAP kinase.

Fungal communication requires the MAK-2 pathway elements STE-20 and RAS-2, the NRC-1 adapter STE-50 and the MAP kinase scaffold HAM-5.

PubMed

Dettmann, Anne; Heilig, Yvonne; Valerius, Oliver; Ludwig, Sarah; Seiler, Stephan

2014-11-01

Intercellular communication is critical for the survival of unicellular organisms as well as for the development and function of multicellular tissues. Cell-to-cell signaling is also required to develop the interconnected mycelial network characteristic of filamentous fungi and is a prerequisite for symbiotic and pathogenic host colonization achieved by molds. Somatic cell-cell communication and subsequent cell fusion is governed by the MAK-2 mitogen activated protein kinase (MAPK) cascade in the filamentous ascomycete model Neurospora crassa, yet the composition and mode of regulation of the MAK-2 pathway are currently unclear. In order to identify additional components involved in MAK-2 signaling we performed affinity purification experiments coupled to mass spectrometry with strains expressing functional GFP-fusion proteins of the MAPK cascade. This approach identified STE-50 as a regulatory subunit of the Ste11p homolog NRC-1 and HAM-5 as cell-communication-specific scaffold protein of the MAPK cascade. Moreover, we defined a network of proteins consisting of two Ste20-related kinases, the small GTPase RAS-2 and the adenylate cyclase capping protein CAP-1 that function upstream of the MAK-2 pathway and whose signals converge on the NRC-1/STE-50 MAP3K complex and the HAM-5 scaffold. Finally, our data suggest an involvement of the striatin interacting phosphatase and kinase (STRIPAK) complex, the casein kinase 2 heterodimer, the phospholipid flippase modulators YPK-1 and NRC-2 and motor protein-dependent vesicle trafficking in the regulation of MAK-2 pathway activity and function. Taken together, these data will have significant implications for our mechanistic understanding of MAPK signaling and for homotypic cell-cell communication in fungi and higher eukaryotes.

A Crosslinker Based on a Tethered Electrophile for Mapping Kinase-Substrate Networks

PubMed Central

Riel-Mehan, Megan M; Shokat, Kevan M

2014-01-01

SUMMARY Despite the continuing progress made towards mapping kinase signaling networks, there are still many phosphorylation events for which the responsible kinase has not yet been identified. We are interested in addressing this problem through forming covalent crosslinks between a peptide substrate and the corresponding phosphorylating kinase. Previously we reported a dialdehyde-based kinase binding probe capable of such a reaction with a peptide containing a cysteine substituted for the phosphorylatable ser/thr/tyr residue. Here, we examine the yield of a previously reported dialdehyde-based probe, and report that the dialdehyde based probes possesses a significant limitation in terms of crosslinked kinase-substrate product yield. To address this limitation, we develop a crosslinking scheme based on a kinase activity-based probe, and this new cross-linker provides an increase in efficiency and substrate specificity, including in the context of cell lysate. PMID:24746561

Interdomain allosteric regulation of Polo kinase by Aurora B and Map205 is required for cytokinesis

PubMed Central

Kachaner, David; Pinson, Xavier; El Kadhi, Khaled Ben; Normandin, Karine; Talje, Lama; Lavoie, Hugo; Lépine, Guillaume; Carréno, Sébastien; Kwok, Benjamin H.; Hickson, Gilles R.

2014-01-01

Drosophila melanogaster Polo and its human orthologue Polo-like kinase 1 fulfill essential roles during cell division. Members of the Polo-like kinase (Plk) family contain an N-terminal kinase domain (KD) and a C-terminal Polo-Box domain (PBD), which mediates protein interactions. How Plks are regulated in cytokinesis is poorly understood. Here we show that phosphorylation of Polo by Aurora B is required for cytokinesis. This phosphorylation in the activation loop of the KD promotes the dissociation of Polo from the PBD-bound microtubule-associated protein Map205, which acts as an allosteric inhibitor of Polo kinase activity. This mechanism allows the release of active Polo from microtubules of the central spindle and its recruitment to the site of cytokinesis. Failure in Polo phosphorylation results in both early and late cytokinesis defects. Importantly, the antagonistic regulation of Polo by Aurora B and Map205 in cytokinesis reveals that interdomain allosteric mechanisms can play important roles in controlling the cellular functions of Plks. PMID:25332165

Activation of the mitogen-activated protein kinase pathway by bone sialoprotein regulates osteoblast differentiation.

PubMed

Gordon, Jonathan A R; Hunter, Graeme K; Goldberg, Harvey A

2009-01-01

Bone sialoprotein (BSP) is an abundant protein in the extracellular matrix of bone that has been suggested to have several different physiological functions, including the nucleation of hydroxyapatite (HA), promotion of cell attachment and binding of collagen. Studies in our lab have demonstrated that increased expression of BSP in osteoblast cells can increase expression of the osteoblast-related genes Runx2 and Osx as well as alkaline phosphatase and osteocalcin and increase matrix mineralization. To determine the molecular mechanisms responsible for the BSP-mediated increase in osteoblastic differentiation, several functional domain mutants of BSP were expressed in primary rat bone osteoblastic cells, including the contiguous glutamic acid sequences (polyGlu) and the arginine-glycine-aspartic acid (RGD) motif. Markers of osteoblast differentiation, including matrix mineralization and alkaline phosphatase staining, were increased in cells expressing BSP mutants of the polyGlu sequences but not in cells expressing RGD-mutated BSP. We also determined the dependence on integrin-associated pathways in promoting BSP-mediated differentiation responses in osteoblasts by demonstrating the activation of focal adhesion kinase, MAP kinase-associated proteins ERK1/2, ribosomal s6 kinase 2 and the AP-1 protein cFos. Thus, the mechanism regulating osteoblast differentiation by BSP was determined to be dependent on integrin-mediated intracellular signaling pathways. Copyright 2008 S. Karger AG, Basel.

CDK-dependent potentiation of MPS1 kinase activity is essential to the mitotic checkpoint.

PubMed

Morin, Violeta; Prieto, Susana; Melines, Sabrina; Hem, Sonia; Rossignol, Michel; Lorca, Thierry; Espeut, Julien; Morin, Nathalie; Abrieu, Ariane

2012-02-21

Accurate chromosome segregation relies upon a mitotic checkpoint that monitors kinetochore attachment toward opposite spindle poles before enabling chromosome disjunction [1]. The MPS1/TTK protein kinase is a core component of the mitotic checkpoint that lies upstream of MAD2 and BubR1 both at the kinetochore and in the cytoplasm [2, 3]. To gain insight into the mechanisms underlying the regulation of MPS1 kinase, we undertook the identification of Xenopus MPS1 phosphorylation sites by mass spectrometry. We mapped several phosphorylation sites onto MPS1 and we show that phosphorylation of S283 in the noncatalytic region of MPS1 is required for full kinase activity. This phosphorylation potentiates MPS1 catalytic efficiency without impairing its affinity for the substrates. By using Xenopus egg extracts depleted of endogenous MPS1 and reconstituted with single point mutants, we show that phosphorylation of S283 is essential to activate the mitotic checkpoint. This phosphorylation does not regulate the localization of MPS1 to the kinetochore but is required for the recruitment of MAD1/MAD2, demonstrating its role at the kinetochore. Constitutive phosphorylation of S283 lowers the number of kinetochores required to hold the checkpoint, which suggests that CDK-dependent phosphorylation of MPS1 is essential to sustain the mitotic checkpoint when few kinetochores remain unattached. Copyright © 2012 Elsevier Ltd. All rights reserved.

Porcine MAP3K5 analysis: molecular cloning, characterization, tissue expression pattern, and copy number variations associated with residual feed intake.

PubMed

Pu, L; Zhang, L C; Zhang, J S; Song, X; Wang, L G; Liang, J; Zhang, Y B; Liu, X; Yan, H; Zhang, T; Yue, J W; Li, N; Wu, Q Q; Wang, L X

2016-08-12

Mitogen-activated protein kinase kinase kinase 5 (MAP3K5) is essential for apoptosis, proliferation, differentiation, and immune responses, and is a candidate marker for residual feed intake (RFI) in pig. We cloned the full-length cDNA sequence of porcine MAP3K5 by rapid-amplification of cDNA ends. The 5451-bp gene contains a 5'-untranslated region (UTR) (718 bp), a coding region (3738 bp), and a 3'-UTR (995 bp), and encodes a peptide of 1245 amino acids, which shares 97, 99, 97, 93, 91, and 84% sequence identity with cattle, sheep, human, mouse, chicken, and zebrafish MAP3K5, respectively. The deduced MAP3K5 protein sequence contains two conserved domains: a DUF4071 domain and a protein kinase domain. Phylogenetic analysis showed that porcine MAP3K5 forms a separate branch to vicugna and camel MAP3K5. Tissue expression analysis using real-time quantitative polymerase chain reaction (qRT-PCR) revealed that MAP3K5 was expressed in the heart, liver, spleen, lung, kidney, muscle, fat, pancrea, ileum, and stomach tissues. Copy number variation was detected for porcine MAP3K5 and validated by qRT-PCR. Furthermore, a significant increase in average copy number was detected in the low RFI group when compared to the high RFI group in a Duroc pig population. These results provide useful information regarding the influence of MAP3K5 on RFI in pigs.

Regulation of MMP-3 expression and secretion by the chemokine eotaxin-1 in human chondrocytes.

PubMed

Chao, Pin-Zhir; Hsieh, Ming-Shium; Cheng, Chao-Wen; Lin, Yung-Feng; Chen, Chien-Ho

2011-11-25

Osteoarthritis (OA) is characterized by the degradation of articular cartilage, marked by the breakdown of matrix proteins. Studies demonstrated the involvement of chemokines in this process, and some may potentially serve as diagnostic markers and therapeutic targets; however, the underlying signal transductions are not well understood. We investigated the effects of the CC chemokine eotaxin-1 (CCL11) on the matrix metalloproteinase (MMP) expression and secretion in the human chondrocyte cell line SW1353 and primary chondrocytes. Eotaxin-1 significantly induced MMP-3 mRNA expression in a dose-dependent manner. Inhibitors of extracellular signal-regulated kinase (ERK) and p38 kinase were able to repress eotaxin-1-induced MMP-3 expression. On the contrary, Rp-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPs), a competitive cAMP antagonist for cAMP receptors, and H-89, a protein kinase A (PKA) inhibitor, markedly enhanced eotaxin-1-induced MMP-3 expression. These results suggest that MMP-3 expression is specifically mediated by the G protein-coupled eotaxin-1 receptor activities. Interestingly, little amount of MMP-3 protein was detected in the cell lysates of eotaxin-1-treated SW1353 cells, and most of MMP-3 protein was in the culture media. Furthermore we found that the eotaxin-1-dependent MMP-3 protein secretion was regulated by phospholipase C (PLC)-protein kinase C (PKC) cascade and c-Jun N-terminal kinase (JNK)/mitogen-activated protein (MAP) kinase pathways. These data indicate a specific regulation of MMP-3 secretion also by eotaxin-1 receptor activities. Eotaxin-1 not only induces MMP-3 gene expression but also promotes MMP-3 protein secretion through G protein-coupled eotaxin-1 receptor activities. Chemokines, such as eotaxin-1, could be a potential candidate in the diagnosis and treatment of arthritis.

Fibroblast growth factor and cyclic AMP (cAMP) synergistically activate gene expression at a cAMP response element.

PubMed Central

Tan, Y; Low, K G; Boccia, C; Grossman, J; Comb, M J

1994-01-01

Growth factors and cyclic AMP (cAMP) are known to activate distinct intracellular signaling pathways. Fibroblast growth factor (FGF) activates ras-dependent kinase cascades, resulting in the activation of MAP kinases, whereas cAMP activates protein kinase A. In this study, we report that growth factors and cAMP act synergistically to stimulate proenkephalin gene expression. Positive synergy between growth factor- and cAMP-activated signaling pathways on gene expression has not been previously reported, and we suggest that these synergistic interactions represent a useful model for analyzing interactions between these pathways. Transfection and mutational studies indicate that both FGF-dependent gene activation and cAMP-dependent gene activation require cAMP response element 2 (CRE-2), a previously characterized cAMP-dependent regulatory element. Furthermore, multiple copies of this element are sufficient to confer FGF regulation upon a minimal promoter, indicating that FGF and cAMP signaling converge upon transcription factors acting at CRE-2. Among many different ATF/AP-1 factors tested, two factors, ATF-3 and c-Jun, stimulate proenkephalin transcription in an FGF- or Ras-dependent fashion. Finally, we show that ATF-3 and c-Jun form heterodimeric complexes in SK-N-MC cells and that the levels of both proteins are increased in response to FGF but not cAMP. Together, these results indicate that growth factor- and cAMP-dependent signaling pathways converge at CRE-2 to synergistically stimulate gene expression and that ATF-3 and c-Jun regulate proenkephalin transcription in response to both growth factor- and cAMP-dependent intracellular signaling pathways. Images PMID:7935470

Osmostress induces autophosphorylation of Hog1 via a C-terminal regulatory region that is conserved in p38α.

PubMed

Maayan, Inbal; Beenstock, Jonah; Marbach, Irit; Tabachnick, Shira; Livnah, Oded; Engelberg, David

2012-01-01

Many protein kinases require phosphorylation at their activation loop for induction of catalysis. Mitogen-activated protein kinases (MAPKs) are activated by a unique mode of phosphorylation, on neighboring Tyrosine and Threonine residues. Whereas many kinases obtain their activation via autophosphorylation, MAPKs are usually phosphorylated by specific, dedicated, MAPK kinases (MAP2Ks). Here we show however, that the yeast MAPK Hog1, known to be activated by the MAP2K Pbs2, is activated in pbs2Δ cells via an autophosphorylation activity that is induced by osmotic pressure. We mapped a novel domain at the Hog1 C-terminal region that inhibits this activity. Removal of this domain provides a Hog1 protein that is partially independent of MAP2K, namely, partially rescues osmostress sensitivity of pbs2Δ cells. We further mapped a short domain (7 amino acid residues long) that is critical for induction of autophosphorylation. Its removal abolishes autophosphorylation, but maintains Pbs2-mediated phosphorylation. This 7 amino acids stretch is conserved in the human p38α. Similar to the case of Hog1, it's removal from p38α abolishes p38α's autophosphorylation capability, but maintains, although reduces, its activation by MKK6. This study joins a few recent reports to suggest that, like many protein kinases, MAPKs are also regulated via induced autoactivation.

Osmostress Induces Autophosphorylation of Hog1 via a C-Terminal Regulatory Region That Is Conserved in p38α

PubMed Central

Maayan, Inbal; Beenstock, Jonah; Marbach, Irit; Tabachnick, Shira; Livnah, Oded; Engelberg, David

2012-01-01

Many protein kinases require phosphorylation at their activation loop for induction of catalysis. Mitogen-activated protein kinases (MAPKs) are activated by a unique mode of phosphorylation, on neighboring Tyrosine and Threonine residues. Whereas many kinases obtain their activation via autophosphorylation, MAPKs are usually phosphorylated by specific, dedicated, MAPK kinases (MAP2Ks). Here we show however, that the yeast MAPK Hog1, known to be activated by the MAP2K Pbs2, is activated in pbs2Δ cells via an autophosphorylation activity that is induced by osmotic pressure. We mapped a novel domain at the Hog1 C-terminal region that inhibits this activity. Removal of this domain provides a Hog1 protein that is partially independent of MAP2K, namely, partially rescues osmostress sensitivity of pbs2Δ cells. We further mapped a short domain (7 amino acid residues long) that is critical for induction of autophosphorylation. Its removal abolishes autophosphorylation, but maintains Pbs2-mediated phosphorylation. This 7 amino acids stretch is conserved in the human p38α. Similar to the case of Hog1, it’s removal from p38α abolishes p38α’s autophosphorylation capability, but maintains, although reduces, its activation by MKK6. This study joins a few recent reports to suggest that, like many protein kinases, MAPKs are also regulated via induced autoactivation. PMID:22984552

Neurotensin-induced proinflammatory signaling in human colonocytes is regulated by β-arrestins and endothelin-converting enzyme-1-dependent endocytosis and resensitization of neurotensin receptor 1.

PubMed

Law, Ivy Ka Man; Murphy, Jane E; Bakirtzi, Kyriaki; Bunnett, Nigel W; Pothoulakis, Charalabos

2012-04-27

The neuropeptide/hormone neurotensin (NT) mediates intestinal inflammation and cell proliferation by binding of its high affinity receptor, neurotensin receptor-1 (NTR1). NT stimulates IL-8 expression in NCM460 human colonic epithelial cells by both MAP kinase- and NF-κB-dependent pathways. Although the mechanism of NTR1 endocytosis has been studied, the relationship between NTR1 intracellular trafficking and inflammatory signaling remains to be elucidated. In the present study, we show that in NCM460 cells exposed to NT, β-arrestin-1 (βARR1), and β-arrestin-2 (βARR2) translocate to early endosomes together with NTR1. Endothelin-converting enzyme-1 (ECE-1) degrades NT in acidic conditions, and its activity is crucial for NTR1 recycling. Pretreatment of NCM460 cells with the ECE-1 inhibitor SM19712 or gene silencing of βARR1 or βARR2 inhibits NT-stimulated ERK1/2 and JNK phosphorylation, NF-κB p65 nuclear translocation and phosphorylation, and IL-8 secretion. Furthermore, NT-induced cell proliferation, but not IL-8 transcription, is attenuated by the JNK inhibitor, JNK(AII). Thus, NTR1 internalization and recycling in human colonic epithelial cells involves βARRs and ECE-1, respectively. Our results also indicate that βARRs and ECE-1-dependent recycling regulate MAP kinase and NF-κB signaling as well as cell proliferation in human colonocytes in response to NT.

Pro-life role for c-Jun N-terminal kinase and p38 mitogen-activated protein kinase at rostral ventrolateral medulla in experimental brain stem death.

PubMed

Chang, Alice Y W

2012-11-17

Based on an experimental brain stem death model, we demonstrated previously that activation of the mitogen-activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ mitogen-activated protein kinase signal-interacting kinase 1/2 (MNK1/2) cascade plays a pro-life role in the rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from systemic arterial pressure, which sequentially increases (pro-life) and decreases (pro-death) to reflect progressive dysfunction of central cardiovascular regulation during the advancement towards brain stem death in critically ill patients. The present study assessed the hypothesis that, in addition to ERK1/2, c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), the other two mammalian members of MAPKs that are originally identified as stress-activated protein kinases, are activated specifically by MAPK kinase 4 (MAP2K4) or MAP2K6 and play a pro-life role in RVLM during experimental brain stem death. We further delineated the participation of phosphorylating activating transcriptional factor-2 (ATF-2) and c-Jun, the classical transcription factor activated by JNK or p38MAPK, in this process. An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol) bilaterally into RVLM of Sprague-Dawley rats was used, alongside cardiovascular, pharmacological and biochemical evaluations. Results from ELISA showed that whereas the total JNK, p38MAPK, MAP2K4 and MAP2K6 were not affected, augmented phosphorylation of JNK at Thr183 and Tyr185 and p38MAPK at Thr180 and Tyr182, accompanied by phosphorylation of their upstream activators MAP2K4 at Ser257 and Thr261 and MAP2K6 at Ser207 and Thr211 in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Moreover, the activity of transcription factors ATF-2 at Thr71 and c-Jun at Ser73, rather than Elk-1 at Ser383 in RVLM were also augmented during the pro-life phase. Furthermore, pretreatment by microinjection into the bilateral RVLM of specific JNK inhibitors, JNK inhibitor I (100 pmol) or SP600125 (5 pmol), or specific p38MAPK inhibitors, p38MAPK inhibitor III (500 pmol) or SB203580 (2 nmol), exacerbated the depressor effect and blunted the augmented life-and-death signal exhibited during the pro-life phase. On the other hand, pretreatment with the negative control for JNK or p38MAPK inhibitor, JNK inhibitor I negative control (100 pmol) or SB202474 (2 nmol), was ineffective in the vehicle-controls and Mev-treatment groups. Our results demonstrated that activation of JNK or p38MAPK in RVLM by their upstream activators MAP2K4 or MAP2K6 plays a preferential pro-life role by sustaining the central cardiovascular regulatory machinery during experimental brain stem death via phosphorylation and activation of nuclear transcription factor ATF-2 or c-Jun.

Escitalopram Ameliorates Tau Hyperphosphorylation and Spatial Memory Deficits Induced by Protein Kinase A Activation in Sprague Dawley Rats.

PubMed

Ren, Qing-Guo; Wang, Yan-Juan; Gong, Wei-Gang; Xu, Lin; Zhang, Zhi-Jun

2015-01-01

Here, we investigated the effect of escitalopram pretreatment on protein kinase A (PKA)-induced tau hyperphosphorylation and spatial memory deficits in rats using western blot and behavioral tests, respectively. We demonstrated that escitalopram effectively ameliorated tau hyperphosphorylation and the spatial memory deficits induced by PKA activation. We measured the total and activity-dependent Ser9-phosphorylated levels of glycogen synthase kinase (GSK)-3β in hippocampal extracts. No significant change in the total level of GSK-3β was observed between the different groups. However, compared with forskolin injection alone, pretreatment with escitalopram increased the level of Ser9-phosphorylated GSK-3β. We also demonstrated that escitalopram increased Akt phosphorylation at Ser473 (the active form of Akt). Furthermore, we identified other important kinases and phosphatases, such as protein phosphatase 2A, extracellular signal-regulated kinases 1 and 2, and MAP kinase kinase-1/2, that have previously been reported to play a crucial role in tau phosphorylation; however, we did not detect any significant change in the activation of these kinases or phosphatases in our study. We unexpectedly demonstrated that forskolin caused anxiety-like behavior in rats, and pretreatment with escitalopram did not significantly ameliorate the anxiety-like behavior induced by forskolin. These data provide the first evidence that escitalopram ameliorates forskolin-induced tau hyperphosphorylation and spatial memory impairment in rats; these effects do not occur via the anti-anxiety activity of escitalopram but may involve the Akt/GSK-3β signaling pathway.

DFsn collaborates with Highwire to down-regulate the Wallenda/DLK kinase and restrain synaptic terminal growth

PubMed Central

Wu, Chunlai; Daniels, Richard W; DiAntonio, Aaron

2007-01-01

Background The growth of new synapses shapes the initial formation and subsequent rearrangement of neural circuitry. Genetic studies have demonstrated that the ubiquitin ligase Highwire restrains synaptic terminal growth by down-regulating the MAP kinase kinase kinase Wallenda/dual leucine zipper kinase (DLK). To investigate the mechanism of Highwire action, we have identified DFsn as a binding partner of Highwire and characterized the roles of DFsn in synapse development, synaptic transmission, and the regulation of Wallenda/DLK kinase abundance. Results We identified DFsn as an F-box protein that binds to the RING-domain ubiquitin ligase Highwire and that can localize to the Drosophila neuromuscular junction. Loss-of-function mutants for DFsn have a phenotype that is very similar to highwire mutants – there is a dramatic overgrowth of synaptic termini, with a large increase in the number of synaptic boutons and branches. In addition, synaptic transmission is impaired in DFsn mutants. Genetic interactions between DFsn and highwire mutants indicate that DFsn and Highwire collaborate to restrain synaptic terminal growth. Finally, DFsn regulates the levels of the Wallenda/DLK kinase, and wallenda is necessary for DFsn-dependent synaptic terminal overgrowth. Conclusion The F-box protein DFsn binds the ubiquitin ligase Highwire and is required to down-regulate the levels of the Wallenda/DLK kinase and restrain synaptic terminal growth. We propose that DFsn and Highwire participate in an evolutionarily conserved ubiquitin ligase complex whose substrates regulate the structure and function of synapses. PMID:17697379

PRAK, a novel protein kinase regulated by the p38 MAP kinase.

PubMed Central

New, L; Jiang, Y; Zhao, M; Liu, K; Zhu, W; Flood, L J; Kato, Y; Parry, G C; Han, J

1998-01-01

We have identified and cloned a novel serine/ threonine kinase, p38-regulated/activated protein kinase (PRAK). PRAK is a 471 amino acid protein with 20-30% sequence identity to the known MAP kinase-regulated protein kinases RSK1/2/3, MNK1/2 and MAPKAP-K2/3. PRAK was found to be expressed in all human tissues and cell lines examined. In HeLa cells, PRAK was activated in response to cellular stress and proinflammatory cytokines. PRAK activity was regulated by p38alpha and p38beta both in vitro and in vivo and Thr182 was shown to be the regulatory phosphorylation site. Activated PRAK in turn phosphorylated small heat shock protein 27 (HSP27) at the physiologically relevant sites. An in-gel kinase assay demonstrated that PRAK is a major stress-activated kinase that can phosphorylate small heat shock protein, suggesting a potential role for PRAK in mediating stress-induced HSP27 phosphorylation in vivo. PMID:9628874

The diurnal oscillation of MAP (mitogen-activated protein) kinase and adenylyl cyclase activities in the hippocampus depends on the suprachiasmatic nucleus.

PubMed

Phan, Trongha X; Phan, Trongha H; Chan, Guy C-K; Sindreu, Carlos B; Eckel-Mahan, Kristin L; Storm, Daniel R

2011-07-20

Consolidation of hippocampus-dependent memory is dependent on activation of the cAMP/Erk/MAPK (mitogen-activated protein kinase) signal transduction pathway in the hippocampus. Recently, we discovered that adenylyl cyclase and MAPK activities undergo a circadian oscillation in the hippocampus and that inhibition of this oscillation impairs contextual memory. This suggests the interesting possibility that the persistence of hippocampus-dependent memory depends upon the reactivation of MAPK in the hippocampus during the circadian cycle. A key unanswered question is whether the circadian oscillation of this signaling pathway is intrinsic to the hippocampus or is driven by the master circadian clock in the suprachiasmatic nucleus (SCN). To address this question, we ablated the SCN of mice by electrolytic lesion and examined hippocampus-dependent memory as well as adenylyl cyclase and MAPK activities. Electrolytic lesion of the SCN 2 d after training for contextual fear memory reduced contextual memory measured 2 weeks after training, indicating that maintenance of contextual memory depends on the SCN. Spatial memory was also compromised in SCN-lesioned mice. Furthermore, the diurnal oscillation of adenylyl cyclase and MAPK activities in the hippocampus was destroyed by lesioning of the SCN. These data suggest that hippocampus-dependent long-term memory is dependent on the SCN-controlled oscillation of the adenylyl cyclase/MAPK pathway in the hippocampus.

PfPK7, an atypical MEK-related protein kinase, reflects the absence of classical three-component MAPK pathways in the human malaria parasite Plasmodium falciparum.

PubMed

Dorin, Dominique; Semblat, Jean-Philippe; Poullet, Patrick; Alano, Pietro; Goldring, J P Dean; Whittle, Christina; Patterson, Shelley; Chakrabarti, Debopam; Doerig, Christian

2005-01-01

Two members of the mitogen-activated protein kinase (MAPK) family have been previously characterized in Plasmodium falciparum, but in vitro attempts at identifying MAP kinase kinase (MAPKK) homologues have failed. Here we report the characterization of a novel plasmodial protein kinase, PfPK7, whose top scores in blastp analysis belong to the MAPKK3/6 subgroup of MAPKKs. However, homology to MAPKKs is restricted to regions of the C-terminal lobe of the kinase domain, whereas the N-terminal region is closer to fungal protein kinase A enzymes (PKA, members of the AGC group of protein kinases). Hence, PfPK7 is a 'composite' enzyme displaying regions of similarity to more than one protein kinase family, similar to a few other plasmodial protein kinases. PfPK7 is expressed in several developmental stages of the parasite, both in the mosquito vector and in the human host. Recombinant PfPK7 displayed kinase activity towards a variety of substrates, but was unable to phosphorylate the two P. falciparum MAPK homologues in vitro, and was insensitive to PKA and MEK inhibitors. Together with the absence of a typical MAPKK activation site in its T-loop, this suggests that PfPK7 is not a MAPKK orthologue, despite the fact that this enzyme is the most 'MAPKK-like' enzyme encoded in the P. falciparum genome. This is consistent with recent observations that the plasmodial MAPKs are not true orthologues of the ERK1/2, p38 or JNK MAPKs, and strengthens the evidence that classical three-component module-dependent MAPK signalling pathways do not operate in malaria parasites, a feature that has not been described in any other eukaryote.

Computation of restoration of ligand response in the random kinetics of a prostate cancer cell signaling pathway.

PubMed

Dana, Saswati; Nakakuki, Takashi; Hatakeyama, Mariko; Kimura, Shuhei; Raha, Soumyendu

2011-01-01

Mutation and/or dysfunction of signaling proteins in the mitogen activated protein kinase (MAPK) signal transduction pathway are frequently observed in various kinds of human cancer. Consistent with this fact, in the present study, we experimentally observe that the epidermal growth factor (EGF) induced activation profile of MAP kinase signaling is not straightforward dose-dependent in the PC3 prostate cancer cells. To find out what parameters and reactions in the pathway are involved in this departure from the normal dose-dependency, a model-based pathway analysis is performed. The pathway is mathematically modeled with 28 rate equations yielding those many ordinary differential equations (ODE) with kinetic rate constants that have been reported to take random values in the existing literature. This has led to us treating the ODE model of the pathways kinetics as a random differential equations (RDE) system in which the parameters are random variables. We show that our RDE model captures the uncertainty in the kinetic rate constants as seen in the behavior of the experimental data and more importantly, upon simulation, exhibits the abnormal EGF dose-dependency of the activation profile of MAP kinase signaling in PC3 prostate cancer cells. The most likely set of values of the kinetic rate constants obtained from fitting the RDE model into the experimental data is then used in a direct transcription based dynamic optimization method for computing the changes needed in these kinetic rate constant values for the restoration of the normal EGF dose response. The last computation identifies the parameters, i.e., the kinetic rate constants in the RDE model, that are the most sensitive to the change in the EGF dose response behavior in the PC3 prostate cancer cells. The reactions in which these most sensitive parameters participate emerge as candidate drug targets on the signaling pathway. 2011 Elsevier Ireland Ltd. All rights reserved.

TAO kinases mediate activation of p38 in response to DNA damage

PubMed Central

Raman, Malavika; Earnest, Svetlana; Zhang, Kai; Zhao, Yingming; Cobb, Melanie H

2007-01-01

Thousand and one amino acid (TAO) kinases are Ste20p-related MAP kinase kinase kinases (MAP3Ks) that activate p38 MAPK. Here we show that the TAO kinases mediate the activation of p38 in response to various genotoxic stimuli. TAO kinases are activated acutely by ionizing radiation, ultraviolet radiation, and hydroxyurea. Full-length and truncated fragments of dominant negative TAOs inhibit the activation of p38 by DNA damage. Inhibition of TAO expression by siRNA also decreases p38 activation by these agents. Cells in which TAO kinases have been knocked down are less capable of engaging the DNA damage-induced G2/M checkpoint and display increased sensitivity to IR. The DNA damage kinase ataxia telangiectasia mutated (ATM) phosphorylates TAOs in vitro; radiation induces phosphorylation of TAO on a consensus site for phosphorylation by the ATM protein kinase in cells; and TAO and p38 activation is compromised in cells from a patient with ataxia telangiectasia that lack ATM. These findings indicate that TAO kinases are regulators of p38-mediated responses to DNA damage and are intermediates in the activation of p38 by ATM. PMID:17396146

Assessment of the Role of MAP Kinase in Mediating Activity-Dependent Transcriptional Activation of the Immediate Early Gene "Arc/Arg3.1" in the Dentate Gyrus in Vivo

ERIC Educational Resources Information Center

Chotiner, Jennifer K.; Nielson, Jessica; Farris, Shannon; Lewandowski, Gail; Huang, Fen; Banos, Karla; de Leon, Ray; Steward, Oswald

2010-01-01

Different physiological and behavioral events activate transcription of "Arc/Arg3.1" in neurons in vivo, but the signal transduction pathways that mediate induction in particular situations remain to be defined. Here, we explore the relationships between induction of "Arc/Arg3.1" transcription in dentate granule cells in vivo and activation of…

Phosphoproteomic characterization of DNA damage response in melanoma cells following MEK/PI3K dual inhibition.

PubMed

Kirkpatrick, Donald S; Bustos, Daisy J; Dogan, Taner; Chan, Jocelyn; Phu, Lilian; Young, Amy; Friedman, Lori S; Belvin, Marcia; Song, Qinghua; Bakalarski, Corey E; Hoeflich, Klaus P

2013-11-26

Targeted therapeutics that block signal transduction through the RAS-RAF-MEK and PI3K-AKT-mTOR pathways offer significant promise for the treatment of human malignancies. Dual inhibition of MAP/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) with the potent and selective small-molecule inhibitors GDC-0973 and GDC-0941 has been shown to trigger tumor cell death in preclinical models. Here we have used phosphomotif antibodies and mass spectrometry (MS) to investigate the effects of MEK/PI3K dual inhibition during the period immediately preceding cell death. Upon treatment, melanoma cell lines responded by dramatically increasing phosphorylation on proteins containing a canonical DNA damage-response (DDR) motif, as defined by a phosphorylated serine or threonine residue adjacent to glutamine, [s/t]Q. In total, >2,000 [s/t]Q phosphorylation sites on >850 proteins were identified by LC-MS/MS, including an extensive network of DDR proteins. Linear mixed-effects modeling revealed 101 proteins in which [s/t]Q phosphorylation was altered significantly in response to GDC-0973/GDC-0941. Among the most dramatic changes, we observed rapid and sustained phosphorylation of sites within the ABCDE cluster of DNA-dependent protein kinase. Preincubation of cells with the inhibitors of the DDR kinases DNA-dependent protein kinase or ataxia-telangiectasia mutated enhanced GDC-0973/GDC-0941-mediated cell death. Network analysis revealed specific enrichment of proteins involved in RNA metabolism along with canonical DDR proteins and suggested a prominent role for this pathway in the response to MEK/PI3K dual inhibition.

Phosphoproteomic characterization of DNA damage response in melanoma cells following MEK/PI3K dual inhibition

PubMed Central

Kirkpatrick, Donald S.; Bustos, Daisy J.; Dogan, Taner; Chan, Jocelyn; Phu, Lilian; Young, Amy; Friedman, Lori S.; Belvin, Marcia; Song, Qinghua; Bakalarski, Corey E.; Hoeflich, Klaus P.

2013-01-01

Targeted therapeutics that block signal transduction through the RAS–RAF–MEK and PI3K–AKT–mTOR pathways offer significant promise for the treatment of human malignancies. Dual inhibition of MAP/ERK kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) with the potent and selective small-molecule inhibitors GDC-0973 and GDC-0941 has been shown to trigger tumor cell death in preclinical models. Here we have used phosphomotif antibodies and mass spectrometry (MS) to investigate the effects of MEK/PI3K dual inhibition during the period immediately preceding cell death. Upon treatment, melanoma cell lines responded by dramatically increasing phosphorylation on proteins containing a canonical DNA damage-response (DDR) motif, as defined by a phosphorylated serine or threonine residue adjacent to glutamine, [s/t]Q. In total, >2,000 [s/t]Q phosphorylation sites on >850 proteins were identified by LC-MS/MS, including an extensive network of DDR proteins. Linear mixed-effects modeling revealed 101 proteins in which [s/t]Q phosphorylation was altered significantly in response to GDC-0973/GDC-0941. Among the most dramatic changes, we observed rapid and sustained phosphorylation of sites within the ABCDE cluster of DNA-dependent protein kinase. Preincubation of cells with the inhibitors of the DDR kinases DNA-dependent protein kinase or ataxia-telangiectasia mutated enhanced GDC-0973/GDC-0941–mediated cell death. Network analysis revealed specific enrichment of proteins involved in RNA metabolism along with canonical DDR proteins and suggested a prominent role for this pathway in the response to MEK/PI3K dual inhibition. PMID:24218548

Phosphorylation of rat liver heterogeneous nuclear ribonucleoproteins A2 and C can be modulated by calmodulin.

PubMed Central

Bosser, R; Faura, M; Serratosa, J; Renau-Piqueras, J; Pruschy, M; Bachs, O

1995-01-01

It was previously reported that the phosphorylation of three proteins of 36, 40 to 42, and 50 kDa by casein kinase 2 is inhibited by calmodulin in nuclear extracts from rat liver cells (R. Bosser, R. Aligué, D. Guerini, N. Agell, E. Carafoli, and O. Bachs, J. Biol. Chem. 268:15477-15483, 1993). By immunoblotting, peptide mapping, and endogenous phosphorylation experiments, the 36- and 40- to 42-kDa proteins have been identified as the A2 and C proteins, respectively, of the heterogeneous nuclear ribonucleoprotein particles. To better understand the mechanism by which calmodulin inhibits the phosphorylation of these proteins, they were purified by using single-stranded DNA chromatography, and the effect of calmodulin on their phosphorylation by casein kinase 2 was analyzed. Results revealed that whereas calmodulin inhibited the phosphorylation of purified A2 and C proteins in a Ca(2+)-dependent manner, it did not affect the casein kinase 2 phosphorylation of a different protein substrate, i.e., beta-casein. These results indicate that the effect of calmodulin was not on casein kinase 2 activity but on specific protein substrates. The finding that the A2 and C proteins can bind to a calmodulin-Sepharose column in a Ca(2+)-dependent manner suggests that this association could prevent the phosphorylation of the proteins by casein kinase 2. Immunoelectron microscopy studies have revealed that such interactions could also occur in vivo, since calmodulin and A2 and C proteins colocalize on the ribonucleoprotein particles in rat liver cell nuclei. PMID:7823935

Down-Regulation by Resveratrol of Basic Fibroblast Growth Factor-Stimulated Osteoprotegerin Synthesis through Suppression of Akt in Osteoblasts

PubMed Central

Kuroyanagi, Gen; Otsuka, Takanobu; Yamamoto, Naohiro; Matsushima-Nishiwaki, Rie; Nakakami, Akira; Mizutani, Jun; Kozawa, Osamu; Tokuda, Haruhiko

2014-01-01

It is firmly established that resveratrol, a natural food compound abundantly found in grape skins and red wine, has beneficial properties for human health. In the present study, we investigated the effect of basic fibroblast growth factor (FGF-2) on osteoprotegerin (OPG) synthesis in osteoblast-like MC3T3-E1 cells and whether resveratrol affects the OPG synthesis. FGF-2 stimulated both the OPG release and the expression of OPG mRNA. Resveratrol significantly suppressed the FGF-2-stimulated OPG release and the mRNA levels of OPG. SRT1720, an activator of SIRT1, reduced the FGF-2-induced OPG release and the OPG mRNA expression. PD98059, an inhibitor of upstream kinase activating p44/p42 mitogen-activated protein (MAP) kinase, had little effect on the FGF-2-stimulated OPG release. On the other hand, SB203580, an inhibitor of p38 MAP kinase, SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), and Akt inhibitor suppressed the OPG release induced by FGF-2. Resveratrol failed to affect the FGF-2-induced phosphorylation of p44/p42 MAP kinase, p38 MAP kinase or SAPK/JNK. The phosphorylation of Akt induced by FGF-2 was significantly suppressed by resveratrol or SRT1720. These findings strongly suggest that resveratrol down-regulates FGF-2-stimulated OPG synthesis through the suppression of the Akt pathway in osteoblasts and that the inhibitory effect of resveratrol is mediated at least in part by SIRT1 activation. PMID:25290095

Does the Loss of Stromal Caveolin-1 Remodel the Tumor Microenvironment by Activating Src-Mediated PEAK1 and PI3K Pathways

DTIC Science & Technology

2016-09-01

Inhibition of MAP kinase pathway prevents plasma protrusions Next we used a selective inhibitor of MAP kinases , PD98059, to address whether we can...from human patients harbor AKT1 and that AKT1 kinase activity is sustained in these particles, nominating them as active signaling platforms...with the extracellular matrix (ECM) and extracellular molecules (2). Though many classic extracellular signaling molecules (e.g., hormones, peptide

The Carboxyl Terminus of v-Abl Protein Can Augment SH2 Domain Function

PubMed Central

Warren, David; Heilpern, Andrew J.; Berg, Kent; Rosenberg, Naomi

2000-01-01

Abelson murine leukemia virus (Ab-MLV) transforms NIH 3T3 and pre-B cells via expression of the v-Abl tyrosine kinase. Although the enzymatic activity of this molecule is absolutely required for transformation, other regions of the protein are also important for this response. Among these are the SH2 domain, involved in phosphotyrosine-dependent protein-protein interactions, and the long carboxyl terminus, which plays an important role in transformation of hematopoietic cells. Important signals are sent from each of these regions, and transformation is most likely orchestrated by the concerted action of these different parts of the protein. To explore this idea, we compared the ability of the v-Src SH2 domain to substitute for that of v-Abl in the full-length P120 v-Abl protein and in P70 v-Abl, a protein that lacks the carboxyl terminus characteristic of Abl family members. Ab-MLV strains expressing P70/S2 failed to transform NIH 3T3 cells and demonstrated a greatly reduced capacity to mediate signaling events associated with the Ras-dependent mitogen-activated protein (MAP) kinase pathway. In contrast, Ab-MLV strains expressing P120/S2 were indistinguishable from P120 with respect to these features. Analyses of additional mutants demonstrated that the last 162 amino acids of the carboxyl terminus were sufficient to restore transformation. These data demonstrate that an SH2 domain with v-Abl substrate specificity is required for NIH 3T3 transformation in the absence of the carboxyl terminus and suggest that cooperativity between the extreme carboxyl terminus and the SH2 domain facilitates the transmission of transforming signals via the MAP kinase pathway. PMID:10775585

Cloning and characterization of the pheromone biosynthesis activating neuropeptide receptor gene in Spodoptera littoralis larvae.

PubMed

Zheng, Lei; Lytle, Christian; Njauw, Ching-Ni; Altstein, Miriam; Martins-Green, Manuela

2007-05-15

In noctuid moths cuticular pigmentation is regulated by the pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) family, which also mediates a variety of other functions in moths and other insects. Numerous studies have shown that these neuropeptides exert their functions through activation of the PBAN receptor (PBAN-R), with subsequent Ca(2+) influx, followed by either activation of cAMP or direct activation of downstream kinases. Recently, several PBAN-Rs have been identified, all of which are from the pheromone gland of adult female moths, but evidence shows that functional PK/PBAN-Rs can also be expressed in insect larvae, where they mediate melanization and possibly other functions (e.g., diapause). Here, we identified a gene encoding a G-protein-coupled receptor from the 5th instar larval tissue of the moth Spodoptera littoralis. The cDNA of this gene contains an open reading frame with a length of 1050 nucleotides, which translates to a 350-amino acid, 42-kDa protein that shares 92% amino acid identity with Helicoverpa zea and Helicoverpa armigera PBAN-R, 81% with Bombyx mori PBAN-R and 72% with Plutella xylostella PBAN-R. The S. littoralis PBAN-R gene was stably expressed in NIH3T3 cells and transiently in HEK293 cells. We show that it mediates the dose-dependent PBAN-induced intracellular Ca(2+) response and activation of the MAP kinase via a PKC-dependent but Galphai-independent signaling mechanism. Other PK/PBAN family peptides (pheromonotropin and a C-terminally PBAN-derived peptide PBAN(28-33)NH(2)) also triggered MAP kinase activation. This receptor, together with the previously cloned PBAN-R, may facilitate our understanding of the cell-specific responses and functional diversities of this diverse neuropeptide family.

An atlas of the human kinome reveals the mutational landscape underlying dysregulated phosphorylation cascades in cancer

PubMed Central

Olow, Aleksandra; Chen, Zhongzhong; Niedner, R. Hannes; Wolf, Denise M.; Yau, Christina; Pankov, Aleksandr; Lee, Evelyn Pei Rong; Brown-Swigart, Lamorna; van’t Veer, Laura J.; Coppé, Jean-Philippe

2016-01-01

Kinase inhibitors are used widely to treat various cancers, but adaptive reprogramming of kinase cascades and activation of feedback loop mechanisms often contribute to therapeutic resistance. Determining comprehensive, accurate maps of kinase circuits may therefore help elucidate mechanisms of response and resistance to kinase inhibitor therapies. In this study, we identified and validated phosphorylatable target sites across human cell and tissue types to generate PhosphoAtlas, a map of 1,733 functionally interconnected proteins comprising the human phospho-reactome. A systematic curation approach was used to distill protein phosphorylation data cross-referenced from 38 public resources. We demonstrated how a catalog of 2,617 stringently verified heptameric peptide regions at the catalytic interface of kinases and substrates could expose mutations that recurrently perturb specific phospho-hubs. In silico mapping of 2,896 nonsynonymous tumor variants identified from thousands of tumor tissues, also revealed that normal and aberrant catalytic interactions co-occur frequently, showing how tumors systematically hijack, as well as spare, particular sub-networks. Overall, our work provides an important new resource for interrogating the human tumor kinome to strategically identify therapeutically actionable kinase networks which drive tumorigenesis. PMID:26921330

Single-well monitoring of protein-protein interaction and phosphorylation-dephosphorylation events.

PubMed

Arcand, Mathieu; Roby, Philippe; Bossé, Roger; Lipari, Francesco; Padrós, Jaime; Beaudet, Lucille; Marcil, Alexandre; Dahan, Sophie

2010-04-20

We combined oxygen channeling assays with two distinct chemiluminescent beads to detect simultaneously protein phosphorylation and interaction events that are usually monitored separately. This novel method was tested in the ERK1/2 MAP kinase pathway. It was first used to directly monitor dissociation of MAP kinase ERK2 from MEK1 upon phosphorylation and to evaluate MAP kinase phosphatase (MKP) selectivity and mechanism of action. In addition, MEK1 and ERK2 were probed with an ATP competitor and an allosteric MEK1 inhibitor, which generated distinct phosphorylation-interaction patterns. Simultaneous monitoring of protein-protein interactions and substrate phosphorylation can provide significant mechanistic insight into enzyme activity and small molecule action.

Direct integrin alphavbeta6-ERK binding: implications for tumour growth.

PubMed

Ahmed, Nuzhat; Niu, Jun; Dorahy, Douglas J; Gu, Xinhua; Andrews, Sarah; Meldrum, Cliff J; Scott, Rodney J; Baker, Mark S; Macreadie, Ian G; Agrez, Michael V

2002-02-21

Blockade of the mitogen-activated protein (MAP) kinase pathway suppresses growth of colon cancer in vivo. Here we demonstrate a direct link between the extracellular signal-regulated kinase ERK2 and the growth-promoting cell adhesion molecule, integrin alphavbeta6, in colon cancer cells. Down-regulation of beta6 integrin subunit expression inhibits tumour growth in vivo and MAP kinase activity in response to serum stimulation. In alphavbeta6-expressing cells ERK2 is bound only to the beta6 subunit. The increase in cytosolic MAP kinase activity upon epidermal growth factor stimulation is all accounted for by beta6-bound ERK. Deletion of the ERK2 binding site on the beta6 cytoplasmic domain inhibits tumour growth and leads to an association between ERK and the beta5 subunit. The physical interaction between integrin alphavbeta6 and ERK2 defines a novel paradigm of integrin-mediated signalling and provides a therapeutic target for cancer treatment.

Molecular pathways of platelet factor 4/CXCL4 signaling.

PubMed

Kasper, Brigitte; Petersen, Frank

2011-01-01

The platelet-derived chemokine CXCL4 takes a specific and unique position within the family of chemotactic cytokines. Today, much attention is directed to CXCL4's capacity to inhibit angiogenesis and to promote innate immune responses, which makes this chemokine an interesting tool and target for potential intervention in tumor growth and inflammation. However, such attempts demand a comprehensive knowledge on the molecular mechanisms and pathways underlying the corresponding cellular functions. At least two structurally different receptors, CXCR3-B and a chondroitin sulfate proteoglycan, are capable of binding CXCL4 and to induce a specific intracellular signaling machinery. While signaling mediated by CXCR3-B involves Gs proteins, elevated cAMP levels, and p38 MAP kinase, signaling via proteoglycans appears to be more complicated and varies strongly between the cell types analyzed. In CXCL4-activated neutrophils and monocytes, tyrosine kinases of the Src family and Syk as well as monomeric GTPases and members of the MAP kinase family have been identified as essential intracellular signals. Most intriguingly, signaling does not proceed in a linear sequence of events but in a repeated activation of certain transducing elements like Rac2 or sphingosine kinase 1. Depending on the downstream targets, such biphasic kinetics either leads to a redundant and prolonged activation of a single pathway or to a timely separated initiation of disparate signals and functions. Results of the studies reviewed here help to understand the molecular basis of CXCL4's functional diversity and provide insights into integrated signaling processes in general. Copyright © 2011 Elsevier GmbH. All rights reserved.

Mitogen-activated protein kinase is required for the behavioral desensitization that occurs after repeated injections of angiotensin II

PubMed Central

Vento, Peter J.; Daniels, Derek

2013-01-01

Angiotensin II (AngII) acts on central angiotensin type 1 (AT1) receptors to increase water and saline intake. Prolonged exposure to AngII in cell culture models results in a desensitization of the AT1 receptor that is thought to involve receptor internalization, and a behavioral correlate of this desensitization has been shown in rats after repeated central injections of AngII. Specifically, rats given repeated injections of AngII drink less water than controls after a subsequent test injection of AngII. Under the same conditions, however, repeated injections of AngII have no effect on AngII-induced saline intake. Given earlier studies indicating that separate intracellular signaling pathways mediate AngII-induced water and saline intake, we hypothesized that the desensitization observed in rats may be incomplete, leaving the receptor able to activate mitogen-activated protein (MAP) kinases (ERK1/2), which play a role in AngII-induced saline intake without affecting water intake. In support of this hypothesis, we found no difference in MAP kinase phosphorylation after an AngII test injection in rats given prior treatment with repeated injections of vehicle, AngII, or Sar1,Ile4,Ile8-AngII (SII), an AngII analog that activates MAP kinase without G protein coupling. In addition, we found that pretreatment with the MAP kinase inhibitor U0126 completely blocked the desensitizing effect of repeated AngII injections on water intake. Furthermore, AngII-induced water intake was reduced similarly by repeated injections of AngII or SII. The results suggest that G protein-independent signaling is sufficient to produce behavioral desensitization of the angiotensin system and that the desensitization requires MAP kinase activation. PMID:22581747

Mitogen-activated protein kinase is required for the behavioural desensitization that occurs after repeated injections of angiotensin II.

PubMed

Vento, Peter J; Daniels, Derek

2012-12-01

Angiotensin II (Ang II) acts on central angiotensin type 1 (AT(1)) receptors to increase water and saline intake. Prolonged exposure to Ang II in cell culture models results in a desensitization of the AT(1) receptor that is thought to involve receptor internalization, and a behavioural correlate of this desensitization has been shown in rats after repeated central injections of Ang II. Specifically, rats given repeated injections of Ang II drink less water than control animals after a subsequent test injection of Ang II. In the same conditions, however, repeated injections of Ang II have no effect on Ang II-induced saline intake. Given earlier studies indicating that separate intracellular signalling pathways mediate Ang II-induced water and saline intake, we hypothesized that the desensitization observed in rats may be incomplete, leaving the receptor able to activate mitogen-activated protein (MAP) kinases (ERK1/2), which play a role in Ang II-induced saline intake without affecting water intake. In support of this hypothesis, we found no difference in MAP kinase phosphorylation after an Ang II test injection in rats given prior treatment with repeated injections of vehicle, Ang II or Sar(1),Ile(4),Ile(8)-Ang II (SII), an Ang II analogue that activates MAP kinase without G protein coupling. In addition, we found that pretreatment with the MAP kinase inhibitor U0126 completely blocked the desensitizing effect of repeated Ang II injections on water intake. Furthermore, Ang II-induced water intake was reduced to a similar extent by repeated injections of Ang II or SII. The results suggest that G protein-independent signalling is sufficient to produce behavioural desensitization of the angiotensin system and that the desensitization requires MAP kinase activation.

Mapping DNA damage-dependent genetic interactions in yeast via party mating and barcode fusion genetics.

PubMed

Díaz-Mejía, J Javier; Celaj, Albi; Mellor, Joseph C; Coté, Atina; Balint, Attila; Ho, Brandon; Bansal, Pritpal; Shaeri, Fatemeh; Gebbia, Marinella; Weile, Jochen; Verby, Marta; Karkhanina, Anna; Zhang, YiFan; Wong, Cassandra; Rich, Justin; Prendergast, D'Arcy; Gupta, Gaurav; Öztürk, Sedide; Durocher, Daniel; Brown, Grant W; Roth, Frederick P

2018-05-28

Condition-dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State-of-the-art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double-mutant strains, does not scale readily to multi-condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG-GI), by which double-mutant strains generated via en masse "party" mating can also be monitored en masse for growth to detect genetic interactions. By using site-specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG-GI enables multiplexed quantitative tracking of double mutants via next-generation sequencing. We applied BFG-GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4NQO), bleomycin, zeocin, and three other DNA-damaging environments. BFG-GI recapitulated known genetic interactions and yielded new condition-dependent genetic interactions. We validated and further explored a subnetwork of condition-dependent genetic interactions involving MAG1 , SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

HAM-5 Functions As a MAP Kinase Scaffold during Cell Fusion in Neurospora crassa

PubMed Central

Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles; Wang, Yuexi; Yang, Feng; Starr, Trevor L.; Camp, David G.; Smith, Richard D.; Glass, N. Louise

2014-01-01

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC-1, MEK-2 and MAK-2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every ∼8 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a recently identified scaffold for the MAK-1 MAP kinase pathway in Sordaria macrospora. How the MAK-2 oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) that can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK-2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM-5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM-5-GFP co-localized with NRC-1, MEK-2 and MAK-2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM-5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK-2 activity influences HAM-5 function/localization. However, MAK-2-GFP showed cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta. Via co-immunoprecipitation experiments, HAM-5 was shown to physically interact with NRC-1, MEK-2 and MAK-2, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members for oscillation and chemotropic interactions during germling and hyphal fusion in N. crassa. The identification of HAM-5 as a scaffold-like protein will help to link the activation of MAK-2 cascade to upstream factors and proteins involved in this intriguing process of fungal communication. PMID:25412208

The X-linked juvenile retinoschisis protein retinoschisin is a novel regulator of mitogen-activated protein kinase signalling and apoptosis in the retina.

