scholarly journals Activation of mitogen-activated protein kinase couples neurotensin receptor stimulation to induction of the primary response gene Krox-24

1996 ◽  
Vol 320 (1) ◽  
pp. 145-151 ◽  
Author(s):  
Caroline POINOT-CHAZEL ◽  
Marielle PORTIER ◽  
Monsif BOUABOULA ◽  
Natalio VITA ◽  
Florence PECCEU ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cho Cells ◽  
Mapk Pathway ◽  
Chinese Hamster ◽  
Structural Features ◽  
Mitogen Activated Protein Kinase ◽  
Kinase Assay ◽  
Transcriptional Level ◽  
Single Class ◽  
Mitogen Activated Protein

Neurotensin (NT) is a neuropeptide that is important in a variety of biological processes such as signal transduction and cell growth. NT effects are mediated by a single class of cell-surface receptors, known as neurotensin receptors (NTRs), which exhibit structural features of the G-protein-coupled receptors superfamily. We investigated NTR signalling properties with Chinese hamster ovary (CHO) cells stably transformed with human NTR (hNTR). First, we showed that NTR stimulation by NT induced the activation of the mitogen-activated protein kinases (MAPKs) in time- and dose-dependent manners. Both p42 and p44 MAPK isoforms were retarded in gel-shift assays, which was consistent with their activation by phosphorylation. In addition we showed that NT caused a prolonged activation of MAPK as measured by in-gel kinase assay. Secondly, we demonstrated that NT induced the expression of the growth-related gene Krox-24 at the protein level, as assessed by Western-blot analysis, and at the transcriptional level, as demonstrated in CHO cells transfected with hNTR and a reporter gene for Krox-24. Activation of MAPK and induction of Krox-24 were both prevented by the NTR antagonist SR 48692, confirming the specific action on NTR. Furthermore we observed coupling of NTR to a mitogenic pathway and Krox-24 induction in the human adenocarcinoma cell line HT29, which naturally expresses NTRs. Considering coupling pathways between NTR stimulation and MAPK activation, we observed a partial inhibition by pertussis toxin (PTX) and a complete blockade by the protein kinase C (PKC) inhibitor GF 109203X. Taken together, these results suggest that (1) stimulation of NTR activates the MAPK pathway by mechanisms involving dual coupling to both PTX-sensitive and PTX-insensitive G-proteins as well as PKC activation, and (2) these effects are associated with the induction of Krox-24, which might be a target of MAPK effector.

scholarly journals Functional coupling of adenosine A2a receptor to inhibition of the mitogen-activated protein kinase cascade in Chinese hamster ovary cells

1996 ◽  
Vol 316 (1) ◽  
pp. 81-86 ◽  
Author(s):  
Daisuke HIRANO ◽  
Yoshiko AOKI ◽  
Hiroyuki OGASAWARA ◽  
Hisashi KODAMA ◽  
Iwao WAGA ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Mitogen Activated Protein Kinase ◽  
Mapk Activation ◽  
A2a Receptor ◽  
Mitogen Activated Protein

Activation of Gs-coupled receptors enhances the increase in cyclic AMP mediated by adenylate cyclases. As it has been shown that cyclic AMP inhibits the epidermal growth factor-activated mitogen-activated protein kinase (MAPK) signalling pathway, stimulation of Gs-coupled receptors may lead to the inhibition of MAPK activation. To investigate the effect of a Gs-coupled receptor on the MAPK cascade, we cloned the adenosine (Ado) A2a receptor from a guinea-pig leucocyte cDNA library, and established Chinese hamster ovary (CHO) cells stably expressing the receptor (CHOAdoA2R). The [3H]5´-N-ethylcarbamoyladenosine (NECA) binding characteristics (Kd = 91.0±5.4 nM, Bmax = 707±11 fmol/mg of protein, n = 3) and NECA-induced cyclic AMP production indicate that the cloned Ado A2a receptor was functionally expressed in the cells. In CHO cells, thrombin induced intracellular Ca2+ increase and MAPK activation through the intrinsic G-coupled receptor. In CHOAdoA2R cells, NECA partially inhibited thrombin-elicited MAPK activation. When combining NECA-treatment with 1,2-bis-(o-aminophenoxy)ethane-N,N,N´,N´-tetra-acetic acid acetoxymethyl ester (BAPTA-AM) loading, a nearly complete inhibition of the MAPK activation occurred. Forskolin also partially inhibited the MAPK activation and synergized with BAPTA-AM, suggesting that partial inhibition of MAPK activation by NECA results from cyclic AMP production via Ado A2a receptor activation. The same synergism of MAPK inhibition between wortmannin and BAPTA-AM was observed, but not between wortmannin and NECA. These results suggest that cyclic AMP production through Ado A2a receptor inhibits thrombin-elicited MAPK activation by a Ca2+-independent/ wortmannin-sensitive pathway in CHO cells.

