MAP kinase genes and colon and rectal cancer

Science.gov (United States)

Slattery, Martha L.

2012-01-01

Mitogen-activated protein kinase (MAPK) pathways regulate many cellular functions including cell proliferation, differentiation, migration and apoptosis. We evaluate genetic variation in the c-Jun-N-terminal kinases, p38, and extracellular regulated kinases 1/2 MAPK-signaling pathways and colon and rectal cancer risk using data from population-based case-control studies (colon: n = 1555 cases, 1956 controls; rectal: n = 754 cases, 959 controls). We assess 19 genes (DUSP1, DUSP2, DUSP4, DUSP6, DUSP7, MAP2K1, MAP3K1, MAP3K2, MAP3K3, MAP3K7, MAP3K9, MAP3K10, MAP3K11, MAPK1, MAPK3, MAPK8, MAPK12, MAPK14 and RAF1). MAP2K1 rs8039880 [odds ratio (OR) = 0.57, 95% confidence interval (CI) = 0.38, 0.83; GG versus AA genotype] and MAP3K9 rs11625206 (OR = 1.41, 95% CI = 1.14, 1.76; recessive model) were associated with colon cancer (P adj value rectal cancer (P adj cancer risk. Genetic variants had unique associations with KRAS, TP53 and CIMP+ tumors. DUSP2 rs1724120 [hazard rate ratio (HRR) = 0.72, 95%CI = 0.54, 0.96; AA versus GG/GA), MAP3K10 rs112956 (HRR = 1.40, 95% CI = 1.10, 1.76; CT/TT versus CC) and MAP3K11 (HRR = 1.76, 95% CI 1.18, 2.62 TT versus GG/GT) influenced survival after diagnosis with colon cancer; MAP2K1 rs8039880 (HRR = 2.53, 95% CI 1.34, 4.79 GG versus AG/GG) and Raf1 rs11923427 (HRR = 0.59 95% CI = 0.40, 0.86; AA versus TT/TA) were associated with rectal cancer survival. These data suggest that genetic variation in the MAPK-signaling pathway influences colorectal cancer risk and survival after diagnosis. Associations may be modified by lifestyle factors that influence inflammation and oxidative stress. PMID:23027623

Partial purification and characterization of a wortmannin-sensitive and insulin-stimulated protein kinase that activates heart 6-phosphofructo-2-kinase.

OpenAIRE

Deprez, J; Bertrand, L; Alessi, D R; Krause, U; Hue, L; Rider, M H

2000-01-01

A wortmannin-sensitive and insulin-stimulated protein kinase (WISK), which phosphorylates and activates cardiac 6-phosphofructo-2-kinase (PFK-2), was partially purified from perfused rat hearts. Immunoblotting showed that WISK was devoid of protein kinase B (PKB), serum- and glucocorticoid-regulated protein kinase and protein kinase Czeta (PKCzeta). Comparison of the inhibition of WISK, PKCalpha and PKCzeta by different protein kinase inhibitors suggested that WISK was not a member of the PKC...

Insulin signaling inhibits the 5-HT2C receptor in choroid plexus via MAP kinase

Directory of Open Access Journals (Sweden)

Guan Kunliang

2003-06-01

Full Text Available Abstract Background G protein-coupled receptors (GPCRs interact with heterotrimeric GTP-binding proteins (G proteins to modulate acute changes in intracellular messenger levels and ion channel activity. In contrast, long-term changes in cellular growth, proliferation and differentiation are often mediated by tyrosine kinase receptors and certain GPCRs by activation of mitogen-activated protein (MAP kinases. Complex interactions occur between these signaling pathways, but the specific mechanisms of such regulatory events are not well-understood. In particular it is not clear whether GPCRs are modulated by tyrosine kinase receptor-MAP kinase pathways. Results Here we describe tyrosine kinase receptor regulation of a GPCR via MAP kinase. Insulin reduced the activity of the 5-HT2C receptor in choroid plexus cells which was blocked by the MAP kinase kinase (MEK inhibitor, PD 098059. We demonstrate that the inhibitory effect of insulin and insulin-like growth factor type 1 (IGF-1 on the 5-HT2C receptor is dependent on tyrosine kinase, RAS and MAP kinase. The effect may be receptor-specific: insulin had no effect on another GPCR that shares the same G protein signaling pathway as the 5-HT2C receptor. This effect is also direct: activated MAP kinase mimicked the effect of insulin, and removing a putative MAP kinase site from the 5-HT2C receptor abolished the effect of insulin. Conclusion These results show that insulin signaling can inhibit 5-HT2C receptor activity and suggest that MAP kinase may play a direct role in regulating the function of a specific GPCR.

Activation of the Cph1-dependent MAP kinase signaling pathway induces white-opaque switching in Candida albicans.

Directory of Open Access Journals (Sweden)

Bernardo Ramírez-Zavala

Full Text Available 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 (Ste11(ΔN467 efficiently stimulated white cells to switch to the opaque phase, a behavior that did not occur in response to pheromone. Ste11(ΔN467-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.

Activation of G-proteins by receptor-stimulated nucleoside diphosphate kinase in Dictyostelium.

Science.gov (United States)

Bominaar, A A; Molijn, A C; Pestel, M; Veron, M; Van Haastert, P J

1993-01-01

Recently, interest in the enzyme nucleoside diphosphate kinase (EC2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase activity on cellular processes might be the result of altered transmembrane signal transduction via guanine nucleotide-binding proteins (G-proteins). In the cellular slime mould Dictyostelium discoideum, extracellular cAMP induces an increase of phospholipase C activity via a surface cAMP receptor and G-proteins. In this paper it is demonstrated that part of the cellular NDP kinase is associated with the membrane and stimulated by cell surface cAMP receptors. The GTP produced by the action of NDP kinase is capable of activating G-proteins as monitored by altered G-protein-receptor interaction and the activation of the effector enzyme phospholipase C. Furthermore, specific monoclonal antibodies inhibit the effect of NDP kinase on G-protein activation. These results suggest that receptor-stimulated NDP kinase contributes to the mediation of hormone action by producing GTP for the activation of GTP-binding proteins. Images PMID:8389692

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

Science.gov (United States)

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

A double-mutant collection targeting MAP kinase related genes in Arabidopsis for studying genetic interactions.

Science.gov (United States)

Su, Shih-Heng; Krysan, Patrick J

2016-12-01

Mitogen-activated protein kinase cascades are conserved in all eukaryotes. In Arabidopsis thaliana there are approximately 80 genes encoding MAP kinase kinase kinases (MAP3K), 10 genes encoding MAP kinase kinases (MAP2K), and 20 genes encoding MAP kinases (MAPK). Reverse genetic analysis has failed to reveal abnormal phenotypes for a majority of these genes. One strategy for uncovering gene function when single-mutant lines do not produce an informative phenotype is to perform a systematic genetic interaction screen whereby double-mutants are created from a large library of single-mutant lines. Here we describe a new collection of 275 double-mutant lines derived from a library of single-mutants targeting genes related to MAP kinase signaling. To facilitate this study, we developed a high-throughput double-mutant generating pipeline using a system for growing Arabidopsis seedlings in 96-well plates. A quantitative root growth assay was used to screen for evidence of genetic interactions in this double-mutant collection. Our screen revealed four genetic interactions, all of which caused synthetic enhancement of the root growth defects observed in a MAP kinase 4 (MPK4) single-mutant line. Seeds for this double-mutant collection are publicly available through the Arabidopsis Biological Resource Center. Scientists interested in diverse biological processes can now screen this double-mutant collection under a wide range of growth conditions in order to search for additional genetic interactions that may provide new insights into MAP kinase signaling. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

DMPD: Macrophage-stimulating protein and RON receptor tyrosine kinase: potentialregulators of macrophage inflammatory activities. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 12472665 Macrophage-stimulating protein and RON receptor tyrosine kinase: potential...:545-53. (.png) (.svg) (.html) (.csml) Show Macrophage-stimulating protein and RON receptor tyrosine kinase:...le Macrophage-stimulating protein and RON receptor tyrosine kinase: potentialregulators of macrophage inflam

The cAMP Signaling and MAP Kinase Pathways in Plant Pathogenic Fungi

NARCIS (Netherlands)

Mehrabi, R.; Zhao, X.; Kim, Y.; Xu, J.R.

2009-01-01

The key components of the well conserved cyclic AMP signaling and MAP kinase pathways have been functionally characterized in the corn smut Ustilago maydis, rice blast fungus Magnaporthe grisea, and a few other fungal pathogens. In general, the cAMP signaling and the MAP kinase cascade homologous to

Stress-induced activation of protein kinase CK2 by direct interaction with p38 mitogen-activated protein kinase

DEFF Research Database (Denmark)

Sayed, M; Kim, S O; Salh, B S

2000-01-01

Protein kinase CK2 has been implicated in the regulation of a wide range of proteins that are important in cell proliferation and differentiation. Here we demonstrate that the stress signaling agents anisomycin, arsenite, and tumor necrosis factor-alpha stimulate the specific enzyme activity of CK2...... in the human cervical carcinoma HeLa cells by up to 8-fold, and this could be blocked by the p38 MAP kinase inhibitor SB203580. We show that p38alpha MAP kinase, in a phosphorylation-dependent manner, can directly interact with the alpha and beta subunits of CK2 to activate the holoenzyme through what appears...

The role of p38 MAP kinase and c-Jun N-terminal protein kinase signaling in the differentiation and apoptosis of immortalized neural stem cells

International Nuclear Information System (INIS)

Yang, Se-Ran; Cho, Sung-Dae; Ahn, Nam-Shik; Jung, Ji-Won; Park, Joon-Suk; Jo, Eun-Hye; Hwang, Jae-Woong; Kim, Sung-Hoon; Lee, Bong-Hee; Kang, Kyung-Sun; Lee, Yong-Soon

2005-01-01

The two distinct members of the mitogen-activated protein (MAP) kinase family c-Jun N-terminal protein kinase (JNK) and p38 MAP kinase, play an important role in central nervous system (CNS) development and differentiation. However, their role and functions are not completely understood in CNS. To facilitate in vitro study, we have established an immortal stem cell line using SV40 from fetal rat embryonic day 17. In these cells, MAP kinase inhibitors (SP600125, SB202190, and PD98059) were treated for 1, 24, 48, and 72 h to examine the roles of protein kinases. Early inhibition of JNK did not alter phenotypic or morphological changes of immortalized cells, however overexpression of Bax and decrease of phosphorylated AKT was observed. The prolonged inhibition of JNK induced polyploidization of immortalized cells, and resulted in differentiation and inhibition of cell proliferation. Moreover, JNK and p38 MAP kinase but not ERK1/2 was activated, and p21, p53, and Bax were overexpressed by prolonged inhibition of JNK. These results indicate that JNK and p38 MAP kinase could play dual roles on cell survival and apoptosis. Furthermore, this established cell line could facilitate study of the role of JNK and p38 MAP kinase on CNS development or differentiation/apoptosis

Phosphorylation sites of Arabidopsis MAP Kinase Substrate 1 (MKS1)

DEFF Research Database (Denmark)

Caspersen, M.B.; Qiu, J.-L.; Zhang, X.

2007-01-01

The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophore......The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified...... phosphopeptide detection. As MAP kinases generally phosphorylate serine or threonine followed by proline (Ser/Thr-Pro), theoretical masses of potentially phosphorylated peptides were calculated and mass spectrometric peaks matching these masses were fragmented and searched for a neutral-loss signal...... at approximately 98 Da indicative of phosphorylation. Additionally, mass spectrometric peaks present in the MPK4-treated MKS1, but not in the control peptide map of untreated MKS1, were fragmented. Fragmentation spectra were subjected to a MASCOT database search which identified three of the twelve Ser-Pro serine...

Identification of p38α MAP kinase inhibitors by pharmacophore based virtual screening

DEFF Research Database (Denmark)

Gangwal, Rahul P; Das, Nihar R; Thanki, Kaushik

2014-01-01

The p38α mitogen-activated protein (MAP) kinase plays a vital role in treating many inflammatory diseases. In the present study, a combined ligand and structure based pharmacophore model was developed to identify potential DFG-in selective p38 MAP kinase inhibitors. Conformations of co...

Free radical-mediated stimulation of tyrosine-specific protein kinase in rat liver plasma membrane

International Nuclear Information System (INIS)

Chan, T.M.; Tatoyan, A.; Cheng, E.; Shargill, N.S.; Pleta, M.

1986-01-01

Incorporation of 32 P from (γ- 32 P)-ATP into endogenous proteins of plasma membranes isolated from rat liver was significantly increased by several naphthoquinones including menadione. This apparent stimulation of membrane-associated protein kinase activity by these compounds was most striking (up to 6-7 fold) when the synthetic copolymers containing glutamate and tyrosine residues (4:1) was used as substrate. Since tyrosine residues are the only possible phosphate acceptor in the copolymers, the quinone-stimulated liver membrane protein kinase is most likely tyrosine specific. Although not required for protein kinase activity, dithiothreitol (DTT) was necessary for its stimulation by these quinonoid compounds. Hydrolysis of ATP was not significantly affected by quinones under the experimental conditions. Both menadione and vitamin k 5 increased phosphorylation of plasma membrane proteins of molecular weight 45 and 60 kd. The stimulatory effect of menadione on protein phosphorylation was prevented by the addition of superoxide dismutase. Dihydroxyfumerate, which spontaneously produces various radical species, and H 2 O 2 , also stimulated tyrosine-specific protein phosphorylation. DTT was also required for their full effect. It, therefore, appears that quinonone stimulation of tyrosine-specific protein phosphorylation is mediated by oxygen radicals

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

Science.gov (United States)

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.

Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

Science.gov (United States)

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

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

Science.gov (United States)

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.

Sucrose nonfermenting AMPK-related kinase (SNARK) mediates contraction-stimulated glucose transport in mouse skeletal muscle

OpenAIRE

Koh, Ho-Jin; Toyoda, Taro; Fujii, Nobuharu; Jung, Michelle M.; Rathod, Amee; Middelbeek, R. Jan-Willem; Lessard, Sarah J.; Treebak, Jonas T.; Tsuchihara, Katsuya; Esumi, Hiroyasu; Richter, Erik A.; Wojtaszewski, Jørgen F. P.; Hirshman, Michael F.; Goodyear, Laurie J.

2010-01-01

The signaling mechanisms that mediate the important effects of contraction to increase glucose transport in skeletal muscle are not well understood, but are known to occur through an insulin-independent mechanism. Muscle-specific knockout of LKB1, an upstream kinase for AMPK and AMPK-related protein kinases, significantly inhibited contraction-stimulated glucose transport. This finding, in conjunction with previous studies of ablated AMPKα2 activity showing no effect on contraction-stimulated...

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

Science.gov (United States)

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.

The C-type lectin OCILRP2 costimulates EL4 T cell activation via the DAP12-Raf-MAP kinase pathway.

Science.gov (United States)

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.

CZK3, a MAP kinase kinase kinase homolog in Cercospora zeae-maydis, regulates cercosporin biosynthesis, fungal development, and pathogenesis.

Science.gov (United States)

Shim, Won-Bo; Dunkle, Larry D

2003-09-01

The fungus Cercospora zeae-maydis causes gray leaf spot of maize and produces cercosporin, a photosensitizing perylenequinone with toxic activity against a broad spectrum of organisms. However, little is known about the biosynthetic pathway or factors that regulate cercosporin production. Analysis of a cDNA subtraction library comprised of genes that are up-regulated during cercosporin synthesis revealed a sequence highly similar to mitogen-activated protein (MAP) kinases in other fungi. Sequencing and conceptual translation of the full-length genomic sequence indicated that the gene, which we designated CZK3, contains a 4,119-bp open reading frame devoid of introns and encodes a 1,373-amino acid sequence that is highly similar to Wis4, a MAP kinase kinase kinase in Schizosaccharomyces pombe. Targeted disruption of CZK3 suppressed expression of genes predicted to participate in cercosporin biosynthesis and abolished cercosporin production. The disrupted mutants grew faster on agar media than the wild type but were deficient in conidiation and elicited only small chlorotic spots on inoculated maize leaves compared with rectangular necrotic lesions incited by the wild type. Complementation of disruptants with the CZK3 open reading frame and flanking sequences restored wild-type levels of conidiation, growth rate, and virulence as well as the ability to produce cercosporin. The results suggest that cercosporin is a virulence factor in C. zeae-maydis during maize pathogenesis, but the pleiotropic effects of CZK3 disruption precluded definitive conclusions.

Src Kinase becomes preferentially associated with the VEGFR, KDR/Flk-1, following VEGF stimulation of vascular endothelial cells

Directory of Open Access Journals (Sweden)

Wang Jing

2002-12-01

Full Text Available Abstract Background The cytoplasmic tyrosine kinase, Src, has been found to play a crucial role in VEGF (vascular endothelial growth factor – dependent vascular permeability involved in angiogenesis. The two main VEGFRs present on vascular endothelial cells are KDR/Flk-1 (kinase insert domain-containing receptor/fetal liver kinase-1 and Flt-1 (Fms-like tyrosine kinase-1. However, to date, it has not been determined which VEGF receptor (VEGFR is involved in binding to and activating Src kinase following VEGF stimulation of the receptors. Results In this report, we demonstrate that Src preferentially associates with KDR/Flk-1 rather than Flt-1 in human umbilical vein endothelial cells (HUVECs, and that VEGF stimulation resulted in an increase of Src activity associated with activated KDR/Flk-1. These findings were determined through immunoprecipitation-kinase experiments and coimmunoprecipitation studies, and were further confirmed by GST-pull-down assays and Far Western studies. However, Fyn and Yes, unlike Src, were found to associate preferentially with Flt-1. Conclusions Thus, Src preferentially associates with KDR/Flk-1, rather than with Flt-1, upon VEGF stimulation in endothelial cells. Our findings further highlight the potential significance of upregulated KDR/Flk-1-associated Src activity in the process of angiogenesis, and help to elucidate more clearly the specific roles and mechanisms involving Src family tyrosine kinase in VEGF-stimulated signal transduction events.

LTB4 stimulates growth of human pancreatic cancer cells via MAPK and PI-3 kinase pathways

International Nuclear Information System (INIS)

Tong, W.-G.; Ding, X.-Z.; Talamonti, Mark S.; Bell, Richard H.; Adrian, Thomas E.

2005-01-01

We have previously shown the importance of LTB4 in human pancreatic cancer. LTB4 receptor antagonists block growth and induce apoptosis in pancreatic cancer cells both in vitro and in vivo. Therefore, we investigated the effect of LTB4 on proliferation of human pancreatic cancer cells and the mechanisms involved. LTB4 stimulated DNA synthesis and proliferation of both PANC-1 and AsPC-1 human pancreatic cancer cells, as measured by thymidine incorporation and cell number. LTB4 stimulated rapid and transient activation of MEK and ERK1/2 kinases. The MEK inhibitors, PD98059 and U0126, blocked LTB4-stimulated ERK1/2 activation and cell proliferation. LTB4 also stimulated phosphorylation of p38 MAPK; however, the p38 MAPK inhibitor, SB203580, failed to block LTB4-stimulated growth. The activity of JNK/SAPK was not affected by LTB4 treatment. Phosphorylation of Akt was also induced by LTB4 and this effect was blocked by the PI-3 kinase inhibitor wortmannin, which also partially blocked LTB4-stimulated cell proliferation. In conclusion, LTB4 stimulates proliferation of human pancreatic cancer cells through MEK/ERK and PI-3 kinase/Akt pathways, while p38 MPAK and JNK/SAPK are not involved

The Drosophila rolled locus encodes a MAP kinase required in the sevenless signal transduction pathway.

OpenAIRE

Biggs, W H; Zavitz, K H; Dickson, B; van der Straten, A; Brunner, D; Hafen, E; Zipursky, S L

1994-01-01

Mitogen-activated protein (MAP) kinases have been proposed to play a critical role in receptor tyrosine kinase (RTK)-mediated signal transduction pathways. Although genetic and biochemical studies of RTK pathways in Caenorhabditis elegans, Drosophila melanogaster and mammals have revealed remarkable similarities, a genetic requirement for MAP kinases in RTK signaling has not been established. During retinal development in Drosophila, the sevenless (Sev) RTK is required for development of the ...

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

Directory of Open Access Journals (Sweden)

Gen Kuroyanagi

2014-10-01

Full Text Available 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.

The C-type lectin OCILRP2 costimulates EL4 T cell activation via the DAP12-Raf-MAP kinase pathway.

Directory of Open Access Journals (Sweden)

Qiang Lou

Full Text Available 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.

The role of p38 MAP kinase in cancer cell apoptosis

International Nuclear Information System (INIS)

Lenassi, M.; Plemenitas, A.

2006-01-01

Background. Cellular behaviour in response to many extracellular stimuli is mediated through MAP kinase signalling pathways. p38 MAP kinase that is represented in mammals by four isoforms (p38α, p38β, p38γ and p38δ) is one of the four main subgroups of MAP kinases. Recent studies show that p38 activation is necessary for cancer cell death initiated by variety of anti-cancer agents. This finding connected cancer therapies previously considered to be mechanistically unrelated and raised the possibility of developing anti-cancer agents that lack the side effects caused by events upstream of p38 MAPK. Many of the details of p38 induced apoptosis still need to be elucidated. Since most of the past studies rely only on the cell culture models, all the results have to be verified using in vivo models. Also very little is known about the role of p38 mediated apoptosis on non-neoplastic cells in response to anti-cancer agents. Conclusion. Although p38 activation of cancer cell apoptosis is a very complex process, recent studies indicate a good starting point for new strategies that would increase the efficiency and decrease the toxicity of proven therapies. (author)

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

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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.

HAM-5 functions as a MAP kinase scaffold during cell fusion in Neurospora crassa.

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Wilfried Jonkers

2014-11-01

Full Text Available 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

Synapses of Amphids Defective (SAD-A) Kinase Promotes Glucose-stimulated Insulin Secretion through Activation of p21-activated Kinase (PAK1) in Pancreatic β-Cells*

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Nie, Jia; Sun, Chao; Faruque, Omar; Ye, Guangming; Li, Jia; Liang, Qiangrong; Chang, Zhijie; Yang, Wannian; Han, Xiao; Shi, Yuguang

2012-01-01

The p21-activated kinase-1 (PAK1) is implicated in regulation of insulin exocytosis as an effector of Rho GTPases. PAK1 is activated by the onset of glucose-stimulated insulin secretion (GSIS) through phosphorylation of Thr-423, a major activation site by Cdc42 and Rac1. However, the kinase(s) that phosphorylates PAK1 at Thr-423 in islet β-cells remains elusive. The present studies identified SAD-A (synapses of amphids defective), a member of AMP-activated protein kinase-related kinases exclusively expressed in brain and pancreas, as a key regulator of GSIS through activation of PAK1. We show that SAD-A directly binds to PAK1 through its kinase domain. The interaction is mediated by the p21-binding domain (PBD) of PAK1 and requires both kinases in an active conformation. The binding leads to direct phosphorylation of PAK1 at Thr-423 by SAD-A, triggering the onset of GSIS from islet β-cells. Consequently, ablation of PAK1 kinase activity or depletion of PAK1 expression completely abolishes the potentiating effect of SAD-A on GSIS. Consistent with its role in regulating GSIS, overexpression of SAD-A in MIN6 islet β-cells significantly stimulated cytoskeletal remodeling, which is required for insulin exocytosis. Together, the present studies identified a critical role of SAD-A in the activation of PAK1 during the onset of insulin exocytosis. PMID:22669945

Synapses of amphids defective (SAD-A) kinase promotes glucose-stimulated insulin secretion through activation of p21-activated kinase (PAK1) in pancreatic β-Cells.

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Nie, Jia; Sun, Chao; Faruque, Omar; Ye, Guangming; Li, Jia; Liang, Qiangrong; Chang, Zhijie; Yang, Wannian; Han, Xiao; Shi, Yuguang

2012-07-27

The p21-activated kinase-1 (PAK1) is implicated in regulation of insulin exocytosis as an effector of Rho GTPases. PAK1 is activated by the onset of glucose-stimulated insulin secretion (GSIS) through phosphorylation of Thr-423, a major activation site by Cdc42 and Rac1. However, the kinase(s) that phosphorylates PAK1 at Thr-423 in islet β-cells remains elusive. The present studies identified SAD-A (synapses of amphids defective), a member of AMP-activated protein kinase-related kinases exclusively expressed in brain and pancreas, as a key regulator of GSIS through activation of PAK1. We show that SAD-A directly binds to PAK1 through its kinase domain. The interaction is mediated by the p21-binding domain (PBD) of PAK1 and requires both kinases in an active conformation. The binding leads to direct phosphorylation of PAK1 at Thr-423 by SAD-A, triggering the onset of GSIS from islet β-cells. Consequently, ablation of PAK1 kinase activity or depletion of PAK1 expression completely abolishes the potentiating effect of SAD-A on GSIS. Consistent with its role in regulating GSIS, overexpression of SAD-A in MIN6 islet β-cells significantly stimulated cytoskeletal remodeling, which is required for insulin exocytosis. Together, the present studies identified a critical role of SAD-A in the activation of PAK1 during the onset of insulin exocytosis.

2-Aminopyridine-Based Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Inhibitors: Assessment of Mechanism-Based Safety.

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Dow, Robert L; Ammirati, Mark; Bagley, Scott W; Bhattacharya, Samit K; Buckbinder, Leonard; Cortes, Christian; El-Kattan, Ayman F; Ford, Kristen; Freeman, Gary B; Guimarães, Cristiano R W; Liu, Shenping; Niosi, Mark; Skoura, Athanasia; Tess, David

2018-04-12

Studies have linked the serine-threonine kinase MAP4K4 to the regulation of a number of biological processes and/or diseases, including diabetes, cancer, inflammation, and angiogenesis. With a majority of the members of our lead series (e.g., 1) suffering from time-dependent inhibition (TDI) of CYP3A4, we sought design avenues that would eliminate this risk. One such approach arose from the observation that carboxylic acid-based intermediates employed in our discovery efforts retained high MAP4K4 inhibitory potency and were devoid of the TDI risk. The medicinal chemistry effort that led to the discovery of this central nervous system-impaired inhibitor together with its preclinical safety profile is described.

The Ste20 Family Kinases MAP4K4, MINK1, and TNIK Converge to Regulate Stress-Induced JNK Signaling in Neurons.

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Larhammar, Martin; Huntwork-Rodriguez, Sarah; Rudhard, York; Sengupta-Ghosh, Arundhati; Lewcock, Joseph W

2017-11-15

The c-Jun- N -terminal kinase (JNK) signaling pathway regulates nervous system development, axon regeneration, and neuronal degeneration after acute injury or in chronic neurodegenerative disease. Dual leucine zipper kinase (DLK) is required for stress-induced JNK signaling in neurons, yet the factors that initiate DLK/JNK pathway activity remain poorly defined. In the present study, we identify the Ste20 kinases MAP4K4, misshapen-like kinase 1 (MINK1 or MAP4K6) and TNIK Traf2- and Nck-interacting kinase (TNIK or MAP4K7), as upstream regulators of DLK/JNK signaling in neurons. Using a trophic factor withdrawal-based model of neurodegeneration in both male and female embryonic mouse dorsal root ganglion neurons, we show that MAP4K4, MINK1, and TNIK act redundantly to regulate DLK activation and downstream JNK-dependent phosphorylation of c-Jun in response to stress. Targeting MAP4K4, MINK1, and TNIK, but not any of these kinases individually, is sufficient to protect neurons potently from degeneration. Pharmacological inhibition of MAP4Ks blocks stabilization and phosphorylation of DLK within axons and subsequent retrograde translocation of the JNK signaling complex to the nucleus. These results position MAP4Ks as important regulators of the DLK/JNK signaling pathway. SIGNIFICANCE STATEMENT Neuronal degeneration occurs in disparate circumstances: during development to refine neuronal connections, after injury to clear damaged neurons, or pathologically during disease. The dual leucine zipper kinase (DLK)/c-Jun- N -terminal kinase (JNK) pathway represents a conserved regulator of neuronal injury signaling that drives both neurodegeneration and axon regeneration, yet little is known about the factors that initiate DLK activity. Here, we uncover a novel role for a subfamily of MAP4 kinases consisting of MAP4K4, Traf2- and Nck-interacting kinase (TNIK or MAP4K7), and misshapen-like kinase 1 (MINK1 or MAP4K6) in regulating DLK/JNK signaling in neurons. Inhibition of

Recent Advances of Colony-Stimulating Factor-1 Receptor (CSF-1R) Kinase and Its Inhibitors.

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El-Gamal, Mohammed I; Al-Ameen, Shahad K; Al-Koumi, Dania M; Hamad, Mawadda G; Jalal, Nouran A; Oh, Chang-Hyun

2018-01-17

Colony stimulation factor-1 receptor (CSF-1R), which is also known as FMS kinase, plays an important role in initiating inflammatory, cancer, and bone disorders when it is overstimulated by its ligand, CSF-1. Innate immunity, as well as macrophage differentiation and survival, are regulated by the stimulation of the CSF-1R. Another ligand, interlukin-34 (IL-34), was recently reported to activate the CSF-1R receptor in a different manner. The relationship between CSF-1R and microglia has been reviewed. Both CSF-1 antibodies and small molecule CSF-1R kinase inhibitors have now been tested in animal models and in humans. In this Perspective, we discuss the role of CSF-1 and IL-34 in producing cancer, bone disorders, and inflammation. We also review the newly discovered and improved small molecule kinase inhibitors and monoclonal antibodies that have shown potent activity toward CSF-1R, reported from 2012 until 2017.

Loss of mitogen-activated protein kinase kinase kinase 4 (MAP3K4 reveals a requirement for MAPK signalling in mouse sex determination.

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Debora Bogani

2009-09-01

Full Text Available 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

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

Science.gov (United States)

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

Deep Brain Stimulation for Pantothenate Kinase-Associated Neurodegeneration

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Pedro J. Garcia-Ruiz

2015-01-01

Full Text Available Pantothenate kinase-associated neurodegeneration (PKAN is usually associated with dystonia, which is typically severe and progressive over time. Pallidal stimulation (GPi DBS has been carried out in selected cases of PKAN with drug-resistant dystonia with variable results. We report a 30-month follow-up study of a 30-year-old woman with PKAN-related dystonia treated with GPi DBS. Postoperatively, the benefit quickly became evident, as the patient exhibited a marked improvement in her dystonia, including her writing difficulty. This result has been maintained up to the present. GPi DBS should be considered in dystonic PKAN patients provided fixed contractures and/or pyramidal symptoms are not present.

RhoA–Rho kinase and Platelet Activating Factor Stimulation of Ovine Fetal Pulmonary Vascular Smooth Muscle Cell Proliferation

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Renteria, Lissette S.; Austin, Monique; Lazaro, Mariecon; Andrews, Mari Ashley; Lustina, Jennessee; Raj, J. Usha; Ibe, Basil O.

2013-01-01

Objectives Platelet Activating Factor (PAF) is produced by pulmonary vascular smooth muscle Cells (PVSMC). We studied effect of Rho kinase on PAF stimulation of PVSMC proliferation in an attempt to understand a role for RhoA/Rho kinase on PAF-induced ovine fetal pulmonary vascular remodeling. Our hypothesis is that PAF acts through Rho kinase, as one of its downstream signaling, to induce arterial (SMC-PA) and venous (SMC-PV) growth in the hypoxic lung environment of the fetus in utero. Materials and methods Rho kinase and MAPK effects on PAF receptor (PAFR)-mediated cell growth and PAFR expression were studied by DNA synthesis, Western and immunocytochemistry. Effects of constructs T19N and G14V on PAF-induced cell proliferation was also studied. Results Hypoxia increased PVSMC proliferation and the Rho kinase inhibitors, Y-27632 and Fasudil (HA-1077) as well as MAPK inhibitors PD 98059 and SB 203580 attenuated PAF stimulation of cell proliferation. RhoA T19N and G14V stimulated cell proliferation, but co-incubation with PAF did not affect proliferative effects of the constructs. PAFR protein expression was significantly down-regulated in both cell types by both Y-27632 and HA-1077 with comparable profiles. Also cells treated with Y-27632 showed less PAF receptor fluorescence with significant disruption of the cell morphology. Conclusions Our results show that Rho kinase nonspecifically modulates PAFR-mediated responses via a translational modification of PAFR protein and suggest that, in vivo, activation of Rho kinase by PAF may be one other pathway to sustain PAFR-mediated PVSMC growth. PMID:24033386

RhoA-Rho kinase and platelet-activating factor stimulation of ovine foetal pulmonary vascular smooth muscle cell proliferation.

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Renteria, L S; Austin, M; Lazaro, M; Andrews, M A; Lustina, J; Raj, J U; Ibe, B O

2013-10-01

Platelet-activating factor (PAF) is produced by pulmonary vascular smooth muscle cells (PVSMC). We studied effects of Rho kinase on PAF stimulation of PVSMC proliferation in an attempt to understand the role of RhoA/Rho kinase on PAF-induced ovine foetal pulmonary vascular remodelling. Our hypothesis is that PAF acts through Rho kinase, as one of its downstream signals, to induce arterial (SMC-PA) and venous (SMC-PV) cell proliferation in the hypoxic lung environment of the foetus, in utero. Rho kinase and MAPK effects on PAF receptor (PAFR)-mediated cell population expansion, and PAFR expression, were studied by DNA synthesis, western blot analysis and immunocytochemistry. Effects of constructs T19N and G14V on PAF-induced cell proliferation were also investigated. Hypoxia increased PVSMC proliferation and Rho kinase inhibitors, Y-27632 and Fasudil (HA-1077) as well as MAPK inhibitors PD 98059 and SB 203580 attenuated PAF stimulation of cell proliferation. RhoA T19N and G14V stimulated cell proliferation, but co-incubation with PAF did not affect proliferative effects of the constructs. PAFR protein expression was significantly downregulated in both cell types by both Y-27632 and HA-1077, with comparable profiles. Also, cells treated with Y-27632 had less PAF receptor fluorescence with significant disruption of cell morphology. Our results show that Rho kinase non-specifically modulated PAFR-mediated responses by a translational modification of PAFR protein, and suggest that, in vivo, activation of Rho kinase by PAF may be a further pathway to sustain PAFR-mediated PVSMC proliferation. © 2013 John Wiley & Sons Ltd.

Trichinella spiralis infection enhances protein kinase C phosphorylation in guinea pig alveolar macrophages.

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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.

Regulation of peripheral inflammation by spinal p38 MAP kinase in rats.

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David L Boyle

2006-09-01

Full Text Available Somatic afferent input to the spinal cord from a peripheral inflammatory site can modulate the peripheral response. However, the intracellular signaling mechanisms in the spinal cord that regulate this linkage have not been defined. Previous studies suggest spinal cord p38 mitogen-activated protein (MAP kinase and cytokines participate in nociceptive behavior. We therefore determined whether these pathways also regulate peripheral inflammation in rat adjuvant arthritis, which is a model of rheumatoid arthritis.Selective blockade of spinal cord p38 MAP kinase by administering the p38 inhibitor SB203580 via intrathecal (IT catheters in rats with adjuvant arthritis markedly suppressed paw swelling, inhibited synovial inflammation, and decreased radiographic evidence of joint destruction. The same dose of SB203580 delivered systemically had no effect, indicating that the effect was mediated by local concentrations in the neural compartment. Evaluation of articular gene expression by quantitative real-time PCR showed that spinal p38 inhibition markedly decreased synovial interleukin-1 and -6 and matrix metalloproteinase (MMP3 gene expression. Activation of p38 required tumor necrosis factor alpha (TNFalpha in the nervous system because IT etanercept (a TNF inhibitor given during adjuvant arthritis blocked spinal p38 phosphorylation and reduced clinical signs of adjuvant arthritis.These data suggest that peripheral inflammation is sensed by the central nervous system (CNS, which subsequently activates stress-induced kinases in the spinal cord via a TNFalpha-dependent mechanism. Intracellular p38 MAP kinase signaling processes this information and profoundly modulates somatic inflammatory responses. Characterization of this mechanism could have clinical and basic research implications by supporting development of new treatments for arthritis and clarifying how the CNS regulates peripheral immune responses.

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

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Horacio Bach

2018-01-01

Full Text Available 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.

Eotaxin induces degranulation and chemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases

DEFF Research Database (Denmark)

Kampen, G T; Stafford, S; Adachi, T

2000-01-01

Eotaxin and other CC chemokines acting via CC chemokine receptor-3 (CCR3) are believed to play an integral role in the development of eosinophilic inflammation in asthma and allergic inflammatory diseases. However, little is known about the intracellular events following agonist binding to CCR3...... and the relationship of these events to the functional response of the cell. The objectives of this study were to investigate CCR3-mediated activation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase-2 (ERK2), p38, and c-jun N-terminal kinase (JNK) in eosinophils and to assess...... the requirement for MAP kinases in eotaxin-induced eosinophil cationic protein (ECP) release and chemotaxis. MAP kinase activation was studied in eotaxin-stimulated eosinophils (more than 97% purity) by Western blotting and immune-complex kinase assays. ECP release was measured by radioimmunoassay. Chemotaxis...

Phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) is an AMPK target participating in contraction-stimulated glucose uptake in skeletal muscle.

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Liu, Yang; Lai, Yu-Chiang; Hill, Elaine V; Tyteca, Donatienne; Carpentier, Sarah; Ingvaldsen, Ada; Vertommen, Didier; Lantier, Louise; Foretz, Marc; Dequiedt, Franck; Courtoy, Pierre J; Erneux, Christophe; Viollet, Benoît; Shepherd, Peter R; Tavaré, Jeremy M; Jensen, Jørgen; Rider, Mark H

2013-10-15

PIKfyve (FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase), the lipid kinase that phosphorylates PtdIns3P to PtdIns(3,5)P2, has been implicated in insulin-stimulated glucose uptake. We investigated whether PIKfyve could also be involved in contraction/AMPK (AMP-activated protein kinase)-stimulated glucose uptake in skeletal muscle. Incubation of rat epitrochlearis muscles with YM201636, a selective PIKfyve inhibitor, reduced contraction- and AICAriboside (5-amino-4-imidazolecarboxamide riboside)-stimulated glucose uptake. Consistently, PIKfyve knockdown in C2C12 myotubes reduced AICAriboside-stimulated glucose transport. Furthermore, muscle contraction increased PtdIns(3,5)P2 levels and PIKfyve phosphorylation. AMPK phosphorylated PIKfyve at Ser307 both in vitro and in intact cells. Following subcellular fractionation, PIKfyve recovery in a crude intracellular membrane fraction was increased in contracting versus resting muscles. Also in opossum kidney cells, wild-type, but not S307A mutant, PIKfyve was recruited to endosomal vesicles in response to AMPK activation. We propose that PIKfyve activity is required for the stimulation of skeletal muscle glucose uptake by contraction/AMPK activation. PIKfyve is a new AMPK substrate whose phosphorylation at Ser307 could promote PIKfyve translocation to endosomes for PtdIns(3,5)P2 synthesis to facilitate GLUT4 (glucose transporter 4) translocation.

The Amoebal MAP Kinase Response to Legionella pneumophila Is Regulated by DupA

OpenAIRE

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

2009-01-01

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...

Presurgical mapping with functional MRI. Comparative study with transcranial magnetic stimulation and intraoperative mapping

Energy Technology Data Exchange (ETDEWEB)

Kaminogo, Makio; Morikawa, Minoru; Ishimaru, Hideki; Ochi, Makoto; Onizuka, Masanori; Shirakawa, Yasushi; Takahashi, Haruki; Shibata, Shobu [Nagasaki Univ. (Japan). School of Medicine

1999-05-01

The thumb movement was evoked by transcranical magnetic stimulation (TCS) for the mapping of the motor cortex. After the placement of the marker determined by TCS on the scalp, fMRI under motor tasks consisting of repetitive grasping was performed. For motor cortex activation, an axial oblique plane to maximize gray matter sampling in the rolandic cortex was employed in order to compare these different mapping techniques more precisely. Sixteen patients with brain tumors were included in this study. In nine patients, fMRI disclosed activation in one restricted gyrus or in the localized area around one restricted sulcus. Of these nine patients, preoperative TCS mapping corresponded closely with fMRI in six, while in the remaining three, the TCS marker fell between 1 and 2 cm apart from the fMRI-activated area. However, in these three patients, intraoperative electrocortical stimulation corresponded with the preoperative mapping with fMRI. In six patients, contiguous two gyri were activated by motor tasks. The TCS marker was disclosed on one of the two activated gyri. Of these six patients, the position of the TCS marker and fMRI-activated site corresponded with each other in four cases. They were found on the same gyrus but there was 1.0-2.0 cm distance between them in two cases. Intraoperative somatosensory evoked potential was monitored in two of these six cases. They corresponded well with the mapping by fMRI and TCS together. In only one patient, no significant activation area was obtained by fMRI because of excessive head motion during motor tasks. The TCS maker in this patients was identical with intraoperative electro-cortical stimulation mapping. (K.H.)

Presurgical mapping with functional MRI. Comparative study with transcranial magnetic stimulation and intraoperative mapping

International Nuclear Information System (INIS)

Kaminogo, Makio; Morikawa, Minoru; Ishimaru, Hideki; Ochi, Makoto; Onizuka, Masanori; Shirakawa, Yasushi; Takahashi, Haruki; Shibata, Shobu

1999-01-01

The thumb movement was evoked by transcranical magnetic stimulation (TCS) for the mapping of the motor cortex. After the placement of the marker determined by TCS on the scalp, fMRI under motor tasks consisting of repetitive grasping was performed. For motor cortex activation, an axial oblique plane to maximize gray matter sampling in the rolandic cortex was employed in order to compare these different mapping techniques more precisely. Sixteen patients with brain tumors were included in this study. In nine patients, fMRI disclosed activation in one restricted gyrus or in the localized area around one restricted sulcus. Of these nine patients, preoperative TCS mapping corresponded closely with fMRI in six, while in the remaining three, the TCS marker fell between 1 and 2 cm apart from the fMRI-activated area. However, in these three patients, intraoperative electrocortical stimulation corresponded with the preoperative mapping with fMRI. In six patients, contiguous two gyri were activated by motor tasks. The TCS marker was disclosed on one of the two activated gyri. Of these six patients, the position of the TCS marker and fMRI-activated site corresponded with each other in four cases. They were found on the same gyrus but there was 1.0-2.0 cm distance between them in two cases. Intraoperative somatosensory evoked potential was monitored in two of these six cases. They corresponded well with the mapping by fMRI and TCS together. In only one patient, no significant activation area was obtained by fMRI because of excessive head motion during motor tasks. The TCS maker in this patients was identical with intraoperative electro-cortical stimulation mapping. (K.H.)

Mitogen activated protein kinases selectively regulate palytoxin-stimulated gene expression in mouse keratinocytes

International Nuclear Information System (INIS)

Zeliadt, Nicholette A.; Warmka, Janel K.; Wattenberg, Elizabeth V.

2003-01-01

We have been investigating how the novel skin tumor promoter palytoxin transmits signals through mitogen activated protein kinases (MAPKs). Palytoxin activates three major MAPKs, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, in a keratinocyte cell line derived from initiated mouse skin (308). We previously showed that palytoxin requires ERK to increase matrix metalloproteinase-13 (MMP-13) gene expression, an enzyme implicated in carcinogenesis. Diverse stimuli require JNK and p38 to increase MMP-13 gene expression, however. We therefore used the JNK and p38 inhibitors SP 600125 and SB 202190, respectively, to investigate the role of these MAPKs in palytoxin-induced MMP-13 gene expression. Surprisingly, palytoxin does not require JNK and p38 to increase MMP-13 gene expression. Accordingly, ERK activation, independent of palytoxin and in the absence of JNK and p38 activation, is sufficient to induce MMP-13 gene expression in 308 keratinocytes. Dexamethasone, a synthetic glucocorticoid that inhibits activator protein-1 (AP-1), blocked palytoxin-stimulated MMP-13 gene expression. Therefore, the AP-1 site present in the promoter of the MMP-13 gene appears to be functional and to play a key role in palytoxin-stimulated gene expression. Previous studies showed that palytoxin simulates an ERK-dependent selective increase in the c-Fos content of AP-1 complexes that bind to the promoter of the MMP-13 gene. JNK and p38 can also modulate c-Fos. Palytoxin does not require JNK or p38 to increase c-Fos binding, however. Altogether, these studies indicate that ERK plays a distinctly essential role in transmitting palytoxin-stimulated signals to specific nuclear targets in keratinocytes derived from initiated mouse skin

Arabidopsis MAP kinase 4 negatively regulates systemic acquired resistance

DEFF Research Database (Denmark)

Petersen, M.; Brodersen, P.; Naested, H.

2000-01-01

Transposon inactivation of Arabidopsis MAP kinase 4 produced the mpk4 mutant exhibiting constitutive systemic acquired resistance (SAR) including elevated salicylic acid (SA) revels, increased resistance to virulent pathogens, and constitutive pathogenesis-related gene expression shown by Northern...... of NPR1. PDF1.2 and THI2.1 gene induction by jasmonate was blocked in mpk4 expressing NahG, suggesting that MPK4 is required for jasmonic acid-responsive gene expression....

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

Energy Technology Data Exchange (ETDEWEB)

Xing, Li; Shieh, Huey S.; Selness, Shaun R.; Devraj, Rajesh V.; Walker, John K.; Devadas, Balekudru; Hope, Heidi R.; Compton, Robert P.; Schindler, John F.; Hirsch, Jeffrey L.; Benson, Alan G.; Kurumbail, Ravi G.; Stegeman, Roderick A.; Williams, Jennifer M.; Broadus, Richard M.; Walden, Zara; Monahan, Joseph B.; Pfizer

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 the 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.

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

Science.gov (United States)

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

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

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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.

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

Energy Technology Data Exchange (ETDEWEB)

Comess, Kenneth M.; Sun, Chaohong; Abad-Zapatero, Cele; Goedken, Eric R.; Gum, Rebecca J.; Borhani, David W.; Argiriadi, Maria; Groebe, Duncan R.; Jia, Yong; Clampit, Jill E.; Haasch, Deanna L.; Smith, Harriet T.; Wang, Sanyi; Song, Danying; Coen, Michael L.; Cloutier, Timothy E.; Tang, Hua; Cheng, Xueheng; Quinn, Christopher; Liu, Bo; Xin, Zhili; Liu, Gang; Fry, Elizabeth H.; Stoll, Vincent; Ng, Teresa I.; Banach, David; Marcotte, Doug; Burns, David J.; Calderwood, David J.; Hajduk, Philip J. (Abbott)

2012-03-02

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 on 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

RhMKK9, a rose MAP KINASE KINASE gene, is involved in rehydration-triggered ethylene production in rose gynoecia.

Science.gov (United States)

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.

Mitogen-Activated Protein Kinase 8 (MAP3K8) Mediates the Signaling Pathway of Estradiol Stimulating Progesterone Production Through G Protein-Coupled Receptor 30 (GPR30) in Mouse Corpus Luteum.

Science.gov (United States)

Liu, Ying; Li, Yueqin; Zhang, Di; Liu, Jiali; Gou, Kemian; Cui, Sheng

2015-05-01

The corpus luteum (CL) is a transient endocrine gland developed from the ovulated follicles, and the most important function is to synthesize and secrete progesterone (P(4)), a key hormone to maintain normal pregnancy and estrous cycle in most mammals. It is known that estrogen has a vital role in stimulating P(4) synthesis in CL, but it still remains unclear about the mechanism of estradiol (E(2)) regulating P(4) production in CL. Our results here first show that all of the CL cells express MAPK 8 (MAP3K8), and the MAP3K8 level is much higher at the midstage than at the early and late stages during CL development. The further functional studies show that the forced inhibition of endogenous MAP3K8 by using MAP3K8 small interfering RNA and MAP3K8 signaling inhibitor (MAP3K8i) in the luteal cells significantly block the P(4) synthesis and neutralize the enhancing effect of E(2) on P(4) production in the CL. In addition, our results here demonstrate that the stimulating effect of E(2) on P(4) synthesis relies on the estrogen no-classical protein-coupled receptor 30, and MAP3K8 is involved in mediating the protein-coupled receptor 30signaling of E(2) affecting P(4) synthesis via stimulating ERK phosphorylation. These novel findings are critical for our understanding the ovary physiology and pathological mechanism.

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

DEFF Research Database (Denmark)

Andreasson, E.; Jenkins, T.; Brodersen, P.

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...

Platelet-activating factor stimulation of tyrosine kinase and its relationship to phospholipase C in rabbit platelets: Studies with genistein and monoclonal antibody to phosphotyrosine

International Nuclear Information System (INIS)

Dhar, A.; Paul, A.K.; Shukla, S.D.

1990-01-01

Platelet-activating factor (PAF) is a proinflammatory lipid that has platelet-stimulating property. PAF receptor-coupled activation of phosphoinositide-specific phospholipase C (PLC) and phosphorylation of several proteins has already been established in our laboratory. To investigate further the molecular mechanism and relationship between activation of PLC and protein phosphorylation, we have used Genistein (a putative inhibitor of tyrosine-specific protein kinases), phosphotyrosine antibody, and phosphoamino acid analysis to probe the involvement of tyrosine kinase in this process. Washed rabbit platelets were loaded with myo-[2-3H]inositol and challenged with PAF (100 nM) after pretreatment with Genistein. PLC-mediated production of radioactive inositol monophosphate, inositol diphosphate, and inositol triphosphate was monitored. PAF alone caused stimulation of PLC activity [( 3H]inositol triphosphate production), whereas pretreatment with Genistein (0.5 mM) diminished PAF-stimulated PLC activity to basal level. Genistein also blocked PAF-stimulated platelet aggregation at this dose. In contrast to Genistein, staurosporine which inhibits protein kinase C, potentiated PAF-stimulated [3H]inositol triphosphate production. Genistein substantially inhibited the combined effects of staurosporine and PAF on inositol triphosphate production. Genistein also reduced PAF-induced phosphorylation of Mr 20,000 and 50,000 proteins. Phorbol 12-myristate 13-acetate-induced Mr 40,000 protein phosphorylation was also affected by Genistein. The above results suggested that Genistein inhibited tyrosine kinase at an early stage of signal transduction by inhibiting PLC. This, in turn, decreased the activation of protein kinase C and, therefore, caused a reduction in Mr 40,000 protein phosphorylation

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

Science.gov (United States)

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

Combined noninvasive language mapping by navigated transcranial magnetic stimulation and functional MRI and its comparison with direct cortical stimulation.

Science.gov (United States)

Ille, Sebastian; Sollmann, Nico; Hauck, Theresa; Maurer, Stefanie; Tanigawa, Noriko; Obermueller, Thomas; Negwer, Chiara; Droese, Doris; Zimmer, Claus; Meyer, Bernhard; Ringel, Florian; Krieg, Sandro M

2015-07-01

Repetitive navigated transcranial magnetic stimulation (rTMS) is now increasingly used for preoperative language mapping in patients with lesions in language-related areas of the brain. Yet its correlation with intraoperative direct cortical stimulation (DCS) has to be improved. To increase rTMS's specificity and positive predictive value, the authors aim to provide thresholds for rTMS's positive language areas. Moreover, they propose a protocol for combining rTMS with functional MRI (fMRI) to combine the strength of both methods. The authors performed multimodal language mapping in 35 patients with left-sided perisylvian lesions by using rTMS, fMRI, and DCS. The rTMS mappings were conducted with a picture-to-trigger interval (PTI, time between stimulus presentation and stimulation onset) of either 0 or 300 msec. The error rates (ERs; that is, the number of errors per number of stimulations) were calculated for each region of the cortical parcellation system (CPS). Subsequently, the rTMS mappings were analyzed through different error rate thresholds (ERT; that is, the ER at which a CPS region was defined as language positive in terms of rTMS), and the 2-out-of-3 rule (a stimulation site was defined as language positive in terms of rTMS if at least 2 out of 3 stimulations caused an error). As a second step, the authors combined the results of fMRI and rTMS in a predefined protocol of combined noninvasive mapping. To validate this noninvasive protocol, they correlated its results to DCS during awake surgery. The analysis by different rTMS ERTs obtained the highest correlation regarding sensitivity and a low rate of false positives for the ERTs of 15%, 20%, 25%, and the 2-out-of-3 rule. However, when comparing the combined fMRI and rTMS results with DCS, the authors observed an overall specificity of 83%, a positive predictive value of 51%, a sensitivity of 98%, and a negative predictive value of 95%. In comparison with fMRI, rTMS is a more sensitive but less specific

Parietotemporal Stimulation Affects Acquisition of Novel Grapheme-Phoneme Mappings in Adult Readers

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Jessica W. Younger

2018-03-01

Full Text Available Neuroimaging work from developmental and reading intervention research has suggested a cause of reading failure may be lack of engagement of parietotemporal cortex during initial acquisition of grapheme-phoneme (letter-sound mappings. Parietotemporal activation increases following grapheme-phoneme learning and successful reading intervention. Further, stimulation of parietotemporal cortex improves reading skill in lower ability adults. However, it is unclear whether these improvements following stimulation are due to enhanced grapheme-phoneme mapping abilities. To test this hypothesis, we used transcranial direct current stimulation (tDCS to manipulate parietotemporal function in adult readers as they learned a novel artificial orthography with new grapheme-phoneme mappings. Participants received real or sham stimulation to the left inferior parietal lobe (L IPL for 20 min before training. They received explicit training over the course of 3 days on 10 novel words each day. Learning of the artificial orthography was assessed at a pre-training baseline session, the end of each of the three training sessions, an immediate post-training session and a delayed post-training session about 4 weeks after training. Stimulation interacted with baseline reading skill to affect learning of trained words and transfer to untrained words. Lower skill readers showed better acquisition, whereas higher skill readers showed worse acquisition, when training was paired with real stimulation, as compared to readers who received sham stimulation. However, readers of all skill levels showed better maintenance of trained material following parietotemporal stimulation, indicating a differential effect of stimulation on initial learning and consolidation. Overall, these results indicate that parietotemporal stimulation can enhance learning of new grapheme-phoneme relationships in readers with lower reading skill. Yet, while parietotemporal function is critical to new

Parietotemporal Stimulation Affects Acquisition of Novel Grapheme-Phoneme Mappings in Adult Readers

Science.gov (United States)

Younger, Jessica W.; Booth, James R.