PubMed

Plössl, Karolina; Weber, Bernhard H F; Friedrich, Ulrike

2017-04-01

X-linked juvenile retinoschisis (XLRS) is a hereditary retinal dystrophy in young males, caused by mutations in the RS1 gene. The function of the encoded protein, termed retinoschisin, and the molecular mechanisms underlying XLRS pathogenesis are still unresolved, although a direct interaction partner of the secreted retinoschisin, the retinal Na/K-ATPase, was recently identified. Earlier gene expression studies in retinoschisin-deficient (Rs1h -/Y ) mice provided a first indication of pathological up-regulation of mitogen-activated protein (MAP) kinase signalling in disease pathogenesis. To further investigate the role for retinoschisin in MAP kinase regulation, we exposed Y-79 cells and murine Rs1h -/Y retinae to recombinant retinoschisin and the XLRS-associated mutant RS1-C59S. Although normal retinoschisin stably bound to retinal cells, RS1-C59S exhibited a strongly reduced binding affinity. Simultaneously, exposure to normal retinoschisin significantly reduced phosphorylation of C-RAF and MAP kinases ERK1/2 in Y-79 cells and murine Rs1h -/Y retinae. Expression of MAP kinase target genes C-FOS and EGR1 was also down-regulated in both model systems. Finally, retinoschisin treatment decreased pro-apoptotic BAX-2 transcript levels in Y-79 cells and Rs1h -/Y retinae. Upon retinoschisin treatment, these cells showed increased resistance against apoptosis, reflected by decreased caspase-3 activity (in Y-79 cells) and increased photoreceptor survival (in Rs1h -/Y retinal explants). RS1-C59S did not influence C-RAF or ERK1/2 activation, C-FOS or EGR1 expression, or apoptosis. Our data imply that retinoschisin is a novel regulator of MAP kinase signalling and exerts an anti-apoptotic effect on retinal cells. We therefore discuss that disturbances of MAP kinase signalling by retinoschisin deficiency could be an initial step in XLRS pathogenesis. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Mitogen-Activated Protein Kinase 14 Promotes AKI

PubMed Central

Husi, Holger; Gonzalez-Lafuente, Laura; Valiño-Rivas, Lara; Fresno, Manuel; Sanz, Ana Belen; Mullen, William; Albalat, Amaya; Mezzano, Sergio; Vlahou, Tonia; Mischak, Harald

2017-01-01

An improved understanding of pathogenic pathways in AKI may identify novel therapeutic approaches. Previously, we conducted unbiased liquid chromatography-tandem mass spectrometry–based protein expression profiling of the renal proteome in mice with acute folate nephropathy. Here, analysis of the dataset identified enrichment of pathways involving NFκB in the kidney cortex, and a targeted data mining approach identified components of the noncanonical NFκB pathway, including the upstream kinase mitogen-activated protein kinase kinase kinase 14 (MAP3K14), the NFκB DNA binding heterodimer RelB/NFκB2, and proteins involved in NFκB2 p100 ubiquitination and proteasomal processing to p52, as upregulated. Immunohistochemistry localized MAP3K14 expression to tubular cells in acute folate nephropathy and human AKI. In vivo, kidney expression levels of NFκB2 p100 and p52 increased rapidly after folic acid injection, as did DNA binding of RelB and NFκB2, detected in nuclei isolated from the kidneys. Compared with wild-type mice, MAP3K14 activity–deficient aly/aly (MAP3K14aly/aly) mice had less kidney dysfunction, inflammation, and apoptosis in acute folate nephropathy and less kidney dysfunction and a lower mortality rate in cisplatin-induced AKI. The exchange of bone marrow between wild-type and MAP3K14aly/aly mice did not affect the survival rate of either group after folic acid injection. In cultured tubular cells, MAP3K14 small interfering RNA targeting decreased inflammation and cell death. Additionally, cell culture and in vivo studies identified the chemokines MCP-1, RANTES, and CXCL10 as MAP3K14 targets in tubular cells. In conclusion, MAP3K14 promotes kidney injury through promotion of inflammation and cell death and is a promising novel therapeutic target. PMID:27620989

Aminopyridine-based c-Jun N-terminal kinase inhibitors with cellular activity and minimal cross-kinase activity.

PubMed

Szczepankiewicz, Bruce G; Kosogof, Christi; Nelson, Lissa T J; Liu, Gang; Liu, Bo; Zhao, Hongyu; Serby, Michael D; Xin, Zhili; Liu, Mei; Gum, Rebecca J; Haasch, Deanna L; Wang, Sanyi; Clampit, Jill E; Johnson, Eric F; Lubben, Thomas H; Stashko, Michael A; Olejniczak, Edward T; Sun, Chaohong; Dorwin, Sarah A; Haskins, Kristi; Abad-Zapatero, Cele; Fry, Elizabeth H; Hutchins, Charles W; Sham, Hing L; Rondinone, Cristina M; Trevillyan, James M

2006-06-15

The c-Jun N-terminal kinases (JNK-1, -2, and -3) are members of the mitogen activated protein (MAP) kinase family of enzymes. They are activated in response to certain cytokines, as well as by cellular stresses including chemotoxins, peroxides, and irradiation. They have been implicated in the pathology of a variety of different diseases with an inflammatory component including asthma, stroke, Alzheimer's disease, and type 2 diabetes mellitus. In this work, high-throughput screening identified a JNK inhibitor with an excellent kinase selectivity profile. Using X-ray crystallography and biochemical screening to guide our lead optimization, we prepared compounds with inhibitory potencies in the low-double-digit nanomolar range, activity in whole cells, and pharmacokinetics suitable for in vivo use. The new compounds were over 1,000-fold selective for JNK-1 and -2 over other MAP kinases including ERK2, p38alpha, and p38delta and showed little inhibitory activity against a panel of 74 kinases.

Pro-life role for c-Jun N-terminal kinase and p38 mitogen-activated protein kinase at rostral ventrolateral medulla in experimental brain stem death

PubMed Central

2012-01-01

Background Based on an experimental brain stem death model, we demonstrated previously that activation of the mitogen-activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ mitogen-activated protein kinase signal-interacting kinase 1/2 (MNK1/2) cascade plays a pro-life role in the rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from systemic arterial pressure, which sequentially increases (pro-life) and decreases (pro-death) to reflect progressive dysfunction of central cardiovascular regulation during the advancement towards brain stem death in critically ill patients. The present study assessed the hypothesis that, in addition to ERK1/2, c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), the other two mammalian members of MAPKs that are originally identified as stress-activated protein kinases, are activated specifically by MAPK kinase 4 (MAP2K4) or MAP2K6 and play a pro-life role in RVLM during experimental brain stem death. We further delineated the participation of phosphorylating activating transcriptional factor-2 (ATF-2) and c-Jun, the classical transcription factor activated by JNK or p38MAPK, in this process. Results An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol) bilaterally into RVLM of Sprague–Dawley rats was used, alongside cardiovascular, pharmacological and biochemical evaluations. Results from ELISA showed that whereas the total JNK, p38MAPK, MAP2K4 and MAP2K6 were not affected, augmented phosphorylation of JNK at Thr183 and Tyr185 and p38MAPK at Thr180 and Tyr182, accompanied by phosphorylation of their upstream activators MAP2K4 at Ser257 and Thr261 and MAP2K6 at Ser207 and Thr211 in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Moreover, the activity of transcription factors ATF-2 at Thr71 and c-Jun at Ser73, rather than Elk-1 at Ser383 in RVLM were also augmented during the pro-life phase. Furthermore, pretreatment by microinjection into the bilateral RVLM of specific JNK inhibitors, JNK inhibitor I (100 pmol) or SP600125 (5 pmol), or specific p38MAPK inhibitors, p38MAPK inhibitor III (500 pmol) or SB203580 (2 nmol), exacerbated the depressor effect and blunted the augmented life-and-death signal exhibited during the pro-life phase. On the other hand, pretreatment with the negative control for JNK or p38MAPK inhibitor, JNK inhibitor I negative control (100 pmol) or SB202474 (2 nmol), was ineffective in the vehicle-controls and Mev-treatment groups. Conclusions Our results demonstrated that activation of JNK or p38MAPK in RVLM by their upstream activators MAP2K4 or MAP2K6 plays a preferential pro-life role by sustaining the central cardiovascular regulatory machinery during experimental brain stem death via phosphorylation and activation of nuclear transcription factor ATF-2 or c-Jun. PMID:23157661

Cardiac effects of 3-iodothyronamine: a new aminergic system modulating cardiac function.

PubMed

Chiellini, Grazia; Frascarelli, Sabina; Ghelardoni, Sandra; Carnicelli, Vittoria; Tobias, Sandra C; DeBarber, Andrea; Brogioni, Simona; Ronca-Testoni, Simonetta; Cerbai, Elisabetta; Grandy, David K; Scanlan, Thomas S; Zucchi, Riccardo

2007-05-01

3-Iodothyronamine T1AM is a novel endogenous thyroid hormone derivative that activates the G protein-coupled receptor known as trace anime-associated receptor 1 (TAAR1). In the isolated working rat heart and in rat cardiomyocytes, T1AM produced a reversible, dose-dependent negative inotropic effect (e.g., 27+/-5, 51+/-3, and 65+/-2% decrease in cardiac output at 19, 25, and 38 microM concentration, respectively). An independent negative chronotropic effect was also observed. The hemodynamic effects of T1AM were remarkably increased in the presence of the tyrosine kinase inhibitor genistein, whereas they were attenuated in the presence of the tyrosine phosphatase inhibitor vanadate. No effect was produced by inhibitors of protein kinase A, protein kinase C, calcium-calmodulin kinase II, phosphatidylinositol-3-kinase, or MAP kinases. Tissue cAMP levels were unchanged. In rat ventricular tissue, Western blot experiments with antiphosphotyrosine antibodies showed reduced phosphorylation of microsomal and cytosolic proteins after perfusion with synthetic T1AM; reverse transcriptase-polymerase chain reaction experiments revealed the presence of transcripts for at least 5 TAAR subtypes; specific and saturable binding of [125I]T1AM was observed, with a dissociation constant in the low micromolar range (5 microM); and endogenous T1AM was detectable by tandem mass spectrometry. In conclusion, our findings provide evidence for the existence of a novel aminergic system modulating cardiac function.

An Extracellular Subtilase Switch for Immune Priming in Arabidopsis

PubMed Central

Mauch-Mani, Brigitte; Gil, Ma José; Vera, Pablo

2013-01-01

In higher eukaryotes, induced resistance associates with acquisition of a priming state of the cells for a more effective activation of innate immunity; however, the nature of the components for mounting this type of immunological memory is not well known. We identified an extracellular subtilase from Arabidopsis, SBT3.3, the overexpression of which enhances innate immune responses while the loss of function compromises them. SBT3.3 expression initiates a durable autoinduction mechanism that promotes chromatin remodeling and activates a salicylic acid(SA)-dependent mechanism of priming of defense genes for amplified response. Moreover, SBT3.3 expression-sensitized plants for enhanced expression of the OXI1 kinase gene and activation of MAP kinases following pathogen attack, providing additional clues for the regulation of immune priming by SBT3.3. Conversely, in sbt3.3 mutant plants pathogen-mediated induction of SA-related defense gene expression is drastically reduced and activation of MAP kinases inhibited. Moreover, chromatin remodeling of defense-related genes normally associated with activation of an immune priming response appear inhibited in sbt3.3 plants, further indicating the importance of the extracellular SBT3.3 subtilase in the establishment of immune priming. Our results also point to an epigenetic control in the regulation of plant immunity, since SBT3.3 is up-regulated and priming activated when epigenetic control is impeded. SBT3.3 represents a new regulator of primed immunity. PMID:23818851

An extracellular subtilase switch for immune priming in Arabidopsis.

PubMed

Ramírez, Vicente; López, Ana; Mauch-Mani, Brigitte; Gil, Ma José; Vera, Pablo

2013-01-01

In higher eukaryotes, induced resistance associates with acquisition of a priming state of the cells for a more effective activation of innate immunity; however, the nature of the components for mounting this type of immunological memory is not well known. We identified an extracellular subtilase from Arabidopsis, SBT3.3, the overexpression of which enhances innate immune responses while the loss of function compromises them. SBT3.3 expression initiates a durable autoinduction mechanism that promotes chromatin remodeling and activates a salicylic acid(SA)-dependent mechanism of priming of defense genes for amplified response. Moreover, SBT3.3 expression-sensitized plants for enhanced expression of the OXI1 kinase gene and activation of MAP kinases following pathogen attack, providing additional clues for the regulation of immune priming by SBT3.3. Conversely, in sbt3.3 mutant plants pathogen-mediated induction of SA-related defense gene expression is drastically reduced and activation of MAP kinases inhibited. Moreover, chromatin remodeling of defense-related genes normally associated with activation of an immune priming response appear inhibited in sbt3.3 plants, further indicating the importance of the extracellular SBT3.3 subtilase in the establishment of immune priming. Our results also point to an epigenetic control in the regulation of plant immunity, since SBT3.3 is up-regulated and priming activated when epigenetic control is impeded. SBT3.3 represents a new regulator of primed immunity.

CERAMIDE SYNTHASE 1 IS REGULATED BY PROTEASOMAL MEDIATED TURNOVER

PubMed Central

Sridevi, Priya; Alexander, Hannah; Laviad, Elad L.; Pewzner-Jung, Yael; Hannink, Mark; Futerman, Anthony H.; Alexander, Stephen

2009-01-01

Ceramide is an important bioactive lipid, intimately involved in many cellular functions, including the regulation of cell death, and in cancer and chemotherapy. Ceramide is synthesized de novo from sphinganine and acyl CoA via a family of 6 ceramide synthase enzymes, each having a unique preference for different fatty acyl CoA substrates and a unique tissue distribution. However, little is known regarding the regulation of these important enzymes. In this study we focus on ceramide synthase 1 (CerS1) which is the most structurally and functionally distinct of the enzymes, and describe a regulatory mechanism that specifically controls the level of CerS1 via ubiquitination and proteasome dependent protein turnover. We show that both endogenous and ectopically expressed CerS1 have rapid basal turnover and that diverse stresses including chemotherapeutic drugs, UV light and DTT can induce CerS1 turnover. The turnover requires CerS1 activity and is regulated by the opposing actions of p38 MAP kinase and protein kinase C (PKC). p38 MAP kinase is a positive regulator of turnover, while PKC is a negative regulator of turnover. CerS1 is phosphorylated in vivo and activation of PKC increases the phosphorylation of the protein. This study reveals a novel and highly specific mechanism by which CerS1 protein levels are regulated and which directly impacts ceramide homeostasis. PMID:19393694

The HOG pathway controls osmotic regulation of transcription via the stress response element (STRE) of the Saccharomyces cerevisiae CTT1 gene.

PubMed Central

Schüller, C; Brewster, J L; Alexander, M R; Gustin, M C; Ruis, H

1994-01-01

The HOG signal pathway of the yeast Saccharomyces cerevisiae is defined by the PBS2 and HOG1 genes encoding members of the MAP kinase kinase and of the MAP kinase family, respectively. Mutations in this pathway (deletions of PBS2 or HOG1, or point mutations in HOG1) almost completely abolish the induction of transcription by osmotic stress that is mediated by stress response elements (STREs). We have demonstrated previously that STREs also mediate induction of transcription by heat shock, nitrogen starvation and oxidative stress. This study shows that they are also activated by low external pH, sorbate, benzoate or ethanol stress. Induction by these other stress signals appears to be HOG pathway independent. HOG1-dependent osmotic induction of transcription of the CTT1 gene encoding the cytosolic catalase T occurs in the presence of a protein synthesis inhibitor and can be detected rapidly after an increase of tyrosine phosphorylation of Hog1p triggered by high osmolarity. Consistent with a role of STREs in the induction of stress resistance, a number of other stress protein genes (e.g. HSP104) are regulated like CTT1. Furthermore, catalase T was shown to be important for viability under severe osmotic stress, and heat shock was demonstrated to provide cross-protection against osmotic stress. Images PMID:7523111

The HOG pathway controls osmotic regulation of transcription via the stress response element (STRE) of the Saccharomyces cerevisiae CTT1 gene.

PubMed

Schüller, C; Brewster, J L; Alexander, M R; Gustin, M C; Ruis, H

1994-09-15

The HOG signal pathway of the yeast Saccharomyces cerevisiae is defined by the PBS2 and HOG1 genes encoding members of the MAP kinase kinase and of the MAP kinase family, respectively. Mutations in this pathway (deletions of PBS2 or HOG1, or point mutations in HOG1) almost completely abolish the induction of transcription by osmotic stress that is mediated by stress response elements (STREs). We have demonstrated previously that STREs also mediate induction of transcription by heat shock, nitrogen starvation and oxidative stress. This study shows that they are also activated by low external pH, sorbate, benzoate or ethanol stress. Induction by these other stress signals appears to be HOG pathway independent. HOG1-dependent osmotic induction of transcription of the CTT1 gene encoding the cytosolic catalase T occurs in the presence of a protein synthesis inhibitor and can be detected rapidly after an increase of tyrosine phosphorylation of Hog1p triggered by high osmolarity. Consistent with a role of STREs in the induction of stress resistance, a number of other stress protein genes (e.g. HSP104) are regulated like CTT1. Furthermore, catalase T was shown to be important for viability under severe osmotic stress, and heat shock was demonstrated to provide cross-protection against osmotic stress.

Structural Bioinformatics-Based Prediction of Exceptional Selectivity of p38 MAP Kinase Inhibitor PH-797804

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xing, Li; Shieh, Huey S.; Selness, Shaun R.

2009-07-24

PH-797804 is a diarylpyridinone inhibitor of p38{alpha} mitogen-activated protein (MAP) kinase derived from a racemic mixture as the more potent atropisomer (aS), first proposed by molecular modeling and subsequently confirmed by experiments. On the basis of structural comparison with a different biaryl pyrazole template and supported by dozens of high-resolution crystal structures of p38{alpha} inhibitor complexes, PH-797804 is predicted to possess a high level of specificity across the broad human kinase genome. We used a structural bioinformatics approach to identify two selectivity elements encoded by the TXXXG sequence motif on the p38{alpha} kinase hinge: (i) Thr106 that serves as themore » gatekeeper to the buried hydrophobic pocket occupied by 2,4-difluorophenyl of PH-797804 and (ii) the bidentate hydrogen bonds formed by the pyridinone moiety with the kinase hinge requiring an induced 180{sup o} rotation of the Met109-Gly110 peptide bond. The peptide flip occurs in p38{alpha} kinase due to the critical glycine residue marked by its conformational flexibility. Kinome-wide sequence mining revealed rare presentation of the selectivity motif. Corroboratively, PH-797804 exhibited exceptionally high specificity against MAP kinases and the related kinases. No cross-reactivity was observed in large panels of kinase screens (selectivity ratio of >500-fold). In cellular assays, PH-797804 demonstrated superior potency and selectivity consistent with the biochemical measurements. PH-797804 has met safety criteria in human phase I studies and is under clinical development for several inflammatory conditions. Understanding the rationale for selectivity at the molecular level helps elucidate the biological function and design of specific p38{alpha} kinase inhibitors.« less

Effects of protein kinase C activators on phorbol ester-sensitive and -resistant EL4 thymoma cells.

PubMed

Sansbury, H M; Wisehart-Johnson, A E; Qi, C; Fulwood, S; Meier, K E

1997-09-01

Phorbol ester-sensitive EL4 murine thymoma cells respond to phorbol 12-myristate 13-acetate with activation of ERK mitogen-activated protein kinases, synthesis of interleukin-2, and death, whereas phorbol ester-resistant variants of this cell line do not exhibit these responses. Additional aspects of the resistant phenotype were examined, using a newly-established resistant cell line. Phorbol ester induced morphological changes, ERK activation, calcium-dependent activation of the c-Jun N-terminal kinase (JNK), interleukin-2 synthesis, and growth inhibition in sensitive but not resistant cells. A series of protein kinase C activators caused membrane translocation of protein kinase C's (PKCs) alpha, eta, and theta in both cell lines. While PKC eta was expressed at higher levels in sensitive than in resistant cells, overexpression of PKC eta did not restore phorbol ester-induced ERK activation to resistant cells. In sensitive cells, PKC activators had similar effects on cell viability and ERK activation, but differed in their abilities to induce JNK activation and interleukin-2 synthesis. PD 098059, an inhibitor of the mitogen activated protein (MAP)/ERK kinase kinase MEK, partially inhibited ERK activation and completely blocked phorbol ester-induced cell death in sensitive cells. Thus MEK and/or ERK activation, but not JNK activation or interleukin-2 synthesis, appears to be required for phorbol ester-induced toxicity. Alterations in phorbol ester response pathways, rather than altered expression of PKC isoforms, appear to confer phorbol ester resistance to EL4 cells.

Tunable signal processing in synthetic MAP kinase cascades.

PubMed

O'Shaughnessy, Ellen C; Palani, Santhosh; Collins, James J; Sarkar, Casim A

2011-01-07

The flexibility of MAPK cascade responses enables regulation of a vast array of cell fate decisions, but elucidating the mechanisms underlying this plasticity is difficult in endogenous signaling networks. We constructed insulated mammalian MAPK cascades in yeast to explore how intrinsic and extrinsic perturbations affect the flexibility of these synthetic signaling modules. Contrary to biphasic dependence on scaffold concentration, we observe monotonic decreases in signal strength as scaffold concentration increases. We find that augmenting the concentration of sequential kinases can enhance ultrasensitivity and lower the activation threshold. Further, integrating negative regulation and concentration variation can decouple ultrasensitivity and threshold from the strength of the response. Computational analyses show that cascading can generate ultrasensitivity and that natural cascades with different kinase concentrations are innately biased toward their distinct activation profiles. This work demonstrates that tunable signal processing is inherent to minimal MAPK modules and elucidates principles for rational design of synthetic signaling systems. Copyright © 2011 Elsevier Inc. All rights reserved.

miR-188 promotes senescence of lineage-negative bone marrow cells by targeting MAP3K3 expression.

PubMed

Zheng, Yue; Liu, Hua; Kong, Ye

2017-08-01

Lineage-negative bone marrow cells (lin-BMCs) have reparative potential for overcoming endothelial dysfunction and reducing cardiovascular risk. Here, we found that miR-188 is upregulated and mitogen-activated protein kinase kinase kinase 3 (MAP3K3) is downregulated in aged lin-BMCs, whereas their expression is reversed in young lin-BMCs. We identified and confirmed MAP3K3 as a direct target of miR-188. MiR-188 overexpression or MAP3K3 silencing in young lin-BMCs increases p16 and p21 expression, enhances cell senescence, and decreases the ability for cell proliferation, migration, and tube formation. Conversely, miR-188 suppression in aged lin-BMCs yields the opposite results. We further found that MAP3K3 is involved in miR-188-induced promotion of lin-BMC senescence. All data reveal that miR-188 induces lin-BMC senescence by targeting MAP3K3 expression, thus, providing new theoretical basis for the prevention and treatment of cardiovascular diseases. © 2017 Federation of European Biochemical Societies.

MAP kinase dependent cyclinE/cdk2 activity promotes DNA replication in early sea urchin embryos

PubMed Central

Kisielewska, J.; Philipova, R.; Huang, J.-Y.; Whitaker, M.

2009-01-01

Sea urchins provide an excellent model for studying cell cycle control mechanisms governing DNA replication in vivo. Fertilization and cell cycle progression are tightly coordinated by Ca2+ signals, but the mechanisms underlying the onset of DNA replication after fertilization remain less clear. In this study we demonstrate that calcium-dependent activation of ERK1 promotes accumulation of cyclinE/cdk2 into the male and female pronucleus and entry into first S-phase. We show that cdk2 activity rises quickly after fertilization to a maximum at 4 min, corresponding in timing to the early ERK1 activity peak. Abolishing MAP kinase activity after fertilization with MEK inhibitor, U0126, substantially reduces the early peak of cdk2 activity and prevents cyclinE and cdk2 accumulation in both sperm pronucleus and zygote nucleus in vivo. Both p27kip1 and roscovitine, cdk2 inhibitors, prevented DNA replication suggesting cdk2 involvement in this process in sea urchin. Inhibition of cdk2 activity using p27kip1 had no effect on the phosphorylation of MBP by ERK, but completely abolished phosphorylation of retinoblastoma protein, a cdk2 substrate, indicating that cdk2 activity is downstream of ERK1 activation. This pattern of regulation of DNA synthesis conforms to the pattern observed in mammalian somatic cells. PMID:19665013

Arginine-vasopressin directly promotes a thermogenic and pro-inflammatory adipokine expression profile in brown adipocytes.

PubMed

Küchler, Sebastian; Perwitz, Nina; Schick, Rafael Reinhold; Klein, Johannes; Westphal, Sören

2010-09-24

Arginine-vasopressin (AVP) - via activation of the hypothalamic-pituitary-adrenal (HPA) axis - may play a role in the regulation of energy homeostasis and related cardiovascular complications. Brown adipose tissue (BAT) - via dissipation of energy in the form of heat - contributes to whole body energy balance. BAT expresses vasopressin receptors. We investigated direct effects of AVP on brown adipose endocrine and metabolic functions. UCP-1 protein expression in differentiated brown adipocytes was induced after acute exposure of adipocytes to AVP. This effect was time-dependent with a maximum increase after 8h. AVP also induced a time- and dose-dependent increase in p38 MAP kinase phosphorylation. Pharmacological inhibition of p38 MAP kinase with SB 202190 abolished the induction of UCP-1 protein expression. Furthermore, while acute AVP treatment enhanced mRNA expression of MCP-1 and IL-6, adiponectin mRNA expression was reduced. Yet, on the level of intracellular glucose uptake, there was no AVP-induced change of adipose insulin-induced glucose uptake. Finally, there was no difference in lipid accumulation between control and AVP-treated cells. Taken together, our data demonstrate direct effects of AVP on thermogenic, inflammatory, and glucoregulatory gene expression in brown adipocytes, thus expanding the hitherto known spectrum of this neuropeptides's biological effects and suggesting a direct adipotropic role as a stress-promoting factor. Copyright 2010 Elsevier B.V. All rights reserved.

A New MAP Kinase Protein Involved in Estradiol-Stimulated Reproduction of the Helminth Parasite Taenia crassiceps

PubMed Central

Escobedo, Galileo; Soldevila, Gloria; Ortega-Pierres, Guadalupe; Chávez-Ríos, Jesús Ramsés; Nava, Karen; Fonseca-Liñán, Rocío; López-Griego, Lorena; Hallal-Calleros, Claudia; Ostoa-Saloma, Pedro; Morales-Montor, Jorge

2010-01-01

MAP kinases (MAPK) are involved in the regulation of cellular processes such as reproduction and growth. In parasites, the role of MAPK has been scarcely studied. Here, we describe the participation of an ERK-like protein in estrogen-dependent reproduction of the helminth parasite Taenia crassiceps. Our results show that 17β-estradiol induces a concentration-dependent increase in the bud number of in vitro cultured cysticerci. If parasites are also incubated in presence of an ERK-inhibitor, the stimulatory effect of estrogen is blocked. The expression of ERK-like mRNA and its corresponding protein was detected in the parasite. The ERK-like protein was over-expressed by all treatments. Nevertheless, a strong induction of phosphorylation of this protein was observed only in response to 17β-estradiol. Cross-contamination by host cells was discarded by flow cytometry analysis. Parasite cells expressing the ERK-like protein were exclusively located at the subtegument tissue by confocal microscopy. Finally, the ERK-like protein was separated by bidimensional electrophoresis and then sequenced, showing the conserved TEY activation motif, typical of all known ERK 1/2 proteins. Our results show that an ERK-like protein is involved in the molecular signalling during the interaction between the host and T. crassiceps, and may be considered as target for anti-helminth drugs design. PMID:20145710

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tsukamoto, Ikuko, E-mail: tukamoto@med.kagawa-u.ac.jp; Sakakibara, Norikazu; Maruyama, Tokumi

Research highlights: {yields} A novel nucleic acid analogue (2Cl-C.OXT-A, m.w. 284) showed angiogenic potency. {yields} It stimulated the tube formation, proliferation and migration of HUVEC in vitro. {yields} 2Cl-C.OXT-A induced the activation of ERK1/2 and MEK in HUVEC. {yields} Angiogenic potency in vivo was confirmed in CAM assay and rabbit cornea assay. {yields} A synthesized small angiogenic agent would have great clinical therapeutic value. -- Abstract: A novel nucleic acid analogue (2Cl-C.OXT-A) significantly stimulated tube formation of human umbilical endothelial cells (HUVEC). Its maximum potency at 100 {mu}M was stronger than that of vascular endothelial growth factor (VEGF), a positivemore » control. At this concentration, 2Cl-C.OXT-A moderately stimulated proliferation as well as migration of HUVEC. To gain mechanistic insights how 2Cl-C.OXT-A promotes angiogenic responses in HUVEC, we performed immunoblot analyses using phospho-specific antibodies as probes. 2Cl-C.OXT-A induced robust phosphorylation/activation of MAP kinase ERK1/2 and an upstream MAP kinase kinase MEK. Conversely, a MEK inhibitor PD98059 abolished ERK1/2 activation and tube formation both enhanced by 2Cl-C.OXT-A. In contrast, MAP kinase responses elicited by 2Cl-C.OXT-A were not inhibited by SU5416, a specific inhibitor of VEGF receptor tyrosine kinase. Collectively these results suggest that 2Cl-C.OXT-A-induces angiogenic responses in HUVEC mediated by a MAP kinase cascade comprising MEK and ERK1/2, but independently of VEGF receptor tyrosine kinase. In vivo assay using chicken chorioallantoic membrane (CAM) and rabbit cornea also suggested the angiogenic potency of 2Cl-C.OXT-A.« less

Pancreatic glucagon-like peptide-1 receptor couples to multiple G proteins and activates mitogen-activated protein kinase pathways in Chinese hamster ovary cells.

PubMed

Montrose-Rafizadeh, C; Avdonin, P; Garant, M J; Rodgers, B D; Kole, S; Yang, H; Levine, M A; Schwindinger, W; Bernier, M

1999-03-01

Chinese hamster ovary (CHO) cells stably expressing the human insulin receptor and the rat glucagon-like peptide-1 (GLP-1) receptor (CHO/GLPR) were used to study the functional coupling of the GLP-1 receptor with G proteins and to examine the regulation of the mitogen-activated protein (MAP) kinase signaling pathway by GLP-1. We showed that ligand activation of GLP-1 receptor led to increased incorporation of GTP-azidoanilide into Gs alpha, Gq/11 alpha, and Gi1,2 alpha, but not Gi3 alpha. GLP-1 increased p38 MAP kinase activity 2.5- and 2.0-fold over the basal level in both CHO/GLPR cells and rat insulinoma cells (RIN 1046-38), respectively. Moreover, GLP-1 induced phosphorylation of the immediate upstream kinases of p38, MKK3/MKK6, in CHO/GLPR and RIN 1046-38 cells. Ligand-stimulated GLP-1 receptor produced 1.45- and 2.7-fold increases in tyrosine phosphorylation of 42-kDa extracellular signal-regulated kinase (ERK) in CHO/GLPR and RIN 1046-38 cells, respectively. In CHO/GLPR cells, these effects of GLP-1 on the ERK and p38 MAP kinase pathways were inhibited by pretreatment with cholera toxin (CTX), but not with pertussis toxin. The combination of insulin and GLP-1 resulted in an additive response (1.6-fold over insulin alone) that was attenuated by CTX. In contrast, the ability of insulin alone to activate these pathways was insensitive to either toxin. Our study indicates a direct coupling between the GLP-1 receptor and several G proteins, and that CTX-sensitive proteins are required for GLP-1-mediated activation of MAP kinases.

Involvement of mitogen-activated protein kinase activation in the signal-transduction pathways of the soya bean oxidative burst.

PubMed Central

Taylor, A T; Kim, J; Low, P S

2001-01-01

The oxidative burst constitutes one of the most rapid defence responses characterized in the Plant Kingdom. We have observed that four distinct elicitors of the soya bean oxidative burst activate kinases of masses approximately 44 kDa and approximately 47 kDa. Evidence that these kinases regulate production of reactive oxygen species include: (i) their rapid activation by oxidative burst elicitors, (ii) their tight temporal correlation between activation/deactivation of the kinases and activation/deactivation of the oxidative burst, (iii) the identical pharmacological profile of kinase activation and oxidant production for 13 commonly used inhibitors, and (iv) the autologous activation of both kinases and oxidant production by calyculin A and cantharidin, two phosphatase inhibitors. Immunological and biochemical studies reveal that the activated 44 kDa and 47 kDa kinases are mitogen-activated protein (MAP) kinase family members. The kinases prefer myelin basic protein as a substrate, and they phosphorylate primarily on threonine residues. The kinases are themselves phosphorylated on tyrosine residues, and this phosphorylation is required for activity. Finally, both kinases are recognized by an antibody against activated MAP kinase immediately after (but not before) cell stimulation by elicitors. Based on these and other observations, a preliminary sequence of signalling steps linking elicitor stimulation, kinase activation and Ca(2+) entry, to initiation of oxidant production, is proposed. PMID:11311144

Enhancement of ligand-dependent down-regulation of glucocorticoid receptor by lipopolysaccharide.

PubMed

Hirasawa, Noriyasu; Yashima, Kazushi; Ishihara, Kenji

2009-10-07

The inhibitory actions of glucocorticoids are often attenuated in inflamed tissues. The aim of the present study was to investigate whether the dexamethasone-induced downregulation of glucocorticoid receptor (GR) expression was enhanced by the stimulation with lipopolysaccharide (LPS). Various cells were stimulated with LPS (1microg/ml) for 30min and then treated with dexamethasone (1microM) for specified periods. The levels of GR and the phosphorylation at Ser211 were determined by Western blot. The effects of kinase inhibitors and a proteasome inhibitor on them were examined. The treatment of NCI-H292 cells with dexamethasone reduced the levels of GR, and the pretreatment with LPS accelerated the reduction. Such an enhancement by LPS of the dexamethasone-induced downregulation was observed in the respiratory epithelial cell lines BEAS-2B and A549, but not in the keratinocyte-like cell line HaCaT, the hematopoietic cell lines U937, THP-1 and Eol-1, or in hepatocytoma HepG2 cells. The treatment with dexamethasone and LPS apparently decreased GR levels in the lungs of BALB/c mice but not in the liver. In NCI-H292 cells, the LPS-enhanced downregulation of GR expression was recovered by the proteasome inhibitor MG-132. SP600125, SB203580 and roscovitine but not U0126 inhibited the LPS-induced enhancement of both the phosphorylation and the downregulation of GR. These findings suggested that the ligand-dependent downregulation of GR expression via the proteasome was apparent in the respiratory epithelial cells and enhanced by lipopolysaccharide via the activation of p38 MAP kinase, c-Jun N-terminal kinase and cyclin-dependent kinases.

Phosphorylation of MAP65-1 by Arabidopsis Aurora Kinases Is Required for Efficient Cell Cycle Progression1[OPEN

PubMed Central

Weimer, Annika K.; Stoppin-Mellet, Virginie; Kosetsu, Ken; Cedeño, Cesyen; Jaquinod, Michel; Njo, Maria; De Milde, Liesbeth; Tompa, Peter; Inzé, Dirk; Beeckman, Tom; Vantard, Marylin

2017-01-01

Aurora kinases are key effectors of mitosis. Plant Auroras are functionally divided into two clades. The alpha Auroras (Aurora1 and Aurora2) associate with the spindle and the cell plate and are implicated in controlling formative divisions throughout plant development. The beta Aurora (Aurora3) localizes to centromeres and likely functions in chromosome separation. In contrast to the wealth of data available on the role of Aurora in other kingdoms, knowledge on their function in plants is merely emerging. This is exemplified by the fact that only histone H3 and the plant homolog of TPX2 have been identified as Aurora substrates in plants. Here we provide biochemical, genetic, and cell biological evidence that the microtubule-bundling protein MAP65-1—a member of the MAP65/Ase1/PRC1 protein family, implicated in central spindle formation and cytokinesis in animals, yeasts, and plants—is a genuine substrate of alpha Aurora kinases. MAP65-1 interacts with Aurora1 in vivo and is phosphorylated on two residues at its unfolded tail domain. Its overexpression and down-regulation antagonistically affect the alpha Aurora double mutant phenotypes. Phospho-mutant analysis shows that Aurora contributes to the microtubule bundling capacity of MAP65-1 in concert with other mitotic kinases. PMID:27879390

Identifying kinase dependency in cancer cells by integrating high-throughput drug screening and kinase inhibition data.

PubMed

Ryall, Karen A; Shin, Jimin; Yoo, Minjae; Hinz, Trista K; Kim, Jihye; Kang, Jaewoo; Heasley, Lynn E; Tan, Aik Choon

2015-12-01

Targeted kinase inhibitors have dramatically improved cancer treatment, but kinase dependency for an individual patient or cancer cell can be challenging to predict. Kinase dependency does not always correspond with gene expression and mutation status. High-throughput drug screens are powerful tools for determining kinase dependency, but drug polypharmacology can make results difficult to interpret. We developed Kinase Addiction Ranker (KAR), an algorithm that integrates high-throughput drug screening data, comprehensive kinase inhibition data and gene expression profiles to identify kinase dependency in cancer cells. We applied KAR to predict kinase dependency of 21 lung cancer cell lines and 151 leukemia patient samples using published datasets. We experimentally validated KAR predictions of FGFR and MTOR dependence in lung cancer cell line H1581, showing synergistic reduction in proliferation after combining ponatinib and AZD8055. KAR can be downloaded as a Python function or a MATLAB script along with example inputs and outputs at: http://tanlab.ucdenver.edu/KAR/. aikchoon.tan@ucdenver.edu. Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Expression of Human CTP Synthetase in Saccharomyces cerevisiae Reveals Phosphorylation by Protein Kinase A*

PubMed Central

Han, Gil-Soo; Sreenivas, Avula; Choi, Mal-Gi; Chang, Yu-Fang; Martin, Shelley S.; Baldwin, Enoch P.; Carman, George M.

2005-01-01

CTP synthetase (EC 6.3.4.2, UTP: ammonia ligase (ADP-forming)) is an essential enzyme in all organisms; it generates the CTP required for the synthesis of nucleic acids and membrane phospholipids. In this work we showed that the human CTP synthetase genes, CTPS1 and CTPS2, were functional in Saccharomyces cerevisiae and complemented the lethal phenotype of the ura7Δ ura8Δ mutant lacking CTP synthetase activity. The expression of the CTPS1-and CTPS2-encoded human CTP synthetase enzymes in the ura7Δ ura8Δ mutant was shown by immunoblot analysis of CTP synthetase proteins, the measurement of CTP synthetase activity, and the synthesis of CTP in vivo. Phosphoamino acid and phosphopeptide mapping analyses of human CTP synthetase 1 isolated from 32Pi-labeled cells revealed that the enzyme was phosphorylated on multiple serine residues in vivo. Activation of protein kinase A activity in yeast resulted in transient increases (2-fold) in the phosphorylation of human CTP synthetase 1 and the cellular level of CTP. Human CTP synthetase 1 was also phosphorylated by mammalian protein kinase A in vitro. Using human CTP synthetase 1 purified from Escherichia coli as a substrate, protein kinase A activity was dose- and time-dependent, and dependent on the concentrations of CTP synthetase1 and ATP. These studies showed that S. cerevisiae was useful for the analysis of human CTP synthetase phosphorylation. PMID:16179339

Yersinia YopP-induced apoptotic cell death in murine dendritic cells is partially independent from action of caspases and exhibits necrosis-like features.

PubMed

Gröbner, Sabine; Autenrieth, Stella E; Soldanova, Irena; Gunst, Dani S J; Schaller, Martin; Bohn, Erwin; Müller, Steffen; Leverkus, Martin; Wesselborg, Sebastian; Autenrieth, Ingo B; Borgmann, Stefan

2006-11-01

Yersinia outer protein P (YopP) is a virulence factor of Yersinia enterocolitica that is injected into the cytosol of host cells where it targets MAP kinase kinases (MKKs) and inhibitor of kappaB kinase (IKK)-beta resulting in inhibition of cytokine production as well as induction of apoptosis in murine macrophages and dendritic cells (DC). Here we show that DC death was only partially prevented by the broad spectrum caspase inhibitor zVAD-fmk, indicating simultaneous caspase-dependent and caspase-independent mechanisms of cell death induction by YopP. Microscopic analyses and measurement of cell size demonstrated necrosis-like morphology of caspase-independent cell death. Application of zVAD-fmk prevented cleavage of procaspases and Bid, decrease of the inner transmembrane mitochondrial potential DeltaPsi(m) and mitochondrial release of cytochrome c. From these data we conclude that YopP-induced activation of the mitochondrial death pathway is mediated upstream via caspases. In conclusion, our results suggest that YopP simultaneously induces caspase-dependent apoptotic and caspase-independent necrosis-like death in DC. However, it has to be resolved if necrosis-like DC death occurs independently from apoptotic events or as an apoptotic epiphenomenon.

Role of Rho/ROCK and p38 MAP kinase pathways in transforming growth factor-beta-mediated Smad-dependent growth inhibition of human breast carcinoma cells in vivo.

PubMed

Kamaraju, Anil K; Roberts, Anita B

2005-01-14

TGF-beta is a multifunctional cytokine known to exert its biological effects through a variety of signaling pathways of which Smad signaling is considered to be the main mediator. At present, the Smad-independent pathways, their interactions with each other, and their roles in TGF-beta-mediated growth inhibitory effects are not well understood. To address these questions, we have utilized a human breast cancer cell line MCF10CA1h and demonstrate that p38 MAP kinase and Rho/ROCK pathways together with Smad2 and Smad3 are necessary for TGF-beta-mediated growth inhibition of this cell line. We show that Smad2/3 are indispensable for TGF-beta-mediated growth inhibition, and that both p38 and Rho/ROCK pathways affect the linker region phosphorylation of Smad2/3. Further, by using Smad3 mutated at the putative phosphorylation sites in the linker region, we demonstrate that phosphorylation at Ser203 and Ser207 residues is required for the full transactivation potential of Smad3, and that these residues are targets of the p38 and Rho/ROCK pathways. We demonstrate that activation of the p38 MAP kinase pathway is necessary for the full transcriptional activation potential of Smad2/Smad3 by TGF-beta, whereas activity of Rho/ROCK is necessary for both down-regulation of c-Myc protein and up-regulation of p21waf1 protein, directly interfering with p21waf1 transcription. Our results not only implicate Rho/ROCK and p38 MAPK pathways as necessary for TGF-beta-mediated growth inhibition, but also demonstrate their individual contributions and the basis for their cooperation with each other.

Virus-specific DNA sequences present in cells which carry the herpes simplex virus thymidine kinase gene.

PubMed

Minson, A C; Darby, G K; Wildy, P

1979-11-01

Two independently derived cell lines which carry the herpes simplex type 2 thymidine kinase gene have been examined for the presence of HSV-2-specific DNA sequences. Both cell lines contained 1 to 3 copies per cell of a sequence lying within map co-ordinates 0.2 to 0.4 of the HSV-2 genome. Revertant cells, which contained no detectable thymidine kinase, did not contain this DNA sequence. The failure of EcoR1-restricted HSV-2 DNA to act as a donor of the thymidine kinase gene in transformation experiments suggests that the gene lies close to the EcoR1 restriction site within this sequence at a map position of approx. 0.3. The HSV-2 kinase gene is therefore approximately co-linear with the HSV-1 gene.

Neurotensin-induced Proinflammatory Signaling in Human Colonocytes Is Regulated by β-Arrestins and Endothelin-converting Enzyme-1-dependent Endocytosis and Resensitization of Neurotensin Receptor 1*

PubMed Central

Law, Ivy Ka Man; Murphy, Jane E.; Bakirtzi, Kyriaki; Bunnett, Nigel W.; Pothoulakis, Charalabos

2012-01-01

The neuropeptide/hormone neurotensin (NT) mediates intestinal inflammation and cell proliferation by binding of its high affinity receptor, neurotensin receptor-1 (NTR1). NT stimulates IL-8 expression in NCM460 human colonic epithelial cells by both MAP kinase- and NF-κB-dependent pathways. Although the mechanism of NTR1 endocytosis has been studied, the relationship between NTR1 intracellular trafficking and inflammatory signaling remains to be elucidated. In the present study, we show that in NCM460 cells exposed to NT, β-arrestin-1 (βARR1), and β-arrestin-2 (βARR2) translocate to early endosomes together with NTR1. Endothelin-converting enzyme-1 (ECE-1) degrades NT in acidic conditions, and its activity is crucial for NTR1 recycling. Pretreatment of NCM460 cells with the ECE-1 inhibitor SM19712 or gene silencing of βARR1 or βARR2 inhibits NT-stimulated ERK1/2 and JNK phosphorylation, NF-κB p65 nuclear translocation and phosphorylation, and IL-8 secretion. Furthermore, NT-induced cell proliferation, but not IL-8 transcription, is attenuated by the JNK inhibitor, JNK(AII). Thus, NTR1 internalization and recycling in human colonic epithelial cells involves βARRs and ECE-1, respectively. Our results also indicate that βARRs and ECE-1-dependent recycling regulate MAP kinase and NF-κB signaling as well as cell proliferation in human colonocytes in response to NT. PMID:22416137

Transcriptome and proteome analysis of tyrosine kinase inhibitor treated canine mast cell tumour cells identifies potentially kit signaling-dependent genes

PubMed Central

2012-01-01

Background Canine mast cell tumour proliferation depends to a large extent on the activity of KIT, a tyrosine kinase receptor. Inhibitors of the KIT tyrosine kinase have recently been introduced and successfully applied as a therapeutic agent for this tumour type. However, little is known on the downstream target genes of this signaling pathway and molecular changes after inhibition. Results Transcriptome analysis of the canine mast cell tumour cell line C2 treated for up to 72 hours with the tyrosine kinase inhibitor masitinib identified significant changes in the expression levels of approximately 3500 genes or 16% of the canine genome. Approximately 40% of these genes had increased mRNA expression levels including genes associated with the pro-proliferative pathways of B- and T-cell receptors, chemokine receptors, steroid hormone receptors and EPO-, RAS and MAP kinase signaling. Proteome analysis of C2 cells treated for 72 hours identified 24 proteins with changed expression levels, most of which being involved in gene transcription, e.g. EIA3, EIA4, TARDBP, protein folding, e.g. HSP90, UCHL3, PDIA3 and protection from oxidative stress, GSTT3, SELENBP1. Conclusions Transcriptome and proteome analysis of neoplastic canine mast cells treated with masitinib confirmed the strong important and complex role of KIT in these cells. Approximately 16% of the total canine genome and thus the majority of the active genes were significantly transcriptionally regulated. Most of these changes were associated with reduced proliferation and metabolism of treated cells. Interestingly, several pro-proliferative pathways were up-regulated, which may represent attempts of masitinib treated cells to activate alternative pro-proliferative pathways. These pathways may contain hypothetical targets for a combination therapy with masitinib to further improve its therapeutic effect. PMID:22747577

Systematic Determination of Human Cyclin Dependent Kinase (CDK)-9 Interactome Identifies Novel Functions in RNA Splicing Mediated by the DEAD Box (DDX)-5/17 RNA Helicases.

PubMed

Yang, Jun; Zhao, Yingxin; Kalita, Mridul; Li, Xueling; Jamaluddin, Mohammad; Tian, Bing; Edeh, Chukwudi B; Wiktorowicz, John E; Kudlicki, Andrzej; Brasier, Allan R

2015-10-01

Inducible transcriptional elongation is a rapid, stereotypic mechanism for activating immediate early immune defense genes by the epithelium in response to viral pathogens. Here, the recruitment of a multifunctional complex containing the cyclin dependent kinase 9 (CDK9) triggers the process of transcriptional elongation activating resting RNA polymerase engaged with innate immune response (IIR) genes. To identify additional functional activity of the CDK9 complex, we conducted immunoprecipitation (IP) enrichment-stable isotope labeling LC-MS/MS of the CDK9 complex in unstimulated cells and from cells activated by a synthetic dsRNA, polyinosinic/polycytidylic acid [poly (I:C)]. 245 CDK9 interacting proteins were identified with high confidence in the basal state and 20 proteins in four functional classes were validated by IP-SRM-MS. These data identified that CDK9 interacts with DDX 5/17, a family of ATP-dependent RNA helicases, important in alternative RNA splicing of NFAT5, and mH2A1 mRNA two proteins controlling redox signaling. A direct comparison of the basal versus activated state was performed using stable isotope labeling and validated by IP-SRM-MS. Recruited into the CDK9 interactome in response to poly(I:C) stimulation are HSPB1, DNA dependent kinases, and cytoskeletal myosin proteins that exchange with 60S ribosomal structural proteins. An integrated human CDK9 interactome map was developed containing all known human CDK9- interacting proteins. These data were used to develop a probabilistic global map of CDK9-dependent target genes that predicted two functional states controlling distinct cellular functions, one important in immune and stress responses. The CDK9-DDX5/17 complex was shown to be functionally important by shRNA-mediated knockdown, where differential accumulation of alternatively spliced NFAT5 and mH2A1 transcripts and alterations in downstream redox signaling were seen. The requirement of CDK9 for DDX5 recruitment to NFAT5 and mH2A1 chromatin target was further demonstrated using chromatin immunoprecipitation (ChIP). These data indicate that CDK9 is a dynamic multifunctional enzyme complex mediating not only transcriptional elongation, but also alternative RNA splicing and potentially translational control. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

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

Molecular mechanisms of liver preconditioning

PubMed Central

Alchera, Elisa; Dal Ponte, Caterina; Imarisio, Chiara; Albano, Emanuele; Carini, Rita

2010-01-01

Ischemia/reperfusion (I/R) injury still represents an important cause of morbidity following hepatic surgery and limits the use of marginal livers in hepatic transplantation. Transient blood flow interruption followed by reperfusion protects tissues against damage induced by subsequent I/R. This process known as ischemic preconditioning (IP) depends upon intrinsic cytoprotective systems whose activation can inhibit the progression of irreversible tissue damage. Compared to other organs, liver IP has additional features as it reduces inflammation and promotes hepatic regeneration. Our present understanding of the molecular mechanisms involved in liver IP is still largely incomplete. Experimental studies have shown that the protective effects of liver IP are triggered by the release of adenosine and nitric oxide and the subsequent activation of signal networks involving protein kinases such as phosphatidylinositol 3-kinase, protein kinase C δ/ε and p38 MAP kinase, and transcription factors such as signal transducer and activator of transcription 3, nuclear factor-κB and hypoxia-inducible factor 1. This article offers an overview of the molecular events underlying the preconditioning effects in the liver and points to the possibility of developing pharmacological approaches aimed at activating the intrinsic protective systems in patients undergoing liver surgery. PMID:21182220

Optimized Target Residence Time: Type I1/2 Inhibitors for p38α MAP Kinase with Improved Binding Kinetics through Direct Interaction with the R-Spine.

PubMed

Wentsch, Heike K; Walter, Niklas M; Bührmann, Mike; Mayer-Wrangowski, Svenja; Rauh, Daniel; Zaman, Guido J R; Willemsen-Seegers, Nicole; Buijsman, Rogier C; Henning, Melanie; Dauch, Daniel; Zender, Lars; Laufer, Stefan

2017-05-02

Skepinone-L was recently reported to be a p38α MAP kinase inhibitor with high potency and excellent selectivity in vitro and in vivo. However, this class of compounds still act as fully ATP-competitive Type I binders which, furthermore, suffer from short residence times at the enzyme. We herein describe a further development with the first Type I1/2 binders for p38α MAP kinase. Type I1/2 inhibitors interfere with the R-spine, inducing a glycine flip and occupying both hydrophobic regions I and II. This design approach leads to prolonged target residence time, binding to both the active and inactive states of the kinase, excellent selectivity, excellent potency on the enzyme level, and low nanomolar activity in a human whole blood assay. This promising binding mode is proven by X-ray crystallography. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Regulation of Nucleocytoplasmic Shuttling of Bruton's Tyrosine Kinase (Btk) through a Novel SH3-Dependent Interaction with Ankyrin Repeat Domain 54 (ANKRD54)

PubMed Central

Hussain, Alamdar; Mohammad, Dara K.; Mohamed, Abdalla J.; Nguyen, Vivian; Metalnikov, Pavel; Colwill, Karen; Pawson, Tony; Nore, Beston F.

2012-01-01

Bruton's tyrosine kinase (Btk), belonging to the Tec family of tyrosine kinases (TFKs), is essential for B-lymphocyte development. Abrogation of Btk signaling causes human X-linked agammaglobulinemia (XLA) and murine X-linked immunodeficiency (Xid). We employed affinity purification of Flag-tagged Btk, combined with tandem mass spectrometry, to capture and identify novel interacting proteins. We here characterize the interaction with ankryin repeat domain 54 protein (ANKRD54), also known as Lyn-interacting ankyrin repeat protein (Liar). While Btk is a nucleocytoplasmic protein, the Liar pool was found to shuttle at a higher rate than Btk. Importantly, our results suggest that Liar mediates nuclear export of both Btk and another TFK, Txk/Rlk. Liar-mediated Btk shuttling was enriched for activation loop, nonphosphorylated Btk and entirely dependent on Btk's SH3 domain. Liar also showed reduced binding to an aspartic acid phosphomimetic SH3 mutant. Three other investigated nucleus-located proteins, Abl, estrogen receptor β (ERβ), and transcription factor T-bet, were all unaffected by Liar. We mapped the interaction site to the C terminus of the Btk SH3 domain. A biotinylated, synthetic Btk peptide, ARDKNGQEGYIPSNYVTEAEDS, was sufficient for this interaction. Liar is the first protein identified that specifically influences the nucleocytoplasmic shuttling of Btk and Txk and belongs to a rare group of known proteins carrying out this activity in a Crm1-dependent manner. PMID:22527282

Therapeutical relevance of MAP-kinase inhibitors in renal diseases: current knowledge and future clinical perspectives.

PubMed

Grande, M Teresa; López-Novoa, José M

2008-01-01

Renal failure, both acute and chronic, represents an important health problem by its social, sanitary and economic aspects. Mitogen-activated protein kinases (MAPK) are a family of mediators involved in the transduction of extracellular stimuli to intracellular responses. The best studied members of this family are extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), Jun NH(2)-terminal kinase (JNK), p38 kinase and extracellular signal regulated kinases 5 (ERK5) also known as big MAP Kinase 1 (BMK1). MAPKs plays a role in regulating renal function and all these pathways have been demonstrated to be activated in many "in vivo" and cellular models or renal failure. As MAP kinases are key regulators in the control of cell proliferation and cell death, many more or less specific inhibitors of these pathways are being developed for the treatment of tumors. The purpose of this review is to examine the data available on the role of MAPKs activation in "in vivo" models of renal failure, as well as in different renal cell types (especially in mesangial cells, podocytes, tubular epithelial cells and fibroblasts) subjected to stress or damage. We have also reviewed the effect of MAPKs inhibition on renal damage, both "in vivo" and "in vitro". Data collected allow to suggest that therapy of chronic and acute renal disease with MAPKs inhibitors is a promising therapeutic area, although much more basic and clinical studies are necessary before this kind of therapy can be used in the everyday clinic.

Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans*

PubMed Central

Andrusiak, Matthew G.; Jin, Yishi

2016-01-01

Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundworm Caenorhabditis elegans was developed as a system to study genes required for development and nervous system function. The powerful genetics of C. elegans in combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components in C. elegans. PMID:26907690

The role of MAP4K3 in lifespan regulation of Caenorhabditiselegans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khan, Maruf H.; Hart, Matthew J., E-mail: HartMJ@uthscsa.edu; Rea, Shane L., E-mail: reas3@uthscsa.edu

2012-08-24

Highlights: Black-Right-Pointing-Pointer Inhibition of MAP4K3 by RNAi leads to increased mean lifespan in Caenorhabditis elegans. Black-Right-Pointing-Pointer Mutation in the citron homology domain of MAP4K3 leads to increased mean lifespan. Black-Right-Pointing-Pointer Mutation in the kinase domain of MAP4K3 has no significant effect on mean lifespan. -- Abstract: The TOR pathway is a kinase signaling pathway that regulates cellular growth and proliferation in response to nutrients and growth factors. TOR signaling is also important in lifespan regulation - when this pathway is inhibited, either naturally, by genetic mutation, or by pharmacological means, lifespan is extended. MAP4K3 is a Ser/Thr kinase that hasmore » recently been found to be involved in TOR activation. Unexpectedly, the effect of this protein is not mediated via Rheb, the more widely known TOR activation pathway. Given the role of TOR in growth and lifespan control, we looked at how inhibiting MAP4K3 in Caenorhabditiselegans affects lifespan. We used both feeding RNAi and genetic mutants to look at the effect of MAP4K3 deficiency. Our results show a small but significant increase in mean lifespan in MAP4K3 deficient worms. MAP4K3 thus represents a new target in the TOR pathway that can be targeted for pharmacological intervention to control lifespan.« less

GhMAP3K65, a Cotton Raf-Like MAP3K Gene, Enhances Susceptibility to Pathogen Infection and Heat Stress by Negatively Modulating Growth and Development in Transgenic Nicotiana benthamiana.

PubMed

Zhai, Na; Jia, Haihong; Liu, Dongdong; Liu, Shuchang; Ma, Manli; Guo, Xingqi; Li, Han

2017-11-21

Mitogen-activated protein kinase kinase kinases (MAP3Ks), the top components of MAPK cascades, modulate many biological processes, such as growth, development and various environmental stresses. Nevertheless, the roles of MAP3Ks remain poorly understood in cotton. In this study, GhMAP3K65 was identified in cotton, and its transcription was inducible by pathogen infection, heat stress, and multiple signalling molecules. Silencing of GhMAP3K65 enhanced resistance to pathogen infection and heat stress in cotton. In contrast, overexpression of GhMAP3K65 enhanced susceptibility to pathogen infection and heat stress in transgenic Nicotiana benthamiana . The expression of defence-associated genes was activated in transgenic N. benthamiana plants after pathogen infection and heat stress, indicating that GhMAP3K65 positively regulates plant defence responses. Nevertheless, transgenic N. benthamiana plants impaired lignin biosynthesis and stomatal immunity in their leaves and repressed vitality of their root systems. In addition, the expression of lignin biosynthesis genes and lignin content were inhibited after pathogen infection and heat stress. Collectively, these results demonstrate that GhMAP3K65 enhances susceptibility to pathogen infection and heat stress by negatively modulating growth and development in transgenic N. benthamiana plants.