scholarly journals Activation of B-Raf and Regulation of the Mitogen-activated Protein Kinase Pathway by the Go α chain

2000 ◽  
Vol 11 (4) ◽  
pp. 1129-1142 ◽  
Author(s):  
Valeria Antonelli ◽  
Francesca Bernasconi ◽  
Yung H. Wong ◽  
Lucia Vallar
Keyword(s):  
Protein Kinase ◽  
Egf Receptor ◽  
Mapk Pathway ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Mitogen Activated Protein Kinase ◽  
Phosphatidylinositol 3 Kinase ◽  
Ovary Cells ◽  
Mapk Activity ◽  
Mitogen Activated Protein

Many receptors coupled to the pertussis toxin-sensitive Gi/o proteins stimulate the mitogen-activated protein kinase (MAPK) pathway. The role of the α chains of these G proteins in MAPK activation is poorly understood. We investigated the ability of Gαo to regulate MAPK activity by transient expression of the activated mutant Gαo-Q205L in Chinese hamster ovary cells. Gαo-Q205L was not sufficient to activate MAPK but greatly enhanced the response to the epidermal growth factor (EGF) receptor. This effect was not associated with changes in the state of tyrosine phosphorylation of the EGF receptor. Gαo-Q205L also potentiated MAPK stimulation by activated Ras. In Chinese hamster ovary cells, EGF receptors activate B-Raf but not Raf-1 or A-Raf. We found that expression of activated Gαo stimulated B-Raf activity independently of the activation of the EGF receptor or Ras. Inactivation of protein kinase C and inhibition of phosphatidylinositol-3 kinase abolished both B-Raf activation and EGF receptor-dependent MAPK stimulation by Gαo. Moreover, Gαo-Q205L failed to affect MAPK activation by fibroblast growth factor receptors, which stimulate Raf-1 and A-Raf but not B-Raf activity. These results suggest that Gαo can regulate the MAPK pathway by activating B-Raf through a mechanism that requires a concomitant signal from tyrosine kinase receptors or Ras to efficiently stimulate MAPK activity. Further experiments showed that receptor-mediated activation of Gαo caused a B-Raf response similar to that observed after expression of the mutant subunit. The finding that Gαo induces Ras-independent and protein kinase C- and phosphatidylinositol-3 kinase-dependent activation of B-Raf and conditionally stimulates MAPK activity provides direct evidence for intracellular signals connecting this G protein subunit to the MAPK pathway.

scholarly journals Expression of transgenes enriched in rare codons is enhanced by the MAPK pathway

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jackson Peterson ◽  
Siqi Li ◽  
Erin Kaltenbrun ◽  
Ozgun Erdogan ◽  
Christopher M. Counter
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Rare Codons ◽  
Synonymous Codons ◽  
Multiple Components ◽  
Human Genes ◽  
Regulatory Module ◽  
Mitogen Activated Protein

AbstractThe ability to translate three nucleotide sequences, or codons, into amino acids to form proteins is conserved across all organisms. All but two amino acids have multiple codons, and the frequency that such synonymous codons occur in genomes ranges from rare to common. Transcripts enriched in rare codons are typically associated with poor translation, but in certain settings can be robustly expressed, suggestive of codon-dependent regulation. Given this, we screened a gain-of-function library for human genes that increase the expression of a GFPrare reporter encoded by rare codons. This screen identified multiple components of the mitogen activated protein kinase (MAPK) pathway enhancing GFPrare expression. This effect was reversed with inhibitors of this pathway and confirmed to be both codon-dependent and occur with ectopic transcripts naturally coded with rare codons. Finally, this effect was associated, at least in part, with enhanced translation. We thus identify a potential regulatory module that takes advantage of the redundancy in the genetic code to modulate protein expression.

scholarly journals Ghrelin exerts a proliferative effect on a rat pituitary somatotroph cell line via the mitogen-activated protein kinase pathway

2004 ◽  
pp. 233-240 ◽  
Author(s):  
AM Nanzer ◽  
S Khalaf ◽  
AM Mozid ◽  
RC Fowkes ◽  
MV Patel ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Kinase ◽  
Cell Line ◽  
Kinase Inhibitor ◽  
Thymidine Incorporation ◽  
Mapk Pathway ◽  
Mitogen Activated Protein Kinase ◽  
Gh3 Cells ◽  
Rat Pituitary ◽  
Mitogen Activated Protein