2018-01-01

Neuroimaging work from developmental and reading intervention research has suggested a cause of reading failure may be lack of engagement of parietotemporal cortex during initial acquisition of grapheme-phoneme (letter-sound) mappings. Parietotemporal activation increases following grapheme-phoneme learning and successful reading intervention. Further, stimulation of parietotemporal cortex improves reading skill in lower ability adults. However, it is unclear whether these improvements following stimulation are due to enhanced grapheme-phoneme mapping abilities. To test this hypothesis, we used transcranial direct current stimulation (tDCS) to manipulate parietotemporal function in adult readers as they learned a novel artificial orthography with new grapheme-phoneme mappings. Participants received real or sham stimulation to the left inferior parietal lobe (L IPL) for 20 min before training. They received explicit training over the course of 3 days on 10 novel words each day. Learning of the artificial orthography was assessed at a pre-training baseline session, the end of each of the three training sessions, an immediate post-training session and a delayed post-training session about 4 weeks after training. Stimulation interacted with baseline reading skill to affect learning of trained words and transfer to untrained words. Lower skill readers showed better acquisition, whereas higher skill readers showed worse acquisition, when training was paired with real stimulation, as compared to readers who received sham stimulation. However, readers of all skill levels showed better maintenance of trained material following parietotemporal stimulation, indicating a differential effect of stimulation on initial learning and consolidation. Overall, these results indicate that parietotemporal stimulation can enhance learning of new grapheme-phoneme relationships in readers with lower reading skill. Yet, while parietotemporal function is critical to new learning, its role in

Negative regulation of MAP kinase signaling in Drosophila by Ptp61F/PTP1B.

Science.gov (United States)

Tchankouo-Nguetcheu, Stéphane; Udinotti, Mario; Durand, Marjorie; Meng, Tzu-Ching; Taouis, Mohammed; Rabinow, Leonard

2014-10-01

PTP1B is an important negative regulator of insulin and other signaling pathways in mammals. However, the role of PTP1B in the regulation of RAS-MAPK signaling remains open to deliberation, due to conflicting evidence from different experimental systems. The Drosophila orthologue of mammalian PTP1B, PTP61F, has until recently remained largely uncharacterized. To establish the potential role of PTP61F in the regulation of signaling pathways in Drosophila and particularly to help resolve its fundamental function in RAS-MAPK signaling, we generated a new allele of Ptp61F as well as employed both RNA interference and overexpression alleles. Our results validate recent data showing that the activity of insulin and Abl kinase signaling is increased in Ptp61F mutants and RNA interference lines. Importantly, we establish negative regulation of the RAS/MAPK pathway by Ptp61F activity in whole animals. Of particular interest, our results document the modulation of hyperactive MAP kinase activity by Ptp61F alleles, showing that the phosphatase intervenes to directly or indirectly regulate MAP kinase itself.

Hsp90 inhibition differentially destabilises MAP kinase and TGF-beta signalling components in cancer cells revealed by kinase-targeted chemoproteomics

International Nuclear Information System (INIS)

Haupt, Armin; Dahl, Andreas; Lappe, Michael; Lehrach, Hans; Gonzalez, Cayetano; Drewes, Gerard; Lange, Bodo MH; Joberty, Gerard; Bantscheff, Marcus; Fröhlich, Holger; Stehr, Henning; Schweiger, Michal R; Fischer, Axel; Kerick, Martin; Boerno, Stefan T

2012-01-01

The heat shock protein 90 (Hsp90) is required for the stability of many signalling kinases. As a target for cancer therapy it allows the simultaneous inhibition of several signalling pathways. However, its inhibition in healthy cells could also lead to severe side effects. This is the first comprehensive analysis of the response to Hsp90 inhibition at the kinome level. We quantitatively profiled the effects of Hsp90 inhibition by geldanamycin on the kinome of one primary (Hs68) and three tumour cell lines (SW480, U2OS, A549) by affinity proteomics based on immobilized broad spectrum kinase inhibitors ('kinobeads'). To identify affected pathways we used the KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway classification. We combined Hsp90 and proteasome inhibition to identify Hsp90 substrates in Hs68 and SW480 cells. The mutational status of kinases from the used cell lines was determined using next-generation sequencing. A mutation of Hsp90 candidate client RIPK2 was mapped onto its structure. We measured relative abundances of > 140 protein kinases from the four cell lines in response to geldanamycin treatment and identified many new potential Hsp90 substrates. These kinases represent diverse families and cellular functions, with a strong representation of pathways involved in tumour progression like the BMP, MAPK and TGF-beta signalling cascades. Co-treatment with the proteasome inhibitor MG132 enabled us to classify 64 kinases as true Hsp90 clients. Finally, mutations in 7 kinases correlate with an altered response to Hsp90 inhibition. Structural modelling of the candidate client RIPK2 suggests an impact of the mutation on a proposed Hsp90 binding domain. We propose a high confidence list of Hsp90 kinase clients, which provides new opportunities for targeted and combinatorial cancer treatment and diagnostic applications

Resveratrol stimulates AMP kinase activity in neurons.

Science.gov (United States)

Dasgupta, Biplab; Milbrandt, Jeffrey

2007-04-24

Resveratrol is a polyphenol produced by plants that has multiple beneficial activities similar to those associated with caloric restriction (CR), such as increased life span and delay in the onset of diseases associated with aging. CR improves neuronal health, and the global beneficial effects of CR have been postulated to be mediated by the nervous system. One key enzyme thought to be activated during CR is the AMP-activated kinase (AMPK), a sensor of cellular energy levels. AMPK is activated by increases in the cellular AMP:ATP ratio, whereupon it functions to help preserve cellular energy. In this regard, the regulation of dietary food intake by hypothalamic neurons is mediated by AMPK. The suppression of nonessential energy expenditure by activated AMPK along with the CR mimetic and neuroprotective properties of resveratrol led us to hypothesize that neuronal activation of AMPK could be an important component of resveratrol activity. Here, we show that resveratrol activated AMPK in Neuro2a cells and primary neurons in vitro as well as in the brain. Resveratrol and the AMPK-activating compound 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) promoted robust neurite outgrowth in Neuro2a cells, which was blocked by genetic and pharmacologic inhibition of AMPK. Resveratrol also stimulated mitochondrial biogenesis in an AMPK-dependent manner. Resveratrol-stimulated AMPK activity in neurons depended on LKB1 activity but did not require the NAD-dependent protein deacetylase SIRT1 during this time frame. These findings suggest that neuronal activation of AMPK by resveratrol could affect neuronal energy homeostasis and contribute to the neuroprotective effects of resveratrol.

Anti-inflammatory effect of longan seed extract in carrageenan stimulated Sprague-Dawley rats

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Ching-Hsiao Lee

2016-08-01

Full Text Available Objective(s: Longan seeds have been used as a folk medicine in China. Longan seed extract (LSE is known for antioxidative, antiproliferative, hypoglycemic, and hypouremic effects. However, its anti-inflammatory effect has not been shown. Materials and Methods: In this study, Sprague-Dawley (SD rats were given LSE orally (vehicle, 10, and 30 mg/kg for 3 days to its test anti-inflammatory effect by injecting λ-carrageenan (CARR in the right hind paw or lipopolysaccharide (LPS, IP. For the positive control, animals were given aspirin (20 mg/kg orally and treated likewise. Serum or tissue samples from treated rats were collected after 3 hr of stimulation. Regarding the in vitro study, BV2 microglial cells were stimulated with LPS in the presence of LSE or normal saline for 10 min or 24 hr for Western blot and ELISA assay, respectively. Results: LSE reduced CARR-induced edema in the experimental animals. LSE also reduced LPS/CARR-induced nitric oxide (NO, interleukin-1β (IL1β, IL6, and COX2 productions. These inflammatory factors were also reduced dose dependently by LSE in LPS-stimulated BV2 cells. Furthermore, Western blot analysis revealed that LSE inhibited LPS activated c-Jun NH2-terminal protein kinase (JNK, extracellular signal-regulated kinases (ERKs, and p38 MAP kinases signaling pathways, caspase-3, inducible NO synthase, and COX2 expressions. Conclusion: LSE pretreatment suppressed CARR- and LPS-induced inflammations and these effects might be through the inhibition of MAP kinases signaling pathways and inflammatory factors.

Tiam1-Rac1 Axis Promotes Activation of p38 MAP Kinase in the Development of Diabetic Retinopathy: Evidence for a Requisite Role for Protein Palmitoylation

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Rajakrishnan Veluthakal

2015-04-01

Full Text Available Background/Aims: Evidence in multiple tissues, including retina, suggests generation of reactive oxygen species (ROS and the ensuing oxidative stress as triggers for mitochondrial defects and cell apoptosis. We recently reported novel roles for Tiam1-Rac1-Nox2 axis in retinal mitochondrial dysfunction and cell death leading to the development of diabetic retinopathy. Herein, we tested the hypothesis that activation of p38 MAP kinase, a stress kinase, represents the downstream signaling event to Rac1-Nox2 activation in diabetes-induced metabolic stress leading to capillary cell apoptosis. Methods: Activation of p38 MAP kinase was quantified by Western blotting in retinal endothelial cells incubated with high glucose (20 mM for up to 96 hours, a duration where mitochondrial dysfunction and capillary cell apoptosis can be observed. NSC23766 and 2-bromopalmitate (2-BP were used to assess the roles of Tiam1-Rac1 and palmitoylation pathways, respectively. Results: Activation of p38 MAP kinase was observed as early as 3 hours after high glucose exposure, and continued until 96 hours. Consistent with this, p38 MAP kinase activation was significantly higher in the retina from diabetic mice compared to age-matched normal mice. NSC23766 markedly attenuated hyperglycemia-induced activation of p38 MAP kinase. Lastly, 2-BP inhibited glucose-induced Rac1, Nox2 and p38 MAP kinase activation in endothelial cells. Conclusions: Tiam1-Rac1-mediated activation of Nox2 and p38 MAP kinase constitutes early signaling events leading to mitochondrial dysfunction and the development of diabetic retinopathy. Our findings also provide the first evidence to implicate novel roles for protein palmitoylation in this signaling cascade.

Mechanism of activation of glycogen phosphorylase by fructose in the liver. Stimulation of phosphorylase kinase related to the consumption of adenosine triphosphate.

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Van de Werve, G; Hers, H G

1979-01-15

1. A dose-dependent activation of phosphorylase and consumption of ATP was observed in isolated hepatocytes incubated in the presence of fructose; histone kinase and phosphorylase kinase activities were unchanged at doses of this sugar that were fully effective on phosphorylase. The activation of phosphorylase by fructose was also observed in cells incubated in a Ca2+-free medium as well as in the livers of rats in vivo. 2. In a liver high-speed supernatant, fructose, tagatose and sorbose stimulated the activity of phosphorylase kinase; this effect was dependent on the presence of K+ ions, which are required for the activity of fructokinase; it was accompanied by the transformation of ATP into ADP. In the presence of hexokinase, glucose also stimulated phosphorylase kinase, both in an Na+ or a K+ medium. 3. The activities of partially purified muscle or liver phosphorylase kinase were unchanged in the presence of fructose. 4. Some properties of liver phosphorylase kinase are described, including a high molecular weight and an inhibition at ATP/Mg ratios above 0.5, as well as an effect of ATP concentration on the hysteretic behaviour of this enzyme. 5. The effect of fructose on the activation of phosphorylase is discussed in relation to the comsumption of ATP.

Pediatric awake craniotomy and intra-operative stimulation mapping.

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Balogun, James A; Khan, Osaama H; Taylor, Michael; Dirks, Peter; Der, Tara; Carter Snead Iii, O; Weiss, Shelly; Ochi, Ayako; Drake, James; Rutka, James T

2014-11-01

The indications for operating on lesions in or near areas of cortical eloquence balance the benefit of resection with the risk of permanent neurological deficit. In adults, awake craniotomy has become a versatile tool in tumor, epilepsy and functional neurosurgery, permitting intra-operative stimulation mapping particularly for language, sensory and motor cortical pathways. This allows for maximal tumor resection with considerable reduction in the risk of post-operative speech and motor deficits. We report our experience of awake craniotomy and cortical stimulation for epilepsy and supratentorial tumors located in and around eloquent areas in a pediatric population (n=10, five females). The presenting symptom was mainly seizures and all children had normal neurological examinations. Neuroimaging showed lesions in the left opercular (n=4) and precentral or peri-sylvian regions (n=6). Three right-sided and seven left-sided awake craniotomies were performed. Two patients had a history of prior craniotomy. All patients had intra-operative mapping for either speech or motor or both using cortical stimulation. The surgical goal for tumor patients was gross total resection, while for all epilepsy procedures, focal cortical resections were completed without any difficulty. None of the patients had permanent post-operative neurologic deficits. The patient with an epileptic focus over the speech area in the left frontal lobe had a mild word finding difficulty post-operatively but this improved progressively. Follow-up ranged from 6 to 27 months. Pediatric awake craniotomy with intra-operative mapping is a precise, safe and reliable method allowing for resection of lesions in eloquent areas. Further validations on larger number of patients will be needed to verify the utility of this technique in the pediatric population. Copyright © 2014 Elsevier Ltd. All rights reserved.

Mapping for Acute Transvenous Phrenic Nerve Stimulation Study (MAPS Study).

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Dekker, Lukas R C; Gerritse, Bart; Scheiner, Avram; Kornet, Lilian

2017-03-01

Central sleep apnea syndrome, correlated with the occurrence of heart failure, is characterized by periods of insufficient ventilation during sleep. This acute study in 15 patients aims to map the venous system and determine if diaphragmatic movement can be achieved by phrenic nerve stimulation at various locations within the venous system. Subjects underwent a scheduled catheter ablation procedure. During the procedural waiting time, one multielectrode electrophysiology catheter was subsequently placed at the superior and inferior vena cava and the junctions of the left jugular and left brachiocephalic vein and right jugular and right brachiocephalic vein, for phrenic nerve stimulation (1-2 seconds ON/2-3 seconds OFF, 40 Hz, pulse width 210 μs). Diaphragmatic movement was assessed manually and by a breathing mask. During a follow-up assessment between 2 and 4 weeks postprocedure, occurrence of adverse events was assessed. In all patients diaphragmatic movement was induced at one or more locations using a median threshold of at least 2 V and maximally 7.5 V (i.e., e 3.3 mA, 14.2 mA). The lowest median current to obtain diaphragmatic stimulation without discomfort was found for the right brachiocephalic vein (4.7 mA). In 12/15 patients diaphragmatic movement could be induced without any discomfort, but in three patients hiccups occurred. Diaphragmatic stimulation from the brachiocephalic and caval veins is feasible. Potential side effects should be eliminated by adapting the stimulation pattern. This information could be used to design a catheter, combining cardiac pacing with enhancing diaphragm movement during a sleep apnea episode. © 2017 Wiley Periodicals, Inc.

The MAP kinase Pmk1 and protein kinase A are required for rotenone resistance in the fission yeast, Schizosaccharomyces pombe

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Wang, Yiwei; Gulis, Galina; Buckner, Scott; Johnson, P. Connor; Sullivan, Daniel [Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487 (United States); Busenlehner, Laura [Department of Chemistry, The University of Alabama, Tuscaloosa, AL 35487 (United States); Marcus, Stevan, E-mail: smarcus@bama.ua.edu [Department of Biological Sciences, The University of Alabama, Tuscaloosa, AL 35487 (United States)

2010-08-20

Research highlights: {yields} Rotenone induces generation of ROS and mitochondrial fragmentation in fission yeast. {yields} The MAPK Pmk1 and PKA are required for rotenone resistance in fission yeast. {yields} Pmk1 and PKA are required for ROS clearance in rotenone treated fission yeast cells. {yields} PKA plays a role in ROS clearance under normal growth conditions in fission yeast. -- Abstract: Rotenone is a widely used pesticide that induces Parkinson's disease-like symptoms in rats and death of dopaminergic neurons in culture. Although rotenone is a potent inhibitor of complex I of the mitochondrial electron transport chain, it can induce death of dopaminergic neurons independently of complex I inhibition. Here we describe effects of rotenone in the fission yeast, Schizosaccharomyces pombe, which lacks complex I and carries out rotenone-insensitive cellular respiration. We show that rotenone induces generation of reactive oxygen species (ROS) as well as fragmentation of mitochondrial networks in treated S. pombe cells. While rotenone is only modestly inhibitory to growth of wild type S. pombe cells, it is strongly inhibitory to growth of mutants lacking the ERK-type MAP kinase, Pmk1, or protein kinase A (PKA). In contrast, cells lacking the p38 MAP kinase, Spc1, exhibit modest resistance to rotenone. Consistent with these findings, we provide evidence that Pmk1 and PKA, but not Spc1, are required for clearance of ROS in rotenone treated S. pombe cells. Our results demonstrate the usefulness of S. pombe for elucidating complex I-independent molecular targets of rotenone as well as mechanisms conferring resistance to the toxin.

The MAP kinase Pmk1 and protein kinase A are required for rotenone resistance in the fission yeast, Schizosaccharomyces pombe

International Nuclear Information System (INIS)

Wang, Yiwei; Gulis, Galina; Buckner, Scott; Johnson, P. Connor; Sullivan, Daniel; Busenlehner, Laura; Marcus, Stevan

2010-01-01

Research highlights: → Rotenone induces generation of ROS and mitochondrial fragmentation in fission yeast. → The MAPK Pmk1 and PKA are required for rotenone resistance in fission yeast. → Pmk1 and PKA are required for ROS clearance in rotenone treated fission yeast cells. → PKA plays a role in ROS clearance under normal growth conditions in fission yeast. -- Abstract: Rotenone is a widely used pesticide that induces Parkinson's disease-like symptoms in rats and death of dopaminergic neurons in culture. Although rotenone is a potent inhibitor of complex I of the mitochondrial electron transport chain, it can induce death of dopaminergic neurons independently of complex I inhibition. Here we describe effects of rotenone in the fission yeast, Schizosaccharomyces pombe, which lacks complex I and carries out rotenone-insensitive cellular respiration. We show that rotenone induces generation of reactive oxygen species (ROS) as well as fragmentation of mitochondrial networks in treated S. pombe cells. While rotenone is only modestly inhibitory to growth of wild type S. pombe cells, it is strongly inhibitory to growth of mutants lacking the ERK-type MAP kinase, Pmk1, or protein kinase A (PKA). In contrast, cells lacking the p38 MAP kinase, Spc1, exhibit modest resistance to rotenone. Consistent with these findings, we provide evidence that Pmk1 and PKA, but not Spc1, are required for clearance of ROS in rotenone treated S. pombe cells. Our results demonstrate the usefulness of S. pombe for elucidating complex I-independent molecular targets of rotenone as well as mechanisms conferring resistance to the toxin.

Torilin Inhibits Inflammation by Limiting TAK1-Mediated MAP Kinase and NF-κB Activation

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Mehari Endale

2017-01-01

Full Text Available Torilin, a sesquiterpene isolated from the fruits of Torilis japonica, has shown antimicrobial, anticancer, and anti-inflammatory properties. However, data on the mechanism of torilin action against inflammation is limited. This study aimed at determining the anti-inflammatory property of torilin in LPS-induced inflammation using in vitro model of inflammation. We examined torilin’s effect on expression levels of inflammatory mediators and cytokines in LPS-stimulated RAW 264.7 macrophages. The involvement of NF-kB and AP-1, MAP kinases, and adaptor proteins were assessed. Torilin strongly inhibited LPS-induced NO release, iNOS, PGE2, COX-2, NF-α, IL-1β, IL-6, and GM-CSF gene and protein expressions. In addition, MAPKs were also suppressed by torilin pretreatment. Involvement of ERK1/2, P38MAPK, and JNK1/2 was further confirmed by PD98059, SB203580, and SP600125 mediated suppression of iNOS and COX-2 proteins. Furthermore, torilin attenuated NF-kB and AP-1 translocation, DNA binding, and reporter gene transcription. Interestingly, torilin inhibited TAK1 kinase activation with the subsequent suppression of MAPK-mediated JNK, p38, ERK1/2, and AP-1 (ATF-2 and c-jun activation and IKK-mediated I-κBα degradation, p65/p50 activation, and translocation. Together, the results revealed the suppression of NF-κB and AP-1 regulated inflammatory mediator and cytokine expressions, suggesting the test compound’s potential as a candidate anti-inflammatory agent.

Intra-operative multi-site stimulation: Expanding methodology for cortical brain mapping of language functions.

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Gonen, Tal; Gazit, Tomer; Korn, Akiva; Kirschner, Adi; Perry, Daniella; Hendler, Talma; Ram, Zvi

2017-01-01

Direct cortical stimulation (DCS) is considered the gold-standard for functional cortical mapping during awake surgery for brain tumor resection. DCS is performed by stimulating one local cortical area at a time. We present a feasibility study using an intra-operative technique aimed at improving our ability to map brain functions which rely on activity in distributed cortical regions. Following standard DCS, Multi-Site Stimulation (MSS) was performed in 15 patients by applying simultaneous cortical stimulations at multiple locations. Language functioning was chosen as a case-cognitive domain due to its relatively well-known cortical organization. MSS, performed at sites that did not produce disruption when applied in a single stimulation point, revealed additional language dysfunction in 73% of the patients. Functional regions identified by this technique were presumed to be significant to language circuitry and were spared during surgery. No new neurological deficits were observed in any of the patients following surgery. Though the neuro-electrical effects of MSS need further investigation, this feasibility study may provide a first step towards sophistication of intra-operative cortical mapping.

Induced overexpression of protein kinase D1 stimulates mitogenic signaling in human pancreatic carcinoma PANC-1 cells.

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Kisfalvi, Krisztina; Hurd, Cliff; Guha, Sushovan; Rozengurt, Enrique

2010-05-01

Neurotensin (NT) stimulates protein kinase D1 (PKD1), extracellular signal regulated kinase (ERK), c-Jun N-terminal Kinase (JNK), and DNA synthesis in the human pancreatic adenocarcinoma cell line PANC-1. To determine the effect of PKD1 overexpression on these biological responses, we generated inducible stable PANC-1 clones that express wild-type (WT) or kinase-dead (K618N) forms of PKD1 in response to the ecdysone analog ponasterone-A (PonA). NT potently stimulated c-Jun Ser(63) phosphorylation in both wild type and clonal derivatives of PANC-1 cells. PonA-induced expression of WT, but not K618N PKD1, rapidly blocked NT-mediated c-Jun Ser(63) phosphorylation either at the level of or upstream of MKK4, a dual-specificity kinase that leads to JNK activation. This is the first demonstration that PKD1 suppresses NT-induced JNK/cJun activation in PANC-1 cells. In contrast, PKD1 overexpression markedly increased the duration of NT-induced ERK activation in these cells. The reciprocal influence of PKD1 signaling on pro-mitogenicERK and pro-apopotic JNK/c-Jun pathways prompted us to examine whether PKD1 overexpression promotes DNA synthesis and proliferation of PANC-1 cells. Our results show that PKD1 overexpression increased DNA synthesis and cell numbers of PANC-1 cells cultured in regular dishes or in polyhydroxyethylmethacrylate [Poly-(HEMA)]-coated dishes to eliminate cell adhesion (anchorage-independent growth). Furthermore, PKD1 overexpression markedly enhanced DNA synthesis induced by NT (1-10 nM). These results indicate that PKD1 mediates mitogenic signaling in PANC-1 and suggests that this enzyme could be a novel target for the development of therapeutic drugs that restrict the proliferation of these cells.

A probabilistic map of the human ventral sensorimotor cortex using electrical stimulation.

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Breshears, Jonathan D; Molinaro, Annette M; Chang, Edward F

2015-08-01

The human ventral sensorimotor cortex (vSMC) is involved in facial expression, mastication, and swallowing, as well as the dynamic and highly coordinated movements of human speech production. However, vSMC organization remains poorly understood, and previously published population-driven maps of its somatotopy do not accurately reflect the variability across individuals in a quantitative, probabilistic fashion. The goal of this study was to describe the responses to electrical stimulation of the vSMC, generate probabilistic maps of function in the vSMC, and quantify the variability across individuals. Photographic, video, and stereotactic MRI data of intraoperative electrical stimulation of the vSMC were collected for 33 patients undergoing awake craniotomy. Stimulation sites were converted to a 2D coordinate system based on anatomical landmarks. Motor, sensory, and speech stimulation responses were reviewed and classified. Probabilistic maps of stimulation responses were generated, and spatial variance was quantified. In 33 patients, the authors identified 194 motor, 212 sensory, 61 speech-arrest, and 27 mixed responses. Responses were complex, stereotyped, and mostly nonphysiological movements, involving hand, orofacial, and laryngeal musculature. Within individuals, the presence of oral movement representations varied; however, the dorsal-ventral order was always preserved. The most robust motor responses were jaw (probability 0.85), tongue (0.64), lips (0.58), and throat (0.52). Vocalizations were seen in 6 patients (0.18), more dorsally near lip and dorsal throat areas. Sensory responses were spatially dispersed; however, patients' subjective reports were highly precise in localization within the mouth. The most robust responses included tongue (0.82) and lips (0.42). The probability of speech arrest was 0.85, highest 15-20 mm anterior to the central sulcus and just dorsal to the sylvian fissure, in the anterior precentral gyrus or pars opercularis. The

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

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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

MgSlt2, a cellular integrity MAP kinase of the fungal wheat pathogen Mycosphaerella graminicola, is dispensable for penetration but essential for invasive growth

NARCIS (Netherlands)

Mehrabi, R.; Lee, van der T.A.J.; Waalwijk, C.; Kema, G.H.J.

2006-01-01

Among expressed sequence tag libraries of Mycosphaerella graminicola isolate IPO323, we identified a full-length cDNA clone with high homology to the mitogen-activated protein (MAP) kinase Slt2 in Saccharomyces cerevisiae. This MAP kinase consists of a 1,242-bp open reading frame, and encodes a

Electrical stimulation with periodic alternating intervals stimulates neuronal cells to produce neurotrophins and cytokines through activation of mitogen-activated protein kinase pathways.

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Yamamoto, Kenta; Yamamoto, Toshiro; Honjo, Kenichi; Ichioka, Hiroaki; Oseko, Fumishige; Kishida, Tsunao; Mazda, Osam; Kanamura, Narisato

2015-12-01

Peripheral neuropathy is a representative complication of dental surgery. Electrical therapy, based on electrical stimulation with periodic alternating intervals (ES-PAI), may promote nerve regeneration after peripheral nerve injury in a non-invasive manner, potentially providing an effective therapy for neuropathy. This study aimed to analyze the molecular mechanisms underlying the nerve recovery stimulated by ES-PAI. In brief, ES-PAI was applied to a neuronal cell line, Neuro2A, at various intensities using the pulse generator apparatus, FREUDE. Cell viability, neurotrophin mRNA expression, and cytokine production were examined using a tetrazolium-based assay, real-time RT-PCR, and ELISA, respectively. Mitogen-activated protein kinase (MAPK) signaling was assessed using flow cytometry. It was found that ES-PAI increased the viability of cells and elevated expression of nerve growth factor (NGF) and neurotrophin-3 (NT-3); ESPAI also augmented vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) expression, which was restored by addition of p38 inhibitors. Phosphorylation of p38 and extracellular signal-regulated kinase 1/2 (ERK-1/2) was augmented by ES-PAI. Hence, ES-PAI may ameliorate peripheral neuropathy by promoting neuronal cell proliferation and production of neurogenic factors by activating p38 and ERK-1/2 pathways. © 2015 Eur J Oral Sci.

Role of protein kinase C and epidermal growth factor receptor signalling in growth stimulation by neurotensin in colon carcinoma cells

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Dajani Olav

2011-10-01

Full Text Available Abstract Background Neurotensin has been found to promote colon carcinogenesis in rats and mice, and proliferation of human colon carcinoma cell lines, but the mechanisms involved are not clear. We have examined signalling pathways activated by neurotensin in colorectal and pancreatic carcinoma cells. Methods Colon carcinoma cell lines HCT116 and HT29 and pancreatic adenocarcinoma cell line Panc-1 were cultured and stimulated with neurotensin or epidermal growth factor (EGF. DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA. Levels and phosphorylation of proteins in signalling pathways were assessed by Western blotting. Results Neurotensin stimulated the phosphorylation of both extracellular signal-regulated kinase (ERK and Akt in all three cell lines, but apparently did so through different pathways. In Panc-1 cells, neurotensin-induced phosphorylation of ERK, but not Akt, was dependent on protein kinase C (PKC, whereas an inhibitor of the β-isoform of phosphoinositide 3-kinase (PI3K, TGX221, abolished neurotensin-induced Akt phosphorylation in these cells, and there was no evidence of EGF receptor (EGFR transactivation. In HT29 cells, in contrast, the EGFR tyrosine kinase inhibitor gefitinib blocked neurotensin-stimulated phosphorylation of both ERK and Akt, indicating transactivation of EGFR, independently of PKC. In HCT116 cells, neurotensin induced both a PKC-dependent phosphorylation of ERK and a metalloproteinase-mediated transactivation of EGFR that was associated with a gefitinib-sensitive phosphorylation of the downstream adaptor protein Shc. The activation of Akt was also inhibited by gefitinib, but only partly, suggesting a mechanism in addition to EGFR transactivation. Inhibition of PKC blocked neurotensin-induced DNA synthesis in HCT116 cells. Conclusions While acting predominantly through PKC in Panc-1 cells and via EGFR transactivation in HT29 cells, neurotensin used both these pathways in HCT116

Role of protein kinase C and epidermal growth factor receptor signalling in growth stimulation by neurotensin in colon carcinoma cells

International Nuclear Information System (INIS)

Müller, Kristin M; Tveteraas, Ingun H; Aasrum, Monica; Ødegård, John; Dawood, Mona; Dajani, Olav; Christoffersen, Thoralf; Sandnes, Dagny L

2011-01-01

Neurotensin has been found to promote colon carcinogenesis in rats and mice, and proliferation of human colon carcinoma cell lines, but the mechanisms involved are not clear. We have examined signalling pathways activated by neurotensin in colorectal and pancreatic carcinoma cells. Colon carcinoma cell lines HCT116 and HT29 and pancreatic adenocarcinoma cell line Panc-1 were cultured and stimulated with neurotensin or epidermal growth factor (EGF). DNA synthesis was determined by incorporation of radiolabelled thymidine into DNA. Levels and phosphorylation of proteins in signalling pathways were assessed by Western blotting. Neurotensin stimulated the phosphorylation of both extracellular signal-regulated kinase (ERK) and Akt in all three cell lines, but apparently did so through different pathways. In Panc-1 cells, neurotensin-induced phosphorylation of ERK, but not Akt, was dependent on protein kinase C (PKC), whereas an inhibitor of the β-isoform of phosphoinositide 3-kinase (PI3K), TGX221, abolished neurotensin-induced Akt phosphorylation in these cells, and there was no evidence of EGF receptor (EGFR) transactivation. In HT29 cells, in contrast, the EGFR tyrosine kinase inhibitor gefitinib blocked neurotensin-stimulated phosphorylation of both ERK and Akt, indicating transactivation of EGFR, independently of PKC. In HCT116 cells, neurotensin induced both a PKC-dependent phosphorylation of ERK and a metalloproteinase-mediated transactivation of EGFR that was associated with a gefitinib-sensitive phosphorylation of the downstream adaptor protein Shc. The activation of Akt was also inhibited by gefitinib, but only partly, suggesting a mechanism in addition to EGFR transactivation. Inhibition of PKC blocked neurotensin-induced DNA synthesis in HCT116 cells. While acting predominantly through PKC in Panc-1 cells and via EGFR transactivation in HT29 cells, neurotensin used both these pathways in HCT116 cells. In these cells, neurotensin-induced activation of ERK

A high-resolution computational localization method for transcranial magnetic stimulation mapping.

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Aonuma, Shinta; Gomez-Tames, Jose; Laakso, Ilkka; Hirata, Akimasa; Takakura, Tomokazu; Tamura, Manabu; Muragaki, Yoshihiro

2018-05-15

Transcranial magnetic stimulation (TMS) is used for the mapping of brain motor functions. The complexity of the brain deters determining the exact localization of the stimulation site using simplified methods (e.g., the region below the center of the TMS coil) or conventional computational approaches. This study aimed to present a high-precision localization method for a specific motor area by synthesizing computed non-uniform current distributions in the brain for multiple sessions of TMS. Peritumoral mapping by TMS was conducted on patients who had intra-axial brain neoplasms located within or close to the motor speech area. The electric field induced by TMS was computed using realistic head models constructed from magnetic resonance images of patients. A post-processing method was implemented to determine a TMS hotspot by combining the computed electric fields for the coil orientations and positions that delivered high motor-evoked potentials during peritumoral mapping. The method was compared to the stimulation site localized via intraoperative direct brain stimulation and navigated TMS. Four main results were obtained: 1) the dependence of the computed hotspot area on the number of peritumoral measurements was evaluated; 2) the estimated localization of the hand motor area in eight non-affected hemispheres was in good agreement with the position of a so-called "hand-knob"; 3) the estimated hotspot areas were not sensitive to variations in tissue conductivity; and 4) the hand motor areas estimated by this proposal and direct electric stimulation (DES) were in good agreement in the ipsilateral hemisphere of four glioma patients. The TMS localization method was validated by well-known positions of the "hand-knob" in brains for the non-affected hemisphere, and by a hotspot localized via DES during awake craniotomy for the tumor-containing hemisphere. Copyright © 2018 Elsevier Inc. All rights reserved.

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

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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.

Role of Glycogen Synthase Kinase-3β in APP Hyperphosphorylation Induced by NMDA Stimulation in Cortical Neurons

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Xanthi Antoniou

2010-01-01

Full Text Available The phosphorylation of Amyloid Precursor Protein (APP at Thr668 plays a key role in APP metabolism that is highly relevant to AD. The c-Jun-N-terminal kinase (JNK, glycogen synthase kinase-3β (GSK-3β and cyclin-dependent kinase 5 (Cdk5 can all be responsible for this phosphorylation. These kinases are activated by excitotoxic stimuli fundamental hallmarks of AD. The exposure of cortical neurons to a high dose of NMDA (100 μM for 30’-45’ led to an increase of P-APP Thr668. During NMDA stimulation APP hyperphosphorylation has to be assigned to GSK-3β activity, since addition of L803-mts, a substrate competitive inhibitor of GSK-3β reduced APP phosphorylation induced by NMDA. On the contrary, inhibition of JNK and Cdk5 with D-JNKI1 and Roscovitine respectively did not prevent NMDA-induced P-APP increase. These data show a tight connection, in excitotoxic conditions, between APP metabolism and the GSK-3β signaling pathway.

IGF-I Stimulates Cooperative Interaction between the IGF-I Receptor and CSK Homologous Kinase that Regulates SHPS-1 Phosphorylation in Vascular Smooth Muscle Cells

Science.gov (United States)

Radhakrishnan, Yashwanth; Shen, Xinchun; Maile, Laura A.; Xi, Gang

2011-01-01

IGF-I plays an important role in smooth muscle cell proliferation and migration. In vascular smooth muscle cells cultured in 25 mm glucose, IGF-I stimulated a significant increase in Src homology 2 domain containing protein tyrosine phosphatase substrate-1 (SHPS-1) phosphorylation compared with 5 mm glucose and this increase was required for smooth muscle cell proliferation. A proteome-wide screen revealed that carboxyl-terminal SRC kinase homologous kinase (CTK) bound directly to phosphotyrosines in the SHPS-1 cytoplasmic domain. Because the kinase(s) that phosphorylates these tyrosines in response to IGF-I is unknown, we determined the roles of IGF-I receptor (IGF-IR) and CTK in mediating SHPS-1 phosphorylation. After IGF-I stimulation, CTK was recruited to IGF-IR and subsequently to phospho-SHPS-1. Expression of an IGF-IR mutant that eliminated CTK binding reduced CTK transfer to SHPS-1, SHPS-1 phosphorylation, and cell proliferation. IGF-IR phosphorylated SHPS-1, which provided a binding site for CTK. CTK recruitment to SHPS-1 resulted in a further enhancement of SHPS-1 phosphorylation. CTK knockdown also impaired IGF-I-stimulated SHPS-1 phosphorylation and downstream signaling. Analysis of specific tyrosines showed that mutation of tyrosines 428/452 in SHPS-1 to phenylalanine reduced SHPS-1 phosphorylation but allowed CTK binding. In contrast, the mutation of tyrosines 469/495 inhibited IGF-IR-mediated the phosphorylation of SHPS-1 and CTK binding, suggesting that IGF-IR phosphorylated Y469/495, allowing CTK binding, and that CTK subsequently phosphorylated Y428/452. Based on the above findings, we conclude that after IGF-I stimulation, CTK is recruited to IGF-IR and its recruitment facilitates CTK's subsequent association with phospho-SHPS-1. This results in the enhanced CTK transfer to SHPS-1, and the two kinases then fully phosphorylate SHPS-1, which is necessary for IGF-I stimulated cellular proliferation. PMID:21799000

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

Science.gov (United States)

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

A chimeric cyclic interferon-α2b peptide induces apoptosis by sequential activation of phosphatidylinositol 3-kinase, protein kinase Cδ and p38 MAP kinase.

Science.gov (United States)

Blank, V C; Bertucci, L; Furmento, V A; Peña, C; Marino, V J; Roguin, L P

2013-06-10

We have previously demonstrated that tyrosine phosphorylation of STAT1/3 and p38 mitogen-activated protein kinase (p38 MAPK) activation are involved in the apoptotic response triggered by a chimeric cyclic peptide of the interferon-α2b (IFN-α2b) in WISH cells. Since the peptide also induced serine phosphorylation of STAT proteins, in the present study we examined the kinase involved in serine STAT1 phosphorylation and the signaling effectors acting upstream such activation. We first found that p38 MAPK is involved in serine STAT1 phosphorylation, since a reduction of phophoserine-STAT1 levels was evident after incubating WISH cells with cyclic peptide in the presence of a p38 pharmacological inhibitor or a dominant-negative p38 mutant. Next, we demonstrated that the peptide induced activation of protein kinase Cδ (PKCδ). Based on this finding, the role of this kinase was then evaluated. After incubating WISH cells with a PKCδ inhibitor or after decreasing PKCδ expression levels by RNA interference, both peptide-induced serine STAT1 and p38 phosphorylation levels were significantly decreased, indicating that PKCδ functions as an upstream regulator of p38. We also showed that PKCδ and p38 activation stimulated by the peptide was inhibited by a specific pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) or by a dominant-negative p85 PI3K-regulatory subunit, suggesting that PI3K is upstream in the signaling cascade. In addition, the role of PI3K and PKCδ in cyclic peptide-induced apoptosis was examined. Both signaling effectors were found to regulate the antiproliferative activity and the apoptotic response triggered by the cyclic peptide in WISH cells. In conclusion, we herein demonstrated that STAT1 serine phosphorylation is mediated by the sequential activation of PI3K, PKCδ and p38 MAPK. This signaling cascade contributes to the antitumor effect induced by the chimeric IFN-α2b cyclic peptide in WISH cells. Copyright © 2013 Elsevier Inc

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

Science.gov (United States)

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.

Stimulated mitogen-activated protein kinase is necessary but not sufficient for the mitogenic response to angiotensin II. A role for phospholipase D.

Science.gov (United States)

Wilkie, N; Morton, C; Ng, L L; Boarder, M R

1996-12-13

Activation of the mitogen-activated protein kinase (MAPK) cascade has been widely associated with cell proliferation; previous studies have shown that angiotensin II (AII), acting on 7-transmembrane G protein-coupled receptors, stimulates the MAPK pathway. In this report we investigate whether the MAPK pathway is required for the mitogenic response to AII stimulation of vascular smooth muscle cells derived from the hypertensive rat (SHR-VSM). AII stimulates the phosphorylation of MAPK, as determined by Western blot specific for the tyrosine 204 phosphorylated form of the protein. This MAPK phosphorylation was inhibited by the presence of the inhibitor of MAPK kinase activation, PD 098059. Using a peptide kinase assay shown to measure the p42 and p44 isoforms of MAPK, the stimulated response to AII was inhibited by PD 098059 with an IC50 of 15.6 +/- 1.6 microM. The AII stimulation of [3H]thymidine incorporation was inhibited by PD 098059 with an IC50 of 17.8 +/- 3.1 microM. PD 098059 had no effect on AII-stimulated phospholipase C or phospholipase D (PLD) activity. When the SHR-VSM cells were stimulated with phorbol ester, there was an activation of MAPK similar in size and duration to the response to AII, but there was no significant enhancement of [3H]thymidine incorporation. There was also no activation of PLD by phorbol ester, while AII produced a robust PLD response. Diversion of the product of the PLD reaction by 1-butanol caused a partial loss of the [3H]thymidine response; this did not occur with tertiary butanol, which did not interfere with the PLD reaction. These results show that in these cells the MAPK cascade is required but not sufficient for the mitogenic response to AII, and suggest that the full mitogenic response requires both MAPK in conjunction with other signaling components, one of which is PLD.

Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei

Science.gov (United States)

Li, H.; Roux, S. J.

1992-01-01

Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.

Cyclic AMP (cAMP)-mediated stimulation of adipocyte differentiation requires the synergistic action of Epac- and cAMP-dependent protein kinase-dependent processes

DEFF Research Database (Denmark)

Petersen, Rasmus Koefoed; Madsen, Lise; Pedersen, Lone Møller

2008-01-01

AMP-dependent stimulation of adipocyte differentiation. Epac, working via Rap, acted synergistically with cAMP-dependent protein kinase (protein kinase A [PKA]) to promote adipogenesis. The major role of PKA was to down-regulate Rho and Rho-kinase activity, rather than to enhance CREB phosphorylation. Suppression of Rho......-kinase impaired proadipogenic insulin/insulin-like growth factor 1 signaling, which was restored by activation of Epac. This interplay between PKA and Epac-mediated processes not only provides novel insight into the initiation and tuning of adipocyte differentiation, but also demonstrates a new mechanism of c......AMP signaling whereby cAMP uses both PKA and Epac to achieve an appropriate cellular response....

Identification of a new adapter protein that may link the common beta subunit of the receptor for granulocyte/macrophage colony-stimulating factor, interleukin (IL)-3, and IL-5 to phosphatidylinositol 3-kinase.

Science.gov (United States)

Jücker, M; Feldman, R A

1995-11-17

Binding of human granulocyte/macrophage colony-stimulating factor (hGM-CSF) to its receptor induces the rapid activation of phosphatidylinositol-3 kinase (PI 3-kinase). As hGM-CSF receptor (hGMR) does not contain a consensus sequence for binding of PI 3-kinase, hGMR must use a distinct mechanism for its association with and activation of PI 3-kinase. Here, we describe the identification of a tyrosine-phosphorylated protein of 76-85 kDa (p80) that associates with the common beta subunit of hGMR and with the SH2 domains of the p85 subunit of PI 3-kinase in hGM-CSF-stimulated cells. Src/Yes and Lyn were tightly associated with the p80.PI 3-kinase complex, suggesting that p80 and other phosphotyrosyl proteins present in the complex were phosphorylated by Src family kinases. Tyrosine phosphorylation of p80 was only detected in hGM-CSF or human interleukin-3-stimulated cells, suggesting that activation of p80 might be specific for signaling via the common beta subunit. We postulate that p80 functions as an adapter protein that may participate in linking the hGM-CSF receptor to the PI 3-kinase signaling pathway.

Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

Energy Technology Data Exchange (ETDEWEB)

Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald, E-mail: gerald.thiel@uks.eu

2015-03-01

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified.

Resveratrol upregulates Egr-1 expression and activity involving extracellular signal-regulated protein kinase and ternary complex factors

International Nuclear Information System (INIS)

Rössler, Oliver G.; Glatzel, Daniel; Thiel, Gerald

2015-01-01

Many intracellular functions have been attributed to resveratrol, a polyphenolic phytoalexin found in grapes and in other plants. Here, we show that resveratrol induces the expression of the transcription factor Egr-1 in human embryonic kidney cells. Using a chromosomally embedded Egr-1-responsive reporter gene, we show that the Egr-1 activity was significantly elevated in resveratrol-treated cells, indicating that the newly synthesized Egr-1 protein was biologically active. Stimulus-transcription coupling leading to the resveratrol-induced upregulation of Egr-1 expression and activity requires the protein kinases Raf and extracellular signal-regulated protein kinase ERK, while MAP kinase phosphatase-1 functions as a nuclear shut-off device that interrupts the signaling cascade connecting resveratrol stimulation with enhanced Egr-1 expression. On the transcriptional level, Elk-1, a key transcriptional regulator of serum response element-driven gene transcription, connects the intracellular signaling cascade elicited by resveratrol with transcription of the Egr-1 gene. These data were corroborated by the observation that stimulation of the cells with resveratrol increased the transcriptional activation potential of Elk-1. The SRE as well as the GC-rich DNA binding site of Egr-1 function as resveratrol-responsive elements. Thus, resveratrol regulates gene transcription via activation of the stimulus-regulated protein kinases Raf and ERK and the stimulus-responsive transcription factors TCF and Egr-1. - Highlights: • The plant polyphenol resveratrol upregulates Egr-1 expression and activity. • The stimulation of Egr-1 requires the protein kinases ERK and Raf. • Resveratrol treatment upregulates the transcriptional activation potential of Elk-1. • Resveratrol-induced stimulation of Egr-1 requires ternary complex factors. • Two distinct resveratrol-responsive elements were identified

Functional Redundancy of ERK1 and ERK2 MAP Kinases during Development

Directory of Open Access Journals (Sweden)

Christophe Frémin

2015-08-01

Full Text Available ERK1 and ERK2 are the effector kinases of the ERK1/2 MAP-kinase signaling pathway, which plays a central role in transducing signals controlling cell proliferation, differentiation, and survival. Deregulated activity of the ERK1/2 pathway is linked to a group of developmental syndromes and contributes to the pathogenesis of various human diseases. One fundamental question that remains unaddressed is whether ERK1 and ERK2 have evolved unique physiological functions or whether they are used redundantly to reach a threshold of global ERK activity. Here, we show that the extent of development of the mouse placenta and embryo bearing different combinations of Erk1 and Erk2 alleles is strictly correlated with total ERK1/2 activity. We further demonstrate that transgenic expression of ERK1 fully rescues the embryonic and placental developmental defects associated with the loss of ERK2. We conclude that ERK1 and ERK2 exert redundant functions in mouse development.

Regulation of the MAP kinase cascade in PC12 cells: B-Raf activates MEK-1 (MAP kinase or ERK kinase) and is inhibited by cAMP

DEFF Research Database (Denmark)

Peraldi, P; Frödin, M; Barnier, J V

1995-01-01

AMP inhibits B-Raf autokinase activity as well as its ability to phosphorylate and activate MEK-1. This inhibition is likely to be due to a direct effect since we found that PKA phosphorylates B-Raf in vitro. Further, we show that B-Raf binds to p21ras, but more important, this binding to p21ras is virtually...... abolished with B-Raf from PC12 cells treated with CPT-cAMP. Hence, these data indicate that the PKA-mediated phosphorylation of B-Raf hampers its interaction with p21ras, which is responsible for the PKA-mediated decrease in B-Raf activity. Finally, our work suggests that in PC12 cells, cAMP stimulates MAP...

Extrasynaptic N-methyl-D-aspartate (NMDA) receptor stimulation induces cytoplasmic translocation of the CDKL5 kinase and its proteasomal degradation.

Science.gov (United States)

Rusconi, Laura; Kilstrup-Nielsen, Charlotte; Landsberger, Nicoletta

2011-10-21

Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients with epileptic encephalopathy characterized by early onset intractable epilepsy, including infantile spasms and other types of seizures, severe developmental delay, and often the development of Rett syndrome-like features. Despite its clear involvement in proper brain development, CDKL5 functions are still far from being understood. In this study, we analyzed the subcellular localization of the endogenous kinase in primary murine hippocampal neurons. CDKL5 was localized both in nucleus and cytoplasm and, conversely to proliferating cells, did not undergo constitutive shuttling between these compartments. Nevertheless, glutamate stimulation was able to induce the exit of the kinase from the nucleus and its subsequent accumulation in the perinuclear cytoplasm. Moreover, we found that sustained glutamate stimulation promoted CDKL5 proteasomal degradation. Both events were mediated by the specific activation of extrasynaptic pool of N-methyl-d-aspartate receptors. Proteasomal degradation was also induced by withdrawal of neurotrophic factors and hydrogen peroxide treatment, two different paradigms of cell death. Altogether, our results indicate that both subcellular localization and expression of CDKL5 are modulated by the activation of extrasynaptic N-methyl-D-aspartate receptors and suggest regulation of CDKL5 by cell death pathways.

Extrasynaptic N-Methyl-d-aspartate (NMDA) Receptor Stimulation Induces Cytoplasmic Translocation of the CDKL5 Kinase and Its Proteasomal Degradation*

Science.gov (United States)

Rusconi, Laura; Kilstrup-Nielsen, Charlotte; Landsberger, Nicoletta

2011-01-01

Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) have been found in patients with epileptic encephalopathy characterized by early onset intractable epilepsy, including infantile spasms and other types of seizures, severe developmental delay, and often the development of Rett syndrome-like features. Despite its clear involvement in proper brain development, CDKL5 functions are still far from being understood. In this study, we analyzed the subcellular localization of the endogenous kinase in primary murine hippocampal neurons. CDKL5 was localized both in nucleus and cytoplasm and, conversely to proliferating cells, did not undergo constitutive shuttling between these compartments. Nevertheless, glutamate stimulation was able to induce the exit of the kinase from the nucleus and its subsequent accumulation in the perinuclear cytoplasm. Moreover, we found that sustained glutamate stimulation promoted CDKL5 proteasomal degradation. Both events were mediated by the specific activation of extrasynaptic pool of N-methyl-d-aspartate receptors. Proteasomal degradation was also induced by withdrawal of neurotrophic factors and hydrogen peroxide treatment, two different paradigms of cell death. Altogether, our results indicate that both subcellular localization and expression of CDKL5 are modulated by the activation of extrasynaptic N-methyl-d-aspartate receptors and suggest regulation of CDKL5 by cell death pathways. PMID:21832092

Phosphorylation and activation of p42 and p44 mitogen-activated protein kinase are required for the P2 purinoceptor stimulation of endothelial prostacyclin production.

Science.gov (United States)

Patel, V; Brown, C; Goodwin, A; Wilkie, N; Boarder, M R

1996-11-15

Extracellular ATP and ADP, released from platelets and other sites stimulate the endothelial production of prostacyclin (PGI2) by acting on G-protein-coupled P2Y2 and P2Y2 purinoceptors, contributing to the maintenance of a non-thrombogenic surface. The mechanism, widely described as being dependent on elevated cytosolic [Ca2+], also requires protein tyrosine phosphorylation. Here we show that activation of both these P2 receptor types leads to the tyrosine phosphorylation and activation of both the p42 and p44 forms of mitogen-activated protein kinase (MAPK). 2-Methylthio-ATP and UTP, selectively activating P2Y1 and P2Y2 purinoceptors respectively, and ATP, a non-selective agonist at these two receptors, stimulate the tyrosine phosphorylation of both p42mapk and p44mapk, as revealed by Western blots with an antiserum specific for the tyrosine-phosphorylated forms of the enzymes. By using separation on Resource Q columns, peptide kinase activity associated with the phosphorylated MAPK enzymes distributes into two peaks, one mainly p42mapk and one mainly p44mapk, both of which are stimulated by ATP with respect to kinase activity and phospho-MAPK immunoreactivity. Stimulation of P2Y1 or P2Y2 purinoceptors leads to a severalfold increase in PGI2 efflux; this was blocked in a dose-dependent manner by the selective MAPK kinase inhibitor PD98059. This drug also blocked the agonist-stimulated increase in phospho-MAPK immunoreactivity for both p42mapk and p44mapk but left the phospholipase C response to P2 agonists essentially unchanged. Olomoucine has been reported to inhibit p44mapk activity. Here we show that in the same concentration range olomoucine inhibits activity in both peaks from the Resource Q column and also the agonist stimulation of 6-keto-PGF1, but has no effect on agonist-stimulated phospho-MAPK immunoreactivity. These results provide direct evidence for the involvement of p42 and p44 MAPK in the PGI2 response of intact endothelial cells: we have shown

A calcium-dependent protein kinase can inhibit a calmodulin-stimulated Ca2+ pump (ACA2) located in the endoplasmic reticulum of Arabidopsis

Science.gov (United States)

Hwang, I.; Sze, H.; Harper, J. F.; Evans, M. L. (Principal Investigator)

2000-01-01

The magnitude and duration of a cytosolic Ca(2+) release can potentially be altered by changing the rate of Ca(2+) efflux. In plant cells, Ca(2+) efflux from the cytoplasm is mediated by H(+)/Ca(2+)-antiporters and two types of Ca(2+)-ATPases. ACA2 was recently identified as a calmodulin-regulated Ca(2+)-pump located in the endoplasmic reticulum. Here, we show that phosphorylation of its N-terminal regulatory domain by a Ca(2+)-dependent protein kinase (CDPK isoform CPK1), inhibits both basal activity ( approximately 10%) and calmodulin stimulation ( approximately 75%), as shown by Ca(2+)-transport assays with recombinant enzyme expressed in yeast. A CDPK phosphorylation site was mapped to Ser(45) near a calmodulin binding site, using a fusion protein containing the N-terminal domain as an in vitro substrate for a recombinant CPK1. In a full-length enzyme, an Ala substitution for Ser(45) (S45/A) completely blocked the observed CDPK inhibition of both basal and calmodulin-stimulated activities. An Asp substitution (S45/D) mimicked phosphoinhibition, indicating that a negative charge at this position is sufficient to account for phosphoinhibition. Interestingly, prior binding of calmodulin blocked phosphorylation. This suggests that, once ACA2 binds calmodulin, its activation state becomes resistant to phosphoinhibition. These results support the hypothesis that ACA2 activity is regulated as the balance between the initial kinetics of calmodulin stimulation and CDPK inhibition, providing an example in plants for a potential point of crosstalk between two different Ca(2+)-signaling pathways.