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

Timing Is Everything: Highly Specific and Transient Expression of a MAP Kinase Determines Auxin-Induced Leaf Venation Patterns in Arabidopsis

PubMed Central

Stanko, Vera; Giuliani, Concetta; Retzer, Katarzyna; Djamei, Armin; Wahl, Vanessa; Wurzinger, Bernhard; Wilson, Cathal; Heberle-Bors, Erwin; Teige, Markus; Kragler, Friedrich

2014-01-01

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules present in all eukaryotes. In plants, MAPK cascades were shown to regulate cell division, developmental processes, stress responses, and hormone pathways. The subgroup A of Arabidopsis MAPKs consists of AtMPK3, AtMPK6, and AtMPK10. AtMPK3 and AtMPK6 are activated by their upstream MAP kinase kinases (MKKs) AtMKK4 and AtMKK5 in response to biotic and abiotic stress. In addition, they were identified as key regulators of stomatal development and patterning. AtMPK10 has long been considered as a pseudo-gene, derived from a gene duplication of AtMPK6. Here we show that AtMPK10 is expressed highly but very transiently in seedlings and at sites of local auxin maxima leaves. MPK10 encodes a functional kinase and interacts with the upstream MAP kinase kinase (MAPKK) AtMKK2. mpk10 mutants are delayed in flowering in long-day conditions and in continuous light. Moreover, cotyledons of mpk10 and mkk2 mutants have reduced vein complexity, which can be reversed by inhibiting polar auxin transport (PAT). Auxin does not affect AtMPK10 expression while treatment with the PAT inhibitor HFCA extends the expression in leaves and reverses the mpk10 mutant phenotype. These results suggest that the AtMKK2–AtMPK10 MAPK module regulates venation complexity by altering PAT efficiency. PMID:25064848

Heat-shock inactivation of the TFIIH-associated kinase and change in the phosphorylation sites on the C-terminal domain of RNA polymerase II.

PubMed

Dubois, M F; Vincent, M; Vigneron, M; Adamczewski, J; Egly, J M; Bensaude, O

1997-02-15

The C-terminal domain (CTD) of the RNA polymerase II largest subunit (RPB1) plays a central role in transcription. The CTD is unphosphorylated when the polymerase assembles into a preinitiation complex of transcription and becomes heavily phosphorylated during promoter clearance and entry into elongation of transcription. A kinase associated to the general transcription factor TFIIH, in the preinitiation complex, phosphorylates the CTD. The TFIIH-associated CTD kinase activity was found to decrease in extracts from heat-shocked HeLa cells compared to unstressed cells. This loss of activity correlated with a decreased solubility of the TFIIH factor. The TFIIH-kinase impairment during heat-shock was accompanied by the disappearance of a particular phosphoepitope (CC-3) on the RPB1 subunit. The CC-3 epitope was localized on the C-terminal end of the CTD and generated in vitro when the RPB1 subunit was phosphorylated by the TFIIH-associated kinase but not by another CTD kinase such as MAP kinase. In apparent discrepancy, the overall RPB1 subunit phosphorylation increased during heat-shock. The decreased activity in vivo of the TFIIH kinase might be compensated by a stress-activated CTD kinase such as MAP kinase. These results also suggest that heat-shock gene transcription may have a weak requirement for TFIIH kinase activity.

Heat-shock inactivation of the TFIIH-associated kinase and change in the phosphorylation sites on the C-terminal domain of RNA polymerase II.

PubMed Central

Dubois, M F; Vincent, M; Vigneron, M; Adamczewski, J; Egly, J M; Bensaude, O

1997-01-01

The C-terminal domain (CTD) of the RNA polymerase II largest subunit (RPB1) plays a central role in transcription. The CTD is unphosphorylated when the polymerase assembles into a preinitiation complex of transcription and becomes heavily phosphorylated during promoter clearance and entry into elongation of transcription. A kinase associated to the general transcription factor TFIIH, in the preinitiation complex, phosphorylates the CTD. The TFIIH-associated CTD kinase activity was found to decrease in extracts from heat-shocked HeLa cells compared to unstressed cells. This loss of activity correlated with a decreased solubility of the TFIIH factor. The TFIIH-kinase impairment during heat-shock was accompanied by the disappearance of a particular phosphoepitope (CC-3) on the RPB1 subunit. The CC-3 epitope was localized on the C-terminal end of the CTD and generated in vitro when the RPB1 subunit was phosphorylated by the TFIIH-associated kinase but not by another CTD kinase such as MAP kinase. In apparent discrepancy, the overall RPB1 subunit phosphorylation increased during heat-shock. The decreased activity in vivo of the TFIIH kinase might be compensated by a stress-activated CTD kinase such as MAP kinase. These results also suggest that heat-shock gene transcription may have a weak requirement for TFIIH kinase activity. PMID:9016617

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: I. Biochemical analysis.

PubMed

Okuno, S; Kitani, T; Fujisawa, H

2001-10-01

Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr(177) and Thr(196), respectively, by the upstream Ca(2+)/calmodulin-dependent protein kinases CaM-kinase kinase alpha and beta, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase alpha is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase alpha by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase alpha toward PKIV peptide, which contains the sequence surrounding Thr(196) of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca(2+)/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca(2+)/calmodulin. Six phosphorylation sites on CaM-kinase kinase alpha, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca(2+)/calmodulin suppresses phosphorylation on Ser(52), Ser(74), Thr(108), and Ser(458) by PKA, but accelerates phosphorylation on Ser(475). The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.

LPS-induced chemokine expression in both MyD88-dependent and -independent manners is regulated by Cot/Tpl2-ERK axis in macrophages.

PubMed

Bandow, Kenjiro; Kusuyama, Joji; Shamoto, Mitsuo; Kakimoto, Kyoko; Ohnishi, Tomokazu; Matsuguchi, Tetsuya

2012-05-21

LPS signaling is mediated through MyD88-dependent and -independent pathways, activating NF-?B, MAP kinases and IRF3. Cot/Tpl2 is an essential upstream kinase in LPS-mediated activation of ERKs. Here we explore the roles of MyD88 and Cot/Tpl2 in LPS-induced chemokine expression by studying myd88(-/-) and cot/tpl2(-/-) macrophages. Among the nine LPS-responsive chemokines examined, mRNA induction of ccl5, cxcl10, and cxcl13 is mediated through the MyD88-independent pathway. Notably, Cot/Tpl2-ERK signaling axis exerts negative effects on the expression of these three chemokines. In contrast, LPS-induced gene expression of ccl2, ccl7, cxcl2, cxcl3, ccl8, and cxcl9 is mediated in the MyD88-dependent manner. The Cot/Tpl2-ERK axis promotes the expression of the first four and inhibits the expression of the latter two. Thus, LPS induces expression of multiple chemokines through various signaling pathways in macrophages. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Species differences in the effects of prostanoids on MAP kinase phosphorylation, myosin light chain phosphorylation and contraction in bovine and cat iris sphincter smooth muscle.

PubMed

Kaddour-Djebbar, I; Ansari, H R; Akhtar, R A; Abdel-Latif, A A

2005-01-01

There is evidence from our own laboratory and that of others that EP-receptor ligands are strong contractile agonists in bovine iris sphincter and that FP-receptor agonists are strong contractile agonists in cat iris sphincter. Here, we have investigated the effects of prostaglandin (PG) receptor agonists of the FP-, EP-, TP- and DP-class on myosin light chain (MLC) phosphorylation, p42/p44 MAP kinase phosphorylation and contraction in the iris sphincter of bovine and cat. Using three signal transduction mechanism assays, namely MLC phosphorylation, MAP kinase phosphorylation and contraction, we demonstrated that in bovine iris sphincter the rank order of potency of the PG agonists in the contractile and MLC phosphorylation assays is as follows: E2>U46619>F2alpha>D2, and in cat F2alpha>D2>E2>U46619. In the MAP kinase assay, in bovine iris sphincter the rank order of potency is E2>F2alpha and in cat F2alpha>E2. These conclusions are supported by the following findings: (1) In the contractile assay, in the bovine sphincter the EC50s for PGF2alpha, PGE2, U46619 and PGD2 were found to be 1.4x10(-7), 5.0x10(-9), 9.0x10(-9) and 1.3x10(-6)M, respectively, and the corresponding values in the cat were 1.9x10(-8), 2.3x10(-7), 1.5x10(-6) and 6.9x10(-8)M, respectively. (2) In the MLC phophorylation assay, in the bovine sphincter PGF2alpha, PGE2, U46619 and PGD2 increased MLC phophorylation by 118%, 165%, 153% and 72%, respectively, and the corresponding values in cat were 175%, 99%, 90% and 95%, respectively. (3) In the MAP kinase assay, in the bovine iris sphincter PGF2alpha and PGE2, increased MAP kinase phosphorylation by 276% and 328%, respectively, and the corresponding values in cat were 308% and 245%, respectively. The data presented demonstrate pronounced species differences in the effects of the prostanoids on the MLC kinase signaling pathway in bovine and cat irides and furthermore confirm the existence of FP-receptors in that of the bovine.

Discovery and Characterization of Non-ATP Site Inhibitors of the Mitogen Activated Protein (MAP) Kinases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele

Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites onmore » the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38{alpha} (involved in the formation of TNF{alpha} and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional {sup 1}H/{sup 13}C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38{alpha} both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.« less

Regulation of MMP-3 expression and secretion by the chemokine eotaxin-1 in human chondrocytes

PubMed Central

2011-01-01

Background Osteoarthritis (OA) is characterized by the degradation of articular cartilage, marked by the breakdown of matrix proteins. Studies demonstrated the involvement of chemokines in this process, and some may potentially serve as diagnostic markers and therapeutic targets; however, the underlying signal transductions are not well understood. Methods We investigated the effects of the CC chemokine eotaxin-1 (CCL11) on the matrix metalloproteinase (MMP) expression and secretion in the human chondrocyte cell line SW1353 and primary chondrocytes. Results Eotaxin-1 significantly induced MMP-3 mRNA expression in a dose-dependent manner. Inhibitors of extracellular signal-regulated kinase (ERK) and p38 kinase were able to repress eotaxin-1-induced MMP-3 expression. On the contrary, Rp-adenosine-3',5'-cyclic monophosphorothioate (Rp-cAMPs), a competitive cAMP antagonist for cAMP receptors, and H-89, a protein kinase A (PKA) inhibitor, markedly enhanced eotaxin-1-induced MMP-3 expression. These results suggest that MMP-3 expression is specifically mediated by the G protein-coupled eotaxin-1 receptor activities. Interestingly, little amount of MMP-3 protein was detected in the cell lysates of eotaxin-1-treated SW1353 cells, and most of MMP-3 protein was in the culture media. Furthermore we found that the eotaxin-1-dependent MMP-3 protein secretion was regulated by phospholipase C (PLC)-protein kinase C (PKC) cascade and c-Jun N-terminal kinase (JNK)/mitogen-activated protein (MAP) kinase pathways. These data indicate a specific regulation of MMP-3 secretion also by eotaxin-1 receptor activities. Conclusions Eotaxin-1 not only induces MMP-3 gene expression but also promotes MMP-3 protein secretion through G protein-coupled eotaxin-1 receptor activities. Chemokines, such as eotaxin-1, could be a potential candidate in the diagnosis and treatment of arthritis. PMID:22114952

Protein kinase inhibitor peptide (PKI): a family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function.

PubMed

Dalton, George D; Dewey, William L

2006-02-01

Signal transduction cascades involving cAMP-dependent protein kinase are highly conserved among a wide variety of organisms. Given the universal nature of this enzyme it is not surprising that cAMP-dependent protein kinase plays a critical role in numerous cellular processes. This is particularly evident in the nervous system where cAMP-dependent protein kinase is involved in neurotransmitter release, gene transcription, and synaptic plasticity. Protein kinase inhibitor peptide (PKI) is an endogenous thermostable peptide that modulates cAMP-dependent protein kinase function. PKI contains two distinct functional domains within its amino acid sequence that allow it to: (1) potently and specifically inhibit the activity of the free catalytic subunit of cAMP-dependent protein kinase and (2) export the free catalytic subunit of cAMP-dependent protein kinase from the nucleus. Three distinct PKI isoforms (PKIalpha, PKIbeta, PKIgamma) have been identified and each isoform is expressed in the brain. PKI modulates neuronal synaptic activity, while PKI also is involved in morphogenesis and symmetrical left-right axis formation. In addition, PKI also plays a role in regulating gene expression induced by cAMP-dependent protein kinase. Future studies should identify novel physiological functions for endogenous PKI both in the nervous system and throughout the body. Most interesting will be the determination whether functional differences exist between individual PKI isoforms which is an intriguing possibility since these isoforms exhibit: (1) cell-type specific tissue expression patterns, (2) different potencies for the inhibition of cAMP-dependent protein kinase activity, and (3) expression patterns that are hormonally, developmentally and cell-cycle regulated. Finally, synthetic peptide analogs of endogenous PKI will continue to be invaluable tools that are used to elucidate the role of cAMP-dependent protein kinase in a variety of cellular processes throughout the nervous system and the rest of the body.

How MAP kinase modules function as robust, yet adaptable, circuits.

PubMed

Tian, Tianhai; Harding, Angus

2014-01-01

Genetic and biochemical studies have revealed that the diversity of cell types and developmental patterns evident within the animal kingdom is generated by a handful of conserved, core modules. Core biological modules must be robust, able to maintain functionality despite perturbations, and yet sufficiently adaptable for random mutations to generate phenotypic variation during evolution. Understanding how robust, adaptable modules have influenced the evolution of eukaryotes will inform both evolutionary and synthetic biology. One such system is the MAP kinase module, which consists of a 3-tiered kinase circuit configuration that has been evolutionarily conserved from yeast to man. MAP kinase signal transduction pathways are used across eukaryotic phyla to drive biological functions that are crucial for life. Here we ask the fundamental question, why do MAPK modules follow a conserved 3-tiered topology rather than some other number? Using computational simulations, we identify a fundamental 2-tiered circuit topology that can be readily reconfigured by feedback loops and scaffolds to generate diverse signal outputs. When this 2-kinase circuit is connected to proximal input kinases, a 3-tiered modular configuration is created that is both robust and adaptable, providing a biological circuit that can regulate multiple phenotypes and maintain functionality in an uncertain world. We propose that the 3-tiered signal transduction module has been conserved through positive selection, because it facilitated the generation of phenotypic variation during eukaryotic evolution.

How MAP kinase modules function as robust, yet adaptable, circuits

PubMed Central

Tian, Tianhai; Harding, Angus

2014-01-01

Genetic and biochemical studies have revealed that the diversity of cell types and developmental patterns evident within the animal kingdom is generated by a handful of conserved, core modules. Core biological modules must be robust, able to maintain functionality despite perturbations, and yet sufficiently adaptable for random mutations to generate phenotypic variation during evolution. Understanding how robust, adaptable modules have influenced the evolution of eukaryotes will inform both evolutionary and synthetic biology. One such system is the MAP kinase module, which consists of a 3-tiered kinase circuit configuration that has been evolutionarily conserved from yeast to man. MAP kinase signal transduction pathways are used across eukaryotic phyla to drive biological functions that are crucial for life. Here we ask the fundamental question, why do MAPK modules follow a conserved 3-tiered topology rather than some other number? Using computational simulations, we identify a fundamental 2-tiered circuit topology that can be readily reconfigured by feedback loops and scaffolds to generate diverse signal outputs. When this 2-kinase circuit is connected to proximal input kinases, a 3-tiered modular configuration is created that is both robust and adaptable, providing a biological circuit that can regulate multiple phenotypes and maintain functionality in an uncertain world. We propose that the 3-tiered signal transduction module has been conserved through positive selection, because it facilitated the generation of phenotypic variation during eukaryotic evolution. PMID:25483189

Trivalent dimethylarsenic compound induces histone H3 phosphorylation and abnormal localization of Aurora B kinase in HepG2 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Suzuki, Toshihide, E-mail: toshi-su@pharm.teikyo-u.ac.j; Miyazaki, Koichi; Kita, Kayoko

2009-12-15

Trivalent dimethylarsinous acid [DMA(III)] has been shown to induce mitotic abnormalities, such as centrosome abnormality, multipolar spindles, multipolar division, and aneuploidy, in several cell lines. In order to elucidate the mechanisms underlying these mitotic abnormalities, we investigated DMA(III)-mediated changes in histone H3 phosphorylation and localization of Aurora B kinase, which is a key molecule in cell mitosis. DMA(III) caused the phosphorylation of histone H3 (ser10) and was distributed predominantly in mitotic cells, especially in prometaphase cells. By contrast, most of the phospho-histone H3 was found to be localized in interphase cells after treatment with inorganic arsenite [iAs(III)], suggesting the involvementmore » of a different pathway in phosphorylation. DMA(III) activated Aurora B kinase and slightly activated ERK MAP kinase. Phosphorylation of histone H3 by DMA(III) was effectively reduced by ZM447439 (Aurora kinase inhibitor) and slightly reduced by U0126 (MEK inhibitor). By contrast, iAs(III)-dependent histone H3 phosphorylation was markedly reduced by U0126. Aurora B kinase is generally localized in the midbody during telophase and plays an important role in cytokinesis. However, in some cells treated with DMA(III), Aurora B was not localized in the midbody of telophase cells. These findings suggested that DMA(III) induced a spindle abnormality, thereby activating the spindle assembly checkpoint (SAC) through the Aurora B kinase pathway. In addition, cytokinesis was not completed because of the abnormal localization of Aurora B kinase by DMA(III), thereby resulting in the generation of multinucleated cells. These results provide insight into the mechanism of arsenic tumorigenesis.« less

Registered Report: COT drives resistance to RAF inhibition through MAP kinase pathway reactivation.

PubMed

Sharma, Vidhu; Young, Lisa; Cavadas, Miguel; Owen, Kate

2016-03-21

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered Report describes the proposed replication plan of key experiments from "COT drives resistance to RAF inhibition through MAPK pathway reactivation" by Johannessen and colleagues, published in Nature in 2010 (Johannessen et al., 2010). The key experiments to be replicated are those reported in Figures 3B, 3D-E, 3I, and 4E-F. In Figures 3B, D-E, RPMI-7951 and OUMS023 cells were reported to exhibit robust ERK/MEK activity concomitant with reduced growth sensitivity in the presence of the BRAF inhibitor PLX4720. MAP3K8 (COT/TPL2) directly regulated MEK/ERK phosphorylation, as the treatment of RPMI-7951 cells with a MAP3K8 kinase inhibitor resulted in a dose-dependent suppression of MEK/ERK activity (Figure 3I). In contrast, MAP3K8-deficient A375 cells remained sensitive to BRAF inhibition, exhibiting reduced growth and MEK/ERK activity during inhibitor treatment. To determine if RAF and MEK inhibitors together can overcome single-agent resistance, MAP3K8-expressing A375 cells treated with PLX4720 along with MEK inhibitors significantly inhibited both cell viability and ERK activation compared to treatment with PLX4720 alone, as reported in Figures 4E-F. The Reproducibility Project: Cancer Biology is collaboration between the Center for Open Science and Science Exchange and the results of the replications will be published in eLife.

A MAP4 kinase related to Ste20 is a nutrient-sensitive regulator of mTOR signalling

PubMed Central

Findlay, Greg M.; Yan, Lijun; Procter, Julia; Mieulet, Virginie; Lamb, Richard F.

2007-01-01

The mTOR (mammalian target of rapamycin) signalling pathway is a key regulator of cell growth and is controlled by growth factors and nutrients such as amino acids. Although signalling pathways from growth factor receptors to mTOR have been elucidated, the pathways mediating signalling by nutrients are poorly characterized. Through a screen for protein kinases active in the mTOR signalling pathway in Drosophila we have identified a Ste20 family member (MAP4K3) that is required for maximal S6K (S6 kinase)/4E-BP1 [eIF4E (eukaryotic initiation factor 4E)-binding protein 1] phosphorylation and regulates cell growth. Importantly, MAP4K3 activity is regulated by amino acids, but not the growth factor insulin and is not regulated by the mTORC1 inhibitor rapamycin. Our results therefore suggest a model whereby nutrients signal to mTORC1 via activation of MAP4K3. PMID:17253963

Functional characterization of the MKC1 gene of Candida albicans, which encodes a mitogen-activated protein kinase homolog related to cell integrity.

PubMed Central

Navarro-García, F; Sánchez, M; Pla, J; Nombela, C

1995-01-01

Mitogen-activated protein (MAP) kinases represent a group of serine/threonine protein kinases playing a central role in signal transduction processes in eukaryotic cells. Using a strategy based on the complementation of the thermosensitive autolytic phenotype of slt2 null mutants, we have isolated a Candida albicans homolog of Saccharomyces cerevisiae MAP kinase gene SLT2 (MPK1), which is involved in the recently outlined PKC1-controlled signalling pathway. The isolated gene, named MKC1 (MAP kinase from C. albicans), coded for a putative protein, Mkc1p, of 58,320 Da that displayed all the characteristic domains of MAP kinases and was 55% identical to S. cerevisiae Slt2p (Mpk1p). The MKC1 gene was deleted in a diploid Candida strain, and heterozygous and homozygous strains, in both Ura+ and Ura- backgrounds, were obtained to facilitate the analysis of the function of the gene. Deletion of the two alleles of the MKC1 gene gave rise to viable cells that grew at 28 and 37 degrees C but, nevertheless, displayed a variety of phenotypic traits under more stringent conditions. These included a low growth yield and a loss of viability in cultures grown at 42 degrees C, a high sensitivity to thermal shocks at 55 degrees C, an enhanced susceptibility to caffeine that was osmotically remediable, and the formation of a weak cell wall with a very low resistance to complex lytic enzyme preparations. The analysis of the functions downstream of the MKC1 gene should contribute to understanding of the connection of growth and morphogenesis in pathogenic fungi. PMID:7891715

The Dictyostelium MAP kinase kinase DdMEK1 regulates chemotaxis and is essential for chemoattractant-mediated activation of guanylyl cyclase.

PubMed Central

Ma, H; Gamper, M; Parent, C; Firtel, R A

1997-01-01

We have identified a MAP kinase kinase (DdMEK1) that is required for proper aggregation in Dictyostelium. Null mutations produce extremely small aggregate sizes, resulting in the formation of slugs and terminal fruiting bodies that are significantly smaller than those of wild-type cells. Time-lapse video microscopy and in vitro assays indicate that the cells are able to produce cAMP waves that move through the aggregation domains. However, these cells are unable to undergo chemotaxis properly during aggregation in response to the chemoattractant cAMP or activate guanylyl cyclase, a known regulator of chemotaxis in Dictyostelium. The activation of guanylyl cyclase in response to osmotic stress is, however, normal. Expression of putative constitutively active forms of DdMEK1 in a ddmek1 null background is capable, at least partially, of complementing the small aggregate size defect and the ability to activate guanylyl cyclase. However, this does not result in constitutive activation of guanylyl cyclase, suggesting that DdMEK1 activity is necessary, but not sufficient, for cAMP activation of guanylyl cyclase. Analysis of a temperature-sensitive DdMEK1 mutant suggests that DdMEK1 activity is required throughout aggregation at the time of guanylyl cyclase activation, but is not essential for proper morphogenesis during the later multicellular stages. The activation of the MAP kinase ERK2, which is essential for chemoattractant activation of adenylyl cyclase, is not affected in ddmek1 null strains, indicating that DdMEK1 does not regulate ERK2 and suggesting that at least two independent MAP kinase cascades control aggregation in Dictyostelium. PMID:9250676

A Phosphorylation Switch Regulates the Transcriptional Activation of Cell Cycle Regulator p21 by Histone Deacetylase Inhibitors*

PubMed Central

Simboeck, Elisabeth; Sawicka, Anna; Zupkovitz, Gordin; Senese, Silvia; Winter, Stefan; Dequiedt, Franck; Ogris, Egon; Di Croce, Luciano; Chiocca, Susanna; Seiser, Christian

2010-01-01

Histone deacetylase inhibitors induce cell cycle arrest and apoptosis in tumor cells and are, therefore, promising anti-cancer drugs. The cyclin-dependent kinase inhibitor p21 is activated in histone deacetylase (HDAC) inhibitor-treated tumor cells, and its growth-inhibitory function contributes to the anti-tumorigenic effect of HDAC inhibitors. We show here that induction of p21 by trichostatin A involves MAP kinase signaling. Activation of the MAP kinase signaling pathway by growth factors or stress signals results in histone H3 serine 10 phosphorylation at the p21 promoter and is crucial for acetylation of the neighboring lysine 14 and recruitment of activated RNA polymerase II in response to trichostatin A treatment. In non-induced cells, the protein phosphatase PP2A is associated with the p21 gene and counteracts its activation. Induction of p21 is linked to simultaneous acetylation and phosphorylation of histone H3. The dual modification mark H3S10phK14ac at the activated p21 promoter is recognized by the phospho-binding protein 14-3-3ζ, which protects the phosphoacetylation mark from being processed by PP2A. Taken together we have revealed a cross-talk of reversible phosphorylation and acetylation signals that controls the activation of p21 by HDAC inhibitors and identify the phosphatase PP2A as chromatin-associated transcriptional repressor in mammalian cells. PMID:20952396

Anti-inflammatory effects of secondary metabolites of marine Pseudomonas sp. in human neutrophils are through inhibiting P38 MAPK, JNK, and calcium pathways.

PubMed

Yang, Shun-Chin; Sung, Ping-Jyun; Lin, Chwan-Fwu; Kuo, Jimmy; Chen, Chun-Yu; Hwang, Tsong-Long

2014-01-01

Activated neutrophils play a significant role in the pathogenesis of many inflammatory diseases. The metabolites of marine microorganisms are increasingly employed as sources for developing new drugs; however, very few marine drugs have been studied in human neutrophils. Herein, we showed that secondary metabolites of marine Pseudomonas sp. (N11) significantly inhibited superoxide anion generation and elastase release in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-activated human neutrophils, with IC50 values of 0.67±0.38 µg/ml and 0.84±0.12 µg/ml, respectively. In cell-free systems, neither superoxide anion-scavenging effect nor inhibition of elastase activity was associated with the suppressive effects of N11. N11 inhibited the phosphorylation of p38 MAP kinase and JNK, but not Erk and Akt, in FMLP-induced human neutrophils. Also, N11 dose-dependently attenuated the transient elevation of intracellular calcium concentration in activated neutrophils. In contrast, N11 failed to alter phorbol myristate acetate-induced superoxide anion generation, and the inhibitory effects of N11 were not reversed by protein kinase A inhibitor. In conclusion, the anti-inflammatory effects of N11 on superoxide anion generation and elastase release in activated human neutrophils are through inhibiting p38 MAP kinase, JNK, and calcium pathways. Our results suggest that N11 has the potential to be developed to treat neutrophil-mediated inflammatory diseases.

Genetic control of an epigenetic cell degeneration syndrome in Podospora anserina.

PubMed

Haedens, Vicki; Malagnac, Fabienne; Silar, Philippe

2005-06-01

Filamentous fungi frequently present degenerative processes, whose molecular basis is very often unknown. Here, we present three mutant screens that result in the identification of 29 genes that directly or indirectly control Crippled Growth (CG), an epigenetic cell degeneration of the filamentous ascomycete Podospora anserina. Two of these genes were previously shown to encode a MAP kinase kinase kinase and an NADPH oxidase involved in a signal transduction cascade that participates in stationary phase differentiations, fruiting body development and defence against fungal competitors. The numerous genes identified can be incorporated in a model in which CG results from the sustained activation of the MAP kinase cascade. Our data also emphasize the complex regulatory network underlying three interconnected processes in P. anserina: sexual reproduction, defence against competitors, and cell degeneration.

Context Specificity of Stress-activated Mitogen-activated Protein (MAP) Kinase Signaling: The Story as Told by Caenorhabditis elegans.

PubMed

Andrusiak, Matthew G; Jin, Yishi

2016-04-08

Stress-associated p38 and JNK mitogen-activated protein (MAP) kinase signaling cascades trigger specific cellular responses and are involved in multiple disease states. At the root of MAP kinase signaling complexity is the differential use of common components on a context-specific basis. The roundwormCaenorhabditis eleganswas developed as a system to study genes required for development and nervous system function. The powerful genetics ofC. elegansin combination with molecular and cellular dissections has led to a greater understanding of how p38 and JNK signaling affects many biological processes under normal and stress conditions. This review focuses on the studies revealing context specificity of different stress-activated MAPK components inC. elegans. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Stress and vascular responses: atheroprotective effect of laminar fluid shear stress in endothelial cells: possible role of mitogen-activated protein kinases.

PubMed

Yoshizumi, Masanori; Abe, Jun-Ichi; Tsuchiya, Koichiro; Berk, Bradford C; Tamaki, Toshiaki

2003-03-01

Atherosclerosis preferentially occurs in areas of turbulent blood flow and low fluid shear stress, whereas laminar blood flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent findings suggest a steady laminar blood flow decreases EC apoptosis and inhibits TNF-mediated EC activation. EC apoptosis or activation is suggested to be involved in plaque erosion, which may lead to platelet aggregation. TNF-alpha regulates gene expression in ECs, in part, by stimulating mitogen-activated protein (MAP) kinases, which phosphorylate transcription factors. We hypothesized that steady laminar flow inhibits cytokine-mediated activation of MAP kinases in ECs. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm(2)) on TNF-alpha-stimulated activity of three MAP kinases in human umbilical vein ECs (HUVEC): extracellular signal-regulated kinase (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. TNF-alpha activated ERK1/2, JNK, and p38 maximally at 15 min in HUVEC. Pre-exposing HUVEC for 10 min to flow inhibited TNF-alpha activation of JNK, but showed no significant effect on ERK1/2 or p38 activation. Incubation of HUVEC with PD98059, a specific ERK1/2 inhibitor, blocked the flow-mediated inhibition of TNF activation of JNK. Transfection studies with dominant-negative constructs of the protein kinase MEK5 suggested an important role for big mitogen-activated protein kinase 1 (BMK1) in flow-mediated regulation of EC activation by TNF-alpha. Understanding the mechanisms by which steady laminar flow regulates JNK activation by cytokines may provide insight into the atheroprotective mechanisms induced by laminar blood flow.

Adhesion-Dependent Redistribution of MAP Kinase and MEK Promotes Muscarinic Receptor-Mediated Signaling to the Nucleus

PubMed Central

Slack, Barbara E.; Siniaia, Marina S.

2008-01-01

The mitogen-activated protein kinases (MAPKs) are activated by extracellular signals, and translocate to the nucleus where they modulate transcription. Integrin-mediated cell adhesion to extracellular matrix (ECM) proteins is required for efficient transmission of MAPK-based signals initiated by growth factors. However, the modulation of G protein-coupled receptor (GPCR) signaling by adhesion is less well understood. In the present study we assessed the impact of cell adhesion on MAPK activation by muscarinic M3 receptors. The muscarinic agonist carbachol more efficiently promoted stress fiber formation and tyrosine phosphorylation of focal adhesion-associated proteins in M3 receptor-expressing cells adherent to fibronectin or collagen type I, as compared to polylysine. Overall MAPK activation was robust in cells adherent to all three substrata. However, total levels of MAPK and mitogen-activated protein kinase kinase (MEK) in the nucleus were significantly greater in cells adherent to ECM proteins for 2.5 hours, and levels of activated MAPK and MEK in the nuclei of these cells were higher following carbachol stimulation, relative to levels in cells adherent to polylysine. MEK inhibitors did not prevent adhesion-dependent translocation of MAPK and MEK to the nucleus, and increased nuclear phospho-MEK levels in carbachol-stimulated cells. The results suggest that adhesion of cells to ECM triggers the redistribution of MAPK and MEK to the nucleus, possibly as a result of the cytoskeletal rearrangements that accompany cell spreading. This may represent a mechanism for priming the nucleus with MEK and MAPK, leading to more rapid and pronounced increases in intranuclear phospho-MAPK upon GPCR stimulation. PMID:15779001

Pharmacological activity and protein phosphorylation caused by nitric oxide-releasing microparticles.

PubMed

Yoo, Jin-Wook; Choe, Eun-Sang; Ahn, Sung-Min; Lee, Chi H

2010-01-01

Nitric oxide (NO)-releasing microparticles were developed as a potential treatment option against various blood flow irregulations including sexual dysfunction, atherosclerosis and metal stent-induced restenosis. Polymeric microparticles containing diethylenetriamine diazeniumdiolate (DETA NONOate), a NO donor, were prepared using modified double-emulsion solvent evaporation method to maximize the loading efficacy and stability of DETA NONOate. The pharmacological effects of the NO-releasing microparticles were evaluated by examining the changes in the vaginal blood flow in rats. The effects of NO on the phosphorylation of protein kinase C (PKC) and mitogen activated protein (MAP) kinases in excised vaginal mucosa, such as extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and p38, were examined using immunoblotting technique to determine whether NO activates PKC, which subsequently plays an integral role in the formation of PKC-MAP kinase modules. The viability of vagina cells (VK2E6E7) upon exposure to NO-releasing microparticles was examined for cytotoxicity assessment. In contrast to rapid and short-term effects of non-formulated DETA NONOate, microparticles containing DETA NONOate exerted beneficial effects on the blood flow (148+/-13%) for an extended period of time, inducing a significant change at 5 min after its application and the maximum blood flow of 172+/-23% at 120 min. The enhanced vaginal blood flow was maintained for up to 210 min and gradually returned to the baseline afterward. The results of Western immunoblotting study displayed differential expression of MAP kinases (ERK1/2 and JNK) upon NO treatment, clearly demonstrating that PKC is involved in the blood flow regulation process. There were no significant changes in cell viability in vaginal cells upon exposure to NO-releasing microparticles as compared with the control. The results of this work supported that NO-releasing microparticles could improve the vaginal blood flow without causing cytotoxic effects and PKC-MAP kinase modules are involved in the NO-induced blood flow regulation process.

Mice lacking MKP-1 and MKP-5 Reveal Hierarchical Regulation of Regenerative Myogenesis.

PubMed

Shi, Hao; Gatzke, Florian; Molle, Julia M; Lee, Han Bin; Helm, Emma T; Oldham, Jessie J; Zhang, Lei; Gerrard, David E; Bennett, Anton M

2015-11-12

The relative contribution of the MAP kinase phosphatases (MKPs) in the integration of MAP kinase-dependent signaling during regenerative myogenesis has yet to be fully investigated. MKP-1 and MKP-5 maintain skeletal muscle homeostasis by providing positive and negative effects on regenerative myogenesis, respectively. In order to define the hierarchical contributions of MKP-1 and MKP-5 in the regulation of regenerative myogenesis we genetically ablated both MKPs in mice. MKP-1/MKP 5-deficient double-knockout (MKP1/5- DKO) mice were viable, and upon skeletal muscle injury, were severely impaired in their capacity to regenerate skeletal muscle. Satellite cells were fewer in number in MKP1/5-DKO mice and displayed a reduced proliferative capacity as compared with those derived from wild-type mice. MKP1/5-DKO mice exhibited increased inflammation and the macrophage M1 to M2 transition during the resolution of inflammation was impaired following injury. These results demonstrate that the actions of MKP-1 to positively regulate myogenesis predominate over those of MKP-5, which negatively regulates myogenesis. Hence, MKP-1 and MKP-5 function to maintain skeletal muscle homeostasis through non-overlapping and opposing signaling pathways.

Mice lacking MKP-1 and MKP-5 Reveal Hierarchical Regulation of Regenerative Myogenesis

PubMed Central

Shi, Hao; Gatzke, Florian; Molle, Julia M.; Lee, Han Bin; Helm, Emma T.; Oldham, Jessie J.; Zhang, Lei; Gerrard, David E.; Bennett, Anton M.

2015-01-01

The relative contribution of the MAP kinase phosphatases (MKPs) in the integration of MAP kinase-dependent signaling during regenerative myogenesis has yet to be fully investigated. MKP-1 and MKP-5 maintain skeletal muscle homeostasis by providing positive and negative effects on regenerative myogenesis, respectively. In order to define the hierarchical contributions of MKP-1 and MKP-5 in the regulation of regenerative myogenesis we genetically ablated both MKPs in mice. MKP-1/MKP 5-deficient double-knockout (MKP1/5- DKO) mice were viable, and upon skeletal muscle injury, were severely impaired in their capacity to regenerate skeletal muscle. Satellite cells were fewer in number in MKP1/5-DKO mice and displayed a reduced proliferative capacity as compared with those derived from wild-type mice. MKP1/5-DKO mice exhibited increased inflammation and the macrophage M1 to M2 transition during the resolution of inflammation was impaired following injury. These results demonstrate that the actions of MKP-1 to positively regulate myogenesis predominate over those of MKP-5, which negatively regulates myogenesis. Hence, MKP-1 and MKP-5 function to maintain skeletal muscle homeostasis through non-overlapping and opposing signaling pathways. PMID:27064463

Herbal medicine Inchin-ko-to (TJ-135) prevents liver fibrosis and enzyme-altered lesions in rat liver cirrhosis induced by a choline-deficient L-amino acid-defined diet.

PubMed

Sakaida, Isao; Tsuchiya, Masako; Kawaguchi, Kotarou; Kimura, Teruaki; Terai, Shuji; Okita, Kiwamu

2003-06-01

The herbal medicine Inchin-ko-to (TJ-135), extract power from three herbs, has recently been reported possessing anti-apoptotic activity. The aim of this study was to investigate whether TJ-135 has any influence on the development of preneoplastic lesions as well as liver fibrosis. The effects of the TJ-135 were examined using the choline-deficient L-amino acid-defined diet-induced liver fibrosis model. In addition, the effect of TJ-135 on mitogen-activated protein (MAP) kinase, type III procollagen mRNA expression and the medium N-terminal procollagen III propeptide (PIIINP) concentration in a hepatic stellate cell line (LI90) were examined. TJ-135 prevented fibrosis in a dose-dependent manner up to 1.5% (w/w). TJ-135 also reduced the expression of type III procollagen mRNA in the liver, as well as the number of activated stellate cells. Furthermore, TJ-135 reduced the area of preneoplastic lesions in the liver. With LI90 cells, TJ-135 reduced MAP kinase (ERK and JNK but not P38) activities resulting in reduced type III procollagen mRNA and PIIINP concentrations in the medium in a dose-dependent manner. These results indicate that although TJ-135 has anti-apoptotic activity, TJ-135 does not increase preneoplastic lesions but significantly reduces liver fibrosis through the inhibition of stellate cell activation without a reduction of hepatocyte cell death.

Pneumolysin-induced CXCL8 production by nasopharyngeal epithelial cells is dependent on calcium flux and MAPK activation via Toll-like receptor 4.

PubMed

Dogan, Semih; Zhang, Qibo; Pridmore, Alison C; Mitchell, Timothy J; Finn, Adam; Murdoch, Craig

2011-01-01

The natural niche of Streptococcus pneumoniae is the nasopharyngeal mucosa and nasopharyngeal carriage of pneumococci is widely prevalent. Pneumolysin (Ply), a pore-forming protein produced by S. pneumonia, may be important in driving the innate immune response of the nasopharynx. We studied the Ply-induced production of CXCL8 by nasopharyngeal cells and further analysed the mechanism of this induction. Detroit nasopharyngeal cells were stimulated with supernatants derived from bacterial cultures of Ply-deficient, wild-type pneumococci and recombinant Ply, and CXCL8 measured by ELISA. The role of MAP kinase family members in Ply-induced CXCL8 production was analysed using specific inhibitors, NF-κB activity was measured by immunoblot and Ply-mediated TLR4 activation analysed by a CXCL8 promotor luciferase assay. Ply significantly increased production of CXCL8 in Detroit and primary nasal cells. Flow cytometric analysis showed that Detroit cells express cell surface TLR4. CXCL8 production was dependent on changes in the intracellular Ca(2+) levels and also by NF-κB via activation of TLR4, and MAP kinase signalling. Ply induces production of CXCL8 by nasopharyngeal cells using signalling mechanisms involving Ca(2+) mobilisation and activation of MAPK and NF-κB via TLR4. This may be important in regulating nasopharyngeal immunity against pneumococcal colonization. Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.

MAP kinase-signaling controls nuclear translocation of tripeptidyl-peptidase II in response to DNA damage and oxidative stress.

PubMed

Preta, Giulio; de Klark, Rainier; Chakraborti, Shankhamala; Glas, Rickard

2010-08-27

Reactive oxygen species (ROS) are a continuous hazard in eukaroytic cells by their ability to cause damage to biomolecules, in particular to DNA. Previous data indicated that the cytosolic serine peptidase tripeptidyl-peptidase II (TPPII) translocates into the nucleus of most tumor cell lines in response to gamma-irradiation and ROS production; an event that promoted p53 expression as well as caspase-activation. We here observed that nuclear translocation of TPPII was dependent on signaling by MAP kinases, including p38MAPK. Further, this was caused by several types of DNA-damaging drugs, a DNA cross-linker (cisplatinum), an inhibitor of topoisomerase II (etoposide), and to some extent also by nucleoside-analogues (5-fluorouracil, hydroxyurea). In the minority of tumor cell lines where TPPII was not translocated into the nucleus in response to DNA damage we observed reduced intracellular ROS levels, and the expression levels of redox defense systems were increased. Further, treatment with the ROS-inducer gamma-hexa-chloro-cyclohexane (gamma-HCH, lindane), an inhibitor of GAP junctions, restored nuclear translocation of TPPII in these cell lines upon gamma-irradiation. Moreover, blocking nuclear translocation of TPPII in etoposide-treated cells, by using a peptide-derived inhibitor (Z-Gly-Leu-Ala-OH), attenuated expression of gamma-H2AX in gamma-irradiated melanoma cells. Our results indicated a role for TPPII in MAPK-dependent DNA damage signaling. Copyright 2010 Elsevier Inc. All rights reserved.

MAP3K1 May be a Promising Susceptibility Gene for Type 2 Diabetes Mellitus in an Iranian Population

PubMed Central

Torkamandi, Shahram; Bastami, Milad; Ghaedi, Hamid; Moghadam, Fateme; Mirfakhraie, Reza; Omrani, Mir Davood

2016-01-01

Considering that MAPK (mitogen- activated protein kinase) signaling pathway has an important role in the progression of inflammatory cytokine secretion in type 2 diabetes mellitus (T2DM), we have recently investigated the reported genetic polymorphism from genome wide association study in MAP3K1 (mitogen-activated protein kinase kinase kinase 1) in diabetes as an important member of MAPK signaling. This study aimed to investigate the possible association of rs10461617 at the upstream of MAP3K1 gene in an Iranian case-control study with the risk of T2DM. The study population was comprised of 342 unrelated Iranian individuals including 177 patients with T2DM and 165 unrelated healthy control subjects. Genotyping was performed using PCR-RFLP and confirmed with sequencing. In a logistic regression analysis, the rs10461617A allele was associated with a significantly higher risk of T2DM assuming the log- additive model (OR: 1.44, 95% CI: 1.01-2.05, P = 0.039). In conclusion, we provided the first evidence for the association of rs10461617 at the upstream of MAP3K1 with the risk of T2DM in an Iranian population. PMID:27942499

Role of Arg228 in the phosphorylation of galactokinase: the mechanism of GHMP kinases by quantum mechanics/molecular mechanics studies.

PubMed

Huang, Meilan; Li, Xiaozhou; Zou, Jian-Wei; Timson, David J

2013-07-16

GHMP kinases are a group of structurally related small molecule kinases. They have been found in all kingdoms of life and are mostly responsible for catalyzing the ATP-dependent phosphorylation of intermediary metabolites. Although the GHMP kinases are of clinical, pharmaceutical, and biotechnological importance, the mechanism of GHMP kinases is controversial. A catalytic base mechanism was suggested for mevalonate kinase that has a structural feature of the γ-phosphate of ATP close to an aspartate residue; however, for one GHMP family member, homoserine kinase, where the residue acting as general base is absent, a direct phosphorylation mechanism was suggested. Furthermore, it was proposed by some authors that all the GHMP kinases function by a similar mechanism. This controversy in mechanism has limited our ability to exploit these enzymes as drug targets and in biotechnology. Here the phosphorylation reaction mechanism of the human galactokinase, a member of the GHMP kinase family, was investigated using molecular dynamics simulations and density functional theory-based quantum mechanics/molecular mechanics calculations (B3LYP-D/AMBER99). The reaction coordinates were localized by potential energy scan using an adiabatic mapping method. Our results indicate that a highly conserved Glu174 captures Arg105 in the proximity of the α-phosphate of ATP, forming a H-bond network; therefore, the mobility of ATP in the large oxyanion hole is restricted. Arg228 functions to stabilize the negative charge developed at the β,γ-bridging oxygen of the ATP during bond cleavage. The reaction occurs via a direct phosphorylation mechanism, and the Asp186 in the proximity of ATP does not directly participate in the reaction pathway. Because Arg228 is not conserved among GHMP kinases, reagents which form interactions with Arg228, and therefore can interrupt its function in phosphorylation, may be developed into potential selective inhibitors for galactokinase.

Midazolam suppresses interleukin-1β-induced interleukin-6 release from rat glial cells

PubMed Central

2011-01-01

Background Peripheral-type benzodiazepine receptor (PBR) expression levels are low in normal human brain, but their levels increase in inflammation, brain injury, neurodegenerative states and gliomas. It has been reported that PBR functions as an immunomodulator. The mechanisms of action of midazolam, a benzodiazepine, in the immune system in the CNS remain to be fully elucidated. We previously reported that interleukin (IL)-1β stimulates IL-6 synthesis from rat C6 glioma cells and that IL-1β induces phosphorylation of inhibitory kappa B (IκB), p38 mitogen-activated protein (MAP) kinase, stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2, and signal transducer and activator of transcription (STAT)3. It has been shown that p38 MAP kinase is involved in IL-1β-induced IL-6 release from these cells. In the present study, we investigated the effect of midazolam on IL-1β-induced IL-6 release from C6 cells, and the mechanisms of this effect. Methods Cultured C6 cells were stimulated by IL-1β. IL-6 release from C6 cells was measured using an enzyme-linked immunosorbent assay, and phosphorylation of IκB, the MAP kinase superfamily, and STAT3 was analyzed by Western blotting. Results Midazolam, but not propofol, inhibited IL-1β-stimulated IL-6 release from C6 cells. The IL-1β-stimulated levels of IL-6 were suppressed by wedelolactone (an inhibitor of IκB kinase), SP600125 (an inhibitor of SAPK/JNK), and JAK inhibitor I (an inhibitor of JAK 1, 2 and 3). However, IL-6 levels were not affected by PD98059 (an inhibitor of MEK1/2). Midazolam markedly suppressed IL-1β-stimulated STAT3 phosphorylation without affecting the phosphorylation of p38 MAP kinase, SAPK/JNK or IκB. Conclusion These results strongly suggest that midazolam inhibits IL-1β-induced IL-6 release in rat C6 glioma cells via suppression of STAT3 activation. Midazolam may affect immune system function in the CNS. PMID:21682888

Regulation of Cell Cycle and Stress Responses to Hydrostatic Pressure in Fission Yeast

PubMed Central

George, Vinoj T.; Brooks, Gavin

2007-01-01

We have investigated the cellular responses to hydrostatic pressure by using the fission yeast Schizosaccharomyces pombe as a model system. Exposure to sublethal levels of hydrostatic pressure resulted in G2 cell cycle delay. This delay resulted from Cdc2 tyrosine-15 (Y-15) phosphorylation, and it was abrogated by simultaneous disruption of the Cdc2 kinase regulators Cdc25 and Wee1. However, cell cycle delay was independent of the DNA damage, cytokinesis, and cell size checkpoints, suggesting a novel mechanism of Cdc2-Y15 phosphorylation in response to hydrostatic pressure. Spc1/Sty1 mitogen-activated protein (MAP) kinase, a conserved member of the eukaryotic stress-activated p38, mitogen-activated protein (MAP) kinase family, was rapidly activated after pressure stress, and it was required for cell cycle recovery under these conditions, in part through promoting polo kinase (Plo1) phosphorylation on serine 402. Moreover, the Spc1 MAP kinase pathway played a key role in maintaining cell viability under hydrostatic pressure stress through the bZip transcription factor, Atf1. Further analysis revealed that prestressing cells with heat increased barotolerance, suggesting adaptational cross-talk between these stress responses. These findings provide new insight into eukaryotic homeostasis after exposure to pressure stress. PMID:17699598

Association analysis between mitogen-activated protein/extracellular signal-regulated kinase (MEK) gene polymorphisms and depressive disorder in the Han Chinese population.

PubMed

Hu, Yingyan; Hong, Wu; Smith, Alicia; Yu, Shunying; Li, Zezhi; Wang, Dongxiang; Yuan, Chengmei; Cao, Lan; Wu, Zhiguo; Huang, Jia; Fralick, Drew; Phillips, Michael Robert; Fang, Yiru

2017-11-01

Recent research findings suggest that BDNF and BDNF signaling pathways participate in the development of major depressive disorder. Mitogen-activated extracellular signal-regulated kinase (MEK) is the most important kinase in the extracellular signal-regulated kinase pathway, and the extracellular signal-regulated kinase pathway is the key signaling pathway of BDNF, so it may play a role in development of depressive disorder. The aim of this study is to investigate the association between polymorphisms of the MAP2K1 (also known as MEK) gene and depressive disorder. Three single nucleotide polymorphisms (SNPs), were significantly associated with depressive disorder: rs1549854 (p = 0.006), rs1432441 (p = 0.025), and rs7182853 (p = 0.039). When subdividing the sample by gender, two of the SNPs remained statistically associated with depressive disorder in females: rs1549854 (p = 0.013) and rs1432441 (p = 0.04). The rs1549854 and rs1432441 polymorphisms of the MAP2K1 gene may be associated with major depressive disorder, especially in females. This study is the first to report that the MAP2K1 gene may be a genetic marker for depressive disorder. Copyright © 2017 Elsevier B.V. All rights reserved.

The facilitated component of intestinal glucose absorption

PubMed Central

Kellett, George L

2001-01-01

Over the last decade, a debate has developed about the mechanism of the passive or ‘diffusive’ component of intestinal glucose absorption and, indeed, whether it even exists. Pappenheimer and colleagues have proposed that paracellular solvent drag contributes a passive component, which, at high concentrations of sugars similar to those in the jejunal lumen immediately after a meal, is severalfold greater than the active component mediated by the Na+-glucose cotransporter SGLT1. On the other hand, Ferraris & Diamond maintain that the kinetics of glucose absorption can be explained solely in terms of SGLT1 and that a passive or paracellular component plays little, if any, part. Recently, we have provided new evidence that the passive component of glucose absorption exists, but is in fact facilitated since it is mediated by the rapid, glucose-dependent activation and recruitment of the facilitative glucose transporter GLUT2 to the brush-border membrane; regulation involves a protein kinase C (PKC)-dependent pathway activated by glucose transport through SGLT1 and also involves mitogen-activated protein kinase (MAP kinase) signalling pathways. This topical review seeks to highlight the significant points of the debate, to show how our proposals on GLUT2 impact on different aspects of the debate and to look at the regulatory events that are likely to be involved in the short-term regulation of sugar absorption during the assimilation of a meal. PMID:11251042

Autophosphorylation-based calcium (Ca2+) sensitivity priming and Ca2+/Calmodulin inhibition of Arabidopsis thaliana Ca2+-dependent protein kinase 28 (CPK28)

USDA-ARS?s Scientific Manuscript database

Plant calcium (Ca2+) dependent protein kinases (CPKs) are composed of a dual specificity (Ser/Thr and Tyr) kinase domain tethered to a Calmodulin-like domain (CLD) via an autoinhibitory junction (J) and represent the primary Ca2+-dependent protein kinase activities in plant systems. While regulation...

Bioinformatics-driven discovery of rational combination for overcoming EGFR-mutant lung cancer resistance to EGFR therapy.

PubMed

Kim, Jihye; Vasu, Vihas T; Mishra, Rangnath; Singleton, Katherine R; Yoo, Minjae; Leach, Sonia M; Farias-Hesson, Eveline; Mason, Robert J; Kang, Jaewoo; Ramamoorthy, Preveen; Kern, Jeffrey A; Heasley, Lynn E; Finigan, James H; Tan, Aik Choon

2014-09-01

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer death in the United States. Targeted tyrosine kinase inhibitors (TKIs) directed against the epidermal growth factor receptor (EGFR) have been widely and successfully used in treating NSCLC patients with activating EGFR mutations. Unfortunately, the duration of response is short-lived, and all patients eventually relapse by acquiring resistance mechanisms. We performed an integrative systems biology approach to determine essential kinases that drive EGFR-TKI resistance in cancer cell lines. We used a series of bioinformatics methods to analyze and integrate the functional genetics screen and RNA-seq data to identify a set of kinases that are critical in survival and proliferation in these TKI-resistant lines. By connecting the essential kinases to compounds using a novel kinase connectivity map (K-Map), we identified and validated bosutinib as an effective compound that could inhibit proliferation and induce apoptosis in TKI-resistant lines. A rational combination of bosutinib and gefitinib showed additive and synergistic effects in cancer cell lines resistant to EGFR TKI alone. We have demonstrated a bioinformatics-driven discovery roadmap for drug repurposing and development in overcoming resistance in EGFR-mutant NSCLC, which could be generalized to other cancer types in the era of personalized medicine. K-Map can be accessible at: http://tanlab.ucdenver.edu/kMap. Supplementary data are available at Bioinformatics online. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Inhibition of benzopyrene diol epoxide-induced apoptosis by cadmium (II) is AP-1-independent: role of extracelluler signal related kinase

PubMed Central

Mukherjee, Jagat J.; Gupta, Suresh K.; Kumar, Subodh

2010-01-01

Cadmium, a major metal constituent of tobacco smoke, elicits synergistic enhancement of cell transformation when combined with benzo[a]pyrene (BP) or other PAHs. The mechanism underlying this synergism is not clearly understood. We observed that (+/−)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE), an ultimate carcinogen of BP, induces apoptosis in promotion sensitive mouse epidermal JB6 Cl41 cells at non-cytotoxic concentrations. BPDE also activates AP-1 several folds in AP-1 reporter JB6 cells. Cadmium at non-cytotoxic concentrations inhibits both AP-1 activation and apoptosis in response to BPDE. Since AP-1 is known to be involved in stress-induced apoptosis we investigated whether inhibition of AP-1 by cadmium has any role in the inhibition of BPDE-induced apoptosis. MAP kinases (particularly ERKs, p38 and JNKs) are known to have important role in DNA damage-induced AP-1 activation. We observed that ERK and JNK, but not p38 MAP kinase, are involved in BPDE-induced AP-1 activation. Effect of cadmium on MAP kinases and the effect of inhibition of above three MAP kinases on BPDE-induced AP-1 activation and apoptosis indicate that AP-1 is probably not involved in BPDE-induced apoptosis. Cadmium up-regulates BPDE-activated ERKs and ERK inhibition by U0126 relieves cadmium-mediated inhibition of BPDE-induced apoptosis. We suggest that cadmium inhibits BPDE-induced apoptosis not involving AP-1 but probably through a different mechanism by up-regulating ERK which is known to promote cell survival. PMID:18093576

Modulation of GABAergic receptor binding by activation of calcium and calmodulin-dependent kinase II membrane phosphorylation.

PubMed

Churn, S B; DeLorenzo, R J

1998-10-26

gamma-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system (CNS). Because of the important role that GABA plays in the CNS, alteration of GABAA receptor function would significantly affect neuronal excitability. Protein phosphorylation is a major mechanism for regulating receptor function in the brain and has been implicated in modulating GABAA receptor function. Therefore, this study was initiated to determine the role of calmodulin-dependent kinase II (CaM kinase II) membrane phosphorylation on GABAA receptor binding. Synaptosomal membrane fractions were tested for CaM kinase II activity towards endogenous substrates. In addition, muscimol binding was evaluated under equilibrium conditions in synaptosomal membrane fractions subjected to either basal (Mg2+ alone) or maximal CaM kinase II-dependent phosphorylation. Activation of endogenous CaM kinase II-dependent phosphorylation resulted in a significant enhancement of the apparent Bmax for muscimol binding without significantly altering the apparent binding affinity. The enhanced muscimol binding could be increased further by the addition of exogenous CaM kinase II to synaptosomal membrane fractions. Co-incubation with inhibitors of kinase activity during the phosphorylation reactions blocked the CaM kinase II-dependent increase in muscimol binding. The data support the hypothesis that activation of CaM kinase II-dependent phosphorylation caused an increased GABAA receptor binding and may play an important role in modulating the function of this inhibitory receptor/chloride ion channel complex. Copyright 1998 Elsevier Science B.V.

A superoxide anion-scavenger, 1,3-selenazolidin-4-one suppresses serum deprivation-induced apoptosis in PC12 cells by activating MAP kinase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nishina, Atsuyoshi, E-mail: nishina@yone.ac.jp; Kimura, Hirokazu; Kozawa, Kunihisa

Synthetic organic selenium compounds, such as ebselen, may show glutathione peroxidase-like antioxidant activity and have a neurotrophic effect. We synthesized 1,3-selenazolidin-4-ones, new types of synthetic organic selenium compounds (five-member ring compounds), to study their possible applications as antioxidants or neurotrophic-like molecules. Their superoxide radical scavenging effects were assessed using the quantitative, highly sensitive method of real-time kinetic chemiluminescence. At 166 {mu}M, the O{sub 2}{sup -} scavenging activity of 1,3-selenazolidin-4-ones ranged from 0 to 66.2%. 2-[3-(4-Methoxyphenyl)-4-oxo-1,3-selenazolidin-2-ylidene]malononitrile (compound b) showed the strongest superoxide anion-scavenging activity among the 6 kinds of 2-methylene-1,3-selenazolidin-4-ones examined. Compound b had a 50% inhibitory concentration (IC{sub 50}) atmore » 92.4 {mu}M and acted as an effective and potentially useful O{sub 2}{sup -} scavenger in vitro. The effect of compound b on rat pheochromocytome cell line PC12 cells was compared with that of ebselen or nerve growth factor (NGF) by use of the MTT [3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay. When ebselen was added at 100 {mu}M or more, toxicity toward PC12 cells was evident. On the contrary, compound b suppressed serum deprivation-induced apoptosis in PC12 cells more effectively at a concentration of 100 {mu}M. The activity of compound b to phosphorylate mitogen-activated protein kinase/extracellular signal-regulated protein kinase (ERK) 1/2 (MAP kinase) in PC12 cells was higher than that of ebselen, and the former at 100 {mu}M induced the phosphorylation of MAP kinase to a degree similar to that induced by NGF. From these results, we conclude that this superoxide anion-scavenger, compound b, suppressed serum deprivation-induced apoptosis by promoting the phosphorylation of MAP kinase. -- Highlights: Black-Right-Pointing-Pointer We newly synthesized 1,3-selenazolidin-4-ones to study their possible applications. Black-Right-Pointing-Pointer Among new compounds, compound b showed the strongest SOSA. Black-Right-Pointing-Pointer Compound b suppressed serum deprivation-induced apoptosis in PC12 cells. Black-Right-Pointing-Pointer Compound b suppressed apoptosis by promoting the activation of MAP kinase.« less

Involvement of endogenous antioxidant systems in the protective activity of pituitary adenylate cyclase-activating polypeptide against hydrogen peroxide-induced oxidative damages in cultured rat astrocytes.

PubMed

Douiri, Salma; Bahdoudi, Seyma; Hamdi, Yosra; Cubì, Roger; Basille, Magali; Fournier, Alain; Vaudry, Hubert; Tonon, Marie-Christine; Amri, Mohamed; Vaudry, David; Masmoudi-Kouki, Olfa

2016-06-01

Astroglial cells possess an array of cellular defense mechanisms, including superoxide dismutase (SOD) and catalase antioxidant enzymes, to prevent damages caused by oxidative stress. Nevertheless, astroglial cell viability and functionality can be affected by significant oxidative stress. We have previously shown that pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent glioprotective agent that prevents hydrogen peroxide (H2 O2 )-induced apoptosis in cultured astrocytes. The purpose of this study was to investigate the potential protective effect of PACAP against oxidative-generated alteration of astrocytic antioxidant systems. Incubation of cells with subnanomolar concentrations of PACAP inhibited H2 O2 -evoked reactive oxygen species accumulation, mitochondrial respiratory burst, and caspase-3 mRNA level increase. PACAP also stimulated SOD and catalase activities in a concentration-dependent manner, and counteracted the inhibitory effect of H2 O2 on the activity of these two antioxidant enzymes. The protective action of PACAP against H2 O2 -evoked inhibition of antioxidant systems in astrocytes was protein kinase A, PKC, and MAP-kinase dependent. In the presence of H2 O2 , the SOD blocker NaCN and the catalase inhibitor 3-aminotriazole, both suppressed the protective effects of PACAP on SOD and catalase activities, mitochondrial function, and cell survival. Taken together, these results indicate that the anti-apoptotic effect of PACAP on astroglial cells can account for the activation of endogenous antioxidant enzymes and reduction in respiration rate, thus preserving mitochondrial integrity and preventing caspase-3 expression provoked by oxidative stress. Considering its powerful anti-apoptotic and anti-oxidative properties, the PACAPergic signaling system should thus be considered for the development of new therapeutical approaches to cure various pathologies involving oxidative neurodegeneration. We propose the following cascade for the glioprotective action of Pituitary adenylate cyclase-activating polypeptide (PACAP) against H2 O2 -induced astrocyte damages and cell apoptosis in cultured rat astrocytes. PACAP, through activation of its receptor, PAC1-R, and the protein kinase A (PKA), protein kinase C (PKC), and MAP-kinases signaling pathways, prevents accumulation of ROS and inhibition of SOD and catalase activities. This allows the preservation of mitochondrial membrane integrity and the reduction in caspase-3 activation induced by H2 O2 . These data may lead to the development of new strategies for cerebral injury treatment. Cat, catalase; Cyt. C, cytochrome C; SOD, superoxide dismutase. © 2016 International Society for Neurochemistry.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhan, Yu; Abi Saab, Widian F.; Modi, Nidhi

Mixed lineage kinase 3 (MLK3) is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates MAPK signaling pathways and regulates cellular responses such as proliferation, migration and apoptosis. Here we report high levels of total and phospho-MLK3 in ovarian cancer cell lines in comparison to immortalized nontumorigenic ovarian epithelial cell lines. Using small interfering RNA (siRNA)-mediated gene silencing, we determined that MLK3 is required for the invasion of SKOV3 and HEY1B ovarian cancer cells. Furthermore, mlk3 silencing substantially reduced matrix metalloproteinase (MMP)-1, -2, -9 and -12 gene expression and MMP-2 and -9 activities in SKOV3 and HEY1B ovarian cancer cells.more » MMP-1, -2, -9 and-12 expression, and MLK3-induced activation of MMP-2 and MMP-9 requires both extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) activities. In addition, inhibition of activator protein-1 (AP-1) reduced MMP-1, MMP-9 and MMP-12 gene expression. Collectively, these findings establish MLK3 as an important regulator of MMP expression and invasion in ovarian cancer cells. -- Highlights: Black-Right-Pointing-Pointer Ovarian cancer cell lines have high levels of total and phosphorylated MLK3. Black-Right-Pointing-Pointer MLK3 is required for MMP expression and activity in ovarian cancer cells. Black-Right-Pointing-Pointer MLK3 is required for invasion of SKOV3 and HEY1B ovarian cancer cells. Black-Right-Pointing-Pointer MLK3-dependent regulation of MMP-2 and MMP-9 activities requires ERK and JNK.« less

Target of rapamycin complex 2 signals to downstream effector yeast protein kinase 2 (Ypk2) through adheres-voraciously-to-target-of-rapamycin-2 protein 1 (Avo1) in Saccharomyces cerevisiae.

PubMed

Liao, Hsien-Ching; Chen, Mei-Yu

2012-02-24

The conserved Ser/Thr kinase target of rapamycin (TOR) serves as a central regulator in controlling cell growth-related functions. There exist two distinct TOR complexes, TORC1 and TORC2, each coupling to specific downstream effectors and signaling pathways. In Saccharomyces cerevisiae, TORC2 is involved in regulating actin organization and maintaining cell wall integrity. Ypk2 (yeast protein kinase 2), a member of the cAMP-dependent, cGMP-dependent, and PKC (AGC) kinase family, is a TORC2 substrate known to participate in actin and cell wall regulation. Employing avo3(ts) mutants with defects in TORC2 functions that are suppressible by active Ypk2, we investigated the molecular interactions involved in mediating TORC2 signaling to Ypk2. GST pulldown assays in yeast lysates demonstrated physical interactions between Ypk2 and components of TORC2. In vitro binding assays revealed that Avo1 directly binds to Ypk2. In avo3(ts) mutants, the TORC2-Ypk2 interaction was reduced and could be restored by AVO1 overexpression, highlighting the important role of Avo1 in coupling TORC2 to Ypk2. The interaction was mapped to an internal region (amino acids 600-840) of Avo1 and a C-terminal region of Ypk2. Ypk2(334-677), a truncated form of Ypk2 containing the Avo1-interacting region, was able to interfere with Avo1-Ypk2 interaction in vitro. Overexpressing Ypk2(334-677) in yeast cells resulted in a perturbation of TORC2 functions, causing defective cell wall integrity, aberrant actin organization, and diminished TORC2-dependent Ypk2 phosphorylation evidenced by the loss of an electrophoretic mobility shift. Together, our data support the conclusion that the direct Avo1-Ypk2 interaction is crucial for TORC2 signaling to the downstream Ypk2 pathway.

[Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells].

PubMed

Sheremet, Ia A; Emets, A I; Azmi, A; Vissenberg, K; Verbelen, J-P; Blium, Ia B

2012-01-01

In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

Mechanotransduction in skeletal muscle

PubMed Central

Burkholder, Thomas J.

2007-01-01

Mechanical signals are critical to the development and maintenance of skeletal muscle, but the mechanisms that convert these shape changes to biochemical signals is not known. When a deformation is imposed on a muscle, changes in cellular and molecular conformations link the mechanical forces with biochemical signals, and the close integration of mechanical signals with electrical, metabolic, and hormonal signaling may disguise the aspect of the response that is specific to the mechanical forces. The mechanically induced conformational change may directly activate downstream signaling and may trigger messenger systems to activate signaling indirectly. Major effectors of mechanotransduction include the ubiquitous mitogen activated protein kinase (MAP) and phosphatidylinositol-3’ kinase (PI-3K), which have well described receptor dependent cascades, but the chain of events leading from mechanical stimulation to biochemical cascade is not clear. This review will discuss the mechanics of biological deformation, loading of cellular and molecular structures, and some of the principal signaling mechanisms associated with mechanotransduction. PMID:17127292

Mechanotransduction in skeletal muscle.

PubMed

Burkholder, Thomas J

2007-01-01

Mechanical signals are critical to the development and maintenance of skeletal muscle, but the mechanisms that convert these shape changes to biochemical signals is not known. When a deformation is imposed on a muscle, changes in cellular and molecular conformations link the mechanical forces with biochemical signals, and the close integration of mechanical signals with electrical, metabolic, and hormonal signaling may disguise the aspect of the response that is specific to the mechanical forces. The mechanically induced conformational change may directly activate downstream signaling and may trigger messenger systems to activate signaling indirectly. Major effectors of mechanotransduction include the ubiquitous mitogen activated protein kinase (MAP) and phosphatidylinositol-3' kinase (PI-3K), which have well described receptor dependent cascades, but the chain of events leading from mechanical stimulation to biochemical cascade is not clear. This review will discuss the mechanics of biological deformation, loading of cellular and molecular structures, and some of the principal signaling mechanisms associated with mechanotransduction.

Truncation- and motif-based pan-cancer analysis reveals tumor-suppressing kinases.

PubMed

Hudson, Andrew M; Stephenson, Natalie L; Li, Cynthia; Trotter, Eleanor; Fletcher, Adam J; Katona, Gitta; Bieniasz-Krzywiec, Patrycja; Howell, Matthew; Wirth, Chris; Furney, Simon; Miller, Crispin J; Brognard, John

2018-04-17

A major challenge in cancer genomics is identifying "driver" mutations from the many neutral "passenger" mutations within a given tumor. To identify driver mutations that would otherwise be lost within mutational noise, we filtered genomic data by motifs that are critical for kinase activity. In the first step of our screen, we used data from the Cancer Cell Line Encyclopedia and The Cancer Genome Atlas to identify kinases with truncation mutations occurring within or before the kinase domain. The top 30 tumor-suppressing kinases were aligned, and hotspots for loss-of-function (LOF) mutations were identified on the basis of amino acid conservation and mutational frequency. The functional consequences of new LOF mutations were biochemically validated, and the top 15 hotspot LOF residues were used in a pan-cancer analysis to define the tumor-suppressing kinome. A ranked list revealed MAP2K7, an essential mediator of the c-Jun N-terminal kinase (JNK) pathway, as a candidate tumor suppressor in gastric cancer, despite its mutational frequency falling within the mutational noise for this cancer type. The majority of mutations in MAP2K7 abolished its catalytic activity, and reactivation of the JNK pathway in gastric cancer cells harboring LOF mutations in MAP2K7 or the downstream kinase JNK suppressed clonogenicity and growth in soft agar, demonstrating the functional relevance of inactivating the JNK pathway in gastric cancer. Together, our data highlight a broadly applicable strategy to identify functional cancer driver mutations and define the JNK pathway as tumor-suppressive in gastric cancer. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa

DOE PAGES

Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles; ...

2014-11-20

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC1, MEK2 and MAK2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every 4 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a protein of unknown biochemical function. How this oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) thatmore » can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM5-GFP co-localized with NRC1, MEK2 and MAK2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK2 activity influences HAM5 function/localization. However, MAK2-GFP showed only cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta, as observed in wild type germlings. Via co-immunoprecipitation experiments, HAM5 was shown to physically interact with MAK2, MEK2 and NRC1, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members during oscillation and chemotropic interactions during both germling and hyphal fusion in N. crassa. The identification of HAM5 as a scaffold-like protein will help to link the activation of MAK2 to upstream factors and other proteins involved in this intriguing process of fungal communication.« less

HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jonkers, Wilfried; Leeder, Abigail C.; Ansong, Charles

Cell fusion in genetically identical Neurospora crassa germlings and in hyphae is a highly regulated process involving the activation of a conserved MAP kinase cascade that includes NRC1, MEK2 and MAK2. During chemotrophic growth in germlings, the MAP kinase cascade members localize to conidial anastomosis tube (CAT) tips every 4 minutes, perfectly out of phase with another protein that is recruited to the tip: SOFT, a protein of unknown biochemical function. How this oscillation process is initiated, maintained and what proteins regulate the MAP kinase cascade is currently unclear. A global phosphoproteomics approach using an allele of mak-2 (mak-2Q100G) thatmore » can be specifically inhibited by the ATP analog 1NM-PP1 was utilized to identify MAK2 kinase targets in germlings that were potentially involved in this process. One such putative target was HAM5, a protein of unknown biochemical function. Previously, Δham-5 mutants were shown to be deficient for hyphal fusion. Here we show that HAM5-GFP co-localized with NRC1, MEK2 and MAK2 and oscillated with identical dynamics from the cytoplasm to CAT tips during chemotropic interactions. In the Δmak-2 strain, HAM5-GFP localized to punctate complexes that did not oscillate, but still localized to the germling tip, suggesting that MAK2 activity influences HAM5 function/localization. However, MAK2-GFP showed only cytoplasmic and nuclear localization in a Δham-5 strain and did not localize to puncta, as observed in wild type germlings. Via co-immunoprecipitation experiments, HAM5 was shown to physically interact with MAK2, MEK2 and NRC1, suggesting that it functions as a scaffold/transport hub for the MAP kinase cascade members during oscillation and chemotropic interactions during both germling and hyphal fusion in N. crassa. The identification of HAM5 as a scaffold-like protein will help to link the activation of MAK2 to upstream factors and other proteins involved in this intriguing process of fungal communication.« less

Neuroprotective and Blood-Retinal Barrier-Preserving Effects of Cannabidiol in Experimental Diabetes

PubMed Central

El-Remessy, Azza B.; Al-Shabrawey, Mohamed; Khalifa, Yousuf; Tsai, Nai-Tse; Caldwell, Ruth B.; Liou, Gregory I.

2006-01-01

Diabetic retinopathy is characterized by blood-retinal barrier (BRB) breakdown and neurotoxicity. These pathologies have been associated with oxidative stress and proinflammatory cytokines, which may operate by activating their downstream target p38 MAP kinase. In the present study, the protective effects of a nonpsychotropic cannabinoid, cannabidiol (CBD), were examined in streptozotocin-induced diabetic rats after 1, 2, or 4 weeks. Retinal cell death was determined by terminal dUTP nick-end labeling assay; BRB function by quantifying extravasation of bovine serum albumin-fluorescein; and oxidative stress by assays for lipid peroxidation, dichlorofluorescein fluorescence, and tyrosine nitration. Experimental diabetes induced significant increases in oxidative stress, retinal neuronal cell death, and vascular permeability. These effects were associated with increased levels of tumor necrosis factor-α, vascular endothelial growth factor, and intercellular adhesion molecule-1 and activation of p38 MAP kinase, as assessed by enzyme-linked immunosorbent assay, immunohistochemistry, and/or Western blot. CBD treatment significantly reduced oxidative stress; decreased the levels of tumor necrosis factor-α, vascular endothelial growth factor, and intercellular adhesion molecule-1; and prevented retinal cell death and vascular hyperpermeability in the diabetic retina. Consistent with these effects, CBD treatment also significantly inhibited p38 MAP kinase in the diabetic retina. These results demonstrate that CBD treatment reduces neurotoxicity, inflammation, and BRB breakdown in diabetic animals through activities that may involve inhibition of p38 MAP kinase. PMID:16400026

Kinase Pathway Dependence in Primary Human Leukemias Determined by Rapid Inhibitor Screening

PubMed Central

Tyner, Jeffrey W.; Yang, Wayne F.; Bankhead, Armand; Fan, Guang; Fletcher, Luke B.; Bryant, Jade; Glover, Jason M.; Chang, Bill H.; Spurgeon, Stephen E.; Fleming, William H.; Kovacsovics, Tibor; Gotlib, Jason R.; Oh, Stephen T.; Deininger, Michael W.; Zwaan, C. Michel; Den Boer, Monique L.; van den Heuvel-Eibrink, Marry M.; O’Hare, Thomas; Druker, Brian J.; Loriaux, Marc M.

2012-01-01

Kinases are dysregulated in most cancer but the frequency of specific kinase mutations is low, indicating a complex etiology in kinase dysregulation. Here we report a strategy to rapidly identify functionally important kinase targets, irrespective of the etiology of kinase pathway dysregulation, ultimately enabling a correlation of patient genetic profiles to clinically effective kinase inhibitors. Our methodology assessed the sensitivity of primary leukemia patient samples to a panel of 66 small-molecule kinase inhibitors over 3 days. Screening of 151 leukemia patient samples revealed a wide diversity of drug sensitivities, with 70% of the clinical specimens exhibiting hypersensitivity to one or more drugs. From this data set, we developed an algorithm to predict kinase pathway dependence based on analysis of inhibitor sensitivity patterns. Applying this algorithm correctly identified pathway dependence in proof-of-principle specimens with known oncogenes, including a rare FLT3 mutation outside regions covered by standard molecular diagnostic tests. Interrogation of all 151 patient specimens with this algorithm identified a diversity of gene targets and signaling pathways that could aid prioritization of deep sequencing data sets, permitting a cumulative analysis to understand kinase pathway dependence within leukemia subsets. In a proof-of-principle case, we showed that in vitro drug sensitivity could predict both a clinical response and the development of drug resistance. Taken together, our results suggested that drug target scores derived from a comprehensive kinase inhibitor panel could predict pathway dependence in cancer cells while simultaneously identifying potential therapeutic options. PMID:23087056

Characterization of PsMPK2, the first C1 subgroup MAP kinase from pea (Pisum sativum L.).

PubMed

Ortiz-Masia, Dolores; Perez-Amador, Miguel A; Carbonell, Pablo; Aniento, Fernando; Carbonell, Juan; Marcote, Maria J

2008-05-01

Mitogen-activated protein kinase (MAPK) cascades play a key role in plant growth and development as well as in biotic and abiotic stress responses. They are classified according to their sequence homology into four major groups (A-D). A large amount of information about MAPKs in groups A and B is available but few data of the C group have been reported. In this study, a C1 subgroup MAP kinase cDNA, PsMPK2, was isolated from Pisum sativum. PsMPK2 is expressed in vegetative (root and leaf) and reproductive (stamen, pistil and fruit) organs. Expression of PsMPK2 in Arabidopsis thaliana shows that mechanical injury and other stress signals as abscisic acid, jasmonic acid and hydrogen peroxide increase its kinase activity, extending previous results indicating that C1 subgroup MAPKs may be involved in the response to stress.

[Application of Kohonen Self-Organizing Feature Maps in QSAR of human ADMET and kinase data sets].

PubMed

Hegymegi-Barakonyi, Bálint; Orfi, László; Kéri, György; Kövesdi, István

2013-01-01

QSAR predictions have been proven very useful in a large number of studies for drug design, such as kinase inhibitor design as targets for cancer therapy, however the overall predictability often remains unsatisfactory. To improve predictability of ADMET features and kinase inhibitory data, we present a new method using Kohonen's Self-Organizing Feature Map (SOFM) to cluster molecules based on explanatory variables (X) and separate dissimilar ones. We calculated SOFM clusters for a large number of molecules with human ADMET and kinase inhibitory data, and we showed that chemically similar molecules were in the same SOFM cluster, and within such clusters the QSAR models had significantly better predictability. We used also target variables (Y, e.g. ADMET) jointly with X variables to create a novel type of clustering. With our method, cells of loosely coupled XY data could be identified and separated into different model building sets.

Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase

NASA Astrophysics Data System (ADS)

Wagner, John A.; Cozens, Alison L.; Schulman, Howard; Gruenert, Dieter C.; Stryer, Lubert; Gardner, Phyllis

1991-02-01

CYSTIC fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase1,2 and protein kinase C3,4. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels1-4. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2+-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2+-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.

Phosphoinositide 3-kinase-dependent Ras activation by tauroursodesoxycholate in rat liver.

PubMed Central

Kurz, A K; Block, C; Graf, D; Dahl, S V; Schliess, F; Häussinger, D

2000-01-01

Ursodesoxycholic acid, widely used for the treatment of cholestatic liver disease, causes choleretic, anti-apoptotic and immunomodulatory effects. Here the effects on choleresis of its taurine conjugate tauroursodesoxycholate (TUDC), which is present in the enterohepatic circulation, were correlated with the activation of important elements of intracellular signal transduction in cultured rat hepatocytes and perfused rat liver. TUDC induced a time- and concentration-dependent activation of the small GTP-binding protein Ras and of phosphoinositide 3-kinase (PI 3-kinase) in cultured hepatocytes. Ras activation was dependent on PI 3-kinase activity, without the involvement of protein kinase C- and genistein-sensitive tyrosine kinases. Ras activation by TUDC was followed by an activation of the mitogen-activated protein kinases extracellular-signal-regulated kinase-1 (Erk-1) and Erk-2. In perfused rat liver, PI 3-kinase inhibitors largely abolished the stimulatory effect of TUDC on taurocholate excretion, suggesting an important role for a PI 3-kinase/Ras/Erk pathway in the choleretic effect of TUDC. PMID:10926845

Rapid synthesis of VX-745: p38 MAP kinase inhibition in Werner syndrome cells.

PubMed

Bagley, Mark C; Davis, Terence; Dix, Matthew C; Rokicki, Michal J; Kipling, David

2007-09-15

The p38 mitogen-activated protein kinase inhibitor VX-745 is prepared rapidly and efficiently in a four-step sequence using a combination of conductive heating and microwave-mediated steps. Its inhibitory activity was confirmed in hTERT immortalized HCA2 and WS dermal fibroblasts at 0.5-1.0 microM concentration by ELISA and immunoblot assay, and displays excellent kinase selectivity over the related stress-activated kinase JNK.

A single amino acid substitution makes ERK2 susceptible to pyridinyl imidazole inhibitors of p38 MAP kinase.

PubMed Central

Fox, T.; Coll, J. T.; Xie, X.; Ford, P. J.; Germann, U. A.; Porter, M. D.; Pazhanisamy, S.; Fleming, M. A.; Galullo, V.; Su, M. S.; Wilson, K. P.

1998-01-01

Mitogen-activated protein (MAP) kinases are serine/threonine kinases that mediate intracellular signal transduction pathways. Pyridinyl imidazole compounds block pro-inflammatory cytokine production and are specific p38 kinase inhibitors. ERK2 is related to p38 in sequence and structure, but is not inhibited by pyridinyl imidazole inhibitors. Crystal structures of two pyridinyl imidazoles complexed with p38 revealed these compounds bind in the ATP site. Mutagenesis data suggested a single residue difference at threonine 106 between p38 and other MAP kinases is sufficient to confer selectivity of pyridinyl imidazoles. We have changed the equivalent residue in human ERK2, Q105, into threonine and alanine, and substituted four additional ATP binding site residues. The single residue change Q105A in ERK2 enhances the binding of SB202190 at least 25,000-fold compared to wild-type ERK2. We report enzymatic analyses of wild-type ERK2 and the mutant proteins, and the crystal structure of a pyridinyl imidazole, SB203580, bound to an ERK2 pentamutant, I103L, Q105T, D106H, E109G. T110A. These ATP binding site substitutions induce low nanomolar sensitivity to pyridinyl imidazoles. Furthermore, we identified 5-iodotubercidin as a potent ERK2 inhibitor, which may help reveal the role of ERK2 in cell proliferation. PMID:9827991

Phosphorylation of Dopamine Transporter Serine 7 Modulates Cocaine Analog Binding*

PubMed Central

Moritz, Amy E.; Foster, James D.; Gorentla, Balachandra K.; Mazei-Robison, Michelle S.; Yang, Jae-Won; Sitte, Harald H.; Blakely, Randy D.; Vaughan, Roxanne A.

2013-01-01

As an approach to elucidating dopamine transporter (DAT) phosphorylation characteristics, we examined in vitro phosphorylation of a recombinant rat DAT N-terminal peptide (NDAT) using purified protein kinases. We found that NDAT becomes phosphorylated at single distinct sites by protein kinase A (Ser-7) and calcium-calmodulin-dependent protein kinase II (Ser-13) and at multiple sites (Ser-4, Ser-7, and Ser-13) by protein kinase C (PKC), implicating these residues as potential sites of DAT phosphorylation by these kinases. Mapping of rat striatal DAT phosphopeptides by two-dimensional thin layer chromatography revealed basal and PKC-stimulated phosphorylation of the same peptide fragments and comigration of PKC-stimulated phosphopeptide fragments with NDAT Ser-7 phosphopeptide markers. We further confirmed by site-directed mutagenesis and mass spectrometry that Ser-7 is a site for PKC-stimulated phosphorylation in heterologously expressed rat and human DATs. Mutation of Ser-7 and nearby residues strongly reduced the affinity of rat DAT for the cocaine analog (−)-2β-carbomethoxy-3β-(4-fluorophenyl) tropane (CFT), whereas in rat striatal tissue, conditions that promote DAT phosphorylation caused increased CFT affinity. Ser-7 mutation also affected zinc modulation of CFT binding, with Ala and Asp substitutions inducing opposing effects. These results identify Ser-7 as a major site for basal and PKC-stimulated phosphorylation of native and expressed DAT and suggest that Ser-7 phosphorylation modulates transporter conformational equilibria, shifting the transporter between high and low affinity cocaine binding states. PMID:23161550

Impact of PTEN on the expression of insulin-like growth factors (IGFs) and IGF-binding proteins in human gastric adenocarcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yi, Ho-Keun; Kim, Sun-Young; Hwang, Pyoung-Han

2005-05-13

PTEN is a tumor suppressor gene that is frequently mutated or deleted in a variety of human cancers including human gastric cancer. PTEN functions primarily as a lipid phosphatase and plays a key role in the regulation of the PI3 kinase/Akt pathway, thereby modulating cell proliferation and cell survival. On the other hand, the IGF system plays an important role in cell proliferation and cell survival via the PI3 kinase/Akt and MAP kinase pathways in many cancer cells. To characterize the impact of PTEN on the IGF-IGFR-IGFBP axis in gastric cancer, we overexpressed PTEN using an adenovirus gene transfer systemmore » in human gastric adenocarcinoma cells, SNU-484 and SNU-663, which lack PTEN. Overexpression of PTEN inhibited serum-induced as well as IGF-I-induced cell proliferation as compared to control cells. PTEN overexpression resulted in a significant decrease in the expression of IGF-I, -II, and IGF-IR. Interestingly, amongst the six IGFBPs, only IGFBP-3 was upregulated by PTEN, whereas IGFBP-4 and -6 were reduced. The IGFBP-3 promoter activity assay and Western immunoblotting demonstrate that PTEN regulates IGFBP-3 at the transcriptional level. In addition, the PI3 kinase inhibitor, LY294002, upregulates IGFBP-3 expression but downregulates IGF-I and IGF-II, indicating that PTEN controls IGFBP-3 and IGFs by an Akt-dependent pathway. These findings suggest that PTEN may inhibit antiapoptotic IGF actions not only by blocking the IGF-IGFR-induced Akt activity, but also by regulating expression of components of the IGF system, in particular, upregulation of IGFBP-3, which is known to exert antiproliferative effects through IGF-dependent and IGF-independent mechanisms in cancer cells.« less

The citrus postharvest pathogen Penicillium digitatum depends on the PdMpkB kinase for developmental and virulence functions.

PubMed

Ma, Haijie; Sun, Xuepeng; Wang, Mingshuang; Gai, Yunpeng; Chung, Kuang-Ren; Li, Hongye

2016-11-07

The postharvest pathogen Penicillium digitatum causes green mold decay on citrus fruit, resulting in severe economic losses. To explore possible factors involved in fungal pathogenesis, phenotypic characterization of the budding yeast Fus3/Kiss1 mitogen-activated protein (MAP) kinase homolog was carried out. The P. digitatum MAP kinase B coding gene, designated PdMpkB, was functionally inactivated via homologous recombination. The fungal strain (∆PdMpkB) carrying a PdMpkBdeletion demonstrated altered gene expression profiles, reduced growth and conidiogenesis, elevated resistance to osmotic stress, and failed to induce green mold decay on citrus fruit. ∆PdMpkB was more resistant to CaCl2, NaCl and sorbitol than its progenitor strain, indicating a negative regulatory function of PdMpkB in osmotic stress adaptation. Fungal infection assays on citrus fruit revealed that ∆PdMpkB proliferated poorly within host tissues, induced water-soaking lesions, failed to break through host cuticle layers and thus, failed to produce aerial hyphae and conidia. Introduction of a functional copy of PdMpkB into a null mutant restored all defective phenotypes. Transcriptome analysis revealed that inactivation of PdMpkB impacted expression of the genes associated with cell wall-degrading enzyme activities, carbohydrate and amino acid metabolisms, conidial formation, and numerous metabolic processes. Our results define pivotal roles of the PdMpkB-mediated signaling pathway in developmental and pathological functions in the citrus postharvest pathogen P. digitatum. Copyright © 2016 Elsevier B.V. All rights reserved.

Functional Mapping of Protein Kinase A Reveals Its Importance in Adult Schistosoma mansoni Motor Activity

PubMed Central

de Saram, Paulu S. R.; Ressurreição, Margarida; Davies, Angela J.; Rollinson, David; Emery, Aidan M.; Walker, Anthony J.

2013-01-01

Cyclic AMP (cAMP)-dependent protein kinase/protein kinase A (PKA) is the major transducer of cAMP signalling in eukaryotic cells. Here, using laser scanning confocal microscopy and ‘smart’ anti-phospho PKA antibodies that exclusively detect activated PKA, we provide a detailed in situ analysis of PKA signalling in intact adult Schistosoma mansoni, a causative agent of debilitating human intestinal schistosomiasis. In both adult male and female worms, activated PKA was consistently found associated with the tegument, oral and ventral suckers, oesophagus and somatic musculature. In addition, the seminal vesicle and gynaecophoric canal muscles of the male displayed activated PKA whereas in female worms activated PKA localized to the ootype wall, the ovary, and the uterus particularly around eggs during expulsion. Exposure of live worms to the PKA activator forskolin (50 µM) resulted in striking PKA activation in the central and peripheral nervous system including at nerve endings at/near the tegument surface. Such neuronal PKA activation was also observed without forskolin treatment, but only in a single batch of worms. In addition, PKA activation within the central and peripheral nervous systems visibly increased within 15 min of worm-pair separation when compared to that observed in closely coupled worm pairs. Finally, exposure of adult worms to forskolin induced hyperkinesias in a time and dose dependent manner with 100 µM forskolin significantly increasing the frequency of gross worm movements to 5.3 times that of control worms (P≤0.001). Collectively these data are consistent with PKA playing a central part in motor activity and neuronal communication, and possibly interplay between these two systems in S. mansoni. This study, the first to localize a protein kinase when exclusively in an activated state in adult S. mansoni, provides valuable insight into the intricacies of functional protein kinase signalling in the context of whole schistosome physiology. PMID:23326613

Neurotensin-induced Erk1/2 phosphorylation and growth of human colonic cancer cells are independent from growth factors receptors activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Massa, Fabienne; Tormo, Aurelie; Beraud-Dufour, Sophie

2011-10-14

Highlights: {yields} We compare intracellular pathways of NT and EGF in HT29 cells. {yields} NT does not transactivate EGFR. {yields} Transactivation of EGFR is not a general rule in cancer cell growth. -- Abstract: Neurotensin (NT) promotes the proliferation of human colonic cancer cells by undefined mechanisms. We already demonstrated that, in the human colon adenocarcinoma cell line HT29, the effects of NT were mediated by a complex formed between the NT receptor-1 (NTSR1) and-3 (NTSR3). Here we examined cellular mechanisms that led to NT-induced MAP kinase phosphorylation and growth factors receptors transactivation in colonic cancer cells and proliferation inmore » HT29 cells. With the aim to identify upstream signaling involved in NT-elicited MAP kinase activation, we found that the stimulatory effects of the peptide were totally independent from the activation of the epidermal growth factor receptor (EGFR) both in the HT29 and the HCT116 cells. NT was unable to promote phosphorylation of EGFR and to compete with EGF for its binding to the receptor. Pharmacological approaches allowed us to differentiate EGF and NT signaling in HT29 cells since only NT activation of Erk1/2 was shown to be sensitive to PKC inhibitors and since only NT increased the intracellular level of calcium. We also observed that NT was not able to transactivate Insulin-like growth factor receptor. Our findings indicate that, in the HT29 and HCT116 cell lines, NT stimulates MAP kinase phosphorylation and cell growth by a pathway which does not involve EGF system but rather NT receptors which transduce their own intracellular effectors. These results indicate that depending on the cell line used, blocking EGFR is not the general rule to inhibit NT-induced cancer cell proliferation.« less

Functionally Redundant RXLR Effectors from Phytophthora infestans Act at Different Steps to Suppress Early flg22-Triggered Immunity

PubMed Central

Fraiture, Malou; Liu, Xiaoyu; Boevink, Petra C.; Gilroy, Eleanor M.; Chen, Ying; Kandel, Kabindra; Sessa, Guido; Birch, Paul R. J.; Brunner, Frédéric

2014-01-01

Genome sequences of several economically important phytopathogenic oomycetes have revealed the presence of large families of so-called RXLR effectors. Functional screens have identified RXLR effector repertoires that either compromise or induce plant defense responses. However, limited information is available about the molecular mechanisms underlying the modes of action of these effectors in planta. The perception of highly conserved pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs), such as flg22, triggers converging signaling pathways recruiting MAP kinase cascades and inducing transcriptional re-programming, yielding a generic anti-microbial response. We used a highly synchronizable, pathogen-free protoplast-based assay to identify a set of RXLR effectors from Phytophthora infestans (PiRXLRs), the causal agent of potato and tomato light blight that manipulate early stages of flg22-triggered signaling. Of thirty-three tested PiRXLR effector candidates, eight, called Suppressor of early Flg22-induced Immune response (SFI), significantly suppressed flg22-dependent activation of a reporter gene under control of a typical MAMP-inducible promoter (pFRK1-Luc) in tomato protoplasts. We extended our analysis to Arabidopsis thaliana, a non-host plant species of P. infestans. From the aforementioned eight SFI effectors, three appeared to share similar functions in both Arabidopsis and tomato by suppressing transcriptional activation of flg22-induced marker genes downstream of post-translational MAP kinase activation. A further three effectors interfere with MAMP signaling at, or upstream of, the MAP kinase cascade in tomato, but not in Arabidopsis. Transient expression of the SFI effectors in Nicotiana benthamiana enhances susceptibility to P. infestans and, for the most potent effector, SFI1, nuclear localization is required for both suppression of MAMP signaling and virulence function. The present study provides a framework to decipher the molecular mechanisms underlying the manipulation of host MAMP-triggered immunity (MTI) by P. infestans and to understand the basis of host versus non-host resistance in plants towards P. infestans. PMID:24763622

Chromosome mapping of the human arrestin (SAG), {beta}-arrestin 2 (ARRB2), and {beta}-adrenergic receptor kinase 2 (ADRBK2) genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Calabrese, G.; Sallese, M.; Stornaiuolo, A.

1994-09-01

Two types of proteins play a major role in determining homologous desensitization of G-coupled receptors: {beta}-adrenergic receptor kinase ({beta}ARK), which phosphorylates the agonist-occupied receptor and its functional cofactor, {beta}-arrestin. Both {beta}ARK and {beta}-arrestin are members of multigene families. The family of G-protein-coupled receptor kinases includes rhodopsin kinase, {beta}ARK1, {beta}ARK2, IT11-A (GRK4), GRK5, and GRK6. The arrestin/{beta}-arrestin gene family includes arrestin (also known as S-antigen), {beta}-arrestin 1, and {beta}-arrestin 2. Here we report the chromosome mapping of the human genes for arrestin (SAG), {beta}arrestin 2 (ARRB2), and {beta}ARK2 (ADRBK2) by fluorescence in situ hybridization (FISH). FISH results confirmed the assignment ofmore » the gene coding for arrestin (SAG) to chromosome 2 and allowed us to refine its localization to band q37. The gene coding for {beta}-arrestin 2 (ARRB2) was mapped to chromosome 17p13 and that coding for {beta}ARK2 (ADRBK2) to chromosome 22q11. 17 refs., 1 fig.« less

Epileptogenesis causes an N-methyl-d-aspartate receptor/Ca2+-dependent decrease in Ca2+/calmodulin-dependent protein kinase II activity in a hippocampal neuronal culture model of spontaneous recurrent epileptiform discharges.

PubMed

Blair, Robert E; Sombati, Sompong; Churn, Severn B; Delorenzo, Robert J

2008-06-24

Alterations in the function of Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) have been observed in both in vivo and in vitro models of epileptogenesis; however the molecular mechanism mediating the effects of epileptogenesis on CaM kinase II has not been elucidated. This study was initiated to evaluate the molecular pathways involved in causing the long-lasting decrease in CaM kinase II activity in the hippocampal neuronal culture model of low Mg2+-induced spontaneous recurrent epileptiform discharges (SREDs). We show here that the decrease in CaM kinase II activity associated with SREDs in hippocampal cultures involves a Ca2+/N-methyl-d-aspartate (NMDA) receptor-dependent mechanism. Low Mg2+-induced SREDs result in a significant decrease in Ca2+/calmodulin-dependent substrate phosphorylation of the synthetic peptide autocamtide-2. Reduction of extracellular Ca2+ levels (0.2 mM in treatment solution) or the addition of dl-2-amino-5-phosphonovaleric acid (APV) 25 microM blocked the low Mg2+-induced decrease in CaM kinase II-dependent substrate phosphorylation. Antagonists of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid receptor or L-type voltage sensitive Ca2+ channel had no effect on the low Mg2+-induced decrease in CaM kinase II-dependent substrate phosphorylation. The results of this study demonstrate that the decrease in CaM kinase II activity associated with this model of epileptogenesis involves a selective Ca2+/NMDA receptor-dependent mechanism and may contribute to the production and maintenance of SREDs in this model.

Epileptogenesis causes an N-methyl-d-aspartate receptor/Ca2+-dependent decrease in Ca2+/calmodulin-dependent protein kinase II activity in a hippocampal neuronal culture model of spontaneous recurrent epileptiform discharges

PubMed Central

Blair, Robert E.; Sombati, Sompong; Churn, Severn B.; DeLorenzo, Robert J.

2008-01-01

Alterations in the function of Ca2+/calmodulin-dependent protein kinase II (CaM Kinase II) have been observed in both in vivo and in vitro models of epileptogenesis; however the molecular mechanism mediating the effects of epileptogenesis on CaM Kinase II have not been elucidated. This study was initiated to evaluate the molecular pathways involved in causing the long lasting decrease in CaM Kinase II activity in the hippocampal neuronal culture model of low Mg2+ induced spontaneous recurrent epileptiform discharges (SREDs). We show here that the decrease in CaM kinase II activity associated with SREDs in hippocampal cultures involves a Ca2+/N-methyl-d-aspartate (NMDA) receptor-dependent mechanism. Low Mg2+ induced SREDs results in a significant decrease in Ca2+/calmodulin-dependent substrate phosphorylation of the synthetic peptide autocamtide-2. Reduction of extracellular Ca2+ levels (0.2 mM in treatment solution) or the addition of DL-2-amino-5-phosphonovaleric acid (APV) 25 µM blocked the low Mg2+ induced decrease in CaM kinase II-dependent substrate phosphorylation. Antagonists of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainic acid receptor or L-type voltage sensitive Ca2+ channel had no effect on the low Mg2+ induced decrease in CaM kinase II-dependent substrate phosphorylation. The results of this study demonstrate that the decrease in CaM kinase II activity associated with this model of epileptogenesis involves a selective Ca2+/NMDA receptor-dependent mechanism and may contribute to the production and maintenance of SREDs in this model. PMID:18495112

Crystal structure of casein kinase-1, a phosphate-directed protein kinase.

PubMed Central

Xu, R M; Carmel, G; Sweet, R M; Kuret, J; Cheng, X

1995-01-01

The structure of a truncated variant of casein kinase-1 from Schizosaccharomyces pombe, has been determined in complex with MgATP at 2.0 A resolution. The model resembles the 'closed', ATP-bound conformations of the cyclin-dependent kinase 2 and the cAMP-dependent protein kinase, with clear differences in the structure of surface loops that impart unique features to casein kinase-1. The structure is of unphosphorylated, active conformation of casein kinase-1 and the peptide-binding site is fully accessible to substrate. Images PMID:7889932

Phosphorylation of ERK/MAP Kinase Is Required for Long-Term Potentiation in Anatomically Restricted Regions of the Lateral Amygdala in Vivo

ERIC Educational Resources Information Center

Schafe, Glenn E.; Swank, Michael W.; Rodriguez, Sarina M.; Debiec, Jacek; Doyere, Valerie

2008-01-01

We have previously shown that the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/ MAPK) is transiently activated in anatomically restricted regions of the lateral amygdala (LA) following Pavlovian fear conditioning and that blockade of ERK/MAPK activation in the LA impairs both fear memory consolidation and long-term…

HOG MAP kinase regulation of alternariol biosynthesis in Alternaria alternata is important for substrate colonization.

PubMed

Graf, Eva; Schmidt-Heydt, Markus; Geisen, Rolf

2012-07-16

Strains of the genus Alternaria are ubiquitously present and frequently found on fruits, vegetables and cereals. One of the most commonly found species from this genus is A. alternata which is able to produce the mycotoxin alternariol among others. To date only limited knowledge is available about the regulation of the biosynthesis of alternariol, especially under conditions relevant to food. Tomatoes are a typical substrate of A. alternata and have a high water activity. On the other hand cereals with moderate water activity are also frequently colonized by A. alternata. In the current analysis it was demonstrated that even minor changes in the osmotic status of the substrate affect the alternariol biosynthesis of strains from vegetables resulting in nearly complete inhibition. High osmolarity in the environment is usually transmitted to the transcriptional level of downstream regulated genes by the HOG signal cascade (high osmolarity glycerol cascade) which is a MAP kinase transduction pathway. The phosphorylation status of the A. alternata HOG (AaHOG) was determined. Various concentrations of NaCl induce the phosphorylation of AaHOG in a concentration, time and strain dependent manner. A strain with a genetically inactivated aahog gene was no longer able to produce alternariol indicating that the activity of the aahog gene is required for alternariol biosynthesis. Further experiments revealed that the biosynthesis of alternariol is important for the fungus to colonize tomato tissue. The tight water activity dependent regulation of alternariol biosynthesis ensures alternariol biosynthesis at conditions which indicate an optimal colonization substrate for the fungus. Copyright © 2012 Elsevier B.V. All rights reserved.

Cocaine Effects on Dopaminergic Transmission Depend on a Balance between Sigma-1 and Sigma-2 Receptor Expression.

PubMed

Aguinaga, David; Medrano, Mireia; Vega-Quiroga, Ignacio; Gysling, Katia; Canela, Enric I; Navarro, Gemma; Franco, Rafael

2018-01-01

Sigma σ 1 and σ 2 receptors are targets of cocaine. Despite sharing a similar name, the two receptors are structurally unrelated and their physiological role is unknown. Cocaine increases the level of dopamine, a key neurotransmitter in CNS motor control and reward areas. While the drug also affects dopaminergic signaling by allosteric modulations exerted by σ 1 R interacting with dopamine D 1 and D 2 receptors, the potential regulation of dopaminergic transmission by σ 2 R is also unknown. We here demonstrate that σ 2 R may form heteroreceptor complexes with D 1 but not with D 2 receptors. Remarkably σ 1 , σ 2 , and D 1 receptors may form heterotrimers with particular signaling properties. Determination of cAMP levels, MAP kinase activation and label-free assays demonstrate allosteric interactions within the trimer. Importantly, the presence of σ 2 R induces bias in signal transduction as σ 2 R ligands increase cAMP signaling whereas reduce MAP kinase activation. These effects, which are opposite to those exerted via σ 1 R, suggest that the D 1 receptor-mediated signaling depends on the degree of trimer formation and the differential balance of sigma receptor and heteroreceptor expression in acute versus chronic cocaine consumption. Although the physiological role is unknown, the heteroreceptor complex formed by σ 1 , σ 2 , and D 1 receptors arise as relevant to convey the cocaine actions on motor control and reward circuits and as a key factor in acquisition of the addictive habit.

Cocaine Effects on Dopaminergic Transmission Depend on a Balance between Sigma-1 and Sigma-2 Receptor Expression

PubMed Central

Aguinaga, David; Medrano, Mireia; Vega-Quiroga, Ignacio; Gysling, Katia; Canela, Enric I.; Navarro, Gemma; Franco, Rafael

2018-01-01

Sigma σ1 and σ2 receptors are targets of cocaine. Despite sharing a similar name, the two receptors are structurally unrelated and their physiological role is unknown. Cocaine increases the level of dopamine, a key neurotransmitter in CNS motor control and reward areas. While the drug also affects dopaminergic signaling by allosteric modulations exerted by σ1R interacting with dopamine D1 and D2 receptors, the potential regulation of dopaminergic transmission by σ2R is also unknown. We here demonstrate that σ2R may form heteroreceptor complexes with D1 but not with D2 receptors. Remarkably σ1, σ2, and D1 receptors may form heterotrimers with particular signaling properties. Determination of cAMP levels, MAP kinase activation and label-free assays demonstrate allosteric interactions within the trimer. Importantly, the presence of σ2R induces bias in signal transduction as σ2R ligands increase cAMP signaling whereas reduce MAP kinase activation. These effects, which are opposite to those exerted via σ1R, suggest that the D1 receptor-mediated signaling depends on the degree of trimer formation and the differential balance of sigma receptor and heteroreceptor expression in acute versus chronic cocaine consumption. Although the physiological role is unknown, the heteroreceptor complex formed by σ1, σ2, and D1 receptors arise as relevant to convey the cocaine actions on motor control and reward circuits and as a key factor in acquisition of the addictive habit. PMID:29483862

Loss of MAP3K1 enhances proliferation and apoptosis during retinal development

PubMed Central

Mongan, Maureen; Wang, Jingcai; Liu, Hongshan; Fan, Yunxia; Jin, Chang; Kao, Winston Y.-W.; Xia, Ying

2011-01-01

Precise coordination of progenitor cell proliferation and differentiation is essential for proper organ morphogenesis and function during mammalian development. The mitogen-activated protein kinase kinase kinase 1 (MAP3K1) has a well-established role in anterior eyelid development, as Map3k1-knockout mice have defective embryonic eyelid closure and an `eye-open at birth' (EOB) phenotype. Here, we show that MAP3K1 is highly expressed in the posterior of the developing eye and is required for retina development. The MAP3K1-deficient mice exhibit increased proliferation and apoptosis, and Müller glial cell overproduction in the developing retinas. Consequently, the retinas of these mice show localized rosette-like arrangements in the outer nuclear layer, and develop abnormal vascularization, broken down retinal pigment epithelium, photoreceptor loss and early onset of retinal degeneration. Although the retinal defect is associated with increased cyclin D1 and CDK4/6 expression, and RB phosphorylation and E2F-target gene upregulation, it is independent of the EOB phenotype and of JNK. The retinal developmental defect still occurs in knockout mice that have undergone tarsorrhaphy, but is absent in compound mutant Map3k1+/ΔKDJnk1–/– and Map3k1+/ΔKDJnk+/–Jnk2+/– mice that have EOB and reduced JNK signaling. Our results unveil a novel role for MAP3K1 in which it crosstalks with the cell cycle regulatory pathways in the prevention of retina malformation and degeneration. PMID:21862560

Nobiletin and its related flavonoids with CRE-dependent transcription-stimulating and neuritegenic activities.

PubMed

Nagase, Hiroyuki; Omae, Naoki; Omori, Akiko; Nakagawasai, Osamu; Tadano, Takeshi; Yokosuka, Akihito; Sashida, Yutaka; Mimaki, Yoshihiro; Yamakuni, Tohru; Ohizumi, Yasushi

2005-12-02

cAMP response element (CRE) transcription is dysregulated in neurodegenerative disorders in the central nervous system (CNS), including polyglutamine diseases. As the first step to find natural compounds with protective action against neurodegeneration in the CNS, we here examined whether six citrus flavonoids, namely nobiletin, 5-demethylnobiletin, tangeretin, sinensetin, 6-demethoxytangeretin, and 6-demethoxynobiletin, stimulated CRE-dependent transcription and induced neurite outgrowth in PC12D cells. Among the compounds, nobiletin most potently enhanced CRE-dependent transcription and neurite outgrowth by activating ERK/MAP kinase-dependent signalling to increase CREB phosphorylation. The transcription and neurite outgrowth were stimulated by nobiletin in a concentration-dependent manner, with a strong correlation between them. Furthermore, a 11-day oral administration of nobiletin rescued impaired memory in olfactory-bulbectomized mice documented to be accompanied by a cholinergic neurodegeneration. These results suggest that nobiletin with the activity to improve impaired memory may become a potential leading compound for drug development for neurodegenerative disorders exhibiting the dysregulated CRE-dependent transcription.

Method of empirical dependences in estimation and prediction of activity of creatine kinase isoenzymes in cerebral ischemia

NASA Astrophysics Data System (ADS)

Sergeeva, Tatiana F.; Moshkova, Albina N.; Erlykina, Elena I.; Khvatova, Elena M.

2016-04-01

Creatine kinase is a key enzyme of energy metabolism in the brain. There are known cytoplasmic and mitochondrial creatine kinase isoenzymes. Mitochondrial creatine kinase exists as a mixture of two oligomeric forms - dimer and octamer. The aim of investigation was to study catalytic properties of cytoplasmic and mitochondrial creatine kinase and using of the method of empirical dependences for the possible prediction of the activity of these enzymes in cerebral ischemia. Ischemia was revealed to be accompanied with the changes of the activity of creatine kinase isoenzymes and oligomeric state of mitochondrial isoform. There were made the models of multiple regression that permit to study the activity of creatine kinase system in cerebral ischemia using a calculating method. Therefore, the mathematical method of empirical dependences can be applied for estimation and prediction of the functional state of the brain by the activity of creatine kinase isoenzymes in cerebral ischemia.

Allosteric activation of apicomplexan calcium-dependent protein kinases

DOE PAGES

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

2015-08-24

Calcium-dependent protein kinases (CDPKs) comprise the major group of Ca 2+-regulated kinases in plants and protists. It has long been assumed that CDPKs are activated, like other Ca 2+-regulated kinases, by derepression of the kinase domain (KD). However, we found that removal of the autoinhibitory domain from Toxoplasma gondii CDPK1 is not sufficient for kinase activation. From a library of heavy chain-only antibody fragments (VHHs), we isolated an antibody (1B7) that binds TgCDPK1 in a conformation-dependent manner and potently inhibits it. We uncovered the molecular basis for this inhibition by solving the crystal structure of the complex and simulating, throughmore » molecular dynamics, the effects of 1B7–kinase interactions. In contrast to other Ca 2+-regulated kinases, the regulatory domain of TgCDPK1 plays a dual role, inhibiting or activating the kinase in response to changes in Ca 2+ concentrations. We propose that the regulatory domain of TgCDPK1 acts as a molecular splint to stabilize the otherwise inactive KD. This dependence on allosteric stabilization reveals a novel susceptibility in this important class of parasite enzymes.« less

The role of hybrid ubiquitin chains in the MyD88 and other innate immune signalling pathways.

PubMed

Cohen, Philip; Strickson, Sam

2017-07-01

The adaptor protein MyD88 is required for signal transmission by toll-like receptors and receptors of the interleukin-1 family of cytokines. MyD88 signalling triggers the formation of Lys63-linked and Met1-linked ubiquitin (K63-Ub, M1-Ub) chains within minutes. The K63-Ub chains, which are formed by the E3 ubiquitin ligases TRAF6, Pellino1 and Pellino2, activate TAK1, the master kinase that switches on mitogen-activated protein (MAP) kinase cascades and initiates activation of the canonical IκB kinase (IKK) complex. The M1-Ub chains, which are formed by the linear ubiquitin chain assembly complex (LUBAC), bind to the NEMO (NF-κB essential modulator) component of the IKK complex and are required for TAK1 to activate IKKs, but not MAP kinases. An essential E3 ligase-independent role of TRAF6 is to recruit LUBAC into the MyD88 signalling complex, where it recognises preformed K63-Ub chains attached to protein components of these complexes, such as IRAK1 (IL-1 receptor-associated kinase), producing ubiquitin chains containing both types of linkage, termed K63/M1-Ub hybrids. The formation of K63/M1-Ub hybrids, which is a feature of several innate immune signalling pathways, permits the co-recruitment of proteins that interact with either K63-Ub or M1-Ub chains. Two likely roles for K63/M1-Ub hybrids are to facilitate the TAK1-dependent activation of the IKK complex and to prevent the hyperactivation of these kinases by recruiting A20 and A20-binding inhibitor of NF-κB1 (ABIN1). These proteins restrict activation of the TAK1 and IKK complexes, probably by competing with them for binding to K63/M1-Ub hybrids. The formation of K63/M1-Ub hybrids may also regulate the rate at which the ubiquitin linkages in these chains are hydrolysed. The IKK-catalysed phosphorylation of some of its substrates permits their recognition by the E3 ligase SCF βTRCP , leading to their Lys48-linked ubiquitylation and proteasomal degradation. Innate immune signalling is therefore controlled by the formation and destruction of three different types of ubiquitin linkage.