Identification and analysis of a novel protein-tyrosine kinase from bovine thymus

International Nuclear Information System (INIS)

Zioncheck, T.F.; Harrison, M.L.; Geahlen, R.L.

1986-01-01

A cytosolic protein-tyrosine kinase has been identified and purified to near homogeneity from calf thymus by using the phosphorylation of the tyrosine-containing peptide angiotensin I as an assay. Specific peptide phosphorylating activity was enhanced by carrying out the assay at high ionic strength (2M NaCl). The inclusion of NaCl at this concentration acts to stimulate endogenous protein-tyrosine kinase activity while simultaneously inhibiting other endogenous kinases. The purification procedure involved extraction of the enzyme from calf-thymus and sequential chromatography on columns of DEAE-cellulose, heparin-agarose, casein-sepharose, butylagarose, and Sephadex G-75. Analysis of the most highly purified preparations by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single Coomassie blue-stained band of 41 KDa. This molecular weight was consistent with results obtained from gel filtration, indicating that the enzyme exists as a monomer. The enzyme has also been found to catalyze an autophosphorylation reaction. Incubation of the enzyme with Mn 2+ and [γ- 32 P]ATP led to its modification on a tyrosine residue. Phosphopeptide mapping experiments indicated that the 41 KDa kinase was distinct from p56, the major membrane-associated protein-tyrosine kinase in T lymphocytes

Accurate motor mapping in awake common marmosets using micro-electrocorticographical stimulation and stochastic threshold estimation

DEFF Research Database (Denmark)

Kosugi, Akito; Takemi, Mitsuaki; Tia, Banty

2018-01-01

OBJECTIVE: Motor map has been widely used as an indicator of motor skills and learning, cortical injury, plasticity, and functional recovery. Cortical stimulation mapping using epidural electrodes is recently adopted for animal studies. However, several technical limitations still remain. Test-re...

The phosphatidylinositol 3-kinase inhibitor, wortmannin, inhibits insulin-induced activation of phosphatidylcholine hydrolysis and associated protein kinase C translocation in rat adipocytes.

Science.gov (United States)

Standaert, M L; Avignon, A; Yamada, K; Bandyopadhyay, G; Farese, R V

1996-02-01

We questioned whether phosphatidylinositol 3-kinase (PI 3-kinase) and protein kinase C (PKC) function as interrelated signalling mechanisms during insulin action in rat adipocytes. Insulin rapidly activated a phospholipase D that hydrolyses phosphatidylcholine (PC), and this activation was accompanied by increases in diacylglycerol and translocative activation of PKC-alpha and PKC-beta in the plasma membrane. Wortmannin, an apparently specific PI 3-kinase inhibitor, inhibited insulin-stimulated, phospholipase D-dependent PC hydrolysis and subsequent translocation of PKC-alpha and PKC-beta to the plasma membrane. Wortmannin did not inhibit PKC directly in vitro, or the PKC-dependent effects of phorbol esters on glucose transport in intact adipocytes. The PKC inhibitor RO 31-8220 did not inhibit PI 3-kinase directly or its activation in situ by insulin, but inhibited both insulin-stimulated and phorbol ester-stimulated glucose transport. Our findings suggest that insulin acts through PI 3-kinase to activate a PC-specific phospholipase D and causes the translocative activation of PKC-alpha and PKC-beta in plasma membranes of rat adipocytes.

The difference between electrical microstimulation and direct electrical stimulation - towards new opportunities for innovative functional brain mapping?

Science.gov (United States)

Vincent, Marion; Rossel, Olivier; Hayashibe, Mitsuhiro; Herbet, Guillaume; Duffau, Hugues; Guiraud, David; Bonnetblanc, François

2016-04-01

Both electrical microstimulation (EMS) and direct electrical stimulation (DES) of the brain are used to perform functional brain mapping. EMS is applied to animal fundamental neuroscience experiments, whereas DES is performed in the operating theatre on neurosurgery patients. The objective of the present review was to shed new light on electrical stimulation techniques in brain mapping by comparing EMS and DES. There is much controversy as to whether the use of DES during wide-awake surgery is the 'gold standard' for studying the brain function. As part of this debate, it is sometimes wrongly assumed that EMS and DES induce similar effects in the nervous tissues and have comparable behavioural consequences. In fact, the respective stimulation parameters in EMS and DES are clearly different. More surprisingly, there is no solid biophysical rationale for setting the stimulation parameters in EMS and DES; this may be due to historical, methodological and technical constraints that have limited the experimental protocols and prompted the use of empirical methods. In contrast, the gap between EMS and DES highlights the potential for new experimental paradigms in electrical stimulation for functional brain mapping. In view of this gap and recent technical developments in stimulator design, it may now be time to move towards alternative, innovative protocols based on the functional stimulation of peripheral nerves (for which a more solid theoretical grounding exists).

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

Science.gov (United States)

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.

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

Science.gov (United States)

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

2016-01-01

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. PMID:27834677

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

International Nuclear Information System (INIS)

Nishina, Atsuyoshi; Kimura, Hirokazu; Kozawa, Kunihisa; Sommen, Geoffroy; Nakamura, Takao; Heimgartner, Heinz; Koketsu, Mamoru; Furukawa, Shoei

2011-01-01

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 μM, the O 2 − 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 50 ) at 92.4 μM and acted as an effective and potentially useful O 2 − 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 μM 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 μM. 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 μM 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: ► We newly synthesized 1,3-selenazolidin-4-ones to study their possible applications. ► Among new

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

Energy Technology Data Exchange (ETDEWEB)

Nishina, Atsuyoshi, E-mail: nishina@yone.ac.jp [Yonezawa Women' s Junior College, 6-15-1 Tohrimachi, Yonezawa, Yamagata 992-0025 (Japan); Kimura, Hirokazu; Kozawa, Kunihisa [Gunma Prefectural Institute of Public Health and Environmental Sciences, 378 Kamioki, Maebashi, Gunma 371-0052 (Japan); Sommen, Geoffroy [Lonza Braine SA, Chaussee de Tubize 297, B-1420 Braine l' Alleud (Belgium); Nakamura, Takao [Department of Biomedical Information Engineering, Graduate School of Medical Science, Yamagata University, Yamagata 990-9585 (Japan); Heimgartner, Heinz [University of Zuerich, Institut of Organic Chemistry, Winterthurerstrasse 190, CH-8057 Zuerich (Switzerland); Koketsu, Mamoru [Department of Materials Science and Technology, Faculty of Engineering, Gifu University, Gifu 501-1193 (Japan); Furukawa, Shoei [Laboratory of Molecular Biology, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585 (Japan)

2011-12-15

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}) at 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

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

Science.gov (United States)

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).

Language mapping using high gamma electrocorticography, fMRI, and TMS versus electrocortical stimulation.

Science.gov (United States)

Babajani-Feremi, Abbas; Narayana, Shalini; Rezaie, Roozbeh; Choudhri, Asim F; Fulton, Stephen P; Boop, Frederick A; Wheless, James W; Papanicolaou, Andrew C

2016-03-01

The aim of the present study was to compare localization of the language cortex using cortical stimulation mapping (CSM), high gamma electrocorticography (hgECoG), functional magnetic resonance imaging (fMRI), and transcranial magnetic stimulation (TMS). Language mapping using CSM, hgECoG, fMRI, and TMS were compared in nine patients with epilepsy. Considering CSM as reference, we compared language mapping approaches based on hgECoG, fMRI, and TMS using their sensitivity, specificity, and the results of receiver operating characteristic (ROC) analyses. Our results show that areas involved in language processing can be identified by hgECoG, fMRI, and TMS. The average sensitivity/specificity of hgECoG, fMRI, and TMS across all patients was 100%/85%, 50%/80%, and 67%/66%, respectively. The average area under the ROC curve of hgECoG, fMRI, and TMS across CSM-positive patients was 0.98, 0.76, and 0.68, respectively. There is considerable concordance between CSM, hgECoG, fMRI, and TMS language mapping. Our results reveal that hgECoG, fMRI, and TMS are valuable tools for presurgical language mapping. Language mapping on the basis of hgECoG, fMRI, and TMS can provide important additional information, therefore, these methods can be used in conjunction with CSM or as an alternative, when the latter is deemed impractical. Copyright © 2015 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.

The interaction between tropomyosin-related kinase B receptors and serine kinases modulates acetylcholine release in adult neuromuscular junctions.

Science.gov (United States)

Santafé, Manel M; Garcia, Neus; Tomàs, Marta; Obis, Teresa; Lanuza, Maria A; Besalduch, Nuria; Tomàs, Josep

2014-02-21

We conducted an electrophysiological study of the functional link between the tropomyosin-related kinase B (trkB) receptor signaling mechanism and serine-threonine kinases, both protein kinase C (PKC) and protein kinase A (PKA). We describe their coordinated role in transmitter release at the neuromuscular junction (NMJ) of the Levator auris longus muscle of the adult mouse. The trkB receptor normally seems to be coupled to stimulate ACh release because inhibiting the trkB receptor with K-252a results in a significant reduction in the size of EPPs. We found that the intracellular PKC pathway can operate as in basal conditions (to potentiate ACh release) without the involvement of the trkB receptor function, although the trkB pathway needs an operative PKC pathway if it is to couple to the release mechanism and potentiate it. To actively stimulate PKA (which also results in ACh release potentiation), the operativity of trkB is a necessary condition, and one effect of trkB may be PKA stimulation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

p38 mitogen-activated protein kinase up-regulates NF-κB transcriptional activation through RelA phosphorylation during stretch-induced myogenesis

International Nuclear Information System (INIS)

Ji, Guoping; Liu, Dongxu; Liu, Jing; Gao, Hui; Yuan, Xiao; Shen, Gang

2010-01-01

p38 MAPK and nuclear factor-B (NF-B) signaling pathways play an indispensable role in the control of skeletal myogenesis. The specific contribution of these signaling pathways to the response of myoblast to the mechanical stimulation and the molecular mechanisms underlying this response remain unresolved. Using an established in vitro model, we now show that p38 MAP kinase activity regulates the transcriptional activation of NF-κB in response to mechanical stimulation of myoblasts. Furthermore, SB203580 blocked stretch-induced NF-κB activation during myogenesis, not through down-regulation of degradation of IκB-α, and consequent translocation of the p65 subunit of NF-κB to the nucleus. It is likely that stretch-induced NF-κB activation by phosphorylation of p65 NF-κB. Moreover, depletion of p38α using siRNA significantly reduces stretch-induced phosphorylation of RelA and NF-κB activity. These results provides the first evidence of a cross-talk between p38 MAPK and NF-κB signaling pathways during stretch-induced myogenesis, with phosphorylation of RelA being one of the effectors of this promyogenic mechanism. The α isoform of p38MAP kinase regulates the transcriptional activation of NF-κB following stimulation with cyclic stretch.

p38 mitogen-activated protein kinase up-regulates NF-{kappa}B transcriptional activation through RelA phosphorylation during stretch-induced myogenesis

Energy Technology Data Exchange (ETDEWEB)

Ji, Guoping [Department of Orthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Research Institute of Stomatology, Shanghai 200011 (China); Liu, Dongxu [Department of Orthodontics, College of Stomatology, Shandong University, Jinan, Shandong Province 250012 (China); Liu, Jing [Department of Orthodontics, The Affiliated Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong Province 266075 (China); Gao, Hui [Department of Orthodontics, Tianjin Stomatological Hospital, Tianjin 300041 (China); Yuan, Xiao, E-mail: yuanxiaoqd@163.com [Department of Orthodontics, The Affiliated Qingdao Municipal Hospital, Qingdao University, Qingdao, Shandong Province 266075 (China); Shen, Gang, E-mail: ganshen2007@163.com [Department of Orthodontics, College of Stomatology, Ninth People' s Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai Research Institute of Stomatology, Shanghai 200011 (China)

2010-01-01

p38 MAPK and nuclear factor-B (NF-B) signaling pathways play an indispensable role in the control of skeletal myogenesis. The specific contribution of these signaling pathways to the response of myoblast to the mechanical stimulation and the molecular mechanisms underlying this response remain unresolved. Using an established in vitro model, we now show that p38 MAP kinase activity regulates the transcriptional activation of NF-{kappa}B in response to mechanical stimulation of myoblasts. Furthermore, SB203580 blocked stretch-induced NF-{kappa}B activation during myogenesis, not through down-regulation of degradation of I{kappa}B-{alpha}, and consequent translocation of the p65 subunit of NF-{kappa}B to the nucleus. It is likely that stretch-induced NF-{kappa}B activation by phosphorylation of p65 NF-{kappa}B. Moreover, depletion of p38{alpha} using siRNA significantly reduces stretch-induced phosphorylation of RelA and NF-{kappa}B activity. These results provides the first evidence of a cross-talk between p38 MAPK and NF-{kappa}B signaling pathways during stretch-induced myogenesis, with phosphorylation of RelA being one of the effectors of this promyogenic mechanism. The {alpha} isoform of p38MAP kinase regulates the transcriptional activation of NF-{kappa}B following stimulation with cyclic stretch.

Repeated mapping of cortical language sites by preoperative navigated transcranial magnetic stimulation compared to repeated intraoperative DCS mapping in awake craniotomy

Science.gov (United States)

2014-01-01

Background Repetitive navigated transcranial magnetic stimulation (rTMS) was recently described for mapping of human language areas. However, its capability of detecting language plasticity in brain tumor patients was not proven up to now. Thus, this study was designed to evaluate such data in order to compare rTMS language mapping to language mapping during repeated awake surgery during follow-up in patients suffering from language-eloquent gliomas. Methods Three right-handed patients with left-sided gliomas (2 opercular glioblastomas, 1 astrocytoma WHO grade III of the angular gyrus) underwent preoperative language mapping by rTMS as well as intraoperative language mapping provided via direct cortical stimulation (DCS) for initial as well as for repeated Resection 7, 10, and 15 months later. Results Overall, preoperative rTMS was able to elicit clear language errors in all mappings. A good correlation between initial rTMS and DCS results was observed. As a consequence of brain plasticity, initial DCS and rTMS findings only corresponded with the results obtained during the second examination in one out of three patients thus suggesting changes of language organization in two of our three patients. Conclusions This report points out the usefulness but also the limitations of preoperative rTMS language mapping to detect plastic changes in language function or for long-term follow-up prior to DCS even in recurrent gliomas. However, DCS still has to be regarded as gold standard. PMID:24479694

Optimal timing of pulse onset for language mapping with navigated repetitive transcranial magnetic stimulation.

Science.gov (United States)

Krieg, Sandro M; Tarapore, Phiroz E; Picht, Thomas; Tanigawa, Noriko; Houde, John; Sollmann, Nico; Meyer, Bernhard; Vajkoczy, Peter; Berger, Mitchel S; Ringel, Florian; Nagarajan, Srikantan

2014-10-15

Within the primary motor cortex, navigated transcranial magnetic stimulation (nTMS) has been shown to yield maps strongly correlated with those generated by direct cortical stimulation (DCS). However, the stimulation parameters for repetitive nTMS (rTMS)-based language mapping are still being refined. For this purpose, the present study compares two rTMS protocols, which differ in the timing of pulse train onset relative to picture presentation onset during object naming. Results were the correlated with DCS language mapping during awake surgery. Thirty-two patients with left-sided perisylvian tumors were examined by rTMS prior to awake surgery. Twenty patients underwent rTMS pulse trains starting at 300 ms after picture presentation onset (delayed TMS), whereas another 12 patients received rTMS pulse trains starting at the picture presentation onset (ONSET TMS). These rTMS results were then evaluated for correlation with intraoperative DCS results as gold standard in terms of differential consistencies in receiver operating characteristics (ROC) statistics. Logistic regression analysis by protocols and brain regions were conducted. Within and around Broca's area, there was no difference in sensitivity (onset TMS: 100%, delayed TMS: 100%), negative predictive value (NPV) (onset TMS: 100%, delayed TMS: 100%), and positive predictive value (PPV) (onset TMS: 55%, delayed TMS: 54%) between the two protocols compared to DCS. However, specificity differed significantly (onset TMS: 67%, delayed TMS: 28%). In contrast, for posterior language regions, such as supramarginal gyrus, angular gyrus, and posterior superior temporal gyrus, early pulse train onset stimulation showed greater specificity (onset TMS: 92%, delayed TMS: 20%), NPV (onset TMS: 92%, delayed TMS: 57%) and PPV (onset TMS: 75%, delayed TMS: 30%) with comparable sensitivity (onset TMS: 75%, delayed TMS: 70%). Logistic regression analysis also confirmed the greater fit of the predictions by rTMS that had the

Inhibition of colony-stimulating-factor-1 signaling in vivo with the orally bioavailable cFMS kinase inhibitor GW2580.

Science.gov (United States)

Conway, James G; McDonald, Brad; Parham, Janet; Keith, Barry; Rusnak, David W; Shaw, Eva; Jansen, Marilyn; Lin, Peiyuan; Payne, Alan; Crosby, Renae M; Johnson, Jennifer H; Frick, Lloyd; Lin, Min-Hwa Jasmine; Depee, Scott; Tadepalli, Sarva; Votta, Bart; James, Ian; Fuller, Karen; Chambers, Timothy J; Kull, Frederick C; Chamberlain, Stanley D; Hutchins, Jeff T

2005-11-01

Colony-stimulating-factor-1 (CSF-1) signaling through cFMS receptor kinase is increased in several diseases. To help investigate the role of cFMS kinase in disease, we identified GW2580, an orally bioavailable inhibitor of cFMS kinase. GW2580 completely inhibited human cFMS kinase in vitro at 0.06 microM and was inactive against 26 other kinases. GW2580 at 1 microM completely inhibited CSF-1-induced growth of mouse M-NFS-60 myeloid cells and human monocytes and completely inhibited bone degradation in cultures of human osteoclasts, rat calvaria, and rat fetal long bone. In contrast, GW2580 did not affect the growth of mouse NS0 lymphoblastoid cells, human endothelial cells, human fibroblasts, or five human tumor cell lines. GW2580 also did not affect lipopolysaccharide (LPS)-induced TNF, IL-6, and prostaglandin E2 production in freshly isolated human monocytes and mouse macrophages. After oral administration, GW2580 blocked the ability of exogenous CSF-1 to increase LPS-induced IL-6 production in mice, inhibited the growth of CSF-1-dependent M-NFS-60 tumor cells in the peritoneal cavity, and diminished the accumulation of macrophages in the peritoneal cavity after thioglycolate injection. Unexpectedly, GW2580 inhibited LPS-induced TNF production in mice, in contrast to effects on monocytes and macrophages in vitro. In conclusion, GW2580's selective inhibition of monocyte growth and bone degradation is consistent with cFMS kinase inhibition. The ability of GW2580 to chronically inhibit CSF-1 signaling through cFMS kinase in normal and tumor cells in vivo makes GW2580 a useful tool in assessing the role of cFMS kinase in normal and disease processes.

Somatotopic Map and Inter- and Intra-Digit Distance in Brodmann Area 2 by Pressure Stimulation

Science.gov (United States)

Choi, Mi-Hyun; Kim, Sung-Phil; Kim, Hyung-Sik; Gim, Seon-Young; Kim, Woo-Ram; Mun, Kyung-Ryul; Lim, Dae-Woon; Lee, Bongsoo; Chung, Soon-Cheol

2016-01-01

The somatotopic representation of the tactile stimulation on the finger in the brain is an essential part of understanding the human somatosensory system as well as rehabilitation and other clinical therapies. Many studies have used vibrotactile stimulations and reported finger somatotopic representations in the Brodmann area 3 (BA 3). On the contrary, few studies investigated finger somatotopic representation using pressure stimulations. Therefore, the present study aimed to find a comprehensive somatotopic representation (somatotopic map and inter- and intra-digit distance) within BA 2 of humans that could describe tactile stimulations on different joints across the fingers by applying pressure stimulation to three joints-the first (p1), second (p2), and third (p3) joints-of four fingers (index, middle, ring, and little finger). Significant differences were observed in the inter-digit distance between the first joints (p1) of the index and little fingers, and between the third joints (p3) of the index and little fingers. In addition, a significant difference was observed in the intra-digit distance between p1 and p3 of the little finger. This study suggests that a somatotopic map and inter- and intra-digit distance could be found in BA 2 in response to pressure stimulation on finger joints. PMID:27452859

Effects of phorbol ester on mitogen-activated protein kinase kinase activity in wild-type and phorbol ester-resistant EL4 thymoma cells.

Science.gov (United States)

Gause, K C; Homma, M K; Licciardi, K A; Seger, R; Ahn, N G; Peterson, M J; Krebs, E G; Meier, K E

1993-08-05

Phorbol ester-sensitive and -resistant EL4 thymoma cell lines differ in their ability to activate mitogen-activated protein kinase (MAPK) in response to phorbol ester. Treatment of wild-type EL4 cells with phorbol ester results in the rapid activations of MAPK and pp90rsk kinase, a substrate for MAPK, while neither kinase is activated in response to phorbol ester in variant EL4 cells. This study examines the activation of MAPK kinase (MAPKK), an activator of MAPK, in wild-type and variant EL4 cells. Phosphorylation of a 40-kDa substrate, identified as MAPK, was observed following in vitro phosphorylation reactions using cytosolic extracts or Mono Q column fractions prepared from phorbol ester-treated wild-type EL4 cells. MAPKK activity coeluted with a portion of the inactive MAPK upon Mono Q anion-exchange chromatography, permitting detection of the MAPKK activity in fractions containing both kinases. This MAPKK activity was present in phorbol ester-treated wild-type cells, but not in phorbol ester-treated variant cells or in untreated wild-type or variant cells. The MAPKK from wild-type cells was able to activate MAPK prepared from either wild-type or variant cells. MAPKK activity could be stimulated in both wildtype and variant EL4 cells in response to treatment of cells with okadaic acid. These results indicate that the failure of variant EL4 cells to activate MAP kinase in response to phorbol ester is due to a failure to activate MAPKK. Therefore, the step that confers phorbol ester resistance to variant EL4 cells lies between the activation of protein kinase C and the activation of MAPKK.

Computational-experimental approach to drug-target interaction mapping: A case study on kinase inhibitors.

Directory of Open Access Journals (Sweden)

Anna Cichonska

2017-08-01

Full Text Available Due to relatively high costs and labor required for experimental profiling of the full target space of chemical compounds, various machine learning models have been proposed as cost-effective means to advance this process in terms of predicting the most potent compound-target interactions for subsequent verification. However, most of the model predictions lack direct experimental validation in the laboratory, making their practical benefits for drug discovery or repurposing applications largely unknown. Here, we therefore introduce and carefully test a systematic computational-experimental framework for the prediction and pre-clinical verification of drug-target interactions using a well-established kernel-based regression algorithm as the prediction model. To evaluate its performance, we first predicted unmeasured binding affinities in a large-scale kinase inhibitor profiling study, and then experimentally tested 100 compound-kinase pairs. The relatively high correlation of 0.77 (p < 0.0001 between the predicted and measured bioactivities supports the potential of the model for filling the experimental gaps in existing compound-target interaction maps. Further, we subjected the model to a more challenging task of predicting target interactions for such a new candidate drug compound that lacks prior binding profile information. As a specific case study, we used tivozanib, an investigational VEGF receptor inhibitor with currently unknown off-target profile. Among 7 kinases with high predicted affinity, we experimentally validated 4 new off-targets of tivozanib, namely the Src-family kinases FRK and FYN A, the non-receptor tyrosine kinase ABL1, and the serine/threonine kinase SLK. Our sub-sequent experimental validation protocol effectively avoids any possible information leakage between the training and validation data, and therefore enables rigorous model validation for practical applications. These results demonstrate that the kernel

Hierarchical modeling of activation mechanisms in the ABL and EGFR kinase domains: thermodynamic and mechanistic catalysts of kinase activation by cancer mutations.

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Anshuman Dixit

2009-08-01

Full Text Available Structural and functional studies of the ABL and EGFR kinase domains have recently suggested a common mechanism of activation by cancer-causing mutations. However, dynamics and mechanistic aspects of kinase activation by cancer mutations that stimulate conformational transitions and thermodynamic stabilization of the constitutively active kinase form remain elusive. We present a large-scale computational investigation of activation mechanisms in the ABL and EGFR kinase domains by a panel of clinically important cancer mutants ABL-T315I, ABL-L387M, EGFR-T790M, and EGFR-L858R. We have also simulated the activating effect of the gatekeeper mutation on conformational dynamics and allosteric interactions in functional states of the ABL-SH2-SH3 regulatory complexes. A comprehensive analysis was conducted using a hierarchy of computational approaches that included homology modeling, molecular dynamics simulations, protein stability analysis, targeted molecular dynamics, and molecular docking. Collectively, the results of this study have revealed thermodynamic and mechanistic catalysts of kinase activation by major cancer-causing mutations in the ABL and EGFR kinase domains. By using multiple crystallographic states of ABL and EGFR, computer simulations have allowed one to map dynamics of conformational fluctuations and transitions in the normal (wild-type and oncogenic kinase forms. A proposed multi-stage mechanistic model of activation involves a series of cooperative transitions between different conformational states, including assembly of the hydrophobic spine, the formation of the Src-like intermediate structure, and a cooperative breakage and formation of characteristic salt bridges, which signify transition to the active kinase form. We suggest that molecular mechanisms of activation by cancer mutations could mimic the activation process of the normal kinase, yet exploiting conserved structural catalysts to accelerate a conformational transition

The MAP kinase-activated protein kinase Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen Candida albicans.

Science.gov (United States)

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.

Arabidopsis MAP Kinase 4 regulates gene expression via transcription factor release in the nucleus

DEFF Research Database (Denmark)

Qiu, Jin-Long; Fiil, Berthe Katrine; Petersen, Klaus

2008-01-01

kinase 4 (MPK4) exists in nuclear complexes with the WRKY33 transcription factor. This complex depends on the MPK4 substrate MKS1. Challenge with Pseudomonas syringae or flagellin leads to the activation of MPK4 and phosphorylation of MKS1. Subsequently, complexes with MKS1 and WRKY33 are released from...... MPK4, and WRKY33 targets the promoter of PHYTOALEXIN DEFICIENT3 (PAD3) encoding an enzyme required for the synthesis of antimicrobial camalexin. Hence, wrky33 mutants are impaired in the accumulation of PAD3 mRNA and camalexin production upon infection. That WRKY33 is an effector of MPK4 is further...... supported by the suppression of PAD3 expression in mpk4-wrky33 double mutant backgrounds. Our data establish direct links between MPK4 and innate immunity and provide an example of how a plant MAP kinase can regulate gene expression by releasing transcription factors in the nucleus upon activation....

Sphingosine kinase-1 is a hypoxia-regulated gene that stimulates migration of human endothelial cells

International Nuclear Information System (INIS)

Schwalm, Stephanie; Doell, Frauke; Roemer, Isolde; Bubnova, Svetlana; Pfeilschifter, Josef; Huwiler, Andrea

2008-01-01

Sphingosine kinases (SK) catalyze the production of sphingosine-1-phosphate which in turn regulates cell responses such as proliferation and migration. Here, we show that exposure of the human endothelial cell line EA.hy 926 to hypoxia stimulates a increased SK-1, but not SK-2, mRNA, protein expression, and activity. This effect was due to stimulated SK-1 promoter activity which contains two putative hypoxia-inducible factor-responsive-elements (HRE). By deletion of one of the two HREs, hypoxia-induced promoter activation was abrogated. Furthermore, hypoxia upregulated the expression of HIF-1α and HIF-2α, and both contributed to SK-1 gene transcription as shown by selective depletion of HIF-1α or HIF-2α by siRNA. The hypoxia-stimulated SK-1 upregulation was functionally coupled to increased migration since the selective depletion of SK-1, but not of SK-2, by siRNAs abolished the migratory response. In summary, these data show that hypoxia upregulates SK-1 activity and results in an accelerated migratory capacity of endothelial cells. SK-1 may thus serve as an attractive therapeutic target to treat diseases associated with increased endothelial migration and angiogenesis such as cancer growth and progression

Structural coupling of SH2-kinase domains links Fes and Abl substrate recognition and kinase activation.

Science.gov (United States)

Filippakopoulos, Panagis; Kofler, Michael; Hantschel, Oliver; Gish, Gerald D; Grebien, Florian; Salah, Eidarus; Neudecker, Philipp; Kay, Lewis E; Turk, Benjamin E; Superti-Furga, Giulio; Pawson, Tony; Knapp, Stefan

2008-09-05

The SH2 domain of cytoplasmic tyrosine kinases can enhance catalytic activity and substrate recognition, but the molecular mechanisms by which this is achieved are poorly understood. We have solved the structure of the prototypic SH2-kinase unit of the human Fes tyrosine kinase, which appears specialized for positive signaling. In its active conformation, the SH2 domain tightly interacts with the kinase N-terminal lobe and positions the kinase alphaC helix in an active configuration through essential packing and electrostatic interactions. This interaction is stabilized by ligand binding to the SH2 domain. Our data indicate that Fes kinase activation is closely coupled to substrate recognition through cooperative SH2-kinase-substrate interactions. Similarly, we find that the SH2 domain of the active Abl kinase stimulates catalytic activity and substrate phosphorylation through a distinct SH2-kinase interface. Thus, the SH2 and catalytic domains of active Fes and Abl pro-oncogenic kinases form integrated structures essential for effective tyrosine kinase signaling.

A genome-wide RNAi screen reveals MAP kinase phosphatases as key ERK pathway regulators during embryonic stem cell differentiation.

Directory of Open Access Journals (Sweden)

Shen-Hsi Yang

Full Text Available Embryonic stem cells and induced pluripotent stem cells represent potentially important therapeutic agents in regenerative medicine. Complex interlinked transcriptional and signaling networks control the fate of these cells towards maintenance of pluripotency or differentiation. In this study we have focused on how mouse embryonic stem cells begin to differentiate and lose pluripotency and, in particular, the role that the ERK MAP kinase and GSK3 signaling pathways play in this process. Through a genome-wide siRNA screen we have identified more than 400 genes involved in loss of pluripotency and promoting the onset of differentiation. These genes were functionally associated with the ERK and/or GSK3 pathways, providing an important resource for studying the roles of these pathways in controlling escape from the pluripotent ground state. More detailed analysis identified MAP kinase phosphatases as a focal point of regulation and demonstrated an important role for these enzymes in controlling ERK activation kinetics and subsequently determining early embryonic stem cell fate decisions.

Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.

Science.gov (United States)

Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

2003-08-01

DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.

Identification and characterization of an ABA-activated MAP kinase cascade in Arabidopsis thaliana

KAUST Repository

Danquah, Agyemang

2015-04-01

Summary Abscisic acid (ABA) is a major phytohormone involved in important stress-related and developmental plant processes. Recent phosphoproteomic analyses revealed a large set of ABA-triggered phosphoproteins as putative mitogen-activated protein kinase (MAPK) targets, although the evidence for MAPKs involved in ABA signalling is still scarce. Here, we identified and reconstituted in vivo a complete ABA-activated MAPK cascade, composed of the MAP3Ks MAP3K17/18, the MAP2K MKK3 and the four C group MAPKs MPK1/2/7/14. In planta, we show that ABA activation of MPK7 is blocked in mkk3-1 and map3k17mapk3k18 plants. Coherently, both mutants exhibit hypersensitivity to ABA and altered expression of a set of ABA-dependent genes. A genetic analysis further reveals that this MAPK cascade is activated by the PYR/PYL/RCAR-SnRK2-PP2C ABA core signalling module through protein synthesis of the MAP3Ks, unveiling an atypical mechanism for MAPK activation in eukaryotes. Our work provides evidence for a role of an ABA-induced MAPK pathway in plant stress signalling. Significance Statement We report in this article the identification of a complete MAPK module, composed of MAP3K17/18, MKK3 and MPK1/2/7/14, which is activated by ABA through the ABA core signalling complex. We showed that the activation of this module requires the MAP3K protein synthesis which occurs after hours of stress treatment, suggesting that the pathway is involved in a delayed wave of cellular responses to ABA and drought. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

Identification and characterization of an ABA-activated MAP kinase cascade in Arabidopsis thaliana

KAUST Repository

Danquah, Agyemang; Zé licourt, Axel de; Boudsocq, Marie; Neubauer, Jorinde; Frei Dit Frey, Nicolas; Leonhardt, Nathalie; Pateyron, Sté phanie; Gwinner, Frederik; Tamby, Jean Philippe; Ortiz-Masià , Dolores; Marcote, Marí a Jesú s; Hirt, Heribert; Colcombet, Jean

2015-01-01

Summary Abscisic acid (ABA) is a major phytohormone involved in important stress-related and developmental plant processes. Recent phosphoproteomic analyses revealed a large set of ABA-triggered phosphoproteins as putative mitogen-activated protein kinase (MAPK) targets, although the evidence for MAPKs involved in ABA signalling is still scarce. Here, we identified and reconstituted in vivo a complete ABA-activated MAPK cascade, composed of the MAP3Ks MAP3K17/18, the MAP2K MKK3 and the four C group MAPKs MPK1/2/7/14. In planta, we show that ABA activation of MPK7 is blocked in mkk3-1 and map3k17mapk3k18 plants. Coherently, both mutants exhibit hypersensitivity to ABA and altered expression of a set of ABA-dependent genes. A genetic analysis further reveals that this MAPK cascade is activated by the PYR/PYL/RCAR-SnRK2-PP2C ABA core signalling module through protein synthesis of the MAP3Ks, unveiling an atypical mechanism for MAPK activation in eukaryotes. Our work provides evidence for a role of an ABA-induced MAPK pathway in plant stress signalling. Significance Statement We report in this article the identification of a complete MAPK module, composed of MAP3K17/18, MKK3 and MPK1/2/7/14, which is activated by ABA through the ABA core signalling complex. We showed that the activation of this module requires the MAP3K protein synthesis which occurs after hours of stress treatment, suggesting that the pathway is involved in a delayed wave of cellular responses to ABA and drought. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

The Fyn tyrosine kinase binds Irs-1 and forms a distinct signaling complex during insulin stimulation.

Science.gov (United States)

Sun, X J; Pons, S; Asano, T; Myers, M G; Glasheen, E; White, M F

1996-05-03

Irs-proteins link the receptors for insulin/IGF-1, growth hormones, and several interleukins and interferons to signaling proteins that contain Src homology-2 (SH2). To identify new Irs-1-binding proteins, we screened a mouse embryo expression library with recombinant [32P]Irs-1, which revealed a specific association between p59fyn and Irs-1. The SH2 domain in p59fyn bound to phosphorylated Tyr895 and Tyr1172, which are located in YXX(L/I) motifs. Mutation of p59fyn at the COOH-terminal tyrosine phosphorylation site (Tyr531) enhanced its binding to Irs-1 during insulin stimulation. Binding experiments with various SH2 protein revealed that Grb-2 was largely excluded from Irs-1 complexes containing p59fyn, whereas Grb-2 and p85 occurred in the same Irs-1 complex. By comparison with the insulin receptor, p59fyn kinase phosphorylated a unique cohort of tyrosine residues in Irs-1. These results outline a role for p59fyn or other related Src-kinases during insulin and cytokine signaling.

Association of a Network of Interferon-Stimulated Genes with a Locus Encoding a Negative Regulator of Non-conventional IKK Kinases and IFNB1

Directory of Open Access Journals (Sweden)

Saloua Jeidane

2016-10-01

Full Text Available Functional genomic analysis of gene expression in mice allowed us to identify a quantitative trait locus (QTL linked in trans to the expression of 190 gene transcripts and in cis to the expression of only two genes, one of which was Ypel5. Most of the trans-expression QTL genes were interferon-stimulated genes (ISGs, and their expression in mouse macrophage cell lines was stimulated in an IFNB1-dependent manner by Ypel5 silencing. In human HEK293T cells, YPEL5 silencing enhanced the induction of IFNB1 by pattern recognition receptors and phosphorylation of TBK1/IKBKE kinases, whereas co-immunoprecipitation experiments revealed that YPEL5 interacted physically with IKBKE. We thus found that the Ypel5 gene (contained in a locus linked to a network of ISGs in mice is a negative regulator of IFNB1 production and innate immune responses that interacts functionally and physically with TBK1/IKBKE kinases.

A mitotically inheritable unit containing a MAP kinase module.

Science.gov (United States)

Kicka, Sébastien; Bonnet, Crystel; Sobering, Andrew K; Ganesan, Latha P; Silar, Philippe

2006-09-05

Prions are novel kinds of hereditary units, relying solely on proteins, that are infectious and inherited in a non-Mendelian fashion. To date, they are either based on autocatalytic modification of a 3D conformation or on autocatalytic cleavage. Here, we provide further evidence that in the filamentous fungus Podospora anserina, a MAP kinase cascade is probably able to self-activate and generate C, a hereditary unit that bears many similarities to prions and triggers cell degeneration. We show that in addition to the MAPKKK gene, both the MAPKK and MAPK genes are necessary for the propagation of C, and that overexpression of MAPK as that of MAPKKK facilitates the appearance of C. We also show that a correlation exists between the presence of C and localization of the MAPK inside nuclei. These data emphasize the resemblance between prions and a self-positively regulated cascade in terms of their transmission. This thus further expands the concept of protein-base inheritance to regulatory networks that have the ability to self-activate.

Chitin and stress induced protein kinase activation

DEFF Research Database (Denmark)

Kenchappa, Chandra Shekar; Azevedo da Silva, Raquel; Bressendorff, Simon

2017-01-01

The assays described here are pertinent to protein kinase studies in any plant. They include an immunoblot phosphorylation/activation assay and an in-gel activity assay for MAP kinases (MPKs) using the general protein kinase substrate myelin basic protein. They also include a novel in-gel peptide...... substrate assay for Snf1-related kinase family 2 members (SnRK2s). This kinase family-specific assay overcomes some limitations of in-gel assays and permits the identification of different types of kinase activities in total protein extracts....

The role of phosphatidylinositol 3-kinase in neural cell adhesion molecule-mediated neuronal differentiation and survival

DEFF Research Database (Denmark)

Ditlevsen, Dorte K; Køhler, Lene B; Pedersen, Martin Volmer

2003-01-01

The neural cell adhesion molecule, NCAM, is known to stimulate neurite outgrowth from primary neurones and PC12 cells presumably through signalling pathways involving the fibroblast growth factor receptor (FGFR), protein kinase A (PKA), protein kinase C (PKC), the Ras-mitogen activated protein...... kinase (MAPK) pathway and an increase in intracellular Ca2+ levels. Stimulation of neurones with the synthetic NCAM-ligand, C3, induces neurite outgrowth through signalling pathways similar to the pathways activated through physiological, homophilic NCAM-stimulation. We present here data indicating...... that phosphatidylinositol 3-kinase (PI3K) is required for NCAM-mediated neurite outgrowth from PC12-E2 cells and from cerebellar and dopaminergic neurones in primary culture, and that the thr/ser kinase Akt/protein kinase B (PKB) is phosphorylated downstream of PI3K after stimulation with C3. Moreover, we present data...

Transgenic Analysis of the Leishmania MAP Kinase MPK10 Reveals an Auto-inhibitory Mechanism Crucial for Stage-Regulated Activity and Parasite Viability

DEFF Research Database (Denmark)

Cayla, M.; Rachidi, N.; Leclercq, O.

2014-01-01

Protozoan pathogens of the genus Leishmania have evolved unique signaling mechanisms that can sense changes in the host environment and trigger adaptive stage differentiation essential for host cell infection. The signaling mechanisms underlying parasite development remain largely elusive even...... though Leishmania mitogen-activated protein kinases (MAPKs) have been linked previously to environmentally induced differentiation and virulence. Here, we unravel highly unusual regulatory mechanisms for Leishmania MAP kinase 10 (MPK10). Using a transgenic approach, we demonstrate that MPK10 is stage...... at position 395 that could be implicated in kinase regulation. Finally, we uncovered a feedback loop that limits MPK10 activity through dephosphorylation of the tyrosine residue of the TxY motif. Together our data reveal novel aspects of protein kinase regulation in Leishmania, and propose MPK10...

Inhibition of the MEK-1/p42 MAP kinase reduces aryl hydrocarbon receptor-DNA interactions

International Nuclear Information System (INIS)

Yim, Sujin; Oh, Myoungsuk; Choi, Su Mi; Park, Hyunsung

2004-01-01

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) induces expression of the cytochrome P450 1A1 gene, cyp1a1, by binding to its receptor, aryl hydrocarbon receptor (AhR). TCDD-bound AhR translocates to the nucleus and forms a heterodimer with its partner protein, AhR nuclear translocator (Arnt). The AhR/Arnt heterodimer then binds to the dioxin-response elements (DREs) in the cyp1a1 enhancer and stimulates transcription of cyp1a1. We tested whether kinase pathways are involved in this process by treating Hepa1c1c7 cells with kinase inhibitors. The MEK-1 inhibitor PD98059 reduced TCDD-induced transcription of cyp1a1. TCDD treatment results in phosphorylation of p44/p42 mitogen-activated protein kinase (MAPK), a substrate of MEK-1. Overexpression of dominant negative form of p42 MAPK suppressed TCDD-dependent transcription of a reporter gene controlled by dioxin-response elements (DREs), and pretreatment with PD98059 also blocked this transcription. PD98059 pretreatment also inhibited TCDD-induced DRE binding of the AhR/Arnt heterodimer. Together these results indicate that TCDD activates the MEK-1/p44/p42 MAPK pathway, which in turn activates AhR and so facilitates binding of AhR to the cyp1a1 DRE

Gene regulation by MAP kinase cascades

DEFF Research Database (Denmark)

Fiil, Berthe Katrine; Petersen, Klaus; Petersen, Morten

2009-01-01

Mitogen-activated protein kinase (MAPK) cascades are signaling modules that transduce extracellular stimuli to a range of cellular responses. Research in yeast and metazoans has shown that MAPK-mediated phosphorylation directly or indirectly regulates the activity of transcription factors. Plant ...

SH2/SH3 adaptor proteins can link tyrosine kinases to a Ste20-related protein kinase, HPK1.

Science.gov (United States)

Anafi, M; Kiefer, F; Gish, G D; Mbamalu, G; Iscove, N N; Pawson, T

1997-10-31

Ste20-related protein kinases have been implicated as regulating a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain, and in transfected cells activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Here we have investigated candidate upstream regulators that might interact with HPK1. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 bound in vitro to specific proline-rich motifs in the HPK1 tail and functioned synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation did not affect the binding of Grb2 to HPK1 but induced recruitment of the Grb2.HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulated the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases.

Deciphering the Arginine-binding preferences at the substrate-binding groove of Ser/Thr kinases by computational surface mapping.

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Avraham Ben-Shimon

2011-11-01

Full Text Available 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.

Phosphatidic acid accumulation and catecholamine release in adrenal chromaffin cells: stimulation by high potassium and by nicotine, and effect of a diacylglycerol kinase inhibitor R 59 022.

Science.gov (United States)

Owen, P J; Jones, J A; Boarder, M R

1991-09-01

Using primary cultures of bovine adrenal chromaffin cells labelled with 32Pi, we show that stimulation with bradykinin, nicotine, or a depolarising concentration of potassium stimulates the accumulation of [32P]phosphatidic acid. The effects of nicotine and potassium are smaller than the effect of bradykinin, and are dependent entirely on extracellular calcium. The diacylglycerol kinase inhibitor R 59 022 attenuates the formation of phosphatidic acid by nicotine and depolarising concentrations of potassium. This inhibitor also blocks the nicotine and potassium stimulation of noradrenaline release from chromaffin cells. Using 45Ca2+ influx studies, we show that the nicotine-evoked calcium influx is also attenuated by R 59 022. These observations contrast with those in another report in which we showed that bradykinin stimulation of either [32P]phosphatidic acid accumulation or noradrenaline release is not affected by R 59 022. It is likely that the calcium influx produced by nicotine and depolarising potassium is blocked by R 59 022 by a mechanism that is independent of its ability to block diacylglycerol kinase. The nicotine- and potassium-stimulated [32P]phosphatidic acid accumulation is a consequence of this calcium influx and presumably reflects calcium activation of either phospholipase C or phospholipase D.

Mapping organelle motion reveals a vesicular conveyor belt spatially replenishing secretory vesicles in stimulated chromaffin cells.

Science.gov (United States)

Maucort, Guillaume; Kasula, Ravikiran; Papadopulos, Andreas; Nieminen, Timo A; Rubinsztein-Dunlop, Halina; Meunier, Frederic A

2014-01-01

How neurosecretory cells spatially adjust their secretory vesicle pools to replenish those that have fused and released their hormonal content is currently unknown. Here we designed a novel set of image analyses to map the probability of tracked organelles undergoing a specific type of movement (free, caged or directed). We then applied our analysis to time-lapse z-stack confocal imaging of secretory vesicles from bovine Chromaffin cells to map the global changes in vesicle motion and directionality occurring upon secretagogue stimulation. We report a defined region abutting the cortical actin network that actively transports secretory vesicles and is dissipated by actin and microtubule depolymerizing drugs. The directionality of this "conveyor belt" towards the cell surface is activated by stimulation. Actin and microtubule networks therefore cooperatively probe the microenvironment to transport secretory vesicles to the periphery, providing a mechanism whereby cells globally adjust their vesicle pools in response to secretagogue stimulation.

Double dissociation between syntactic gender and picture naming processing: a brain stimulation mapping study.

Science.gov (United States)

Vidorreta, Jose Garbizu; Garcia, Roser; Moritz-Gasser, Sylvie; Duffau, Hugues

2011-03-01

Neural foundations of syntactic gender processing remain poorly understood. We used electrostimulation mapping in nine right-handed awake patients during surgery for a glioma within the left hemisphere, to study whether the cortico-subcortical structures involved in naming versus syntactic gender processing are common or distinct. In French, the article determines the grammatical gender. Thus, the patient was asked to perform a picture naming task and to give the appropriate article for each picture, with and without stimulation. Cortical stimulation elicited reproducible syntactic gender disturbances in six patients, in the inferior frontal gyrus (three cases), and in the posterior middle temporal gyrus (three cases). Interestingly, no naming disorders were generated during stimulation of the syntactic sites, while cortical areas inducing naming disturbances never elicited grammatical gender errors when stimulated. Moreover, at the subcortical level, stimulation of the white matter lateral to the caudate nucleus induced gender errors in three patients, with no naming disorders. Using cortico-subcortical electrical mapping in awake patients, we demonstrate for the first time (1) a double dissociation between syntactic gender and naming processing, supporting independent network model rather than serial theory, (2) the involvement of the left inferior frontal gyrus, especially the pars triangularis, and the posterior left middle temporal gyrus in grammatical gender processing, (3) the existence of white matter pathways, likely a sub-part of the left superior longitudinal fasciculus, underlying a large-scale distributed cortico-subcortical circuit which might selectively sub-serve syntactic gender processing, even if interconnected with parallel sub-networks involved in naming (semantic and phonological) processing. Copyright © 2010 Wiley-Liss, Inc.

Wada-test, functional magnetic resonance imaging and direct electrical stimulation - brain mapping methods

International Nuclear Information System (INIS)

Minkin, K.; Tanova, R.; Busarski, A.; Penkov, M.; Penev, L.; Hadjidekov, V.

2009-01-01

Modern neurosurgery requires accurate preoperative and intraoperative localization of brain pathologies but also of brain functions. The presence of individual variations in healthy subjects and the shift of brain functions in brain diseases provoke the introduction of various methods for brain mapping. The aim of this paper was to analyze the most widespread methods for brain mapping: Wada-test, functional magnetic resonance imaging (fMRI) and intraoperative direct electrical stimulation (DES). This study included 4 patients with preoperative brain mapping using Wada-test and fMRI. Intraoperative mapping with DES during awake craniotomy was performed in one case. The histopathological diagnosis was low-grade glioma in 2 cases, cortical dysplasia (1 patient) and arteriovenous malformation (1 patient). The brain mapping permits total lesion resection in three of four patients. There was no new postoperative deficit despite surgery near or within functional brain areas. Brain plasticity provoking shift of eloquent areas from their usual locations was observed in two cases. The brain mapping methods allow surgery in eloquent brain areas recognized in the past as 'forbidden areas'. Each method has advantages and disadvantages. The precise location of brain functions and pathologies frequently requires combination of different brain mapping methods. (authors)

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

Science.gov (United States)

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.

Mitogen activated protein kinase kinase kinase 3 (MAP3K3/MEKK3) overexpression is an early event in esophageal tumorigenesis and is a predictor of poor disease prognosis

International Nuclear Information System (INIS)

Hasan, Raghibul; Sharma, Rinu; Saraya, Anoop; Chattopadhyay, Tushar K; DattaGupta, Siddartha; Walfish, Paul G; Chauhan, Shyam S; Ralhan, Ranju

2014-01-01

Mitogen-activated protein kinase kinase kinase3 (MAP3K3/MEKK3) was identified to be differentially expressed in esophageal squamous cell carcinoma (ESCC) using cDNA microarrays by our laboratory. Here in we determined the clinical significance of MEKK3 in ESCC. Immunohistochemical analysis of MEKK3 expression was carried out in archived tissue sections from 93 ESCCs, 47 histologically normal and 61 dysplastic esophageal tissues and correlated with clinicopathological parameters and disease prognosis over up to 7.5 years for ESCC patients. MEKK3 expression was significantly increased in esophageal dysplasia and ESCC in comparison with normal mucosa (p trend < 0.001). Kaplan Meier survival analysis showed significantly reduced median disease free survival median DFS = 10 months in patients with MEKK3 positive ESCCs compared to patients with no immunopositivity (median DFS = 19 months, p = 0.04). ESCC patients with MEKK3 positive and lymph node positive tumors had median DFS = 9 months, as compared to median DFS = 21 months in patients who did not show the alterations (p = 0.01). In multivariate Cox regression analysis, combination of MEKK3 overexpression and node positivity [p = 0.015, hazard ratio (HR) = 2.082, 95% CI = 1.154 - 3.756] emerged as important predictor of reduced disease free survival and poor prognosticator for ESCC patients. Alterations in MEKK3 expression occur in early stages of development of ESCC and are sustained during disease progression; MEKK3 in combination with lymph node positivity has the potential to serve as adverse prognosticator in ESCC

Calcium-dependent but calmodulin-independent protein kinase from soybean

International Nuclear Information System (INIS)

Harmon, A.C.; Putnam-Evans, C.; Cormier, M.J.

1987-01-01

A calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar (≥ 2 micromolar). The protein kinase activity was stimulated 100-fold by ≥ 10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (≤ 2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H1 in a calcium-dependent manner. Following electroblotting onto nitrocellulose, the kinase bound 45 Ca 2+ in the presence of KCl and MgCl 2 , which indicated that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calcium-dependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity

A novel approach for monitoring writing interferences during navigated transcranial magnetic stimulation mappings of writing related cortical areas.

Science.gov (United States)

Rogić Vidaković, Maja; Gabelica, Dragan; Vujović, Igor; Šoda, Joško; Batarelo, Nikolina; Džimbeg, Andrija; Zmajević Schönwald, Marina; Rotim, Krešimir; Đogaš, Zoran

2015-11-30

It has recently been shown that navigated repetitive transcranial magnetic stimulation (nTMS) is useful in preoperative neurosurgical mapping of motor and language brain areas. In TMS mapping of motor cortices the evoked responses can be quantitatively monitored by electromyographic (EMG) recordings. No such setup exists for monitoring of writing during nTMS mappings of writing related cortical areas. We present a novel approach for monitoring writing during nTMS mappings of motor writing related cortical areas. To our best knowledge, this is the first demonstration of quantitative monitoring of motor evoked responses from hand by EMG, and of pen related activity during writing with our custom made pen, together with the application of chronometric TMS design and patterned protocol of rTMS. The method was applied in four healthy subjects participating in writing during nTMS mapping of the premotor cortical area corresponding to BA 6 and close to the superior frontal sulcus. The results showed that stimulation impaired writing in all subjects. The corresponding spectra of measured signal related to writing movements was observed in the frequency band 0-20 Hz. Magnetic stimulation affected writing by suppressing normal writing frequency band. The proposed setup for monitoring of writing provides additional quantitative data for monitoring and the analysis of rTMS induced writing response modifications. The setup can be useful for investigation of neurophysiologic mechanisms of writing, for therapeutic effects of nTMS, and in preoperative mapping of language cortical areas in patients undergoing brain surgery. Copyright © 2015 Elsevier B.V. All rights reserved.

ACTIVATION OF G-PROTEINS BY RECEPTOR-STIMULATED NUCLEOSIDE DIPHOSPHATE KINASE IN DICTYOSTELIUM

NARCIS (Netherlands)

Bominaar, Anthony A.; Molijn, Anco C.; Pestel, Martine; Veron, Michel; Haastert, Peter J.M. van

Recently, interest in the enzyme nucleoside diphosphate kinase (EC 2.7.4.6) has increased as a result of its possible involvement in cell proliferation and development. Since NDP kinase is one of the major sources of GTP in cells, it has been suggested that the effects of an altered NDP kinase

The role of MAP kinases in the induction of iNOS expression in neutrophils exposed to NDMA: the involvement transcription factors.