The role of hybrid ubiquitin chains in the MyD88 and other innate immune signalling pathways

PubMed Central

Cohen, Philip; Strickson, Sam

2017-01-01

The adaptor protein MyD88 is required for signal transmission by toll-like receptors and receptors of the interleukin-1 family of cytokines. MyD88 signalling triggers the formation of Lys63-linked and Met1-linked ubiquitin (K63-Ub, M1-Ub) chains within minutes. The K63-Ub chains, which are formed by the E3 ubiquitin ligases TRAF6, Pellino1 and Pellino2, activate TAK1, the master kinase that switches on mitogen-activated protein (MAP) kinase cascades and initiates activation of the canonical IκB kinase (IKK) complex. The M1-Ub chains, which are formed by the linear ubiquitin chain assembly complex (LUBAC), bind to the NEMO (NF-κB essential modulator) component of the IKK complex and are required for TAK1 to activate IKKs, but not MAP kinases. An essential E3 ligase-independent role of TRAF6 is to recruit LUBAC into the MyD88 signalling complex, where it recognises preformed K63-Ub chains attached to protein components of these complexes, such as IRAK1 (IL-1 receptor-associated kinase), producing ubiquitin chains containing both types of linkage, termed K63/M1-Ub hybrids. The formation of K63/M1-Ub hybrids, which is a feature of several innate immune signalling pathways, permits the co-recruitment of proteins that interact with either K63-Ub or M1-Ub chains. Two likely roles for K63/M1-Ub hybrids are to facilitate the TAK1-dependent activation of the IKK complex and to prevent the hyperactivation of these kinases by recruiting A20 and A20-binding inhibitor of NF-κB1 (ABIN1). These proteins restrict activation of the TAK1 and IKK complexes, probably by competing with them for binding to K63/M1-Ub hybrids. The formation of K63/M1-Ub hybrids may also regulate the rate at which the ubiquitin linkages in these chains are hydrolysed. The IKK-catalysed phosphorylation of some of its substrates permits their recognition by the E3 ligase SCFβTRCP, leading to their Lys48-linked ubiquitylation and proteasomal degradation. Innate immune signalling is therefore controlled by the formation and destruction of three different types of ubiquitin linkage. PMID:28475177

In vitro mapping of Myotonic Dystrophy (DM) gene promoter

DOE Office of Scientific and Technical Information (OSTI.GOV)

Storbeck, C.J.; Sabourin, L.; Baird, S.

1994-09-01

The Myotonic Dystrophy Kinase (DMK) gene has been cloned and shared homology to serine/threonine protein kinases. Overexpression of this gene in stably transfected mouse myoblasts has been shown to inhibit fusion into myotubes while myoblasts stably transfected with an antisense construct show increased fusion potential. These experiments, along with data showing that the DM gene is highly expressed in muscle have highlighted the possibility of DMK being involved in myogenesis. The promoter region of the DM gene lacks a consensus TATA box and CAAT box, but harbours numerous transcription binding sites. Clones containing extended 5{prime} upstream sequences (UPS) of DMKmore » only weakly drive the reporter gene chloramphenicol acetyl transferase (CAT) when transfected into C2C12 mouse myoblasts. However, four E-boxes are present in the first intron of the DM gene and transient assays show increased expression of the CAT gene when the first intron is present downstream of these 5{prime} UPS in an orientation dependent manner. Comparison between mouse and human sequence reveals that the regions in the first intron where the E-boxes are located are highly conserved. The mapping of the promoter and the importance of the first intron in the control of DMK expression will be presented.« less

Synthesis and biological evaluation of 5-(fluoro-substituted-6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)imidazoles as inhibitors of transforming growth factor-β type I receptor kinase.

PubMed

Krishnaiah, Maddeboina; Jin, Cheng Hua; Sheen, Yhun Yhong; Kim, Dae-Kee

2015-11-15

To further optimize a clinical candidate 5 (EW-7197), a series of 5-(3-, 4-, or 5-fluoro-substituted-6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)imidazoles 19a-l have been synthesized and evaluated for their TGF-β type I receptor kinase (ALK5) and p38α MAP kinase inhibitory activity in an enzyme assay. The 5-(5-fluoro-substituted-6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)imidazoles 19h-l displayed the similar level of potency to that of 5 against both ALK5 (IC50=7.68-13.70 nM) and p38α MAP kinase (IC50=1240-3370 nM). Among them, 19j inhibited ALK5 with IC50 value of 7.68 nM in a kinase assay and displayed 82% inhibition at 100 nM in a luciferase reporter assay. Copyright © 2015 Elsevier Ltd. All rights reserved.

Nat1 promotes translation of specific proteins that induce differentiation of mouse embryonic stem cells.

PubMed

Sugiyama, Hayami; Takahashi, Kazutoshi; Yamamoto, Takuya; Iwasaki, Mio; Narita, Megumi; Nakamura, Masahiro; Rand, Tim A; Nakagawa, Masato; Watanabe, Akira; Yamanaka, Shinya

2017-01-10

Novel APOBEC1 target 1 (Nat1) (also known as "p97," "Dap5," and "Eif4g2") is a ubiquitously expressed cytoplasmic protein that is homologous to the C-terminal two thirds of eukaryotic translation initiation factor 4G (Eif4g1). We previously showed that Nat1-null mouse embryonic stem cells (mES cells) are resistant to differentiation. In the current study, we found that NAT1 and eIF4G1 share many binding proteins, such as the eukaryotic translation initiation factors eIF3 and eIF4A and ribosomal proteins. However, NAT1 did not bind to eIF4E or poly(A)-binding proteins, which are critical for cap-dependent translation initiation. In contrast, compared with eIF4G1, NAT1 preferentially interacted with eIF2, fragile X mental retardation proteins (FMR), and related proteins and especially with members of the proline-rich and coiled-coil-containing protein 2 (PRRC2) family. We also found that Nat1-null mES cells possess a transcriptional profile similar, although not identical, to the ground state, which is established in wild-type mES cells when treated with inhibitors of the ERK and glycogen synthase kinase 3 (GSK3) signaling pathways. In Nat1-null mES cells, the ERK pathway is suppressed even without inhibitors. Ribosome profiling revealed that translation of mitogen-activated protein kinase kinase kinase 3 (Map3k3) and son of sevenless homolog 1 (Sos1) is suppressed in the absence of Nat1 Forced expression of Map3k3 induced differentiation of Nat1-null mES cells. These data collectively show that Nat1 is involved in the translation of proteins that are required for cell differentiation.

Regulation of angiotensin II-induced neuromodulation by MARCKS in brain neurons.

PubMed

Lu, D; Yang, H; Lenox, R H; Raizada, M K

1998-07-13

Angiotensin II (Ang II) exerts chronic stimulatory actions on tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH), and the norepinephrine transporter (NET), in part, by influencing the transcription of their genes. These neuromodulatory actions of Ang II involve Ras-Raf-MAP kinase signal transduction pathways (Lu, D., H. Yang, and M.K. Raizada. 1997. J. Cell Biol. 135:1609-1617). In this study, we present evidence to demonstrate participation of another signaling pathway in these neuronal actions of Ang II. It involves activation of protein kinase C (PKC)beta subtype and phosphorylation and redistribution of myristoylated alanine-rich C kinase substrate (MARCKS) in neurites. Ang II caused a dramatic redistribution of MARCKS from neuronal varicosities to neurites. This was accompanied by a time-dependent stimulation of its phosphorylation, that was mediated by the angiotensin type 1 receptor subtype (AT1). Incubation of neurons with PKCbeta subtype specific antisense oligonucleotide (AON) significantly attenuated both redistribution and phosphorylation of MARCKS. Furthermore, depletion of MARCKS by MARCKS-AON treatment of neurons resulted in a significant decrease in Ang II-stimulated accumulation of TH and DbetaH immunoreactivities and [3H]NE uptake activity in synaptosomes. In contrast, mRNA levels of TH, DbetaH, and NET were not influenced by MARKS-AON treatment. MARCKS pep148-165, which contains PKC phosphorylation sites, inhibited Ang II stimulation of MARCKS phosphorylation and reduced the amount of TH, DbetaH, and [3H]NE uptake in neuronal synaptosomes. These observations demonstrate that phosphorylation of MARCKS by PKCbeta and its redistribution from varicosities to neurites is important in Ang II-induced synaptic accumulation of TH, DbetaH, and NE. They suggest that a coordinated stimulation of transcription of TH, DbetaH, and NET, mediated by Ras-Raf-MAP kinase followed by their transport mediated by PKCbeta-MARCKS pathway are key in persistent stimulation of Ang II's neuromodulatory actions.

SALT-RESPONSIVE ERF1 Regulates Reactive Oxygen Species–Dependent Signaling during the Initial Response to Salt Stress in Rice[W

PubMed Central

Schmidt, Romy; Mieulet, Delphine; Hubberten, Hans-Michael; Obata, Toshihiro; Hoefgen, Rainer; Fernie, Alisdair R.; Fisahn, Joachim; San Segundo, Blanca; Guiderdoni, Emmanuel; Schippers, Jos H.M.; Mueller-Roeber, Bernd

2013-01-01

Early detection of salt stress is vital for plant survival and growth. Still, the molecular processes controlling early salt stress perception and signaling are not fully understood. Here, we identified SALT-RESPONSIVE ERF1 (SERF1), a rice (Oryza sativa) transcription factor (TF) gene that shows a root-specific induction upon salt and hydrogen peroxide (H2O2) treatment. Loss of SERF1 impairs the salt-inducible expression of genes encoding members of a mitogen-activated protein kinase (MAPK) cascade and salt tolerance–mediating TFs. Furthermore, we show that SERF1-dependent genes are H2O2 responsive and demonstrate that SERF1 binds to the promoters of MAPK KINASE KINASE6 (MAP3K6), MAPK5, DEHYDRATION-RESPONSIVE ELEMENT BINDING2A (DREB2A), and ZINC FINGER PROTEIN179 (ZFP179) in vitro and in vivo. SERF1 also directly induces its own gene expression. In addition, SERF1 is a phosphorylation target of MAPK5, resulting in enhanced transcriptional activity of SERF1 toward its direct target genes. In agreement, plants deficient for SERF1 are more sensitive to salt stress compared with the wild type, while constitutive overexpression of SERF1 improves salinity tolerance. We propose that SERF1 amplifies the reactive oxygen species–activated MAPK cascade signal during the initial phase of salt stress and translates the salt-induced signal into an appropriate expressional response resulting in salt tolerance. PMID:23800963

Tpl2 kinase regulates T cell interferon-γ production and host resistance to Toxoplasma gondii

PubMed Central

Watford, Wendy T.; Hissong, Bruce D.; Durant, Lydia R.; Yamane, Hidehiro; Muul, Linda M.; Kanno, Yuka; Tato, Cristina M.; Ramos, Haydeé L.; Berger, Alan E.; Mielke, Lisa; Pesu, Marko; Solomon, Benjamin; Frucht, David M.; Paul, William E.; Sher, Alan; Jankovic, Dragana; Tsichlis, Philip N.; O'Shea, John J.

2008-01-01

Tpl2 (Tumor progression locus 2), also known as Cot/MAP3K8, is a hematopoietically expressed serine-threonine kinase. Tpl2 is known to have critical functions in innate immunity in regulating tumor necrosis factor–α, Toll-like receptor, and G protein–coupled receptor signaling; however, our understanding of its physiological role in T cells is limited. We investigated the potential roles of Tpl2 in T cells and found that it was induced by interleukin-12 in human and mouse T cells in a Stat4-dependent manner. Deficiency of Tpl2 was associated with impaired interferon (IFN)-γ production. Accordingly, Tpl2−/− mice had impaired host defense against Toxoplasma gondii with reduced parasite clearance and decreased IFN-γ production. Furthermore, reconstitution of Rag2−/− mice with Tpl2-deficient T cells followed by T. gondii infection recapitulated the IFN-γ defect seen in the Tpl2-deficient mice, confirming a T cell–intrinsic defect. CD4+ T cells isolated from Tpl2−/− mice showed poor induction of T-bet and failure to up-regulate Stat4 protein, which is associated with impaired TCR-dependent extracellular signal-regulated kinase activation. These data underscore the role of Tpl2 as a regulator of T helper cell lineage decisions and demonstrate that Tpl2 has an important functional role in the regulation of Th1 responses. PMID:19001140

A fine structure genomic map of the region of 12q13 containing SAS and CDK4

DOE Office of Scientific and Technical Information (OSTI.GOV)

Linder, C.Y.; Elkahloun, A.G.; Su, Y.A.

1994-09-01

We have recently adapted a method, originally described by Rackwitz, to the rapid restriction mapping of multiple cosmid DNA samples. Linearization of the cosmids at the lambda cohesive site using lambda terminase is followed by partial digestion with selected restriction enzymes and hybridization to oligonucleotides specific for the right or left hand termini. Partial digestions are performed in a microtiter plate thus allowing up to 12 cosmid clones to be digested with one restriction enzyme. We have applied this rapid restriction mapping method to cosmids derived from a region of chromosome 12q13 that has recently been shown to be amplifiedmore » in a variety of cancers including malignant fibrous histiocytoma, fibrosarcoma, liposarcoma, osteosarcoma and brain tumors. A small segment of this amplification unit containing three genes, SAS (a membrane protein), CDK4 (a cyclin dependent kinase) and OS-9 (a recently described cDNA) has been analyzed with the system described above. This fine structure genomic map will be useful for completing the expression map of this region as well as characterizing its pattern of amplification in tumor specimens.« less

2′,5′-Dihydroxychalcone-induced glutathione is mediated by oxidative stress and kinase signaling pathways

PubMed Central

Kachadourian, Remy; Pugazhenthi, Subbiah; Velmurugan, Kalpana; Backos, Donald S.; Franklin, Christopher C.; McCord, Joe M.; Day, Brian J.

2011-01-01

Hydroxychalcones are naturally occurring compounds that continue to attract considerable interest due to their anti-inflammatory and anti-angiogenic properties. They have been reported to inhibit the synthesis of the inducible nitric oxide (NO) synthase and to induce the expression of heme oxygenase-1 (HO-1). This study examines the mechanisms by which 2′,5′-dihydroxychalcone (2′,5′-DHC) induces an increase in cellular glutathione (GSH) levels using a cell line stably expressing a luciferase reporter gene driven by antioxidant response elements (MCF-7/AREc32). 2′,5′-DHC-induced increase in cellular GSH levels was partially inhibited by the catalytic antioxidant MnTDE-1,3-IP5+, suggesting that reactive oxygen species (ROS) mediate the antioxidant adaptive response. 2′,5′-DHC treatment induced the phosphorylation of c-Jun N-terminal kinase (JNK) pathway that was also inhibited by MnTDE-1,3-IP5+. These findings suggest a ROS-dependent activation of the AP-1 transcriptional response. However, while 2′,5′-DHC triggered the NF-E2-related factor 2 (Nrf2) transcriptional response, co-treatment with MnTDE-1,3-IP5+ did not decrease 2′,5′-DHC-induced Nrf2/ARE activity, showing that this pathway is not dependent on ROS. Moreover, pharmacological inhibitors of mitogen-activated protein (MAP) kinase pathways showed a role for JNK and p38MAPK in mediating the 2′,5′-DHC-induced Nrf2 response. These findings suggest that the 2′,5′-DHC-induced increase in GSH levels results from a combination of ROS-dependent and ROS-independent pathways. PMID:21712085

PEBP1, a RAF kinase inhibitory protein, negatively regulates starvation-induced autophagy by direct interaction with LC3.

PubMed

Noh, Hae Sook; Hah, Young-Sool; Zada, Sahib; Ha, Ji Hye; Sim, Gyujin; Hwang, Jin Seok; Lai, Trang Huyen; Nguyen, Huynh Quoc; Park, Jae-Yong; Kim, Hyun Joon; Byun, June-Ho; Hahm, Jong Ryeal; Kang, Kee Ryeon; Kim, Deok Ryong

2016-11-01

Autophagy plays a critical role in maintaining cell homeostasis in response to various stressors through protein conjugation and activation of lysosome-dependent degradation. MAP1LC3B/LC3B (microtubule- associated protein 1 light chain 3 β) is conjugated with phosphatidylethanolamine (PE) in the membranes and regulates initiation of autophagy through interaction with many autophagy-related proteins possessing an LC3-interacting region (LIR) motif, which is composed of 2 hydrophobic amino acids (tryptophan and leucine) separated by 2 non-conserved amino acids (WXXL). In this study, we identified a new putative LIR motif in PEBP1/RKIP (phosphatidylethanolamine binding protein 1) that was originally isolated as a PE-binding protein and also a cellular inhibitor of MAPK/ERK signaling. PEBP1 was specifically bound to PE-unconjugated LC3 in cells, and mutation (WXXL mutated to AXXA) of this LIR motif disrupted its interaction with LC3 proteins. Interestingly, overexpression of PEBP1 significantly inhibited starvation-induced autophagy by activating the AKT and MTORC1 (mechanistic target of rapamycin [serine/threonine kinase] complex 1) signaling pathway and consequently suppressing the ULK1 (unc-51 like autophagy activating kinase 1) activity. In contrast, ablation of PEBP1 expression dramatically promoted the autophagic process under starvation conditions. Furthermore, PEBP1 lacking the LIR motif highly stimulated starvation-induced autophagy through the AKT-MTORC1-dependent pathway. PEBP1 phosphorylation at Ser153 caused dissociation of LC3 from the PEBP1-LC3 complex for autophagy induction. PEBP1-dependent suppression of autophagy was not associated with the MAPK pathway. These findings suggest that PEBP1 can act as a negative mediator in autophagy through stimulation of the AKT-MTORC1 pathway and direct interaction with LC3.

Novel 3-Substituted 7-Phenylpyrrolo[3,2-f]quinolin-9(6H)-ones as Single Entities with Multitarget Antiproliferative Activity.

PubMed

Carta, Davide; Bortolozzi, Roberta; Hamel, Ernest; Basso, Giuseppe; Moro, Stefano; Viola, Giampietro; Ferlin, Maria Grazia

2015-10-22

A series of chemically modified 7-phenylpyrrolo[3,2-f]quinolinones was synthesized and evaluated as anticancer agents. Among them, the most cytotoxic (subnanomolar GI50 values) amidic derivative 5f was shown to act as an inhibitor of tubulin polymerization (IC50, 0.99 μM) by binding to the colchicine site with high affinity. Moreover, 5f induced cell cycle arrest in the G2/M phase of the cell cycle in a concentration dependent manner, followed by caspase-dependent apoptotic cell death. Compound 5f also showed lower toxicity in nontumoral cells, suggesting selectivity toward cancer cells. Additional experiments revealed that 5f inhibited the enzymatic activity of multiple kinases, including AURKA, FLT3, GSK3A, MAP3K, MEK, RSK2, RSK4, PLK4, ULK1, and JAK1. Computational studies showed that 5f can be properly accommodated in the colchicine binding site of tubulin as well as in the ATP binding clefts of all examined kinases. Our data indicate that the excellent antiproliferative profile of 5f may be derived from its interactions with multiple cellular targets.

Accurate calculation of mutational effects on the thermodynamics of inhibitor binding to p38α MAP kinase: a combined computational and experimental study.

PubMed

Zhu, Shun; Travis, Sue M; Elcock, Adrian H

2013-07-09

A major current challenge for drug design efforts focused on protein kinases is the development of drug resistance caused by spontaneous mutations in the kinase catalytic domain. The ubiquity of this problem means that it would be advantageous to develop fast, effective computational methods that could be used to determine the effects of potential resistance-causing mutations before they arise in a clinical setting. With this long-term goal in mind, we have conducted a combined experimental and computational study of the thermodynamic effects of active-site mutations on a well-characterized and high-affinity interaction between a protein kinase and a small-molecule inhibitor. Specifically, we developed a fluorescence-based assay to measure the binding free energy of the small-molecule inhibitor, SB203580, to the p38α MAP kinase and used it measure the inhibitor's affinity for five different kinase mutants involving two residues (Val38 and Ala51) that contact the inhibitor in the crystal structure of the inhibitor-kinase complex. We then conducted long, explicit-solvent thermodynamic integration (TI) simulations in an attempt to reproduce the experimental relative binding affinities of the inhibitor for the five mutants; in total, a combined simulation time of 18.5 μs was obtained. Two widely used force fields - OPLS-AA/L and Amber ff99SB-ILDN - were tested in the TI simulations. Both force fields produced excellent agreement with experiment for three of the five mutants; simulations performed with the OPLS-AA/L force field, however, produced qualitatively incorrect results for the constructs that contained an A51V mutation. Interestingly, the discrepancies with the OPLS-AA/L force field could be rectified by the imposition of position restraints on the atoms of the protein backbone and the inhibitor without destroying the agreement for other mutations; the ability to reproduce experiment depended, however, upon the strength of the restraints' force constant. Imposition of position restraints in corresponding simulations that used the Amber ff99SB-ILDN force field had little effect on their ability to match experiment. Overall, the study shows that both force fields can work well for predicting the effects of active-site mutations on small molecule binding affinities and demonstrates how a direct combination of experiment and computation can be a powerful strategy for developing an understanding of protein-inhibitor interactions.

Analysis of Activated Platelet-Derived Growth Factor β Receptor and Ras-MAP Kinase Pathway in Equine Sarcoid Fibroblasts

PubMed Central

Altamura, Gennaro; Corteggio, Annunziata; Nasir, Lubna; Yuan, Zheng Qiang; Roperto, Franco; Borzacchiello, Giuseppe

2013-01-01

Equine sarcoids are skin tumours of fibroblastic origin affecting equids worldwide. Bovine papillomavirus type-1 (BPV-1) and, less commonly, type-2 are recognized as etiological factors of sarcoids. The transforming activity of BPV is related to the functions of its major oncoprotein E5 which binds to the platelet-derived growth factor β receptor (PDGFβR) causing its phosphorylation and activation. In this study, we demonstrate, by coimmunoprecipitation and immunoblotting, that in equine sarcoid derived cell lines PDGFβR is phosphorylated and binds downstream molecules related to Ras-mitogen-activated protein kinase-ERK pathway thus resulting in Ras activation. Imatinib mesylate is a tyrosine kinase receptors inhibitor which selectively inhibits the activation of PDGFβR in the treatment of several human and animal cancers. Here we show that imatinib inhibits receptor phosphorylation, and cell viability assays demonstrate that this drug decreases sarcoid fibroblasts viability in a dose-dependent manner. This study contributes to a better understanding of the molecular mechanisms involved in the pathology of sarcoids and paves the way to a new therapeutic approach for the treatment of this common equine skin neoplasm. PMID:23936786

Bisphosphonates inactivate human EGFRs to exert antitumor actions

PubMed Central

Yuen, Tony; Stachnik, Agnes; Iqbal, Jameel; Sgobba, Miriam; Gupta, Yogesh; Lu, Ping; Colaianni, Graziana; Ji, Yaoting; Zhu, Ling-Ling; Kim, Se-Min; Li, Jianhua; Liu, Peng; Izadmehr, Sudeh; Sangodkar, Jaya; Bailey, Jack; Latif, Yathin; Mujtaba, Shiraz; Epstein, Solomon; Davies, Terry F.; Bian, Zhuan; Zallone, Alberta; Aggarwal, Aneel K.; Haider, Shozeb; New, Maria I.; Sun, Li; Narla, Goutham; Zaidi, Mone

2014-01-01

Bisphosphonates are the most commonly prescribed medicines for osteoporosis and skeletal metastases. The drugs have also been shown to reduce cancer progression, but only in certain patient subgroups, suggesting that there is a molecular entity that mediates bisphosphonate action on tumor cells. Using connectivity mapping, we identified human epidermal growth factor receptors (human EGFR or HER) as a potential new molecular entity for bisphosphonate action. Protein thermal shift and cell-free kinase assays, together with computational modeling, demonstrated that N-containing bisphosphonates directly bind to the kinase domain of HER1/2 to cause a global reduction in downstream signaling. By doing so, the drugs kill lung, breast, and colon cancer cells that are driven by activating mutations or overexpression of HER1. Knocking down HER isoforms thus abrogates cell killing by bisphosphonates, establishing complete HER dependence and ruling out a significant role for other receptor tyrosine kinases or the enzyme farnesyl pyrophosphate synthase. Consistent with this finding, colon cancer cells expressing low levels of HER do not respond to bisphosphonates. The results suggest that bisphosphonates can potentially be repurposed for the prevention and therapy of HER family-driven cancers. PMID:25453081

The p110α and p110β Isoforms of Class I Phosphatidylinositol 3-Kinase Are Involved in Toll-Like Receptor 5 Signaling in Epithelial Cells

PubMed Central

Ivison, Sabine M.; Khan, Mohammed A. S.; Graham, Nicholas R.; Shobab, Leila A.; Yao, Yu; Kifayet, Arnawaz; Sly, Laura M.; Steiner, Theodore S.

2010-01-01

Background. Bacterial flagellin triggers inflammation in mammalian cells via Toll-like receptor (TLR) 5. Release of the chemokine IL-8 in response to flagellin involves NF-κB, p38 MAP kinase, and phosphatidylinositol 3-kinase (PI3K). However, PI3K has been reported to be either pro- or anti-inflammatory in different model systems. We hypothesized that this could be due to different activities of the p110α and β isoforms of PI3K. Results. PI3K and Akt were rapidly activated in Caco-2 colon carcinoma cells by flagellin. Using a plasmid-based shRNA delivery system and novel p110 isoform-specific inhibitors, we found that flagellin-induced IL-8 production was dependent on both p110α and p110β. However in the mouse, inhibition of p110β but not p110α reduced the increase of serum IL-6 levels induced by intraperitoneal injection of flagellin. Conclusions. These data demonstrate that the p110α and β isoforms of class IA PI3K are both required for the proinflammatory response to flagellin. PMID:20953381

Paroxetine-induced apoptosis in human osteosarcoma cells: Activation of p38 MAP kinase and caspase-3 pathways without involvement of [Ca{sup 2+}]{sub i} elevation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chou, C.-T.; Department of Biological Sciences, National Sun Yat-sen University, 804, Taiwan; He Shiping

2007-02-01

Selective serotonin reuptake inhibitors (SSRIs), a group of antidepressants, are generally used for treatment of various mood and anxiety disorders. There has been much research showing the anti-tumor and cytotoxic activities of some antidepressants; but the detailed mechanisms were unclear. In cultured human osteosarcoma cells (MG63), paroxetine reduced cell viability in a concentration- and time-dependent manner. Paroxetine caused apoptosis as assessed by propidium iodide-stained cells and increased caspase-3 activation. Although immunoblotting data revealed that paroxetine could activate the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun NH{sub 2}-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), only SB203580 (a p38more » MAPK inhibitor) partially prevented cells from apoptosis. Paroxetine also induced [Ca{sup 2+}]{sub i} increases which involved the mobilization of intracellular Ca{sup 2+} stored in the endoplasmic reticulum and Ca{sup 2+} influx from extracellular medium. However, pretreatment with BAPTA/AM, a Ca{sup 2+} chelator, to prevent paroxetine-induced [Ca{sup 2+}]{sub i} increases did not protect cells from death. The results suggest that in MG63 cells, paroxetine caused Ca{sup 2+}-independent apoptosis via inducing p38 MAPK-associated caspase-3 activation.« less

New leads for selective GSK-3 inhibition: pharmacophore mapping and virtual screening studies.

PubMed

Patel, Dhilon S; Bharatam, Prasad V

2006-01-01

Glycogen Synthase Kinase-3 is a regulatory serine/threonine kinase, which is being targeted for the treatment of a number of human diseases including type-2 diabetes mellitus, neurodegenerative diseases, cancer and chronic inflammation. Selective GSK-3 inhibition is an important requirement owing to the possibility of side effects arising from other kinases. A pharmacophore mapping strategy is employed in this work to identify new leads for selective GSK-3 inhibition. Ligands known to show selective GSK-3 inhibition were employed in generating a pharmacophore map using distance comparison method (DISCO). The derived pharmacophore map was validated using (i) important interactions involved in selective GSK-3 inhibitions, and (ii) an in-house database containing different classes of GSK-3 selective, non-selective and inactive molecules. New Lead identification was carried out by performing virtual screening using validated pharmacophoric query and three chemical databases namely NCI, Maybridge and Leadquest. Further data reduction was carried out by employing virtual filters based on (i) Lipinski's rule of 5 (ii) van der Waals bumps and (iii) restricting the number of rotatable bonds to seven. Final screening was carried out using FlexX based molecular docking study.

New leads for selective GSK-3 inhibition: pharmacophore mapping and virtual screening studies

NASA Astrophysics Data System (ADS)

Patel, Dhilon S.; Bharatam, Prasad V.

2006-01-01

Glycogen Synthase Kinase-3 is a regulatory serine/threonine kinase, which is being targeted for the treatment of a number of human diseases including type-2 diabetes mellitus, neurodegenerative diseases, cancer and chronic inflammation. Selective GSK-3 inhibition is an important requirement owing to the possibility of side effects arising from other kinases. A pharmacophore mapping strategy is employed in this work to identify new leads for selective GSK-3 inhibition. Ligands known to show selective GSK-3 inhibition were employed in generating a pharmacophore map using distance comparison method (DISCO). The derived pharmacophore map was validated using (i) important interactions involved in selective GSK-3 inhibitions, and (ii) an in-house database containing different classes of GSK-3 selective, non-selective and inactive molecules. New Lead identification was carried out by performing virtual screening using validated pharmacophoric query and three chemical databases namely NCI, Maybridge and Leadquest. Further data reduction was carried out by employing virtual filters based on (i) Lipinski's rule of 5 (ii) van der Waals bumps and (iii) restricting the number of rotatable bonds to seven. Final screening was carried out using FlexX based molecular docking study.

Skeletal muscle Ca(2+)-independent kinase activity increases during either hypertrophy or running

NASA Technical Reports Server (NTRS)

Fluck, M.; Waxham, M. N.; Hamilton, M. T.; Booth, F. W.

2000-01-01

Spikes in free Ca(2+) initiate contractions in skeletal muscle cells, but whether and how they might signal to transcription factors in skeletal muscles of living animals is unknown. Since previous studies in non-muscle cells have shown that serum response factor (SRF) protein, a transcription factor, is phosphorylated rapidly by Ca(2+)/calmodulin (CaM)-dependent protein kinase after rises in intracellular Ca(2+), we measured enzymatic activity that phosphorylates SRF (designated SRF kinase activity). Homogenates from 7-day-hypertrophied anterior latissimus dorsi muscles of roosters had more Ca(2+)-independent SRF kinase activity than their respective control muscles. However, no differences were noted in Ca(2+)/CaM-dependent SRF kinase activity between control and trained muscles. To determine whether the Ca(2+)-independent and Ca(2+)/CaM-dependent forms of Ca(2+)/CaM-dependent protein kinase II (CaMKII) might contribute to some of the SRF kinase activity, autocamtide-3, a synthetic substrate that is specific for CaMKII, was employed. While the Ca(2+)-independent form of CaMKII was increased, like the Ca(2+)-independent form of SRF kinase, no alteration in CaMKII occurred at 7 days of stretch overload. These observations suggest that some of SRF phosphorylation by skeletal muscle extracts could be due to CaMKII. To determine whether this adaptation was specific to the exercise type (i.e., hypertrophy), similar measurements were made in the white vastus lateralis muscle of rats that had completed 2 wk of voluntary running. Although Ca(2+)-independent SRF kinase was increased, no alteration occurred in Ca(2+)/CaM-dependent SRF kinase activity. Thus any role of Ca(2+)-independent SRF kinase signaling has downstream modulators specific to the exercise phenotype.

Bilirubin induces a calcium-dependent inhibition of multifunctional Ca2+/calmodulin-dependent kinase II activity in vitro.

PubMed

Churn, S B; DeLorenzo, R J; Shapiro, S M

1995-12-01

Excessive bilirubin levels in newborn infants result in long-term neurologic deficits that remain after bilirubin levels return to normal. Much of the observed neurologic deficits can be attributed to bilirubin-induced, delayed neuronal cell death. Inhibition of calcium/calmodulin-dependent kinase II (CaM kinase II) activity that precedes cell death is observed in conditions such as seizure activity, stroke, and glutamate excitotoxicity. Because neonatal bilirubin exposure results in neuronal loss in developing brain systems, we tested whether bilirubin exposure would induce an immediate inhibition of CaM activity, in vitro. P-81 filtration assay of basal and calcium-stimulated kinase activity was performed under standard kinase assay conditions. Bilirubin and/or albumin was added to the reaction vessels to determine the effect of these agents on kinase activity. Bilirubin exposure resulted in a concentration-dependent inhibition of CaM kinase II activity (IC50 = 16.78 microM). At concentrations above 50 microM, bilirubin exposure resulted in a 71 +/- 8% (mean +/- SD) inhibition of kinase activity (p < 0.001, t test, n = 10). Bilirubin exposure did not result in kinase inhibition if excessive bilirubin was removed by albumin binding before stimulation of kinase activity (106.9 +/- 9.6% control activity, n = 5). However, removal of bilirubin by binding with albumin after calcium addition did not restore kinase activity. (36.1 +/- 3.8% control activity, n = 5). Thus, once inhibition was observed, the activity could not be restored by addition of albumin. The data suggest that bilirubin exposure resulted in a calcium-dependent inhibition of CaM kinase II activity that, once induced, was not reversible by removing bilirubin by the addition of albumin. Because inhibition of CaM kinase II activity has been correlated with delayed neuronal cell death in many neuropathologic conditions, bilirubin-induced inhibition of this enzyme may be a cellular mechanism by which bilirubin exposure results in delayed neuronal cell death in developing brain.

Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

PubMed

Toubiana, Julie; Rossi, Anne-Lise; Belaidouni, Nadia; Grimaldi, David; Pene, Frederic; Chafey, Philippe; Comba, Béatrice; Camoin, Luc; Bismuth, Georges; Claessens, Yann-Erick; Mira, Jean-Paul; Chiche, Jean-Daniel

2015-10-01

TLR2 has a prominent role in host defense against a wide variety of pathogens. Stimulation of TLR2 triggers MyD88-dependent signaling to induce NF-κB translocation, and activates a Rac1-PI 3-kinase dependent pathway that leads to transactivation of NF-κB through phosphorylation of the P65 NF-κB subunit. This transactivation pathway involves tyrosine phosphorylations. The role of the tyrosine kinases in TLR signaling is controversial, with discrepancies between studies using only chemical inhibitors and knockout mice. Here, we show the involvement of the tyrosine-kinase Lyn in TLR2-dependent activation of NF-κB in human cellular models, by using complementary inhibition strategies. Stimulation of TLR2 induces the formation of an activation cluster involving TLR2, CD14, PI 3-kinase and Lyn, and leads to the activation of AKT. Lyn-dependent phosphorylation of the p110 catalytic subunit of PI 3-kinase is essential to the control of PI 3-kinase biological activity upstream of AKT and thereby to the transactivation of NF-κB. Thus, Lyn kinase activity is crucial in TLR2-mediated activation of the innate immune response in human mononuclear cells. © The Author(s) 2015.

Adenosine mimetics as inhibitors of NAD+-dependent histone deacetylases, from kinase to sirtuin inhibition.

PubMed

Trapp, Johannes; Jochum, Anne; Meier, Rene; Saunders, Laura; Marshall, Brett; Kunick, Conrad; Verdin, Eric; Goekjian, Peter; Sippl, Wolfgang; Jung, Manfred

2006-12-14

NAD+-dependent histone deacetylases, sirtuins, cleave acetyl groups from lysines of histones and other proteins to regulate their activity. Identification of potent selective inhibitors would help to elucidate sirtuin biology and could lead to useful therapeutic agents. NAD+ has an adenosine moiety that is also present in the kinase cofactor ATP. Kinase inhibitors based upon adenosine mimesis may thus also target NAD+-dependent enzymes. We present a systematic approach using adenosine mimics from one cofactor class (kinase inhibitors) as a viable method to generate new lead structures in another cofactor class (sirtuin inhibitors). Our findings have broad implications for medicinal chemistry and specifically for sirtuin inhibitor design. Our results also raise a question as to whether selectivity profiling for kinase inhibitors should be limited to ATP-dependent targets.

Cloning and characterization of GETS-1, a goldfish Ets family member that functions as a transcriptional repressor in muscle.

PubMed

Goldman, D; Sapru, M K; Stewart, S; Plotkin, J; Libermann, T A; Wasylyk, B; Guan, K

1998-10-15

An Ets transcription factor family member, GETS-1, was cloned from a goldfish retina cDNA library. GETS-1 contains a conserved Ets DNA-binding domain at its N-terminus and is most similar to ternary complex factor (TCF) serum-response-factor protein-1a (SAP-1a). GETS-1 is expressed in many tissues, but is enriched in retina and brain. As with the TCFs SAP-1a and ets-related protein (ERP), overexpression of the GETS-1 promoter suppresses nicotinic acetylcholine receptor epsilon-subunit gene expression in cultured muscle cells. A consensus Ets binding site sequence in the promoter of the epsilon-subunit gene is required for GETS-1-mediated repression. GETS-1 repressor activity is abrogated by overexpression of an activated Ras/mitogen-activated protein kinase (MAP kinase) or by mutation of Ser-405, a MAP kinase phosphorylation site in GETS-1. Fusion proteins created between GETS-1 and the Gal4 DNA-binding domain show that, like other TCFs, GETS-1 contains a C-terminal activation domain that is activated by a Ras/MAP kinase signalling cascade. Interestingly, mutation of Ser-405 located within this activation domain abrogated transcriptional activation of the fusion protein.

Role of Gab1 in Heart, Placenta, and Skin Development and Growth Factor- and Cytokine-Induced Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Activation

PubMed Central

Itoh, Motoyuki; Yoshida, Yuichi; Nishida, Keigo; Narimatsu, Masahiro; Hibi, Masahiko; Hirano, Toshio

2000-01-01

Gab1 is a member of the Gab/DOS (Daughter of Sevenless) family of adapter molecules, which contain a pleckstrin homology (PH) domain and potential binding sites for SH2 and SH3 domains. Gab1 is tyrosine phosphorylated upon stimulation of various cytokines, growth factors, and antigen receptors in cell lines and interacts with signaling molecules, such as SHP-2 and phosphatidylinositol 3-kinase, although its biological roles have not yet been established. To reveal the functions of Gab1 in vivo, we generated mice lacking Gab1 by gene targeting. Gab1-deficient embryos died in utero and displayed developmental defects in the heart, placenta, and skin, which were similar to phenotypes observed in mice lacking signals of the hepatocyte growth factor/scatter factor, platelet-derived growth factor, and epidermal growth factor pathways. Consistent with these observations, extracellular signal-regulated kinase mitogen-activated protein (ERK MAP) kinases were activated at much lower levels in cells from Gab1-deficient embryos in response to these growth factors or to stimulation of the cytokine receptor gp130. These results indicate that Gab1 is a common player in a broad range of growth factor and cytokine signaling pathways linking ERK MAP kinase activation. PMID:10779359

Nicotinic modulation of hippocampal cell signaling and associated effects on learning and memory.

PubMed

Kutlu, Munir Gunes; Gould, Thomas J

2016-03-01

The hippocampus is a key brain structure involved in synaptic plasticity associated with long-term declarative memory formation. Importantly, nicotine and activation of nicotinic acetylcholine receptors (nAChRs) can alter hippocampal plasticity and these changes may occur through modulation of hippocampal kinases and transcription factors. Hippocampal kinases such as cAMP-dependent protein kinase (PKA), calcium/calmodulin-dependent protein kinases (CAMKs), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and c-jun N-terminal kinase 1 (JNK1), and the transcription factor cAMP-response element-binding protein (CREB) that are activated either directly or indirectly by nicotine may modulate hippocampal plasticity and in parallel hippocampus-dependent learning and memory. Evidence suggests that nicotine may alter hippocampus-dependent learning by changing the time and magnitude of activation of kinases and transcription factors normally involved in learning and by recruiting additional cell signaling molecules. Understanding how nicotine alters learning and memory will advance basic understanding of the neural substrates of learning and aid in understanding mental disorders that involve cognitive and learning deficits. Copyright © 2015 Elsevier Inc. All rights reserved.

Ets-1 interacts through a similar binding interface with Ku70 and Poly (ADP-Ribose) Polymerase-1.

PubMed

Choul-Li, Souhaila; Legrand, Arnaud J; Vicogne, Dorothée; Villeret, Vincent; Aumercier, Marc

2018-06-18

The Ets-1 transcription factor plays an important role in various physiological and pathological processes. These diverse roles of Ets-1 are likely to depend on its interaction proteins. We have previously showed that Ets-1 interacted with DNA-dependent protein kinase (DNA-PK) complex including its regulatory subunits, Ku70 and Ku86 and with poly (ADP-ribose) polymerase-1 (PARP-1). In this study, the binding domains for the interaction between Ets-1 and these proteins were reported. We demonstrated that the interaction of Ets-1 with DNA-PK was mediated through the Ku70 subunit and was mapped to the C-terminal region of Ets-1 and the C-terminal part of Ku70 including SAP domain. The interactive domains between Ets-1 and PARP-1 have been mapped to the C-terminal region of Ets-1 and the BRCA1 carboxy-terminal (BRCT) domain of PARP-1. The results presented in this study may advance our understanding of the functional link between Ets-1 and its interaction partners, DNA-PK and PARP-1.

A dual-specificity isoform of the protein kinase inhibitor PKI produced by alternate gene splicing.

PubMed

Kumar, Priyadarsini; Walsh, Donal A

2002-03-15

We have previously shown that the protein kinase inhibitor beta (PKIbeta) form of the cAMP-dependent protein kinase inhibitor exists in multiple isoforms, some of which are specific inhibitors of the cAMP-dependent protein kinase, whereas others also inhibit the cGMP-dependent enzyme [Kumar, Van Patten and Walsh (1997), J. Biol. Chem. 272, 20011-20020]. We have now demonstrated that the switch from a cAMP-dependent protein kinase (PKA)-specific inhibitor to one with dual specificity arises as a consequence of alternate gene splicing. We have confirmed using bacterially produced pure protein that a single inhibitor species has dual specificity for both PKA and cGMP-dependent protein kinase (PKG), inhibiting each with very high and closely similar inhibitory potencies. The gene splicing converted a protein with 70 amino acids into one of 109 amino acids, and did not change the inhibitory potency to PKA, but changed it from a protein that had no detectable PKG inhibitory activity to one that now inhibited PKG in the nanomolar range.

ALTERED PHOSPHORYLATION OF MAP KINASE AFTER ACUTE EXPOSURE TO PCB153.

EPA Science Inventory

Long-term potentiation (LTP) is a model of synaptic plasticity believed to encompass the physiological substrate of memory. The mitogen-activated protein kinase (ERK1/2) signalling cascade contributes to synaptic plasticity and to long-term memory formation. Learning and LTP st...

Structure-activity relationships of phenothiazines and related drugs for inhibition of protein kinase C.

PubMed

Aftab, D T; Ballas, L M; Loomis, C R; Hait, W N

1991-11-01

Phenothiazines are known to inhibit the activity of protein kinase C. To identify structural features that determine inhibitory activity against the enzyme, we utilized a semiautomated assay [Anal. Biochem. 187:84-88 (1990)] to compare the potency of greater than 50 phenothiazines and related compounds. Potency was decreased by trifluoro substitution at position 2 on the phenothiazine nucleus and increased by quinoid structures on the nucleus. An alkyl bridge of at least three carbons connecting the terminal amine to the nucleus was required for activity. Primary amines and unsubstituted piperazines were the most potent amino side chains. We selected 7,8-dihydroxychlorpromazine (DHCP) (IC50 = 8.3 microM) and 2-chloro-9-(3-[1-piperazinyl]propylidene)thioxanthene (N751) (IC50 = 14 microM) for further study because of their potency and distinct structural features. Under standard (vesicle) assay conditions, DHCP was noncompetitive with respect to phosphatidylserine and a mixed-type inhibitor with respect to ATP. N751 was competitive with respect to phosphatidylserine and noncompetitive with respect to ATP. Using the mixed micelle assay, DHCP was a competitive inhibitor with respect to both phosphatidylserine and ATP. DHCP was selective for protein kinase C compared with cAMP-dependent protein kinase, calmodulin-dependent protein kinase type II, and casein kinase. N751 was more potent against protein kinase C compared with cAMP-dependent protein kinase and casein kinase but less potent against protein kinase C compared with calmodulin-dependent protein kinase type II. DHCP was analyzed for its ability to inhibit different isoenzymes of protein kinase C, and no significant isozyme selectivity was detected. These data provide important information for the rational design of more potent and selective inhibitors of protein kinase C.

Partial purification and characterization of a Ca(2+)-dependent protein kinase from the green alga, Dunaliella salina

NASA Technical Reports Server (NTRS)

Roux, S. J.

1990-01-01

A calcium-dependent protein kinase was partially purified and characterized from the green alga Dunaliella salina. The enzyme was activated at free Ca2+ concentrations above 10(-7) molar. and half-maximal activation was at about 3 x 10(-7) molar. The optimum pH for its Ca(2+)-dependent activity was 7.5. The addition of various phospholipids and diolein had no effects on enzyme activity and did not alter the sensitivity of the enzyme toward Ca2+. The enzyme was inhibited by calmodulin antagonists, N-(6-aminohexyl)-1-naphthalene sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide in a dose-dependent manner while the protein kinase C inhibitor, sphingosine, had little effect on enzyme activity up to 800 micromolar. Immunoassay showed some calmodulin was present in the kinase preparations. However, it is unlikely the kinase was calmodulin regulated, since it still showed stimulation by Ca2+ in gel assays after being electrophoretically separated from calmodulin by two different methods. This gel method of detection of the enzyme indicated that a protein band with an apparent molecular weight of 40,000 showed protein kinase activity at each one of the several steps in the purification procedure. Gel assay analysis also showed that after native gel isoelectric focusing the partially purified kinase preparations had two bands with calcium-dependent activity, at isoelectric points 6.7 and 7.1. By molecular weight, by isoelectric point, and by a comparative immunoassay, the Dunaliella kinase appears to differ from at least some of the calcium-dependent, but calmodulin and phospholipid independent kinases described from higher plants.

Enhanced IL-1{beta}-induced IL-8 production in cystic fibrosis lung epithelial cells is dependent of both mitogen-activated protein kinases and NF-{kappa}B signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Muselet-Charlier, Celine; Universite Pierre et Marie Curie-Paris 6, Paris, UMR-S719, F-75012; Roque, Telma

2007-06-01

Transcription nuclear factor-{kappa}B (NF-{kappa}B) is hyperactivated in cystic fibrosis (CF) lung epithelial cells, and participates in exaggerated IL-8 production in the CF lung. We recently found that rapid activation of NF-{kappa}B occurred in a CF lung epithelial IB3-1 cell line (CF cells) upon IL-1{beta} stimulation, which was not observed in its CFTR-corrected lung epithelial S9 cell line (corrected cells). To test whether other signaling pathways such as that of mitogen-activated protein kinases (MAPKs) could be involved in IL-1{beta}-induced IL-8 production of CF cells, we investigated ERK1/2, JNK, and p38MAP signaling compared to NF-{kappa}B. Within 30 min, exposure to IL-1{beta} causedmore » high activation of NF-{kappa}B, ERK1/2, p38MAP but not JNK in CF cells compared to corrected cells. Treatment of IL-1{beta}-stimulated CF cells with a series of chemical inhibitors of NF-{kappa}B, ERK1/2, and p38MAP, when used separately, reduced slightly IL-8 production. However, when used together, these inhibitors caused a blockade in IL-1{beta}-induced IL-8 production in CF cells. Understanding of the cross-talk between NF-{kappa}B and MAPKs signaling in CF lung epithelial cells may help in developing new therapeutics to reduce lung inflammation in patients with CF.« less

Alpha-adrenergic blocker mediated osteoblastic stem cell differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Yoon Jung; Lee, Jue Yeon; Research Center, Nano Intelligent Biomedical Engineering Corporation

Highlights: Black-Right-Pointing-Pointer Doxazocin directly up-regulated bone metabolism at a low dose. Black-Right-Pointing-Pointer Doxazocin induced osteoblastic stem cell differentiation without affecting cell proliferation. Black-Right-Pointing-Pointer This osteogenic stem cell differentiation is mediated by ERK-signal dependent pathway. -- Abstract: Recent researches have indicated a role for antihypertensive drugs including alpha- or beta-blockers in the prevention of bone loss. Some epidemiological studies reported the protective effects of those agents on fracture risk. However, there is limited information on the association with those agents especially at the mechanism of action. In the present study, we investigated the effects of doxazosin, an alpha-blocker that is clinicallymore » used for the treatment of benign prostatic hyperplasia (BPH) along with antihypertensive medication, on the osteogenic stem cell differentiation. We found that doxazosin increased osteogenic differentiation of human mesenchymal stem cells, detected by Alizarin red S staining and calcein. Doxazosin not only induced expression of alkaline phosphatase, type I collagen, osteopontin, and osteocalcin, it also resulted in increased phosphorylation of extracellular signal-regulated kinase (ERK1/2), a MAP kinase involved in osteoblastic differentiation. Treatment with U0126, a MAP kinase inhibitor, significantly blocked doxazosin-induced osteoblastic differentiation. Unrelated to activation of osteogenic differentiation by doxazosin, we found that there were no significant changes in adipogenic differentiation or in the expression of adipose-specific genes, including peroxisome proliferator-activated receptor {gamma}, aP2, or LPL. In this report, we suggest that doxazosin has the ability to increase osteogenic cell differentiation via ERK1/2 activation in osteogenic differentiation of adult stem cells, which supports the protective effects of antihypertensive drug on fracture risk and according to our data doxazosin might be useful for application in the field of bone metabolism.« less

GATA-Dependent Glutaminolysis Drives Appressorium Formation in Magnaporthe oryzae by Suppressing TOR Inhibition of cAMP/PKA Signaling

PubMed Central

Marroquin-Guzman, Margarita; Wilson, Richard A.

2015-01-01

Fungal plant pathogens are persistent and global food security threats. To invade their hosts they often form highly specialized infection structures, known as appressoria. The cAMP/ PKA- and MAP kinase-signaling cascades have been functionally delineated as positive-acting pathways required for appressorium development. Negative-acting regulatory pathways that block appressorial development are not known. Here, we present the first detailed evidence that the conserved Target of Rapamycin (TOR) signaling pathway is a powerful inhibitor of appressorium formation by the rice blast fungus Magnaporthe oryzae. We determined TOR signaling was activated in an M. oryzae mutant strain lacking a functional copy of the GATA transcription factor-encoding gene ASD4. Δasd4 mutant strains could not form appressoria and expressed GLN1, a glutamine synthetase-encoding orthologue silenced in wild type. Inappropriate expression of GLN1 increased the intracellular steady-state levels of glutamine in Δasd4 mutant strains during axenic growth when compared to wild type. Deleting GLN1 lowered glutamine levels and promoted appressorium formation by Δasd4 strains. Furthermore, glutamine is an agonist of TOR. Treating Δasd4 mutant strains with the specific TOR kinase inhibitor rapamycin restored appressorium development. Rapamycin was also shown to induce appressorium formation by wild type and Δcpka mutant strains on non-inductive hydrophilic surfaces but had no effect on the MAP kinase mutant Δpmk1. When taken together, we implicate Asd4 in regulating intracellular glutamine levels in order to modulate TOR inhibition of appressorium formation downstream of cPKA. This study thus provides novel insight into the metabolic mechanisms that underpin the highly regulated process of appressorium development. PMID:25901357

Silibinin induces apoptosis of HT29 colon carcinoma cells through early growth response-1 (EGR-1)-mediated non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) up-regulation.

PubMed

Woo, Seon Min; Min, Kyoung-Jin; Kim, Shin; Park, Jong-Wook; Kim, Dong Eun; Chun, Kyung-Soo; Kim, Young Ho; Lee, Tae-Jin; Kim, Sang Hyun; Choi, Yung Hyun; Chang, Jong-Soo; Kwon, Taeg Kyu

2014-03-25

Silibinin, an effective anti-cancer and chemopreventive agent, has been shown to exert multiple effects on cancer cells, including inhibition of both cell proliferation and migration. However, the molecular mechanisms responsible for these effects are not fully understood. We observed that silibinin significantly induced the expression of the non-steroidal anti-inflammatory drug-activated gene-1 (NAG-1) in both p53 wild-type and p53-null cancer cell lines, suggesting that silibinin-induced NAG-1 up-regulation is p53-independent manner. Silibinin up-regulates early growth response-1 (EGR-1) expression. The ectopic expression of EGR-1 significantly increased NAG-1 promoter activity and NAG-1 protein expression in a dose-dependent manner. Furthermore, down-regulation of EGR-1 expression using siRNA markedly reduced silibinin-mediated NAG-1 expression, suggesting that the expression of EGR-1 is critical for silibinin-induced NAG-1 expression. We also observed that reactive oxygen species (ROS) are generated by silibinin; however, ROS did not affect silibinin-induced NAG-1 expression and apoptosis. In addition, we demonstrated that the mitogen-activated protein kinase (MAP kinase) signal transduction pathway is involved in silibinin-induced NAG-1 expression. Inhibitors of p38 MAP kinase (SB203580) attenuated silibinin-induced NAG-1 expression. Furthermore, we found that siRNA-mediated knockdown of NAG-1 attenuated silibinin-induced apoptosis. Collectively, the results of this study demonstrate for the first time that up-regulation of NAG-1 contributes to silibinin-induced apoptosis in cancer cells. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Regulation of fear extinction versus other affective behaviors by discrete cortical scaffolding complexes associated with NR2B and PKA signaling

PubMed Central

Corcoran, K A; Leaderbrand, K; Jovasevic, V; Guedea, A L; Kassam, F; Radulovic, J

2015-01-01

In patients suffering from post-traumatic stress disorder (PTSD), fear evoked by trauma-related memories lasts long past the traumatic event and it is often complicated by general anxiety and depressed mood. This poses a treatment challenge, as drugs beneficial for some symptoms might exacerbate others. For example, in preclinical studies, antagonists of the NR2B subunit of N-methyl-d-aspartate receptors and activators of cAMP-dependent protein kinase (PKA) act as potent antidepressants and anxiolytics, but they block fear extinction. Using mice, we attempted to overcome this problem by interfering with individual NR2B and PKA signaling complexes organized by scaffolding proteins. We infused cell-permeable Tat peptides that displaced either NR2B from receptor for activated C kinase 1 (RACK1), or PKA from A-kinase anchor proteins (AKAPs) or microtubule-associated proteins (MAPs). The infusions were targeted to the retrosplenial cortex, an area involved in both fear extinction of remotely acquired memories and in mood regulation. Tat-RACK1 and Tat-AKAP enhanced fear extinction, all peptides reduced anxiety and none affected baseline depression-like behavior. However, disruption of PKA complexes distinctively interfered with the rapid antidepressant actions of the N-methyl-D-aspartate receptors antagonist MK-801 in that Tat-MAP2 blocked, whereas Tat-AKAP completely inverted the effect of MK-801 from antidepressant to depressant. These effects were unrelated to the MK-801-induced changes of brain-derived neurotrophic factor messenger RNA levels. Together, the findings suggest that NR2B–RACK1 complexes specifically contribute to fear extinction, and may provide a target for the treatment of PTSD. AKAP-PKA, on the other hand, appears to modulate fear extinction and antidepressant responses in opposite directions. PMID:26460481

Regulation of fear extinction versus other affective behaviors by discrete cortical scaffolding complexes associated with NR2B and PKA signaling.

PubMed

Corcoran, K A; Leaderbrand, K; Jovasevic, V; Guedea, A L; Kassam, F; Radulovic, J

2015-10-13

In patients suffering from post-traumatic stress disorder (PTSD), fear evoked by trauma-related memories lasts long past the traumatic event and it is often complicated by general anxiety and depressed mood. This poses a treatment challenge, as drugs beneficial for some symptoms might exacerbate others. For example, in preclinical studies, antagonists of the NR2B subunit of N-methyl-d-aspartate receptors and activators of cAMP-dependent protein kinase (PKA) act as potent antidepressants and anxiolytics, but they block fear extinction. Using mice, we attempted to overcome this problem by interfering with individual NR2B and PKA signaling complexes organized by scaffolding proteins. We infused cell-permeable Tat peptides that displaced either NR2B from receptor for activated C kinase 1 (RACK1), or PKA from A-kinase anchor proteins (AKAPs) or microtubule-associated proteins (MAPs). The infusions were targeted to the retrosplenial cortex, an area involved in both fear extinction of remotely acquired memories and in mood regulation. Tat-RACK1 and Tat-AKAP enhanced fear extinction, all peptides reduced anxiety and none affected baseline depression-like behavior. However, disruption of PKA complexes distinctively interfered with the rapid antidepressant actions of the N-methyl-D-aspartate receptors antagonist MK-801 in that Tat-MAP2 blocked, whereas Tat-AKAP completely inverted the effect of MK-801 from antidepressant to depressant. These effects were unrelated to the MK-801-induced changes of brain-derived neurotrophic factor messenger RNA levels. Together, the findings suggest that NR2B-RACK1 complexes specifically contribute to fear extinction, and may provide a target for the treatment of PTSD. AKAP-PKA, on the other hand, appears to modulate fear extinction and antidepressant responses in opposite directions.

Brd4 modulates the innate immune response through Mnk2-eIF4E pathway-dependent translational control of IκBα.

PubMed

Bao, Yan; Wu, Xuewei; Chen, Jinjing; Hu, Xiangming; Zeng, Fuxing; Cheng, Jianjun; Jin, Hong; Lin, Xin; Chen, Lin-Feng

2017-05-16

Bromodomain-containing factor Brd4 has emerged as an important transcriptional regulator of NF-κB-dependent inflammatory gene expression. However, the in vivo physiological function of Brd4 in the inflammatory response remains poorly defined. We now demonstrate that mice deficient for Brd4 in myeloid-lineage cells are resistant to LPS-induced sepsis but are more susceptible to bacterial infection. Gene-expression microarray analysis of bone marrow-derived macrophages (BMDMs) reveals that deletion of Brd4 decreases the expression of a significant amount of LPS-induced inflammatory genes while reversing the expression of a small subset of LPS-suppressed genes, including MAP kinase-interacting serine/threonine-protein kinase 2 ( Mknk2 ). Brd4 -deficient BMDMs display enhanced Mnk2 expression and the corresponding eukaryotic translation initiation factor 4E (eIF4E) activation after LPS stimulation, leading to an increased translation of IκBα mRNA in polysomes. The enhanced newly synthesized IκBα reduced the binding of NF-κB to the promoters of inflammatory genes, resulting in reduced inflammatory gene expression and cytokine production. By modulating the translation of IκBα via the Mnk2-eIF4E pathway, Brd4 provides an additional layer of control for NF-κB-dependent inflammatory gene expression and inflammatory response.

AMP-activated protein kinase-mediated feedback phosphorylation controls the Ca2+/calmodulin (CaM) dependence of Ca2+/CaM-dependent protein kinase kinase β.

PubMed

Nakanishi, Akihiro; Hatano, Naoya; Fujiwara, Yuya; Sha'ri, Arian; Takabatake, Shota; Akano, Hiroki; Kanayama, Naoki; Magari, Masaki; Nozaki, Naohito; Tokumitsu, Hiroshi

2017-12-01

The Ca 2+ /calmodulin-dependent protein kinase kinase β (CaMKKβ)/5'-AMP-activated protein kinase (AMPK) phosphorylation cascade affects various Ca 2+ -dependent metabolic pathways and cancer growth. Unlike recombinant CaMKKβ that exhibits higher basal activity (autonomous activity), activation of the CaMKKβ/AMPK signaling pathway requires increased intracellular Ca 2+ concentrations. Moreover, the Ca 2+ /CaM dependence of CaMKKβ appears to arise from multiple phosphorylation events, including autophosphorylation and activities furnished by other protein kinases. However, the effects of proximal downstream kinases on CaMKKβ activity have not yet been evaluated. Here, we demonstrate feedback phosphorylation of CaMKKβ at multiple residues by CaMKKβ-activated AMPK in addition to autophosphorylation in vitro , leading to reduced autonomous, but not Ca 2+ /CaM-activated, CaMKKβ activity. MS analysis and site-directed mutagenesis of AMPK phosphorylation sites in CaMKKβ indicated that Thr 144 phosphorylation by activated AMPK converts CaMKKβ into a Ca 2+ /CaM-dependent enzyme as shown by completely Ca 2+ /CaM-dependent CaMKK activity of a phosphomimetic T144E CaMKKβ mutant. CaMKKβ mutant analysis indicated that the C-terminal domain (residues 471-587), including the autoinhibitory region, plays an important role in stabilizing an inactive conformation in a Thr 144 phosphorylation-dependent manner. Furthermore, immunoblot analysis with anti-phospho-Thr 144 antibody revealed phosphorylation of Thr 144 in CaMKKβ in transfected COS-7 cells that was further enhanced by exogenous expression of AMPKα. These results indicate that AMPK-mediated feedback phosphorylation of CaMKKβ regulates the CaMKKβ/AMPK signaling cascade and may be physiologically important for intracellular maintenance of Ca 2+ -dependent AMPK activation by CaMKKβ. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: II. Mutational analysis.

PubMed

Kitani, T; Okuno, S; Fujisawa, H

2001-10-01

We previously reported that rat brain Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) IV is inactivated by cAMP-dependent protein kinase (PKA) [Kameshita, I. and Fujisawa, H. (1991) Biochem. Biophys. Res. Commun. 180, 191-196]. In the preceding paper, we demonstrated that changes in the activity of CaM-kinase IV by PKA results from the phosphorylation of CaM-kinase kinase alpha by PKA and identified six phosphorylation sites, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA. In the present study, a causal relationship between the phosphorylation and change in the activity toward PKIV peptide has been studied using mutant enzymes with amino acid substitutions at the six phosphorylation sites. The following conclusions can be drawn from the experimental results: (i) Phosphorylation of Ser74 and/or unidentified sites causes an increase in activity; (ii) phosphorylation of Thr(108) or Ser(458) causes a decrease in the activity; (iii) the inhibitory effect of the phosphorylation of Thr(108) is canceled by the stimulatory effect of the phosphorylation, but that of Ser(458) is not; and (iv) the inhibitory effects of Thr(108) and Ser(458) are synergistic. In contrast to the activity toward PKIV peptide, the activity toward CaM-kinase IV appears to be decreased by the phosphorylation of Thr(108), but not significantly affected by the phosphorylation of Ser(458).

Trichinella spiralis infection enhances protein kinase C phosphorylation in guinea pig alveolar macrophages.

PubMed

Dzik, J M; Zieliński, Z; Cieśla, J; Wałajtys-Rode, E

2010-03-01

To learn more about the signalling pathways involved in superoxide anion production in guinea pig alveolar macrophages, triggered by Trichinella spiralis infection, protein level and phosphorylation of mitogen activated protein (MAP) kinases and protein kinase C (PKC) were investigated. Infection with T. spiralis, the nematode having 'lung phase' during colonization of the host, enhances PKC phosphorylation in guinea pig alveolar macrophages. Isoenzymes beta and delta of PKC have been found significantly phosphorylated, although their location was not changed as a consequence of T. spiralis infection. Neither in macrophages from T. spiralis-infected guinea pig nor in platelet-activating factor (PAF)-stimulated macrophages from uninfected animals, participation of MAP kinases in respiratory burst activation was statistically significant. The parasite antigens seem to act through macrophage PAF receptors, transducing a signal for enhanced NADPH oxidase activity, as stimulating effect of newborn larvae homogenate on respiratory burst was abolished by specific PAF receptor antagonist CV 6209. A suppressive action of T. spiralis larvae on host alveolar macrophage innate immunological response was reflected by diminished protein level of ERK2 kinase and suppressed superoxide anion production, in spite of high level of PKC phosphorylation.

Deciphering the Arginine-Binding Preferences at the Substrate-Binding Groove of Ser/Thr Kinases by Computational Surface Mapping

PubMed Central

Ben-Shimon, Avraham; Niv, Masha Y.

2011-01-01

Protein kinases are key signaling enzymes that catalyze the transfer of γ-phosphate from an ATP molecule to a phospho-accepting residue in the substrate. Unraveling the molecular features that govern the preference of kinases for particular residues flanking the phosphoacceptor is important for understanding kinase specificities toward their substrates and for designing substrate-like peptidic inhibitors. We applied ANCHORSmap, a new fragment-based computational approach for mapping amino acid side chains on protein surfaces, to predict and characterize the preference of kinases toward Arginine binding. We focus on positions P−2 and P−5, commonly occupied by Arginine (Arg) in substrates of basophilic Ser/Thr kinases. The method accurately identified all the P−2/P−5 Arg binding sites previously determined by X-ray crystallography and produced Arg preferences that corresponded to those experimentally found by peptide arrays. The predicted Arg-binding positions and their associated pockets were analyzed in terms of shape, physicochemical properties, amino acid composition, and in-silico mutagenesis, providing structural rationalization for previously unexplained trends in kinase preferences toward Arg moieties. This methodology sheds light on several kinases that were described in the literature as having non-trivial preferences for Arg, and provides some surprising departures from the prevailing views regarding residues that determine kinase specificity toward Arg. In particular, we found that the preference for a P−5 Arg is not necessarily governed by the 170/230 acidic pair, as was previously assumed, but by several different pairs of acidic residues, selected from positions 133, 169, and 230 (PKA numbering). The acidic residue at position 230 serves as a pivotal element in recognizing Arg from both the P−2 and P−5 positions. PMID:22125489

Association of Common Genetic Variants in the MAP4K4 Locus with Prediabetic Traits in Humans

PubMed Central

Ketterer, Caroline; Heni, Martin; Machicao, Fausto; Guilherme, Adilson; Grallert, Harald; Schulze, Matthias B.; Boeing, Heiner; Stefan, Norbert; Fritsche, Andreas; Czech, Michael P.; Häring, Hans-Ulrich

2012-01-01

Mitogen-activated protein kinase kinase kinase kinase 4 (MAP4K4) is expressed in all diabetes-relevant tissues and mediates cytokine-induced insulin resistance. We investigated whether common single nucleotide polymorphisms (SNPs) in the MAP4K4 locus associate with glucose intolerance, insulin resistance, impaired insulin release, or elevated plasma cytokines. The best hit was tested for association with type 2 diabetes. Subjects (N = 1,769) were recruited from the Tübingen Family (TÜF) study for type 2 diabetes and genotyped for tagging SNPs. In a subgroup, cytokines were measured. Association with type 2 diabetes was tested in a prospective case-cohort study (N = 2,971) derived from the EPIC-Potsdam study. Three SNPs (rs6543087, rs17801985, rs1003376) revealed nominal and two SNPs (rs11674694, rs11678405) significant associations with 2-hour glucose levels. SNPs rs6543087 and rs11674694 were also nominally associated with decreased insulin sensitivity. Another two SNPs (rs2236936, rs2236935) showed associations with reduced insulin release, driven by effects in lean subjects only. Three SNPs (rs11674694, rs13003883, rs2236936) revealed nominal associations with IL-6 levels. SNP rs11674694 was significantly associated with type 2 diabetes. In conclusion, common variation in MAP4K4 is associated with insulin resistance and β-cell dysfunction, possibly via this gene’s role in inflammatory signalling. This variation’s impact on insulin sensitivity may be more important since its effect on insulin release vanishes with increasing BMI. PMID:23094072

MAP kinase-signaling controls nuclear translocation of tripeptidyl-peptidase II in response to DNA damage and oxidative stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Preta, Giulio; Klark, Rainier de; Chakraborti, Shankhamala

2010-08-27

Research highlights: {yields} Nuclear translocation of TPPII occurs in response to different DNA damage inducers. {yields} Nuclear accumulation of TPPII is linked to ROS and anti-oxidant enzyme levels. {yields} MAPKs control nuclear accumulation of TPPII. {yields} Inhibited nuclear accumulation of TPPII decreases DNA damage-induced {gamma}-H2AX expression. -- Abstract: Reactive oxygen species (ROS) are a continuous hazard in eukaroytic cells by their ability to cause damage to biomolecules, in particular to DNA. Previous data indicated that the cytosolic serine peptidase tripeptidyl-peptidase II (TPPII) translocates into the nucleus of most tumor cell lines in response to {gamma}-irradiation and ROS production; an eventmore » that promoted p53 expression as well as caspase-activation. We here observed that nuclear translocation of TPPII was dependent on signaling by MAP kinases, including p38MAPK. Further, this was caused by several types of DNA-damaging drugs, a DNA cross-linker (cisplatinum), an inhibitor of topoisomerase II (etoposide), and to some extent also by nucleoside-analogues (5-fluorouracil, hydroxyurea). In the minority of tumor cell lines where TPPII was not translocated into the nucleus in response to DNA damage we observed reduced intracellular ROS levels, and the expression levels of redox defense systems were increased. Further, treatment with the ROS-inducer {gamma}-hexa-chloro-cyclohexane ({gamma}-HCH, lindane), an inhibitor of GAP junctions, restored nuclear translocation of TPPII in these cell lines upon {gamma}-irradiation. Moreover, blocking nuclear translocation of TPPII in etoposide-treated cells, by using a peptide-derived inhibitor (Z-Gly-Leu-Ala-OH), attenuated expression of {gamma}-H2AX in {gamma}-irradiated melanoma cells. Our results indicated a role for TPPII in MAPK-dependent DNA damage signaling.« less

The C-Type Lectin OCILRP2 Costimulates EL4 T Cell Activation via the DAP12-Raf-MAP Kinase Pathway

PubMed Central

Lou, Qiang; Zhang, Wei; Liu, Guangchao; Ma, Yuanfang

2014-01-01

OCILRP2 is a typical Type-II transmembrane protein that is selectively expressed in activated T lymphocytes, dendritic cells, and B cells and functions as a novel co-stimulator of T cell activation. However, the signaling pathways underlying OCILRP2 in T cell activation are still not completely understood. In this study, we found that the knockdown of OCILRP2 expression with shRNA or the blockage of its activity by an anti-OCILRP2 antagonist antibody reduced CD3/CD28-costimulated EL4 T cell viability and IL-2 production, inhibit Raf1, MAPK3, and MAPK8 activation, and impair NFAT and NF-κB transcriptional activities. Furthermore, immunoprecipitation results indicated that OCILRP2 could interact with the DAP12 protein, an adaptor containing an intracellular ITAM motif that can transduce signals to induce MAP kinase activation for T cell activation. Our data reveal that after binding with DAP12, OCILRP2 activates the Raf-MAP kinase pathways, resulting in T cell activation. PMID:25411776

The C-type lectin OCILRP2 costimulates EL4 T cell activation via the DAP12-Raf-MAP kinase pathway.

PubMed

Lou, Qiang; Zhang, Wei; Liu, Guangchao; Ma, Yuanfang

2014-01-01

OCILRP2 is a typical Type-II transmembrane protein that is selectively expressed in activated T lymphocytes, dendritic cells, and B cells and functions as a novel co-stimulator of T cell activation. However, the signaling pathways underlying OCILRP2 in T cell activation are still not completely understood. In this study, we found that the knockdown of OCILRP2 expression with shRNA or the blockage of its activity by an anti-OCILRP2 antagonist antibody reduced CD3/CD28-costimulated EL4 T cell viability and IL-2 production, inhibit Raf1, MAPK3, and MAPK8 activation, and impair NFAT and NF-κB transcriptional activities. Furthermore, immunoprecipitation results indicated that OCILRP2 could interact with the DAP12 protein, an adaptor containing an intracellular ITAM motif that can transduce signals to induce MAP kinase activation for T cell activation. Our data reveal that after binding with DAP12, OCILRP2 activates the Raf-MAP kinase pathways, resulting in T cell activation.

Fission yeast Csk1 is a CAK-activating kinase (CAKAK).

PubMed Central

Hermand, D; Pihlak, A; Westerling, T; Damagnez, V; Vandenhaute, J; Cottarel, G; Mäkelä, T P

1998-01-01

Cell cycle progression is dependent on the sequential activity of cyclin-dependent kinases (CDKs). For full activity, CDKs require an activating phosphorylation of a conserved residue (corresponding to Thr160 in human CDK2) carried out by the CDK-activating kinase (CAK). Two distinct CAK kinases have been described: in budding yeast Saccharomyces cerevisiae, the Cak1/Civ1 kinase is responsible for CAK activity. In several other species including human, Xenopus, Drosophila and fission yeast Schizosaccharomyces pombe, CAK has been identified as a complex homologous to CDK7-cyclin H (Mcs6-Mcs2 in fission yeast). Here we identify the fission yeast Csk1 kinase as an in vivo activating kinase of the Mcs6-Mcs2 CAK defining Csk1 as a CAK-activating kinase (CAKAK). PMID:9857180

Stimulation of dihydroxyacetone and glycerol kinase activity in Streptococcus faecalis by phosphoenolpyruvate-dependent phosphorylation catalyzed by enzyme I and HPr of the phosphotransferase systems

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deutscher, J.; Sauerwald, H.

1986-06-01

Recently a report was given of the phosphoenolpyruvate (PEP)-dependent phosphorylation of a 55-kilodalton protein of Streptococus faecalis catalyzed by enzyme I and histidine-containing protein (HPr) of the phosphotransferase system. The purified 55-kilodalton protein was found to exhibit dihydroxyacetone kinase activity. Glycerol was six times more slowly phosphorylated than dihydroxyacetone. The K/sub m/s were found to 0.7 mM for ATP, 0.45 mM for dihydroxyacetone, and 0.9 MM for glycerol. PEP-dependent phosphorylation of dihydroxyacetone kinase stimulated phosphorylation of both substrates about 10-fold. Fructose 1,6-diphosphate at concentrations higher than 2 mM inhibited the activity of phosphorylated and unphosphorylated dihydroxyacetone kinase in a noncompetitivemore » manner. The rate of PEP-dependent phosphorylation of dihydroxyacetone kinase was about 200-fold slower than the phosphorylation rate of III proteins (also called enzyme III or factor III), which so far have been considered the only phosphoryl acceptors of histidyl-phosphorylated HPr. P-Dihydroxyacetone kinase was found to be able to transfer its phosphoryl group in a backward reaction to HPr. Following (/sup 32/P)PEP-dependent phosphorylation and tryptic digestion of dihydroxyacetone kinase, the authors isolated a labeled peptide composed of 37 amino acids, as determined by amino acid analysis. The single histidyl residue of this peptide most likely carries the phosphoryl group in phosphorylated dihydroxyacetone kinase.« less

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.

NIK and Cot cooperate to trigger NF-kappaB p65 phosphorylation.

PubMed

Wittwer, Tobias; Schmitz, M Lienhard

2008-06-27

The serine/threonine kinase Cot triggers NF-kappaB-dependent transactivation and activation of various MAPKinases. Here we identify Cot as a novel p65 interacting protein kinase. Cot expression induces p65 phosphorylation at serines 536 and 468 in dependence from its kinase function. Accordingly, shRNA-mediated knockdown of Cot expression interferes with TNF-induced NF-kappaB-dependent gene expression. Also the C-terminally truncated, oncogenic form of Cot is able to trigger p65 phosphorylation. In vitro kinase assays and dominant negative mutants revealed that NIK functions downstream of Cot to mediate p65 phosphorylation.

Identification and characterization of a novel serine-threonine kinase gene from the Xp22 region.

PubMed

Montini, E; Andolfi, G; Caruso, A; Buchner, G; Walpole, S M; Mariani, M; Consalez, G; Trump, D; Ballabio, A; Franco, B

1998-08-01

Eukaryotic protein kinases are part of a large and expanding family of proteins. Through our transcriptional mapping effort in the Xp22 region, we have isolated and sequenced the full-length transcript of STK9, a novel cDNA highly homologous to serine-threonine kinases. A number of human genetic disorders have been mapped to the region where STK9 has been localized including Nance-Horan (NH) syndrome, oral-facial-digital syndrome type 1 (OFD1), and a novel locus for nonsyndromic sensorineural deafness (DFN6). To evaluate the possible involvement of STK9 in any of the above-mentioned disorders, a 2416-bp full-length cDNA was assembled. The entire genomic structure of the gene, which is composed of 20 coding exons, was determined. Northern analysis revealed a transcript larger than 9.5 kb in several tissues including brain, lung, and kidney. The mouse homologue (Stk9) was identified and mapped in the mouse in the region syntenic to human Xp. This location is compatible with the location of the Xcat mutant, which shows congenital cataracts very similar to those observed in NH patients. Sequence homologies, expression pattern, and mapping information in both human and mouse make STK9 a candidate gene for the above-mentioned disorders. Copyright 1998 Academic Press.

The role of MAP kinases in the induction of iNOS expression in neutrophils exposed to NDMA: the involvement transcription factors.

PubMed

Ratajczak-Wrona, W; Jablonska, E; Garley, M; Jablonski, J; Radziwon, P; Iwaniuk, A

2013-01-01

The role of MAP kinases in the activation of AP-1 (c-Jun, c-Fos) and NF-κB p65 engaged in the regulation of iNOS expression in human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was analyzed in the study. The study included a group of 20 healthy individuals. Isolated human PMN were incubated in the presence of NDMA. Selective MAP kinases inhibitors were used. The expression of proteins in the cytoplasmic and nuclear fractions was assessed using Western blot method. The results show that NDMA intensifies iNOS, c-Jun, NF-κB p65 and IκB-α expression in the analyzed PMNs. The blocking of the p38 pathway led to lower iNOS expression, and higher expression of c-Jun and c-Fos in the cytoplasmic fraction, and also lower c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. A decrease in iNOS expression in the cytoplasmic fraction, and also c-Jun in both fractions of the examined cells, was observed as a result of JNK pathway inhibition. The blocking of the ERK5 pathway led to higher iNOS, c-Jun and c-Fos expression in the cytoplasmic fraction, and higher c-Jun expression in the nuclear fraction of PMNs exposed to NDMA. The study also demonstrated that blocking of the p38 and JNK pathways resulted in higher expression of NF-κB p65 and IκB-α in the cytoplasmic fraction and their lower expression in the nuclear fraction of these cells. Our data indicate the role of MAP kinases p38 and JNK in the activation of c-Jun and NF-κB p65 transcription factors engaged in the regulation of iNOS expression in human neutrophils exposed to NDMA. However ERK5 kinase is not involved in the regulation of iNOS and NO production by those cells.

Calcium/calmodulin-dependent kinase II phosphorylation of the GABAA receptor alpha1 subunit modulates benzodiazepine binding.

PubMed

Churn, Severn B; Rana, Aniruddha; Lee, Kangmin; Parsons, J Travis; De Blas, Angel; Delorenzo, Robert J

2002-09-01

gamma-Aminobutyric acid (GABA) is the primary neurotransmitter that is responsible for the fast inhibitory synaptic transmission in the central nervous system. A major post-translational mechanism that can rapidly regulate GABAAR function is receptor phosphorylation. This study was designed to test the effect of endogenous calcium and calmodulin-dependent kinase II (CaM kinase II) activation on both allosteric modulator binding and GABAA receptor subunit phosphorylation. Endogenous CaM kinase II activity was stimulated, and GABAA receptors were subsequently analyzed for bothallosteric modulator binding properties and immunoprecipitated and analyzed for subunit phosphorylation levels. A significant increase in allosteric-modulator binding of the GABAAR was observed under conditions maximal for CaM kinase II activation. In addition, CaM kinase II activation resulted in a direct increase in phosphorylation of the GABAA receptor alpha1 subunit. The data suggest that the CaM kinase II-dependent phosphorylation of the GABAA receptor alpha1 subunit modulated allosteric modulator binding to the GABAA receptor.

Mapping pathological phenotypes in a mouse model of CDKL5 disorder.

PubMed

Amendola, Elena; Zhan, Yang; Mattucci, Camilla; Castroflorio, Enrico; Calcagno, Eleonora; Fuchs, Claudia; Lonetti, Giuseppina; Silingardi, Davide; Vyssotski, Alexei L; Farley, Dominika; Ciani, Elisabetta; Pizzorusso, Tommaso; Giustetto, Maurizio; Gross, Cornelius T

2014-01-01

Mutations in cyclin-dependent kinase-like 5 (CDKL5) cause early-onset epileptic encephalopathy, a neurodevelopmental disorder with similarities to Rett Syndrome. Here we describe the physiological, molecular, and behavioral phenotyping of a Cdkl5 conditional knockout mouse model of CDKL5 disorder. Behavioral analysis of constitutive Cdkl5 knockout mice revealed key features of the human disorder, including limb clasping, hypoactivity, and abnormal eye tracking. Anatomical, physiological, and molecular analysis of the knockout uncovered potential pathological substrates of the disorder, including reduced dendritic arborization of cortical neurons, abnormal electroencephalograph (EEG) responses to convulsant treatment, decreased visual evoked responses (VEPs), and alterations in the Akt/rpS6 signaling pathway. Selective knockout of Cdkl5 in excitatory and inhibitory forebrain neurons allowed us to map the behavioral features of the disorder to separable cell-types. These findings identify physiological and molecular deficits in specific forebrain neuron populations as possible pathological substrates in CDKL5 disorder.

τ kinases in the rat heat shock model: Possible implications for Alzheimer disease

PubMed Central

Shanavas, Alikunju; Papasozomenos, Sozos Ch.

2000-01-01

We have previously shown, by using the phosphate-dependent anti-τ antibodies Tau-1 and PHF-1, that heat shock induces rapid dephosphorylation of τ followed by hyperphosphorylation in female rats. In this study, we analyzed in forebrain homogenates from female Sprague–Dawley rats the activities of extracellular signal regulated kinase 1/2 (ERK1/2), c-Jun NH2-terminal kinase (JNK), glycogen synthase kinase-3β (GSK-3β), cyclin-dependent kinase 5 (Cdk5), cAMP-dependent protein kinase A (PKA), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) at 0 (n = 5), 3 (n = 4), 6 (n = 5), and 12 (n = 5) h after heat shock and in non-heat-shocked controls (n = 5). Immunoprecipitation kinase assays at 0 h showed suppression of the activities of all kinases except of GSK-3β, which showed increased activity. At 3–6 h, the activities of ERK1/2, JNK, Cdk5, and GSK-3β toward selective substrates were increased; however, only JNK, Cdk5, and GSK-3β but not ERK1/2 were overactivated toward purified bovine τ. At 3–6 h, kinase assays specific for PKA and CaMKII showed no increased activity toward either τ or selective substrates. All of eight anti-τ antibodies tested showed dephosphorylation at 0 h and hyperphosphorylation at 3–6 h, except for 12E8, which showed hyperphosphorylation also at 0 h. Immunoblot analysis using activity-dependent antibodies against ERK1/2, JNK, and GSK-3β confirmed the above data. Increased activation and inhibition of kinases after heat shock were statistically significant in comparison with controls. Because τ is hyperphosphorylated in Alzheimer disease these findings suggest that JNK, GSK-3β, and Cdk5 may play a role in its pathogenesis. PMID:11121021

RSK3 is required for concentric myocyte hypertrophy in an activated Raf1 model for Noonan syndrome.

PubMed

Passariello, Catherine L; Martinez, Eliana C; Thakur, Hrishikesh; Cesareo, Maria; Li, Jinliang; Kapiloff, Michael S

2016-04-01

Noonan syndrome (NS) is a congenital disorder resulting from mutations of the Ras-Raf signaling pathway. Hypertrophic cardiomyopathy associated with RAF1 "RASopathy" mutations is a major risk factor for heart failure and death in NS and has been attributed to activation of MEK1/2-ERK1/2 mitogen-activated protein kinases. We recently discovered that type 3 p90 ribosomal S6 kinase (RSK3) is an ERK effector that is required, like ERK1/2, for concentric myocyte hypertrophy in response to pathological stress such as pressure overload. In order to test whether RSK3 also contributes to NS-associated hypertrophic cardiomyopathy, RSK3 knock-out mice were crossed with mice bearing the Raf1(L613V) human NS mutation. We confirmed that Raf1(L613V) knock-in confers a NS-like phenotype, including cardiac hypertrophy. Active RSK3 was increased in Raf1(L613V) mice. Constitutive RSK3 gene deletion prevented the Raf1(L613V)-dependent concentric growth in width of the cardiac myocyte and attenuated cardiac hypertrophy in female mice. These results are consistent with RSK3 being an important mediator of ERK1/2-dependent growth in RASopathy. In conjunction with previously published data showing that RSK3 is important for pathological remodeling of the heart, these data suggest that targeting of this downstream MAP-kinase pathway effector should be considered in the treatment of RASopathy-associated hypertrophic cardiomyopathy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mice haploinsufficient for Map2k7, a gene involved in neurodevelopment and risk for schizophrenia, show impaired attention, a vigilance decrement deficit and unstable cognitive processing in an attentional task: impact of minocycline.

PubMed

Openshaw, R L; Thomson, D M; Penninger, J M; Pratt, J A; Morris, B J

2017-01-01

Members of the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein (MAP) kinases, and the upstream kinase MKK7, have all been strongly linked with synaptic plasticity and with the development of the neocortex. However, the impact of disruption of this pathway on cognitive function is unclear. In the current study, we test the hypothesis that reduced MKK7 expression is sufficient to cause cognitive impairment. Attentional function in mice haploinsufficient for Map2k7 (Map2k7 +/- mice) was investigated using the five-choice serial reaction time task (5-CSRTT). Once stable performance had been achieved, Map2k7 +/- mice showed a distinctive attentional deficit, in the form of an increased number of missed responses, accompanied by a more pronounced decrement in performance over time and elevated intra-individual reaction time variability. When performance was reassessed after administration of minocycline-a tetracycline antibiotic currently showing promise for the improvement of attentional deficits in patients with schizophrenia-signs of improvement in attentional performance were detected. Overall, Map2k7 haploinsufficiency causes a distinctive pattern of cognitive impairment strongly suggestive of an inability to sustain attention, in accordance with those seen in psychiatric patients carrying out similar tasks. This may be important for understanding the mechanisms of cognitive dysfunction in clinical populations and highlights the possibility of treating some of these deficits with minocycline.

Activation of c-jun N-terminal kinase upon influenza A virus (IAV) infection is independent of pathogen-related receptors but dependent on amino acid sequence variations of IAV NS1.

PubMed

Nacken, Wolfgang; Anhlan, Darisuren; Hrincius, Eike R; Mostafa, Ahmed; Wolff, Thorsten; Sadewasser, Anne; Pleschka, Stephan; Ehrhardt, Christina; Ludwig, Stephan

2014-08-01

A hallmark cell response to influenza A virus (IAV) infections is the phosphorylation and activation of c-jun N-terminal kinase (JNK). However, so far it is not fully clear which molecules are involved in the activation of JNK upon IAV infection. Here, we report that the transfection of influenza viral-RNA induces JNK in a retinoic acid-inducible gene I (RIG-I)-dependent manner. However, neither RIG-I-like receptors nor MyD88-dependent Toll-like receptors were found to be involved in the activation of JNK upon IAV infection. Viral JNK activation may be blocked by addition of cycloheximide and heat shock protein inhibitors during infection, suggesting that the expression of an IAV-encoded protein is responsible for JNK activation. Indeed, the overexpression of nonstructural protein 1 (NS1) of certain IAV subtypes activated JNK, whereas those of some other subtypes failed to activate JNK. Site-directed mutagenesis experiments using NS1 of the IAV H7N7, H5N1, and H3N2 subtypes identified the amino acid residue phenylalanine (F) at position 103 to be decisive for JNK activation. Cleavage- and polyadenylation-specific factor 30 (CPSF30), whose binding to NS1 is stabilized by the amino acids F103 and M106, is not involved in JNK activation. Conclusively, subtype-specific sequence variations in the IAV NS1 protein result in subtype-specific differences in JNK signaling upon IAV infection. Influenza A virus (IAV) infection leads to the activation or modulation of multiple signaling pathways. Here, we demonstrate for the first time that the c-jun N-terminal kinase (JNK), a long-known stress-activated mitogen-activated protein (MAP) kinase, is activated by RIG-I when cells are treated with IAV RNA. However, at the same time, nonstructural protein 1 (NS1) of IAV has an intrinsic JNK-activating property that is dependent on IAV subtype-specific amino acid variations around position 103. Our findings identify two different and independent pathways that result in the activation of JNK in the course of an IAV infection. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Activation of c-jun N-Terminal Kinase upon Influenza A Virus (IAV) Infection Is Independent of Pathogen-Related Receptors but Dependent on Amino Acid Sequence Variations of IAV NS1

PubMed Central

Nacken, Wolfgang; Anhlan, Darisuren; Hrincius, Eike R.; Mostafa, Ahmed; Wolff, Thorsten; Sadewasser, Anne; Pleschka, Stephan; Ehrhardt, Christina

2014-01-01

ABSTRACT A hallmark cell response to influenza A virus (IAV) infections is the phosphorylation and activation of c-jun N-terminal kinase (JNK). However, so far it is not fully clear which molecules are involved in the activation of JNK upon IAV infection. Here, we report that the transfection of influenza viral-RNA induces JNK in a retinoic acid-inducible gene I (RIG-I)-dependent manner. However, neither RIG-I-like receptors nor MyD88-dependent Toll-like receptors were found to be involved in the activation of JNK upon IAV infection. Viral JNK activation may be blocked by addition of cycloheximide and heat shock protein inhibitors during infection, suggesting that the expression of an IAV-encoded protein is responsible for JNK activation. Indeed, the overexpression of nonstructural protein 1 (NS1) of certain IAV subtypes activated JNK, whereas those of some other subtypes failed to activate JNK. Site-directed mutagenesis experiments using NS1 of the IAV H7N7, H5N1, and H3N2 subtypes identified the amino acid residue phenylalanine (F) at position 103 to be decisive for JNK activation. Cleavage- and polyadenylation-specific factor 30 (CPSF30), whose binding to NS1 is stabilized by the amino acids F103 and M106, is not involved in JNK activation. Conclusively, subtype-specific sequence variations in the IAV NS1 protein result in subtype-specific differences in JNK signaling upon IAV infection. IMPORTANCE Influenza A virus (IAV) infection leads to the activation or modulation of multiple signaling pathways. Here, we demonstrate for the first time that the c-jun N-terminal kinase (JNK), a long-known stress-activated mitogen-activated protein (MAP) kinase, is activated by RIG-I when cells are treated with IAV RNA. However, at the same time, nonstructural protein 1 (NS1) of IAV has an intrinsic JNK-activating property that is dependent on IAV subtype-specific amino acid variations around position 103. Our findings identify two different and independent pathways that result in the activation of JNK in the course of an IAV infection. PMID:24872593

Epidermal growth factor receptor (EGFR) transactivation by estrogen via the G-protein-coupled receptor, GPR30: a novel signaling pathway with potential significance for breast cancer.

PubMed

Filardo, Edward J

2002-02-01

The biological and biochemical effects of estrogen have been ascribed to its known receptors, which function as ligand-inducible transcription factors. However, estrogen also triggers rapid activation of classical second messengers (cAMP, calcium, and inositol triphosphate) and stimulation of intracellular signaling cascades mitogen-activated protein kinase (MAP K), PI3K and eNOS. These latter events are commonly activated by membrane receptors that either possess intrinsic tyrosine kinase activity or couple to heterotrimeric G-proteins. We have shown that estrogen transactivates the epidermal growth factor receptor (EGFR) to MAP K signaling axis via the G-protein-coupled receptor (GPCR), GPR30, through the release of surface-bound proHB-EGF from estrogen receptor (ER)-negative human breast cancer cells [Molecular Endocrinology 14 (2000) 1649]. This finding is consistent with a growing body of evidence suggesting that transactivation of EGFRs by GPCRs is a recurrent theme in cell signaling. GPCR-mediated transactivation of EGFRs by estrogen provides a previously unappreciated mechanism of cross-talk between estrogen and serum growth factors, and explains prior data reporting the EGF-like effects of estrogen. This novel mechanism by which estrogen activates growth factor-dependent signaling and its implications for breast cancer biology are discussed further in this review.

TRAF2 multitasking in TNF receptor-induced signaling to NF-κB, MAP kinases and cell death.

PubMed

Borghi, Alice; Verstrepen, Lynn; Beyaert, Rudi

2016-09-15

Tumor Necrosis Factor (TNF) is a potent inflammatory cytokine that exerts its functions through the activation of two distinct receptors, TNFR1 and TNFR2. Both receptors can activate canonical NF-κB and JNK MAP kinase signaling, while TNFR2 can also activate non-canonical NF-κB signaling, leading to numerous changes in gene expression that drive inflammation, cell proliferation and cell survival. On the other hand, TNFR1 also activates signaling pathways leading to cell death by either apoptosis or necroptosis, depending on the cellular context. A key player in TNFR1- and TNFR2-induced signaling is the RING finger protein TRAF2, which is recruited to both receptors upon their stimulation. TRAF2 exerts multiple receptor-specific functions but also mediates cross-talk between TNFR1 and TNFR2, dictating the outcome of TNF stimulation. In this review, we provide an overview of the positive and negative regulatory role of TRAF2 in different TNFR1 and TNFR2 signaling pathways. We discuss the underlying molecular mechanism of action, distinguishing between TRAF2 scaffold and E3 ubiquitin ligase functions, and the regulation of TRAF2 by specific post-translational modifications. Finally, we elaborate on some possible strategies to modulate TRAF2 function in the context of therapeutic targeting in autoimmunity and cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

Kinase Substrate Sensor (KISS), a Mammalian In Situ Protein Interaction Sensor*

PubMed Central

Lievens, Sam; Gerlo, Sarah; Lemmens, Irma; De Clercq, Dries J. H.; Risseeuw, Martijn D. P.; Vanderroost, Nele; De Smet, Anne-Sophie; Ruyssinck, Elien; Chevet, Eric; Van Calenbergh, Serge; Tavernier, Jan

2014-01-01

Probably every cellular process is governed by protein-protein interaction (PPIs), which are often highly dynamic in nature being modulated by in- or external stimuli. Here we present KISS, for KInase Substrate Sensor, a mammalian two-hybrid approach designed to map intracellular PPIs and some of the dynamic features they exhibit. Benchmarking experiments indicate that in terms of sensitivity and specificity KISS is on par with other binary protein interaction technologies while being complementary with regard to the subset of PPIs it is able to detect. We used KISS to evaluate interactions between different types of proteins, including transmembrane proteins, expressed at their native subcellular location. In situ analysis of endoplasmic reticulum stress-induced clustering of the endoplasmic reticulum stress sensor ERN1 and ligand-dependent β-arrestin recruitment to GPCRs illustrated the method's potential to study functional PPI modulation in complex cellular processes. Exploring its use as a tool for in cell evaluation of pharmacological interference with PPIs, we showed that reported effects of known GPCR antagonists and PPI inhibitors are properly recapitulated. In a three-hybrid setup, KISS was able to map interactions between small molecules and proteins. Taken together, we established KISS as a sensitive approach for in situ analysis of protein interactions and their modulation in a changing cellular context or in response to pharmacological challenges. PMID:25154561

The gene for creatine kinase, mitochondrial 2 (sarcomeric; CKMT2), maps to chromosome 5q13. 3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Richard, I.; Devaud, C.; Cherif, D.

1993-10-01

YAC clones for the creatine kinase, mitochrondial 2 (sarcomeric; CKMT2), gene were isolated. One of these YACs was localized on chromosome 5q13.3 by fluorescence in situ hybridization. A polymorphic dinucleotide repeat (heterozygosity 0.77) was identified within the seventh intron of the CKMT2 gene. Genotyping of CEPH families allowed positioning of CKMT2 on the multipoint map of chromosome 5 between D5S424 and D5S428, distal to spinal muscular atrophy (SMA) (5q12-q14). 8 refs., 1 fig., 2 tabs.

Activation of the JNK pathway is essential for transformation by the Met oncogene.

PubMed

Rodrigues, G A; Park, M; Schlessinger, J

1997-05-15

The Met/Hepatocyte Growth Factor (HGF) receptor tyrosine kinase is oncogenically activated through a rearrangement that creates a hybrid gene Tpr-Met. The resultant chimeric p65(Tpr-Met) protein is constitutively phosphorylated on tyrosine residues in vivo and associates with a number of SH2-containing signaling molecules including the p85 subunit of PI-3 kinase and the Grb2 adaptor protein, which couples receptor tyrosine kinases to the Ras signaling pathway. Mutation of the binding site for Grb2 impairs the ability of Tpr-Met oncoprotein to transform fibroblasts, suggesting that the activation of the Ras/MAP kinase signaling pathway through Grb2 may be essential for cellular transformation. To test this hypothesis dominant-negative mutants of Grb2 with deletions of the SH3 domains were introduced into Tpr-Met transformed fibroblasts. Cells overexpressing the mutants were found to be morphologically reverted and exhibited reduced growth in soft agar. Surprisingly, the Grb2 mutants blocked activation of the JNK/SAPK but not MAP kinase activity induced by the Tpr-Met oncoprotein. Additionally, cells expressing dominant-negative Grb2 mutants had reduced PI-3-kinase activity and dominant-negative mutants of Rac1 blocked both Tpr-Met-induced transformation and activation of JNK. These experiments reveal a novel link between Met and the JNK pathway, which is essential for transformation by this oncogene.

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed Central

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-01-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes. PMID:9121442

Megakaryocytic differentiation induced by constitutive activation of mitogen-activated protein kinase kinase.

PubMed

Whalen, A M; Galasinski, S C; Shapiro, P S; Nahreini, T S; Ahn, N G

1997-04-01

The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic stem cells. Phorbol esters, which initiate megakaryocyte differentiation in this cell line, caused a rapid increase in extracellular-signal-regulated kinase (ERK), which remained elevated for 2 h and returned to near-basal levels by 24 h. In the absence of extracellular stimuli, ERK could be activated by expression of constitutively active mutants of mitogen-activated protein (MAP) kinase kinase (MKK), resulting in cell adhesion and spreading, increased cell size, inhibition of cell growth, and induction of the platelet-specific integrin alphaIIb beta3, all hallmarks of megakaryocytic differentiation. In contrast, expression of wild-type MKK had little effect. In addition, constitutively active MKK suppressed the expression of an erythroid marker, alpha-globin, indicating the ability to suppress cellular responses necessary for alternative cell lineages. The MKK inhibitor PD98059 blocked MKK/ERK activation and cellular responses to phorbol ester, demonstrating that activation of MKK is necessary and sufficient to induce a differentiation program along the megakaryocyte lineage. Thus, the MAP kinase cascade, which promotes cell growth and proliferation in many cell types, instead inhibits cell proliferation and initiates lineage-specific differentiation in K562 cells, establishing a model system to investigate the mechanisms by which this signal transduction pathway specifies cell fate and developmental processes.

The MAP kinase-activated protein kinase Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen Candida albicans.

PubMed

Li, Xichuan; Du, Wei; Zhao, Jingwen; Zhang, Lilin; Zhu, Zhiyan; Jiang, Linghuo

2010-06-01

Rck2p is the Hog1p-MAP kinase-activated protein kinase required for the attenuation of protein synthesis in response to an osmotic challenge in Saccharomyces cerevisiae. Rck2p also regulates rapamycin sensitivity in both S. cerevisiae and Candida albicans. In this study, we demonstrate that the deletion of CaRCK2 renders C. albicans cells sensitive to, and CaRck2p translocates from the cytosol to the nucleus in response to, cell wall stresses caused by Congo red, Calcoflor White, elevated heat and zymolyase. However, the kinase activity of CaRck2p is not required for the cellular response to these cell wall stresses. Furthermore, transcripts of cell wall protein-encoding genes CaBGL2, CaHWP1 and CaXOG1 are reduced in C. albicans cells lacking CaRCK2. The deletion of CaRCK2 also reduces the in vitro filamentation of C. albicans and its virulence in a mouse model of systemic candidasis. The kinase activity of CaRck2p is required for the virulence, but not for the in vitro filamentation, in C. albicans. Therefore, Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen C. albicans.

Separating myoblast differentiation from muscle cell fusion using IGF-I and the p38 MAP kinase inhibitor SB202190.

PubMed

Gardner, Samantha; Gross, Sean M; David, Larry L; Klimek, John E; Rotwein, Peter

2015-10-01

The p38 MAP kinases play critical roles in skeletal muscle biology, but the specific processes regulated by these kinases remain poorly defined. Here we find that activity of p38α/β is important not only in early phases of myoblast differentiation, but also in later stages of myocyte fusion and myofibrillogenesis. By treatment of C2 myoblasts with the promyogenic growth factor insulin-like growth factor (IGF)-I, the early block in differentiation imposed by the p38 chemical inhibitor SB202190 could be overcome. Yet, under these conditions, IGF-I could not prevent the later impairment of muscle cell fusion, as marked by the nearly complete absence of multinucleated myofibers. Removal of SB202190 from the medium of differentiating myoblasts reversed the fusion block, as multinucleated myofibers were detected several hours later and reached ∼90% of the culture within 30 h. Analysis by quantitative mass spectroscopy of proteins that changed in abundance following removal of the inhibitor revealed a cohort of upregulated muscle-enriched molecules that may be important for both myofibrillogenesis and fusion. We have thus developed a model system that allows separation of myoblast differentiation from muscle cell fusion and should be useful in identifying specific steps regulated by p38 MAP kinase-mediated signaling in myogenesis. Copyright © 2015 the American Physiological Society.

Construction of phosphorylation interaction networks by text mining of full-length articles using the eFIP system.

PubMed

Tudor, Catalina O; Ross, Karen E; Li, Gang; Vijay-Shanker, K; Wu, Cathy H; Arighi, Cecilia N

2015-01-01

Protein phosphorylation is a reversible post-translational modification where a protein kinase adds a phosphate group to a protein, potentially regulating its function, localization and/or activity. Phosphorylation can affect protein-protein interactions (PPIs), abolishing interaction with previous binding partners or enabling new interactions. Extracting phosphorylation information coupled with PPI information from the scientific literature will facilitate the creation of phosphorylation interaction networks of kinases, substrates and interacting partners, toward knowledge discovery of functional outcomes of protein phosphorylation. Increasingly, PPI databases are interested in capturing the phosphorylation state of interacting partners. We have previously developed the eFIP (Extracting Functional Impact of Phosphorylation) text mining system, which identifies phosphorylated proteins and phosphorylation-dependent PPIs. In this work, we present several enhancements for the eFIP system: (i) text mining for full-length articles from the PubMed Central open-access collection; (ii) the integration of the RLIMS-P 2.0 system for the extraction of phosphorylation events with kinase, substrate and site information; (iii) the extension of the PPI module with new trigger words/phrases describing interactions and (iv) the addition of the iSimp tool for sentence simplification to aid in the matching of syntactic patterns. We enhance the website functionality to: (i) support searches based on protein roles (kinases, substrates, interacting partners) or using keywords; (ii) link protein entities to their corresponding UniProt identifiers if mapped and (iii) support visual exploration of phosphorylation interaction networks using Cytoscape. The evaluation of eFIP on full-length articles achieved 92.4% precision, 76.5% recall and 83.7% F-measure on 100 article sections. To demonstrate eFIP for knowledge extraction and discovery, we constructed phosphorylation-dependent interaction networks involving 14-3-3 proteins identified from cancer-related versus diabetes-related articles. Comparison of the phosphorylation interaction network of kinases, phosphoproteins and interactants obtained from eFIP searches, along with enrichment analysis of the protein set, revealed several shared interactions, highlighting common pathways discussed in the context of both diseases. © The Author(s) 2015. Published by Oxford University Press.

Modulation of skeletal muscle fiber type by mitogen-activated protein kinase signaling.

PubMed

Shi, Hao; Scheffler, Jason M; Pleitner, Jonathan M; Zeng, Caiyun; Park, Sungkwon; Hannon, Kevin M; Grant, Alan L; Gerrard, David E

2008-08-01

Skeletal muscle is composed of diverse fiber types, yet the underlying molecular mechanisms responsible for this diversification remain unclear. Herein, we report that the extracellular signal-regulated kinase (ERK) 1/2 pathway, but not p38 or c-Jun NH(2)-terminal kinase (JNK), is preferentially activated in fast-twitch muscles. Pharmacological blocking of ERK1/2 pathway increased slow-twitch fiber type-specific reporter activity and repressed those associated with the fast-twitch fiber phenotype in vitro. Overexpression of a constitutively active ERK2 had an opposite effect. Inhibition of ERK signaling in cultured myotubes increased slow-twitch fiber-specific protein accumulation while repressing those characteristic of fast-twitch fibers. Overexpression of MAP kinase phosphatase-1 (MKP1) in mouse and rat muscle fibers containing almost exclusively type IIb or IIx fast myosin heavy chain (MyHC) isoforms induced de novo synthesis of the slower, more oxidative type IIa and I MyHCs in a time-dependent manner. Conversion to the slower phenotype was confirmed by up-regulation of slow reporter gene activity and down-regulation of fast reporter activities in response to forced MKP1 expression in vivo. In addition, activation of ERK2 signaling induced up-regulation of fast-twitch fiber program in soleus. These data suggest that the MAPK signaling, most likely the ERK1/2 pathway, is necessary to preserve the fast-twitch fiber phenotype with a concomitant repression of slow-twitch fiber program.

Fibroblast growth factor 2 and cyclic AMP synergistically regulate bone sialoprotein gene expression.

PubMed

Shimizu, Emi; Nakayama, Youhei; Nakajima, Yu; Kato, Naoko; Takai, Hideki; Kim, Dong-Soon; Arai, Masato; Saito, Ryoichiro; Sodek, Jaro; Ogata, Yorimasa

2006-07-01

Bone sialoprotein (BSP) is a noncollagenous protein of the mineralized bone extracellular matrix. We here report that FGF2 and cAMP act synergistically to stimulate BSP gene expression. Treatment of ROS 17/2.8 cells with either 10 ng/ml FGF2 or 1 microM FSK for 6 h resulted in 5.4- and 8.2-fold increases, respectively, in the levels of BSP mRNA. However, in the presence of both FGF2 and forskolin (FGF/FSK), BSP mRNA levels were increased synergistically by 20.4-fold. Using a luciferase reporter construct, encompassing BSP promoter nucleotides -116 to +60, transcription was also increased synergistically by 15.0-fold with FGF/FSK, compared to stimulations of 2.6- and 5.3-fold, respectively, for FGF2 and FSK alone. Transcriptional stimulation by FGF/FSK abrogated in constructs included 2 bp mutations in the inverted CCAAT, CRE, FRE and Pit-1 elements. Whereas the FRE-protein complex was increased by FGF2 and FGF/FSK, the Pit-1-protein complex was decreased by FSK and FGF/FSK. Notably, transcriptional activity induced by FGF/FSK was blocked by protein kinase A, tyrosine kinase and MEK inhibitors. These studies indicate that the combinatorial effects of FGF and FSK act through PKA, tyrosine kinase and MAP-kinase-dependent pathways, which target the inverted CCAAT, CRE, FRE and Pit-1 elements in the BSP gene to synergistically increase BSP expression.

Predicting a small molecule-kinase interaction map: A machine learning approach

PubMed Central

2011-01-01

Background We present a machine learning approach to the problem of protein ligand interaction prediction. We focus on a set of binding data obtained from 113 different protein kinases and 20 inhibitors. It was attained through ATP site-dependent binding competition assays and constitutes the first available dataset of this kind. We extract information about the investigated molecules from various data sources to obtain an informative set of features. Results A Support Vector Machine (SVM) as well as a decision tree algorithm (C5/See5) is used to learn models based on the available features which in turn can be used for the classification of new kinase-inhibitor pair test instances. We evaluate our approach using different feature sets and parameter settings for the employed classifiers. Moreover, the paper introduces a new way of evaluating predictions in such a setting, where different amounts of information about the binding partners can be assumed to be available for training. Results on an external test set are also provided. Conclusions In most of the cases, the presented approach clearly outperforms the baseline methods used for comparison. Experimental results indicate that the applied machine learning methods are able to detect a signal in the data and predict binding affinity to some extent. For SVMs, the binding prediction can be improved significantly by using features that describe the active site of a kinase. For C5, besides diversity in the feature set, alignment scores of conserved regions turned out to be very useful. PMID:21708012

3D-QSAR and molecular docking study on bisarylmaleimide series as glycogen synthase kinase 3, cyclin dependent kinase 2 and cyclin dependent kinase 4 inhibitors: an insight into the criteria for selectivity.

PubMed

Dessalew, Nigus; Bharatam, Prasad V

2007-07-01

Selective glycogen synthase kinase 3 (GSK3) inhibition over cyclin dependent kinases such as cyclin dependent kinase 2 (CDK2) and cyclin dependent kinase 4 (CDK4) is an important requirement for improved therapeutic profile of GSK3 inhibitors. The concepts of selectivity and additivity fields have been employed in developing selective CoMFA models for these related kinases. Initially, sets of three individual CoMFA models were developed, using 36 compounds of bisarylmaleimide series to correlate with the GSK3, CDK2 and CDK4 inhibitory potencies. These models showed a satisfactory statistical significance: CoMFA-GSK3 (r(2)(con), r(2)(cv): 0.931, 0.519), CoMFA-CDK2 (0.937, 0.563), and CoMFA-CDK4 (0.892, 0.725). Three different selective CoMFA models were then developed using differences in pIC(50) values. These three models showed a superior statistical significance: (i) CoMFA-Selective1 (r(2)(con), r(2)(cv): 0.969, 0.768), (ii) CoMFA-Selective 2 (0.974, 0.835) and (iii) CoMFA-Selective3 (0.963, 0.776). The selective models were found to outperform the individual models in terms of the quality of correlation and were found to be more informative in pinpointing the structural basis for the observed quantitative differences of kinase inhibition. An in-depth comparative investigation was carried out between the individual and selective models to gain an insight into the selectivity criterion. To further validate this approach, a set of new compounds were designed which show selectivity and were docked into the active site of GSK3, using FlexX based incremental construction algorithm.

Differential Requirements for Src-Family Kinases in SYK or ZAP70-Mediated SLP-76 Phosphorylation in Lymphocytes

PubMed Central

Fasbender, Frank; Claus, Maren; Wingert, Sabine; Sandusky, Mina; Watzl, Carsten

2017-01-01

In a synthetic biology approach using Schneider (S2) cells, we show that SLP-76 is directly phosphorylated at tyrosines Y113 and Y128 by SYK in the presence of ITAM-containing adapters such as CD3ζ, DAP12, or FcεRγ. This phosphorylation was dependent on at least one functional ITAM and a functional SH2 domain within SYK. Inhibition of Src-kinases by inhibitors PP1 and PP2 did not reduce SLP-76 phosphorylation in S2 cells, suggesting an ITAM and SYK dependent, but Src-kinase independent signaling pathway. This direct ITAM/SYK/SLP-76 signaling pathway therefore differs from previously described ITAM signaling. However, the SYK-family kinase ZAP70 required the additional co-expression of the Src-family kinases Fyn or Lck to efficiently phosphorylate SLP-76 in S2 cells. This difference in Src-family kinase dependency of SYK versus ZAP70-mediated ITAM-based signaling was further demonstrated in human lymphocytes. ITAM signaling in ZAP70-expressing T cells was dependent on the activity of Src-family kinases. In contrast, Src-family kinases were partially dispensable for ITAM signaling in SYK-expressing B cells or in natural killer cells, which express SYK and ZAP70. This demonstrates that SYK can signal using a Src-kinase independent ITAM-based signaling pathway, which may be involved in calibrating the threshold for lymphocyte activation. PMID:28736554

Differential Requirements for Src-Family Kinases in SYK or ZAP70-Mediated SLP-76 Phosphorylation in Lymphocytes.

PubMed

Fasbender, Frank; Claus, Maren; Wingert, Sabine; Sandusky, Mina; Watzl, Carsten

2017-01-01

In a synthetic biology approach using Schneider (S2) cells, we show that SLP-76 is directly phosphorylated at tyrosines Y113 and Y128 by SYK in the presence of ITAM-containing adapters such as CD3ζ, DAP12, or FcεRγ. This phosphorylation was dependent on at least one functional ITAM and a functional SH2 domain within SYK. Inhibition of Src-kinases by inhibitors PP1 and PP2 did not reduce SLP-76 phosphorylation in S2 cells, suggesting an ITAM and SYK dependent, but Src-kinase independent signaling pathway. This direct ITAM/SYK/SLP-76 signaling pathway therefore differs from previously described ITAM signaling. However, the SYK-family kinase ZAP70 required the additional co-expression of the Src-family kinases Fyn or Lck to efficiently phosphorylate SLP-76 in S2 cells. This difference in Src-family kinase dependency of SYK versus ZAP70-mediated ITAM-based signaling was further demonstrated in human lymphocytes. ITAM signaling in ZAP70-expressing T cells was dependent on the activity of Src-family kinases. In contrast, Src-family kinases were partially dispensable for ITAM signaling in SYK-expressing B cells or in natural killer cells, which express SYK and ZAP70. This demonstrates that SYK can signal using a Src-kinase independent ITAM-based signaling pathway, which may be involved in calibrating the threshold for lymphocyte activation.