Science.gov (United States)

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.

Mapping the follicle-stimulating hormone-induced signalling networks

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Pauline eGloaguen

2011-10-01

Full Text Available Follicle-stimulating hormone (FSH is a central regulator of male and female reproductive function. Over the last decade, there has been a growing perception of the complexity associated with FSH-induced cellular signalling. It is now clear that the canonical Gs/cAMP/PKA pathway is not the sole mechanism that must be considered in FSH biological actions. In parallel, consistent with the emerging concept of biased agonism, several examples of ligand-mediated selective signalling pathway activation by gonadotropin receptors have been reported. In this context, it is important to gain an integrative view of the signalling pathways induced by FSH and how they interconnect to form a network. In this review, we propose a first attempt at building topological maps of various pathways known to be involved in the FSH-induced signalling network. We discuss the multiple facets of FSH-induced signalling and how they converge to the hormone integrated biological response. Despite of their incompleteness, these maps of the FSH-induced signalling network represent a first step towards gaining a system-level comprehension of this hormone’s actions, which may ultimately facilitate the discovery of novel regulatory processes and therapeutic strategies for infertilities and non-steroidal contraception.

Src-family-tyrosine kinase Lyn is critical for TLR2-mediated NF-κB activation through the PI 3-kinase signaling pathway.

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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.

Effect of lipopolysaccharide and chlorpromazine on glucocorticoid receptor-mediated gene transcription and immunoreactivity: a possible involvement of p38-MAP kinase

Czech Academy of Sciences Publication Activity Database

Basta-Kaim, A.; Budziszewska, B.; Jaworska-Feil, L.; Tetich, M.; Kubera, M.; Zajícová, Alena; Holáň, Vladimír; Lasoń, W.

2004-01-01

Roč. 14, č. 6 (2004), s. 521-528 ISSN 0924-977X R&D Projects: GA MŠk LN00A026 Institutional research plan: CEZ:AV0Z5052915 Keywords : p38-MAP kinase, cytokines, gene transcription Subject RIV: EC - Immunology Impact factor: 3.545, year: 2004

Adiponectin and AMP kinase activator stimulate proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells

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Yamauchi Mika

2007-11-01

Full Text Available Abstract Background Adiponectin is a key mediator of the metabolic syndrome that is caused by visceral fat accumulation. Adiponectin and its receptors are known to be expressed in osteoblasts, but their actions with regard to bone metabolism are still unclear. In this study, we investigated the effects of adiponectin on the proliferation, differentiation, and mineralization of osteoblastic MC3T3-E1 cells. Results Adiponectin receptor type 1 (AdipoR1 mRNA was detected in the cells by RT-PCR. The adenosine monophosphate-activated protein kinase (AMP kinase was phosphorylated by both adiponectin and a pharmacological AMP kinase activator, 5-amino-imidazole-4-carboxamide-riboside (AICAR, in the cells. AdipoR1 small interfering RNA (siRNA transfection potently knocked down the receptor mRNA, and the effect of this knockdown persisted for as long as 10 days after the transfection. The transfected cells showed decreased expressions of type I collagen and osteocalcin mRNA, as determined by real-time PCR, and reduced ALP activity and mineralization, as determined by von Kossa and Alizarin red stainings. In contrast, AMP kinase activation by AICAR (0.01–0.5 mM in wild-type MC3T3-E1 cells augmented their proliferation, differentiation, and mineralization. BrdU assay showed that the addition of adiponectin (0.01–1.0 μg/ml also promoted their proliferation. Osterix, but not Runx-2, appeared to be involved in these processes because AdipoR1 siRNA transfection and AICAR treatments suppressed and enhanced osterix mRNA expression, respectively. Conclusion Taken together, this study suggests that adiponectin stimulates the proliferation, differentiation, and mineralization of osteoblasts via the AdipoR1 and AMP kinase signaling pathways in autocrine and/or paracrine fashions.

MAP kinase cascades in Arabidopsis innate immunity

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Rasmussen, Magnus Wohlfahrt; Roux, Milena Edna; Petersen, Morten

2012-01-01

Plant mitogen-activated protein kinase (MAPK) cascades generally transduce extracellular stimuli into cellular responses. These stimuli include the perception of pathogen-associated molecular patterns (PAMPs) by host transmembrane pattern recognition receptors which trigger MAPK-dependent innate ...

Induction of MAP Kinase Homologues during Growth and Morphogenetic Development of Karnal Bunt (Tilletia indica) under the Influence of Host Factor(s) from Wheat Spikes

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Gupta, Atul K.; Seneviratne, J. M.; Joshi, G. K.; Kumar, Anil

2012-01-01

Signaling pathways that activate different mitogen-activated protein kinases (MAPKs) in response to certain environmental conditions, play important role in mating type switching (Fus3) and pathogenicity (Pmk1) in many fungi. In order to determine the roles of such regulatory genes in Tilletia indica, the causal pathogen of Karnal bunt (KB) of wheat, semi-quantitative and quantitative RT-PCR was carried out to isolate and determine the expression of MAP kinase homologues during fungal growth and development under in vitro culture. Maximum expression of TiFus3 and TiPmk1 genes were observed at 14th and 21st days of culture and decreased thereafter. To investigate whether the fungus alters the expression levels of same kinases upon interaction with plants, cultures were treated with 1% of host factors (extracted from S-2 stage of wheat spikes). Such treatment induced the expression of MAPks in time dependent manner compared to the absence of host factors. These results suggest that host factor(s) provide certain signal(s) which activate TiFus3 and TiPmk1 during morphogenetic development of T. indica. The results also provides a clue about the role of host factors in enhancing the disease potential due to induction of MAP kinases involved in fungal development and pathogenecity. PMID:22547988

Biphasic Estradiol-induced AKT Phosphorylation Is Modulated by PTEN via MAP Kinase in HepG2 Cells

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Marino, Maria; Acconcia, Filippo; Trentalance, Anna

2003-01-01

We reported previously in HepG2 cells that estradiol induces cell cycle progression throughout the G1–S transition by the parallel stimulation of both PKC-α and ERK signaling molecules. The analysis of the cyclin D1 gene expression showed that only the MAP kinase pathway was involved. Here, the presence of rapid/nongenomic, estradiol-regulated, PI3K/AKT signal transduction pathway, its modulation by the levels of the tumor suppressor PTEN, its cross-talk with the ERK pathway, and its involvement in DNA synthesis and cyclin D1 gene promoter activity have all been studied in HepG2 cells. 17β-Estradiol induced the rapid and biphasic phosphorylation of AKT. These phosphorylations were independent of each other, being the first wave of activation independent of the estrogen receptor (ER), whereas the second was dependent on ER. Both activations were dependent on PI3K activity; furthermore, the ERK pathway modulated AKT phosphorylation by acting on the PTEN levels. The results showed that the PI3K pathway, as well as ER, were strongly involved in both G1–S progression and cyclin D1 promoter activity by acting on its proximal region (-254 base pairs). These data indicate that in HepG2 cells, different rapid/nongenomic estradiol-induced signal transduction pathways modulate the multiple steps of G1–S phase transition. PMID:12808053

Salmonella Typhimurium type III secretion effectors stimulate innate immune responses in cultured epithelial cells.

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Vincent M Bruno

2009-08-01

Full Text Available Recognition of conserved bacterial products by innate immune receptors leads to inflammatory responses that control pathogen spread but that can also result in pathology. Intestinal epithelial cells are exposed to bacterial products and therefore must prevent signaling through innate immune receptors to avoid pathology. However, enteric pathogens are able to stimulate intestinal inflammation. We show here that the enteric pathogen Salmonella Typhimurium can stimulate innate immune responses in cultured epithelial cells by mechanisms that do not involve receptors of the innate immune system. Instead, S. Typhimurium stimulates these responses by delivering through its type III secretion system the bacterial effector proteins SopE, SopE2, and SopB, which in a redundant fashion stimulate Rho-family GTPases leading to the activation of mitogen-activated protein (MAP kinase and NF-kappaB signaling. These observations have implications for the understanding of the mechanisms by which Salmonella Typhimurium induces intestinal inflammation as well as other intestinal inflammatory pathologies.

Impairment of preoperative language mapping by lesion location: a functional magnetic resonance imaging, navigated transcranial magnetic stimulation, and direct cortical stimulation study.

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Ille, Sebastian; Sollmann, Nico; Hauck, Theresa; Maurer, Stefanie; Tanigawa, Noriko; Obermueller, Thomas; Negwer, Chiara; Droese, Doris; Boeckh-Behrens, Tobias; Meyer, Bernhard; Ringel, Florian; Krieg, Sandro M

2015-08-01

Language mapping by repetitive navigated transcranial magnetic stimulation (rTMS) is increasingly used and has already replaced functional MRI (fMRI) in some institutions for preoperative mapping of neurosurgical patients. Yet some factors affect the concordance of both methods with direct cortical stimulation (DCS), most likely by lesions affecting cortical oxygenation levels. Therefore, the impairment of the accuracy of rTMS and fMRI was analyzed and compared with DCS during awake surgery in patients with intraparenchymal lesions. Language mapping was performed by DCS, rTMS, and fMRI using an object-naming task in 27 patients with left-sided perisylvian lesions, and the induced language errors of each method were assigned to the cortical parcellation system. Subsequently, the receiver operating characteristics were calculated for rTMS and fMRI and compared with DCS as ground truth for regions with (w/) and without (w/o) the lesion in the mapped regions. The w/ subgroup revealed a sensitivity of 100% (w/o 100%), a specificity of 8% (w/o 5%), a positive predictive value of 34% (w/o: 53%), and a negative predictive value (NPV) of 100% (w/o: 100%) for the comparison of rTMS versus DCS. Findings for the comparison of fMRI versus DCS within the w/ subgroup revealed a sensitivity of 32% (w/o: 62%), a specificity of 88% (w/o: 60%), a positive predictive value of 56% (w/o: 62%), and a NPV of 73% (w/o: 60%). Although strengths and weaknesses exist for both rTMS and fMRI, the results show that rTMS is less affected by a brain lesion than fMRI, especially when performing mapping of language-negative cortical regions based on sensitivity and NPV.

p21-activated Kinase1(PAK1) can promote ERK activation in a kinase independent manner

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Wang, Zhipeng; Fu, Meng; Wang, Lifeng

2013-01-01

204) although phosphorylation of b-Raf (Ser445) and c-Raf (Ser 338) remained unchanged. Furthermore, increased activation of the PAK1 activator Rac1 induced the formation of a triple complex of Rac1, PAK1 and Mek1, independent of the kinase activity of PAK1. These data suggest that PAK1 can stimulate...... MEK activity in a kinase independent manner, probably by serving as a scaffold to facilitate interaction of c-Raf....

LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5

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John von Freyend, Simona; Rosenqvist, Heidi; Fink, Annette

2010-01-01

The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified...... LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric...... kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co...

Doxazosin stimulates galectin-3 expression and collagen synthesis in HL-1 cardiomyocytes independent of protein kinase C pathway

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Xiaoqian Qian

2016-12-01

Full Text Available Doxazosin, a drug commonly prescribed for hypertension and prostate disease, increases heart failure risk. However, the underlying mechanism remains unclear. Galectin-3 is an important mediator that plays a pathogenic role in cardiac hypertrophy and heart failure. In the present study, we investigated whether doxazosin could stimulate galectin-3 expression and collagen synthesis in cultured HL-1 cardiomyocytes. We found that doxazosin dose-dependently induced galectin-3 protein expression, with a statistically significant increase in expression with a dose as low as 0.01 μM. Doxazosin upregulated collagen I and α-smooth muscle actin (α-SMA protein levels and also induced apoptotic protein caspase-3 in HL-1 cardiomyocytes. Although we previously reported that activation of protein kinase C (PKC stimulates galectin-3 expression, blocking the PKC pathway with the PKC inhibitor chelerythrine did not prevent doxazosin-induced galectin-3 and collagen expression. Consistently, doxazosin treatment did not alter total and phosphorylated PKC. These results suggest that doxazosin-stimulated galectin-3 is independent of PKC pathway. To determine if the α1-adrenergic pathway is involved, we pretreated the cells with the irreversible α-adrenergic receptor blocker phenoxybenzamine and found that doxazosin-stimulated galectin-3 and collagen expression was similar to controls, suggesting that doxazosin acts independently of α1-adrenergic receptor blockade. Collectively, we show a novel effect of doxazosin on cardiomycytes by stimulating heart fibrosis factor galectin-3 expression. The mechanism of action of doxazosin is not mediated through either activation of the PKC pathway or antagonism of α1-adrenergic receptors.

Protein kinases mediate increment of the phosphorylation of cyclic AMP -responsive element binding protein in spinal cord of rats following capsaicin injection

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Li Junfa

2005-09-01

Full Text Available Abstract Background Strong noxious stimuli cause plastic changes in spinal nociceptive neurons. Intracellular signal transduction pathways from cellular membrane to nucleus, which may further regulate gene expression by critical transcription factors, convey peripheral stimulation. Cyclic AMP-responsive element binding protein (CREB is a well-characterized stimulus-induced transcription factor whose activation requires phosphorylation of the Serine-133 residue. Phospho-CREB can further induce gene transcription and strengthen synaptic transmission by the activation of the protein kinase cascades. However, little is known about the mechanisms by which CREB phosphorylation is regulated by protein kinases during nociception. This study was designed to use Western blot analysis to investigate the role of mitogen-activated protein (MAP/extracellular signal-regulated kinase (ERK kinase (MEK 1/2, PKA and PKC in regulating the phosphorylation of CREB in the spinal cord of rats following intraplantar capsaicin injection. Results We found that capsaicin injection significantly increased the phosphorylation level of CREB in the ipsilateral side of the spinal cord. Pharmacological manipulation of MEK 1/2, PKA and PKC with their inhibitors (U0126, H89 and NPC 15473, respectively significantly blocked this increment of CREB phosphorylation. However, the expression of CREB itself showed no change in any group. Conclusion These findings suggest that the activation of intracellular MAP kinase, PKA and PKC cascades may contribute to the regulation of phospho-CREB in central nociceptive neurons following peripheral painful stimuli.

Regulation of myosin light chain kinase during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

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Shelly Woody

Full Text Available Myosin II (MyoII is required for insulin-responsive glucose transporter 4 (GLUT4-mediated glucose uptake in 3T3-L1 adipocytes. Our previous studies have shown that insulin signaling stimulates phosphorylation of the regulatory light chain (RLC of MyoIIA via myosin light chain kinase (MLCK. The experiments described here delineate upstream regulators of MLCK during insulin-stimulated glucose uptake. Since 3T3-L1 adipocytes express two MyoII isoforms, we wanted to determine which isoform was required for insulin-stimulated glucose uptake. Using a siRNA approach, we demonstrate that a 60% decrease in MyoIIA protein expression resulted in a 40% inhibition of insulin-stimulated glucose uptake. We also show that insulin signaling stimulates the phosphorylation of MLCK. We further show that MLCK can be activated by calcium as well as signaling pathways. We demonstrate that adipocytes treated with the calcium chelating agent, 1,2-b (iso-aminophenoxy ethane-N,N,N',N'-tetra acetic acid, (BAPTA (in the presence of insulin impaired the insulin-induced phosphorylation of MLCK by 52% and the RLC of MyoIIA by 45% as well as impairing the recruitment of MyoIIA to the plasma membrane when compared to cells treated with insulin alone. We further show that the calcium ionophore, A23187 alone stimulated the phosphorylation of MLCK and the RLC associated with MyoIIA to the same extent as insulin. To identify signaling pathways that might regulate MLCK, we examined ERK and CaMKII. Inhibition of ERK2 impaired phosphorylation of MLCK and insulin-stimulated glucose uptake. In contrast, while inhibition of CaMKII did inhibit phosphorylation of the RLC associated with MyoIIA, inhibition of CAMKIIδ did not impair MLCK phosphorylation or translocation to the plasma membrane or glucose uptake. Collectively, our results are the first to delineate a role for calcium and ERK in the activation of MLCK and thus MyoIIA during insulin-stimulated glucose uptake in 3T3-L1 adipocytes.

Contractions activate hormone-sensitive lipase in rat muscle by protein kinase C and mitogen-activated protein kinase

DEFF Research Database (Denmark)

Donsmark, Morten; Langfort, Jozef; Holm, Cecilia

2003-01-01

and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction......Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline......-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50% by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None...

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

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

2009-02-15

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

Negative regulatory role of PI3-kinase in TNF-induced tumor necrosis.

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Matschurat, Susanne; Blum, Sabine; Mitnacht-Kraus, Rita; Dijkman, Henry B P M; Kanal, Levent; De Waal, Robert M W; Clauss, Matthias

2003-10-20

Tissue factor is the prime initiator of blood coagulation. Expression of tissue factor in tumor endothelial cells leads to thrombus formation, occlusion of vessels and development of hemorrhagic infarctions in the tumor tissue, often followed by regression of the tumor. Tumor cells produce endogenous vascular endothelial growth factor (VEGF), which sensitizes endothelial cells for systemically administered tumor necrosis factor alpha (TNF alpha) and synergistically enhances the TNF-induced expression of tissue factor. We have analyzed the pathways involved in the induction of tissue factor in human umbilical cord vein endothelial cells (HUVECs) after combined stimulation with TNF and VEGF. By using specific low molecular weight inhibitors, we demonstrated that protein kinase C (PKC), p44/42 and p38 mitogen-activated protein (MAP) kinases, and stress-activated protein kinase (JNK) are essentially involved in the induction of tissue factor. In contrast, the application of wortmannin, an inhibitor of phosphatidylinositol 3 (PI3)-kinase, led to strongly enhanced expression of tissue factor in TNF- and VEGF-treated cells, implicating a negative regulatory role for PI3-kinase. In vivo, the application of wortmannin promoted the formation of TNF-induced hemorrhages and intratumoral necroses in murine meth A tumors. The co-injection of wortmannin lowered the effective dose of applied TNF. Therefore, it is conceivable that the treatment of TNF-sensitive tumors with a combination of TNF and wortmannin will ensure the selective damage of the tumor endothelium and minimize the risk of systemic toxicity of TNF. TNF-treatment in combination with specific inhibition of PI3-kinase is a novel concept in anti-cancer therapy. Copyright 2003 Wiley-Liss, Inc.

MAP17 Is a Necessary Activator of Renal Na+/Glucose Cotransporter SGLT2

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Coady, Michael J.; El Tarazi, Abdulah; Santer, René; Bissonnette, Pierre; Sasseville, Louis J.; Calado, Joaquim; Lussier, Yoann; Dumayne, Christopher; Bichet, Daniel G.

2017-01-01

The renal proximal tubule reabsorbs 90% of the filtered glucose load through the Na+-coupled glucose transporter SGLT2, and specific inhibitors of SGLT2 are now available to patients with diabetes to increase urinary glucose excretion. Using expression cloning, we identified an accessory protein, 17 kDa membrane-associated protein (MAP17), that increased SGLT2 activity in RNA-injected Xenopus oocytes by two orders of magnitude. Significant stimulation of SGLT2 activity also occurred in opossum kidney cells cotransfected with SGLT2 and MAP17. Notably, transfection with MAP17 did not change the quantity of SGLT2 protein at the cell surface in either cell type. To confirm the physiologic relevance of the MAP17–SGLT2 interaction, we studied a cohort of 60 individuals with familial renal glucosuria. One patient without any identifiable mutation in the SGLT2 coding gene (SLC5A2) displayed homozygosity for a splicing mutation (c.176+1G>A) in the MAP17 coding gene (PDZK1IP1). In the proximal tubule and in other tissues, MAP17 is known to interact with PDZK1, a scaffolding protein linked to other transporters, including Na+/H+ exchanger 3, and to signaling pathways, such as the A-kinase anchor protein 2/protein kinase A pathway. Thus, these results provide the basis for a more thorough characterization of SGLT2 which would include the possible effects of its inhibition on colocalized renal transporters. PMID:27288013

Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases.

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Schlaepfer, D D; Hunter, T

1996-10-01

Focal adhesion kinase (FAK) is a nonreceptor protein-tyrosine kinase (PTK) that associates with integrin receptors and participates in extracellular matrix-mediated signal transduction events. We showed previously that the c-Src nonreceptor PTK and the Grb2 SH2/SH3 adaptor protein bound directly to FAK after fibronectin stimulation (D. D. Schlaepfer, S.K. Hanks, T. Hunter, and P. van der Geer, Nature [London] 372:786-791, 1994). Here, we present evidence that c-Src association with FAK is required for Grb2 binding to FAK. Using a tryptic phosphopeptide mapping approach, the in vivo phosphorylation of the Grb2 binding site on FAK (Tyr-925) was detected after fibronectin stimulation of NIH 3T3 cells and was constitutively phosphorylated in v-Src-transformed NIH 3T3 cells. In vitro, c-Src phosphorylated FAK Tyr-925 in a glutathione S-transferase-FAK C-terminal domain fusion protein, whereas FAK did not. Using epitope-tagged FAK constructs, transiently expressed in human 293 cells, we determined the effect of site-directed mutations on c-Src and Grb2 binding to FAK. Mutation of FAK Tyr-925 disrupted Grb2 binding, whereas mutation of the c-Src binding site on FAK (Tyr-397) disrupted both c-Src and Grb2 binding to FAK in vivo. These results support a model whereby Src-family PTKs are recruited to FAK and focal adhesions following integrin-induced autophosphorylation and exposure of FAK Tyr-397. Src-family binding and phosphorylation of FAK at Tyr-925 creates a Grb2 SH2-domain binding site and provides a link to the activation of the Ras signal transduction pathway. In Src-transformed cells, this pathway may be constitutively activated as a result of FAK Tyr-925 phosphorylation in the absence of integrin stimulation.

Molecular Basis of Meiotic Maturation and Apoptosis of Oocytes, Sperm-Oocyte Interactions and Early Cleavage of Embryos in Mice, Role of Phosphatidylinositol 3-Kinase, Mos, Fas-Fas Ligand, Integrinα6 and MAP Kinase

OpenAIRE

Yumi Hoshino; Ken-ichi Yamanaka; Ikuo Tomioka; Noritaka Fukunaga; Mehdi Abbasi; Eimei Sato

2005-01-01

The interaction between molecular biology and embryology made an extensive progress in the research on gametogenesis, fertilization and early embryogenesis in mice. In this article, molecules involving in meiotic maturation and apoptosis of oocytes, sperm-oocyte interactions and early cleavage of fertilized embryos in mice are described including our recent following experiments. 1) Phosphatidylinositol 3-kinase and Akt participate in the follicle stimulating hormone-induced meiotic maturatio...

Protein kinase N2 regulates AMP kinase signaling and insulin responsiveness of glucose metabolism in skeletal muscle.

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Ruby, Maxwell A; Riedl, Isabelle; Massart, Julie; Åhlin, Marcus; Zierath, Juleen R

2017-10-01

Insulin resistance is central to the development of type 2 diabetes and related metabolic disorders. Because skeletal muscle is responsible for the majority of whole body insulin-stimulated glucose uptake, regulation of glucose metabolism in this tissue is of particular importance. Although Rho GTPases and many of their affecters influence skeletal muscle metabolism, there is a paucity of information on the protein kinase N (PKN) family of serine/threonine protein kinases. We investigated the impact of PKN2 on insulin signaling and glucose metabolism in primary human skeletal muscle cells in vitro and mouse tibialis anterior muscle in vivo. PKN2 knockdown in vitro decreased insulin-stimulated glucose uptake, incorporation into glycogen, and oxidation. PKN2 siRNA increased 5'-adenosine monophosphate-activated protein kinase (AMPK) signaling while stimulating fatty acid oxidation and incorporation into triglycerides and decreasing protein synthesis. At the transcriptional level, PKN2 knockdown increased expression of PGC-1α and SREBP-1c and their target genes. In mature skeletal muscle, in vivo PKN2 knockdown decreased glucose uptake and increased AMPK phosphorylation. Thus, PKN2 alters key signaling pathways and transcriptional networks to regulate glucose and lipid metabolism. Identification of PKN2 as a novel regulator of insulin and AMPK signaling may provide an avenue for manipulation of skeletal muscle metabolism. Copyright © 2017 the American Physiological Society.

MAP kinases in inflammatory bowel disease

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Coskun, Mehmet; Olsen, Jørgen; Seidelin, Jakob Benedict

2011-01-01

The mammalian family of mitogen-activated protein kinases (MAPKs) is activated by diverse extracellular and intracellular stimuli, and thereby they play an essential role in connecting cell-surface receptors to changes in transcriptional programs. The MAPK signaling pathways regulate a wide range...... these signaling pathways have been exploited for the development of therapeutics and discuss the current knowledge of potential MAPK inhibitors and their anti-inflammatory effects in clinical trials related to IBD....

Diacylglycerol kinase ζ regulates RhoA activation via a kinase-independent scaffolding mechanism

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Ard, Ryan; Mulatz, Kirk; Abramovici, Hanan

2012-01-01

, but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase Cα (PKCα......GDI and was required for efficient interaction of PKCα and RhoA. DGKζ-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGKζ-null cells. Collectively our findings suggest...

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

Science.gov (United States)

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.

Calcitonin causes a sustained inhibition of protein kinase C-stimulated bone resorption in contrast to the transient inhibition of parathyroid hormone-induced bone resorption

International Nuclear Information System (INIS)

Ransjoe, M.; Lerner, U.H.

1990-01-01

Calcitonin is a well known inhibitor of osteoclastic bone resortion, both in vivo and in vitro. However, it is also known that calcitonin has only a transient inhibitory effect on bone resorption. The mechanism for this so-called ''escape from inhibition'' phenomenon is not clear. In the present study, the inhibitory effect of calcitonin on phorbol ester-induced bone resorption was examined in cultured neonatal mouse calvaria. Bone resorption was assessed as the release of radioactivity from bones prelabelled in vivo with 45 Ca. Two proteon kinase C-activating phorbol esters, phorbol-12-myristate-13-acetate and phorbol-12,13-dibutyrate, both stimulated 45 Ca release in 120-h cultures at a concentration of 10 nmul/l. Calcitonin (30 nmol/l) inhibited phorbol esterstimulated bone resorption without any ''escape from inhibition''. This was in contrast to the transient inhibitory effect of calcitonin on bone resorption stimulated by parathyroid hormone (10 nmol/l), prostaglandin E 2 (2 μmol/l), and bradykinin (1 μmol/l). Our results suggest that activation of protein kinase C produces a sustained inhibitory effect of calcitonin on bone resorption. (author)

The Role of PAS Kinase in PASsing the Glucose Signal

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Julianne H. Grose

2010-06-01

Full Text Available PAS kinase is an evolutionarily conserved nutrient responsive protein kinase that regulates glucose homeostasis. Mammalian PAS kinase is activated by glucose in pancreatic beta cells, and knockout mice are protected from obesity, liver triglyceride accumulation, and insulin resistance when fed a high-fat diet. Yeast PAS kinase is regulated by both carbon source and cell integrity stress and stimulates the partitioning of glucose toward structural carbohydrate biosynthesis. In our current model for PAS kinase regulation, a small molecule metabolite binds the sensory PAS domain and activates the enzyme. Although bona fide PAS kinase substrates are scarce, in vitro substrate searches provide putative targets for exploration.

Stimulation of JNK Phosphorylation by the PTTH in Prothoracic Glands of the Silkworm, Bombyx mori

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Shi-Hong Gu

2018-02-01

Full Text Available In this study, phosphorylation of c-Jun N-terminal kinase (JNK by the prothoracicotropic hormone (PTTH was investigated in prothoracic glands (PGs of the silkworm, Bombyx mori. Results showed that JNK phosphorylation was stimulated by the PTTH in time- and dose-dependent manners. In vitro activation of JNK phosphorylation in PGs by the PTTH was also confirmed in an in vivo experiment, in which a PTTH injection greatly increased JNK phosphorylation in PGs of day-6 last instar larvae. JNK phosphorylation caused by PTTH stimulation was greatly inhibited by U73122, a potent and specific inhibitor of phospholipase C (PLC and an increase in JNK phosphorylation was also detected when PGs were treated with agents (either A23187 or thapsigargin that directly elevated the intracellular Ca2+ concentration, thereby indicating involvement of PLC and Ca2+. Pretreatment with an inhibitor (U0126 of mitogen-activated protein kinase (MAPK/extracellular signal-regulated kinase (ERK kinase (MEK and an inhibitor (LY294002 of phosphoinositide 3-kinase (PI3K failed to significantly inhibit PTTH-stimulated JNK phosphorylation, indicating that ERK and PI3K were not related to JNK. We further investigated the effect of modulation of the redox state on JNK phosphorylation. In the presence of either an antioxidant (N-acetylcysteine, NAC or diphenylene iodonium (DPI, PTTH-stimulated JNK phosphorylation was blocked. The JNK kinase inhibitor, SP600125, markedly inhibited PTTH-stimulated JNK phosphorylation and ecdysteroid synthesis. The kinase assay of JNK in PGs confirmed its stimulation by PTTH and inhibition by SP600125. Moreover, PTTH treatment did not affect JNK or Jun mRNA expressions. Based on these findings, we concluded that PTTH stimulates JNK phosphorylation in Ca2+- and PLC-dependent manners and that the redox-regulated JNK signaling pathway is involved in PTTH-stimulated ecdysteroid synthesis in B. mori PGs.

Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

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Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P.; Taub, Dennis D.

2014-01-01

Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levelsand impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

Impaired intracortical transmission in G2019S leucine rich-repeat kinase Parkinson patients.

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Ponzo, Viviana; Di Lorenzo, Francesco; Brusa, Livia; Schirinzi, Tommaso; Battistini, Stefania; Ricci, Claudia; Sambucci, Manolo; Caltagirone, Carlo; Koch, Giacomo

2017-05-01

A mutation in leucine-rich repeat kinase 2 is the most common cause of hereditary Parkinson's disease (PD), yet the neural mechanisms and the circuitry potentially involved are poorly understood. We used different transcranial magnetic stimulation protocols to explore in the primary motor cortex the activity of intracortical circuits and cortical plasticity (long-term potentiation) in patients with the G2019S leucine-rich repeat kinase 2 gene mutation when compared with idiopathic PD patients and age-matched healthy subjects. Paired pulse transcranial magnetic stimulation was used to investigate short intracortical inhibition and facilitation and short afferent inhibition. Intermittent theta burst stimulation, a form of repetitive transcranial magnetic stimulation, was used to test long-term potentiation-like cortical plasticity. Leucine-rich repeat kinase 2 and idiopathic PD were tested both in ON and in OFF l-dopa therapy. When compared with idiopathic PD and healthy subjects, leucine-rich repeat kinase 2 PD patients showed a remarkable reduction of short intracortical inhibition in both ON and in OFF l-dopa therapy. This reduction was paralleled by an increase of intracortical facilitation in OFF l-dopa therapy. Leucine-rich repeat kinase 2 PD showed abnormal long-term potentiation-like cortical plasticity in ON l-dopa therapy. The motor cortex in leucine-rich repeat kinase 2 mutated PD patients is strongly disinhibited and hyperexcitable. These abnormalities could be a result of an impairment of inhibitory (gamma-Aminobutyric acid) transmission eventually related to altered neurotransmitter release. © 2017 International Parkinson and Movement Disorder Society. © 2017 International Parkinson and Movement Disorder Society.

STATE OF JNK AND P38 MAP-KINASE SYSTEM IN BLOOD monon uclea r le ucocytes DUR ING INFLAMMATION

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N. Y. Chasovskih

2009-01-01

Full Text Available Abstract. Pogrammed cell death of peripheral blood mononuclear leucocytes from patients with acute inflammatory diseases (non-nosocomial pneumonia, acute appendicitis was investigated under ex vivo conditions, upon cultivation of the cells with selective inhibitors of JNK (SP600125 and р38 МАРК (ML3403. In vitro addition of SP600125 and ML3403 under oxidative stress conditions prevents increase of annexinpositive mononuclear cells numbers, thus suggesting JNK and р38 МАР-kinases to be involved into oxidative mechanisms of apoptosis deregulation. A role of JNK in IL-8 production by mononuclear leucocytes was revealed in cases of acute inflammation. Regulatory effect of JNK and p38 MAP-kinases can be mediated through activation of redox-sensitive apoptogenic signal transduction systems, as well as due to changes in cellular cytokine-producing function.

Cell-type-specific activation of mitogen-activated protein kinases in PAN-induced progressive renal disease in rats

International Nuclear Information System (INIS)

Park, Sang-Joon; Jeong, Kyu-Shik

2004-01-01

We examined the time-course activation and the cell-type specific role of MAP kinases in puromycin aminonucleoside (PAN)-induced renal disease. The maximal activation of c-Jun-NH 2 -terminal kinase (JNK), extracellular signal regulated kinase (ERK), and p38 MAP kinase was detected on Days 52, 38, and 38 after PAN-treatment, respectively. p-JNK was localized in mesangial and proximal tubular cells at the early renal injury. It was expressed, therefore, in the inflammatory cells of tubulointerstitial lesions. While, p-ERK was markedly increased in the glomerular regions and macrophages p-p38 was observed in glomerular endothelial cells, tubular cells, and some inflammatory cells. The results show that the activation of MAP kinases in the early renal injury by PAN-treatment involves cellular changes such as cell proliferation or apoptosis in renal native cells. The activation of MAP kinases in infiltrated inflammatory cells and fibrotic cells plays an important role in destructive events such as glomerulosclerosis and tubulointerstitial fibrosis

Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival.

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Daniel Thomas

Full Text Available The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K, promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting

Caffeine and contraction synergistically stimulate 5'-AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscle.

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Tsuda, Satoshi; Egawa, Tatsuro; Kitani, Kazuto; Oshima, Rieko; Ma, Xiao; Hayashi, Tatsuya

2015-10-01

5'-Adenosine monophosphate-activated protein kinase (AMPK) has been identified as a key mediator of contraction-stimulated insulin-independent glucose transport in skeletal muscle. Caffeine acutely stimulates AMPK in resting skeletal muscle, but it is unknown whether caffeine affects AMPK in contracting muscle. Isolated rat epitrochlearis muscle was preincubated and then incubated in the absence or presence of 3 mmol/L caffeine for 30 or 120 min. Electrical stimulation (ES) was used to evoke tetanic contractions during the last 10 min of the incubation period. The combination of caffeine plus contraction had additive effects on AMPKα Thr(172) phosphorylation, α-isoform-specific AMPK activity, and 3-O-methylglucose (3MG) transport. In contrast, caffeine inhibited basal and contraction-stimulated Akt Ser(473) phosphorylation. Caffeine significantly delayed muscle fatigue during contraction, and the combination of caffeine and contraction additively decreased ATP and phosphocreatine contents. Caffeine did not affect resting tension. Next, rats were given an intraperitoneal injection of caffeine (60 mg/kg body weight) or saline, and the extensor digitorum longus muscle was dissected 15 min later. ES of the sciatic nerve was performed to evoke tetanic contractions for 5 min before dissection. Similar to the findings from isolated muscles incubated in vitro, the combination of caffeine plus contraction in vivo had additive effects on AMPK phosphorylation, AMPK activity, and 3MG transport. Caffeine also inhibited basal and contraction-stimulated Akt phosphorylation in vivo. These findings suggest that caffeine and contraction synergistically stimulate AMPK activity and insulin-independent glucose transport, at least in part by decreasing muscle fatigue and thereby promoting energy consumption during contraction. © 2015 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological

Induction of activator protein (AP)-1 and nuclear factor-kappaB by CD28 stimulation involves both phosphatidylinositol 3-kinase and acidic sphingomyelinase signals.

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Edmead, C E; Patel, Y I; Wilson, A; Boulougouris, G; Hall, N D; Ward, S G; Sansom, D M

1996-10-15

A major obstacle in understanding the signaling events that follow CD28 receptor ligation arises from the fact that CD28 acts as a costimulus to TCR engagement, making it difficult to assess the relative contribution of CD28 signals as distinct from those of the TCR. To overcome this problem, we have exploited the observation that activated human T cell blasts can be stimulated via the CD28 surface molecule in the absence of antigenic challenge; thus, we have been able to observe the response of normal T cells to CD28 activation in isolation. Using this system, we observed that CD28 stimulation by B7-transfected CHO cells induced a proliferative response in T cells that was not accompanied by measurable IL-2 production. However, subsequent analysis of transcription factor generation revealed that B7 stimulation induced both activator protein-1 (AP-1) and nuclear factor-kappaB (NF-kappaB) complexes, but not NF-AT. In contrast, engagement of the TCR by class II MHC/superantigen, either with or without CD28 ligation, resulted in the induction of NF-AT, AP-1, and NF-kappaB as well as IL-2 production. Using selective inhibitors, we investigated the signaling pathways involved in the CD28-mediated induction of AP-1 and NF-kappaB. This revealed that NF-kappaB generation was sensitive to chloroquine, an inhibitor of acidic sphingomyelinase, but not to the phosphatidylinositol 3-kinase inhibitor, wortmannin. In contrast, AP-1 generation was inhibited by wortmannin and was also variably sensitive to chloroquine. These data suggest that in activated normal T cells, CD28-derived signals can stimulate proliferation at least in part via NF-kappaB and AP-1 generation, and that this response uses both acidic sphingomyelinase and phosphatidylinositol 3-kinase-linked pathways.

Caffeine and contraction synergistically stimulate 5′-AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal muscle

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Tsuda, Satoshi; Egawa, Tatsuro; Kitani, Kazuto; Oshima, Rieko; Ma, Xiao; Hayashi, Tatsuya

2015-01-01

5′-Adenosine monophosphate-activated protein kinase (AMPK) has been identified as a key mediator of contraction-stimulated insulin-independent glucose transport in skeletal muscle. Caffeine acutely stimulates AMPK in resting skeletal muscle, but it is unknown whether caffeine affects AMPK in contracting muscle. Isolated rat epitrochlearis muscle was preincubated and then incubated in the absence or presence of 3 mmol/L caffeine for 30 or 120 min. Electrical stimulation (ES) was used to evoke tetanic contractions during the last 10 min of the incubation period. The combination of caffeine plus contraction had additive effects on AMPKα Thr172 phosphorylation, α-isoform-specific AMPK activity, and 3-O-methylglucose (3MG) transport. In contrast, caffeine inhibited basal and contraction-stimulated Akt Ser473 phosphorylation. Caffeine significantly delayed muscle fatigue during contraction, and the combination of caffeine and contraction additively decreased ATP and phosphocreatine contents. Caffeine did not affect resting tension. Next, rats were given an intraperitoneal injection of caffeine (60 mg/kg body weight) or saline, and the extensor digitorum longus muscle was dissected 15 min later. ES of the sciatic nerve was performed to evoke tetanic contractions for 5 min before dissection. Similar to the findings from isolated muscles incubated in vitro, the combination of caffeine plus contraction in vivo had additive effects on AMPK phosphorylation, AMPK activity, and 3MG transport. Caffeine also inhibited basal and contraction-stimulated Akt phosphorylation in vivo. These findings suggest that caffeine and contraction synergistically stimulate AMPK activity and insulin-independent glucose transport, at least in part by decreasing muscle fatigue and thereby promoting energy consumption during contraction. PMID:26471759

PET Mapping for Brain-Computer Interface Stimulation of the Ventroposterior Medial Nucleus of the Thalamus in Rats with Implanted Electrodes.

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Zhu, Yunqi; Xu, Kedi; Xu, Caiyun; Zhang, Jiacheng; Ji, Jianfeng; Zheng, Xiaoxiang; Zhang, Hong; Tian, Mei

2016-07-01

Brain-computer interface (BCI) technology has great potential for improving the quality of life for neurologic patients. This study aimed to use PET mapping for BCI-based stimulation in a rat model with electrodes implanted in the ventroposterior medial (VPM) nucleus of the thalamus. PET imaging studies were conducted before and after stimulation of the right VPM. Stimulation induced significant orienting performance. (18)F-FDG uptake increased significantly in the paraventricular thalamic nucleus, septohippocampal nucleus, olfactory bulb, left crus II of the ansiform lobule of the cerebellum, and bilaterally in the lateral septum, amygdala, piriform cortex, endopiriform nucleus, and insular cortex, but it decreased in the right secondary visual cortex, right simple lobule of the cerebellum, and bilaterally in the somatosensory cortex. This study demonstrated that PET mapping after VPM stimulation can identify specific brain regions associated with orienting performance. PET molecular imaging may be an important approach for BCI-based research and its clinical applications. © 2016 by the Society of Nuclear Medicine and Molecular Imaging, Inc.

Inducible Activation of ERK5 MAP Kinase Enhances Adult Neurogenesis in the Olfactory Bulb and Improves Olfactory Function

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Wang, Wenbin; Lu, Song; Li, Tan; Pan, Yung-Wei; Zou, Junhui; Abel, Glen M.; Xu, Lihong; Storm, Daniel R.

2015-01-01

Recent discoveries have suggested that adult neurogenesis in the subventricular zone (SVZ) and olfactory bulb (OB) may be required for at least some forms of olfactory behavior in mice. However, it is unclear whether conditional and selective enhancement of adult neurogenesis by genetic approaches is sufficient to improve olfactory function under physiological conditions or after injury. Furthermore, specific signaling mechanisms regulating adult neurogenesis in the SVZ/OB are not fully defined. We previously reported that ERK5, a MAP kinase selectively expressed in the neurogenic regions of the adult brain, plays a critical role in adult neurogenesis in the SVZ/OB. Using a site-specific knock-in mouse model, we report here that inducible and targeted activation of the endogenous ERK5 in adult neural stem/progenitor cells enhances adult neurogenesis in the OB by increasing cell survival and neuronal differentiation. This conditional ERK5 activation also improves short-term olfactory memory and odor-cued associative olfactory learning under normal physiological conditions. Furthermore, these mice show enhanced recovery of olfactory function and have more adult-born neurons after a zinc sulfate-induced lesion of the main olfactory epithelium. We conclude that ERK5 MAP kinase is an important endogenous signaling pathway regulating adult neurogenesis in the SVZ/OB, and that conditional activation of endogenous ERK5 is sufficient to enhance adult neurogenesis in the OB thereby improving olfactory function both under normal conditions and after injury. PMID:25995470

A Myb transcription factor of Phytophthora sojae, regulated by MAP kinase PsSAK1, is required for zoospore development.

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Meng Zhang

Full Text Available PsSAK1, a mitogen-activated protein (MAP kinase from Phytophthora sojae, plays an important role in host infection and zoospore viability. However, the downstream mechanism of PsSAK1 remains unclear. In this study, the 3'-tag digital gene expression (DGE profiling method was applied to sequence the global transcriptional sequence of PsSAK1-silenced mutants during the cysts stage and 1.5 h after inoculation onto susceptible soybean leaf tissues. Compared with the gene expression levels of the recipient P. sojae strain, several candidates of Myb family were differentially expressed (up or down in response to the loss of PsSAK1, including of a R2R3-type Myb transcription factor, PsMYB1. qRT-PCR indicated that the transcriptional level of PsMYB1 decreased due to PsSAK1 silencing. The transcriptional level of PsMYB1 increased during sporulating hyphae, in germinated cysts, and early infection. Silencing of PsMYB1 results in three phenotypes: a no cleavage of the cytoplasm into uninucleate zoospores or release of normal zoospores, b direct germination of sporangia, and c afunction in zoospore-mediated plant infection. Our data indicate that the PsMYB1 transcription factor functions downstream of MAP kinase PsSAK1 and is required for zoospore development of P. sojae.

PI3 kinase is important for Ras, MEK and Erk activation of Epo-stimulated human erythroid progenitors

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Schmidt Enrico K

2004-05-01

Full Text Available Abstract Background Erythropoietin is a multifunctional cytokine which regulates the number of erythrocytes circulating in mammalian blood. This is crucial in order to maintain an appropriate oxygen supply throughout the body. Stimulation of primary human erythroid progenitors (PEPs with erythropoietin (Epo leads to the activation of the mitogenic kinases (MEKs and Erks. How this is accomplished mechanistically remained unclear. Results Biochemical studies with human cord blood-derived PEPs now show that Ras and the class Ib enzyme of the phosphatidylinositol-3 kinase (PI3K family, PI3K gamma, are activated in response to minimal Epo concentrations. Surprisingly, three structurally different PI3K inhibitors block Ras, MEK and Erk activation in PEPs by Epo. Furthermore, Erk activation in PEPs is insensitive to the inhibition of Raf kinases but suppressed upon PKC inhibition. In contrast, Erk activation induced by stem cell factor, which activates c-Kit in the same cells, is sensitive to Raf inhibition and insensitive to PI3K and PKC inhibitors. Conclusions These unexpected findings contrast with previous results in human primary cells using Epo at supraphysiological concentrations and open new doors to eventually understanding how low Epo concentrations mediate the moderate proliferation of erythroid progenitors under homeostatic blood oxygen levels. They indicate that the basal activation of MEKs and Erks in PEPs by minimal concentrations of Epo does not occur through the classical cascade Shc/Grb2/Sos/Ras/Raf/MEK/Erk. Instead, MEKs and Erks are signal mediators of PI3K, probably the recently described PI3K gamma, through a Raf-independent signaling pathway which requires PKC activity. It is likely that higher concentrations of Epo that are induced by hypoxia, for example, following blood loss, lead to additional mitogenic signals which greatly accelerate erythroid progenitor proliferation.

Cyclic AMP-dependent protein kinase interferes with GTP γS stimulated IP3 formation in differentiated HL-60 cell membranes

International Nuclear Information System (INIS)

Misaki, Naoyuki; Imaizumi, Taro; Watanabe, Yashuiro

1989-01-01

The effects of addition of activated cyclic AMP-dependent protein kinase (PKA) on the function of islet-activating protein (IAP)-sensitive GTP-binding (G) protein were studied in the plasma membranes of 3 H-inositol-labeled differentiated human leukemic (HL-60) cells. Pretreatment of the membranes with activated PKA in the presence of MgATP for 15 min. at 37 degree C decreased GTP γS-stimulated inositol trisphosphate (IP 3 ) formation by about 30%, but had no influence on Ca 2+ -stimulated IP 3 formation. And autoradiography in the phosphorylation experiments of solubilized HL-60 cell membranes by PKA showed some 32 P incorporated bands, and among them one of the major bands showed the migration at 40 kDa supporting that the G protein coupling with PI response was phosphorylated by PKA. These results showed that pretreatment with activated PKA inhibited the mediating function of the G protein between the fMLP receptor and phospholipase C by its phosphorylation

Human interleukin 1β stimulates islet insulin release by a mechanism not dependent on changes in phospholipase C and protein kinase C activities or Ca2+ handling

International Nuclear Information System (INIS)

Welsh, N.; Nilsson, T.; Hallberg, A.; Arkhammar, P.; Berggren, P.-O.; Sandler, S.

1989-01-01

Isolated islets from adult rats or obese hyperglycemic (ob/ob) mice were incubated with human recombinant interleukin 1β in order to study whether the acute effects of the cytokine on islet insulin release are associated with changes in islet phospholipase C activity, Ca 2+ handling or protein phosphorylation. The cytokine stimulated insulin release both at low and high glucose concentrations during one hour incubations. In shortterm incubations ( 2+ concentration at rest nor that observed subsequent to stimulation with a high concentration of glucose. Furthermore, the endogenous protein kinase C activity, as visualized by immunoprecipitation of a 32 P-labelled substrate for this enzyme, was not altered by interleukin 1β. Separation of 32 P-labelled proteins by means of 2-dimensional gel electrophoresis failed to reveal any specific effects of the cytokine on the total protein phosphorylation activity. These results suggest that the stimulatory effects on insulin release exerted by interleukin 1β are not caused by acute activation of phospholipase C and protein kinase C or by an alternation of islet Ca 2+ handling of the B-cells. (author)

Activation of protein kinase C inhibits synthesis and release of decidual prolactin

International Nuclear Information System (INIS)

Harman, I.; Costello, A.; Ganong, B.; Bell, R.M.; Handwerger, S.

1986-01-01

Activation of calcium-activated, phospholipid-dependent protein kinase C by diacylglycerol and phorbol esters has been shown to mediate release of hormones in many systems. To determine whether protein kinase C activation is also involved in the regulation of prolactin release from human decidual, the authors have examined the effects of various acylglycerols and phorbol esters on the synthesis and release of prolactin from cultured human decidual cells. sn-1,2-Dioctanolyglycerol (diC 8 ), which is known to stimulate protein kinase C in other systems, inhibited prolactin release in a dose-dependent manner with maximal inhibition of 53.1% at 100 μM. Diolein (100 μM), which also stimulates protein kinase C activity in some systems, inhibited prolactin release by 21.3%. Phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 4β-phorbol 12,13-dibutyrate, which activate protein kinase C in other systems, also inhibited the release of prolactin, which the protein kinase C inactivate 4α-phorbol-12,13-didecanoate was without effect. The inhibition of prolactin release was secondary to a decrease in prolactin synthesis. Although diC 8 and PMA inhibited the synthesis and release of prolactin, these agents had no effect on the synthesis or release of trichloroacetic acid-precipitable [ 35 S]methionine-labeled decidual proteins and did not cause the release of the cytosolic enzymes lactic dehydrogenase and alkaline phosphatase. DiC 8 and PMA stimulates the specific activity of protein kinase C in decidual tissue by 14.6 and 14.0-fold, respectively. The inhibition of the synthesis and release of prolactin by diC 8 and phorbol esters strongly implicates protein kinase C in the regulation of the production and release of prolactin from the decidua

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

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Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do, E-mail: ydjung@chonnam.ac.kr

2012-03-01

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

International Nuclear Information System (INIS)

Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho; Jung, Young Do

2012-01-01

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H 2 O 2 ) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H 2 O 2 increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells treated with nicotine

The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans.

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Serena A D'Souza

2016-04-01

Full Text Available The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle's plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue.

Staphylococcal PknB as the First Prokaryotic Representative of the Proline-Directed Kinases

NARCIS (Netherlands)

Miller, Malgorzata; Donat, Stefanie; Rakette, Sonja; Stehle, Thilo; Kouwen, Thijs R. H. M.; Diks, Sander H.; Dreisbach, Annette; Reilman, Ewoud; Gronau, Katrin; Becher, Doerte; Peppelenbosch, Maikel P.; van Dijl, Jan Maarten; Ohlsen, Knut

2010-01-01

In eukaryotic cell types, virtually all cellular processes are under control of proline-directed kinases and especially MAP kinases. Serine/threonine kinases in general were originally considered as a eukaryote-specific enzyme family. However, recent studies have revealed that orthologues of

Sphingosine-1-phosphate stimulates rat primary chondrocyte proliferation

International Nuclear Information System (INIS)

Kim, Mi-Kyoung; Lee, Ha Young; Kwak, Jong-Young; Park, Joo-In; Yun, Jeanho; Bae, Yoe-Sik

2006-01-01

Rat primary chondrocytes express the sphingosine-1-phosphate (S1P) receptor, S1P 2 , S1P 3 , S1P 4 , but not S1P 1 . When chondrocytes were stimulated with S1P or phytosphingosine-1-phosphate (PhS1P, an S1P 1 - and S1P 4 -selective agonist), phospholipase C-mediated cytosolic calcium increase was dramatically induced. S1P and PhS1P also stimulated two kinds of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK) and p38 kinase in chondrocytes. In terms of the two phospholipids-mediated functional modulation of chondrocytes, S1P and PhS1P stimulated cellular proliferation. The two phospholipids-induced chondrocyte proliferations were almost completely blocked by PD98059 but not by SB203580, suggesting that ERK but not p38 kinase is essentially required for the proliferation. Pertussis toxin almost completely inhibited the two phospholipids-induced cellular proliferation and ERK activation, indicating the crucial role of G i protein. This study demonstrates the physiological role of two important phospholipids (S1P and PhS1P) on the modulation of rat primary chondrocyte proliferation, and the crucial role played by ERK in the process

CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.

Science.gov (United States)

Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U

2000-09-12

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

CIKS, a connection to IκB kinase and stress-activated protein kinase

Science.gov (United States)

Leonardi, Antonio; Chariot, Alain; Claudio, Estefania; Cunningham, Kirk; Siebenlist, Ulrich

2000-01-01

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins. PMID:10962033

Contraction-associated translocation of protein kinase C in rat skeletal muscle

DEFF Research Database (Denmark)

Richter, Erik; Cleland, P J; Rattigan, S

1987-01-01

Electrical stimulation of the sciatic nerve of the anaesthetized rat in vivo led to a time-dependent translocation of protein kinase C from the muscle cytosol to the particulate fraction. Maximum activity of protein kinase C in the particulate fraction occurred after 2 min of intermittent short...... tetanic contractions of the gastrocnemius-plantaris-soleus muscle group and coincided with the loss of activity from the cytosol. Translocation of protein kinase C may imply a role for this kinase in contraction-initiated changes in muscle metabolism....

Stimulation of phosphatidylcholine breakdown and diacylglycerol production by growth factors in Swiss-3T3 cells.