Chronic inhibition of Ca(2+)/calmodulin kinase II activity in the pilocarpine model of epilepsy.

PubMed

Churn, S B; Kochan, L D; DeLorenzo, R J

2000-09-01

The development of symptomatic epilepsy is a model of long-term plasticity changes in the central nervous system. The rat pilocarpine model of epilepsy was utilized to study persistent alterations in calcium/calmodulin-dependent kinase II (CaM kinase II) activity associated with epileptogenesis. CaM kinase II-dependent substrate phosphorylation and autophosphorylation were significantly inhibited for up to 6 weeks following epileptogenesis in both the cortex and hippocampus, but not in the cerebellum. The net decrease in CaM kinase II autophosphorylation and substrate phosphorylation was shown to be due to decreased kinase activity and not due to increased phosphatase activity. The inhibition in CaM kinase II activity and the development of epilepsy were blocked by pretreating seizure rats with MK-801 indicating that the long-lasting decrease in CaM kinase II activity was dependent on N-methyl-D-aspartate receptor activation. In addition, the inhibition of CaM kinase II activity was associated in time and regional localization with the development of spontaneous recurrent seizure activity. The decrease in enzyme activity was not attributed to a decrease in the alpha or beta kinase subunit protein expression level. Thus, the significant inhibition of the enzyme occurred without changes in kinase protein expression, suggesting a long-lasting, post-translational modification of the enzyme. This is the first published report of a persistent, post-translational alteration of CaM kinase II activity in a model of epilepsy characterized by spontaneous recurrent seizure activity.

Y-box binding protein-1 serine 102 is a downstream target of p90 ribosomal S6 kinase in basal-like breast cancer cells

PubMed Central

Stratford, Anna L; Fry, Christopher J; Desilets, Curtis; Davies, Alastair H; Cho, Yong Y; Li, Yvonne; Dong, Zigang; Berquin, Isabelle M; Roux, Philippe P; Dunn, Sandra E

2008-01-01

Introduction Basal-like breast cancers (BLBC) frequently overexpress the epidermal growth factor receptor (EGFR) and subsequently have high levels of signaling through the MAP kinase pathway, which is thought to contribute to their aggressive behavior. While we have previously reported the expression of Y-box binding protein-1 (YB-1) in 73% of BLBC, it is unclear whether it can be regulated by a component of the MAP kinase signaling pathway. Phosphorylation of YB-1 at the serine 102 residue is required for transcriptional activation of growth-enhancing genes, such as EGFR. Using Motifscan we identified p90 ribosomal S6 kinase (RSK) as a potential candidate for activating YB-1. Methods Inhibition of RSK1 and RSK2 was achieved using siRNA and the small molecule SL0101. RSK1, RSK2, activated RSK and kinase-dead RSK were expressed in HCC1937 cells. Kinase assays were performed to illustrate direct phosphorylation of YB-1 by RSK. The impact of inhibiting RSK on YB-1 function was measured by luciferase assays and chromatin immunoprecipitation. Results Using an in vitro kinase assay, RSK1 and RSK2 were shown to directly phosphorylate YB-1. Interestingly, they were more effective activators of YB-1 than AKT or another novel YB-1 kinase, PKCα. Phosphorylation of YB-1 (serine 102 residue) is blocked by inhibition of the MAP kinase pathway or by perturbing RSK1/RSK2 with siRNA or SL0101. In immortalized breast epithelial cells where RSK is active yet AKT is not, YB-1 is phosphorylated. Supporting this observation, RSK2-/- mouse embryo fibroblasts lose the ability to phosphorylate YB-1 in response to epidermal growth factor. This subsequently interfered with the ability of YB-1 to regulate the expression of EGFR. The RSK inhibitor SL0101 decreased the ability of YB-1 to bind the promoter, transactivate and ultimately reduce EGFR expression. In concordance with these results the expression of constitutively active RSK1 increased YB-1 phosphorylation, yet the kinase-dead RSK did not. Conclusions We therefore conclude that RSK1/RSK2 are novel activators of YB-1, able to phosphorylate the serine 102 residue. This provides a newly described mechanism whereby YB-1 is activated in breast cancer. This implicates the EGFR/RSK/YB-1 pathway as an important component of BLBC, providing an important opportunity for therapeutic intervention. PMID:19036157

The activation of directional stem cell motility by green light-emitting diode irradiation.

PubMed

Ong, Wei-Kee; Chen, How-Foo; Tsai, Cheng-Ting; Fu, Yun-Ju; Wong, Yi-Shan; Yen, Da-Jen; Chang, Tzu-Hao; Huang, Hsien-Da; Lee, Oscar Kuang-Sheng; Chien, Shu; Ho, Jennifer Hui-Chun

2013-03-01

Light-emitting diode (LED) irradiation is potentially a photostimulator to manipulate cell behavior by opsin-triggered phototransduction and thermal energy supply in living cells. Directional stem cell motility is critical for the efficiency and specificity of stem cells in tissue repair. We explored that green LED (530 nm) irradiation directed the human orbital fat stem cells (OFSCs) to migrate away from the LED light source through activation of extracellular signal-regulated kinases (ERK)/MAP kinase/p38 signaling pathway. ERK inhibitor selectively abrogated light-driven OFSC migration. Phosphorylation of these kinases as well as green LED irradiation-induced cell migration was facilitated by increasing adenosine triphosphate (ATP) production in OFSCs after green LED exposure, and which was thermal stress-independent mechanism. OFSCs, which are multi-potent mesenchymal stem cells isolated from human orbital fat tissue, constitutionally express three opsins, i.e. retinal pigment epithelium-derived rhodopsin homolog (RRH), encephalopsin (OPN3) and short-wave-sensitive opsin 1 (OPN1SW). However, only two non-visual opsins, i.e. RRH and OPN3, served as photoreceptors response to green LED irradiation-induced OFSC migration. In conclusion, stem cells are sensitive to green LED irradiation-induced directional cell migration through activation of ERK signaling pathway via a wavelength-dependent phototransduction. Copyright © 2012 Elsevier Ltd. All rights reserved.

Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36.

PubMed

Avey, Denis; Tepper, Sarah; Pifer, Benjamin; Bahga, Amritpal; Williams, Hunter; Gillen, Joseph; Li, Wenwei; Ogden, Sarah; Zhu, Fanxiu

2016-07-01

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies. KSHV ORF36 encodes a serine/threonine viral protein kinase, which is conserved throughout all herpesviruses. Although several studies have identified the viral and cellular substrates of conserved herpesvirus protein kinases (CHPKs), the precise functions of KSHV ORF36 during lytic replication remain elusive. Here, we report that ORF36 interacts with another lytic protein, ORF45, in a manner dependent on ORF36 kinase activity. We mapped the regions of ORF36 and ORF45 involved in the binding. Their association appears to be mediated by electrostatic interactions, since deletion of either the highly basic N terminus of ORF36 or an acidic patch of ORF45 abolished the binding. In addition, the dephosphorylation of ORF45 protein dramatically reduced its association with ORF36. Importantly, ORF45 enhances both the stability and kinase activity of ORF36. Consistent with previous studies of CHPK homologs, we detected ORF36 protein in extracellular virions. To investigate the roles of ORF36 in the context of KSHV lytic replication, we used bacterial artificial chromosome mutagenesis to engineer both ORF36-null and kinase-dead mutants. We found that ORF36-null/mutant virions are moderately defective in viral particle production and are further deficient in primary infection. In summary, our results uncover a functionally important interaction between ORF36 and ORF45 and indicate a significant role of ORF36 in the production of infectious progeny virions. Kaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus with a significant public health burden. KSHV ORF36 encodes a serine/threonine viral protein kinase, whose functions throughout the viral life cycle have not been elucidated. Here, we report that ORF36 interacts with another KSHV protein, ORF45. We mapped the regions of ORF36 and ORF45 involved in their association and further characterized the consequences of this interaction. We engineered ORF36 mutant viruses in order to investigate the functional roles of ORF36 in the context of KSHV lytic replication, and we confirmed that ORF36 is a component of KSHV virions. Moreover, we found that ORF36 mutants are defective in virion production and primary infection. In summary, we discovered and characterized a functionally important interaction between KSHV ORF36 and ORF45, and our results suggest a significant role of ORF36 in the production of infectious progeny virions, a process critical for KSHV pathogenesis. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Subthreshold IKK activation modulates the effector functions of primary mast cells and allows specific targeting of transformed mast cells

PubMed Central

Drube, Sebastian; Beyer, Mandy; Rothe, Mandy; Rabenhorst, Anja; Göpfert, Christiane; Meininger, Isabel; Diamanti, Michaela A.; Stegner, David; Häfner, Norman; Böttcher, Martin; Reinecke, Kirstin; Herdegen, Thomas; Greten, Florian R.; Nieswandt, Bernhard; Hartmann, Karin; Krämer, Oliver H.; Kamradt, Thomas

2015-01-01

Mast cell differentiation and proliferation depends on IL-3. IL-3 induces the activation of MAP-kinases and STATs and consequently induces proliferation and survival. Dysregulation of IL-3 signaling pathways also contribute to inflammation and tumorigenesis. We show here that IL-3 induces a SFK- and Ca2+-dependent activation of the inhibitor of κB kinases 2 (IKK2) which results in mast cell proliferation and survival but does not induce IκBα-degradation and NFκB activation. Therefore we propose the term “subthreshold IKK activation”. This subthreshold IKK activation also primes mast cells for enhanced responsiveness to IL-33R signaling. Consequently, co-stimulation with IL-3 and IL-33 increases IKK activation and massively enhances cytokine production induced by IL-33. We further reveal that in neoplastic mast cells expressing constitutively active Ras, subthreshold IKK activation is associated with uncontrolled proliferation. Consequently, pharmacological IKK inhibition reduces tumor growth selectively by inducing apoptosis in vivo. Together, subthreshold IKK activation is crucial to mediate the full IL-33-induced effector functions in primary mast cells and to mediate uncontrolled proliferation of neoplastic mast cells. Thus, IKK2 is a new molecularly defined target structure. PMID:25749030

1,2,6-Thiadiazinones as Novel Narrow Spectrum Calcium/Calmodulin-Dependent Protein Kinase Kinase 2 (CaMKK2) Inhibitors.

PubMed

Asquith, Christopher R M; Godoi, Paulo H; Couñago, Rafael M; Laitinen, Tuomo; Scott, John W; Langendorf, Christopher G; Oakhill, Jonathan S; Drewry, David H; Zuercher, William J; Koutentis, Panayiotis A; Willson, Timothy M; Kalogirou, Andreas S

2018-05-19

We demonstrate for the first time that 4 H -1,2,6-thiadiazin-4-one (TDZ) can function as a chemotype for the design of ATP-competitive kinase inhibitors. Using insights from a co-crystal structure of a 3,5-bis(arylamino)-4 H -1,2,6-thiadiazin-4-one bound to calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), several analogues were identified with micromolar activity through targeted displacement of bound water molecules in the active site. Since the TDZ analogues showed reduced promiscuity compared to their 2,4-dianilinopyrimidine counter parts, they represent starting points for development of highly selective kinase inhibitors.

Anti-inflammatory activity of 4-methoxyhonokiol is a function of the inhibition of iNOS and COX-2 expression in RAW 264.7 macrophages via NF-kappaB, JNK and p38 MAPK inactivation.

PubMed

Zhou, Hong Yu; Shin, Eun Myoung; Guo, Lian Yu; Youn, Ui Joung; Bae, KiHwan; Kang, Sam Sik; Zou, Li Bo; Kim, Yeong Shik

2008-05-31

The extracts or constituents from the bark of Magnolia (M.) obovata are known to have many pharmacological activities. 4-Methoxyhonokiol, a neolignan compound isolated from the stem bark of M. obovata, was found to exhibit a potent anti-inflammatory effect in different experimental models. Pretreatment with 4-methoxyhonokiol (i.p.) dose-dependently inhibited the dye leakage and paw swelling in an acetic-acid-induced vascular permeability assay and a carrageenan-induced paw edema assay in mice, respectively. In the lipopolysaccharide (LPS)-induced systemic inflammation model, 4-methoxyhonokiol significantly inhibited plasma nitric oxide (NO) release in mice. To identify the mechanisms underlying this anti-inflammatory action, we investigated the effect of 4-methoxyhonokiol on LPS-induced responses in a murine macrophage cell line, RAW 264.7. The results demonstrated that 4-methoxyhonokiol significantly inhibited LPS-induced NO production as well as the protein and mRNA expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, 4-methoxyhonokiol inhibited LPS-mediated nuclear factor-kappaB (NF-kappaB) activation via the prevention of inhibitor kappaB (IkappaB) phosphorylation and degradation. 4-Methoxyhonokiol had no effect on the LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), whereas it attenuated the phosphorylation of p38 mitogen-activated protein kinase (p38 MAPK) and c-Jun NH2-terminal kinase (JNK) in a concentration-dependent manner. Taken together, our data suggest that 4-methoxyhonokiol is an active anti-inflammatory constituent of the bark of M. obovata, and that its anti-inflammatory property might be a function of the inhibition of iNOS and COX-2 expression via down-regulation of the JNK and p38 MAP kinase signal pathways and inhibition of NF-kappaB activation in RAW 264.7 macrophages.

TAB2 Is Essential for Prevention of Apoptosis in Fetal Liver but Not for Interleukin-1 Signaling

PubMed Central

Sanjo, Hideki; Takeda, Kiyoshi; Tsujimura, Tohru; Ninomiya-Tsuji, Jun; Matsumoto, Kunihiro; Akira, Shizuo

2003-01-01

The proinflammatory cytokine interleukin-1 (IL-1) transmits a signal via several critical cytoplasmic proteins such as MyD88, IRAKs and TRAF6. Recently, serine/threonine kinase TAK1 and TAK1 binding protein 1 and 2 (TAB1/2) have been identified as molecules involved in IL-1-induced TRAF6-mediated activation of AP-1 and NF-κB via mitogen-activated protein (MAP) kinases and IκB kinases, respectively. However, their physiological functions remain to be clarified. To elucidate their roles in vivo, we generated TAB2-deficient mice. The TAB2 deficiency was embryonic lethal due to liver degeneration and apoptosis. This phenotype was similar to that of NF-κB p65-, IKKβ-, and NEMO/IKKγ-deficient mice. However, the IL-1-induced activation of NF-κB and MAP kinases was not impaired in TAB2-deficient embryonic fibroblasts. These findings demonstrate that TAB2 is essential for embryonic development through prevention of liver apoptosis but not for the IL-1 receptor-mediated signaling pathway. PMID:12556483

Protooncogene TCL1b functions as an Akt kinase co-activator that exhibits oncogenic potency in vivo

PubMed Central

Hashimoto, M; Suizu, F; Tokuyama, W; Noguchi, H; Hirata, N; Matsuda-Lennikov, M; Edamura, T; Masuzawa, M; Gotoh, N; Tanaka, S; Noguchi, M

2013-01-01

Protooncogene T-cell leukemia 1 (TCL1), which is implicated in human T-cell prolymphocytic leukemia (T-PLL), interacts with Akt and enhances its kinase activity, functioning as an Akt kinase co-activator. Two major isoforms of TCL1 Protooncogenes (TCL1 and TCL1b) are present adjacent to each other on human chromosome 14q.32. In human T-PLL, both TCL1 and TCL1b are activated by chromosomal translocation. Moreover, TCL1b-transgenic mice have never been created. Therefore, it remains unclear whether TCL1b itself, independent of TCL1, exhibits oncogenicity. In co-immunoprecipitation assays, both ectopic and endogenous TCL1b interacted with Akt. In in vitro Akt kinase assays, TCL1b enhanced Akt kinase activity in dose- and time-dependent manners. Bioinformatics approaches utilizing multiregression analysis, cluster analysis, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway mapping, Venn diagrams and Gene Ontology (GO) demonstrated that TCL1b showed highly homologous gene-induction signatures similar to Myr-Akt or TCL1. TCL1b exhibited oncogenicity in in vitro colony-transformation assay. Further, two independent lines of β-actin promoter-driven TCL1b-transgenic mice developed angiosarcoma on the intestinal tract. Angiosarcoma is a rare form of cancer in humans with poor prognosis. Using immunohistochemistry, 11 out of 13 human angiosarcoma samples were positively stained with both anti-TCL1b and anti-phospho-Akt antibodies. Consistently, in various cancer tissues, 69 out of 146 samples were positively stained with anti-TCL1b, out of which 46 were positively stained with anti-phospho-Akt antibodies. Moreover, TCL1b structure-based inhibitor ‘TCL1b-Akt-in' inhibited Akt kinase activity in in vitro kinase assays and PDGF (platelet-derived growth factor)-induced Akt kinase activities—in turn, ‘TCL1b-Akt-in' inhibited cellular proliferation of sarcoma. The current study disclosed TCL1b bears oncogenicity and hence serves as a novel therapeutic target for human neoplastic diseases. PMID:24042734

Comprehensive Logic Based Analyses of Toll-Like Receptor 4 Signal Transduction Pathway

PubMed Central

Padwal, Mahesh Kumar; Sarma, Uddipan; Saha, Bhaskar

2014-01-01

Among the 13 TLRs in the vertebrate systems, only TLR4 utilizes both Myeloid differentiation factor 88 (MyD88) and Toll/Interleukin-1 receptor (TIR)-domain-containing adapter interferon-β-inducing Factor (TRIF) adaptors to transduce signals triggering host-protective immune responses. Earlier studies on the pathway combined various experimental data in the form of one comprehensive map of TLR signaling. But in the absence of adequate kinetic parameters quantitative mathematical models that reveal emerging systems level properties and dynamic inter-regulation among the kinases/phosphatases of the TLR4 network are not yet available. So, here we used reaction stoichiometry-based and parameter independent logical modeling formalism to build the TLR4 signaling network model that captured the feedback regulations, interdependencies between signaling kinases and phosphatases and the outcome of simulated infections. The analyses of the TLR4 signaling network revealed 360 feedback loops, 157 negative and 203 positive; of which, 334 loops had the phosphatase PP1 as an essential component. The network elements' interdependency (positive or negative dependencies) in perturbation conditions such as the phosphatase knockout conditions revealed interdependencies between the dual-specific phosphatases MKP-1 and MKP-3 and the kinases in MAPK modules and the role of PP2A in the auto-regulation of Calmodulin kinase-II. Our simulations under the specific kinase or phosphatase gene-deficiency or inhibition conditions corroborated with several previously reported experimental data. The simulations to mimic Yersinia pestis and E. coli infections identified the key perturbation in the network and potential drug targets. Thus, our analyses of TLR4 signaling highlights the role of phosphatases as key regulatory factors in determining the global interdependencies among the network elements; uncovers novel signaling connections; identifies potential drug targets for infections. PMID:24699232

Functional roles of Cot/Tpl2 in mast cell responses to lipopolysaccharide and FcεRI-clustering.

PubMed

Chiba, Norika; Kakimoto, Kyoko; Masuda, Akio; Matsuguchi, Tetsuya

2010-11-05

Cot/Tpl2, a member of MAP kinase kinase kinase (MAPKKK), is indispensable for the ERK activation, as well as the production of TNF-α, IL-1β, IL-23, and PGE(2) in lipopolysaccharide (LPS)-stimulated macrophages. However, the expression and the functional roles of Cot/Tpl2 in mast cells have not been elucidated. The administration of LPS impairs allergic airway inflammation in a mast cell-dependent manner, and LPS stimulates mast cells to produce not only pro-inflammatory cytokines, such as IL-6 and TNF-α, but also Th2-type cytokines, such as IL-5, IL-10 and IL-13. Here, we examine the role of Cot/Tpl2 by using bone marrow-derived mast cells (BMMCs) from cot/tpl2 gene-deficient mice. Phosphorylation of ERKs was significantly decreased, whereas that of JNKs and p38 kinase was normal in LPS-stimulated cot/tpl2(-/-) BMMCs compared with wild-type counterparts. LPS-induced mRNA increase was significantly impaired for IL-5, IL-10, IL-13, and TNF-α, but was normal for IL-6, in cot/tpl2(-/-) BMMCs. On the other hand, degranulation by FcεRI-clustering from cot/tpl2(-/-) BMMCs was significantly enhanced compared with the WT control. Although the phosphorylation of ERKs and p38 kinase by FcεRI-clustering was similar in WT and cot/tpl2(-/-) BMMCs, the phosphorylation of Syk was significantly enhanced in cot/tpl2(-/-) BMMCs, which seemed to be due to the increased protein concentration of Syk. These results imply the functional importance of Cot/Tpl2 in mast cells during the course of allergic diseases such as asthma. Copyright © 2010 Elsevier Inc. All rights reserved.

Ethylene Rapidly Up-Regulates the Activities of Both Monomeric GTP-Binding Proteins and Protein Kinase(s) in Epicotyls of Pea1

PubMed Central

Moshkov, Igor E.; Novikova, Galina V.; Mur, Luis A.J.; Smith, Aileen R.; Hall, Michael A.

2003-01-01

It is demonstrated that, in etiolated pea (Pisum sativum) epicotyls, ethylene affects the activation of both monomeric GTP-binding proteins (monomeric G-proteins) and protein kinases. For monomeric G-proteins, the effect may be a rapid (2 min) and bimodal up-regulation, a transiently unimodal activation, or a transient down-regulation. Pretreatment with 1-methylcyclopropene abolishes the response to ethylene overall. Immunoprecipitation studies indicate that some of the monomeric G-proteins affected may be of the Rab class. Protein kinase activity is rapidly up-regulated by ethylene, the effect is inhibited by 1-methylcyclopropene, and the activation is bimodal. Immunoprecipitation indicates that the kinase(s) are of the MAP kinase ERK1 group. It is proposed that the data support the hypothesis that a transduction chain exists that is separate and antagonistic to that currently revealed by studies on Arabidopsis mutants. PMID:12692330

CR108, a novel vitamin K3 derivative induces apoptosis and breast tumor inhibition by reactive oxygen species and mitochondrial dysfunction.

PubMed

Yang, Chun-Ru; Liao, Wei-Siang; Wu, Ya-Hui; Murugan, Kaliyappan; Chen, Chinpiao; Chao, Jui-I

2013-12-15

Vitamin K3 derivatives have been shown to exert anticancer activities. Here we show a novel vitamin K3 derivative (S)-2-(2-hydroxy-3-methylbutylthio)naphthalene-1,4-dione, which is named as CR108 that induces apoptosis and tumor inhibition through reactive oxygen species (ROS) and mitochondrial dysfunction in human breast cancer. CR108 is more effective on the breast cancer cell death than other vitamin K3 derivatives. Moreover, CR108 induced apoptosis in both the non-HER-2-overexpressed MCF-7 and HER-2-overexpressed BT-474 breast cancer cells. CR108 caused the loss of mitochondrial membrane potential, cytochrome c released from mitochondria to cytosol, and cleaved PARP proteins for apoptosis induction. CR108 markedly increased ROS levels in breast cancer cells. N-acetylcysteine (NAC), a general ROS scavenger, completely blocked the CR108-induced ROS levels, mitochondrial dysfunction and apoptosis. Interestingly, CR108 increased the phosphorylation of p38 MAP kinase but conversely inhibited the survivin protein expression. NAC treatment prevented the activation of p38 MAP kinase and rescued the survivin protein levels. SB202190, a specific p38 MAP kinase inhibitor, recovered the survivin protein levels and attenuated the cytotoxicity of CR108-treated cells. Furthermore, CR108 inhibited the xenografted human breast tumor growth in nude mice. Together, we demonstrate that CR108 is a novel vitamin K3 derivative that induces apoptosis and tumor inhibition by ROS production and mitochondrial dysfunction and associates with the phosphorylation of p38 MAP kinase and the inhibition of survivin in the human breast cancer. © 2013.

WRKY transcription factors.

PubMed

Rushton, Paul J; Somssich, Imre E; Ringler, Patricia; Shen, Qingxi J

2010-05-01

WRKY transcription factors are one of the largest families of transcriptional regulators in plants and form integral parts of signalling webs that modulate many plant processes. Here, we review recent significant progress in WRKY transcription factor research. New findings illustrate that WRKY proteins often act as repressors as well as activators, and that members of the family play roles in both the repression and de-repression of important plant processes. Furthermore, it is becoming clear that a single WRKY transcription factor might be involved in regulating several seemingly disparate processes. Mechanisms of signalling and transcriptional regulation are being dissected, uncovering WRKY protein functions via interactions with a diverse array of protein partners, including MAP kinases, MAP kinase kinases, 14-3-3 proteins, calmodulin, histone deacetylases, resistance proteins and other WRKY transcription factors. WRKY genes exhibit extensive autoregulation and cross-regulation that facilitates transcriptional reprogramming in a dynamic web with built-in redundancy. 2010 Elsevier Ltd. All rights reserved.

Chromosomal localization of the mouse Src-like adapter protein (Slap) gene and its putative human homolog SLA

DOE Office of Scientific and Technical Information (OSTI.GOV)

Angrist, M.; Chakravarti, A.; Wells, D.E.

1995-12-10

Molecules containing Src-homology 2 (SH2) and Src-homology 3 (SH3) domains are critical components of signal transduction pathways that serve to relay signals originating from the cell surface to the interior of the cell. Src-like adapter protein (SLAP) is a recently described adapter protein that binds activated the Eck receptor protein-tyrosine kinase. Although SLAP bears a striking homology to the SH3 and SH2 domains of the Src family of nonreceptor tyrosine kinases, it does not contain a tyrosine kinase catalytic domain. In this report, the Slap gene was mapped by linkage analysis to mouse chromosome 15, while its putative human homologmore » (SLA) was identified and mapped to human 8q22.3-qter using a panel of somatic cell hybrids. 10 refs., 2 figs.« less

RhMKK9, a rose MAP KINASE KINASE gene, is involved in rehydration-triggered ethylene production in rose gynoecia.

PubMed

Chen, Jiwei; Zhang, Qian; Wang, Qigang; Feng, Ming; Li, Yang; Meng, Yonglu; Zhang, Yi; Liu, Guoqin; Ma, Zhimin; Wu, Hongzhi; Gao, Junping; Ma, Nan

2017-02-23

Flower opening is an important process in the life cycle of flowering plants and is influenced by various endogenous and environmental factors. Our previous work demonstrated that rose (Rosa hybrida) flowers are highly sensitive to dehydration during flower opening and the water recovery process after dehydration induced ethylene production rapidly in flower gynoecia. In addition, this temporal- and spatial-specific ethylene production is attributed to a transient but robust activation of the rose MAP KINASE6-ACC SYNTHASE1 (RhMPK6-RhACS1) cascade in gynoecia. However, the upstream component of RhMPK6-RhACS1 is unknown, although RhMKK9 (MAP KINASE KINASE9), a rose homologue of Arabidopsis MKK9, could activate RhMPK6 in vitro. In this study, we monitored RhMKK2/4/5/9 expression, the potential upstream kinase to RhMPK6, in rose gynoecia during dehydration and rehydration. We found only RhMKK9 was rapidly and strongly induced by rehydration. Silencing of RhMKK9 significantly decreased rehydration-triggered ethylene production. Consistently, the expression of several ethylene-responsive genes was down regulated in the petals of RhMKK9-silenced flowers. Moreover, we detected the DNA methylation level in the promoter and gene body of RhMKK9 by Chop-PCR. The results showed that rehydration specifically elevated the DNA methylation level on the RhMKK9 gene body, whereas it resulted in hypomethylation in its promoter. Our results showed that RhMKK9 possibly acts as the upstream component of the RhMKK9-RhMPK6-RhACS1 cascade and is responsible for water recovery-triggered ethylene production in rose gynoecia, and epigenetic DNA methylation is involved in the regulation of RhMKK9 expression by rehydration.

Purification and cDNA cloning of SAPKK3, the major activator of RK/p38 in stress- and cytokine-stimulated monocytes and epithelial cells.

PubMed Central

Cuenda, A; Alonso, G; Morrice, N; Jones, M; Meier, R; Cohen, P; Nebreda, A R

1996-01-01

Two chromatographically distinct stress-activated protein kinase kinases (SAPKKs) have been identified in several mammalian cells, termed SAPKK2 and SAPKK3, which activate the MAP kinase family member RK/p38 but not JNK/SAPK in vitro. Here we demonstrate that SAPKK2 is identical or very closely related to the MAP kinase kinase family member MKK3. However, under our assay conditions, SAPKK3 was the major activator of RK/p38 detected in extracts prepared from stress- or interleukin-1-stimulated epithelial (KB) cells, from bacterial lipopolysaccharide and tumour necrosis factor alpha-stimulated THP1 monocytes or from rabbit skeletal muscle. The activated form of SAPKK3 was purified from muscle to near homogeneity, and tryptic peptide sequences were used to clone human and murine cDNAs encoding this enzyme. Human SAPKK3 comprised 334 amino acids and was 78% identical to MKK3. The murine and human SAPKK3 were 97% identical in their amino acid sequences. We also cloned a different murine cDNA that appears to encode a SAPKK3 protein truncated at the N-terminus. SAPKK3 is identical to the recently cloned MKK6. Images PMID:8861944

Alkyl isothiocyanates suppress epidermal growth factor receptor kinase activity but augment tyrosine kinase activity.

PubMed

Nomura, Takahiro; Uehara, Yoshimasa; Kawajiri, Hiroo; Ryoyama, Kazuo; Yamori, Takao; Fuke, Yoko

2009-10-01

We have reported the in vitro and in vivo anticancer activities of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) derived from a Japanese spice, wasabi. In order to obtain some clues about the mechanism of the anticancer activity, we have studied the effect of alkyl isothiocyanates (MITCs) on protein kinase activities. The anti-autophosphorylation activity of MITCs with respect to the epidermal growth factor (EGF)-stimulated receptor kinase of A431 epidermoid carcinoma cells was examined by incorporation of radioactive ATP into an acid-insoluble fraction. Their anti-phosphorylation activity with respect to the non-receptor protein kinase was analyzed by a standard SDS-PAGE method. All the tested MITCs interfered with the EGF-stimulated receptor kinase activity in a dose-dependent manner, although their effects were less than 1/10 of that of erbstatin in microg/ml. On the other hand, the MITCs did not interfere with non-receptor kinases (kinase A, kinase C, tyrosine kinase and calmodulin dependent kinase III), but enhanced non-receptor tyrosine kinase. A possible anticancer mechanism of MITCs may involve the suppression of EGF receptor kinase activity and augmentation of non-receptor PTK.

Upregulation of PEDF expression by PARP inhibition contributes to the decrease in hyperglycemia-induced apoptosis in HUVECs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen Haibing; Department of Ophthalmology, Anhui Provincial Hospital, Hefei; Jia Weiping

2008-05-02

Poly(ADP-ribose)polymerase (PARP) inhibitors decrease angiogenesis through reducing vascular endothelium growth factor (VEGF) induced proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs). In contrast to VEGF, pigment epithelium-derived factor (PEDF) has been demonstrated to act as a strong endogenous inhibitor of angiogenesis. Here, we show that PARP inhibition with a specific inhibitor PJ-34 or specific PARP antisense oligonucleotide upregulates hyperglycemia-induced PEDF expression in HUVECs in a dose-dependent manner. This results in the retard of activation of p38 MAP kinase and the concomitant decrease in cell apoptosis. These results give the first direct demonstration that PEDF might representmore » a target for PARP inhibition treatment and the effects of PEDF on endothelial cells growth are context dependent.« less

Role played by Disabled-2 in albumin induced MAP Kinase signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diwakar, Ramaswamy; Pearson, Alexander L.; Colville-Nash, Paul

2008-02-15

Albumin has been shown to activate the mitogen activated protein kinase (MAPK) pathway in proximal tubular cells (PTECs) of the kidney. Megalin, the putative receptor for albumin has potential signalling properties. However, the mechanisms by which megalin signals are unclear. The adaptor phosphoprotein Disabled-2 (Dab2) is known to interact with the cytoplasmic tail of megalin and may be involved in albumin-mediated MAPK signalling. In this study, we investigated the role of Dab2 in albumin-mediated MAPK signalling and further studied the role of Dab2 in albumin-induced TGF{beta}-1 secretion, a MAPK dependent event. We used RNA interference to knockdown Dab2 protein abundancemore » in HKC-8 cells a model of human PTECs. Albumin activated ERK1,2 and Elk-1 in a MEK-1 dependent manner and resulted in secretion of TGF{beta}-1. In the absence of albumin, knockdown of Dab2 resulted in a trend towards increase in pERK1,2 consistent with its putative role as an inhibitor of cell proliferation. However albumin-induced ERK1,2 activation was completely abolished by Dab2 knockdown. Dab2 knockdown did not however result in inhibition of albumin-induced TGF{beta}-1 secretion. These results suggest that Dab2 is a ligand dependent bi-directional regulator of ERK1,2 activity by demonstrating that in addition to its more traditional role as an inhibitor of ERK1,2 it may also activate ERK1,2.« less

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.

Structures of apicomplexan calcium-dependent protein kinases reveal mechanism of activation by calcium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wernimont, Amy K; Artz, Jennifer D.; Jr, Patrick Finerty

2010-09-21

Calcium-dependent protein kinases (CDPKs) have pivotal roles in the calcium-signaling pathway in plants, ciliates and apicomplexan parasites and comprise a calmodulin-dependent kinase (CaMK)-like kinase domain regulated by a calcium-binding domain in the C terminus. To understand this intramolecular mechanism of activation, we solved the structures of the autoinhibited (apo) and activated (calcium-bound) conformations of CDPKs from the apicomplexan parasites Toxoplasma gondii and Cryptosporidium parvum. In the apo form, the C-terminal CDPK activation domain (CAD) resembles a calmodulin protein with an unexpected long helix in the N terminus that inhibits the kinase domain in the same manner as CaMKII. Calcium bindingmore » triggers the reorganization of the CAD into a highly intricate fold, leading to its relocation around the base of the kinase domain to a site remote from the substrate binding site. This large conformational change constitutes a distinct mechanism in calcium signal-transduction pathways.« less

Characterization of Mitogen-Activated Protein Kinase Expression in Nucleus Accumbens and Hippocampus of Rats Subjected to Food Selection in the Cafeteria Diet Protocol.

PubMed

Sarro-Ramírez, Andrea; Sánchez, Daniel; Tejeda-Padrón, Alma; Buenfil-Canto, Linda Vianey; Valladares-García, Jorge; Pacheco-Pantoja, Elda; Arias-Carrión, Oscar; Murillo-Rodríguez, Eric

2016-01-01

Obesity is a world-wide health problem that requires different experimental perspectives to understand the onset of this disease, including the neurobiological basis of food selection. From a molecular perspective, obesity has been related with activity of several endogenous molecules, including the mitogenactivated protein kinases (MAP-K). The aim of this study was to characterize MAP-K expression in hedonic and learning and memory brain-associated areas such as nucleus accumbens (AcbC) and hippocampus (HIPP) after food selection. We show that animals fed with cafeteria diet during 14 days displayed an increase in p38 MAP-K activity in AcbC if chose cheese. Conversely, a diminution was observed in animals that preferred chocolate in AcbC. Also, a decrease of p38 MAP-K phosphorylation was found in HIPP in rats that selected either cheese or chocolate. Our data demonstrate a putative role of MAP-K expression in food selection. These findings advance our understanding of neuromolecular basis engaged in obesity.

An unusual MAP kinase is required for efficient penetration of the plant surface by Ustilago maydis

PubMed Central

Brachmann, Andreas; Schirawski, Jan; Müller, Philip; Kahmann, Regine

2003-01-01

In Ustilago maydis, pathogenic development is controlled by a heterodimer of the two homeodomain proteins bW and bE. We have identified by RNA fingerprinting a b-regulated gene, kpp6, which encodes an unusual MAP kinase. Kpp6 is similar to a number of other fungal MAP kinases involved in mating and pathogenicity, but contains an additional N-terminal domain unrelated to other proteins. Transcription of the kpp6 gene yields two transcripts differing in length, but encoding proteins of identical mass. One transcript is upregulated by the bW/bE heterodimer, while the other is induced after pheromone stimulation. kpp6 deletion mutants are attenuated in pathogenicity. kpp6T355A,Y357F mutants carrying a non-activatable allele of kpp6 are more severely compromised in pathogenesis. These strains can still form appressoria, but are defective in the subsequent penetration of the plant cuticle. Kpp6 is expressed during all stages of the sexual life cycle except mature spores. We speculate that Kpp6 may respond to a plant signal and regulate the genes necessary for efficient penetration of plant tissue. PMID:12727886

LOCALIZATION OF THE MOUSE THYMIDINE KINASE GENE TO THE DISTAL PORTION OF CHROMOSOME 11

EPA Science Inventory

We report the regional mapping of the thymidine kinase (tk-1) gene in the mouse using two complementary analyses: 1) investigation of chromosome aberrations associated with tx-1 gene inactivation in the L5178Y TX+/-3.7.2c cell line and (2) in situ molecular hybridization of a clo...

Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases.

PubMed

Dent, P; Chow, Y H; Wu, J; Morrison, D K; Jove, R; Sturgill, T W

1994-10-01

Mitogen-activated protein (MAP) kinase kinases (MKKs) are dual-specificity protein kinases which activate p42mapk and p44mapk by phosphorylation of regulatory tyrosine and threonine residues. cDNAs for two isotypes of MKK, MKK1 and MKK2, have been isolated from several species. Here we describe construction of recombinant baculoviruses for high-level expression of histidine-tagged rat MKK1 and MKK2, and procedures for production of nearly homogeneous MKK1 and MKK2 fusion proteins, in both inactive and active forms. Co-infection of Sf9 cells with either MKK1 or MKK2 virus together with recombinant viruses for Raf-1, pp60src (Y527F) and c-Ha-Ras resulted in activations of 250-fold and 150-fold for MKK1 and MKK2 respectively. Specific activities towards kinase-defective p42mapk were of the order of several hundred nanomoles of phosphate transferred/min per mg of MKK protein. The Michaelis constants for both enzymes were approx. 1 microM. Preparations of activated MKK were apparently free of Raf-1 as assessed by Western blotting. Raf-1 phosphorylated MKK1 on one major tryptic phosphopeptide, the phosphorylation of which increased with time. This phosphopeptide contained only phosphoserine and possessed neutral overall charge at pH 1.9 on two-dimensional peptide mapping. Phosphorylation of MKK1 by Raf-1 correlated with activation and reached a plateau of approximately 2 mol/mol.

Phosphorylation and mRNA Splicing of Collapsin Response Mediator Protein-2 Determine Inhibition of Rho-associated Protein Kinase (ROCK) II Function in Carcinoma Cell Migration and Invasion*

PubMed Central

Morgan-Fisher, Marie; Couchman, John R.; Yoneda, Atsuko

2013-01-01

The Rho-associated protein kinases (ROCK I and II) are central regulators of important cellular processes such as migration and invasion downstream of the GTP-Rho. Recently, we reported collapsin response mediator protein (CRMP)-2 as an endogenous ROCK II inhibitor. To reveal how the CRMP-2-ROCK II interaction is controlled, we further mapped the ROCK II interaction site of CRMP-2 and examined whether phosphorylation states of CRMP-2 affected the interaction. Here, we show that an N-terminal fragment of the long CRMP-2 splice variant (CRMP-2L) alone binds ROCK II and inhibits colon carcinoma cell migration and invasion. Furthermore, the interaction of CRMP-2 and ROCK II is partially regulated by glycogen synthase kinase (GSK)-3 phosphorylation of CRMP-2, downstream of PI3K. Inhibition of PI3K reduced interaction of CRMP-2 with ROCK II, an effect rescued by simultaneous inhibition of GSK3. Inhibition of PI3K also reduced colocalization of ROCK II and CRMP-2 at the cell periphery in human breast carcinoma cells. Mimicking GSK3 phosphorylation of CRMP-2 significantly reduced CRMP-2 binding of recombinant full-length and catalytic domain of ROCK II. These data implicate GSK3 in the regulation of ROCK II-CRMP-2 interactions. Using phosphorylation-mimetic and -resistant CRMP-2L constructs, it was revealed that phosphorylation of CRMP-2L negatively regulates its inhibitory function in ROCK-dependent haptotactic cell migration, as well as invasion of human colon carcinoma cells. Collectively, the presented data show that CRMP-2-dependent regulation of ROCK II activity is mediated through interaction of the CRMP-2L N terminus with the ROCK II catalytic domain as well as by GSK3-dependent phosphorylation of CRMP-2. PMID:24036111

A chemical-genetic approach for functional analysis of plant protein kinases

PubMed Central

Salomon, Dor; Bonshtien, Arale

2009-01-01

Plant genomes encode hundreds of protein kinases, yet only for a small fraction of them precise functions and phosphorylation targets have been identified. Recently, we applied a chemical-genetic approach to sensitize the tomato serine/threonine kinase Pto to analogs of PP1, an ATP-competitive and cell-permeable small-molecule inhibitor. The Pto kinase confers resistance to Pst bacteria by activating immune responses upon specific recognition of bacterial effectors. By using PP1 analogs in combination with the analog-sensitive Pto, we shed new light on the role of Pto kinase activity in effector recognition and signal transduction. Here we broaden the use of this chemical-genetic approach to another defense-related plant protein kinase, the MAP kinase LeMPK3. In addition, we show that analog-sensitive but not wild-type kinases are able to use unnatural N6-modified ATP analogs as phosphodonors that can be exploited for tagging direct phosphorylation targets of the kinase of interest. Thus, sensitization of kinases to analogs of the small-molecule inhibitor PP1 and ATP can be an effective tool for the discovery of cellular functions and phosphorylation substrates of plant protein kinases. PMID:19820342

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hien, Tran Thi; Kim, Nak Doo; Pokharel, Yuba Raj

2010-08-01

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

Transcription factor REST negatively influences the protein kinase C-dependent up-regulation of human mu-opioid receptor gene transcription.

PubMed

Bedini, Andrea; Baiula, Monica; Carbonari, Gioia; Spampinato, Santi

2010-01-01

Mu-opioid receptor expression increases during neurogenesis, regulates the survival of maturing neurons and is implicated in ischemia-induced neuronal death. The repressor element 1 silencing transcription factor (REST), a regulator of a subset of genes in differentiating and post-mitotic neurons, is involved in its transcriptional repression. Extracellular signaling molecules and mechanisms that control the human mu-opioid receptor (hMOR) gene transcription are not clearly understood. We examined the role of protein kinase C (PKC) on hMOR transcription in a model of neuronal cells and in the context of the potential influence of REST. In native SH-SY5Y neuroblastoma cells, PKC activation with phorbol 12-myristate 13-acetate (PMA, 16 nM, 24h) down-regulated hMOR transcription and concomitantly elevated the REST binding activity to repressor element 1 of the hMOR promoter. In contrast, PMA activated hMOR gene transcription when REST expression was knocked down by an antisense strategy or by retinoic acid-induced cell differentiation. PMA acts through a PKC-dependent pathway requiring downstream MAP kinases and the transcription factor AP-1. In a series of hMOR-luciferase promoter/reporter constructs transfected into SH-SY5Y cells and PC12 cells, PMA up-regulated hMOR transcription in PC12 cells lacking REST, and in SH-SY5Y cells either transfected with constructs deficient in the REST DNA binding element or when REST was down-regulated in retinoic acid-differentiated cells. These findings help explain how hMOR transcription is regulated and may clarify its contribution to epigenetic modifications and reprogramming of differentiated neuronal cells exposed to PKC-activating agents. Copyright 2009 Elsevier Ltd. All rights reserved.

The disintegrin, trimucrin, suppresses LPS-induced activation of phagocytes primarily through blockade of NF-κB and MAPK activation.

PubMed

Hung, Yu-Chun; Hsu, Chun-Chieh; Chung, Ching-Hu; Huang, Tur-Fu

2016-07-01

In addition to antiplatelet activity, disintegrin, a small-mass RGD-containing polypeptide, has been shown to exert anti-inflammatory effects but the mechanism involved remains unclear. In this study, we report that trimucrin, a disintegrin from the venom of Trimeresurus mucrosquamatus, inhibits lipopolysaccharide (LPS)-induced stimulation of THP-1 and RAW 264.7 cells. We also investigate the underlying mechanism. Trimucrin decreased the release of proinflammatory cytokines including tumor necrosis factor α (TNFα), interleukin-6 (IL-6), nitric oxide, and reactive oxygen species (ROS), and inhibited the adhesion and migration of LPS-activated phagocytes. Trimucrin significantly blocked the expression of nuclear factor kappaB (NF-κB)-related downstream inducible enzymes such as inducible nitric oxide synthase (iNOS) and COX-2. In addition, its anti-inflammatory effect was associated with the decreased mitogen-activated protein kinase (MAPK) phosphorylation. Furthermore, trimucrin concentration dependently inhibited LPS-induced phosphorylation of focal adhesion kinase (FAK), PI3K, and Akt. Trimucrin also reversed the DNA-binding activity of NF-κB by suppressing the LPS-induced nuclear translocation of p65 and the cytosolic IκB release. Flow cytometric analyses showed that trimucrin bound to cells in a concentration-dependent manner. The anti-αVβ3 mAb also specifically decreased the binding of fluorescein isothiocyanate (FITC)-conjugated trimucrin. Binding assays demonstrated that integrin αVβ3 was the binding site for trimucrin on THP-1 and RAW 264.7 cells. In conclusion, we showed that trimucrin decreases the inflammatory reaction through the attenuation of iNOS expression and nitric oxide (NO) production by blocking MAP kinase and the NF-κB activation in LPS-stimulated THP-1 and RAW 264.7 cells.

STK33 kinase inhibitor BRD-8899 has no effect on KRAS-dependent cancer cell viability.

PubMed

Luo, Tuoping; Masson, Kristina; Jaffe, Jacob D; Silkworth, Whitney; Ross, Nathan T; Scherer, Christina A; Scholl, Claudia; Fröhling, Stefan; Carr, Steven A; Stern, Andrew M; Schreiber, Stuart L; Golub, Todd R

2012-02-21

Approximately 30% of human cancers harbor oncogenic gain-of-function mutations in KRAS. Despite interest in KRAS as a therapeutic target, direct blockade of KRAS function with small molecules has yet to be demonstrated. Based on experiments that lower mRNA levels of protein kinases, KRAS-dependent cancer cells were proposed to have a unique requirement for the serine/threonine kinase STK33. Thus, it was suggested that small-molecule inhibitors of STK33 might have therapeutic benefit in these cancers. Here, we describe the development of selective, low nanomolar inhibitors of STK33's kinase activity. The most potent and selective of these, BRD8899, failed to kill KRAS-dependent cells. While several explanations for this result exist, our data are most consistent with the view that inhibition of STK33's kinase activity does not represent a promising anti-KRAS therapeutic strategy.

STK33 kinase inhibitor BRD-8899 has no effect on KRAS-dependent cancer cell viability

PubMed Central

Luo, Tuoping; Masson, Kristina; Jaffe, Jacob D.; Silkworth, Whitney; Ross, Nathan T.; Scherer, Christina A.; Scholl, Claudia; Fröhling, Stefan; Carr, Steven A.; Stern, Andrew M.; Schreiber, Stuart L.; Golub, Todd R.

2012-01-01

Approximately 30% of human cancers harbor oncogenic gain-of-function mutations in KRAS. Despite interest in KRAS as a therapeutic target, direct blockade of KRAS function with small molecules has yet to be demonstrated. Based on experiments that lower mRNA levels of protein kinases, KRAS-dependent cancer cells were proposed to have a unique requirement for the serine/threonine kinase STK33. Thus, it was suggested that small-molecule inhibitors of STK33 might have therapeutic benefit in these cancers. Here, we describe the development of selective, low nanomolar inhibitors of STK33’s kinase activity. The most potent and selective of these, BRD8899, failed to kill KRAS-dependent cells. While several explanations for this result exist, our data are most consistent with the view that inhibition of STK33’s kinase activity does not represent a promising anti-KRAS therapeutic strategy. PMID:22323609

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

NASA Technical Reports Server (NTRS)

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

1998-01-01

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

Co-amplification of phosphoinositide 3-kinase enhancer A and cyclin-dependent kinase 4 triggers glioblastoma progression | Office of Cancer Genomics

Cancer.gov

Glioblastoma (GBM) is the most common primary brain tumor and has a dismal prognosis. Amplification of chromosome 12q13-q15 (Cyclin-dependent kinase 4 (CDK4) amplicon) is frequently observed in numerous human cancers including GBM. Phosphoinositide 3-kinase enhancer (PIKE) is a group of GTP-binding proteins that belong to the subgroup of centaurin GTPase family, encoded by CENTG1 located in CDK4 amplicon. However, the pathological significance of CDK4 amplicon in GBM formation remains incompletely understood.

Partial purification and characterization of a Ca(2+)-dependent protein kinase from pea nuclei

NASA Technical Reports Server (NTRS)

Li, H.; Dauwalder, M.; Roux, S. J.

1991-01-01

Almost all the Ca(2+)-dependent protein kinase activity in nuclei purified from etiolated pea (Pisum sativum, L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.3 molar NaCl. This protein kinase can be further purified 80,000-fold by salt fractionation and high performance liquid chromatography, after which it has a high specific activity of about 100 picomoles per minute per microgram in the presence of Ca2+ and reaches half-maximal activation at about 3 x 10(-7) molar free Ca2+, without calmodulin. It is a monomer with a molecular weight near 90,000. It can efficiently use histone III-S, ribosomal S6 protein, and casein as artificial substrates, but it phosphorylates phosvitin only weakly. Its Ca(2+)-dependent kinase activity is half-maximally inhibited by 0.1 millimolar chlorpromazine, by 35 nanomolar K-252a and by 7 nanomolar staurosporine. It is insensitive to sphingosine, an inhibitor of protein kinase C, and to basic polypeptides that block other Ca(2+)-dependent protein kinases. It is not stimulated by exogenous phospholipids or fatty acids. In intact isolated pea nuclei it preferentially phosphorylates several chromatin-associated proteins, with the most phosphorylated protein band being near the same molecular weight (43,000) as a nuclear protein substrate whose phosphorylation has been reported to be stimulated by phytochrome in a calcium-dependent fashion.

SPLICE VARIANT SPECIFIC UPREGULATIONOF CA+2/CALMODULIN DEPENDENT PROTEIN KINASE 1G BY PYRETHROID INSECTICIDES IN VIVO.

EPA Science Inventory

Pyrethroid insecticides induce neurotoxicity in mammals by interfering with ion channel function in excitable neuronal membranes. Previous work demonstrated dose-dependent increases in expression of Ca+2/calmodulin dependent protein kinase (Camk1g) mRNA following acute deltameth...

Ensemble-based modeling and rigidity decomposition of allosteric interaction networks and communication pathways in cyclin-dependent kinases: Differentiating kinase clients of the Hsp90-Cdc37 chaperone

PubMed Central

Stetz, Gabrielle; Tse, Amanda

2017-01-01

The overarching goal of delineating molecular principles underlying differentiation of protein kinase clients and chaperone-based modulation of kinase activity is fundamental to understanding activity of many oncogenic kinases that require chaperoning of Hsp70 and Hsp90 systems to attain a functionally competent active form. Despite structural similarities and common activation mechanisms shared by cyclin-dependent kinase (CDK) proteins, members of this family can exhibit vastly different chaperone preferences. The molecular determinants underlying chaperone dependencies of protein kinases are not fully understood as structurally similar kinases may often elicit distinct regulatory responses to the chaperone. The regulatory divergences observed for members of CDK family are of particular interest as functional diversification among these kinases may be related to variations in chaperone dependencies and can be exploited in drug discovery of personalized therapeutic agents. In this work, we report the results of a computational investigation of several members of CDK family (CDK5, CDK6, CDK9) that represented a broad repertoire of chaperone dependencies—from nonclient CDK5, to weak client CDK6, and strong client CDK9. By using molecular simulations of multiple crystal structures we characterized conformational ensembles and collective dynamics of CDK proteins. We found that the elevated dynamics of CDK9 can trigger imbalances in cooperative collective motions and reduce stability of the active fold, thus creating a cascade of favorable conditions for chaperone intervention. The ensemble-based modeling of residue interaction networks and community analysis determined how differences in modularity of allosteric networks and topography of communication pathways can be linked with the client status of CDK proteins. This analysis unveiled depleted modularity of the allosteric network in CDK9 that alters distribution of communication pathways and leads to impaired signaling in the client kinase. According to our results, these network features may uniquely define chaperone dependencies of CDK clients. The perturbation response scanning and rigidity decomposition approaches identified regulatory hotspots that mediate differences in stability and cooperativity of allosteric interaction networks in the CDK structures. By combining these synergistic approaches, our study revealed dynamic and network signatures that can differentiate kinase clients and rationalize subtle divergences in the activation mechanisms of CDK family members. The therapeutic implications of these results are illustrated by identifying structural hotspots of pathogenic mutations that preferentially target regions of the increased flexibility to enable modulation of activation changes. Our study offers a network-based perspective on dynamic kinase mechanisms and drug design by unravelling relationships between protein kinase dynamics, allosteric communications and chaperone dependencies. PMID:29095844

Artificial-epitope mapping for CK-MB assay.

PubMed

Tai, Dar-Fu; Ho, Yi-Fang; Wu, Cheng-Hsin; Lin, Tzu-Chieh; Lu, Kuo-Hao; Lin, Kun-Shian

2011-06-07

A quantitative method using artificial antibody to detect creatine kinases was developed. Linear epitope sequences were selected based on an artificial-epitope mapping strategy. Nine different MIPs corresponding to the selected peptides were then fabricated on QCM chips. The subtle conformational changes were also recognized by these chips.

CR108, a novel vitamin K3 derivative induces apoptosis and breast tumor inhibition by reactive oxygen species and mitochondrial dysfunction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Chun-Ru; Liao, Wei-Siang; Wu, Ya-Hui

Vitamin K3 derivatives have been shown to exert anticancer activities. Here we show a novel vitamin K3 derivative (S)-2-(2-hydroxy-3-methylbutylthio)naphthalene-1,4-dione, which is named as CR108 that induces apoptosis and tumor inhibition through reactive oxygen species (ROS) and mitochondrial dysfunction in human breast cancer. CR108 is more effective on the breast cancer cell death than other vitamin K3 derivatives. Moreover, CR108 induced apoptosis in both the non-HER-2-overexpressed MCF-7 and HER-2-overexpressed BT-474 breast cancer cells. CR108 caused the loss of mitochondrial membrane potential, cytochrome c released from mitochondria to cytosol, and cleaved PARP proteins for apoptosis induction. CR108 markedly increased ROS levels inmore » breast cancer cells. N-acetylcysteine (NAC), a general ROS scavenger, completely blocked the CR108-induced ROS levels, mitochondrial dysfunction and apoptosis. Interestingly, CR108 increased the phosphorylation of p38 MAP kinase but conversely inhibited the survivin protein expression. NAC treatment prevented the activation of p38 MAP kinase and rescued the survivin protein levels. SB202190, a specific p38 MAP kinase inhibitor, recovered the survivin protein levels and attenuated the cytotoxicity of CR108-treated cells. Furthermore, CR108 inhibited the xenografted human breast tumor growth in nude mice. Together, we demonstrate that CR108 is a novel vitamin K3 derivative that induces apoptosis and tumor inhibition by ROS production and mitochondrial dysfunction and associates with the phosphorylation of p38 MAP kinase and the inhibition of survivin in the human breast cancer. - Highlights: • CR108 is more effective on the cell death than other vitamin K3 derivatives. • CR108 induces apoptosis and tumor inhibition by ROS and mitochondrial dysfunction. • CR108 induces apoptosis by p38 kinase activation and survivin inhibition. • CR108 is a potent vitamin K3 analog that can develop for breast cancer therapy.« less

The Structure of Arabidopsis thaliana OST1 Provides Insights into the Kinase Regulation Mechanism in Response to Osmotic Stress

PubMed Central

Yunta, Cristina; Martínez-Ripoll, Martín; Zhu, Jian-Kang; Albert, Armando

2013-01-01

SnRK [SNF1 (sucrose non-fermenting-1)-related protein kinase] 2.6 [open stomata 1 (OST1)] is well characterized at molecular and physiological levels to control stomata closure in response to water-deficit stress. OST1 is a member of a family of 10 protein kinases from Arabidopsis thaliana (SnRK2) that integrates abscisic acid (ABA)-dependent and ABA-independent signals to coordinate the cell response to osmotic stress. A subgroup of protein phosphatases type 2C binds OST1 and keeps the kinase dephosphorylated and inactive. Activation of OST1 relies on the ABA-dependent inhibition of the protein phosphatases type 2C and the subsequent self-phosphorylation of the kinase. The OST1 ABA-independent activation depends on a short sequence motif that is conserved among all the members of the SnRK2 family. However, little is known about the molecular mechanism underlying this regulation. The crystallographic structure of OST1 shows that ABA-independent regulation motif stabilizes the conformation of the kinase catalytically essential α C helix, and it provides the basis of the ABA-independent regulation mechanism for the SnRK2 family of protein kinases. PMID:21983340

Cancer Osaka thyroid (Cot) phosphorylates Polo-like kinase (PLK1) at Ser137 but not at Thr210.

PubMed

Wu, Binhui; Jiang, Ping; Mu, Yuguang; Wilmouth, Rupert C

2009-12-01

Cancer Osaka thyroid (Cot) is a proto-oncogenic kinase which belongs to the MAP3K family. A peptide-based substrate screening assay revealed that Cot has the ability to phosphorylate Polo-like kinase 1 (Plk1) at Ser137. Kinase assays with intact Plk1 and peptides surrounding Ser137 and Thr210 indicated further that Cot phosphorylates Ser137 but not Thr210. Additional support came from 3D peptide structure prediction and Cot-Plk1 interaction modeling. In vivo experiments demonstrated that wild type Cot, but not a kinase-dead mutant, has the ability to phosphorylate Ser137. Knockdown of Cot in Hela showed a reduction in the level of phosphorylation of Ser137. These results imply for the first time that Cot might be an upstream kinase of Plk1 and suggest a new mechanism for the regulation of the cellular function of Plk1.

Inhibition of Curcumin on ZAKα Activity Resultant in Apoptosis and Anchorage-Independent Growth in Cancer Cells.

PubMed

Lee, Jin-Sun; Wang, Tsu-Shing; Lin, Ming Cheng; Lin, Wei-Wen; Yang, Jaw-Ji

2017-10-31

Curcumin, a popular yellow pigment of the dietary spice turmeric, has been reported to inhibit cell growth and to induce apoptosis in a wide variety of cancer cells. Although numerous studies have investigated anticancer effects of curcumin, the precise molecular mechanism of action remains unidentified. Whereas curcumin mediates cell survival and apoptosis through mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) signaling cascades, its impact on the upstream regulation of MAPK is unclear. The leucine-zipper and sterile-α motif kinase alpha (ZAKα), a mitogen-activated protein kinase kinase kinase (MAP3K), activates the c-Jun N-terminal kinase (JNK) and NF-κB pathway. This paper investigated the prospective involvement of ZAKα in curcumin-induced effects on cancer cells. Our results suggest that the antitumor activity of curcumin is mediated via a mechanism involving inhibition of ZAKα activity.

Dual Leucine Zipper Kinase Inhibitors for the Treatment of Neurodegeneration.

PubMed

Siu, Michael; Sengupta Ghosh, Arundhati; Lewcock, Joseph W

2018-06-04

Dual leucine zipper kinase (DLK, MAP3K12) is an essential driver of the neuronal stress response that regulates neurodegeneration in models of acute neuronal injury and chronic neurodegenerative diseases such as Alzheimer's, Parkinson's, and ALS. In this review, we provide an overview of DLK signaling mechanisms and describe selected small molecules that have been utilized to inhibit DLK kinase activity in vivo. These compounds represent valuable tools for understanding the role of DLK signaling and evaluating the potential for DLK inhibition as a therapeutic strategy to prevent neuronal degeneration.

Co-factors Required for TLR7- and TLR9- dependent Innate Immune Responses

PubMed Central

Chiang, Chih-yuan; Engel, Alex; Opaluch, Amanda M.; Ramos, Irene; Maestre, Ana M.; Secundino, Ismael; De Jesus, Paul D.; Nguyen, Quy T.; Welch, Genevieve; Bonamy, Ghislain M.C.; Miraglia, Loren J.; Orth, Anthony P.; Nizet, Victor; Fernandez-Sesma, Ana; Zhou, Yingyao; Barton, Gregory M.; Chanda, Sumit K.

2012-01-01

SUMMARY Pathogens commonly utilize endocytic pathways to gain cellular access. The endosomal pattern recognition receptors TLR7 and TLR9 detect pathogen-encoded nucleic acids to initiate MyD88-dependent pro-inflammatory responses to microbial infection. Using genome-wide RNAi screening and integrative systems-based analysis we identify 190 co-factors required for TLR7- and TLR9-directed signaling responses. A set of co-factors were cross-profiled for their activities downstream of several immunoreceptors, and then functionally mapped based on the known architecture of NF-κB signaling pathways. Protein complexes and pathways involved in ubiquitin-protein ligase activities, sphingolipid metabolism, chromatin modifications, and ancient stress responses were found to modulate innate recognition of endosomal nucleic acids. Additionally, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was characterized as necessary for ubiquitin-dependent TLR9 targeting to the endolysosome. Proteins and pathways identified here should prove useful in delineating strategies to manipulate innate responses for treatment of autoimmune disorders and microbial infection. PMID:22423970

Mitogen activated protein kinase 6 and MAP kinase phosphatase 1 are involved in the response of Arabidopsis roots to L-glutamate.

PubMed

López-Bucio, Jesús Salvador; Raya-González, Javier; Ravelo-Ortega, Gustavo; Ruiz-Herrera, León Francisco; Ramos-Vega, Maricela; León, Patricia; López-Bucio, José; Guevara-García, Ángel Arturo

2018-03-01

The function and components of L-glutamate signaling pathways in plants have just begun to be elucidated. Here, using a combination of genetic and biochemical strategies, we demonstrated that a MAPK module is involved in the control of root developmental responses to this amino acid. Root system architecture plays an essential role in plant adaptation to biotic and abiotic factors via adjusting signal transduction and gene expression. L-Glutamate (L-Glu), an amino acid with neurotransmitter functions in animals, inhibits root growth, but the underlying genetic mechanisms are poorly understood. Through a combination of genetic analysis, in-gel kinase assays, detailed cell elongation and division measurements and confocal analysis of expression of auxin, quiescent center and stem cell niche related genes, the critical roles of L-Glu in primary root growth acting through the mitogen-activated protein kinase 6 (MPK6) and the dual specificity serine-threonine-tyrosine phosphatase MKP1 could be revealed. In-gel phosphorylation assays revealed a rapid and dose-dependent induction of MPK6 and MPK3 activities in wild-type Arabidopsis seedlings in response to L-Glu. Mutations in MPK6 or MKP1 reduced or increased root cell division and elongation in response to L-Glu, possibly modulating auxin transport and/or response, but in a PLETHORA1 and 2 independent manner. Our data highlight MPK6 and MKP1 as components of an L-Glu pathway linking the auxin response, and cell division for primary root growth.

Simplified molecular input line entry system-based: QSAR modelling for MAP kinase-interacting protein kinase (MNK1).

PubMed

Begum, S; Achary, P Ganga Raju

2015-01-01

Quantitative structure-activity relationship (QSAR) models were built for the prediction of inhibition (pIC50, i.e. negative logarithm of the 50% effective concentration) of MAP kinase-interacting protein kinase (MNK1) by 43 potent inhibitors. The pIC50 values were modelled with five random splits, with the representations of the molecular structures by simplified molecular input line entry system (SMILES). QSAR model building was performed by the Monte Carlo optimisation using three methods: classic scheme; balance of correlations; and balance correlation with ideal slopes. The robustness of these models were checked by parameters as rm(2), r(*)m(2), [Formula: see text] and randomisation technique. The best QSAR model based on single optimal descriptors was applied to study in vitro structure-activity relationships of 6-(4-(2-(piperidin-1-yl) ethoxy) phenyl)-3-(pyridin-4-yl) pyrazolo [1,5-a] pyrimidine derivatives as a screening tool for the development of novel potent MNK1 inhibitors. The effects of alkyl group, -OH, -NO2, F, Cl, Br, I, etc. on the IC50 values towards the inhibition of MNK1 were also reported.

Comparative studies of a new subfamily of human Ste20-like kinases: homodimerization, subcellular localization, and selective activation of MKK3 and p38.

PubMed

Yustein, Jason T; Xia, Liang; Kahlenburg, J Michelle; Robinson, Dan; Templeton, Dennis; Kung, Hsing-Jien

2003-09-18

The Sterile-20 or Ste20 family of serine/threonine kinases is a group of signaling molecules whose physiological roles within mammalian cells are just starting to be elucidated. Here, in this report we present the characterization of three human Ste20-like kinases with greater than 90% similarity within their catalytic domains that define a novel subfamily of Ste20s. Members of this kinase family include rat thousand and one (TAO1) and chicken KFC (kinase from chicken). For the lack of a consensus nomenclature in the literature, in this report, we shall call this family hKFC (for their homology to chicken KFC) and the three members hKFC-A, hKFC-B, and hKFC-C, respectively. These kinases have many similarities including an aminoterminal kinase domain, a serine-rich region, and a coiled-coil configuration within the C-terminus. All three kinases are able to activate the p38 MAP kinase pathway through the specific activation of the upstream MKK3 kinase. We also offer evidence, both theoretical and biochemical, showing that these kinases can undergo self-association. Despite these similarities, these kinases differ in tissue distribution, apparent subcellular localization, and feature structural differences largely within the carboxyl-terminal sequence.

Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response.

PubMed

Chan, Tung O; Zhang, Jin; Tiegs, Brian C; Blumhof, Brian; Yan, Linda; Keny, Nikhil; Penny, Morgan; Li, Xue; Pascal, John M; Armen, Roger S; Rodeck, Ulrich; Penn, Raymond B

2015-10-01

The Akt protein kinase, also known as protein kinase B, plays key roles in insulin receptor signalling and regulates cell growth, survival and metabolism. Recently, we described a mechanism to enhance Akt phosphorylation that restricts access of cellular phosphatases to the Akt activation loop (Thr(308) in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr(308) dephosphorylation and thus Akt deactivation that depends on intramolecular interactions of Akt C-terminal sequences with its kinase domain. Modifications of amino acids surrounding the Akt1 C-terminal mTORC2 (mammalian target of rapamycin complex 2) phosphorylation site (Ser(473)) increased phosphatase resistance of the phosphorylated activation loop (pThr(308)) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr(308) phosphorylation in a regulated fashion. Collectively, these results suggest that the Akt C-terminal hydrophobic groove is a target for the development of agents that enhance Akt phosphorylation by insulin. © 2015 Authors; published by Portland Press Limited.

Akt kinase C-terminal modifications control activation loop dephosphorylation and enhance insulin response

PubMed Central

Chan, Tung O.; Zhang, Jin; Tiegs, Brian C.; Blumhof, Brian; Yan, Linda; Keny, Nikhil; Penny, Morgan; Li, Xue; Pascal, John M.; Armen, Roger S.; Rodeck, Ulrich; Penn, Raymond B.

2015-01-01

The Akt protein kinase, also known as protein kinase B, plays key roles in insulin receptor signalling and regulates cell growth, survival and metabolism. Recently, we described a mechanism to enhance Akt phosphorylation that restricts access of cellular phosphatases to the Akt activation loop (Thr308 in Akt1 or protein kinase B isoform alpha) in an ATP-dependent manner. In the present paper, we describe a distinct mechanism to control Thr308 dephosphorylation and thus Akt deactivation that depends on intramolecular interactions of Akt C-terminal sequences with its kinase domain. Modifications of amino acids surrounding the Akt1 C-terminal mTORC2 (mammalian target of rapamycin complex 2) phosphorylation site (Ser473) increased phosphatase resistance of the phosphorylated activation loop (pThr308) and amplified Akt phosphorylation. Furthermore, the phosphatase-resistant Akt was refractory to ceramide-dependent dephosphorylation and amplified insulin-dependent Thr308 phosphorylation in a regulated fashion. Collectively, these results suggest that the Akt C-terminal hydrophobic groove is a target for the development of agents that enhance Akt phosphorylation by insulin. PMID:26201515

Salicylates inhibit flavivirus replication independently of blocking nuclear factor kappa B activation.

PubMed

Liao, C L; Lin, Y L; Wu, B C; Tsao, C H; Wang, M C; Liu, C I; Huang, Y L; Chen, J H; Wang, J P; Chen, L K

2001-09-01

Flaviviruses comprise a positive-sense RNA genome that replicates exclusively in the cytoplasm of infected cells. Whether flaviviruses require an activated nuclear factor(s) to complete their life cycle and trigger apoptosis in infected cells remains elusive. Flavivirus infections quickly activate nuclear factor kappa B (NF-kappaB), and salicylates have been shown to inhibit NF-kappaB activation. In this study, we investigated whether salicylates suppress flavivirus replication and virus-induced apoptosis in cultured cells. In a dose-dependent inhibition, we found salicylates within a range of 1 to 5 mM not only restricted flavivirus replication but also abrogated flavivirus-triggered apoptosis. However, flavivirus replication was not affected by a specific NF-kappaB peptide inhibitor, SN50, and a proteosome inhibitor, lactacystin. Flaviviruses also replicated and triggered apoptosis in cells stably expressing IkappaBalpha-DeltaN, a dominant-negative mutant that antagonizes NF-kappaB activation, as readily as in wild-type BHK-21 cells, suggesting that NF-kappaB activation is not essential for either flavivirus replication or flavivirus-induced apoptosis. Salicylates still diminished flavivirus replication and blocked apoptosis in the same IkappaBalpha-DeltaN cells. This inhibition of flaviviruses by salicylates could be partially reversed by a specific p38 mitogen-activated protein (MAP) kinase inhibitor, SB203580. Together, these results show that the mechanism by which salicylates suppress flavivirus infection may involve p38 MAP kinase activity but is independent of blocking the NF-kappaB pathway.

Biosurfactant mannosyl-erythritol lipid inhibits secretion of inflammatory mediators from RBL-2H3 cells.

PubMed

Morita, Yosuke; Tadokoro, Satoshi; Sasai, Masao; Kitamoto, Dai; Hirashima, Naohide

2011-12-01

Biosurfactant mannosyl-erythritol lipids (MELs) are glycolipids produced by microbes that have various biological activities. It has been reported that MELs exhibit excellent surface-activity and also various bioactivities, such as induction of cell differentiation and apoptosis. However, little is known about their action related to drug discovery or drug seeds. We investigated the effects of MELs on the secretion of inflammatory mediators from mast cells that play a central role in allergic responses. Mast cells secrete three kinds of inflammatory mediators and we quantified these secreted mediators by photometer or ELISA. The action mechanisms of MELs were studied by Ca(2+)-sensitive fluorescence dye and Western blotting of phosphorylated proteins. MELs inhibited exocytotic release by antigen stimulation in a dose-dependent manner. We also found that MELs inhibited antigen-induced secretion of leukotriene C(4) and cytokine TNF-α (tumor necrosis factor-α). The inhibitory action of MELs on mediator secretion was mediated by inhibition of Ca(2+) increase, phosphorylation of MAP kinases and SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) that serve as a molecular machinery for exocytotic membrane fusion. MELs have anti-inflammatory action inhibiting the secretion of inflammatory mediators from mast cells. MELs affects two of major intracellular signaling pathways including Ca(2+) increase and MAP kinases. MELs also inhibited the phosphorylation of SNARE proteins that is crucial for not only exocytosis but also intracellular vesicular trafficking. 2011 Elsevier B.V. All rights reserved.

Doxycycline Suppresses Microglial Activation by Inhibiting the p38 MAPK and NF-kB Signaling Pathways.

PubMed

Santa-Cecília, Flávia V; Socias, Benjamin; Ouidja, Mohand O; Sepulveda-Diaz, Julia E; Acuña, Leonardo; Silva, Rangel L; Michel, Patrick P; Del-Bel, Elaine; Cunha, Thiago M; Raisman-Vozari, Rita

2016-05-01

In neurodegenerative diseases, the inflammatory response is mediated by activated glial cells, mainly microglia, which are the resident immune cells of the central nervous system. Activated microglial cells release proinflammatory mediators and neurotoxic factors that are suspected to cause or exacerbate these diseases. We recently demonstrated that doxycycline protects substantia nigra dopaminergic neurons in an animal model of Parkinson's disease. This effect was associated with a reduction of microglial cell activation, which suggests that doxycycline may operate primarily as an anti-inflammatory drug. In the present study, we assessed the anti-inflammatory potential of doxycycline using lipopolysaccharide (LPS)-activated primary microglial cells in culture as a model of neuroinflammation. Doxycycline attenuated the expression of key activation markers in LPS-treated microglial cultures in a concentration-dependent manner. More specifically, doxycycline treatment lowered the expression of the microglial activation marker IBA-1 as well as the production of ROS, NO, and proinflammatory cytokines (TNF-α and IL-1β). In primary microglial cells, we also found that doxycycline inhibits LPS-induced p38 MAP kinase phosphorylation and NF-kB nuclear translocation. The present results indicate that the effect of doxycycline on LPS-induced microglial activation probably occurs via the modulation of p38 MAP kinase and NF-kB signaling pathways. These results support the idea that doxycycline may be useful in preventing or slowing the progression of PD and other neurodegenerative diseases that exhibit altered glia function.

Intracellular TLR22 acts as an inflammation equalizer via suppression of NF-κB and selective activation of MAPK pathway in fish.

PubMed

Ding, Xu; Liang, Yaosi; Peng, Wan; Li, Ruozhu; Lin, Haoran; Zhang, Yong; Lu, Danqi

2018-01-01

TLR22, a typical member of the fish-specific TLRs, is a crucial sensor in virally triggered innate immune signalling retained from natural selection. To elucidate the role of the TLR22-specific signalling cascade mechanism, we provide evidence that the double-stranded (ds) RNA-sensor TLR22 positively regulates the ERK pathway and negatively regulates the JNK, p38 MAP kinase and NF-κB pathway. Here, we show that TLR22 restrains NF-κB activation and IFN (interferon) β and AP-1 (activator protein-1) promoter binding (impairing "primary response" genes (TNF and IL-1)), induces "secondary response" genes (IL-12 and IL-6) and mediates the irregular expression of inflammatory genes. Therefore, TLR22 promotes ERK phosphorylation but impairs the JNK and p38 MAP kinases and IκB phosphorylation. Additionally, TLR22 controls the excessive generation of reactive oxygen species (ROS) to avoid damaging the organism. The specific kinetics of TLR22 depends on its distinct cellular localization. We demonstrate that TLR22 is an intracellular receptor localized in the endosome, and the TLR22-TIR domain is the functional structure inducing the signalling cascade post-viral replication in the body. As mentioned above, our data reveal a novel mechanism whereby TLR22-induced positive adjustment and negative regulation evolved independently to avoid harmful and inappropriate inflammatory responses. Copyright © 2017. Published by Elsevier Ltd.

Genistein inhibits voltage-gated sodium currents in SCG neurons through protein tyrosine kinase-dependent and kinase-independent mechanisms.

PubMed

Jia, Zhanfeng; Jia, Yueqin; Liu, Boyi; Zhao, Zhiying; Jia, Qingzhong; Liang, Huiling; Zhang, Hailin

2008-08-01

Voltage-gated sodium channels play a crucial role in the initiation and propagation of neuronal action potentials. Genistein, an isoflavone phytoestrogen, has long been used as a broad-spectrum inhibitor of protein tyrosine kinases (PTK). In addition, genistein-induced modulation of ion channels has been described previously in the literature. In this study, we investigated the effect of genistein on voltage-gated sodium channels in rat superior cervical ganglia (SCG) neurons. The results show that genistein inhibits Na(+) currents in a concentration-dependent manner, with a concentration of half-maximal effect (IC(50)) at 9.1 +/- 0.9 microM. Genistein positively shifted the voltage dependence of activation but did not affect inactivation of the Na(+) current. The inactive genistein analog daidzein also inhibited Na(+) currents, but was less effective than genistein. The IC(50) for daidzein-induced inhibition was 20.7 +/- 0.1 microM. Vanadate, an inhibitor of protein tyrosine phosphatases, partially but significantly reversed genistein-induced inhibition of Na(+) currents. Other protein tyrosine kinase antagonists such as tyrphostin 23, an erbstatin analog, and PP2 all had small but significant inhibitory effects on Na(+) currents. Among all active and inactive tyrosine kinase inhibitors tested, genistein was the most potent inhibitor of Na(+) currents. These results suggest that genistein inhibits Na(+) currents in rat SCG neurons through two distinct mechanisms: protein tyrosine kinase-independent, and protein tyrosine kinase-dependent mechanisms. Furthermore, the Src kinase family may be involved in the basal phosphorylation of the Na(+) channel.

Molecular docking performance evaluated on the D3R Grand Challenge 2015 drug-like ligand datasets

NASA Astrophysics Data System (ADS)

Selwa, Edithe; Martiny, Virginie Y.; Iorga, Bogdan I.

2016-09-01

The D3R Grand Challenge 2015 was focused on two protein targets: Heat Shock Protein 90 (HSP90) and Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4). We used a protocol involving a preliminary analysis of the available data in PDB and PubChem BioAssay, and then a docking/scoring step using more computationally demanding parameters that were required to provide more reliable predictions. We could evidence that different docking software and scoring functions can behave differently on individual ligand datasets, and that the flexibility of specific binding site residues is a crucial element to provide good predictions.

DNA Protecting Activities of Nymphaea nouchali (Burm. f) Flower Extract Attenuate t-BHP-Induced Oxidative Stress Cell Death through Nrf2-Mediated Induction of Heme Oxygenase-1 Expression by Activating MAP-Kinases

PubMed Central

Ju, Mi-Kyoung

2017-01-01

This study was performed to investigate the antioxidant activities of Nymphaea nouchali flower (NNF) extract and the underlying mechanism using RAW 264.7 cells. The presence of gallic acid, catechin, epicatechin, epigallocatechin, epicatechin gallate, caffeic acid, quercetin, and apigenin in the NNF was confirmed by high-performance liquid chromatography (HPLC). The extract had a very potent capacity to scavenge numerous free radicals. NNF extract was also able to prevent DNA damage and quench cellular reactive oxygen species (ROS) generation induced by tert-Butyl hydroperoxide (t-BHP) with no signs of toxicity. The NNF extract was able to augment the expression of both primary and phase II detoxifying enzyme, resulting in combat the oxidative stress. This is accomplished by phosphorylation of mitogen-activated protein kinase (MAP kinase) (p38 kinase and extracellular signal-regulated kinase (ERK)) followed by enhancing the nuclear translocation of the nuclear factor erythroid 2-related factor 2 (Nrf2). This attenuates cellular ROS generation and confers protection from cell death. Altogether, the results of current study revealed that Nymphaea nouchali flower could be a source of natural phytochemicals that could lead to the development of new therapeutic agents for preventing oxidative stress associated diseases and attenuating disease progression. PMID:28956831

Role of Fatty Acid Kinase in Cellular Lipid Homeostasis and SaeRS-Dependent Virulence Factor Expression in Staphylococcus aureus.

PubMed

Ericson, Megan E; Subramanian, Chitra; Frank, Matthew W; Rock, Charles O

2017-08-01

The SaeRS two-component system is a master activator of virulence factor transcription in Staphylococcus aureus , but the cellular factors that control its activity are unknown. Fatty acid (FA) kinase is a two-component enzyme system required for extracellular FA uptake and SaeRS activity. Here, we demonstrate the existence of an intracellular nonesterified FA pool in S. aureus that is elevated in strains lacking FA kinase activity. SaeRS-mediated transcription is restored in FA kinase-negative strains when the intracellular FA pool is reduced either by growth with FA-depleted bovine serum albumin to extract the FA into the medium or by the heterologous expression of Neisseria gonorrhoeae acyl-acyl carrier protein synthetase to activate FA for phospholipid synthesis. These data show that FAs act as negative regulators of SaeRS signaling, and FA kinase activates SaeRS-dependent virulence factor production by lowering inhibitory FA levels. Thus, FA kinase plays a role in cellular lipid homeostasis by activating FA for incorporation into phospholipid, and it indirectly regulates SaeRS signaling by maintaining a low intracellular FA pool. IMPORTANCE The SaeRS two-component system is a master transcriptional activator of virulence factor production in response to the host environment in S. aureus , and strains lacking FA kinase have severely attenuated SaeRS-dependent virulence factor transcription. FA kinase is required for the activation of exogenous FAs, and it plays a role in cellular lipid homeostasis by recycling cellular FAs into the phospholipid biosynthetic pathway. Activation of the sensor kinase, SaeS, is mediated by its membrane anchor domain, and the FAs which accumulate in FA kinase knockout strains are potent inhibitors of SaeS-dependent signaling. This work identifies FAs as physiological effectors for the SaeRS system and reveals a connection between cellular lipid homeostasis and the regulation of virulence factor transcription. FA kinase is widely distributed in Gram-positive bacteria, suggesting similar roles for FA kinase in these organisms. Copyright © 2017 Ericson et al.

Innate sensing of microbial products promotes wound-induced skin cancer.

PubMed

Hoste, Esther; Arwert, Esther N; Lal, Rohit; South, Andrew P; Salas-Alanis, Julio C; Murrell, Dedee F; Donati, Giacomo; Watt, Fiona M

2015-01-09

The association between tissue damage, chronic inflammation and cancer is well known. However, the underlying mechanisms are unclear. Here we characterize a mouse model in which constitutive epidermal extracellular-signal-regulated kinase-MAP-kinase signalling results in epidermal inflammation, and skin wounding induces tumours. We show that tumour incidence correlates with wound size and inflammatory infiltrate. Ablation of tumour necrosis factor receptor (TNFR)-1/-2, Myeloid Differentiation primary response gene 88 or Toll-like receptor (TLR)-5, the bacterial flagellin receptor, but not other innate immune sensors, in radiosensitive leukocytes protects against tumour formation. Antibiotic treatment inhibits, whereas injection of flagellin induces, tumours in a TLR-5-dependent manner. TLR-5 is also involved in chemical-induced skin carcinogenesis in wild-type mice. Leukocytic TLR-5 signalling mediates upregulation of the alarmin HMGB1 (High Mobility Group Box 1) in wound-induced papillomas. HMGB1 is elevated in tumours of patients with Recessive Dystrophic Epidermolysis Bullosa, a disease characterized by chronic skin damage. We conclude that in our experimental model the combination of bacteria, chronic inflammation and wounding cooperate to trigger skin cancer.

Innate sensing of microbial products promotes wound-induced skin cancer

PubMed Central

Hoste, Esther; Arwert, Esther N.; Lal, Rohit; South, Andrew P.; Salas-Alanis, Julio C.; Murrell, Dedee F.; Donati, Giacomo; Watt, Fiona M.

2015-01-01

The association between tissue damage, chronic inflammation and cancer is well known. However, the underlying mechanisms are unclear. Here we characterize a mouse model in which constitutive epidermal extracellular-signal-regulated kinase-MAP-kinase signalling results in epidermal inflammation, and skin wounding induces tumours. We show that tumour incidence correlates with wound size and inflammatory infiltrate. Ablation of tumour necrosis factor receptor (TNFR)-1/-2, Myeloid Differentiation primary response gene 88 or Toll-like receptor (TLR)-5, the bacterial flagellin receptor, but not other innate immune sensors, in radiosensitive leukocytes protects against tumour formation. Antibiotic treatment inhibits, whereas injection of flagellin induces, tumours in a TLR-5-dependent manner. TLR-5 is also involved in chemical-induced skin carcinogenesis in wild-type mice. Leukocytic TLR-5 signalling mediates upregulation of the alarmin HMGB1 (High Mobility Group Box 1) in wound-induced papillomas. HMGB1 is elevated in tumours of patients with Recessive Dystrophic Epidermolysis Bullosa, a disease characterized by chronic skin damage. We conclude that in our experimental model the combination of bacteria, chronic inflammation and wounding cooperate to trigger skin cancer. PMID:25575023

VSDMIP: virtual screening data management on an integrated platform

NASA Astrophysics Data System (ADS)

Gil-Redondo, Rubén; Estrada, Jorge; Morreale, Antonio; Herranz, Fernando; Sancho, Javier; Ortiz, Ángel R.

2009-03-01

A novel software (VSDMIP) for the virtual screening (VS) of chemical libraries integrated within a MySQL relational database is presented. Two main features make VSDMIP clearly distinguishable from other existing computational tools: (i) its database, which stores not only ligand information but also the results from every step in the VS process, and (ii) its modular and pluggable architecture, which allows customization of the VS stages (such as the programs used for conformer generation or docking), through the definition of a detailed workflow employing user-configurable XML files. VSDMIP, therefore, facilitates the storage and retrieval of VS results, easily adapts to the specific requirements of each method and tool used in the experiments, and allows the comparison of different VS methodologies. To validate the usefulness of VSDMIP as an automated tool for carrying out VS several experiments were run on six protein targets (acetylcholinesterase, cyclin-dependent kinase 2, coagulation factor Xa, estrogen receptor alpha, p38 MAP kinase, and neuraminidase) using nine binary (actives/inactive) test sets. The performance of several VS configurations was evaluated by means of enrichment factors and receiver operating characteristic plots.

N-arachidonoyl L-serine, an endocannabinoid-like brain constituent with vasodilatory properties.

PubMed

Milman, Garry; Maor, Yehoshua; Abu-Lafi, Saleh; Horowitz, Michal; Gallily, Ruth; Batkai, Sandor; Mo, Fong-Ming; Offertaler, Laszlo; Pacher, Pal; Kunos, George; Mechoulam, Raphael

2006-02-14

The endocannabinoid N-arachidonoyl ethanolamine (anandamide), found both in the CNS and in the periphery, plays a role in numerous physiological systems. One might expect that the chemically related N-arachidonoyl-L-serine (ARA-S) could also be formed alongside anandamide. We have now isolated ARA-S from bovine brain and elucidated its structure by comparison with synthetic ARA-S. Contrary to anandamide, ARA-S binds very weakly to cannabinoid CB1 and CB2 or vanilloid TRPV1 (transient receptor potential vanilloid 1) receptors. However, it produces endothelium-dependent vasodilation of rat isolated mesenteric arteries and abdominal aorta and stimulates phosphorylation of p44/42 mitogen-activated protein (MAP) kinase and protein kinase B/Akt in cultured endothelial cells. ARA-S also suppresses LPS-induced formation of TNF-alpha in a murine macrophage cell line and in wild-type mice, as well as in mice deficient in CB1 or CB2 receptors. Many of these effects parallel those reported for abnormal cannabidiol (Abn-CBD), a synthetic agonist of a putative novel cannabinoid-type receptor. Hence, ARA-S may represent an endogenous agonist for this receptor.

Grb2 regulates B-cell maturation, B-cell memory responses and inhibits B-cell Ca2+ signalling.

PubMed

Ackermann, Jochen A; Radtke, Daniel; Maurberger, Anna; Winkler, Thomas H; Nitschke, Lars

2011-04-20

Grb2 is a ubiquitously expressed adaptor protein, which activates Ras and MAP kinases in growth factor receptor signalling, while in B-cell receptor (BCR) signalling this role is controversial. In B cell lines it was shown that Grb2 can inhibit BCR-induced Ca(2+) signalling. Nonetheless, the physiological role of Grb2 in primary B cells is still unknown. We generated a B-cell-specific Grb2-deficient mouse line, which had a severe reduction of mature follicular B cells in the periphery due to a differentiation block and decreased B-cell survival. Moreover, we found several changes in important signalling pathways: enhanced BCR-induced Ca(2+) signalling, alterations in mitogen-activated protein kinase activation patterns and strongly impaired Akt activation, the latter pointing towards a defect in PI3K signalling. Interestingly, B-cell-specific Grb2-deficient mice showed impaired IgG and B-cell memory responses, and impaired germinal centre formation. Thus, Grb2-dependent signalling pathways are crucial for lymphocyte differentiation processes, as well as for control of secondary humoral immune responses.

Genesis of Prolactinomas: Studies Using Estrogen-Treated Animals

PubMed Central

Sarkar, Dipak K.

2010-01-01

Prolactin-secreting adenomas (prolactinomas) are the most prevalent form of pituitary tumors in humans. Our knowledge of the formation of these tumors is limited. Experimental work in animal has uncovered that estradiol exposure leads to prolactinoma formation via orchestrated events involving dopamine D2 receptors, transforming growth factor-β (TGF-β) isoforms and their receptors, as well as factors secondary to TGF-β action. Additionally, these studies determined that TGF-β and b-FGF interact to facilitate the communication between lactotropes and folliculo-stellate cells that is necessary for the mitogenic action of estradiol. The downstream signaling that governs lactotropic cell proliferation involves activation of the MAP kinase p44/42-dependent pathway. PMID:16809921

Phosphorylation and Intramolecular Stabilization of the Ligand Binding Domain in the Nuclear Receptor Steroidogenic Factor 1

PubMed Central

Desclozeaux, Marion; Krylova, Irina N.; Horn, Florence; Fletterick, Robert J.; Ingraham, Holly A.

2002-01-01

Steroidogenic factor 1 (SF-1) is an orphan nuclear receptor with no known ligand. We showed previously that phosphorylation at serine 203 located N′-terminal to the ligand binding domain (LBD) enhanced cofactor recruitment, analogous to the ligand-mediated recruitment in ligand-dependent receptors. In this study, results of biochemical analyses and an LBD helix assembly assay suggest that the SF-1 LBD adopts an active conformation, with helices 1 and 12 packed against the predicted alpha-helical bundle, in the apparent absence of ligand. Fine mapping of the previously defined proximal activation function in SF-1 showed that the activation function mapped fully to helix 1 of the LBD. Limited proteolyses demonstrate that phosphorylation of S203 in the hinge region mimics the stabilizing effects of ligand on the LBD. Moreover, similar effects were observed in an SF-1/thyroid hormone LBD chimera receptor, illustrating that the S203 phosphorylation effects are transferable to a heterologous ligand-dependent receptor. Our collective data suggest that the hinge together with helix 1 is an individualized specific motif, which is tightly associated with its cognate LBD. For SF-1, we find that this intramolecular association and hence receptor activity are further enhanced by mitogen-activated protein kinase phosphorylation, thus mimicking many of the ligand-induced changes observed for ligand-dependent receptors. PMID:12242296

The TRPM7 chanzyme is cleaved to release a chromatin modifying kinase

PubMed Central

Krapivinsky, Grigory; Krapivinsky, Luba; Manasian, Yunona; Clapham, David E.

2014-01-01

SUMMARY TRPM7 is a ubiquitous ion channel and kinase, a unique ‘chanzyme’, required for proper early embryonic development. It conducts Zn2+, Mg2+, Ca2+ as well as monovalent cations, and contains a functional serine/threonine kinase at its carboxyl terminus. Here, we show that in normal tissues and cell lines, the kinase is proteolytically cleaved from the channel domain in a cell type-specific manner. These TRPM7 Cleaved Kinase fragments (M7CKs) translocate to the nucleus and bind multiple components of chromatin remodeling complexes, including Polycomb group proteins. In the nucleus, the kinase phosphorylates specific serines/threonines of histones. M7CK-dependent phosphorylation of H3Ser10 at promoters of TRPM7-dependent genes correlates with their activity. We also demonstrate that cytosolic free [Zn2+] is TRPM7-dependent and regulates M7CK binding to transcription factors containing zinc-finger domains. These findings suggest that TRPM7-mediated modulation of intracellular Zn2+ concentration couples ion channel signaling to epigenetic chromatin covalent modifications that affect gene expression patterns. PMID:24855944

The NADPH Oxidases DUOX1 and NOX2 Play Distinct Roles in Redox Regulation of Epidermal Growth Factor Receptor Signaling.

PubMed

Heppner, David E; Hristova, Milena; Dustin, Christopher M; Danyal, Karamatullah; Habibovic, Aida; van der Vliet, Albert

2016-10-28

The epidermal growth factor receptor (EGFR) plays a critical role in regulating airway epithelial homeostasis and responses to injury. Activation of EGFR is regulated by redox-dependent processes involving reversible cysteine oxidation by reactive oxygen species (ROS) and involves both ligand-dependent and -independent mechanisms, but the precise source(s) of ROS and the molecular mechanisms that control tyrosine kinase activity are incompletely understood. Here, we demonstrate that stimulation of EGFR activation by ATP in airway epithelial cells is closely associated with dynamic reversible oxidation of cysteine residues via sequential sulfenylation and S-glutathionylation within EGFR and the non-receptor-tyrosine kinase Src. Moreover, the intrinsic kinase activity of recombinant Src or EGFR was in both cases enhanced by H 2 O 2 but not by GSSG, indicating that the intermediate sulfenylation is the activating modification. H 2 O 2 -induced increase in EGFR tyrosine kinase activity was not observed with the C797S variant, confirming Cys-797 as the redox-sensitive cysteine residue that regulates kinase activity. Redox-dependent regulation of EGFR activation in airway epithelial cells was found to strongly depend on activation of either the NADPH oxidase DUOX1 or the homolog NOX2, depending on the activation mechanism. Whereas DUOX1 and Src play a primary role in EGFR transactivation by wound-derived signals such as ATP, direct ligand-dependent EGFR activation primarily involves NOX2 with a secondary role for DUOX1 and Src. Collectively, our findings establish that redox-dependent EGFR kinase activation involves a dynamic and reversible cysteine oxidation mechanism and that this activation mechanism variably involves DUOX1 and NOX2. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia*

PubMed Central

Dong, Qian; Ernst, Sarah E.; Ostedgaard, Lynda S.; Shah, Viral S.; Ver Heul, Amanda R.; Welsh, Michael J.; Randak, Christoph O.

2015-01-01

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P1,P5-di(adenosine-5′) pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5′-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5′-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl− channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. PMID:25887396

Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia.

PubMed

Dong, Qian; Ernst, Sarah E; Ostedgaard, Lynda S; Shah, Viral S; Ver Heul, Amanda R; Welsh, Michael J; Randak, Christoph O

2015-05-29

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P(1),P(5)-di(adenosine-5') pentaphosphate (Ap5A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap5A, AMP, and adenosine 5'-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5'-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl(-) channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The ubiquitous mitochondrial creatine kinase gene maps to a conserved region on human chromosome 15q15 and mouse chromosome 2 bands F1-F3

DOE Office of Scientific and Technical Information (OSTI.GOV)

Steeghs, K.; Wieringa, B.; Merkx, G.

1994-11-01

Members of the creatine kinase isoenzyme family (CKs; EC 2.7.3.2) are found in mitochondria and specialized subregions of the cytoplasm and catalyze the reversible exchange of high-energy phosphoryl between ATP and phosphocreatine. At least four functionally active genes, which encode the distinct CK subunits CKB, CKM, CKMT1 (ubiquitous), and CKMT2 (sarcomeric), and a variable number of CKB pseudogenes have been identified. Here, we report the use of a CKMT1 containing phage to map the CKMT1 gene by in situ hybridization on both human and mouse chromosomes.

Amide-based inhibitors of p38alpha MAP kinase. Part 2: design, synthesis and SAR of potent N-pyrimidyl amides.

PubMed

Tester, Richland; Tan, Xuefei; Luedtke, Gregory R; Nashashibi, Imad; Schinzel, Kurt; Liang, Weiling; Jung, Joon; Dugar, Sundeep; Liclican, Albert; Tabora, Jocelyn; Levy, Daniel E; Do, Steven

2010-04-15

Optimization of a tri-substituted N-pyridyl amide led to the discovery of a new class of potent N-pyrimidyl amide based p38alpha MAP kinase inhibitors. Initial SAR studies led to the identification of 5-dihydrofuran as an optimal hydrophobic group. Additional side chain modifications resulted in the introduction of hydrogen bond interactions. Through extensive SAR studies, analogs bearing free amino groups and alternatives to the parent (S)-alpha-methyl benzyl moiety were identified. These compounds exhibited improved cellular activities and maintained balance between p38alpha and CYP3A4 inhibition. Copyright 2010 Elsevier Ltd. All rights reserved.

Genetic disruption of the core circadian clock impairs hippocampus-dependent memory.

PubMed

Wardlaw, Sarah M; Phan, Trongha X; Saraf, Amit; Chen, Xuanmao; Storm, Daniel R

2014-08-01

Perturbing the circadian system by electrolytically lesioning the suprachiasmatic nucleus (SCN) or varying the environmental light:dark schedule impairs memory, suggesting that memory depends on the circadian system. We used a genetic approach to evaluate the role of the molecular clock in memory. Bmal1-/- mice, which are arrhythmic under constant conditions, were examined for hippocampus-dependent memory, LTP at the Schaffer-collateral synapse, and signal transduction activity in the hippocampus. Bmal1-/- mice exhibit impaired contextual fear and spatial memory. Furthermore, LTP in hippocampal slices from Bmal1-/- mice is also significantly decreased relative to that from wild-type mice. Activation of Erk1,2 MAP kinase (MAPK) during training for contextual fear memory and diurnal oscillation of MAPK activity and cAMP in the hippocampus is also lost in Bmal1-/- mice, suggesting that the memory defects are due to reduction of the memory consolidation pathway in the hippocampus. We conclude that critical signaling events in the hippocampus required for memory depend on BMAL1. © 2014 Wardlaw et al.; Published by Cold Spring Harbor Laboratory Press.

Convergent regulation of the lysosomal two-pore channel-2 by Mg2+, NAADP, PI(3,5)P2 and multiple protein kinases

PubMed Central

Jha, Archana; Ahuja, Malini; Patel, Sandip; Brailoiu, Eugen; Muallem, Shmuel

2014-01-01

Lysosomal Ca2+ homeostasis is implicated in disease and controls many lysosomal functions. A key in understanding lysosomal Ca2+ signaling was the discovery of the two-pore channels (TPCs) and their potential activation by NAADP. Recent work concluded that the TPCs function as a PI(3,5)P2 activated channels regulated by mTORC1, but not by NAADP. Here, we identified Mg2+ and the MAPKs, JNK and P38 as novel regulators of TPC2. Cytoplasmic Mg2+ specifically inhibited TPC2 outward current, whereas lysosomal Mg2+ partially inhibited both outward and inward currents in a lysosomal lumen pH-dependent manner. Under controlled Mg2+, TPC2 is readily activated by NAADP with channel properties identical to those in response to PI(3,5)P2. Moreover, TPC2 is robustly regulated by P38 and JNK. Notably, NAADP-mediated Ca2+ release in intact cells is regulated by Mg2+, PI(3,5)P2, and P38/JNK kinases, thus paralleling regulation of TPC2 currents. Our data affirm a key role for TPC2 in NAADP-mediated Ca2+ signaling and link this pathway to Mg2+ homeostasis and MAP kinases, pointing to roles for lysosomal Ca2+ in cell growth, inflammation and cancer. PMID:24502975

Convergent regulation of the lysosomal two-pore channel-2 by Mg²⁺, NAADP, PI(3,5)P₂ and multiple protein kinases.

PubMed

Jha, Archana; Ahuja, Malini; Patel, Sandip; Brailoiu, Eugen; Muallem, Shmuel

2014-03-03

Lysosomal Ca(2+) homeostasis is implicated in disease and controls many lysosomal functions. A key in understanding lysosomal Ca(2+) signaling was the discovery of the two-pore channels (TPCs) and their potential activation by NAADP. Recent work concluded that the TPCs function as a PI(3,5)P2 activated channels regulated by mTORC1, but not by NAADP. Here, we identified Mg(2+) and the MAPKs, JNK and P38 as novel regulators of TPC2. Cytoplasmic Mg(2+) specifically inhibited TPC2 outward current, whereas lysosomal Mg(2+) partially inhibited both outward and inward currents in a lysosomal lumen pH-dependent manner. Under controlled Mg(2+), TPC2 is readily activated by NAADP with channel properties identical to those in response to PI(3,5)P2. Moreover, TPC2 is robustly regulated by P38 and JNK. Notably, NAADP-mediated Ca(2+) release in intact cells is regulated by Mg(2+), PI(3,5)P2, and P38/JNK kinases, thus paralleling regulation of TPC2 currents. Our data affirm a key role for TPC2 in NAADP-mediated Ca(2+) signaling and link this pathway to Mg(2+) homeostasis and MAP kinases, pointing to roles for lysosomal Ca(2+) in cell growth, inflammation and cancer.

SH3 domain-mediated binding of the Drk protein to Dos is an important step in signaling of Drosophila receptor tyrosine kinases.

PubMed

Feller, Stephan M; Wecklein, Heike; Lewitzky, Marc; Kibler, Eike; Raabe, Thomas

2002-08-01

Activation of the Sevenless (Sev) receptor tyrosine kinase (RTK) in the developing Drosophila eye is required for the specification of the R7 photoreceptor cell fate. Daughter of Sevenless (Dos), a putative multi-site adaptor protein, is a substrate of the Sev kinase and is known to associate with the tyrosine phosphatase Corkscrew (Csw). Binding of Csw to Dos depends on the Csw Src homology 2 (SH2) domains and is an essential step for signaling by the Sev RTK. Dos, however, lacks a recognizable phosphotyrosine interaction domain and it was previously unclear how it is recruited to the Sev receptor. Here it is shown that the SH2/SH3 domain adaptor protein Drk can provide this link. Drk binds with its SH2 domain to the autophosphorylated Sev receptor while the C-terminal SH3 domain is able to associate with Dos. The Drk SH3 domain binding motifs on Dos were mapped to two sites which do not conform the known Drk SH3 domain binding motif (PxxPxR) but instead have the consensus PxxxRxxKP. Mutational analysis in vitro and in vivo provided evidence that both Drk binding sites fulfil an important function in the context of Sev and Drosophila epidermal growth factor receptor mediated signaling processes.

Activation of MAP kinases by hexavalent chromium, manganese and nickel in human lung epithelial cells.

PubMed

Tessier, Daniel M; Pascal, Laura E

2006-12-01

Epidemiological studies indicate that workers who perform welding operations are at increased risk for bronchitis, siderosis, occupational asthma and lung cancer due to fume exposure. Welding fumes are a complex chemical mixture, and the metal composition is hypothesized to be an etiological factor in respiratory disease due to this exposure. In the present study, human lung epithelial cells in vitro responded to hexavalent chromium, manganese and nickel over a concentration range of 0.2-200 microM with a significant increase in intracellular phosphoprotein (a measure of stress response pathway activation). The mitogen-activated protein kinases ERK1/2, SAPK/JNK and p38 were activated via phosphorylation following 1-h exposures. Hexavalent chromium up-regulated p-38 phosphorylation 23-fold and SAPK/JNK phosphorylation 17-fold, with a comparatively modest 4-fold increase in ERK1/2 phosphorylation. Manganese caused a two- to four-fold increase in SAPK/JNK and ERK 1/2 phosphorylation, with no observed effects on p38 kinase. Nickel caused increased (two-fold) phosphorylation of ERK 1/2 only, and was not cytotoxic over the tested concentration range. The observed effects of welding fume metals on cellular signaling in lung epithelium demonstrate a potentially significant interplay between stress-response signaling (p38 and SAPK/JNK) and anti-apototic signaling (ERK 1/2) that is dependant on the specific metal or combination of metals involved.

Possible involvement of MAP kinase pathways in acquired metal-tolerance induced by heat in plants.

PubMed

Chen, Po-Yu; Lee, Kuo-Ting; Chi, Wen-Chang; Hirt, Heribert; Chang, Ching-Chun; Huang, Hao-Jen

2008-08-01

Cross tolerance is a phenomenon that occurs when a plant, in resisting one form of stress, develops a tolerance to another form. Pretreatment with nonlethal heat shock has been known to protect cells from metal stress. In this study, we found that the treatment of rice roots with more than 25 muM of Cu(2+) caused cell death. However, heat shock pretreatment attenuated Cu(2+)-induced cell death. The mechanisms of the cross tolerance phenomenon between heat shock and Cu(2+) stress were investigated by pretreated rice roots with the protein synthesis inhibitor cycloheximide (CHX). CHX effectively block heat shock protection, suggesting that protection of Cu(2+)-induced cell death by heat shock was dependent on de novo protein synthesis. In addition, heat pretreatment downregulated ROS production and mitogen-activated protein kinase (MAPK) activities, both of which can be greatly elicited by Cu(2+) stress in rice roots. Moreover, the addition of purified recombinant GST-OsHSP70 fusion proteins inhibited Cu(2+)-enhanced MAPK activities in an in vitro kinase assay. Furthermore, loss of heat shock protection was observed in Arabidopsis mkk2 and mpk6 but not in mpk3 mutants under Cu(2+) stress. Taken together, these results suggest that the interaction of OsHSP70 with MAPKs may contribute to the cellular protection in rice roots from excessive Cu(2+) toxicity.

Mechanisms of cell signaling by nitric oxide and peroxynitrite: from mitochondria to MAP kinases

NASA Technical Reports Server (NTRS)

Levonen, A. L.; Patel, R. P.; Brookes, P.; Go, Y. M.; Jo, H.; Parthasarathy, S.; Anderson, P. G.; Darley-Usmar, V. M.

2001-01-01

Many of the biological and pathological effects of nitric oxide (NO) are mediated through cell signaling pathways that are initiated by NO reacting with metalloproteins. More recently, it has been recognized that the reaction of NO with free radicals such as superoxide and the lipid peroxyl radical also has the potential to modulate redox signaling. Although it is clear that NO can exert both cytotoxic and cytoprotective actions, the focus of this overview are those reactions that could lead to protection of the cell against oxidative stress in the vasculature. This will include the induction of antioxidant defenses such as glutathione, activation of mitogen-activated protein kinases in response to blood flow, and modulation of mitochondrial function and its impact on apoptosis. Models are presented that show the increased synthesis of glutathione in response to shear stress and inhibition of cytochrome c release from mitochondria. It appears that in the vasculature NO-dependent signaling pathways are of three types: (i) those involving NO itself, leading to modulation of mitochondrial respiration and soluble guanylate cyclase; (ii) those that involve S-nitrosation, including inhibition of caspases; and (iii) autocrine signaling that involves the intracellular formation of peroxynitrite and the activation of the mitogen-activated protein kinases. Taken together, NO plays a major role in the modulation of redox cell signaling through a number of distinct pathways in a cellular setting.

Aberrant Ras regulation and reduced p190 tyrosine phosphorylation in cells lacking p120-Gap.

PubMed Central

van der Geer, P; Henkemeyer, M; Jacks, T; Pawson, T

1997-01-01

The Ras guanine nucleotide-binding protein functions as a molecular switch in signalling downstream of protein-tyrosine kinases. Ras is activated by exchange of GDP for GTP and is turned off by hydrolysis of bound GTP to GDP. Ras itself has a low intrinsic GTPase activity that can be stimulated by GTPase-activating proteins (GAPs), including p120-Gap and neurofibromin. These GAPs possess a common catalytic domain but contain distinct regulatory elements that may couple different external signals to control of the Ras pathway. p120-Gap, for example, has two N-terminal SH2 domains that directly recognize phosphotyrosine motifs on activated growth factor receptors and cytoplasmic phosphoproteins. To analyze the role of p120-Gap in Ras regulation in vivo, we have used fibroblasts derived from mouse embryos with a null mutation in the gene for p120-Gap (Gap). Platelet-derived growth factor stimulation of Gap-/- cells led to an abnormally large increase in the level of Ras-GTP and in the duration of mitogen-activated protein (MAP) kinase activation compared with wild-type cells, suggesting that p120-Gap is specifically activated following growth factor stimulation. Induction of DNA synthesis in response to platelet-derived growth factor and morphological transformation by the v-src and EJ-ras oncogenes were not significantly affected by the absence of p120-Gap. However, we found that normal tyrosine phosphorylation of p190-rhoGap, a cytoplasmic protein that associates with the p120-Gap SH2 domains, was dependent on the presence of p120-Gap. Our results suggest that p120-Gap has specific functions in downregulating the Ras/MAP kinase pathway following growth factor stimulation, and in modulating the phosphorylation of p190-rhoGap, but is not required for mitogenic signalling. PMID:9121432

Expression and signaling of G protein-coupled estrogen receptor 1 (GPER) in rat sertoli cells.

PubMed

Lucas, Thaís F G; Royer, Carine; Siu, Erica R; Lazari, Maria Fatima M; Porto, Catarina S

2010-08-01

The aim of the present study was to investigate the expression and signaling of the G protein-coupled estrogen receptor 1 (GPER) in cultured immature rat Sertoli cells--in which we have previously described the classical estrogen receptors (ESR1 and ESR2). Expression of GPER in cultured Sertoli cells from 15-day-old rats was detected by RT-PCR and immunoassays. Gper transcripts also were present in testes from 5-, 15-, and 120-day-old rats. Short-term treatment of Sertoli cells with 17beta-estradiol (E2), the GPER agonist G-1, or the ESR antagonist ICI 182,780 (ICI) rapidly activated MAPK3/1 (ERK1/2), even after down-regulation of ESR1 and ESR2, suggesting a role for GPER in the rapid E2 action in these cells. MAPK3/1 phosphorylation induced by ICI or G-1 was blocked by pertussis toxin, selective inhibitor of the SRC family of protein tyrosine kinases, metalloprotease inhibitor, MAP2K1/2 inhibitor, and epidermal growth factor receptor (EGFR) kinase inhibitor. Furthermore, E2, but not G-1, induced up-regulation of cyclin D1 in the Sertoli cells. This effect was blocked by ICI. E2 and G-1 decreased BAX and increased BCL2 expression and these effects were blocked by MAP2K1/2 inhibitor and EGFR kinase inhibitor. The pretreatment with ICI did not block the effect of E2. Taken together, these results indicate that in Sertoli cells 1) GPER-mediated MAPK3/1 activation occurs via EGFR transactivation through G protein beta gamma subunits that promote SRC-mediated metalloprotease-dependent release of EGFR ligands, which bind to EGFR and lead to MAPK3/1 phosphorylation; 2) E2-ESRs play a role in Sertoli cell proliferation; and 3) E2-GPER may regulate gene expression involved with apoptosis. ESR and GPER may mediate actions important for Sertoli cell function and maintenance of normal testis development and homeostasis.

The Atypical MAP Kinase SWIP-13/ERK8 Regulates Dopamine Transporters through a Rho-Dependent Mechanism

PubMed Central

Bermingham, Daniel P.; Snider, Sam L.; Miller, David M.

2017-01-01

The neurotransmitter dopamine (DA) regulates multiple behaviors across phylogeny, with disrupted DA signaling in humans associated with addiction, attention-deficit/ hyperactivity disorder, schizophrenia, and Parkinson's disease. The DA transporter (DAT) imposes spatial and temporal limits on DA action, and provides for presynaptic DA recycling to replenish neurotransmitter pools. Molecular mechanisms that regulate DAT expression, trafficking, and function, particularly in vivo, remain poorly understood, though recent studies have implicated rho-linked pathways in psychostimulant action. To identify genes that dictate the ability of DAT to sustain normal levels of DA clearance, we pursued a forward genetic screen in Caenorhabditis elegans based on the phenotype swimming-induced paralysis (Swip), a paralytic behavior observed in hermaphrodite worms with loss-of-function dat-1 mutations. Here, we report the identity of swip-13, which encodes a highly conserved ortholog of the human atypical MAP kinase ERK8. We present evidence that SWIP-13 acts presynaptically to insure adequate levels of surface DAT expression and DA clearance. Moreover, we provide in vitro and in vivo evidence supporting a conserved pathway involving SWIP-13/ERK8 activation of Rho GTPases that dictates DAT surface expression and function. SIGNIFICANCE STATEMENT Signaling by the neurotransmitter dopamine (DA) is tightly regulated by the DA transporter (DAT), insuring efficient DA clearance after release. Molecular networks that regulate DAT are poorly understood, particularly in vivo. Using a forward genetic screen in the nematode Caenorhabditis elegans, we implicate the atypical mitogen activated protein kinase, SWIP-13, in DAT regulation. Moreover, we provide in vitro and in vivo evidence that SWIP-13, as well as its human counterpart ERK8, regulate DAT surface availability via the activation of Rho proteins. Our findings implicate a novel pathway that regulates DA synaptic availability and that may contribute to risk for disorders linked to perturbed DA signaling. Targeting this pathway may be of value in the development of therapeutics in such disorders. PMID:28842414