Science.gov (United States)

Price, B D; Morris, J D; Hall, A

1989-01-01

The effect of a number of growth factors on phosphatidylcholine (PtdCho) turnover in Swiss-3T3 cells was studied. Phorbol 12-myristate 13-acetate (PMA), bombesin, platelet-derived growth factor (PDGF) and vasopressin rapidly stimulated PtdCho hydrolysis, diacylglycerol (DAG) production, and PtdCho synthesis. Insulin and prostaglandin F2 alpha (PGF2 alpha) stimulated PtdCho synthesis, but not its breakdown, whereas epidermal growth factor (EGF) and bradykinin were without effect. Stimulation of PtdCho hydrolysis by the above ligands resulted in increased production of phosphocholine and DAG (due to phospholipase C activity) and significant amounts of choline, suggesting activation of a phospholipase D as well. CDP-choline and glycerophosphocholine levels were unchanged. Down-regulation of protein kinase C with PMA (400 nM, 40 h) abolished the stimulation of PtdCho hydrolysis and PtdCho synthesis by PMA, bombesin, PDGF and vasopressin, but not the stimulation of PtdCho synthesis by insulin and PGF2 alpha. PtdCho hydrolysis therefore occurs predominantly by activation of protein kinase C (either by PMA or PtdIns hydrolysis) leading to elevation of DAG levels derived from non-PtdIns(4,5)P2 sources. PtdCho synthesis occurs by both a protein kinase C-dependent pathway (stimulated by PMA, PDGF, bombesin and vasopressin) and a protein kinase C-independent pathway (stimulated by insulin and PGF2 alpha). DAG production from PtdCho hydrolysis is not the primary signal to activate protein kinase C, but may contribute to long-term activation of this kinase. PMID:2690829

Phosphorylation of iRhom2 Controls Stimulated Proteolytic Shedding by the Metalloprotease ADAM17/TACE

Directory of Open Access Journals (Sweden)

Miguel Cavadas

2017-10-01

Full Text Available Cell surface metalloproteases coordinate signaling during development, tissue homeostasis, and disease. TACE (TNF-α-converting enzyme, is responsible for cleavage (“shedding” of membrane-tethered signaling molecules, including the cytokine TNF, and activating ligands of the EGFR. The trafficking of TACE within the secretory pathway requires its binding to iRhom2, which mediates the exit of TACE from the endoplasmic reticulum. An important, but mechanistically unclear, feature of TACE biology is its ability to be stimulated rapidly on the cell surface by numerous inflammatory and growth-promoting agents. Here, we report a role for iRhom2 in TACE stimulation on the cell surface. TACE shedding stimuli trigger MAP kinase-dependent phosphorylation of iRhom2 N-terminal cytoplasmic tail. This recruits 14-3-3 proteins, enforcing the dissociation of TACE from complexes with iRhom2, promoting the cleavage of TACE substrates. Our data reveal that iRhom2 controls multiple aspects of TACE biology, including stimulated shedding on the cell surface.

Crocin Suppresses LPS-Stimulated Expression of Inducible Nitric Oxide Synthase by Upregulation of Heme Oxygenase-1 via Calcium/Calmodulin-Dependent Protein Kinase 4

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Ji-Hee Kim

2014-01-01

Full Text Available Crocin is a water-soluble carotenoid pigment that is primarily used in various cuisines as a seasoning and coloring agent, as well as in traditional medicines for the treatment of edema, fever, and hepatic disorder. In this study, we demonstrated that crocin markedly induces the expression of heme oxygenase-1 (HO-1 which leads to an anti-inflammatory response. Crocin inhibited inducible nitric oxide synthase (iNOS expression and nitric oxide production via downregulation of nuclear factor kappa B activity in lipopolysaccharide- (LPS- stimulated RAW 264.7 macrophages. These effects were abrogated by blocking of HO-1 expression or activity. Crocin also induced Ca2+ mobilization from intracellular pools and phosphorylation of Ca2+/calmodulin-dependent protein kinase 4 (CAMK4. CAMK4 knockdown and kinase-dead mutant inhibited crocin-mediated HO-1 expression, Nrf2 activation, and phosphorylation of Akt, indicating that HO-1 expression is mediated by CAMK4 and that Akt is a downstream mediator of CAMK4 in crocin signaling. Moreover, crocin-mediated suppression of iNOS expression was blocked by CAMK4 inhibition. Overall, these results suggest that crocin suppresses LPS-stimulated expression of iNOS by inducing HO-1 expression via Ca2+/calmodulin-CAMK4-PI3K/Akt-Nrf2 signaling cascades. Our findings provide a novel molecular mechanism for the inhibitory effects of crocin against endotoxin-mediated inflammation.

Non-invasive mapping of calculation function by repetitive navigated transcranial magnetic stimulation.

Science.gov (United States)

Maurer, Stefanie; Tanigawa, Noriko; Sollmann, Nico; Hauck, Theresa; Ille, Sebastian; Boeckh-Behrens, Tobias; Meyer, Bernhard; Krieg, Sandro M

2016-11-01

Concerning calculation function, studies have already reported on localizing computational function in patients and volunteers by functional magnetic resonance imaging and transcranial magnetic stimulation. However, the development of accurate repetitive navigated TMS (rTMS) with a considerably higher spatial resolution opens a new field in cognitive neuroscience. This study was therefore designed to evaluate the feasibility of rTMS for locating cortical calculation function in healthy volunteers, and to establish this technique for future scientific applications as well as preoperative mapping in brain tumor patients. Twenty healthy subjects underwent rTMS calculation mapping using 5 Hz/10 pulses. Fifty-two previously determined cortical spots of the whole hemispheres were stimulated on both sides. The subjects were instructed to perform the calculation task composed of 80 simple arithmetic operations while rTMS pulses were applied. The highest error rate (80 %) for all errors of all subjects was observed in the right ventral precentral gyrus. Concerning division task, a 45 % error rate was achieved in the left middle frontal gyrus. The subtraction task showed its highest error rate (40 %) in the right angular gyrus (anG). In the addition task a 35 % error rate was observed in the left anterior superior temporal gyrus. Lastly, the multiplication task induced a maximum error rate of 30 % in the left anG. rTMS seems feasible as a way to locate cortical calculation function. Besides language function, the cortical localizations are well in accordance with the current literature for other modalities or lesion studies.

The Ser/Thr Protein Kinase Protein-Protein Interaction Map of M. tuberculosis.

Science.gov (United States)

Wu, Fan-Lin; Liu, Yin; Jiang, He-Wei; Luan, Yi-Zhao; Zhang, Hai-Nan; He, Xiang; Xu, Zhao-Wei; Hou, Jing-Li; Ji, Li-Yun; Xie, Zhi; Czajkowsky, Daniel M; Yan, Wei; Deng, Jiao-Yu; Bi, Li-Jun; Zhang, Xian-En; Tao, Sheng-Ce

2017-08-01

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Phospholipid environment alters hormone-sensitivity of the purified insulin receptor kinase.

OpenAIRE

Lewis, R E; Czech, M P

1987-01-01

Insulin receptor kinase, affinity-purified by adsorption and elution from immobilized insulin, is stimulated 2-3-fold by insulin in detergent solution. Reconstitution of the receptor kinase into leaky vesicles containing phosphatidylcholine and phosphatidylethanolamine (1:1, w/w) by detergent removal on Sephadex G-50 results in the complete loss of receptor kinase sensitivity to activation by insulin. Insulin receptors in these vesicles also exhibit an increase in their apparent affinity for ...

Tunable signal processing in synthetic MAP kinase cascades.

Science.gov (United States)

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.

Activation of c-Raf-1 kinase signal transduction pathway in alpha(7) integrin-deficient mice.

Science.gov (United States)

Saher, G; Hildt, E

1999-09-24

Integrin alpha(7)-deficient mice develop a novel form of muscular dystrophy. Here we report that deficiency of alpha(7) integrin causes an activation of the c-Raf-1/mitogen-activated protein (MAP) 2 kinase signal transduction pathway in muscle cells. The observed activation of c-Raf-1/MAP2 kinases is a specific effect, because the alpha(7) integrin deficiency does not cause unspecific stress as determined by measurement of the Hsp72/73 level and activity of the JNK2 kinase. Because an increased level of activated FAK was found in muscle of alpha(7) integrin-deficient mice, the activation of c-Raf-1 kinase is triggered most likely by an integrin-dependent pathway. In accordance with this, in the integrin alpha(7)-deficient mice, part of the integrin beta(1D) variant in muscle is replaced by the beta(1A) variant, which permits the FAK activation. A recent report describes that integrin activity can be down-modulated by the c-Raf-1/MAP2 kinase pathway. Specific activation of the c-Raf-1/MAP2 kinases by cell-permeable peptides in skeletal muscle of rabbits causes degeneration of muscle fibers. Therefore, we conclude that in alpha(7) integrin-deficient mice, the continuous activation of c-Raf-1 kinase causes a permanent reduction of integrin activity diminishing integrin-dependent cell-matrix interactions and thereby contributing to the development of the dystrophic phenotype.

Inducible activation of ERK5 MAP kinase enhances adult neurogenesis in the olfactory bulb and improves olfactory function.

Science.gov (United States)

Wang, Wenbin; Lu, Song; Li, Tan; Pan, Yung-Wei; Zou, Junhui; Abel, Glen M; Xu, Lihong; Storm, Daniel R; Xia, Zhengui

2015-05-20

Recent discoveries have suggested that adult neurogenesis in the subventricular zone (SVZ) and olfactory bulb (OB) may be required for at least some forms of olfactory behavior in mice. However, it is unclear whether conditional and selective enhancement of adult neurogenesis by genetic approaches is sufficient to improve olfactory function under physiological conditions or after injury. Furthermore, specific signaling mechanisms regulating adult neurogenesis in the SVZ/OB are not fully defined. We previously reported that ERK5, a MAP kinase selectively expressed in the neurogenic regions of the adult brain, plays a critical role in adult neurogenesis in the SVZ/OB. Using a site-specific knock-in mouse model, we report here that inducible and targeted activation of the endogenous ERK5 in adult neural stem/progenitor cells enhances adult neurogenesis in the OB by increasing cell survival and neuronal differentiation. This conditional ERK5 activation also improves short-term olfactory memory and odor-cued associative olfactory learning under normal physiological conditions. Furthermore, these mice show enhanced recovery of olfactory function and have more adult-born neurons after a zinc sulfate-induced lesion of the main olfactory epithelium. We conclude that ERK5 MAP kinase is an important endogenous signaling pathway regulating adult neurogenesis in the SVZ/OB, and that conditional activation of endogenous ERK5 is sufficient to enhance adult neurogenesis in the OB thereby improving olfactory function both under normal conditions and after injury. Copyright © 2015 the authors 0270-6474/15/357833-17$15.00/0.

A novel spleen tyrosine kinase inhibitor blocks c-Jun N-terminal kinase-mediated gene expression in synoviocytes

NARCIS (Netherlands)

Cha, Hoon-Suk; Boyle, David L.; Inoue, Tomoyuki; Schoot, Reineke; Tak, Paul P.; Pine, Polly; Firestein, Gary S.

2006-01-01

Spleen tyrosine kinase (Syk) is a key regulator of cell signaling induced by cytokines or Fc receptor engagement. However, the role of Syk in rheumatoid arthritis (RA) is not known yet. We investigated the pathways activated by Syk in tumor necrosis factor-alpha (TNFalpha)-stimulated fibroblast-like

Interleukins 2, 4, 7, and 15 stimulate tyrosine phosphorylation of insulin receptor substrates 1 and 2 in T cells. Potential role of JAK kinases.

Science.gov (United States)

Johnston, J A; Wang, L M; Hanson, E P; Sun, X J; White, M F; Oakes, S A; Pierce, J H; O'Shea, J J

1995-12-01

The signaling molecules insulin receptor substrate (IRS)-1 and the newly described IRS-2 (4PS) molecule are major insulin and interleukin 4 (IL-4)-dependent phosphoproteins. We report here that IL-2, IL-7, and IL-15, as well as IL-4, rapidly stimulate the tyrosine phosphorylation of IRS-1 and IRS-2 in human peripheral blood T cells, NK cells, and in lymphoid cell lines. In addition, we show that the Janus kinases, JAK1 and JAK3, associate with IRS-1 and IRS-2 in T cells. Coexpression studies demonstrate that these kinases can tyrosine-phosphorylate IRS-2, suggesting a possible mechanism by which cytokine receptors may induce the tyrosine phosphorylation of IRS-1 and IRS-2. We further demonstrate that the p85 subunit of phosphoinositol 3-kinase associates with IRS-1 in response to IL-2 and IL-4 in T cells. Therefore, these data indicate that IRS-1 and IRS-2 may have important roles in T lymphocyte activation not only in response to IL-4, but also in response to IL-2, IL-7, and IL-15.

Purification of reversibly oxidized proteins (PROP reveals a redox switch controlling p38 MAP kinase activity.

Directory of Open Access Journals (Sweden)

Dennis J Templeton

2010-11-01

Full Text Available Oxidation of cysteine residues of proteins is emerging as an important means of regulation of signal transduction, particularly of protein kinase function. Tools to detect and quantify cysteine oxidation of proteins have been a limiting factor in understanding the role of cysteine oxidation in signal transduction. As an example, the p38 MAP kinase is activated by several stress-related stimuli that are often accompanied by in vitro generation of hydrogen peroxide. We noted that hydrogen peroxide inhibited p38 activity despite paradoxically increasing the activating phosphorylation of p38. To address the possibility that cysteine oxidation may provide a negative regulatory effect on p38 activity, we developed a biochemical assay to detect reversible cysteine oxidation in intact cells. This procedure, PROP, demonstrated in vivo oxidation of p38 in response to hydrogen peroxide and also to the natural inflammatory lipid prostaglandin J2. Mutagenesis of the potential target cysteines showed that oxidation occurred preferentially on residues near the surface of the p38 molecule. Cysteine oxidation thus controls a functional redox switch regulating the intensity or duration of p38 activity that would not be revealed by immunodetection of phosphoprotein commonly interpreted as reflective of p38 activity.

Receptor density balances signal stimulation and attenuation in membrane-assembled complexes of bacterial chemotaxis signaling proteins

Science.gov (United States)

Besschetnova, Tatiana Y.; Montefusco, David J.; Asinas, Abdalin E.; Shrout, Anthony L.; Antommattei, Frances M.; Weis, Robert M.

2008-01-01

All cells possess transmembrane signaling systems that function in the environment of the lipid bilayer. In the Escherichia coli chemotaxis pathway, the binding of attractants to a two-dimensional array of receptors and signaling proteins simultaneously inhibits an associated kinase and stimulates receptor methylation—a slower process that restores kinase activity. These two opposing effects lead to robust adaptation toward stimuli through a physical mechanism that is not understood. Here, we provide evidence of a counterbalancing influence exerted by receptor density on kinase stimulation and receptor methylation. Receptor signaling complexes were reconstituted over a range of defined surface concentrations by using a template-directed assembly method, and the kinase and receptor methylation activities were measured. Kinase activity and methylation rates were both found to vary significantly with surface concentration—yet in opposite ways: samples prepared at high surface densities stimulated kinase activity more effectively than low-density samples, whereas lower surface densities produced greater methylation rates than higher densities. FRET experiments demonstrated that the cooperative change in kinase activity coincided with a change in the arrangement of the membrane-associated receptor domains. The counterbalancing influence of density on receptor methylation and kinase stimulation leads naturally to a model for signal regulation that is compatible with the known logic of the E. coli pathway. Density-dependent mechanisms are likely to be general and may operate when two or more membrane-related processes are influenced differently by the two-dimensional concentration of pathway elements. PMID:18711126

Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

International Nuclear Information System (INIS)

Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

1986-01-01

Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca 2+ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting 32 P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated 32 P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor

Induction of osteoblast differentiation by selective activation of kinase-mediated actions of the estrogen receptor.

Science.gov (United States)

Kousteni, Stavroula; Almeida, Maria; Han, Li; Bellido, Teresita; Jilka, Robert L; Manolagas, Stavros C

2007-02-01

Estrogens control gene transcription by cis or trans interactions of the estrogen receptor (ER) with target DNA or via the activation of cytoplasmic kinases. We report that selective activation of kinase-mediated actions of the ER with 4-estren-3alpha,17beta-diol (estren) or an estradiol-dendrimer conjugate, each a synthetic compound that stimulates kinase-mediated ER actions 1,000 to 10,000 times more potently than direct DNA interactions, induced osteoblastic differentiation in established cell lines of uncommitted osteoblast precursors and primary cultures of osteoblast progenitors by stimulating Wnt and BMP-2 signaling in a kinase-dependent manner. In sharp contrast, 17beta-estradiol (E(2)) suppressed BMP-2-induced osteoblast progenitor commitment and differentiation. Consistent with the in vitro findings, estren, but not E(2), stimulated Wnt/beta-catenin-mediated transcription in T-cell factor-lacZ transgenic mice. Moreover, E(2) stimulated BMP signaling in mice in which ERalpha lacks DNA binding activity and classical estrogen response element-mediated transcription (ERalpha(NERKI/-)) but not in wild-type controls. This evidence reveals for the first time the existence of a large signalosome in which inputs from the ER, kinases, bone morphogenetic proteins, and Wnt signaling converge to induce differentiation of osteoblast precursors. ER can either induce it or repress it, depending on whether the activating ligand (and presumably the resulting conformation of the receptor protein) precludes or accommodates ERE-mediated transcription.

Wortmannin inhibits both insulin- and contraction-stimulated glucose uptake and transport in rat skeletal muscle

DEFF Research Database (Denmark)

Wojtaszewski, Jørgen; Hansen, B F; Ursø, Birgitte

1996-01-01

The role of phosphatidylinositol (PI) 3-kinase for insulin- and contraction-stimulated muscle glucose transport was investigated in rat skeletal muscle perfused with a cell-free perfusate. The insulin receptor substrate-1-associated PI 3-kinase activity was increased sixfold upon insulin...... stimulation but was unaffected by contractions. In addition, the insulin-stimulated PI 3-kinase activity and muscle glucose uptake and transport in individual muscles were dose-dependently inhibited by wortmannin with one-half maximal inhibition values of approximately 10 nM and total inhibition at 1 micro......M. This concentration of wortmannin also decreased the contraction-stimulated glucose transport and uptake by approximately 30-70% without confounding effects on contractility or on muscle ATP and phosphocreatine concentrations. At higher concentrations (3 and 10 microM), wortmannin completely blocked the contraction...

Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth

Energy Technology Data Exchange (ETDEWEB)

Martin, Claire [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France); Lafosse, Jean-Michel [CHU Toulouse, Hopital Rangueil, Service d' orthopedie et Traumatologie, Toulouse F-31000 (France); Malavaud, Bernard [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France); CHU Toulouse, Hopital Rangueil, Service d' Urologie et de Transplantation Renale, Toulouse F-31000 (France); Cuvillier, Olivier, E-mail: olivier.cuvillier@ipbs.fr [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France)

2010-01-01

Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK.

Calcium/calmodulin-dependent kinase II and nitric oxide synthase 1-dependent modulation of ryanodine receptors during β-adrenergic stimulation is restricted to the dyadic cleft.

Science.gov (United States)

Dries, Eef; Santiago, Demetrio J; Johnson, Daniel M; Gilbert, Guillaume; Holemans, Patricia; Korte, Sanne M; Roderick, H Llewelyn; Sipido, Karin R

2016-10-15

The dyadic cleft, where coupled ryanodine receptors (RyRs) reside, is thought to serve as a microdomain for local signalling, as supported by distinct modulation of coupled RyRs dependent on Ca 2+ /calmodulin-dependent kinase II (CaMKII) activation during high-frequency stimulation. Sympathetic stimulation through β-adrenergic receptors activates an integrated signalling cascade, enhancing Ca 2+ cycling and is at least partially mediated through CaMKII. Here we report that CaMKII activation during β-adrenergic signalling is restricted to the dyadic cleft, where it enhances activity of coupled RyRs thereby contributing to the increase in diastolic events. Nitric oxide synthase 1 equally participates in the local modulation of coupled RyRs. In contrast, the increase in the Ca 2+ content of the sarcoplasmic reticulum and related increase in the amplitude of the Ca 2+ transient are primarily protein kinase A-dependent. The present data extend the concept of microdomain signalling in the dyadic cleft and give perspectives for selective modulation of RyR subpopulations and diastolic events. In cardiac myocytes, β-adrenergic stimulation enhances Ca 2+ cycling through an integrated signalling cascade modulating L-type Ca 2+ channels (LTCCs), phospholamban and ryanodine receptors (RyRs). Ca 2+ /calmodulin-dependent kinase II (CaMKII) and nitric oxide synthase 1 (NOS1) are proposed as prime mediators for increasing RyR open probability. We investigate whether this pathway is confined to the high Ca 2+ microdomain of the dyadic cleft and thus to coupled RyRs. Pig ventricular myocytes are studied under whole-cell voltage-clamp and confocal line-scan imaging with Fluo-4 as a [Ca 2+ ] i indicator. Following conditioning depolarizing pulses, spontaneous RyR activity is recorded as Ca 2+ sparks, which are assigned to coupled and non-coupled RyR clusters. Isoproterenol (ISO) (10 nm) increases Ca 2+ spark frequency in both populations of RyRs. However, CaMKII inhibition reduces

Mechanism of orientation of stimulating currents in magnetic brain stimulation (abstract)

Science.gov (United States)

Ueno, S.; Matsuda, T.

1991-04-01

We made a functional map of the human motor cortex related to the hand and foot areas by stimulating the human brain with a focused magnetic pulse. We observed that each functional area in the cortex has an optimum direction for which stimulating currents can produce neural excitation. The present report focuses on the mechanism which is responsible for producing this anisotropic response to brain stimulation. We first obtained a functional map of the brain related to the left ADM (abductor digiti minimi muscles). When the stimulating currents were aligned in the direction from the left to the right hemisphere, clear EMG (electromyographic) responses were obtained only from the left ADM to magnetic stimulation of both hemisphere. When the stimulating currents were aligned in the direction from the right to the left hemisphere, clear EMG signals were obtained only from the right ADM to magnetic stimulation of both hemisphere. The functional maps of the brain were sensitive to changes in the direction of the stimulating currents. To explain the phenomena obtained in the experiments, we developed a model of neural excitation elicited by magnetic stimulation. When eddy currents which are induced by pulsed magnetic fields flow in the direction from soma to the distal part of neural fiber, depolarized area in the distal part are excited, and the membrane excitation propagates along the nerve fiber. In contrast, when the induced currents flow in the direction from the distal part to soma, hyperpolarized parts block or inhibit neural excitation even if the depolarized parts near the soma can be excited. The model explains our observation that the orientation of the induced current vectors reflect both the functional and anatomical organization of the neural fibers in the brain.

Recognition of ERK MAP kinase by PEA-15 reveals a common docking site within the death domain and death effector domain

OpenAIRE

Hill, Justine M.; Vaidyanathan, Hema; Ramos, Joe W.; Ginsberg, Mark H.; Werner, Milton H.

2002-01-01

PEA-15 is a multifunctional protein that modulates signaling pathways which control cell proliferation and cell death. In particular, PEA-15 regulates the actions of the ERK MAP kinase cascade by binding to ERK and altering its subcellular localization. The three-dimensional structure of PEA-15 has been determined using NMR spectroscopy and its interaction with ERK defined by characterization of mutants that modulate ERK function. PEA-15 is composed of an N-terminal death effector domain (DED...

Skeletal Muscle-specific G Protein-coupled Receptor Kinase 2 Ablation Alters Isolated Skeletal Muscle Mechanics and Enhances Clenbuterol-stimulated Hypertrophy.

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Woodall, Benjamin P; Woodall, Meryl C; Luongo, Timothy S; Grisanti, Laurel A; Tilley, Douglas G; Elrod, John W; Koch, Walter J

2016-10-14

GRK2, a G protein-coupled receptor kinase, plays a critical role in cardiac physiology. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 after cardiac insult exacerbates injury and speeds progression to heart failure. Despite the importance of this kinase in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In this study we generated a novel skeletal muscle-specific GRK2 knock-out (KO) mouse (MLC-Cre:GRK2 fl/fl ) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2 fl/fl mice compared with wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, a β 2 -adrenergic receptor (β 2 AR) agonist, was significantly enhanced in MLC-Cre:GRK2 fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, our study provides the first insights into the role of GRK2 in skeletal muscle physiology and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as β 2 AR-induced hypertrophy. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Skeletal Muscle-specific G Protein-coupled Receptor Kinase 2 Ablation Alters Isolated Skeletal Muscle Mechanics and Enhances Clenbuterol-stimulated Hypertrophy*

Science.gov (United States)

Woodall, Benjamin P.; Woodall, Meryl C.; Luongo, Timothy S.; Grisanti, Laurel A.; Tilley, Douglas G.; Elrod, John W.; Koch, Walter J.

2016-01-01

GRK2, a G protein-coupled receptor kinase, plays a critical role in cardiac physiology. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 after cardiac insult exacerbates injury and speeds progression to heart failure. Despite the importance of this kinase in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In this study we generated a novel skeletal muscle-specific GRK2 knock-out (KO) mouse (MLC-Cre:GRK2fl/fl) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2fl/fl mice compared with wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, a β2-adrenergic receptor (β2AR) agonist, was significantly enhanced in MLC-Cre:GRK2fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, our study provides the first insights into the role of GRK2 in skeletal muscle physiology and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as β2AR-induced hypertrophy. PMID:27566547

Polarization of migrating monocytic cells is independent of PI 3-kinase activity.

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Silvia Volpe

Full Text Available BACKGROUND: Migration of mammalian cells is a complex cell type and environment specific process. Migrating hematopoietic cells assume a rapid amoeboid like movement when exposed to gradients of chemoattractants. The underlying signaling mechanisms remain controversial with respect to localization and distribution of chemotactic receptors within the plasma membrane and the role of PI 3-kinase activity in cell polarization. METHODOLOGY/PRINCIPAL FINDINGS: We present a novel model for the investigation of human leukocyte migration. Monocytic THP-1 cells transfected with the alpha(2A-adrenoceptor (alpha(2AAR display comparable signal transduction responses, such as calcium mobilization, MAP-kinase activation and chemotaxis, to the noradrenaline homologue UK 14'304 as when stimulated with CCL2, which binds to the endogenous chemokine receptor CCR2. Time-lapse video microscopy reveals that chemotactic receptors remain evenly distributed over the plasma membrane and that their internalization is not required for migration. Measurements of intramolecular fluorescence resonance energy transfer (FRET of alpha(2AAR-YFP/CFP suggest a uniform activation of the receptors over the entire plasma membrane. Nevertheless, PI 3-kinase activation is confined to the leading edge. When reverting the gradient of chemoattractant by moving the dispensing micropipette, polarized monocytes--in contrast to neutrophils--rapidly flip their polarization axis by developing a new leading edge at the previous posterior side. Flipping of the polarization axis is accompanied by re-localization of PI-3-kinase activity to the new leading edge. However, reversal of the polarization axis occurs in the absence of PI 3-kinase activation. CONCLUSIONS/SIGNIFICANCE: Accumulation and internalization of chemotactic receptors at the leading edge is dispensable for cell migration. Furthermore, uniformly distributed receptors allow the cells to rapidly reorient and adapt to changes in the

A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

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Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

1996-01-01

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity

DEFF Research Database (Denmark)

Köpper, Frederik; Bierwirth, Cathrin; Schön, Margarete

2013-01-01

knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation...

Tamm-Horsfall Glycoprotein Enhances PMN Phagocytosis by Binding to Cell Surface-Expressed Lactoferrin and Cathepsin G That Activates MAP Kinase Pathway

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Chia-Li Yu

2011-03-01

Full Text Available The molecular basis of polymorphonuclear neutrophil (PMN phagocytosis-enhancing activity (PEA by human purified urinary Tamm-Horsfall glyco- protein (THP has not been elucidated. In this study, we found human THP bound to lactoferrin (LF and cathepsin G (CG expressed on the surface of PMN, identified by a proteomic study with MALDI-TOF- LC/LC/mass spectrometric analysis. Pre-incubation of 10% SDS-PAGE electrophoresed PMN lysates with monoclonal anti-LF or anti-CG antibody reduced the binding with THP. To elucidate the signaling pathway of THP on PMN activation, we found THP enhanced ERK1/2 phosphorylation, reduced p38 MAP kinase phosphorylation, but had no effect on DNA binding of the five NF-kB family members in PMN. To further clarify whether the carbohydrate-side chains or protein-core structure in THP molecule is responsible for THP-PEA, THP was cleaved by different degrading enzymes with carbohydrate specificity (neuraminidase and β-galactosidase, protein specificity (V8 protease and proteinase K or glycoconjugate specificity (carboxylpeptidase Y and O-sialoglycoprotein endopeptidase. We clearly demonstrated that the intact protein-core structure in THP molecule was more important for THP-PEA than carbohydrate-side chains. Putting these results together, we conclude that THP adheres to surface-expressed LF and CG on PMN and transduces signaling via the MAP kinase pathway to enhance PMN phagocytosis.

Inhibition of Src kinase activity attenuates amyloid associated microgliosis in a murine model of Alzheimer’s disease

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Dhawan Gunjan

2012-07-01

Full Text Available Abstract Background Microglial activation is an important histologic characteristic of the pathology of Alzheimer’s disease (AD. One hypothesis is that amyloid beta (Aβ peptide serves as a specific stimulus for tyrosine kinase-based microglial activation leading to pro-inflammatory changes that contribute to disease. Therefore, inhibiting Aβ stimulation of microglia may prove to be an important therapeutic strategy for AD. Methods Primary murine microglia cultures and the murine microglia cell line, BV2, were used for stimulation with fibrillar Aβ1-42. The non-receptor tyrosine kinase inhibitor, dasatinib, was used to treat the cells to determine whether Src family kinase activity was required for the Aβ stimulated signaling response and subsequent increase in TNFα secretion using Western blot analysis and enzyme-linked immunosorbent assay (ELISA, respectively. A histologic longitudinal analysis was performed using an AD transgenic mouse model, APP/PS1, to determine an age at which microglial protein tyrosine kinase levels increased in order to administer dasatinib via mini osmotic pump diffusion. Effects of dasatinib administration on microglial and astroglial activation, protein phosphotyrosine levels, active Src kinase levels, Aβ plaque deposition, and spatial working memory were assessed via immunohistochemistry, Western blot, and T maze analysis. Results Aβ fibrils stimulated primary murine microglia via a tyrosine kinase pathway involving Src kinase that was attenuated by dasatinib. Dasatinib administration to APP/PS1 mice decreased protein phosphotyrosine, active Src, reactive microglia, and TNFα levels in the hippocampus and temporal cortex. The drug had no effect on GFAP levels, Aβ plaque load, or the related tyrosine kinase, Lyn. These anti-inflammatory changes correlated with improved performance on the T maze test in dasatinib infused animals compared to control animals. Conclusions These data suggest that amyloid

Stimulating Graphical Summarization in Late Elementary Education: The Relationship between Two Instructional Mind-Map Approaches and Student Characteristics

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Merchie, Emmelien; Van Keer, Hilde

2016-01-01

This study examined the effectiveness of two instructional mind-mapping approaches to stimulate fifth and sixth graders' graphical summarization skills. Thirty-five fifth- and sixth-grade teachers and 644 students from 17 different elementary schools participated. A randomized quasi-experimental repeated-measures design was set up with two…

The impact of preoperative language mapping by repetitive navigated transcranial magnetic stimulation on the clinical course of brain tumor patients.

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Sollmann, Nico; Ille, Sebastian; Hauck, Theresa; Maurer, Stefanie; Negwer, Chiara; Zimmer, Claus; Ringel, Florian; Meyer, Bernhard; Krieg, Sandro M

2015-04-11

Language mapping by repetitive navigated transcranial magnetic stimulation (rTMS) is used for resection planning in patients suffering from brain lesions within regions known to be involved in language function. Yet we also need data that show whether patients benefit clinically from preoperative rTMS for language mapping. We enrolled 25 patients with language eloquently located brain lesions undergoing preoperative rTMS language mapping (GROUP 1, 2011-2013), with the mapping results not being available for the surgeon, and we matched these patients with 25 subjects who also underwent preoperative rTMS (GROUP 2, 2013-2014), but the mapping results were taken into account during tumor resection. Additionally, cortical language maps were generated by analyzing preoperative rTMS and intraoperative direct cortical stimulation (DCS) data. Mean anterior-posterior (ap) craniotomy extents and overall craniotomy sizes were significantly smaller for the patients in GROUP 2 (Ap: p = 0.0117; overall size: p = 0.0373), and postoperative language deficits were found significantly more frequently for the patients in GROUP 1 (p = 0.0153), although the preoperative language status did not differ between groups (p = 0.7576). Additionally, there was a trend towards fewer unexpected tumor residuals, shorter surgery duration, less peri- or postoperative complications, shorter inpatient stay, and higher postoperative Karnofsky performance status scale (KPS) for the patients in GROUP 2. The present study provides a first hint that the clinical course of patients suffering from brain tumors might be improved by preoperative rTMS language mapping. However, a significant difference between both groups was only found for craniotomy extents and postoperative deficits, but not for other clinical parameters, which only showed a trend toward better results in GROUP 2. Therefore, multicenter trials with higher sample sizes are needed to further investigate the distinct impact of r

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

International Nuclear Information System (INIS)

Arai, Roberto J.; Ogata, Fernando T.; Batista, Wagner L.; Masutani, Hiroshi; Yodoi, Junji; Debbas, Victor; Augusto, Ohara; Stern, Arnold; Monteiro, Hugo P.

2008-01-01

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 of 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

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

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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.

Structural Requirements for Yersinia YopJ Inhibition of MAP Kinase Pathways

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Burdette, Dara; Mukherjee, Sohini; Keitany, Gladys; Goldsmith, Elizabeth; Orth, Kim

2008-01-01

MAPK signaling cascades are evolutionally conserved. The bacterial effector, YopJ, uses the unique activity of Ser/Thr acetylation to inhibit the activation of the MAPK kinase (MKK) and prevent activation by phosphorylation. YopJ is also able to block yeast MAPK signaling pathways using this mechanism. Based on these observations, we performed a genetic screen to isolate mutants in the yeast MKK, Pbs2, that suppress YopJ inhibition. One suppressor contains a mutation in a conserved tyrosine residue and bypasses YopJ inhibition by increasing the basal activity of Pbs2. Mutations on the hydrophobic face of the conserved G α-helix in the kinase domain prevent both binding and acetylation by YopJ. Corresponding mutants in human MKKs showed that they are conserved not only structurally, but also functionally. These studies reveal a conserved binding site found on the superfamily of MAPK kinases while providing insight into the molecular interactions required for YopJ inhibition. PMID:18167536

Structural requirements for Yersinia YopJ inhibition of MAP kinase pathways.

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Yi-Heng Hao

2008-01-01

Full Text Available MAPK signaling cascades are evolutionally conserved. The bacterial effector, YopJ, uses the unique activity of Ser/Thr acetylation to inhibit the activation of the MAPK kinase (MKK and prevent activation by phosphorylation. YopJ is also able to block yeast MAPK signaling pathways using this mechanism. Based on these observations, we performed a genetic screen to isolate mutants in the yeast MKK, Pbs2, that suppress YopJ inhibition. One suppressor contains a mutation in a conserved tyrosine residue and bypasses YopJ inhibition by increasing the basal activity of Pbs2. Mutations on the hydrophobic face of the conserved G alpha-helix in the kinase domain prevent both binding and acetylation by YopJ. Corresponding mutants in human MKKs showed that they are conserved not only structurally, but also functionally. These studies reveal a conserved binding site found on the superfamily of MAPK kinases while providing insight into the molecular interactions required for YopJ inhibition.

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.

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Sabine Gruber

Full Text Available 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.

Ribosomal Protein S6 Kinase (RSK-2 as a central effector molecule in RON receptor tyrosine kinase mediated epithelial to mesenchymal transition induced by macrophage-stimulating protein

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Zhang Rui-Wen

2011-05-01

Full Text Available Abstract Background Epithelial to mesenchymal transition (EMT occurs during cancer cell invasion and malignant metastasis. Features of EMT include spindle-like cell morphology, loss of epithelial cellular markers and gain of mesenchymal phenotype. Activation of the RON receptor tyrosine kinase by macrophage-stimulating protein (MSP has been implicated in cellular EMT program; however, the major signaling determinant(s responsible for MSP-induced EMT is unknown. Results The study presented here demonstrates that RSK2, a downstream signaling protein of the Ras-Erk1/2 pathway, is the principal molecule that links MSP-activated RON signaling to complete EMT. Using MDCK cells expressing RON as a model, a spindle-shape based screen was conducted, which identifies RSK2 among various intracellular proteins as a potential signaling molecule responsible for MSP-induced EMT. MSP stimulation dissociated RSK2 with Erk1/2 and promoted RSK2 nuclear translocation. MSP strongly induced RSK2 phosphorylation in a dose-dependent manner. These effects relied on RON and Erk1/2 phosphorylation, which is significantly potentiated by transforming growth factor (TGF-β1, an EMT-inducing cytokine. Specific RSK inhibitor SL0101 completely prevented MSP-induced RSK phosphorylation, which results in inhibition of MSP-induced spindle-like morphology and suppression of cell migration associated with EMT. In HT-29 cancer cells that barely express RSK2, forced RSK2 expression results in EMT-like phenotype upon MSP stimulation. Moreover, specific siRNA-mediated silencing of RSK2 but not RSK1 in L3.6pl pancreatic cancer cells significantly inhibited MSP-induced EMT-like phenotype and cell migration. Conclusions MSP-induced RSK2 activation is a critical determinant linking RON signaling to cellular EMT program. Inhibition of RSK2 activity may provide a therapeutic opportunity for blocking RON-mediated cancer cell migration and subsequent invasion.

Mapping of arithmetic processing by navigated repetitive transcranial magnetic stimulation in patients with parietal brain tumors and correlation with postoperative outcome.

Science.gov (United States)

Ille, Sebastian; Drummer, Katharina; Giglhuber, Katrin; Conway, Neal; Maurer, Stefanie; Meyer, Bernhard; Krieg, Sandro M

2018-03-26

Preserving functionality is of significant importance during neurosurgical resection of brain tumors. Specialized centers also map further brain functions apart from motor and language functions, such as arithmetic processing (AP). The mapping of AP by navigated repetitive transcranial magnetic stimulation (nrTMS) in healthy volunteers has been demonstrated. The present study aimed to correlate the results of mapping AP with functional patient outcomes. We included 26 patients with parietal brain tumors. Due to preoperative impairment of AP, mapping was not possible in 8 patients (31%). We stimulated 52 cortical sites by nrTMS while patients performed a calculation task. Pre- and postoperatively, patients underwent a standardized number-processing and calculation test (NPCT). Tumor resection was blinded to nrTMS results, and the change in NPCT performance was correlated to resected AP-positive spots as identified by nrTMS. The resection of AP-positive sites correlated with a worsening of the postoperative NPCT result in 12 cases. In 3 cases, no AP-positive sites were resected and the postoperative NPCT result was similar to or better than preoperatively. Also, in 3 cases, the postoperative NPCT result was better than preoperatively, although AP-positive sites were resected. Despite only presenting a low number of cases, nrTMS might be a useful tool for preoperative mapping of AP. However, the reliability of the present results has to be evaluated in a larger series and by intraoperative mapping data. Copyright © 2018 Elsevier Inc. All rights reserved.

Digital field mapping for stimulating Secondary School students in the recognition of geological features and landforms

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Giardino, Marco; Magagna, Alessandra; Ferrero, Elena; Perrone, Gianluigi

2015-04-01

Digital field mapping has certainly provided geoscientists with the opportunity to map and gather data in the field directly using digital tools and software rather than using paper maps, notebooks and analogue devices and then subsequently transferring the data to a digital format for subsequent analysis. But, the same opportunity has to be recognized for Geoscience education, as well as for stimulating and helping students in the recognition of landforms and interpretation of the geological and geomorphological components of a landscape. More, an early exposure to mapping during school and prior to university can optimise the ability to "read" and identify uncertainty in 3d models. During 2014, about 200 Secondary School students (aged 12-15) of the Piedmont region (NW Italy) participated in a research program involving the use of mobile devices (smartphone and tablet) in the field. Students, divided in groups, used the application Trimble Outdoors Navigators for tracking a geological trail in the Sangone Valley and for taking georeferenced pictures and notes. Back to school, students downloaded the digital data in a .kml file for the visualization on Google Earth. This allowed them: to compare the hand tracked trail on a paper map with the digital trail, and to discuss about the functioning and the precision of the tools; to overlap a digital/semitransparent version of the 2D paper map (a Regional Technical Map) used during the field trip on the 2.5D landscape of Google Earth, as to help them in the interpretation of conventional symbols such as contour lines; to perceive the landforms seen during the field trip as a part of a more complex Pleistocene glacial landscape; to understand the classical and innovative contributions from different geoscientific disciplines to the generation of a 3D structural geological model of the Rivoli-Avigliana Morainic Amphitheatre. In 2013 and 2014, some other pilot projects have been carried out in different areas of the

Insulin stimulates phospholipase D-dependent phosphatidylcholine hydrolysis, Rho translocation, de novo phospholipid synthesis, and diacylglycerol/protein kinase C signaling in L6 myotubes.

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Standaert, M L; Bandyopadhyay, G; Zhou, X; Galloway, L; Farese, R V

1996-07-01

Previous studies have provided conflicting findings on whether insulin activates certain, potentially important, phospholipid signaling systems in skeletal muscle preparations. In particular, insulin effects on the hydrolysis of phosphatidylcholine (PC) and subsequent activation of protein kinase C (PKC) have not been apparent in some studies. Presently, we examined insulin effects on phospholipid signaling systems, diacylglycerol (DAG) production, and PKC translocation/activation in L6 myotubes. We found that insulin provoked rapid increases in phospholipase D (PLD)-dependent hydrolysis of PC, as evidenced by increases in choline release and phosphatidylethanol production in cells incubated in the presence of ethanol. In association with PC-PLD activation, Rho, a small G protein that is known to activate PC-PLD activation, translocated from the cytosol to the membrane fraction in response to insulin treatment. PC-PLD activation was also accompanied by increases in total DAG production and increases in the translocation of both PKC enzyme activity and DAG-sensitive PKC-alpha, -beta, -delta, and -epsilon from the cytosol to the membrane fraction. A potential role for PKC or a related protein kinase in insulin action was suggested by the finding that RO 31-8220 inhibited both PKC enzyme activity and insulin-stimulated [3H]2-deoxyglucose uptake. Our findings provide the first evidence that insulin stimulates Rho translocation and activates PC-PLD in L6 skeletal muscle cells. Moreover, this signaling system appears to lead to increases in DAG/PKC signaling, which, along with other related signaling factors, may regulate certain metabolic processes, such as glucose transport, in these cells.

{delta}-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

Energy Technology Data Exchange (ETDEWEB)

Heiss, Anika; Ammer, Hermann [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany); Eisinger, Daniela A., E-mail: eisinger@pharmtox.vetmed.uni-muenchen.de [Institute of Pharmacology, Toxicology and Pharmacy Ludwig-Maximilians-University of Munich Koeniginstrasse 16 80539 Muenchen Federal Republic of Germany (Germany)

2009-07-15

{delta}-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen{sup 2,5}]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G{sub i/o} proteins, because pre-treatment with pertussis toxin, but not over-expression of the G{sub q/11} scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the G{beta}{gamma} scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

δ-Opioid receptor-stimulated Akt signaling in neuroblastoma x glioma (NG108-15) hybrid cells involves receptor tyrosine kinase-mediated PI3K activation

International Nuclear Information System (INIS)

Heiss, Anika; Ammer, Hermann; Eisinger, Daniela A.

2009-01-01

δ-Opioid receptor (DOR) agonists possess cytoprotective properties, an effect associated with activation of the 'pro-survival' kinase Akt. Here we delineate the signal transduction pathway by which opioids induce Akt activation in neuroblastoma x glioma (NG108-15) hybrid cells. Exposure of the cells to both [D-Pen 2,5 ]enkephalin and etorphine resulted in a time- and dose-dependent increase in Akt activity, as measured by means of an activation-specific antibody recognizing phosphoserine-473. DOR-mediated Akt signaling is blocked by the opioid antagonist naloxone and involves inhibitory G i/o proteins, because pre-treatment with pertussis toxin, but not over-expression of the G q/11 scavengers EBP50 and GRK2-K220R, prevented this effect. Further studies with Wortmannin and LY294002 revealed that phophoinositol-3-kinase (PI3K) plays a central role in opioid-induced Akt activation. Opioids stimulate Akt activity through transactivation of receptor tyrosine kinases (RTK), because pre-treatment of the cells with inhibitors for neurotrophin receptor tyrosine kinases (AG879) and the insulin-like growth factor receptor IGF-1 (AG1024), but not over-expression of the Gβγ scavenger phosducin, abolished this effect. Activated Akt translocates to the nuclear membrane, where it promotes GSK3 phosphorylation and prevents caspase-3 cleavage, two key events mediating inhibition of cell apoptosis and enhancement of cell survival. Taken together, these results demonstrate that in NG108-15 hybrid cells DOR agonists possess cytoprotective properties mediated by activation of the RTK/PI3K/Akt signaling pathway.

Pressure overload stimulated cardiac hypertrophy leads to a rapid decrease in the mRNA for creatine kinase

International Nuclear Information System (INIS)

Boheler, K.; Popovich, B.; Dillmann, W.H.

1987-01-01

Cardiac hypertrophy (CH) leads to a decrease in creatine kinase (CK) enzymatic activity. To determine if the mRNA for CK also decreases with CH, they performed the following studies. Cardiac RNA was isolated from rats subjected to either abdominal aortic stenosis (AS) or sham surgery. Through Northern blot analysis, total cardiac RNA was quantitated with a CK specific 32 P-labelled cDNA clone. At 3 and 8 days post-constriction, the mRNA for CK decreases by 54.6 +/- 7% and 65.3 +/- 18% respectively, whereas the heart weight increases by 19% and 37% relative to controls. Further studies indicate that CK mRNA also decreases by 41.8% in hypothyroid rats (Tx) but decreases by a total of 68.1% in Tx rats subjected to 8 days of AS. Pressure overload stimulated CH leads to a rapid decrease in CK mRNA in normal and Tx rats. This CK mRNA decrease may account for the decreased efficiency of contraction seen in CH

Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK and Mitogen-Activated Protein Kinases (MAP Kinases Signaling Pathway in Keratinocytes

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Yun-Hee Choi

2015-11-01

Full Text Available Mycosporine-like amino acids (MAAs are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS. In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH, Mycosporine-glycine (M-Gly, and Porphyra (P334 were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK, extracellular signal-regulated kinases (ERK, and c-Jun N-terminal kinases (JNK. These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies.

Mycosporine-Like Amino Acids Promote Wound Healing through Focal Adhesion Kinase (FAK) and Mitogen-Activated Protein Kinases (MAP Kinases) Signaling Pathway in Keratinocytes

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Choi, Yun-Hee; Yang, Dong Joo; Kulkarni, Atul; Moh, Sang Hyun; Kim, Ki Woo

2015-01-01

Mycosporine-like amino acids (MAAs) are secondary metabolites found in diverse marine, freshwater, and terrestrial organisms. Evidence suggests that MAAs have several beneficial effects on skin homeostasis such as protection against UV radiation and reactive oxygen species (ROS). In addition, MAAs are also involved in the modulation of skin fibroblasts proliferation. However, the regulatory function of MAAs on wound repair in human skin is not yet clearly elucidated. To investigate the roles of MAAs on the wound healing process in human keratinocytes, three MAAs, Shinorine (SH), Mycosporine-glycine (M-Gly), and Porphyra (P334) were purified from Chlamydomonas hedlyei and Porphyra yezoensis. We found that SH, M-Gly, and P334 have significant effects on the wound healing process in human keratinocytes and these effects were mediated by activation of focal adhesion kinases (FAK), extracellular signal-regulated kinases (ERK), and c-Jun N-terminal kinases (JNK). These results suggest that MAAs accelerate wound repair by activating the FAK-MAPK signaling pathways. This study also indicates that MAAs can act as a new wound healing agent and further suggests that MAAs might be a novel biomaterial for wound healing therapies. PMID:26703626

Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility.

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Fang, Jung-Da; Lee, Sheau-Ling

2014-07-01

Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation. Copyright © 2014. Published by Elsevier B.V.

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

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Anne N Shemon

2009-06-01

Full Text Available 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.

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

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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.

Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity.

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Takayama, S; White, M F; Kahn, C R

1988-03-05

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function

Protein kinase Cepsilon is important for migration of neuroblastoma cells

International Nuclear Information System (INIS)

Stensman, Helena; Larsson, Christer

2008-01-01

Migration is important for the metastatic capacity and thus for the malignancy of cancer cells. There is limited knowledge on regulatory factors that promote the migration of neuroblastoma cells. This study investigates the hypothesis that protein kinase C (PKC) isoforms regulate neuroblastoma cell motility. PKC isoforms were downregulated with siRNA or modulated with activators and inhibitors. Migration was analyzed with scratch and transwell assays. Protein phosphorylation and expression levels were measured with Western blot. Stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced migration of SK-N-BE(2)C neuroblastoma cells. Treatment with the general protein kinase C (PKC) inhibitor GF109203X and the inhibitor of classical isoforms Gö6976 inhibited migration while an inhibitor of PKCβ isoforms did not have an effect. Downregulation of PKCε, but not of PKCα or PKCδ, with siRNA led to a suppression of both basal and TPA-stimulated migration. Experiments using PD98059 and LY294002, inhibitors of the Erk and phosphatidylinositol 3-kinase (PI3K) pathways, respectively, showed that PI3K is not necessary for TPA-induced migration. The Erk pathway might be involved in TPA-induced migration but not in migration driven by PKCε. TPA induced phosphorylation of the PKC substrate myristoylated alanine-rich C kinase substrate (MARCKS) which was suppressed by the PKC inhibitors. Treatment with siRNA oligonucleotides against different PKC isoforms before stimulation with TPA did not influence the phosphorylation of MARCKS. PKCε is important for migration of SK-N-BE(2)C neuroblastoma cells. Neither the Erk pathway nor MARCKS are critical downstream targets of PKCε but they may be involved in TPA-mediated migration

Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei

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Li, H.; Roux, S. J.

1992-01-01

A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.

Mapping effective connectivity in the human brain with concurrent intracranial electrical stimulation and BOLD-fMRI.

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Oya, Hiroyuki; Howard, Matthew A; Magnotta, Vincent A; Kruger, Anton; Griffiths, Timothy D; Lemieux, Louis; Carmichael, David W; Petkov, Christopher I; Kawasaki, Hiroto; Kovach, Christopher K; Sutterer, Matthew J; Adolphs, Ralph

2017-02-01

Understanding brain function requires knowledge of how one brain region causally influences another. This information is difficult to obtain directly in the human brain, and is instead typically inferred from resting-state fMRI. Here, we demonstrate the safety and scientific promise of a novel and complementary approach: concurrent electrical stimulation and fMRI (es-fMRI) at 3T in awake neurosurgical patients with implanted depth electrodes. We document the results of safety testing, actual experimental setup, and stimulation parameters, that safely and reliably evoke activation in distal structures through stimulation of amygdala, cingulate, or prefrontal cortex. We compare connectivity inferred from the evoked patterns of activation with that estimated from standard resting-state fMRI in the same patients: while connectivity patterns obtained with each approach are correlated, each method produces unique results. Response patterns were stable over the course of 11min of es-fMRI runs. COMPARISON WITH EXISTING METHOD: es-fMRI in awake humans yields unique information about effective connectivity, complementing resting-state fMRI. Although our stimulations were below the level of inducing any apparent behavioral or perceptual effects, a next step would be to use es-fMRI to modulate task performances. This would reveal the acute network-level changes induced by the stimulation that mediate the behavioral and cognitive effects seen with brain stimulation. es-fMRI provides a novel and safe approach for mapping effective connectivity in the human brain in a clinical setting, and will inform treatments for psychiatric and neurodegenerative disorders that use deep brain stimulation. Copyright © 2016 Elsevier B.V. All rights reserved.

Distinct Molecular Effects of Angiotensin II and Angiotensin III in Rat Astrocytes

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Michelle A. Clark

2013-01-01

Full Text Available It is postulated that central effects of angiotensin (Ang II may be indirect due to rapid conversion to Ang III by aminopeptidase A (APA. Previously, we showed that Ang II and Ang III induced mitogen-activated protein (MAP kinases ERK1/2 and stress-activated protein kinase/Jun-terminal kinases (SAPK/JNK phosphorylation in cultured rat astrocytes. Most importantly, both peptides were equipotent in causing phosphorylation of these MAP kinases. In these studies, we used brainstem and cerebellum astrocytes to determine whether Ang II’s phosphorylation of these MAP kinases is due to the conversion of the peptide to Ang III. We pretreated astrocytes with 10 μM amastatin A or 100 μM glutamate phosphonate, selective APA inhibitors, prior to stimulating with either Ang II or Ang III. Both peptides were equipotent in stimulating ERK1/2 and SAPK/JNK phosphorylation. The APA inhibitors failed to prevent Ang II- and Ang III-mediated phosphorylation of the MAP kinases. Further, pretreatment of astrocytes with the APA inhibitors did not affect Ang II- or Ang III-induced astrocyte growth. These findings suggest that both peptides directly induce phosphorylation of these MAP kinases as well as induce astrocyte growth. These studies establish both peptides as biologically active with similar intracellular and physiological effects.

The molecular architecture of human N-acetylgalactosamine kinase.

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Thoden, James B; Holden, Hazel M

2005-09-23

Galactokinase plays a key role in normal galactose metabolism by catalyzing the conversion of alpha-d-galactose to galactose 1-phosphate. Within recent years, the three-dimensional structures of human galactokinase and two bacterial forms of the enzyme have been determined. Originally, the gene encoding galactokinase in humans was mapped to chromosome 17. An additional gene, encoding a protein with sequence similarity to galactokinase, was subsequently mapped to chromosome 15. Recent reports have shown that this second gene (GALK2) encodes an enzyme with greater activity against GalNAc than galactose. This enzyme, GalNAc kinase, has been implicated in a salvage pathway for the reutilization of free GalNAc derived from the degradation of complex carbohydrates. Here we report the first structural analysis of a GalNAc kinase. The structure of the human enzyme was solved in the presence of MnAMPPNP and GalNAc or MgATP and GalNAc (which resulted in bound products in the active site). The enzyme displays a distinctly bilobal appearance with its active site wedged between the two domains. The N-terminal region is dominated by a seven-stranded mixed beta-sheet, whereas the C-terminal motif contains two layers of anti-parallel beta-sheet. The overall topology displayed by GalNAc kinase places it into the GHMP superfamily of enzymes, which generally function as small molecule kinases. From this investigation, the geometry of the GalNAc kinase active site before and after catalysis has been revealed, and the determinants of substrate specificity have been defined on a molecular level.

Protein kinase Cα phosphorylates a novel argininosuccinate synthase site at serine 328 during calcium-dependent stimulation of endothelial nitric-oxide synthase in vascular endothelial cells.

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Haines, Ricci J; Corbin, Karen D; Pendleton, Laura C; Eichler, Duane C

2012-07-27

Endothelial nitric-oxide synthase (eNOS) utilizes l-arginine as its principal substrate, converting it to l-citrulline and nitric oxide (NO). l-Citrulline is recycled to l-arginine by two enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase, providing the substrate arginine for eNOS and NO production in endothelial cells. Together, these three enzymes, eNOS, AS, and argininosuccinate lyase, make up the citrulline-NO cycle. Although AS catalyzes the rate-limiting step in NO production, little is known about the regulation of AS in endothelial cells beyond the level of transcription. In this study, we showed that AS Ser-328 phosphorylation was coordinately regulated with eNOS Ser-1179 phosphorylation when bovine aortic endothelial cells were stimulated by either a calcium ionophore or thapsigargin to produce NO. Furthermore, using in vitro kinase assay, kinase inhibition studies, as well as protein kinase Cα (PKCα) knockdown experiments, we demonstrate that the calcium-dependent phosphorylation of AS Ser-328 is mediated by PKCα. Collectively, these findings suggest that phosphorylation of AS at Ser-328 is regulated in accordance with the calcium-dependent regulation of eNOS under conditions that promote NO production and are in keeping with the rate-limiting role of AS in the citrulline-NO cycle of vascular endothelial cells.

Downstream of tyrosine kinase/docking protein 6, as a novel substrate of tropomyosin-related kinase C receptor, is involved in neurotrophin 3-mediated neurite outgrowth in mouse cortex neurons

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Yuan Jian

2010-06-01

Full Text Available Abstract Background The downstream of tyrosine kinase/docking protein (Dok adaptor protein family has seven members, Dok1 to Dok7, that act as substrates of multiple receptor tyrosine kinase and non-receptor tyrosine kinase. The tropomyosin-related kinase (Trk receptor family, which has three members (TrkA, TrkB and TrkC, are receptor tyrosine kinases that play pivotal roles in many stages of nervous system development, such as differentiation, migration, axon and dendrite projection and neuron patterning. Upon related neurotrophin growth factor stimulation, dimerisation and autophosphorylation of Trk receptors can occur, recruiting adaptor proteins to mediate signal transduction. Results In this report, by using yeast two-hybrid assays, glutathione S-transferase (GST precipitation assays and coimmunoprecipitation (Co-IP experiments, we demonstrate that Dok6 selectively binds to the NPQY motif of TrkC through its phosphotyrosine-binding (PTB domain in a kinase activity-dependent manner. We further confirmed their interaction by coimmunoprecipitation and colocalisation in E18.5 mouse cortex neurons, which provided more in vivo evidence. Next, we demonstrated that Dok6 is involved in neurite outgrowth in mouse cortex neurons via the RNAi method. Knockdown of Dok6 decreased neurite outgrowth in cortical neurons upon neurotrophin 3 (NT-3 stimulation. Conclusions We conclude that Dok6 interacts with the NPQY motif of the TrkC receptor through its PTB domain in a kinase activity-dependent manner, and works as a novel substrate of the TrkC receptor involved in NT-3-mediated neurite outgrowth in mouse cortex neurons.

Distinct and Overlapping Functions of TEC Kinase and BTK in B Cell Receptor Signaling.

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de Bruijn, Marjolein J W; Rip, Jasper; van der Ploeg, Esmee K; van Greuningen, Lars W; Ta, Van T B; Kil, Laurens P; Langerak, Anton W; Rimmelzwaan, Guus F; Ellmeier, Wilfried; Hendriks, Rudi W; Corneth, Odilia B J

2017-04-15

The Tec tyrosine kinase is expressed in many cell types, including hematopoietic cells, and is a member of the Tec kinase family that also includes Btk. Although the role of Btk in B cells has been extensively studied, the role of Tec kinase in B cells remains largely unclear. It was previously shown that Tec kinase has the ability to partly compensate for loss of Btk activity in B cell differentiation, although the underlying mechanism is unknown. In this study, we confirm that Tec kinase is not essential for normal B cell development when Btk is present, but we also found that Tec-deficient mature B cells showed increased activation, proliferation, and survival upon BCR stimulation, even in the presence of Btk. Whereas Tec deficiency did not affect phosphorylation of phospholipase Cγ or Ca 2+ influx, it was associated with significantly increased activation of the intracellular Akt/S6 kinase signaling pathway upon BCR and CD40 stimulation. The increased S6 kinase phosphorylation in Tec-deficient B cells was dependent on Btk kinase activity, as ibrutinib treatment restored pS6 to wild-type levels, although Btk protein and phosphorylation levels were comparable to controls. In Tec-deficient mice in vivo, B cell responses to model Ags and humoral immunity upon influenza infection were enhanced. Moreover, aged mice lacking Tec kinase developed a mild autoimmune phenotype. Taken together, these data indicate that in mature B cells, Tec and Btk may compete for activation of the Akt signaling pathway, whereby the activating capacity of Btk is limited by the presence of Tec kinase. Copyright © 2017 by The American Association of Immunologists, Inc.

Phosphoinositide 3–kinase γ participates in T cell receptor–induced T cell activation

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Alcázar, Isabela; Marqués, Miriam; Kumar, Amit; Hirsch, Emilio; Wymann, Matthias; Carrera, Ana C.; Barber, Domingo F.

2007-01-01

Class I phosphoinositide 3–kinases (PI3Ks) constitute a family of enzymes that generates 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (Tyr) kinase–associated receptors or G protein–coupled receptors (GPCRs). The class I PI3Ks are divided into two types: class IA p85/p110 heterodimers, which are activated by Tyr kinases, and the class IB p110γ isoform, which is activated by GPCR. Although the T cell receptor (TCR) is a protein Tyr kinase–associated receptor, p110γ deletion affects TCR-induced T cell stimulation. We examined whether the TCR activates p110γ, as well as the consequences of interfering with p110γ expression or function for T cell activation. We found that after TCR ligation, p110γ interacts with Gαq/11, lymphocyte-specific Tyr kinase, and ζ-associated protein. TCR stimulation activates p110γ, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. We show that TCR-stimulated p110γ controls RAS-related C3 botulinum substrate 1 activity, F-actin polarization, and the interaction between T cells and antigen-presenting cells, illustrating a crucial role for p110γ in TCR-induced T cell activation. PMID:17998387

Stimulation of p38 (HOG1) kinase pathway by ionizing radiation results in downstream modulation of ATF/CREB transcription factor activity in NIH-3T3 cells

International Nuclear Information System (INIS)

Stevenson, Mary Ann; Yao Jin

1997-01-01

Purpose/Objective:p38 kinase, a member of the MAP kinase family, is activated in response to stresses such as high osmolarity and UV irradiation as well exposure to cytokines such as IL1β and TNFα. The kinase is part of a signal transduction pathway that leads from receptor activation through a three kinase cascade resulting in the activation of p38. p38 activation then leads to the phosphorylation of target proteins that include transcription factors such as nuclear factor of interleukin 6 and members of the activating transcription factor (ATF) family, and in addition, the stress protein, HSP27, via activation of MAPKAP2 kinase. In the present report, we have investigated the potential role of p38 in the response of NIH-3T3 cells to ionizing radiation. Materials and Methods:NIH-3T3 cells were grown to confluence in DMEM+10%CS and then serum deprived for 24 hours in DMEM+0.1%CS. Radiation exposures were delivered using a Philips RT250 (250Kvp X-ray tube). Activated forms of p38 kinase and ATF/CREB transcription factors were identified using immunoblotting techniques employing activation specific antibodies raised against the phosphorylated forms of the kinases/transcription factors. Kinase activity was directly measured using immunokinase assays. DNA binding of transcription factors to their respective consensus sequences was assayed by EMSA. Results:We found that p38 becomes rapidly phosphorylated and activated by exposure to ionizing radiation. Significantly, p38 is activated to a similar degree and with a similar time course by serum derpviation and entry of cells into a non-proliferating G 0 state, suggesting a causal role for p38 in quiescence. Phosphorylation of p38 directly correlated with phosphorylation and activation of ATF/CREB family members as well as DNA binding by these activated factors. Conclusion:Activation of p38 kinase and downstream transcription factors may play an important role in the response of cells to ionizing radiation. We are

Amide-based inhibitors of p38alpha MAP kinase. Part 2: design, synthesis and SAR of potent N-pyrimidyl amides.

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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.

Acute lymphoid and gastrointestinal toxicity induced by selective p38alpha map kinase and map kinase-activated protein kinase-2 (MK2) inhibitors in the dog.

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Morris, Dale L; O'Neil, Shawn P; Devraj, Rajesh V; Portanova, Joseph P; Gilles, Richard W; Gross, Cindy J; Curtiss, Sandra W; Komocsar, Wendy J; Garner, Debra S; Happa, Fernando A; Kraus, Lori J; Nikula, Kristen J; Monahan, Joseph B; Selness, Shaun R; Galluppi, Gerald R; Shevlin, Kimberly M; Kramer, Jeffrey A; Walker, John K; Messing, Dean M; Anderson, David R; Mourey, Robert J; Whiteley, Laurence O; Daniels, John S; Yang, Jerry Z; Rowlands, Philip C; Alden, Carl L; Davis, John W; Sagartz, John E

2010-06-01

Exposure to moderately selective p38alpha mitogen-activated protein kinase (MAPK) inhibitors in the Beagle dog results in an acute toxicity consisting of mild clinical signs (decreased activity, diarrhea, and fever), lymphoid necrosis and depletion in the gut-associated lymphoid tissue (GALT), mesenteric lymph nodes and spleen, and linear colonic and cecal mucosal hemorrhages. Lymphocyte apoptosis and necrosis in the GALT is the earliest and most prominent histopathologic change observed, followed temporally by neutrophilic infiltration and acute inflammation of the lymph nodes and spleen and multifocal mucosal epithelial necrosis and linear hemorrhages in the colon and cecum. These effects are not observed in the mouse, rat, or cynomolgus monkey. To further characterize the acute toxicity in the dog, a series of in vivo, in vitro, and immunohistochemical studies were conducted to determine the relationship between the lymphoid and gastrointestinal (GI) toxicity and p38 MAPK inhibition. Results of these studies demonstrate a direct correlation between p38alpha MAPK inhibition and the acute lymphoid and gastrointestinal toxicity in the dog. Similar effects were observed following exposure to inhibitors of MAPK-activated protein kinase-2 (MK2), further implicating the role of p38alpha MAPK signaling pathway inhibition in these effects. Based on these findings, the authors conclude that p38alpha MAPK inhibition results in acute lymphoid and GI toxicity in the dog and is unique among the species evaluated in these studies.

Human interleukin 1. beta. stimulates islet insulin release by a mechanism not dependent on changes in phospholipase C and protein kinase C activities or Ca sup 2+ handling

Energy Technology Data Exchange (ETDEWEB)

Welsh, N.; Nilsson, T.; Hallberg, A.; Arkhammar, P.; Berggren, P.-O.; Sandler, S.

1989-01-01

Isolated islets from adult rats or obese hyperglycemic (ob/ob) mice were incubated with human recombinant interleukin 1{beta} in order to study whether the acute effects of the cytokine on islet insulin release are associated with changes in islet phospholipase C activity, Ca{sup 2+} handling or protein phosphorylation. The cytokine stimulated insulin release both at low and high glucose concentrations during one hour incubations. In shortterm incubations (<1 min) interleukin 1{beta} did not affect the production of inositoltrisphosphate. Addition of interleukin 1{beta} affected neither the cytoplasmic free Ca{sup 2+} concentration at rest nor that observed subsequent to stimulation with a high concentration of glucose. Furthermore, the endogenous protein kinase C activity, as visualized by immunoprecipitation of a {sup 32}P-labelled substrate for this enzyme, was not altered by interleukin 1{beta}. Separation of {sup 32}P-labelled proteins by means of 2-dimensional gel electrophoresis failed to reveal any specific effects of the cytokine on the total protein phosphorylation activity. These results suggest that the stimulatory effects on insulin release exerted by interleukin 1{beta} are not caused by acute activation of phospholipase C and protein kinase C or by an alternation of islet Ca{sup 2+} handling of the B-cells. (author).

Involvement of stress-activated protein kinase in the cellular response to 1-beta-D-arabinofuranosylcytosine and other DNA-damaging agents.

Science.gov (United States)

Saleem, A; Datta, R; Yuan, Z M; Kharbanda, S; Kufe, D

1995-12-01

The cellular response to 1-beta-D-arabinofuranosylcytosine (ara-C) includes activation of Jun/AP-1, induction of c-jun transcription, and programmed cell death. The stress-activated protein (SAP) kinases stimulate the transactivation function of c-jun by amino terminal phosphorylation. The present work demonstrates that ara-C activates p54 SAP kinase. The finding that SAP kinase is also activated by alkylating agents (mitomycin C and cisplatinum) and the topoisomerase I inhibitor 9-amino-camptothecin supports DNA damage as an initial signal in this cascade. The results demonstrate that ara-C also induces binding of SAP kinase to the SH2/SH3-containing adapter protein Grb2. SAP kinase binds to the SH3 domains of Grb2, while interaction of the p85 alpha-subunit of phosphatidylinositol 3-kinase complex. The results also demonstrate that ara-C treatment is associated with inhibition of lipid and serine kinase activities of PI 3-kinase. The potential significance of the ara-C-induced interaction between SAP kinase and PI 3-kinase is further supported by the demonstration that Wortmannin, an inhibitor of PI 3-kinase, stimulates SAP kinase activity. The finding that Wortmannin treatment is also associated with internucleosomal DNA fragmentation may support a potential link between PI 3-kinase and regulation of both SAP kinase and programmed cell death.

Follicle-stimulating hormone (FSH) activates extracellular signal-regulated kinase phosphorylation independently of beta-arrestin- and dynamin-mediated FSH receptor internalization

Science.gov (United States)

Piketty, Vincent; Kara, Elodie; Guillou, Florian; Reiter, Eric; Crepieux, Pascale

2006-01-01

Background The follicle-stimulating hormone receptor (FSH-R) is a seven transmembrane spanning receptor (7TMR) which plays a crucial role in male and female reproduction. Upon FSH stimulation, the FSH-R activates the extracellular signal-regulated kinases (ERK). However, the mechanisms whereby the agonist-stimulated FSH-R activates ERK are poorly understood. In order to activate ERK, some 7 TMRs require beta-arrestin-and dynamin-dependent internalization to occur, whereas some others do not. In the present study, we examined the ability of the FSH-activated FSH-R to induce ERK phosphorylation, in conditions where its beta-arrestin- and dynamin-mediated internalization was impaired. Methods Human embryonic kidney (HEK) 293 cells were transiently transfected with the rat FSH-R. Internalization of the FSH-R was manipulated by co-expression of either a beta-arrestin (319–418) dominant negative peptide, either an inactive dynamin K44A mutant or of wild-type beta-arrestin 1 or 2. The outcomes on the FSH-R internalization were assayed by measuring 125I-FSH binding at the cell surface when compared to internalized 125I-FSH binding. The resulting ERK phosphorylation level was visualized by Western blot analysis. Results In HEK 293 cells, FSH stimulated ERK phosphorylation in a dose-dependent manner. Co-transfection of the beta- arrestin (319–418) construct, or of the dynamin K44A mutant reduced FSH-R internalization in response to FSH, without affecting ERK phosphorylation. Likewise, overexpression of wild-type beta-arrestin 1 or 2 significantly increased the FSH-R internalization level in response to FSH, without altering FSH-induced ERK phosphorylation. Conclusion From these results, we conclude that the FSH-R does not require beta-arrestin- nor dynamin-mediated internalization to initiate ERK phosphorylation in response to FSH. PMID:16787538

Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases.

OpenAIRE

Schlaepfer, D D; Hunter, T

1996-01-01

Focal adhesion kinase (FAK) is a nonreceptor protein-tyrosine kinase (PTK) that associates with integrin receptors and participates in extracellular matrix-mediated signal transduction events. We showed previously that the c-Src nonreceptor PTK and the Grb2 SH2/SH3 adaptor protein bound directly to FAK after fibronectin stimulation (D. D. Schlaepfer, S.K. Hanks, T. Hunter, and P. van der Geer, Nature [London] 372:786-791, 1994). Here, we present evidence that c-Src association with FAK is req...

The transmembrane domain of the p75 neurotrophin receptor stimulates phosphorylation of the TrkB tyrosine kinase receptor.

Science.gov (United States)

Saadipour, Khalil; MacLean, Michael; Pirkle, Sean; Ali, Solav; Lopez-Redondo, Maria-Luisa; Stokes, David L; Chao, Moses V

2017-10-06

The function of protein products generated from intramembraneous cleavage by the γ-secretase complex is not well defined. The γ-secretase complex is responsible for the cleavage of several transmembrane proteins, most notably the amyloid precursor protein that results in Aβ, a transmembrane (TM) peptide. Another protein that undergoes very similar γ-secretase cleavage is the p75 neurotrophin receptor. However, the fate of the cleaved p75 TM domain is unknown. p75 neurotrophin receptor is highly expressed during early neuronal development and regulates survival and process formation of neurons. Here, we report that the p75 TM can stimulate the phosphorylation of TrkB (tyrosine kinase receptor B). In vitro phosphorylation experiments indicated that a peptide representing p75 TM increases TrkB phosphorylation in a dose- and time-dependent manner. Moreover, mutagenesis analyses revealed that a valine residue at position 264 in the rat p75 neurotrophin receptor is necessary for the ability of p75 TM to induce TrkB phosphorylation. Because this residue is just before the γ-secretase cleavage site, we then investigated whether the p75(αγ) peptide, which is a product of both α- and γ-cleavage events, could also induce TrkB phosphorylation. Experiments using TM domains from other receptors, EGFR and FGFR1, failed to stimulate TrkB phosphorylation. Co-immunoprecipitation and biochemical fractionation data suggested that p75 TM stimulates TrkB phosphorylation at the cell membrane. Altogether, our results suggest that TrkB activation by p75(αγ) peptide may be enhanced in situations where the levels of the p75 receptor are increased, such as during brain injury, Alzheimer's disease, and epilepsy. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Protein kinase C activation and myosin light chain phosphorylation in 32P-labeled arterial smooth muscle

International Nuclear Information System (INIS)

Singer, H.A.

1990-01-01

Experiments using 32P-labeled strips of swine carotid artery medial smooth muscle were performed to define the relative contribution of myosin light chain (MLC) phosphorylation as an activation mechanism mediating contractile responses stimulated by phorbol dibutyrate (PDB). Tryptic phosphopeptide mapping of phosphorylated MLC indicated that near-maximal force responses were associated with increases in functional MLC phosphorylation of less than 10% of the total MLC content following tonic (45 min) stimulation by PDB. Significant phosphorylation of MLC residues, consistent with the specificity of protein kinase C, occurred in response to high concentrations of PDB (greater than 0.1 microM). Histamine (10 microM)-induced MLC phosphorylation after 2 min (72.5% of total MLC) or 45 min (61.7%) was restricted to serine residues on peptides thought to contain serine19. Although agonist (histamine)-induced responses were eliminated under conditions of Ca2+ depletion, near-maximal force in response to 10 microM PDB (89.4% of a standard KCl response) was associated with monophosphorylation of less than 9% of the total MLC on peptides interpreted as containing serine19. A substantial fraction of this was localized to threonine residues. The quantitative analysis of the relation between PDB-stimulated force and the residues in MLC phosphorylated supports the concept that PDB stimulation results in activation of arterial smooth muscle cross bridges by MLC-phosphorylation-independent mechanisms

Akt Kinase-Mediated Checkpoint of cGAS DNA Sensing Pathway

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Gil Ju Seo

2015-10-01

Full Text Available Upon DNA stimulation, cyclic GMP-AMP synthase (cGAS synthesizes the second messenger cyclic GMP-AMP (cGAMP that binds to the STING, triggering antiviral interferon-β (IFN-β production. However, it has remained undetermined how hosts regulate cGAS enzymatic activity after the resolution of DNA immunogen. Here, we show that Akt kinase plays a negative role in cGAS-mediated anti-viral immune response. Akt phosphorylated the S291 or S305 residue of the enzymatic domain of mouse or human cGAS, respectively, and this phosphorylation robustly suppressed its enzymatic activity. Consequently, expression of activated Akt led to the reduction of cGAMP and IFN-β production and the increase of herpes simplex virus 1 replication, whereas treatment with Akt inhibitor augmented cGAS-mediated IFN-β production. Furthermore, expression of the phosphorylation-resistant cGAS S291A mutant enhanced IFN-β production upon DNA stimulation, HSV-1 infection, and vaccinia virus infection. Our study identifies an Akt kinase-mediated checkpoint to fine-tune hosts’ immune responses to DNA stimulation.

SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

Science.gov (United States)

McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

1992-01-01

The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

Mechanism of polyphosphate kinase from Propionibacterium shermanii

International Nuclear Information System (INIS)

Robinson, N.A.

1986-01-01

Polyphosphate kinase, which catalyzes the reaction shown below, is one of two enzymes which have been reported to catalyze the synthesis of polyphosphate. Purification performed by ammonium sulfate precipitation (0-40% fraction) was followed by chromatography. The enzyme represents 70% of the protein in the hydroxylapatite pool and is stable at this level of purity. The subunit molecular weight was determined by SDS polyacrylamide gel analysis, (83,000 +/- 3000), nondenaturing polyacrylamide gel electrophoresis, (80,000 and 86,000 daltons), gel filtration (Biogel A 0.5m column was 85,000 +/- 4000.) Polyphosphate kinase appears to be a monomeric enzyme of ∼83,000 daltons. Four assays were developed for polyphosphate kinase. Basic proteins such as polylysine stimulate the synthesis of polyphosphate, these proteins cause precipitation of polyphosphate kinase from relatively impure enzyme extracts: Synthesized polyphosphate interacts noncovalently with the basic protein-enzyme precipitate. Efficient synthesis of polyphosphate requires the addition of either phosphate or short chain polyphosphate. Synthesis did occur at 1/10 the rate when neither of these two compounds were included. Initiation, elongation, and termination events of polyphosphate synthesis were examined. Short chain polyphosphate acts as a primer, with [ 32 P] short-chain polyphosphate incorporation into long chain polyphosphate by the kinase

Insulin rapidly stimulates phosphorylation of a 46-kDa membrane protein on tyrosine residues as well as phosphorylation of several soluble proteins in intact fat cells

International Nuclear Information System (INIS)

Haering, H.U.; White, M.F.; Machicao, F.; Ermel, B.; Schleicher, E.; Obermaier, B.

1987-01-01

It is speculated that the transmission of an insulin signal across the plasma membrane of cells occurs through activation of the tyrosine-specific receptor kinase, autophosphorylation of the receptor, and subsequent phosphorylation of unidentified substrates in the cell. In an attempt to identify possible substrates, the authors labeled intact rat fat cells with [ 32 P]orthophosphate and used an antiphosphotyrosine antibody to identify proteins that become phosphorylated on tyrosine residues in an insulin-stimulated way. In the membrane fraction of the fat cells, they found, in addition to the 95-kDa β-subunit of the receptor, a 46-kDa phosphoprotein that is phosphorylated exclusively on tyrosine residues. This protein is not immunoprecipitated by antibodies against different regions of the insulin receptor and its HPLC tryptic peptide map is different from the tryptic peptide map of the insulin receptor, suggesting that it is not derived from the receptor β-subunit. Insulin stimulates the tyrosine phosphorylation of the 46-kDa protein within 150 sec in the intact cell 3- to 4-fold in a dose-dependent way at insulin concentrations between 0.5 nM and 100 nM. Insulin (0.5 nM, 100 nM) stimulated within 2 min the 32 P incorporation into a 116-kDa band, a 62 kDa band, and three bands between 45 kDa and 50 kDa 2- to 10-fold. They suggest that the 46-kDa membrane protein and possibly also the soluble proteins are endogenous substrates of the receptor tyrosine kinase in fat cells and that their phosphorylation is an early step in insulin signal transmission

Rab39a interacts with phosphatidylinositol 3-kinase and negatively regulates autophagy induced by lipopolysaccharide stimulation in macrophages.

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Shintaro Seto

Full Text Available Rab39a has pleiotropic functions in phagosome maturation, inflammatory activation and neuritogenesis. Here, we characterized Rab39a function in membrane trafficking of phagocytosis and autophagy induction in macrophages. Rab39a localized to the periphery of LAMP2-positive vesicles and showed the similar kinetics on the phagosome to that of LAMP1. The depletion of Rab39a did not influence the localization of LAMP2 to the phagosome, but it augments the autophagosome formation and LC3 processing by lipopolysaccharide (LPS stimulation. The augmentation of autophagosome formation in Rab39a-knockdown macrophages was suppressed by Atg5 depletion or an inhibitor for phosphatidylinostol 3-kinase (PI3K. Immunoprecipitation analysis revealed that Rab39a interacts with PI3K and that the amino acid residues from 34(th to 41(st in Rab39a were indispensable for this interaction. These results suggest that Rab39a negatively regulates the LPS-induced autophagy in macrophages.

Whole-brain activity mapping onto a zebrafish brain atlas

Science.gov (United States)

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

CSK negatively regulates nerve growth factor induced neural differentiation and augments AKT kinase activity

International Nuclear Information System (INIS)

Dey, Nandini; Howell, Brian W.; De, Pradip K.; Durden, Donald L.

2005-01-01

Src family kinases are involved in transducing growth factor signals for cellular differentiation and proliferation in a variety of cell types. The activity of all Src family kinases (SFKs) is controlled by phosphorylation at their C-terminal 527-tyrosine residue by C-terminal SRC kinase, CSK. There is a paucity of information regarding the role of CSK and/or specific Src family kinases in neuronal differentiation. Pretreatment of PC12 cells with the Src family kinase inhibitor, PP1, blocked NGF-induced activation of SFKs and obliterated neurite outgrowth. To confirm a role for CSK and specific isoforms of SFKs in neuronal differentiation, we overexpressed active and catalytically dead CSK in the rat pheochromocytoma cell line, PC12. CSK overexpression caused a profound inhibition of NGF-induced activation of FYN, YES, RAS, and ERK and inhibited neurite outgrowth, NGF-stimulated integrin-directed migration and blocked the NGF-induced conversion of GDP-RAC to its GTP-bound active state. CSK overexpression markedly augmented the activation state of AKT following NGF stimulation. In contrast, kinase-dead CSK augmented the activation of FYN, RAS, and ERK and increased neurite outgrowth. These data suggest a distinct requirement for CSK in the regulation of NGF/TrkA activation of RAS, RAC, ERK, and AKT via the differential control of SFKs in the orchestration of neuronal differentiation

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

International Nuclear Information System (INIS)

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

2011-01-01

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

The transcriptional response to the inactivation of the PaMpk1 and PaMpk2 MAP kinase pathways in Podospora anserina.

Science.gov (United States)

Bidard, Frédérique; Coppin, Evelyne; Silar, Philippe

2012-08-01

Transcription pattern during mycelium growth of Podospora anserina was assayed by microarray analysis in wild type and in mutants affected in the MAP kinase genes PaMpk1 and PaMpk2 and in the NADPH oxidase gene PaNox1. 15% of the genes have their expression modified by a factor two or more as growth proceeds in wild type. The genes whose expression is modified during growth in P. anserina are either not conserved or differently regulated in Neurospora crassa and Aspergillus niger, two fungi for which transcriptome data during growth are available. The P. anserina mutants display a similar alteration of their transcriptome profile, with nearly 1000 genes affected similarly in the three mutants, accounting for their similar growth phenotypes. Yet, each mutant has its specific set of modified transcripts, in line with particular phenotypes exhibited by each mutant. Again, there is limited conservation during evolution of the genes regulated at the transcription level by MAP kinases, as indicated by the comparison the P. anserina data, with those of Aspergillus fumigatus and N. crassa, two fungi for which gene expression data are available for mutants of the MAPK pathways. Among the genes regulated in wild type and affected in the mutants, those involved in carbohydrate and secondary metabolisms appear prominent. The vast majority of the genes differentially expressed are of unknown function. Availability of their transcription profile at various stages of development should help to decipher their role in fungal physiology and development. Copyright © 2012 Elsevier Inc. All rights reserved.

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

Science.gov (United States)

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.

Accelerometer-based automatic voice onset detection in speech mapping with navigated repetitive transcranial magnetic stimulation.

Science.gov (United States)

Vitikainen, Anne-Mari; Mäkelä, Elina; Lioumis, Pantelis; Jousmäki, Veikko; Mäkelä, Jyrki P

2015-09-30

The use of navigated repetitive transcranial magnetic stimulation (rTMS) in mapping of speech-related brain areas has recently shown to be useful in preoperative workflow of epilepsy and tumor patients. However, substantial inter- and intraobserver variability and non-optimal replicability of the rTMS results have been reported, and a need for additional development of the methodology is recognized. In TMS motor cortex mappings the evoked responses can be quantitatively monitored by electromyographic recordings; however, no such easily available setup exists for speech mappings. We present an accelerometer-based setup for detection of vocalization-related larynx vibrations combined with an automatic routine for voice onset detection for rTMS speech mapping applying naming. The results produced by the automatic routine were compared with the manually reviewed video-recordings. The new method was applied in the routine navigated rTMS speech mapping for 12 consecutive patients during preoperative workup for epilepsy or tumor surgery. The automatic routine correctly detected 96% of the voice onsets, resulting in 96% sensitivity and 71% specificity. Majority (63%) of the misdetections were related to visible throat movements, extra voices before the response, or delayed naming of the previous stimuli. The no-response errors were correctly detected in 88% of events. The proposed setup for automatic detection of voice onsets provides quantitative additional data for analysis of the rTMS-induced speech response modifications. The objectively defined speech response latencies increase the repeatability, reliability and stratification of the rTMS results. Copyright © 2015 Elsevier B.V. All rights reserved.

The "neuro-mapping locator" software. A real-time intraoperative objective paraesthesia mapping tool to evaluate paraesthesia coverage of the painful zone in patients undergoing spinal cord stimulation lead implantation.

Science.gov (United States)

Guetarni, F; Rigoard, P

2015-03-01

Conventional spinal cord stimulation (SCS) generates paraesthesia, as the efficacy of this technique is based on the relationship between the paraesthesia provided by SCS on the painful zone and an analgesic effect on the stimulated zone. Although this basic postulate is based on clinical evidence, it is clear that this relationship has never been formally demonstrated by scientific studies. There is a need for objective evaluation tools ("transducers") to transpose electrical signals to clinical effects and to guide therapeutic choices. We have developed a software at Poitiers University hospital allowing real-time objective mapping of the paraesthesia generated by SCS lead placement and programming during the implantation procedure itself, on a touch screen interface. The purpose of this article is to describe this intraoperative mapping software, in terms of its concept and technical aspects. The Neuro-Mapping Locator (NML) software is dedicated to patients with failed back surgery syndrome, candidates for SCS lead implantation, to actively participate in the implantation procedure. Real-time geographical localization of the paraesthesia generated by percutaneous or multicolumn surgical SCS lead implanted under awake anaesthesia allows intraoperative lead programming and possibly lead positioning to be modified with the patient's cooperation. Software updates should enable us to refine objectives related to the use of this tool and minimize observational biases. The ultimate goals of NML software should not be limited to optimize one specific device implantation in a patient but also allow to compare instantaneously various stimulation strategies, by characterizing new technical parameters as "coverage efficacy" and "device specificity" on selected subgroups of patients. Another longer-term objective would be to organize these predictive factors into computer science ontologies, which could constitute robust and helpful data for device selection and programming

Transforming growth factor beta 1 increases collagen content, and stimulates procollagen I and tissue inhibitor of metalloproteinase-1 production of dental pulp cells: Role of MEK/ERK and activin receptor-like kinase-5/Smad signaling

Directory of Open Access Journals (Sweden)

Po-Shuen Lin

2017-05-01

Conclusion: These results indicate that TGF-β1 may be involved in the healing/regeneration processes of dental pulp in response to injury by stimulation of collagen and TIMP-1 production. These events are associated with activin receptor-like kinase-5/Smad2/3 and MEK/ERK signaling.

Mitogen-activated protein kinases interacting kinases are autoinhibited by a reprogrammed activation segment.

Science.gov (United States)

Jauch, Ralf; Cho, Min-Kyu; Jäkel, Stefan; Netter, Catharina; Schreiter, Kay; Aicher, Babette; Zweckstetter, Markus; Jäckle, Herbert; Wahl, Markus C

2006-09-06

Autoinhibition is a recurring mode of protein kinase regulation and can be based on diverse molecular mechanisms. Here, we show by crystal structure analysis, nuclear magnetic resonance (NMR)-based nucleotide affinity studies and rational mutagenesis that nonphosphorylated mitogen-activated protein (MAP) kinases interacting kinase (Mnk) 1 is autoinhibited by conversion of the activation segment into an autoinhibitory module. In a Mnk1 crystal structure, the activation segment is repositioned via a Mnk-specific sequence insertion at the N-terminal lobe with the following consequences: (i) the peptide substrate binding site is deconstructed, (ii) the interlobal cleft is narrowed, (iii) an essential Lys-Glu pair is disrupted and (iv) the magnesium-binding loop is locked into an ATP-competitive conformation. Consistently, deletion of the Mnk-specific insertion or removal of a conserved phenylalanine side chain, which induces a blockade of the ATP pocket, increase the ATP affinity of Mnk1. Structural rearrangements required for the activation of Mnks are apparent from the cocrystal structure of a Mnk2 D228G -staurosporine complex and can be modeled on the basis of crystal packing interactions. Our data suggest a novel regulatory mechanism specific for the Mnk subfamily.

Insulin increase in MAP kinase phosphorylation is shifted to early time-points by overexpressing APS, while Akt phosphorylation is not influenced.

Science.gov (United States)

Onnockx, Sheela; Xie, Jingwei; Degraef, Chantal; Erneux, Christophe; Pirson, Isabelle

2009-09-10

Upon insulin stimulation, the adaptor protein APS is recruited to the insulin receptor and tyrosine phosphorylated. APS initiates the insulin-induced TC10 cascade which participates to GLUT4 translocation to the plasma membrane. Nevertheless, the molecular mechanism that governs APS and its SH2 and PH domains action on the insulin transduction cascade is not yet fully understood. Here, we show that APS co-immunoprecipitates with the class I PI 3-kinase regulatory subunit p85, through its SH2 domain but that APS does not modulate neither PtdIns(3,4,5)P3 levels nor Akt phosphorylation provoked by insulin. We have confirmed a previously described positive effect of APS overexpression on insulin-induced MAPK phosphorylation upregulation. Consequently, we analyzed the role of SH2 and PH domains of APS in the APS increased MAPK phosphorylation observed upon insulin stimulation and correlated this with the membrane localization of the protein. The effect observed on MAPK phosphorylation requires the intact PH binding domain of APS as well as its SH2 domain.

Effect of triiodothyronine on rat liver chromatin protein kinase

International Nuclear Information System (INIS)

Kruh, J.; Tichonicky, L.

1976-01-01

1) Injection of triiodothyronine to rats stimulates protein kinase activity in liver chromatin nonhistone proteins. A significant increase was found after two daily injections. A 4-fold increase was observed with the purified enzyme after eight daily injections of the hormone. No variations were observed in cytosol protein kinase activity. Electrophoretic pattern, effect of heat denaturation, effect of p-hydroxymercuribenzoate seem to indicate that the enzyme present in treated rats is not identical to the enzyme in control animals, which suggests that thyroid hormone has induced nuclear protein kinase. Diiodothyronine, 3, 3', 5'-triiodothyronine have no effect on protein kinase. 2) Chromatin non-histone proteins isolated from rats injected with triiodothyronine incorporated more 32 P when incubated with [γ- 32 P]ATP than the chromatin proteins from untreated rats. Thyroidectomy reduced the in vitro 32 P incorporation. It is suggested that some of the biological activity of thyroid hormone could be mediated through its effect on chromatin non-histone proteins. (orig.) [de

Whole-brain activity mapping onto a zebrafish brain atlas.

Science.gov (United States)

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.

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

Science.gov (United States)

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.

Phosphatidylinositol 3-Kinase (PI3K) Activity Bound to Insulin-like Growth Factor-I (IGF-I) Receptor, which Is Continuously Sustained by IGF-I Stimulation, Is Required for IGF-I-induced Cell Proliferation*

Science.gov (United States)

Fukushima, Toshiaki; Nakamura, Yusaku; Yamanaka, Daisuke; Shibano, Takashi; Chida, Kazuhiro; Minami, Shiro; Asano, Tomoichiro; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

2012-01-01

Continuous stimulation of cells with insulin-like growth factors (IGFs) in G1 phase is a well established requirement for IGF-induced cell proliferation; however, the molecular components of this prolonged signaling pathway that is essential for cell cycle progression from G1 to S phase are unclear. IGF-I activates IGF-I receptor (IGF-IR) tyrosine kinase, followed by phosphorylation of substrates such as insulin receptor substrates (IRS) leading to binding of signaling molecules containing SH2 domains, including phosphatidylinositol 3-kinase (PI3K) to IRS and activation of the downstream signaling pathways. In this study, we found prolonged (>9 h) association of PI3K with IGF-IR induced by IGF-I stimulation. PI3K activity was present in this complex in thyrocytes and fibroblasts, although tyrosine phosphorylation of IRS was not yet evident after 9 h of IGF-I stimulation. IGF-I withdrawal in mid-G1 phase impaired the association of PI3K with IGF-IR and suppressed DNA synthesis the same as when PI3K inhibitor was added. Furthermore, we demonstrated that Tyr1316-X-X-Met of IGF-IR functioned as a PI3K binding sequence when this tyrosine is phosphorylated. We then analyzed IGF signaling and proliferation of IGF-IR−/− fibroblasts expressing exogenous mutant IGF-IR in which Tyr1316 was substituted with Phe (Y1316F). In these cells, IGF-I stimulation induced tyrosine phosphorylation of IGF-IR and IRS-1/2, but mutated IGF-IR failed to bind PI3K and to induce maximal phosphorylation of GSK3β and cell proliferation in response to IGF-I. Based on these results, we concluded that PI3K activity bound to IGF-IR, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation. PMID:22767591

Inhibition of insulin-stimulated hydrogen peroxide production prevents stimulation of sodium transport in A6 cell monolayers.

NARCIS (Netherlands)

Markadieu, N.Y.G.; Crutzen, R.; Boom, A.; Erneux, C.; Beauwens, R.

2009-01-01

Insulin-stimulated sodium transport across A6 cell (derived from amphibian distal nephron) monolayers involves the activation of a phosphatidylinositol (PI) 3-kinase. We previously demonstrated that exogenous addition of H2O2 to the incubation medium of A6 cell monolayers provokes an increase in PI

KCl stimulation increases norepinephrine transporter function in PC12 cells.

Science.gov (United States)

Mandela, Prashant; Ordway, Gregory A

2006-09-01

The norepinephrine transporter (NET) plays a pivotal role in terminating noradrenergic signaling and conserving norepinephrine (NE) through the process of re-uptake. Recent evidence suggests a close association between NE release and regulation of NET function. The present study evaluated the relationship between release and uptake, and the cellular mechanisms that govern these processes. KCl stimulation of PC12 cells robustly increased [3H]NE uptake via the NET and simultaneously increased [3H]NE release. KCl-stimulated increases in uptake and release were dependent on Ca2+. Treatment of cells with phorbol-12-myristate-13-acetate (PMA) or okadaic acid decreased [3H]NE uptake but did not block KCl-stimulated increases in [3H]NE uptake. In contrast, PMA increased [3H]NE release and augmented KCl-stimulated release, while okadaic acid had no effects on release. Inhibition of Ca2+-activated signaling cascades with KN93 (a Ca2+ calmodulin-dependent kinase inhibitor), or ML7 and ML9 (myosin light chain kinase inhibitors), reduced [3H]NE uptake and blocked KCl-stimulated increases in uptake. In contrast, KN93, ML7 and ML9 had no effect on KCl-stimulated [3H]NE release. KCl-stimulated increases in [3H]NE uptake were independent of transporter trafficking to the plasma membrane. While increases in both NE release and uptake mediated by KCl stimulation require Ca2+, different intracellular mechanisms mediate these two events.

Differential effects of vasopressin and phenylephrine on protein kinase C-mediated protein phosphorylations in isolated hepatocytes

International Nuclear Information System (INIS)

Cooper, R.H.; Johanson, R.A.; Wiliamson, J.R.

1986-01-01

Receptor-mediated breakdown of inositol lipids produces two intracellular signals, diacylglycerol, which activates protein kinase C, and inositol trisphosphate, which causes release of intracellular vesicular Ca 2+ . This study examined the effects of Ca 2+ -ionophores, vasopressin, phenylephrine, and phorbol ester (PMA) on hepatocyte protein phosphorylations. [ 32 P] Phosphoproteins from hepatocytes prelabeled with 32 P were resolved by 2-dimensional SDS-PAGE and corresponding autoradiographs were quantitated by densitometric analysis. The phosphorylation of five proteins, a plasma membrane bound 16 kDa protein with pI 6.4, a cytosolic 16 kDa protein with pI 5.8, and proteins with Mr's of 36 kDa, 52 kDa, and 68 kDa, could be attributed to phosphorylation by protein kinase C since the phosphorylation was stimulated by PMA. When the vasopressin concentration was varied, low vasopressin stimulated the phosphorylation of only the membrane bound 16 kDa protein of the above set of proteins, while higher vasopressin concentrations were required to stimulate the phosphorylation of all five proteins. Phenylephrine, even at supramaximal concentrations, stimulated the phosphorylation of only the membrane bound 16 kDa protein. These results suggest that phenylephrine is a less potent activator of protein kinase C than vasopressin by virtue of limited or localized diacylglycerol production

Interleukin-1 beta induced synthesis of protein kinase C-delta and protein kinase C-epsilon in EL4 thymoma cells: possible involvement of phosphatidylinositol 3-kinase.

Science.gov (United States)

Varley, C L; Royds, J A; Brown, B L; Dobson, P R

2001-01-01

We present evidence here that the proinflammatory cytokine, interleukin-1 beta (IL-1 beta) stimulates a significant increase in protein kinase C (PKC)-epsilon and PKC-delta protein levels and increases PKC-epsilon, but not PKC-delta, transcripts in EL4 thymoma cells. Incubation of EL4 cells with IL-1 beta induced protein synthesis of PKC-epsilon (6-fold increase) by 7 h and had a biphasic effect on PKC-delta levels with peaks at 4 h (2-fold increase) and 24 h (4-fold increase). At the level of mRNA, PKC-epsilon, but not PKC-delta levels, were induced after incubation of EL4 cells with IL-1 beta. The signalling mechanisms utilized by IL-1 beta to induce the synthesis of these PKC isoforms were investigated. Two phosphatidylinositol (PI) 3-kinase-specific inhibitors, wortmannin and LY294002, inhibited IL-1 beta-induced synthesis of PKC-epsilon. However, the PI 3-kinase inhibitors had little effect on the IL-1 beta-induced synthesis of PKC-delta in these cells. Our results indicate that IL-1 beta induced both PKC-delta and PKC-epsilon expression over different time periods. Furthermore, our evidence suggests that IL-1 beta induction of PKC-epsilon, but not PKC-delta, may occur via the PI 3-kinase pathway. Copyright 2001 S. Karger AG, Basel

MAP kinase pathways in the yeast Saccharomyces cerevisiae

Science.gov (United States)

Gustin, M. C.; Albertyn, J.; Alexander, M.; Davenport, K.; McIntire, L. V. (Principal Investigator)

1998-01-01

A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.

Akt Kinase-Mediated Checkpoint of cGAS DNA Sensing Pathway.

Science.gov (United States)

Seo, Gil Ju; Yang, Aerin; Tan, Brandon; Kim, Sungyoon; Liang, Qiming; Choi, Younho; Yuan, Weiming; Feng, Pinghui; Park, Hee-Sung; Jung, Jae U

2015-10-13

Upon DNA stimulation, cyclic GMP-AMP synthase (cGAS) synthesizes the second messenger cyclic GMP-AMP (cGAMP) that binds to the STING, triggering antiviral interferon-β (IFN-β) production. However, it has remained undetermined how hosts regulate cGAS enzymatic activity after the resolution of DNA immunogen. Here, we show that Akt kinase plays a negative role in cGAS-mediated anti-viral immune response. Akt phosphorylated the S291 or S305 residue of the enzymatic domain of mouse or human cGAS, respectively, and this phosphorylation robustly suppressed its enzymatic activity. Consequently, expression of activated Akt led to the reduction of cGAMP and IFN-β production and the increase of herpes simplex virus 1 replication, whereas treatment with Akt inhibitor augmented cGAS-mediated IFN-β production. Furthermore, expression of the phosphorylation-resistant cGAS S291A mutant enhanced IFN-β production upon DNA stimulation, HSV-1 infection, and vaccinia virus infection. Our study identifies an Akt kinase-mediated checkpoint to fine-tune hosts' immune responses to DNA stimulation. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Ascofuranone stimulates expression of adiponectin and peroxisome proliferator activated receptor through the modulation of mitogen activated protein kinase family members in 3T3-L1, murine pre-adipocyte cell line

International Nuclear Information System (INIS)

Chang, Young-Chae; Cho, Hyun-Ji

2012-01-01

Highlights: ► Ascofuranone increases expression of adiponectin and PPARγ. ► Inhibitors for MEK and JNK increased the expression of adiponectin and PPARγ. ► Ascofuranone significantly suppressed phosho-ERK, while increasing phospho-p38. -- Abstract: Ascofuranone, an isoprenoid antibiotic, was originally isolated as a hypolipidemic substance from a culture broth of the phytopathogenic fungus, Ascochyta visiae. Adiponectin is mainly synthesized by adipocytes. It relieves insulin resistance by decreasing the plasma triglycerides and improving glucose uptake, and has anti-atherogenic properties. Here, we found that ascofuranone increases expression of adiponectin and PPARγ, a major transcription factor for adiponectin, in 3T3-L1, murine pre-adipocytes cell line, without promoting accumulation of lipid droplets. Ascofuranone induced expression of adiponectin, and increases the promoter activity of adiponectin and PPRE, PPAR response element, as comparably as a PPARγ agonist, rosiglitazone, that stimulates lipid accumulation in the preadipocyte cell line. Moreover, inhibitors for MEK and JNK, like ascofuranone, considerably increased the expression of adiponectin and PPARγ, while a p38 inhibitor significantly suppressed. Ascofuranone significantly suppressed ERK phosphorylation, while increasing p38 phosphorylation, during adipocyte differentiation program. These results suggest that ascofuranone regulates the expression of adiponectin and PPARγ through the modulation of MAP kinase family members.

The Structure of the MAP2K MEK6 Reveals an Autoinhibitory Dimer

Energy Technology Data Exchange (ETDEWEB)

Min, Xiaoshan; Akella, Radha; He, Haixia; Humphreys, John M.; Tsutakawa, Susan E.; Lee, Seung-Jae; Tainer, John A.; Cobb, Melanie H.; Goldsmith, Elizabeth J.

2009-07-13

MAP2Ks are dual-specificity protein kinases functioning at the center of three-tiered MAP kinase modules. The structure of the kinase domain of the MAP2K MEK6 with phosphorylation site mimetic aspartic acid mutations (MEK6/{Delta}N/DD) has been solved at 2.3 {angstrom} resolution. The structure reveals an autoinhibited elongated ellipsoidal dimer. The enzyme adopts an inactive conformation, based upon structural queues, despite the phosphomimetic mutations. Gel filtration and small-angle X-ray scattering analysis confirm that the crystallographically observed ellipsoidal dimer is a feature of MEK6/{Delta}N/DD and full-length unphosphorylated wild-type MEK6 in solution. The interface includes the phosphate binding ribbon of each subunit, part of the activation loop, and a rare 'arginine stack' between symmetry-related arginine residues in the N-terminal lobe. The autoinhibited structure likely confers specificity on active MAP2Ks. The dimer may also serve the function in unphosphorylated MEK6 of preventing activation loop phosphorylation by inappropriate kinases.

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

Energy Technology Data Exchange (ETDEWEB)

Hwang, Yong Pil; Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Hien, Tran Thi [College of Pharmacy, Chosun University, Gwangju (Korea, Republic of); Jeong, Myung Ho [Heart Research Center, Chonnam National University Hospital, Gwangju (Korea, Republic of); Jeong, Tae Cheon, E-mail: taecheon@ynu.ac.kr [College of Pharmacy, Yeungnam University, Gyungsan (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

2011-11-15

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

Physical and functional interactions between ZIP kinase and UbcH5

International Nuclear Information System (INIS)

Ohbayashi, Norihiko; Okada, Katsuya; Kawakami, Shiho; Togi, Sumihito; Sato, Noriko; Ikeda, Osamu; Kamitani, Shinya; Muromoto, Ryuta; Sekine, Yuichi; Kawai, Taro; Akira, Shizuo; Matsuda, Tadashi

2008-01-01

Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase that has been implicated in cell death and transcriptional regulation, but its mechanism of regulation remains unknown. In our previous study, we showed that leukemia inhibitory factor stimulated threonine-265 phosphorylation of ZIPK, thereby leading to phosphorylation and activation of signal transducer and activator of transcription 3. Here, we identified UbcH5c as a novel ZIPK-binding partner by yeast two-hybrid screening. Importantly, we found that UbcH5c induced ubiquitination of ZIPK. Small-interfering RNA-mediated reduction of endogenous UbcH5 expression decreased ZIPK ubiquitination. Furthermore, coexpression of UbcH5c with ZIPK influenced promyelocytic leukemia protein nuclear body (PML-NB) formation. These results suggest that UbcH5 regulates ZIPK accumulation in PML-NBs by interacting with ZIPK and stimulating its ubiquitination

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

Science.gov (United States)

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.

Protein kinase D1 stimulates proliferation and enhances tumorigenesis of MCF-7 human breast cancer cells through a MEK/ERK-dependent signaling pathway

International Nuclear Information System (INIS)

Karam, Manale; Legay, Christine; Auclair, Christian; Ricort, Jean-Marc

2012-01-01

Protein kinase D1, PKD1, is a novel serine/threonine kinase whose altered expression and dysregulation in many tumors as well as its activation by several mitogens suggest that this protein could regulate proliferation and tumorigenesis. Nevertheless, the precise signaling pathways used are still unclear and the potential direct role of PKD1 in tumor development and progression has not been yet investigated. In order to clarify the role of PKD1 in cell proliferation and tumorigenesis, we studied the effects of PKD1 overexpression in a human adenocarcinoma breast cancer cell line, MCF-7 cells. We demonstrated that overexpression of PKD1 specifically promotes MCF-7 cell proliferation through accelerating G0/G1 to S phase transition of the cell cycle. Moreover, inhibition of endogenous PKD1 significantly reduced cell proliferation. Taken together, these results clearly strengthen the regulatory role of PKD1 in cell growth. We also demonstrated that overexpression of PKD1 specifically diminished serum- and anchorage-dependence for proliferation and survival in vitro and allowed MCF-7 cells to form tumors in vivo. Thus, all these data highlight the central role of PKD1 in biological processes which are hallmarks of malignant transformation. Analysis of two major signaling pathways implicated in MCF-7 cell proliferation showed that PKD1 overexpression significantly increased ERK1/2 phosphorylation state without affecting Akt phosphorylation. Moreover, PKD1 overexpression-stimulated cell proliferation and anchorage-independent growth were totally impaired by inhibition of the MEK/ERK kinase cascade. However, neither of these effects was affected by blocking the PI 3-kinase/Akt signaling pathway. Thus, the MEK/ERK signaling appears to be a determining pathway mediating the biological effects of PKD1 in MCF-7 cells. Taken together, all these data demonstrate that PKD1 overexpression increases the aggressiveness of MCF-7 breast cancer cells through enhancing their oncogenic

Concept Mapping

Science.gov (United States)

Technology & Learning, 2005

2005-01-01

Concept maps are graphical ways of working with ideas and presenting information. They reveal patterns and relationships and help students to clarify their thinking, and to process, organize and prioritize. Displaying information visually--in concept maps, word webs, or diagrams--stimulates creativity. Being able to think logically teaches…

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

Science.gov (United States)

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

2016-01-01

Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. PMID:26895960

Nuclear localization of Src-family tyrosine kinases is required for growth factor-induced euchromatinization

International Nuclear Information System (INIS)

Takahashi, Akinori; Obata, Yuuki; Fukumoto, Yasunori; Nakayama, Yuji; Kasahara, Kousuke; Kuga, Takahisa; Higashiyama, Yukihiro; Saito, Takashi; Yokoyama, Kazunari K.; Yamaguchi, Naoto

2009-01-01

Src-family kinases (SFKs), which participate in various signaling events, are found at not only the plasma membrane but also several subcellular compartments, including the nucleus. Nuclear structural changes are frequently observed during transcription, cell differentiation, senescence, tumorigenesis, and cell cycle. However, little is known about signal transduction in the alteration of chromatin texture. Here, we develop a pixel imaging method for quantitatively evaluating chromatin structural changes. Growth factor stimulation increases euchromatic hypocondensation and concomitant heterochromatic hypercondensation in G 1 phase, and the levels reach a plateau by 30 min, sustain for at least 5 h and return to the basal levels after 24 h. Serum-activated SFKs in the nucleus were more frequently detected in the euchromatin areas than the heterochromatin areas. Nuclear expression of kinase-active SFKs, but not unrelated Syk kinase, drastically increases both euchromatinization and heterochromatinization in a manner dependent on the levels of nuclear tyrosine phosphorylation. However, growth factor stimulation does not induce chromatin structural changes in SYF cells lacking SFKs, and reintroduction of one SFK member into SYF cells can, albeit insufficiently, induce chromatin structural changes. These results suggest that nuclear tyrosine phosphorylation by SFKs plays an important role in chromatin structural changes upon growth factor stimulation.

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.

Science.gov (United States)

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

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.

Directory of Open Access Journals (Sweden)

Ko-Jen Li

Full Text Available 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

Proliferation-stimulating effect of colony stimulating factor 2 on porcine trophectoderm cells is mediated by activation of phosphatidylinositol 3-kinase and extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase.

Directory of Open Access Journals (Sweden)

Wooyoung Jeong

Full Text Available Colony-stimulating factor 2 (CSF2, also known as granulocyte macrophage colony-stimulating factor, facilitates mammalian embryonic development and implantation. However, biological functions and regulatory mechanisms of action of porcine endometrial CSF2 in peri-implantation events have not been elucidated. The aim of present study was to determine changes in cellular activities induced by CSFs and to access CSF2-induced intracellular signaling in porcine primary trophectoderm (pTr cells. Differences in expression of CSF2 mRNA in endometrium from cyclic and pregnant gilts were evaluated. Endometrial CSF2 mRNA expression increases during the peri-implantation period, Days 10 to 14 of pregnancy, as compared to the estrous cycle. pTr cells obtained in Day 12 of pregnancy were cultured in the presence or absence of CSF2 (20 ng/ml and LY294002 (20 µM, U0126 (20 µM, rapamycin (20 nM, and SB203580 (20 µM. CSF2 in pTr cell culture medium at 20 ng/ml significantly induced phosphorylation of AKT1, ERK1/2, MTOR, p70RSK and RPS6 protein, but not STAT3 protein. Also, the PI3K specific inhibitor (LY294002 abolished CSF2-induced increases in p-ERK1/2 and p-MTOR proteins, as well as CSF2-induced phosphorylation of AKT1. Changes in proliferation and migration of pTr cells in response to CSF2 were examined in dose- and time-response experiments. CSF2 significantly stimulated pTr cell proliferation and, U0126, rapamycin and LY294002 blocked this CSF2-induced proliferation of pTr cells. Collectively, during the peri-implantation phase of pregnancy in pigs, endometrial CSF2 stimulates proliferation of trophectoderm cells by activation of the PI3K-and ERK1/2 MAPK-dependent MTOR signal transduction cascades.

Meliae cortex extract exhibits anti-allergic activity through the inhibition of Syk kinase in mast cells

International Nuclear Information System (INIS)

Lee, Jun Ho; Ko, Na Young; Kim, Nam Wook; Mun, Se Hwan; Kim, Jie Wan; Her, Erk; Kim, Bo Kyung; Seo, Dong Wan; Chang, Hyun Wook; Moon, Tae Chul; Han, Jeung Whan; Kim, Young Mi; Choi, Wahn Soo

2007-01-01

The anti-allergic action of various Oriental medicinal herbs was investigated using in vitro and in vivo experimental models. Of these extracts, the ethanol extract of Meliae cortex (MC) exhibited the most potent activity in mast cells; its IC 50 values were 29 ± 1.5 μg/ml for antigen stimulation and 57 ± 3.4 μg/ml for thapsigargin stimulation. It inhibited compound-48/80-induced systemic anaphylaxis by 52.9% at a dose of 300 mg/kg in mice; it also inhibited the expression of the proinflammatory mediator TNF-α. With regard to its mechanism of action, MC suppressed the activating phosphorylation of Syk, a key enzyme in mast-cell signaling processes and that of Akt in a dose-dependent manner. It also inhibited the MAP kinase ERK1/2, which is critical for the production of inflammatory cytokines in mast cells, as indicated by the suppression of the activating phosphorylation of ERK1/2. Taken together, these results suggest that the anti-allergic activity of MC may be due to the inhibition of histamine secretion and cytokine expression through the Syk inhibition in mast cells

Cyanidin-3-glucoside inhibits UVB-induced oxidative damage and inflammation by regulating MAP kinase and NF-κB signaling pathways in SKH-1 hairless mice skin

International Nuclear Information System (INIS)

Pratheeshkumar, Poyil; Son, Young-Ok; Wang, Xin; Divya, Sasidharan Padmaja; Joseph, Binoy; Hitron, John Andrew; Wang, Lei; Kim, Donghern; Yin, Yuanqin; Roy, Ram Vinod; Lu, Jian; Zhang, Zhuo; Wang, Yitao

2014-01-01

Skin cancer is one of the most commonly diagnosed cancers in the United States. Exposure to ultraviolet-B (UVB) radiation induces inflammation and photocarcinogenesis in mammalian skin. Cyanidin-3-glucoside (C3G), a member of the anthocyanin family, is present in various vegetables and fruits especially in edible berries, and displays potent antioxidant and anticarcinogenic properties. In this study, we have assessed the in vivo effects of C3G on UVB irradiation induced chronic inflammatory responses in SKH-1 hairless mice, a well-established model for UVB-induced skin carcinogenesis. Here, we show that C3G inhibited UVB-induced skin damage and inflammation in SKH-1 hairless mice. Our results indicate that C3G inhibited glutathione depletion, lipid peroxidation and myeloperoxidation in mouse skin by chronic UVB exposure. C3G significantly decreased the production of UVB-induced pro-inflammatory cytokines, such as IL-6 and TNF-α, associated with cutaneous inflammation. Likewise, UVB-induced inflammatory responses were diminished by C3G as observed by a remarkable reduction in the levels of phosphorylated MAP kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, C3G also decreased UVB-induced cyclooxygenase-2 (COX-2), PGE 2 and iNOS levels, which are well-known key mediators of inflammation and cancer. Treatment with C3G inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mice skin. Immunofluorescence assay revealed that topical application of C3G inhibited the expression of 8-hydroxy-2′-deoxyguanosine, proliferating cell nuclear antigen, and cyclin D1 in chronic UVB exposed mouse skin. Collectively, these data indicates that C3G can provide substantial protection against the adverse effects of UVB radiation by modulating UVB-induced MAP kinase and NF-κB signaling pathways. - Highlights: • C3G inhibited UVB-induced oxidative damage and inflammation. • C3G inhibited UVB-induced COX-2, iNOS and PGE 2 production. • C3G inhibited

Cyanidin-3-glucoside inhibits UVB-induced oxidative damage and inflammation by regulating MAP kinase and NF-κB signaling pathways in SKH-1 hairless mice skin

Energy Technology Data Exchange (ETDEWEB)

Pratheeshkumar, Poyil; Son, Young-Ok; Wang, Xin; Divya, Sasidharan Padmaja [Center for Research on Environmental Disease, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Joseph, Binoy [Spinal Cord and Brain Injury Research Center and Department of Physiology, University of Kentucky, Lexington, KY 40536-0509 (United States); Hitron, John Andrew; Wang, Lei [Center for Research on Environmental Disease, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Kim, Donghern [Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Yin, Yuanqin [Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Cancer Institute, The First Affiliated Hospital, China Medical University, Shenyang (China); Roy, Ram Vinod [Center for Research on Environmental Disease, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Lu, Jian [Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Institute of Life Sciences, Jiangsu University, Zhenjiang, Jiangsu 212013 (China); Zhang, Zhuo [Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536 (United States); Wang, Yitao [State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau (China); and others

2014-10-01

Skin cancer is one of the most commonly diagnosed cancers in the United States. Exposure to ultraviolet-B (UVB) radiation induces inflammation and photocarcinogenesis in mammalian skin. Cyanidin-3-glucoside (C3G), a member of the anthocyanin family, is present in various vegetables and fruits especially in edible berries, and displays potent antioxidant and anticarcinogenic properties. In this study, we have assessed the in vivo effects of C3G on UVB irradiation induced chronic inflammatory responses in SKH-1 hairless mice, a well-established model for UVB-induced skin carcinogenesis. Here, we show that C3G inhibited UVB-induced skin damage and inflammation in SKH-1 hairless mice. Our results indicate that C3G inhibited glutathione depletion, lipid peroxidation and myeloperoxidation in mouse skin by chronic UVB exposure. C3G significantly decreased the production of UVB-induced pro-inflammatory cytokines, such as IL-6 and TNF-α, associated with cutaneous inflammation. Likewise, UVB-induced inflammatory responses were diminished by C3G as observed by a remarkable reduction in the levels of phosphorylated MAP kinases, Erk1/2, p38, JNK1/2 and MKK4. Furthermore, C3G also decreased UVB-induced cyclooxygenase-2 (COX-2), PGE{sub 2} and iNOS levels, which are well-known key mediators of inflammation and cancer. Treatment with C3G inhibited UVB-induced nuclear translocation of NF-κB and degradation of IκBα in mice skin. Immunofluorescence assay revealed that topical application of C3G inhibited the expression of 8-hydroxy-2′-deoxyguanosine, proliferating cell nuclear antigen, and cyclin D1 in chronic UVB exposed mouse skin. Collectively, these data indicates that C3G can provide substantial protection against the adverse effects of UVB radiation by modulating UVB-induced MAP kinase and NF-κB signaling pathways. - Highlights: • C3G inhibited UVB-induced oxidative damage and inflammation. • C3G inhibited UVB-induced COX-2, iNOS and PGE{sub 2} production. • C3G

Activation of GABAB receptors inhibits protein kinase B /Glycogen Synthase Kinase 3 signaling

Directory of Open Access Journals (Sweden)

Lu Frances Fangjia

2012-11-01

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

The role of MAP4K3 in lifespan regulation of Caenorhabditiselegans

International Nuclear Information System (INIS)

Khan, Maruf H.; Hart, Matthew J.; Rea, Shane L.

2012-01-01

Highlights: ► Inhibition of MAP4K3 by RNAi leads to increased mean lifespan in Caenorhabditis elegans. ► Mutation in the citron homology domain of MAP4K3 leads to increased mean lifespan. ► 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 has 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.

The role of MAP4K3 in lifespan regulation of Caenorhabditiselegans

Energy Technology Data Exchange (ETDEWEB)

Khan, Maruf H. [Barshop Institute for Longevity and Aging Studies, Department of Physiology, University of Texas Health Science Center, San Antonio, TX 78240 (United States); Hart, Matthew J., E-mail: HartMJ@uthscsa.edu [Barshop Institute for Longevity and Aging Studies, Department of Molecular Medicine, University of Texas Health Science Center, San Antonio, TX 78240 (United States); Rea, Shane L., E-mail: reas3@uthscsa.edu [Barshop Institute for Longevity and Aging Studies, Department of Physiology, University of Texas Health Science Center, San Antonio, TX 78240 (United States)

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 has 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.

Role of Piezo Channels in Ultrasound-stimulated Dental Stem Cells.

Science.gov (United States)

Gao, Qianhua; Cooper, Paul R; Walmsley, A Damien; Scheven, Ben A

2017-07-01

Piezo1 and Piezo2 are mechanosensitive membrane ion channels. We hypothesized that Piezo proteins may play a role in transducing ultrasound-associated mechanical signals and activate downstream mitogen-activated protein kinase (MAPK) signaling processes in dental cells. In this study, the expression and role of Piezo channels were investigated in dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) after treatment with low-intensity pulsed ultrasound (LIPUS). Cell proliferation was evaluated by bromodeoxyuridine incorporation. Western blots were used to analyze the proliferating cell nuclear antigen as well as the transcription factors c-fos and c-jun. Enzyme-linked immunosorbent assay and Western blotting were used to determine the activation of MAPK after LIPUS treatment. Ruthenium red (RR), a Piezo ion channel blocker, was applied to determine the functional role of Piezo proteins in LIPUS-stimulated cell proliferation and MAPK signaling. Western blotting showed the presence of Piezo1 and Piezo2 in both dental cell types. LIPUS treatment significantly increased the level of the Piezo proteins in DPSCs after 24 hours; however, no significant effects were observed in PDLSCs. Treatment with RR significantly inhibited LIPUS-stimulated DPSC proliferation but not PDLSC proliferation. Extracellular signal-related kinase (ERK) 1/2 MAPK was consistently activated in DPSCs over a 24-hour time period after LIPUS exposure, whereas phosphorylated c-Jun N-terminal kinase and p38 mitogen-activated protein kinase MAPK were mainly increased in PDLSCs. RR affected MAPK signaling in both dental cell types with its most prominent effects on ERK1/2/MAPK phosphorylation levels; the significant inhibition of LIPUS-induced stimulation of ERK1/2 activation in DPSCs by RR suggests that stimulation of DPSC proliferation by LIPUS involves Piezo-mediated regulation of ERK1/2 MAPK signaling. This study for the first time supports the role of Piezo ion channels in

Cyclin dependent kinase 5 regulates endocytosis in nerve terminals via dynamin I phosphorylation

International Nuclear Information System (INIS)

Tan, T.C.; Hansra, G.; Calova, V.; Cousin, M.; Robinson, P.J.

2002-01-01

Full text: Synaptic vesicle endocytosis (SVE) in nerve terminals is essential for normal synaptic transmission and for memory retrieval. Dynamin I is a 96kDa nerve terminal phosphoprotein necessary for synaptic vesicle endocytosis in the nerve terminal. Dynamin I is dephosphorylated and rephosphorylated in a cyclical fashion with nerve terminal depolarisation and repolarisation. A number of kinases phosphorylate dynamin I in vitro including PKC, MAP kinase and cdc2. PKC phosphorylates dynamin in the proline rich domain on Ser 795 and is also thought to be the in vivo kinase for dynamin I. Another candidate is the neuron specific kinase cdk5, crucial for CNS development. The aim of this study is to identify the kinase which phosphorylates dynamin I in intact nerve terminals. Here we show that cyclin-dependent kinase 5 (cdk5) phosphorylates dynamin I in the proline-rich tail on Ser-774 or Ser-778. The phosphorylation of these sites but not Ser-795 also occurred in intact nerve terminals suggesting that cdk5 is the physiologically relevant enzyme for dynamin I. Synaptosomes prepared from rat brains (after cervical dislocations) and labelled with 32 Pi, were incubated with 100 M roscovitine (a selective inhibitor of cdks), 10 M Ro 31-8220 (a selective PKC inhibitor) and 100 M PD 98059 (a MEK kinase inhibitor). Dynamin rephosphorylation during repolarisation was reduced in synaptosomes treated with roscovitine and Ro 38-8220 but not in synaptosomes treated with PD 98059. Fluorimetric experiments on intact synaptosomes utilising FM-210 (a fluorescent dye) indicate that endocytosis was reduced in synaptosomes treated with 100 M roscovitine. Our results suggest that dynamin phosphorylation in intact nerve terminals may not be regulated by PKC or MAP kinase and that dynamin phosphorylation by cdk5 may regulate endocytosis. Copyright (2002) Australian Neuroscience Society

Insulin, concanavalin A, EGF, IFG-I and vanadate activate de novo phosphatidic acid and diacylglycerol synthesis, C-kinase, and glucose transport in BC3H-1 myocytes

International Nuclear Information System (INIS)

Cooper, D.R.; Hernandez, H.; Konda, T.S.; Standaert, M.S.; Pollet, R.J.; Farese, R.V.

1987-01-01

The authors have reported that insulin stimulates de novo synthesis of phosphatidic acid (PA) which is metabolized directly to diacylglycerol (DG) in BS3H-1 myocytes; this is accompanied by increases in C-kinase activity in membrane and cytosolic extracts. This pathway may be involved in stimulating glucose transport and other metabolic processes. In this study, the authors have compared the effects of concanavalin A, EGF, IGF-I and sodium orthovanadate to insulin on PA/DG synthesis, C-kinase activity and glucose transport. All were found to be effective in stimulating glucose transport. Additionally, all activators rapidly increased the incorporation of [ 3 H]glycerol into DG and total glycerolipids, although none were as effective as insulin, which increased [ 3 H]DG 400% in 1 minute. Increased incorporation into phospholipids and triacylglycerols and to a lesser extent monoacylglycerol was also noted. They examined effects of concanavalin A and EGF on C-kinase activity and found that both agonists, like insulin, increase C-kinase activity in cytosolic and/or membrane fractions. Their findings raise the possibility that activation of receptors having associated tyrosine kinase activity may provoke some cellular responses through de novo PA/GD synthesis and C-kinase activation

Presenilin dependence of phospholipase C and protein kinase C signaling

DEFF Research Database (Denmark)

Dehvari, Nodi; Cedazo-Minguez, Angel; Isacsson, Ola

2007-01-01

-stimulated phospholipase C (PLC) activity which was gamma-secretase dependent. To further evaluate the dependence of PLC on PSs we measured PLC activity and the activation of variant protein kinase C (PKC) isoforms in mouse embryonic fibroblasts (MEFs) lacking either PS1, PS2, or both. PLC activity and PKCalpha...

Diacylglycerol kinase zeta negatively regulates CXCR4-stimulated T lymphocyte firm arrest to ICAM-1 under shear flow.

Science.gov (United States)

Lee, Dooyoung; Kim, Jiyeon; Beste, Michael T; Koretzky, Gary A; Hammer, Daniel A

2012-06-01

T lymphocyte arrest within microvasculature is an essential process in immune surveillance and the adaptive immune response. Integrins and chemokines coordinately regulate when and where T cells stop under flow via chemokine-triggered inside-out activation of integrins. Diacylglycerol kinases (DGKs) regulate the levels of diacylglycerol (DAG) which in turn determine the activation of guanine nucleotide exchange factors (GEFs) and Ras proximity 1 (Rap1) molecules crucial to the activation of integrin lymphocyte function-associated antigen 1 (LFA-1). However, how the level of DGK regulates chemokine-stimulated LFA-1-mediated T cell arrest under flow is unknown. Using a combination of experiment and computational modeling, we demonstrate that DGKζ is a crucial regulator of CXCL12-triggered T cell arrest on surfaces presenting inter-cellular adhesion molecule 1 (ICAM-1). Using flow chamber assays, we found that the deficiency of DGKζ in T cells significantly increased firm arrest to ICAM-1-coated substrates and shortened the time to stop without altering the rolling velocity. These results suggest that DGKζ levels affect LFA-1-mediated T cell firm arrest, but not P-selectin-mediated rolling during CXCL12 stimulation. We accurately simulated the role of DGKζ in firm arrest of T cells computationally using an Integrated-Signaling Adhesive Dynamics (ISAD). In the absence of DGK catalytic reaction, the model cells rolled for a significantly shorter time before arrest, compared to when DGK molecules were present. Predictions of our model for T cell arrest quantitatively match experimental results. Overall these results demonstrate that DGKζ is a negative regulator of CXCL12-triggered inside-out activation of LFA-1 and firm adhesion of T cells under shear flow.

MAP kinase pathways and calcitonin influence CD44 alternate isoform expression in prostate cancer cells

International Nuclear Information System (INIS)

Robbins, Eric W; Travanty, Emily A; Yang, Kui; Iczkowski, Kenneth A

2008-01-01

Dysregulated expression and splicing of cell adhesion marker CD44 is found in many types of cancer. In prostate cancer (PC) specifically, the standard isoform (CD44s) has been found to be downregulated compared with benign tissue whereas predominant variant isoform CD44v7-10 is upregulated. Mitogen-activated protein kinase pathways and paracrine calcitonin are two common factors linked to dysregulated expression and splicing of CD44 in cancer. Calcitonin has been found to increase proliferation and invasion in PC acting through the protein kinase A pathway. In androgen-independent PC with known high CD44v7-10 expression, CD44 total and CD44v7-10 RNA or protein were assessed in response to exogenous and endogenous calcitonin and to inhibitors of protein kinase A, MEK, JNK, or p38 kinase. Benign cells and calcitonin receptor-negative PC cells were also tested. MEK or p38 but not JNK reduced CD44 total RNA by 40%–65% in cancer and benign cells. Inhibition of protein kinase A reduced CD44 total and v7-10 protein expression. In calcitonin receptor-positive cells only, calcitonin increased CD44 variant RNA and protein by 3 h and persisting to 48 h, apparently dependent on an uninhibited p38 pathway. Cells with constitutive CT expression showed an increase in CD44v7-10 mRNA but a decrease in CD44 total RNA. The MEK pathway increases CD44 RNA, while calcitonin, acting through the protein kinase A and p38 pathway, facilitates variant splicing. These findings could be used in the formulation of therapeutic methods for PC targeting CD44 alternate splicing

Inhibition of autophagic proteolysis by inhibitors of phosphoinositide 3-kinase can interfere with the regulation of glycogen synthesis in isolated hepatocytes

NARCIS (Netherlands)

Dubbelhuis, Peter F.; van Sluijters, Daphne A.; Blommaart, Edward F. C.; Gustafson, Lori A.; van Woerkom, George M.; Herling, Andreas W.; Burger, Hans-Joerg; Meijer, Alfred J.

2002-01-01

Amino acid-induced cell swelling stimulates conversion of glucose into glycogen in isolated hepatocytes. Activation of glycogen synthase (GS) phosphatase, caused by the fall in intracellular chloride accompanying regulatory volume decrease, and activation of phosphoinositide 3-kinase (PI 3-kinase),

Unbinding Kinetics of a p38 MAP Kinase Type II Inhibitor from Metadynamics Simulations.

Science.gov (United States)

Casasnovas, Rodrigo; Limongelli, Vittorio; Tiwary, Pratyush; Carloni, Paolo; Parrinello, Michele

2017-04-05

Understanding the structural and energetic requisites of ligand binding toward its molecular target is of paramount relevance in drug design. In recent years, atomistic free energy calculations have proven to be a valid tool to complement experiments in characterizing the thermodynamic and kinetic properties of protein/ligand interaction. Here, we investigate, through a recently developed metadynamics-based protocol, the unbinding mechanism of an inhibitor of the pharmacologically relevant target p38 MAP kinase. We provide a thorough description of the ligand unbinding pathway identifying the most stable binding mode and other thermodynamically relevant poses. From our simulations, we estimated the unbinding rate as k off = 0.020 ± 0.011 s -1 . This is in good agreement with the experimental value (k off = 0.14 s -1 ). Next, we developed a Markov state model that allowed identifying the rate-limiting step of the ligand unbinding process. Our calculations further show that the solvation of the ligand and that of the active site play crucial roles in the unbinding process. This study paves the way to investigations on the unbinding dynamics of more complex p38 inhibitors and other pharmacologically relevant inhibitors in general, demonstrating that metadynamics can be a powerful tool in designing new drugs with engineered binding/unbinding kinetics.

Partial purification and characterization of a Ca(2+)-dependent protein kinase from pea nuclei

Science.gov (United States)

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.

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

Science.gov (United States)

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

2016-04-15

Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Fast periodic stimulation (FPS): a highly effective approach in fMRI brain mapping.

Science.gov (United States)

Gao, Xiaoqing; Gentile, Francesco; Rossion, Bruno

2018-03-03

Defining the neural basis of perceptual categorization in a rapidly changing natural environment with low-temporal resolution methods such as functional magnetic resonance imaging (fMRI) is challenging. Here, we present a novel fast periodic stimulation (FPS)-fMRI approach to define face-selective brain regions with natural images. Human observers are presented with a dynamic stream of widely variable natural object images alternating at a fast rate (6 images/s). Every 9 s, a short burst of variable face images contrasting with object images in pairs induces an objective face-selective neural response at 0.111 Hz. A model-free Fourier analysis achieves a twofold increase in signal-to-noise ratio compared to a conventional block-design approach with identical stimuli and scanning duration, allowing to derive a comprehensive map of face-selective areas in the ventral occipito-temporal cortex, including the anterior temporal lobe (ATL), in all individual brains. Critically, periodicity of the desired category contrast and random variability among widely diverse images effectively eliminates the contribution of low-level visual cues, and lead to the highest values (80-90%) of test-retest reliability in the spatial activation map yet reported in imaging higher level visual functions. FPS-fMRI opens a new avenue for understanding brain function with low-temporal resolution methods.

Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

Science.gov (United States)

Reedijk, M; Liu, X; van der Geer, P; Letwin, K; Waterfield, M D; Hunter, T; Pawson, T

1992-01-01

Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha. Images PMID:1314163

Oryza sativa (Rice) Hull Extract Inhibits Lipopolysaccharide-Induced Inflammatory Response in RAW264.7 Macrophages by Suppressing Extracellular Signal-regulated Kinase, c-Jun N-terminal Kinase, and Nuclear Factor-κB Activation.

Science.gov (United States)

Ha, Sang Keun; Sung, Jeehye; Choi, Inwook; Kim, Yoonsook

2016-01-01

Rice ( Oryza sativa ) is a major cereal crop in many Asian countries and an important staple food source. Rice hulls have been reported to possess antioxidant activities. In this study, we evaluated the antiinflammatory effects of rice hull extract and associated signal transduction mechanisms in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We found that rice hull extract inhibited nitric oxide (NO) and prostaglandin E 2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively. The release of interleukin-1β and tumor necrosis factor-α was also reduced in a dose-dependent manner. Furthermore, rice hull extract attenuated the activation of nuclear factor-kappa B (NF-κB), as well as the phosphorylation of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), in LPS-stimulated RAW264.7 cells. This suggests that rice hull extract decreases the production of inflammatory mediators by downregulating ERK and JNK and the NF-κB signal pathway in RAW 264.7 cells. Rice hull extract inhibits the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages.Rice hull extract inhibited nitric oxide and prostaglandin E 2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively.Rice hull extract exerted anti-inflammatory effect through inhibition of nuclear factor-kappa B, extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling pathways.Rice hull extract may provide a potential therapeutic approach for inflammatory diseases. Abbreviations used: COX-2: cyclooxygenase-2, ERK: extracellular signal-regulated kinase, IκB: inhibitory kappa B, IL-1β: interleukin-1β, iNOS: inducible NO synthase, JNK: c-Jun N-terminal kinase, LPS: lipopolysaccharide, MAPKs: mitogen-activated protein kinases, NF-κB: nuclear factor-κB, NO: nitric oxide, PGE2: prostaglandin E2, RHE: rice hull extract, ROS: reactive oxygen species

AMP-activated kinase mediates adipose stem cell-stimulated neuritogenesis of PC12 cells.

Science.gov (United States)

Tan, B; Luan, Z; Wei, X; He, Y; Wei, G; Johnstone, B H; Farlow, M; Du, Y

2011-05-05

Adipose tissue stroma contains a population of mesenchymal stem cells, which support repair of damaged tissues through the protective effects of secreted trophic factors. Neurotrophic factors, including nerve growth factor (NGF) have been identified in media collected from cultured adipose-derived stem cells (ASC). We previously demonstrated that administration of cell-free ASC conditioned medium (ASC-CM) at 24 h after injury reduced lesion volume and promoted functional recovery in a rat model of neonatal brain hypoxic-ischemic (HI) injury. The timing of administration well after the peak in neural cell apoptosis in the affected region suggests that regeneration of lost neurons is promoted by factors in ASC-CM. In this study, we determined which of the factors in ASC-CM could induce neurogenesis by testing the ability of the mixture, either whole or after inactivating specific components, to stimulate neurite outgrowth in vitro using the neurogenic cell line PC12. Neuritogenesis in PC12 cells treated with ASC-CM was observed at a level comparable to that observed with purified recombinant NGF. It was observed that NGF in ASC-CM was mainly responsible for inducing PC12 cell neuritogenesis. Interestingly, both ASC-CM and NGF induced PC12 cell neuritogenesis through activation of the AMP-activated kinase (AMPK) pathway which is the central protein involved in controlling many critical functions in response to changes in the cellular energy status. Pharmacological and genetic inhibition of AMPK activity greatly reduced neuritogenesis in PC12 cells. These results suggest that, in addition to possessing neuroprotective properties, ASC-CM mediates repair of damaged tissues through inducing neuronal differentiation via NGF-induced AMPK activation. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Ascofuranone stimulates expression of adiponectin and peroxisome proliferator activated receptor through the modulation of mitogen activated protein kinase family members in 3T3-L1, murine pre-adipocyte cell line

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Chang, Young-Chae, E-mail: ycchang@cu.ac.kr [Research Institute of Biomedical Engineering and Department of Medicine, Catholic University of Daegu School of Medicine, Daegu 705-718 (Korea, Republic of); Cho, Hyun-Ji, E-mail: hjcho.dr@gmail.com [Research Institute of Biomedical Engineering and Department of Medicine, Catholic University of Daegu School of Medicine, Daegu 705-718 (Korea, Republic of)

2012-06-08

Highlights: Black-Right-Pointing-Pointer Ascofuranone increases expression of adiponectin and PPAR{gamma}. Black-Right-Pointing-Pointer Inhibitors for MEK and JNK increased the expression of adiponectin and PPAR{gamma}. Black-Right-Pointing-Pointer Ascofuranone significantly suppressed phosho-ERK, while increasing phospho-p38. -- Abstract: Ascofuranone, an isoprenoid antibiotic, was originally isolated as a hypolipidemic substance from a culture broth of the phytopathogenic fungus, Ascochyta visiae. Adiponectin is mainly synthesized by adipocytes. It relieves insulin resistance by decreasing the plasma triglycerides and improving glucose uptake, and has anti-atherogenic properties. Here, we found that ascofuranone increases expression of adiponectin and PPAR{gamma}, a major transcription factor for adiponectin, in 3T3-L1, murine pre-adipocytes cell line, without promoting accumulation of lipid droplets. Ascofuranone induced expression of adiponectin, and increases the promoter activity of adiponectin and PPRE, PPAR response element, as comparably as a PPAR{gamma} agonist, rosiglitazone, that stimulates lipid accumulation in the preadipocyte cell line. Moreover, inhibitors for MEK and JNK, like ascofuranone, considerably increased the expression of adiponectin and PPAR{gamma}, while a p38 inhibitor significantly suppressed. Ascofuranone significantly suppressed ERK phosphorylation, while increasing p38 phosphorylation, during adipocyte differentiation program. These results suggest that ascofuranone regulates the expression of adiponectin and PPAR{gamma} through the modulation of MAP kinase family members.

Protein kinase C-mediated ATP stimulation of Na(+)-ATPase activity in LLC-PK1 cells involves a P2Y2 and/or P2Y4 receptor.

Science.gov (United States)

Wengert, M; Ribeiro, M C; Abreu, T P; Coutinho-Silva, R; Leão-Ferreira, L R; Pinheiro, A A S; Caruso-Neves, C

2013-07-15

ATP-activated P2Y receptors play an important role in renal sodium excretion. The aim of this study was to evaluate the modulation of ATPase-driven sodium reabsorption in the proximal tubule by ATP or adenosine (Ado). LLC-PK1 cells, a model of porcine proximal tubule cells, were used. ATP (10(-6)M) or Ado (10(-6)M) specifically stimulated Na(+)-ATPase activity without any changes in (Na(+)+K(+))-ATPase activity. Our results show that the Ado effect is mediated by its conversion to ATP. Furthermore, it was observed that the effect of ATP was mimicked by UTP, ATPγS and 2-thio-UTP, an agonist of P2Y2 and P2Y4 receptors. In addition, ATP-stimulated Na(+)-ATPase activity involves protein kinase C (PKC). Our results indicate that ATP-induced stimulation of proximal tubule Na(+)-ATPase activity is mediated by a PKC-dependent P2Y2 and/or P2Y4 pathway. These findings provide new perspectives on the role of the effect of P2Y-mediated extracellular ATP on renal sodium handling. Copyright © 2013 Elsevier Inc. All rights reserved.

Activation of the ATR kinase by the RPA-binding protein ETAA1

DEFF Research Database (Denmark)

Haahr, Peter; Hoffmann, Saskia; Tollenaere, Maxim A X

2016-01-01

Activation of the ATR kinase following perturbations to DNA replication relies on a complex mechanism involving ATR recruitment to RPA-coated single-stranded DNA via its binding partner ATRIP and stimulation of ATR kinase activity by TopBP1. Here, we discovered an independent ATR activation pathway...... in vertebrates, mediated by the uncharacterized protein ETAA1 (Ewing's tumour-associated antigen 1). Human ETAA1 accumulates at DNA damage sites via dual RPA-binding motifs and promotes replication fork progression and integrity, ATR signalling and cell survival after genotoxic insults. Mechanistically...

Accurate calculation of mutational effects on the thermodynamics of inhibitor binding to p38α MAP kinase: a combined computational and experimental study.

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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

Cytochalasin B augments diacylglycerol levels in stimulated neutrophils

International Nuclear Information System (INIS)

Honeycutt, P.J.; Niedel, J.

1986-01-01

Diacylglycerol (DG) has gained wide acceptance as an important second messenger and intracellular activator of protein kinase C, but few studies have directly measured DG levels in cells or tissues. The authors measured the mass of DG in lipid extracts from normal human neutrophils by quantitative conversion of DG to [ 32 P] phosphatidic acid using E. coli DG kinase. The chemotactic peptide N-formyl-Met-Leu-Phe (fMLP) stimulated a transient 30% rise in DG that was maximal at 30 to 45 sec and returned to the basal level of 150 picomoles/10 7 cells by one min. This initial peak was followed by a slower, more prolonged 30% increase in DG that was maximal at 20 min. Cytochalasin B (CB) augments many biological responses of neutrophils to fMLP, including superoxide production and lysosomal enzyme release. CB alone caused no change in basal DG levels, but in the presence of CB, fMLP stimulated a rapid, large, and persistent DG response. DG levels increased to 290% of basal at 5 min with a t1/2 = 45 sec. The DG response to fMLP was maximal at 5 to 10 μm CB and 1 μM fMLP. The DG response to optimal fMLP and CB concentrations was decreased 40% by an fMLP antagonist, and no response was elicited by an inactive fMLP analog and CB. Protein kinase C has been implicated in fMLP-stimulated superoxide production and lysosomal enzyme release. These data are consistent with the hypothesis that CB may effect augmentation of biological responses by increasing DG levels

Novel mechanisms of sildenafil in pulmonary hypertension involving cytokines/chemokines, MAP kinases and Akt.

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Tamas Kiss

Full Text Available Pulmonary arterial hypertension (PH is associated with high mortality due to right ventricular failure and hypoxia, therefore to understand the mechanism by which pulmonary vascular remodeling initiates these processes is very important. We used a well-characterized monocrotaline (MCT-induced rat PH model, and analyzed lung morphology, expression of cytokines, mitogen-activated protein kinase (MAPK phosphorylation, and phosphatidylinositol 3-kinase-Akt (PI-3k-Akt pathway and nuclear factor (NF-κB activation in order to elucidate the mechanisms by which sildenafil's protective effect in PH is exerted. Besides its protective effect on lung morphology, sildenafil suppressed multiple cytokines involved in neutrophil and mononuclear cells recruitment including cytokine-induced neutrophil chemoattractant (CINC-1, CINC-2α/β, tissue inhibitor of metalloproteinase (TIMP-1, interleukin (IL-1α, lipopolysaccharide induced CXC chemokine (LIX, monokine induced by gamma interferon (MIG, macrophage inflammatory protein (MIP-1α, and MIP-3α. NF-κB activation and phosphorylation were also attenuated by sildenafil. Furthermore, sildenafil reduced extracellular signal-regulated kinase (ERK1/2 and p38 MAPK activation while enhanced activation of the cytoprotective Akt pathway in PH. These data suggest a beneficial effect of sildenafil on inflammatory and kinase signaling mechanisms that substantially contribute to its protective effects, and may have potential implications in designing future therapeutic strategies in the treatment of pulmonary hypertension.

Crystal structure of an SH2-kinase construct of c-Abl and effect of the SH2 domain on kinase activity.

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Lorenz, Sonja; Deng, Patricia; Hantschel, Oliver; Superti-Furga, Giulio; Kuriyan, John

2015-06-01

Constitutive activation of the non-receptor tyrosine kinase c-Abl (cellular Abelson tyrosine protein kinase 1, Abl1) in the Bcr (breakpoint cluster region)-Abl1 fusion oncoprotein is the molecular cause of chronic myeloid leukaemia (CML). Recent studies have indicated that an interaction between the SH2 (Src-homology 2) domain and the N-lobe (N-terminal lobe) of the c-Abl kinase domain (KD) has a critical role in leukaemogenesis [Grebien et al. (2011) Cell 147, 306-319; Sherbenou et al. (2010) Blood 116, 3278-3285]. To dissect the structural basis of this phenomenon, we studied c-Abl constructs comprising the SH2 and KDs in vitro. We present a crystal structure of an SH2-KD construct bound to dasatinib, which contains the relevant interface between the SH2 domain and the N-lobe of the KD. We show that the presence of the SH2 domain enhances kinase activity moderately and that this effect depends on contacts in the SH2/N-lobe interface and is abrogated by specific mutations. Consistently, formation of the interface decreases slightly the association rate of imatinib with the KD. That the effects are small compared with the dramatic in vivo consequences suggests an important function of the SH2-N-lobe interaction might be to help disassemble the auto-inhibited conformation of c-Abl and promote processive phosphorylation, rather than substantially stimulate kinase activity.

Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation.

Science.gov (United States)

Miralem, Tihomir; Lerner-Marmarosh, Nicole; Gibbs, Peter E M; Jenkins, Jermaine L; Heimiller, Chelsea; Maines, Mahin D

2016-08-01

Biliverdin reductase A (BVR) and Akt isozymes have overlapping pleiotropic functions in the insulin/PI3K/MAPK pathway. Human BVR (hBVR) also reduces the hemeoxygenase activity product biliverdin to bilirubin and is directly activated by insulin receptor kinase (IRK). Akt isoenzymes (Akt1-3) are downstream of IRK and are activated by phosphatidylinositol-dependent kinase 1 (PDK1) phosphorylating T(308) before S(473) autophosphorylation. Akt (RxRxxSF) and PDK1 (RFxFPxFS) binding motifs are present in hBVR. Phosphorylation of glycogen synthase kinase 3 (GSK3) isoforms α/β by Akts inhibits their activity; nonphosphorylated GSK3β inhibits activation of various genes. We examined the role of hBVR in PDK1/Akt1/GSK3 signaling and Akt1 in hBVR phosphorylation. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. hBVR and Akt1 coimmunoprecipitated, and in-cell Förster resonance energy transfer (FRET) and glutathione S-transferase pulldown analyses identified Akt1 pleckstrin homology domain as the interactive domain. hBVR activates phosphorylation of Akt1 at S(473) independent of hBVR's kinase competency. Site-directed mutagenesis, mass spectrometry, and kinetic analyses identified S(230) in hBVR (225)RNRYLSF sequence as the Akt1 target. Underlined amino acids are the essential residues of the signaling motifs. In cells, hBVR-activated Akt1 increased both GSK3α/β and forkhead box of the O class transcription class 3 (FoxO3) phosphorylation and inhibited total GSK3 activity; depletion of hBVR released inhibition and stimulated glucose uptake. Immunoprecipitation analysis showed that PDK1 and hBVR interact through hBVR's PDK1 binding (161)RFGFPAFS motif and formation of the PDK1/hBVR/Akt1 complex. sihBVR blocked complex formation. Findings identify hBVR as a previously unknown coactivator of Akt1 and as a key mediator of Akt1/GSK3 pathway, as well as define a key role for hBVR in Akt1 activation by PDK1.-Miralem, T., Lerner

Extracellular signal regulated kinase 5 mediates signals triggered by the novel tumor promoter palytoxin

International Nuclear Information System (INIS)

Charlson, Aaron T.; Zeliadt, Nicholette A.; Wattenberg, Elizabeth V.

2009-01-01

Palytoxin is classified as a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type skin tumor because it does not bind to or activate protein kinase C. Palytoxin is thus a novel tool for investigating alternative signaling pathways that may affect carcinogenesis. We previously showed that palytoxin activates three major members of the mitogen activated protein kinase (MAPK) family, extracellular signal regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. Here we report that palytoxin also activates another MAPK family member, called ERK5, in HeLa cells and in keratinocytes derived from initiated mouse skin (308 cells). By contrast, TPA does not activate ERK5 in these cell lines. The major cell surface receptor for palytoxin is the Na+,K+-ATPase. Accordingly, ouabain blocked the ability of palytoxin to activate ERK5. Ouabain alone did not activate ERK5. ERK5 thus represents a divergence in the signaling pathways activated by these two agents that bind to the Na+,K+-ATPase. Cycloheximide, okadaic acid, and sodium orthovanadate did not mimic the effect of palytoxin on ERK5. These results indicate that the stimulation of ERK5 by palytoxin is not simply due to inhibition of protein synthesis or inhibition of serine/threonine or tyrosine phosphatases. Therefore, the mechanism by which palytoxin activates ERK5 differs from that by which it activates ERK1/2, JNK, and p38. Finally, studies that used pharmacological inhibitors and shRNA to block ERK5 action indicate that ERK5 contributes to palytoxin-stimulated c-Fos gene expression. These results suggest that ERK5 can act as an alternative mediator for transmitting diverse tumor promoter-stimulated signals.

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

Directory of Open Access Journals (Sweden)

Tariq Hussain

2018-01-01

Full Text Available 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.

Membrane depolarization-induced RhoA/Rho-associated kinase activation and sustained contraction of rat caudal arterial smooth muscle involves genistein-sensitive tyrosine phosphorylation

Science.gov (United States)

Mita, Mitsuo; Tanaka, Hitoshi; Yanagihara, Hayato; Nakagawa, Jun-ichi; Hishinuma, Shigeru; Sutherland, Cindy; Walsh, Michael P.; Shoji, Masaru

2013-01-01

Rho-associated kinase (ROK) activation plays an important role in K+-induced contraction of rat caudal arterial smooth muscle (Mita et al., Biochem J. 2002; 364: 431–40). The present study investigated a potential role for tyrosine kinase activity in K+-induced RhoA activation and contraction. The non-selective tyrosine kinase inhibitor genistein, but not the src family tyrosine kinase inhibitor PP2, inhibited K+-induced sustained contraction (IC50 = 11.3 ± 2.4 µM). Genistein (10 µM) inhibited the K+-induced increase in myosin light chain (LC20) phosphorylation without affecting the Ca2+ transient. The tyrosine phosphatase inhibitor vanadate induced contraction that was reversed by genistein (IC50 = 6.5 ± 2.3 µM) and the ROK inhibitor Y-27632 (IC50 = 0.27 ± 0.04 µM). Vanadate also increased LC20 phosphorylation in a genistein- and Y-27632-dependent manner. K+ stimulation induced translocation of RhoA to the membrane, which was inhibited by genistein. Phosphorylation of MYPT1 (myosin-targeting subunit of myosin light chain phosphatase) was significantly increased at Thr855 and Thr697 by K+ stimulation in a genistein- and Y-27632-sensitive manner. Finally, K+ stimulation induced genistein-sensitive tyrosine phosphorylation of proteins of ∼55, 70 and 113 kDa. We conclude that a genistein-sensitive tyrosine kinase, activated by the membrane depolarization-induced increase in [Ca2+]i, is involved in the RhoA/ROK activation and sustained contraction induced by K+. Ca2+ sensitization, myosin light chain phosphatase, RhoA, Rho-associated kinase, tyrosine kinase PMID:24133693

The Insect Neuropeptide PTTH Activates Receptor Tyrosine Kinase Torso to Initiate Metamorphosis

DEFF Research Database (Denmark)

Rewitz, Kim; Yamanaka, Naoki; Gilbert, Lawrence

2009-01-01

Holometabolous insects undergo complete metamorphosis to become sexually mature adults. Metamorphosis is initiated by brain-derived prothoracicotropic hormone (PTTH), which stimulates the production of the molting hormone ecdysone via an incompletely defined signaling pathway. Here we demonstrate...... in the prothoracic gland (PG), and its loss phenocopies the removal of PTTH. The activation of Torso by PTTH stimulates extracellular signal–regulated kinase (ERK) phosphorylation, and the loss of ERK in the PG phenocopies the loss of PTTH and Torso. We conclude that PTTH initiates metamorphosis by activation...

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

Science.gov (United States)

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.

Kinase fusions are frequent in Spitz tumors and spitzoid melanomas

Science.gov (United States)

Esteve-Puig, Rosaura; Botton, Thomas; Yeh, Iwei; Lipson, Doron; Otto, Geoff; Brennan, Kristina; Murali, Rajmohan; Garrido, Maria; Miller, Vincent A.; Ross, Jeffrey S; Berger, Michael F.; Sparatta, Alyssa; Palmedo, Gabriele; Cerroni, Lorenzo; Busam, Klaus J.; Kutzner, Heinz; Cronin, Maureen T; Stephens, Philip J; Bastian, Boris C.

2014-01-01

Spitzoid neoplasms are a group of melanocytic tumors with distinctive histopathologic features. They include benign tumors (Spitz nevi), malignant tumors (spitzoid melanomas), and tumors with borderline histopathologic features and uncertain clinical outcome (atypical Spitz tumors). Their genetic underpinnings are poorly understood, and alterations in common melanoma-associated oncogenes are typically absent. Here we show that spitzoid neoplasms harbor kinase fusions of ROS1 (17%), NTRK1 (16%), ALK (10%), BRAF (5%), and RET (3%) in a mutually exclusive pattern. The chimeric proteins are constitutively active, stimulate oncogenic signaling pathways, are tumorigenic, and are found in the entire biologic spectrum of spitzoid neoplasms, including 55% of Spitz nevi, 56% of atypical Spitz tumors, and 39% of spitzoid melanomas. Kinase inhibitors suppress the oncogenic signaling of the fusion proteins in vitro. In summary, kinase fusions account for the majority of oncogenic aberrations in spitzoid neoplasms, and may serve as therapeutic targets for metastatic spitzoid melanomas. PMID:24445538

Expression of human choline kinase in NIH 3T3 fibroblasts increases the mitogenic potential of insulin and insulin-like growth factor I.

Science.gov (United States)

Chung, T; Huang, J S; Mukherjee, J J; Crilly, K S; Kiss, Z

2000-05-01

In mammalian cells, growth factors, oncogenes, and carcinogens stimulate phosphocholine (PCho) synthesis by choline kinase (CK), suggesting that PCho may regulate cell growth. To validate the role of PCho in mitogenesis, we determined the effects of insulin, insulin-like growth factor I (IGF-I), and other growth factors on DNA synthesis in NIH 3T3 fibroblast sublines highly expressing human choline kinase (CK) without increasing phosphatidylcholine synthesis. In serum-starved CK expressor cells, insulin and IGF-I stimulated DNA synthesis, p70 S6 kinase (p70 S6K) activity, phosphatidylinositol 3-kinase (PI3K) activity, and activating phosphorylation of p42/p44 mitogen-activated protein kinases (MAPK) to greater extents than in the corresponding vector control cells. Furthermore, the CK inhibitor hemicholinium-3 (HC-3) inhibited insulin- and IGF-I-induced DNA synthesis in the CK overexpressors, but not in the vector control cells. The results indicate that high cellular levels of PCho potentiate insulin- and IGF-I-induced DNA synthesis by MAPK- and p70 S6K-regulated mechanisms.

A review of monopolar motor mapping and a comprehensive guide to continuous dynamic motor mapping for resection of motor eloquent brain tumors.

Science.gov (United States)

Schucht, P; Seidel, K; Jilch, A; Beck, J; Raabe, A

2017-06-01

Monopolar mapping of motor function differs from the most commonly used method of intraoperative mapping, i.e. bipolar direct electrical stimulation at 50-60Hz (Penfield technique mapping). Most importantly, the monopolar probe emits a radial, homogenous electrical field different to the more focused inter-tip bipolar electrical field. Most users combine monopolar stimulation with the short train technique, also called high frequency stimulation, or train-of-five techniques. It consists of trains of four to nine monopolar rectangular electrical pulses of 200-500μs pulse length with an inter stimulus interval of 2-4msec. High frequency short train stimulation triggers a time-locked motor-evoked potential response, which has a defined latency and an easily quantifiable amplitude. In this way, motor thresholds might be used to evaluate a current-to-distance relation. The homogeneous electrical field and the current-to-distance approximation provide the surgeon with an estimate of the remaining distance to the corticospinal tract, enabling the surgeon to adjust the speed of resection as the corticospinal tract is approached. Furthermore, this stimulation paradigm is associated with a lower incidence of intraoperative seizures, allowing continuous stimulation. Hence, monopolar mapping is increasingly used as part of a strategy of continuous dynamic mapping: ergonomically integrated into the surgeon's tools, the monopolar probe reliably provides continuous/uninterrupted feedback on motor function. As part of this strategy, motor mapping is not any longer a time consuming interruption of resection but rather a radar-like, real-time information system on the spatial relationship of the current resection site to eloquent motor structures. Copyright © 2017. Published by Elsevier Masson SAS.

Construction and use of a zebrafish heart voltage and calcium optical mapping system, with integrated electrocardiogram and programmable electrical stimulation

Science.gov (United States)

Lin, Eric; Craig, Calvin; Lamothe, Marcel; Sarunic, Marinko V.; Beg, Mirza Faisal

2015-01-01

Zebrafish are increasingly being used as a model of vertebrate cardiology due to mammalian-like cardiac properties in many respects. The size and fecundity of zebrafish make them suitable for large-scale genetic and pharmacological screening. In larger mammalian hearts, optical mapping is often used to investigate the interplay between voltage and calcium dynamics and to investigate their respective roles in arrhythmogenesis. This report outlines the construction of an optical mapping system for use with zebrafish hearts, using the voltage-sensitive dye RH 237 and the calcium indicator dye Rhod-2 using two industrial-level CCD cameras. With the use of economical cameras and a common 532-nm diode laser for excitation, the rate dependence of voltage and calcium dynamics within the atrial and ventricular compartments can be simultaneously determined. At 140 beats/min, the atrial action potential duration was 36 ms and the transient duration was 53 ms. With the use of a programmable electrical stimulator, a shallow rate dependence of 3 and 4 ms per 100 beats/min was observed, respectively. In the ventricle the action potential duration was 109 ms and the transient duration was 124 ms, with a steeper rate dependence of 12 and 16 ms per 100 beats/min. Synchronous electrocardiograms and optical mapping recordings were recorded, in which the P-wave aligns with the atrial voltage peak and R-wave aligns with the ventricular peak. A simple optical pathway and imaging chamber are detailed along with schematics for the in-house construction of the electrocardiogram amplifier and electrical stimulator. Laboratory procedures necessary for zebrafish heart isolation, cannulation, and loading are also presented. PMID:25740339

Differential Contribution of the Guanylyl Cyclase-Cyclic GMP-Protein Kinase G Pathway to the Proliferation of Neural Stem Cells Stimulated by Nitric Oxide

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Bruno P. Carreira

2012-02-01

Full Text Available Nitric oxide (NO is an important inflammatory mediator involved in the initial boost in the proliferation of neural stem cells following brain injury. However, the mechanisms underlying the proliferative effect of NO are still unclear. The aim of this work was to investigate whether cyclic GMP (cGMP and the cGMP-dependent kinase (PKG are involved in the proliferative effect triggered by NO in neural stem cells. For this purpose, cultures of neural stem cells isolated from the mouse subventricular zone (SVZ were used. We observed that long-term exposure to the NO donor (24 h, NOC-18, increased the proliferation of SVZ cells in a cGMP-dependent manner, since the guanylate cyclase inhibitor, ODQ, prevented cell proliferation. Similarly to NOC-18, the cGMP analogue, 8-Br-cGMP, also increased cell proliferation. Interestingly, shorter exposures to NO (6 h increased cell proliferation in a cGMP-independent manner via the ERK/MAP kinase pathway. The selective inhibitor of PKG, KT5823, prevented the proliferative effect induced by NO at 24 h but not at 6 h. In conclusion, the proliferative effect of NO is initially mediated by the ERK/MAPK pathway, and at later stages by the GC/cGMP/PKG pathway. Thus, our work shows that NO induces neural stem cell proliferation by targeting these two pathways in a biphasic manner.

Validating computationally predicted TMS stimulation areas using direct electrical stimulation in patients with brain tumors near precentral regions.

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Opitz, Alexander; Zafar, Noman; Bockermann, Volker; Rohde, Veit; Paulus, Walter

2014-01-01

The spatial extent of transcranial magnetic stimulation (TMS) is of paramount interest for all studies employing this method. It is generally assumed that the induced electric field is the crucial parameter to determine which cortical regions are excited. While it is difficult to directly measure the electric field, one usually relies on computational models to estimate the electric field distribution. Direct electrical stimulation (DES) is a local brain stimulation method generally considered the gold standard to map structure-function relationships in the brain. Its application is typically limited to patients undergoing brain surgery. In this study we compare the computationally predicted stimulation area in TMS with the DES area in six patients with tumors near precentral regions. We combine a motor evoked potential (MEP) mapping experiment for both TMS and DES with realistic individual finite element method (FEM) simulations of the electric field distribution during TMS and DES. On average, stimulation areas in TMS and DES show an overlap of up to 80%, thus validating our computational physiology approach to estimate TMS excitation volumes. Our results can help in understanding the spatial spread of TMS effects and in optimizing stimulation protocols to more specifically target certain cortical regions based on computational modeling.

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.

Science.gov (United States)

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.

Endothelial microparticle formation by angiotensin II is mediated via Ang II receptor type I/NADPH oxidase/ Rho kinase pathways targeted to lipid rafts.

Science.gov (United States)

Burger, Dylan; Montezano, Augusto C; Nishigaki, Nobuhiro; He, Ying; Carter, Anthony; Touyz, Rhian M

2011-08-01

Circulating microparticles are increased in cardiovascular disease and may themselves promote oxidative stress and inflammation. Molecular mechanisms underlying their formation and signaling are unclear. We investigated the role of reactive oxygen species (ROS), Rho kinase, and lipid rafts in microparticle formation and examined their functional significance in endothelial cells (ECs). Microparticle formation from angiotensin II (Ang II)-stimulated ECs and apolipoprotein E(-/-) mice was assessed by annexin V or by CD144 staining and electron microscopy. Ang II promoted microparticle formation and increased EC O(2)(-) generation and Rho kinase activity. Ang II-stimulated effects were inhibited by irbesartan (Ang II receptor type I blocker) and fasudil (Rho kinase inhibitor). Methyl-β-cyclodextrin and nystatin, which disrupt lipid rafts/caveolae, blocked microparticle release. Functional responses, assessed in microparticle-stimulated ECs, revealed increased O(2)(-) production, enhanced vascular cell adhesion molecule/platelet-EC adhesion molecule expression, and augmented macrophage adhesion. Inhibition of epidermal growth factor receptor blocked the prooxidative and proinflammatory effects of microparticles. In vitro observations were confirmed in apolipoprotein E(-/-) mice, which displayed vascular inflammation and high levels of circulating endothelial microparticles, effects that were reduced by apocynin. We demonstrated direct actions of Ang II on endothelial microparticle release, mediated through NADPH oxidase, ROS, and Rho kinase targeted to lipid rafts. Microparticles themselves stimulated endothelial ROS formation and inflammatory responses. Our findings suggest a feedforward system whereby Ang II promotes EC injury through its own endothelial-derived microparticles.

Analysis of the Mitogen-activated protein kinase kinase 4 (MAP2K4) tumor suppressor gene in ovarian cancer

International Nuclear Information System (INIS)

Davis, Sally J; Choong, David YH; Ramakrishna, Manasa; Ryland, Georgina L; Campbell, Ian G; Gorringe, Kylie L

2011-01-01

MAP2K4 is a putative tumor and metastasis suppressor gene frequently found to be deleted in various cancer types. We aimed to conduct a comprehensive analysis of this gene to assess its involvement in ovarian cancer. We screened for mutations in MAP2K4 using High Resolution Melt analysis of 149 primary ovarian tumors and methylation at the promoter using Methylation-Specific Single-Stranded Conformation Polymorphism analysis of 39 tumors. We also considered the clinical impact of changes in MAP2K4 using publicly available expression and copy number array data. Finally, we used siRNA to measure the effect of reducing MAP2K4 expression in cell lines. In addition to 4 previously detected homozygous deletions, we identified a homozygous 16 bp truncating deletion and a heterozygous 4 bp deletion, each in one ovarian tumor. No promoter methylation was detected. The frequency of MAP2K4 homozygous inactivation was 5.6% overall, and 9.8% in high-grade serous cases. Hemizygous deletion of MAP2K4 was observed in 38% of samples. There were significant correlations of copy number and expression in three microarray data sets. There was a significant correlation between MAP2K4 expression and overall survival in one expression array data set, but this was not confirmed in an independent set. Treatment of JAM and HOSE6.3 cell lines with MAP2K4 siRNA showed some reduction in proliferation. MAP2K4 is targeted by genetic inactivation in ovarian cancer and restricted to high grade serous and endometrioid carcinomas in our cohort

Deep brain stimulation, brain maps and personalized medicine: lessons from the human genome project.

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Fins, Joseph J; Shapiro, Zachary E

2014-01-01

Although the appellation of personalized medicine is generally attributed to advanced therapeutics in molecular medicine, deep brain stimulation (DBS) can also be so categorized. Like its medical counterpart, DBS is a highly personalized intervention that needs to be tailored to a patient's individual anatomy. And because of this, DBS like more conventional personalized medicine, can be highly specific where the object of care is an N = 1. But that is where the similarities end. Besides their differing medical and surgical provenances, these two varieties of personalized medicine have had strikingly different impacts. The molecular variant, though of a more recent vintage has thrived and is experiencing explosive growth, while DBS still struggles to find a sustainable therapeutic niche. Despite its promise, and success as a vetted treatment for drug resistant Parkinson's Disease, DBS has lagged in broadening its development, often encountering regulatory hurdles and financial barriers necessary to mount an adequate number of quality trials. In this paper we will consider why DBS-or better yet neuromodulation-has encountered these challenges and contrast this experience with the more successful advance of personalized medicine. We will suggest that personalized medicine and DBS's differential performance can be explained as a matter of timing and complexity. We believe that DBS has struggled because it has been a journey of scientific exploration conducted without a map. In contrast to molecular personalized medicine which followed the mapping of the human genome and the Human Genome Project, DBS preceded plans for the mapping of the human brain. We believe that this sequence has given personalized medicine a distinct advantage and that the fullest potential of DBS will be realized both as a cartographical or electrophysiological probe and as a modality of personalized medicine.

The insulin and IGF1 receptor kinase domains are functional dimers in the activated state

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Cabail, M. Zulema; Li, Shiqing; Lemmon, Eric; Bowen, Mark E.; Hubbard, Stevan R.; Miller, W. Todd

2015-03-01

The insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R) are highly related receptor tyrosine kinases with a disulfide-linked homodimeric architecture. Ligand binding to the receptor ectodomain triggers tyrosine autophosphorylation of the cytoplasmic domains, which stimulates catalytic activity and creates recruitment sites for downstream signalling proteins. Whether the two phosphorylated tyrosine kinase domains within the receptor dimer function independently or cooperatively to phosphorylate protein substrates is not known. Here we provide crystallographic, biophysical and biochemical evidence demonstrating that the phosphorylated kinase domains of IR and IGF1R form a specific dimeric arrangement involving an exchange of the juxtamembrane region proximal to the kinase domain. In this dimer, the active position of α-helix C in the kinase N lobe is stabilized, which promotes downstream substrate phosphorylation. These studies afford a novel strategy for the design of small-molecule IR agonists as potential therapeutic agents for type 2 diabetes.

FvBck1, a Component of Cell Wall Integrity MAP Kinase Pathway, is Required for Virulence and Oxidative Stress Response in Sugarcane Pokkah Boeng Pathogen

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Chengkang eZhang

2015-10-01

Full Text Available Fusarium verticillioides (formerly F. moniliforme is suggested as one of the causal agents of Pokkah Boeng, a serious disease of sugarcane worldwide. Currently, detailed molecular and physiological mechanism of pathogenesis is unknown. In this study, we focused on cell wall integrity MAPK pathway as one of the potential signaling mechanisms associated with Pokkah Boeng pathogenesis. We identified FvBCK1 gene that encodes a MAP kinase kinase kinase homolog and determined that it is not only required for growth, micro- and macro-conidia production, and cell wall integrity but also for response to osmotic and oxidative stresses. The deletion of FvBCK1 caused a significant reduction in virulence and FB1 production, a carcinogenic mycotoxin produced by the fungus. Moreover, we found the expression levels of three genes, which are known to be involved in superoxide scavenging, were down regulated in the mutant. We hypothesized that the loss of superoxide scavenging capacity was one of the reasons for reduced virulence, but overexpression of catalase or peroxidase gene failed to restore the virulence defect in the deletion mutant. When we introduced Magnaporthe oryzae MCK1 into the FvBck1 deletion mutant, while certain phenotypes were restored, the complemented strain failed to gain full virulence. In summary, FvBck1 plays a diverse role in F. verticillioides, and detailed investigation of downstream signaling pathways will lead to a better understanding of how this MAPK pathway regulates Pokkah Boeng on sugarcane.

Skeletal myocyte hypertrophy requires mTOR kinase activity and S6K1

International Nuclear Information System (INIS)

Park, In-Hyun; Erbay, Ebru; Nuzzi, Paul; Chen Jie

2005-01-01

The protein kinase mammalian target of rapamycin (mTOR) is a central regulator of cell proliferation and growth, with the ribosomal subunit S6 kinase 1 (S6K1) as one of the key downstream signaling effectors. A critical role of mTOR signaling in skeletal muscle differentiation has been identified recently, and an unusual regulatory mechanism independent of mTOR kinase activity and S6K1 is revealed. An mTOR pathway has also been reported to regulate skeletal muscle hypertrophy, but the regulatory mechanism is not completely understood. Here, we report the investigation of mTOR's function in insulin growth factor I (IGF-I)-induced C2C12 myotube hypertrophy. Added at a later stage when rapamycin no longer had any effect on normal myocyte differentiation, rapamycin completely blocked myocyte hypertrophy as measured by myotube diameter. Importantly, a concerted increase of average myonuclei per myotube was observed in IGF-I-stimulated myotubes, which was also inhibited by rapamycin added at a time when it no longer affected normal differentiation. The mTOR protein level, its catalytic activity, its phosphorylation on Ser2448, and the activity of S6K1 were all found increased in IGF-I-stimulated myotubes compared to unstimulated myotubes. Using C2C12 cells stably expressing rapamycin-resistant forms of mTOR and S6K1, we provide genetic evidence for the requirement of mTOR and its downstream effector S6K1 in the regulation of myotube hypertrophy. Our results suggest distinct mTOR signaling mechanisms in different stages of skeletal muscle development: While mTOR regulates the initial myoblast differentiation in a kinase-independent and S6K1-independent manner, the hypertrophic function of mTOR requires its kinase activity and employs S6K1 as a downstream effector

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

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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.

Cerebral cortex activation mapping upon electrical muscle stimulation by 32-channel time-domain functional near-infrared spectroscopy.

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Re, Rebecca; Muthalib, Makii; Contini, Davide; Zucchelli, Lucia; Torricelli, Alessandro; Spinelli, Lorenzo; Caffini, Matteo; Ferrari, Marco; Quaresima, Valentina; Perrey, Stephane; Kerr, Graham

2013-01-01

The application of different EMS current thresholds on muscle activates not only the muscle but also peripheral sensory axons that send proprioceptive and pain signals to the cerebral cortex. A 32-channel time-domain fNIRS instrument was employed to map regional cortical activities under varied EMS current intensities applied on the right wrist extensor muscle. Eight healthy volunteers underwent four EMS at different current thresholds based on their individual maximal tolerated intensity (MTI), i.e., 10 % < 50 % < 100 % < over 100 % MTI. Time courses of the absolute oxygenated and deoxygenated hemoglobin concentrations primarily over the bilateral sensorimotor cortical (SMC) regions were extrapolated, and cortical activation maps were determined by general linear model using the NIRS-SPM software. The stimulation-induced wrist extension paradigm significantly increased activation of the contralateral SMC region according to the EMS intensities, while the ipsilateral SMC region showed no significant changes. This could be due in part to a nociceptive response to the higher EMS current intensities and result also from increased sensorimotor integration in these cortical regions.

Knockout of the predominant conventional PKC isoform, PKCalpha, in mouse skeletal muscle does not affect contraction-stimulated glucose uptake

DEFF Research Database (Denmark)

Jensen, Thomas E; Maarbjerg, Stine J; Rose, Adam J

2009-01-01

Conventional (c) protein kinase C (PKC) activity has been shown to increase with skeletal muscle contraction, and numerous studies using primarily pharmacological inhibitors have implicated cPKCs in contraction-stimulated glucose uptake. Here, to confirm that cPKC activity is required for contrac...... working on other parts of contraction-induced signaling or the remaining cPKC isoforms are sufficient for stimulating glucose uptake during contractions.......Conventional (c) protein kinase C (PKC) activity has been shown to increase with skeletal muscle contraction, and numerous studies using primarily pharmacological inhibitors have implicated cPKCs in contraction-stimulated glucose uptake. Here, to confirm that cPKC activity is required...... for contraction-stimulated glucose uptake in mouse muscles, contraction-stimulated glucose uptake ex vivo was first evaluated in the presence of three commonly used cPKC inhibitors (calphostin C, Gö-6976, and Gö-6983) in incubated mouse soleus and extensor digitorum longus (EDL) muscles. All potently inhibited...

Brain and Music: An Intraoperative Stimulation Mapping Study of a Professional Opera Singer.

Science.gov (United States)

Riva, Marco; Casarotti, Alessandra; Comi, Alessandro; Pessina, Federico; Bello, Lorenzo

2016-09-01

Music is one of the most sophisticated and fascinating functions of the brain. Yet, how music is instantiated within the brain is not fully characterized. Singing is a peculiar aspect of music, in which both musical and linguistic skills are required to provide a merged vocal output. Identifying the neural correlates of this process is relevant for both clinical and research purposes. An adult white man with a presumed left temporal glioma was studied. He is a professional opera singer. A tailored music evaluation, the Montreal Battery of Evaluation of Amusia, was performed preoperatively and postoperatively, with long-term follow-up. Intraoperative stimulation mapping (ISM) with awake surgery with a specific music evaluation battery was used to identify and preserve the cortical and subcortical structures subserving music, along with standard motor-sensory and language mapping. A total resection of a grade I glioma was achieved. The Montreal Battery of Evaluation of Amusia reported an improvement in musical scores after the surgery. ISM consistently elicited several types of errors in the superior temporal gyrus and, to a lesser extent, in the inferior frontal operculum. Most errors occurred during score reading; fewer errors were elicited during the assessment of rhythm. No spontaneous errors were recorded. These areas did not overlap with eloquent sites for counting or naming. ISM and a tailored music battery enabled better characterization of a specific network within the brain subserving score reading independently from speech with long-term clinical impact. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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Md Badrul Alam

2017-09-01

Full Text Available 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.

Protein kinase C and extracellular signal-regulated kinase regulate movement, attachment, pairing and egg release in Schistosoma mansoni.

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Margarida Ressurreição

2014-06-01

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

1,2-Diacylglycerols, but not phorbol esters, activate a potential inhibitory pathway for protein kinase C in GH3 pituitary cells. Evidence for involvement of a sphingomyelinase.

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Kolesnick, R N; Clegg, S

1988-05-15

It has been suggested that sphingoid bases may serve as physiologic inhibitors of protein kinase C. Because 1,2-diacylglycerols, but not phorbol esters, enhance sphingomyelin degradation via a sphingomyelinase in GH3 pituitary cells (Kolesnick, R. N. (1987) J. Biol. Chem. 262, 16759-16762), the effects of phorbol esters, 1,2-diacylglycerols, and sphingomyelinase on protein kinase C activation were assessed. Under basal conditions, the inactive cytosolic form of protein kinase C predominated. 1,2-Diacylglycerols stimulated transient protein kinase C redistribution to the membrane. 1,2-Dioctanoylglycerol (200 micrograms/ml) reduced cytosolic protein kinase C activity to 67% of control from 72 to 48 pmol.min-1.10(6) cells-1 and enhanced membrane-bound activity to 430% of control from 6 to 25 pmol.min-1.10(6) cells-1 after 4 min of stimulation. Thereafter, protein kinase C activity returned to the cytosol. In contrast, the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulated redistribution to the membrane without return to the cytosol. Exogenous sphingomyelinase reduced membrane-bound protein kinase C activity to 30% of control, yet did not alter cytosolic activity. Sphingomyelinase, added after phorbol ester-induced redistribution was completed, restored activity to the cytosol. In these studies, TPA (10(-8) M) reduced cytosolic activity to 62% of control and elevated membrane-bound protein kinase C activity to 650% of control. Sphingomyelinase restored cytosolic activity to 84% of control and reduced membrane-bound activity to 297% of control. Similarly, the free sphingoid bases, sphingosine, sphinganine, and phytosphingosine, reversed phorbol ester-induced protein kinase C redistribution. Since 1,2-diacylglycerols activate a sphingomyelinase and sphingomyelinase action can reverse protein kinase C activation, these studies suggest that a pathway involving a sphingomyelinase might comprise a physiologic negative effector system for protein kinase C

Calcium-Dependent Protein Kinases from Arabidopsis show substrate specificity differences in an analysis of 103 substrates

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Amy eCurran

2011-08-01

Full Text Available 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.

Protein kinase Cα deletion causes hypotension and decreased vascular contractility.

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Wynne, Brandi M; McCarthy, Cameron G; Szasz, Theodora; Molina, Patrick A; Chapman, Arlene B; Webb, R Clinton; Klein, Janet D; Hoover, Robert S

2018-03-01

Protein kinase Cα (PKCα) is a critical regulator of multiple cell signaling pathways including gene transcription, posttranslation modifications and activation/inhibition of many signaling kinases. In regards to the control of blood pressure, PKCα causes increased vascular smooth muscle contractility, while reducing cardiac contractility. In addition, PKCα has been shown to modulate nephron ion transport. However, the role of PKCα in modulating mean arterial pressure (MAP) has not been investigated. In this study, we used a whole animal PKCα knock out (PKC KO) to test the hypothesis that global PKCα deficiency would reduce MAP, by a reduction in vascular contractility. Radiotelemetry measurements of ambulatory blood pressure (day/night) were obtained for 18 h/day during both normal chow and high-salt (4%) diet feedings. PKCα mice had a reduced MAP, as compared with control, which was not normalized with high-salt diet (14 days). Metabolic cage studies were performed to determine urinary sodium excretion. PKC KO mice had a significantly lower diastolic, systolic and MAP as compared with control. No significant differences in urinary sodium excretion were observed between the PKC KO and control mice, whether fed normal chow or high-salt diet. Western blot analysis showed a compensatory increase in renal sodium chloride cotransporter expression. Both aorta and mesenteric vessels were removed for vascular reactivity studies. Aorta and mesenteric arteries from PKC KO mice had a reduced receptor-independent relaxation response, as compared with vessels from control. Vessels from PKC KO mice exhibited a decrease in maximal contraction, compared with controls. Together, these data suggest that global deletion of PKCα results in reduced MAP due to decreased vascular contractility.

Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

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Xu, Qifang; Malecka, Kimberly L.; Fink, Lauren; Jordan, E. Joseph; Duffy, Erin; Kolander, Samuel; Peterson, Jeffrey; Dunbrack, Roland L.

2016-01-01

Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Crystal structures of several homomeric protein kinase complexes have a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer located in the active site of another monomer, a structural complex that we call an “autophosphorylation complex.” We developed and applied a structural bioinformatics method to identify all such autophosphorylation kinase complexes in X-ray crystallographic structures in the Protein Data Bank (PDB). We identified 15 autophosphorylation complexes in the PDB, of which 5 complexes had not previously been described in the publications describing the crystal structures. These 5 consist of tyrosine residues in the N-terminal juxtamembrane regions of colony stimulating factor 1 receptor (CSF1R, Tyr561) and EPH receptor A2 (EPHA2, Tyr594), tyrosine residues in the activation loops of the SRC kinase family member LCK (Tyr394) and insulin-like growth factor 1 receptor (IGF1R, Tyr1166), and a serine in a nuclear localization signal region of CDC-like kinase 2 (CLK2, Ser142). Mutations in the complex interface may alter autophosphorylation activity and contribute to disease; therefore we mutated residues in the autophosphorylation complex interface of LCK and found that two mutations impaired autophosphorylation (T445V and N446A) and mutation of Pro447 to Ala, Gly, or Leu increased autophosphorylation. The identified autophosphorylation sites are conserved in many kinases, suggesting that, by homology, these complexes may provide insight into autophosphorylation complex interfaces of kinases that are relevant drug targets. PMID:26628682

Alteration of sodium, potassium-adenosine triphosphatase activity in rabbit ciliary processes by cyclic adenosine monophosphate-dependent protein kinase

International Nuclear Information System (INIS)

Delamere, N.A.; Socci, R.R.; King, K.L.

1990-01-01

The response of sodium, potassium-adenosine triphosphatase (Na,K-ATPase) to cyclic adenosine monophosphate (cAMP)-dependent protein kinase was examined in membranes obtained from rabbit iris-ciliary body. In the presence of the protein kinase together with 10(-5) M cAMP, Na,K-ATPase activity was reduced. No change in Na,K-ATPase activity was detected in response to the protein kinase without added cAMP. Likewise cAMP alone did not alter Na,K-ATPase activity. Reduction of Na,K-ATPase activity was also observed in the presence of the cAMP-dependent protein kinase catalytic subunit. The response of the enzyme to the kinase catalytic subunit was also examined in membranes obtained from rabbit ciliary processes. In the presence of 8 micrograms/ml of the catalytic subunit, ciliary process Na,K-ATPase activity was reduced by more than 50%. To examine whether other ATPases were suppressed by the protein kinase, calcium-stimulated ATPase activity was examined; its activity was stimulated by the catalytic subunit. To test whether the response of the ciliary process Na,K-ATPase is unique, experiments were also performed using membrane preparations from rabbit lens epithelium or rabbit kidney; the catalytic subunit significantly reduced the activity of Na,K-ATPase from the kidney but not the lens. These Na,K-ATPase studies suggest that in the iris-ciliary body, cAMP may alter sodium pump activity. In parallel 86Rb uptake studies, we observed that ouabain-inhibitable potassium uptake by intact pieces of iris-ciliary body was reduced by exogenous dibutryl cAMP or by forskolin

Increased expression of interleukin-1β in triglyceride-induced macrophage cell death is mediated by p38 MAP kinase.

Science.gov (United States)

Sung, Ho Joong; Son, Sin Jee; Yang, Seung-ju; Rhee, Ki-Jong; Kim, Yoon Suk

2012-07-01

Triglycerides (TG) are implicated in the development of atherosclerosis through formation of foam cells and induction of macrophage cell death. In this study, we report that addition of exogenous TG induced cell death in phorbol 12-myristate 13-acetate-differentiated THP-1 human macrophages. TG treatment induced a dramatic decrease in interleukin-1β (IL-1β) mRNA expression in a dose- and time-dependent manner. The expression of granulocyte macrophage colony-stimulating factor and platelet endothelial cell adhesion molecule remained unchanged. To identify signaling pathways involved in TG-induced downregulation of IL-1β, we added p38 MAPK, protein kinase C (PKC) or c-Raf1 specific inhibitors. We found that inhibition of p38 MAPK alleviated the TG-induced downregulation of IL-1β, whereas inhibition of PKC and c-Raf1 had no effect. This is the first report showing decreased IL-1β expression during TG-induced cell death in a human macrophage line. Our results suggest that downregulation of IL-1β expression by TG-treated macrophages may play a role during atherogenesis.

PRO40 is a scaffold protein of the cell wall integrity pathway, linking the MAP kinase module to the upstream activator protein kinase C.

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Ines Teichert

2014-09-01

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

2,2',4,4'-Tetrachlorobiphenyl upregulates cyclooxygenase-2 in HL-60 cells via p38 mitogen-activated protein kinase and NF-κB

International Nuclear Information System (INIS)

Bezdecny, Steven A.; Karmaus, Peer; Roth, Robert A.; Ganey, Patricia E.

2007-01-01

Polychlorinated biphenyls (PCBs) are ubiquitous, persistent environmental contaminants that affect a number of cellular systems, including neutrophils. Among the effects caused by the noncoplanar PCB 2,2',4,4'-tetrachlorobiphenyl (2244-TCB) in granulocytic HL-60 cells are increases in superoxide anion production, activation of phospholipase A 2 with subsequent release of arachidonic acid (AA) and upregulation of the inflammatory gene cyclooxygenase-2 (COX-2). The objective of this study was to determine the signal transduction pathways involved in the upregulation of COX-2 by 2244-TCB. Treatment of HL-60 cells with 2244-TCB led to increased expression of COX-2 mRNA. This increase was prevented by the transcriptional inhibitor actinomycin D in cells pretreated with 2244-TCB for 10 min. The increase in COX-2 mRNA was associated with release of 3 H-AA, phosphorylation of p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinases, increased levels of nuclear NF-κB and increased superoxide anion production. Bromoenol lactone, an inhibitor of the calcium-independent phospholipase A 2 , reduced 3 H-AA release but had no effect on COX-2 mRNA, protein or activity. Pretreatment with SB-202190 or SB-203580, inhibitors of the p38 MAP kinase pathway, prevented the 2244-TCB-mediated induction of COX-2 and phosphorylation of p38 and ERK MAP kinases. These inhibitors did not alter 3 H-AA release. Treatment with PD 98059 or U 0126, inhibitors of the MAP/ERK (MEK) pathway, prevented the 2244-TCB-mediated activation of ERK but had no effect on COX-2 induction or p38 phosphorylation. 2244-TCB treatment did not affect c-Jun N-terminal kinase (JNK) phosphorylation. 2244-TCB exposure increased the amount of nuclear NF-κB. This increase was prevented by pretreatment with p38 MAP kinase inhibitors, but not by pretreatment with MEK inhibitors. Pretreatment with inhibitors of NF-κB prevented the 2244-TCB-mediated induction of COX-2 mRNA. 2244-TCB

Glycogen synthase kinase 3β promotes liver innate immune activation by restraining AMP-activated protein kinase activation.

Science.gov (United States)

Zhou, Haoming; Wang, Han; Ni, Ming; Yue, Shi; Xia, Yongxiang; Busuttil, Ronald W; Kupiec-Weglinski, Jerzy W; Lu, Ling; Wang, Xuehao; Zhai, Yuan

2018-02-13

Glycogen synthase kinase 3β (Gsk3β [Gsk3b]) is a ubiquitously expressed kinase with distinctive functions in different types of cells. Although its roles in regulating innate immune activation and ischaemia and reperfusion injuries (IRIs) have been well documented, the underlying mechanisms remain ambiguous, in part because of the lack of cell-specific tools in vivo. We created a myeloid-specific Gsk3b knockout (KO) strain to study the function of Gsk3β in macrophages in a murine liver partial warm ischaemia model. Compared with controls, myeloid Gsk3b KO mice were protected from IRI, with diminished proinflammatory but enhanced anti-inflammatory immune responses in livers. In bone marrow-derived macrophages, Gsk3β deficiency resulted in an early reduction of Tnf gene transcription but sustained increase of Il10 gene transcription on Toll-like receptor 4 stimulation in vitro. These effects were associated with enhanced AMP-activated protein kinase (AMPK) activation, which led to an accelerated and higher level of induction of the novel innate immune negative regulator small heterodimer partner (SHP [Nr0b2]). The regulatory function of Gsk3β on AMPK activation and SHP induction was confirmed in wild-type bone marrow-derived macrophages with a Gsk3 inhibitor. Furthermore, we found that this immune regulatory mechanism was independent of Gsk3β Ser9 phosphorylation and the phosphoinositide 3-kinase-Akt signalling pathway. In vivo, myeloid Gsk3β deficiency facilitated SHP upregulation by ischaemia-reperfusion in liver macrophages. Treatment of Gsk3b KO mice with either AMPK inhibitor or SHP small interfering RNA before the onset of liver ischaemia restored liver proinflammatory immune activation and IRI in these otherwise protected hosts. Additionally, pharmacological activation of AMPK protected wild-type mice from liver IRI, with reduced proinflammatory immune activation. Inhibition of the AMPK-SHP pathway by liver ischaemia was demonstrated in tumour resection

Requirement for tyrosine phosphatase during serotonergic neuromodulation by protein kinase C.

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Catarsi, S; Drapeau, P

1997-08-01

Tyrosine kinases and phosphatases are abundant in the nervous system, where they signal cellular differentiation, mediate the responses to growth factors, and direct neurite outgrowth during development. Tyrosine phosphorylation can also alter ion channel activity, but its physiological significance remains unclear. In an identified leech mechanosensory neuron, the ubiquitous neuromodulator serotonin increases the activity of a cation channel by activating protein kinase C (PKC), resulting in membrane depolarization and modulation of the receptive field properties. We observed that the effects on isolated neurons and channels were blocked by inhibiting tyrosine phosphatases. Serotonergic stimulation of PKC thus activates a tyrosine phosphatase activity associated with the channels, which reverses their constitutive inhibition by tyrosine phosphorylation, representing a novel form of neuromodulation.

Activation of AMP-activated protein kinase attenuates hepatocellular carcinoma cell adhesion stimulated by adipokine resistin

International Nuclear Information System (INIS)

Yang, Chen-Chieh; Chang, Shun-Fu; Chao, Jian-Kang; Lai, Yi-Liang; Chang, Wei-En; Hsu, Wen-Hsiu; Kuo, Wu-Hsien

2014-01-01

Resistin, adipocyte-secreting adipokine, may play critical role in modulating cancer pathogenesis. The aim of this study was to investigate the effects of resistin on HCC adhesion to the endothelium, and the mechanism underlying these resistin effects. Human SK-Hep1 cells were used to study the effect of resistin on intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressions as well as NF-κB activation, and hence cell adhesion to human umbilical vein endothelial cells (HUVECs). 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR), an AMP-activated protein kinase (AMPK) activator, was used to determine the regulatory role of AMPK on HCC adhesion to the endothelium in regard to the resistin effects. Treatment with resistin increased the adhesion of SK-Hep1 cells to HUVECs and concomitantly induced NF-κB activation, as well as ICAM-1 and VCAM-1 expressions in SK-Hep1 cells. Using specific blocking antibodies and siRNAs, we found that resistin-induced SK-Hep1 cell adhesion to HUVECs was through NF-κB-regulated ICAM-1 and VCAM-1 expressions. Moreover, treatment with AICAR demonstrated that AMPK activation in SK-Hep1 cells significantly attenuates the resistin effect on SK-Hep1 cell adhesion to HUVECs. These results clarify the role of resistin in inducing HCC adhesion to the endothelium and demonstrate the inhibitory effect of AMPK activation under the resistin stimulation. Our findings provide a notion that resistin play an important role to promote HCC metastasis and implicate AMPK may be a therapeutic target to against HCC metastasis

Interaction with Shc prevents aberrant Erk activation in the absence of extracellular stimuli

KAUST Repository

Suen, KinMan

2013-05-01

Control mechanisms that prevent aberrant signaling are necessary to maintain cellular homeostasis. We describe a new mechanism by which the adaptor protein Shc directly binds the MAP kinase Erk, thus preventing its activation in the absence of extracellular stimuli. The Shc-Erk complex restricts Erk nuclear translocation, restraining Erk-dependent transcription of genes, including those responsible for oncogenic growth. The complex forms through unique binding sites on both the Shc PTB domain and the N-terminal lobe of Erk. Upon receptor tyrosine kinase stimulation, a conformational change within Shc - induced through interaction with the phosphorylated receptor - releases Erk, allowing it to fulfill its role in signaling. Thus, in addition to its established role in promoting MAP kinase signaling in stimulated cells, Shc negatively regulates Erk activation in the absence of growth factors and thus could be considered a tumor suppressor in human cells. © 2013 Nature America, Inc. All rights reserved.

Interaction with Shc prevents aberrant Erk activation in the absence of extracellular stimuli

KAUST Repository

Suen, KinMan; Lin, Chichuan; George, Roger R.; Melo, Fernando A.; Biggs, Eleanor R.; Ahmed, Zamal; Drake, Melanie N.; Arur, Swathi; Arold, Stefan T.; Ladbury, John E S D

2013-01-01

Control mechanisms that prevent aberrant signaling are necessary to maintain cellular homeostasis. We describe a new mechanism by which the adaptor protein Shc directly binds the MAP kinase Erk, thus preventing its activation in the absence of extracellular stimuli. The Shc-Erk complex restricts Erk nuclear translocation, restraining Erk-dependent transcription of genes, including those responsible for oncogenic growth. The complex forms through unique binding sites on both the Shc PTB domain and the N-terminal lobe of Erk. Upon receptor tyrosine kinase stimulation, a conformational change within Shc - induced through interaction with the phosphorylated receptor - releases Erk, allowing it to fulfill its role in signaling. Thus, in addition to its established role in promoting MAP kinase signaling in stimulated cells, Shc negatively regulates Erk activation in the absence of growth factors and thus could be considered a tumor suppressor in human cells. © 2013 Nature America, Inc. All rights reserved.

Inhibition of Vascular Smooth Muscle Growth via Signaling Crosstalk between AMP-Activated Protein Kinase and cAMP-Dependent Protein Kinase

Directory of Open Access Journals (Sweden)

Joshua Daniel Stone

2012-10-01

Full Text Available Abnormal vascular smooth muscle (VSM growth is central in the pathophysiology of vascular disease yet fully effective therapies to curb this growth are lacking. Recent findings from our lab and others support growth control of VSM by adenosine monophosphate (AMP-based approaches including the metabolic sensor AMP-activated protein kinase (AMPK and cAMP-dependent protein kinase (PKA. Molecular crosstalk between AMPK and PKA has been previously suggested, yet the extent to which this occurs and its biological significance in VSM remains unclear. Considering their common AMP backbone and similar signaling characteristics, we hypothesized that crosstalk exists between AMPK and PKA in the regulation of VSM growth. Using rat primary VSM cells, the AMPK agonist AICAR increased AMPK activity and phosphorylation of the catalytic Thr172 site on AMPK. Interestingly, AICAR also phosphorylated a suspected PKA-inhibitory Ser485 site on AMPK, and these cumulative events were reversed by the PKA inhibitor PKI suggesting possible PKA-mediated regulation of AMPK. AICAR also increased PKA activity in a reversible fashion. The cAMP stimulator forskolin increased PKA activity and completely ameliorated Ser/Thr protein phosphatase-2C activity, suggesting a potential mechanism of AMPK modulation by PKA since inhibition of PKA by PKI reduced AMPK activity. Functionally, AMPK inhibited serum-stimulated cell cycle progression and cellular proliferation; however, PKA failed to do so. Moreover, AMPK and PKA reduced PDGF-β-stimulated VSM cell migration. Collectively, these results show that AMPK is capable of reducing VSM growth in both anti-proliferative and anti-migratory fashions. Furthermore, these data suggest that AMPK may be modulated by PKA and that positive feedback may exist between these two systems. These findings reveal a discrete nexus between AMPK and PKA in VSM and provide basis for metabolically-directed targets in reducing pathologic VSM growth.

Bacillus subtilis Two-Component System Sensory Kinase DegS Is Regulated by Serine Phosphorylation in Its Input Domain

DEFF Research Database (Denmark)

Jers, Carsten; Kobir, Ahasanul; Søndergaard, Elsebeth Oline

2011-01-01

Bacillus subtilis two-component system DegS/U is well known for the complexity of its regulation. The cytosolic sensory kinase DegS does not receive a single predominant input signal like most two-component kinases, instead it integrates a wide array of metabolic inputs that modulate its activity......S phosphorylation can be carried out by at least two B. subtilis Hanks-type kinases in vitro, and this stimulates the phosphate transfer towards DegU. The consequences of this process were studied in vivo, using phosphomimetic (Ser76Asp) and non-phosphorylatable (Ser76Ala) mutants of DegS. In a number...

Subunits of the Snf1 kinase heterotrimer show interdependence for association and activity.

Science.gov (United States)

Elbing, Karin; Rubenstein, Eric M; McCartney, Rhonda R; Schmidt, Martin C

2006-09-08

The Snf1 kinase and its mammalian orthologue, the AMP-activated protein kinase (AMPK), function as heterotrimers composed of a catalytic alpha-subunit and two non-catalytic subunits, beta and gamma. The beta-subunit is thought to hold the complex together and control subcellular localization whereas the gamma-subunit plays a regulatory role by binding to and blocking the function of an auto-inhibitory domain (AID) present in the alpha-subunit. In addition, catalytic activity requires phosphorylation by a distinct upstream kinase. In yeast, any one of three Snf1-activating kinases, Sak1, Tos3, or Elm1, can fulfill this role. We have previously shown that Sak1 is the only Snf1-activating kinase that forms a stable complex with Snf1. Here we show that the formation of the Sak1.Snf1 complex requires the beta- and gamma-subunits in vivo. However, formation of the Sak1.Snf1 complex is not necessary for glucose-regulated phosphorylation of the Snf1 activation loop. Snf1 kinase purified from cells lacking the beta-subunits do not contain any gamma-subunit, indicating that the Snf1 kinase does not form a stable alphagamma dimer in vivo. In vitro kinase assays using purified full-length and truncated Snf1 proteins demonstrate that the kinase domain, which lacks the AID, is significantly more active than the full-length Snf1 protein. Addition of purified beta- and gamma-subunits could stimulate the kinase activity of the full-length alpha-subunit but only when all three subunits were present, suggesting an interdependence of all three subunits for assembly of a functional complex.

Modulation of type II TGF-β receptor degradation by integrin-linked kinase.

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Vi, Linda; Boo, Stellar; Sayedyahossein, Samar; Singh, Randeep K; McLean, Sarah; Di Guglielmo, Gianni M; Dagnino, Lina

2015-03-01

Cutaneous responses to injury, infection, and tumor formation involve the activation of resident dermal fibroblasts and subsequent transition to myofibroblasts. The key for induction of myofibroblast differentiation is the activation of transforming growth factor-β (TGF-β) receptors and stimulation of integrins and their associated proteins, including integrin-linked kinase (ILK). Cross-talk processes between TGF-β and ILK are crucial for myofibroblast formation, as ILK-deficient dermal fibroblasts exhibit impaired responses to TGF-β receptor stimulation. We now show that ILK associates with type II TGF-β receptors (TβRII) in ligand- and receptor kinase activity-independent manners. In cells with targeted Ilk gene inactivation, cellular levels of TβRII are decreased, through mechanisms that involve enhanced ubiquitination and proteasomal degradation. Partitioning of TGF-β receptors into membrane has been linked to proteasome-dependent receptor degradation. We found that interfering with membrane raft formation in ILK-deficient cells restored TβRII levels and signaling. These observations support a model whereby ILK functions in fibroblasts to direct TβRII away from degradative pathways during their differentiation into myofibroblasts.

Language and motor function thresholds during pediatric extra-operative electrical cortical stimulation brain mapping.

Science.gov (United States)

Zea Vera, Alonso; Aungaroon, Gewalin; Horn, Paul S; Byars, Anna W; Greiner, Hansel M; Tenney, Jeffrey R; Arthur, Todd M; Crone, Nathan E; Holland, Katherine D; Mangano, Francesco T; Arya, Ravindra

2017-10-01

To examine current thresholds and their determinants for language and motor mapping with extra-operative electrical cortical stimulation (ECS). ECS electrocorticograph recordings were reviewed to determine functional thresholds. Predictors of functional thresholds were found with multivariable analyses. In 122 patients (age 11.9±5.4years), average minimum, frontal, and temporal language thresholds were 7.4 (± 3.0), 7.8 (± 3.0), and 7.4 (± 3.1) mA respectively. Average minimum, face, upper and lower extremity motor thresholds were 5.4 (± 2.8), 6.1 (± 2.8), 4.9 (± 2.3), and 5.3 (± 3.3) mA respectively. Functional and after-discharge (AD)/seizure thresholds were significantly related. Minimum, frontal, and temporal language thresholds were higher than AD thresholds at all ages. Minimum motor threshold was higher than minimum AD threshold up to 8.0years of age, face motor threshold was higher than frontal AD threshold up to 11.8years age, and lower subsequently. UE motor thresholds remained below frontal AD thresholds throughout the age range. Functional thresholds are frequently above AD thresholds in younger children. These findings raise concerns about safety and neurophysiologic validity of ECS mapping. Functional and AD/seizure thresholds relationships suggest individual differences in cortical excitability which cannot be explained by clinical variables. Copyright © 2017 International Federation of Clinical Neurophysiology. Published by Elsevier B.V. All rights reserved.

Interplay between calcineurin and the Slt2 MAP-kinase in mediating cell wall integrity, conidiation and virulence in the insect fungal pathogen Beauveria bassiana.

Science.gov (United States)

Huang, Shuaishuai; He, Zhangjiang; Zhang, Shiwei; Keyhani, Nemat O; Song, Yulin; Yang, Zhi; Jiang, Yahui; Zhang, Wenli; Pei, Yan; Zhang, Yongjun

2015-10-01

The entomopathogenic fungus, Beauveria bassiana, is of environmental and economic importance as an insect pathogen, currently used for the biological control of a number of pests. Cell wall integrity and conidiation are critical parameters for the ability of the fungus to infect insects and for production of the infectious propagules. The contribution of calcineurin and the Slt2 MAP kinase to cell wall integrity and development in B. bassiana was investigated. Gene knockouts of either the calcineurin CNA1 subunit or the Slt2 MAP kinase resulted in decreased tolerance to calcofluor white and high temperature. In contrast, the Δcna1 strain was more tolerant to Congo red but more sensitive to osmotic stress (NaCl, sorbitol) than the wild type, whereas the Δslt2 strain had the opposite phenotype. Changes in cell wall structure and composition were seen in the Δslt2 and Δcna1 strains during growth under cell wall stress as compared to the wild type. Both Δslt2 and Δcna1 strains showed significant alterations in growth, conidiation, and viability. Elevation of intracellular ROS levels, and decreased conidial hydrophobicity and adhesion to hydrophobic surfaces, were also seen for both mutants, as well as decreased virulence. Under cell wall stress conditions, inactivation of Slt2 significantly repressed CN-mediated phosphatase activity suggesting some level of cross talk between the two pathways. Comparative transcriptome profiling of the Δslt2 and Δcna1 strains revealed alterations in the expression of distinct gene sets, with overlap in transcripts involved in cell wall integrity, stress response, conidiation and virulence. These data illustrate convergent and divergent phenotypes and targets of the calcineurin and Slt2 pathways in B. bassiana. Copyright © 2015 Elsevier Inc. All rights reserved.

Prostaglandin E2 stimulates normal bronchial epithelial cell growth through induction of c-Jun and PDK1, a kinase implicated in oncogenesis.

Science.gov (United States)

Fan, Yu; Wang, Ye; Wang, Ke

2015-12-18

Cyclooxygenase-2-derived prostaglandin E2 (PGE2), a bioactive eicosanoid, has been implicated in many biological processes including reproduction, inflammation and tumor growth. We previously showed that PGE2 stimulated lung cancer cell growth and progression through PGE2 receptor EP2/EP4-mediated kinase signaling pathways. However, the role of PGE2 in controlling lung airway epithelial cell phenotype remains unknown. We evaluated the effects of c-Jun and 3-phosphoinositede dependent protein kinase-1 (PDK1) in mediating epithelial cell hyperplasia induced by PGE2. The bronchial epithelial cell lines BEAS-2B and HBEc14-KT were cultured and then treated with PGE2. PDK1 small interfering RNA (siRNA) and a PDK1 inhibitor, an antagonist of the PGE2 receptor subtype EP4 and EP4 siRNA, c-Jun siRNA, and overexpressions of c-Jun and PDK1 have been used to evaluate the effects on cell proliferation. We demonstrated that PGE2 increased normal bronchial epithelial cell proliferation through induction of PDK1, an ankyrin repeat-containing Ser/Thr kinase implicated in the induction of apoptosis and the suppression of tumor growth. PDK1 siRNA and a PDK1 inhibitor blocked the effects of PGE2 on normal cell growth. The PGE2-induced PDK1 expression was blocked by an antagonist of the PGE2 receptor subtype EP4 and by EP4 siRNA. In addition, we showed that induction of PDK1 by PGE2 was associated with induction of the transcription factor, c-Jun protein. Silencing of c-Jun using siRNA and point mutations of c-Jun sites in the PDK1 gene promoter resulted in blockade of PDK1 expression and promoter activity induced by PGE2. In contrast, overexpression of c-Jun induced PDK1 gene promoter activity and expression followed increased cell proliferation. PGE2 increases normal bronchial epithelial cell proliferation through increased PDK1 gene expression that is dependent on EP4 and induction of c-Jun. Therewith, our data suggest a new role of c-Jun and PDK1 in mediating epithelial cell

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

Science.gov (United States)

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

2016-03-21

Seed dormancy has fundamental importance in plant survival and crop production; however, the mechanisms regulating dormancy remain unclear [1-3]. Seed dormancy levels generally decrease during domestication to ensure that crops successfully germinate in the field. However, reduction of seed dormancy can cause devastating losses in cereals like wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) due to pre-harvest sprouting, the germination of mature seed (grain) on the mother plant when rain occurs before harvest. Understanding the mechanisms of dormancy can facilitate breeding of crop varieties with the appropriate levels of seed dormancy [4-8]. Barley is a model crop [9, 10] and has two major seed dormancy quantitative trait loci (QTLs), SD1 and SD2, on chromosome 5H [11-19]. We detected a QTL designated Qsd2-AK at SD2 as the single major determinant explaining the difference in seed dormancy between the dormant cultivar "Azumamugi" (Az) and the non-dormant cultivar "Kanto Nakate Gold" (KNG). Using map-based cloning, we identified the causal gene for Qsd2-AK as Mitogen-activated Protein Kinase Kinase 3 (MKK3). The dormant Az allele of MKK3 is recessive; the N260T substitution in this allele decreases MKK3 kinase activity and appears to be causal for Qsd2-AK. The N260T substitution occurred in the immediate ancestor allele of the dormant allele, and the established dormant allele became prevalent in barley cultivars grown in East Asia, where the rainy season and harvest season often overlap. Our findings show fine-tuning of seed dormancy during domestication and provide key information for improving pre-harvest sprouting tolerance in barley and wheat. Copyright © 2016 Elsevier Ltd. All rights reserved.

The threshold of cortical electrical stimulation for mapping sensory and motor functional areas.

Science.gov (United States)

Guojun, Zhang; Duanyu, Ni; Fu, Paul; Lixin, Cai; Tao, Yu; Wei, Du; Liang, Qiao; Zhiwei, Ren

2014-02-01

This study aimed to investigate the threshold of cortical electrical stimulation (CES) for functional brain mapping during surgery for the treatment of rolandic epilepsy. A total of 21 patients with rolandic epilepsy who underwent surgical treatment at the Beijing Institute of Functional Neurosurgery between October 2006 and March 2008 were included in this study. Their clinical data were retrospectively collected and analyzed. The thresholds of CES for motor response, sensory response, and after discharge production along with other threshold-related factors were investigated. The thresholds (mean ± standard deviation) for motor response, sensory response, and after discharge production were 3.48 ± 0.87, 3.86 ± 1.31, and 4.84 ± 1.38 mA, respectively. The threshold for after discharge production was significantly higher than those of both the motor and sensory response (both pstimulation frequency of 50 Hz and a pulse width of 0.2 ms, the threshold of sensory and motor responses were similar, and the threshold of after discharge production was higher than that of sensory and motor response. Copyright © 2013 Elsevier Ltd. All rights reserved.

Dual Regulation of a Chimeric Plant Serine/Threonine Kinase by Calcium and Calcium/Calmodulin

Science.gov (United States)

Takezawa, D.; Ramachandiran, S.; Paranjape, V.; Poovaiah, B. W.

1996-01-01

A chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) gene characterized by a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain was recently cloned from plants. The Escherichia coli-expressed CCaMK phosphorylates various protein and peptide substrates in a Ca(2+)/calmodulin-dependent manner. The calmodulin-binding region of CCAMK has similarity to the calmodulin-binding region of the alpha-subunit of multifunctional Ca(2+)/calmodulin-dependent protein kinase (CaMKII). CCaMK exhibits basal autophosphorylation at the threonine residue(s) (0.098 mol of P-32/mol) that is stimulated 3.4-fold by Ca(2+) (0.339 mol of P-32/mol), while calmodulin inhibits Ca(2+)-stimulated autophosphorylation to the basal level. A deletion mutant lacking the visinin-like domain did not show Ca(2+)-simulated autophosphorylation activity but retained Ca(2+)/calmodulin-dependent protein kinase activity at a reduced level. Ca(2+)-dependent mobility shift assays using E.coli-expressed protein from residues 358-520 revealed that Ca(2+) binds to the visinin-like domain. Studies with site-directed mutants of the visinin-like domain indicated that EF-hands II and III are crucial for Ca(2+)-induced conformational changes in the visinin-like domain. Autophosphorylation of CCaMK increases Ca(2+)/calmodulin-dependent protein kinase activity by about 5-fold, whereas it did not affect its C(2+)-independent activity. This report provides evidence for the existence of a protein kinase in plants that is modulated by Ca(2+) and Ca(2+)/calmodulin. The presence of a visinin-like Ca(2+)-binding domain in CCaMK adds an additional Ca(2+)-sensing mechanism not previously known to exist in the Ca(2+)/calmodulin-mediated signaling cascade in plants.

Metazoan-like signaling in a unicellular receptor tyrosine kinase

Directory of Open Access Journals (Sweden)

Schultheiss Kira P

2013-02-01

Full Text Available Abstract Background Receptor tyrosine kinases (RTKs are crucial components of signal transduction systems in multicellular animals. Surprisingly, numerous RTKs have been identified in the genomes of unicellular choanoflagellates and other protists. Here, we report the first biochemical study of a unicellular RTK, namely RTKB2 from Monosiga brevicollis. Results We cloned, expressed, and purified the RTKB2 kinase, and showed that it is enzymatically active. The activity of RTKB2 is controlled by autophosphorylation, as in metazoan RTKs. RTKB2 possesses six copies of a unique domain (designated RM2 in its C-terminal tail. An isolated RM2 domain (or a synthetic peptide derived from the RM2 sequence served as a substrate for RTKB2 kinase. When phosphorylated, the RM2 domain bound to the Src homology 2 domain of MbSrc1 from M. brevicollis. NMR structural studies of the RM2 domain indicated that it is disordered in solution. Conclusions Our results are consistent with a model in which RTKB2 activation stimulates receptor autophosphorylation within the RM2 domains. This leads to recruitment of Src-like kinases (and potentially other M. brevicollis proteins and further phosphorylation, which may serve to increase or dampen downstream signals. Thus, crucial features of signal transduction circuitry were established prior to the evolution of metazoans from their unicellular ancestors.

Defective TCR stimulation in anergized type 2 T helper cells correlates with abrogated p56(lck) and ZAP-70 tyrosine kinase activities.

Science.gov (United States)

Faith, A; Akdis, C A; Akdis, M; Simon, H U; Blaser, K

1997-07-01

Development of IgE-mediated allergic conditions is dependent on the secretion of a Th2 cytokine pattern, including IL-4, IL-5, and IL-13. The induction of anergy would be one mechanism to abrogate cytokine secretion by Th2 cells, which may be pivotal to the allergic response. We demonstrate here that incubation of cloned human CD4+ phospholipase A2 (PLA)-specific Th2 cells with antigenic peptide, in the absence of professional APC, results in a state of nonresponsiveness. The anergic T cells failed to proliferate or secrete IL-4 in response to optimal stimulation with PLA and autologous, professional APC. Secretion of IL-5 and IL-13, however, was only partially inhibited. The anergic state of the Th2 cells was not associated with CD3 or CD28 down-regulation. However, anergy did appear to be closely related to alterations in signaling pathways, mediated through the TCR, of the cells. In contrast to untreated Th2 cells, anergized Th2 cells failed to respond to anti-CD3 mAb with either increased tyrosine kinase activity or increased levels of tyrosine phosphorylation of p56(lck) or ZAP70. A strong and sustained intracellular calcium flux, observed in untreated Th2 cells in response to anti-CD3 mAb, was absent in anergic Th2 cells. Furthermore, the induction of anergy seems to represent an active process, associated with increased levels of basal tyrosine kinase activity, cytokine production, and CD25 up-regulation in anergic Th2 cells. Together, our results indicate that anergy in Th2 cells is associated with defective transmembrane signaling through the TCR.

Autoregulation of kinase dephosphorylation by ATP binding in AGC protein kinases.

Science.gov (United States)

Chan, Tung O; Pascal, John M; Armen, Roger S; Rodeck, Ulrich

2012-02-01

AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non- ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.

Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

International Nuclear Information System (INIS)

Crowe, David L; Ohannessian, Arthur

2004-01-01

Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK). Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK). Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC) lines. Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway

Recruitment of focal adhesion kinase and paxillin to β1 integrin promotes cancer cell migration via mitogen activated protein kinase activation

Directory of Open Access Journals (Sweden)

Ohannessian Arthur

2004-05-01

Full Text Available Abstract Background Integrin-extracellular matrix interactions activate signaling cascades such as mitogen activated protein kinases (MAPK. Integrin binding to extracellular matrix increases tyrosine phosphorylation of focal adhesion kinase (FAK. Inhibition of FAK activity by expression of its carboxyl terminus decreases cell motility, and cells from FAK deficient mice also show reduced migration. Paxillin is a focal adhesion protein which is also phosphorylated on tyrosine. FAK recruitment of paxillin to the cell membrane correlates with Shc phosphorylation and activation of MAPK. Decreased FAK expression inhibits papilloma formation in a mouse skin carcinogenesis model. We previously demonstrated that MAPK activation was required for growth factor induced in vitro migration and invasion by human squamous cell carcinoma (SCC lines. Methods Adapter protein recruitment to integrin subunits was examined by co-immunoprecipitation in SCC cells attached to type IV collagen or plastic. Stable clones overexpressing FAK or paxillin were created using the lipofection technique. Modified Boyden chambers were used for invasion assays. Results In the present study, we showed that FAK and paxillin but not Shc are recruited to the β1 integrin cytoplasmic domain following attachment of SCC cells to type IV collagen. Overexpression of either FAK or paxillin stimulated cancer cell migration on type IV collagen and invasion through reconstituted basement membrane which was dependent on MAPK activity. Conclusions We concluded that recruitment of focal adhesion kinase and paxillin to β1 integrin promoted cancer cell migration via the mitogen activated protein kinase pathway.

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

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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.

SAD-A kinase controls islet β-cell size and function as a mediator of mTORC1 signaling.

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Nie, Jia; Liu, Xiaolei; Lilley, Brendan N; Zhang, Hai; Pan, Y Albert; Kimball, Scot R; Zhang, Jun; Zhang, Weiping; Wang, Li; Jefferson, Leonard S; Sanes, Joshua R; Han, Xiao; Shi, Yuguang