OBJECTIVES: Ghrelin is a brain-gut peptide with GH-releasing and appetite-inducing activities and a widespread tissue distribution. Ghrelin is the endogenous ligand of the GH secretagogue receptor type 1a (GHS-R1a), and both ghrelin and the GHS-R1a are expressed in the pituitary. There are conflicting data regarding the effects of ghrelin on cell proliferation. A positive effect on proliferation and activation of the mitogen-activated protein kinase (MAPK) pathway has been found in hepatoma, adipose, cardiomyocyte and prostate cell lines. However, ghrelin has also been shown to have anti-proliferative effects on breast, lung and thyroid cell lines. We therefore examined the effect of ghrelin on the rat pituitary cell line GH3. METHODS: RT-PCR was used for the detection of GHS-R1a and pre-proghrelin mRNA expression in GH3 cells. The effect of ghrelin on cell proliferation was studied using [(3)H]thymidine incorporation; cell counting and the activation of the MAPK pathway were studied using immunoblotting and inhibitors of the extracellular signal-regulated kinase 1 and 2 (ERK 1/2), protein kinase C (PKC) and tyrosine phosphatase pathways. RESULTS: GHS-R1a and ghrelin mRNA expression were detected in GH3 cells. Ghrelin, at 10(-10) to 10(-6) M concentrations, significantly increased [(3)H]thymidine incorporation (at 10(-9) M, 183+/-13% (means+/-s.e.m.) compared with untreated controls), while 12-phorbol 13-myristate acetate (PMA) at 10(-7) M (used as a positive control) caused a 212+/-14% increase. A reproducible stimulatory effect of desoctanoyl ghrelin was also observed on [(3)H]thymidine incorporation (135+/-5%; P<0.01 at 10(-9) M compared with control), as well as on the cell count (control 6.8 x 10(4)+/-8.7 x 10(3) cells/ml vs desoctanoyl ghrelin (10(-9) M) 1.04 x 10(5)+/-7.5 x 10(3) cells/ml; P<0.01). Ghrelin caused a significant increase in phosphorylated ERK 1/2 in immunoblotting, while desoctanoyl ghrelin showed a smaller but also significant stimulatory effect. The positive effect of ghrelin and desoctanoyl ghrelin on [(3)H]thymidine incorporation was abolished by the MAPK kinase inhibitor U0126, the PKC inhibitor GF109203X and the tyrosine kinase inhibitor tyrphostin 23, suggesting that the ghrelin-induced cell proliferation of GH3 cells is mediated both via a PKC-MAPK-dependent pathway and via a tyrosine kinase-dependent pathway. This could also be clearly demonstrated by Western blot analysis, where a transient increase in ERK 1/2 phosphorylation by ghrelin was attenuated by all three inhibitors. CONCLUSION: We have shown a novel role for ghrelin in stimulating the proliferation of a somatotroph pituitary tumour cell line, suggesting that ERK activation is involved in mediating the effects of ghrelin on cell proliferation. Desoctanoyl ghrelin showed a similar effect. As ghrelin has been shown to be expressed in both normal and adenomatous pituitary tissue, locally produced ghrelin may play a role in pituitary tumorigenesis via an autocrine/paracrine pathway.

Endothelins stimulate mitogen-activated protein kinase cascade through either ETA or ETB

1994 ◽  
Vol 267 (4) ◽  
pp. C1130-C1135 ◽  
Author(s):  
Y. Wang ◽  
P. M. Rose ◽  
M. L. Webb ◽  
M. J. Dunn
Keyword(s):  
Protein Kinase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Mapk Cascade ◽  
Mitogen Activated Protein Kinase ◽  
Thymidine Uptake ◽  
Mapk Activation ◽  
Transfected Cells ◽  
Gel Retardation ◽  
Mitogen Activated Protein

Endothelin (ET) has been shown to activate mitogen-activated protein kinase (MAPK). However, it has been unclear which of the ET receptors is coupled to MAPK activation. In the present study, we conducted experiments to determine which ET receptor is linked to MAPK activation. We found that both human ETA and ETB were coupled to the MAPK cascade in ETA or ETB cDNA-transfected Chinese hamster ovary cells. ET-1 was more potent than ET-3 in the activation of p42 MAPK, induction of MAPK kinase (MAPKK) gel retardation and uptake of [3H]thymidine in ETA-transfected cells, whereas sarafotoxin (S6c) showed no stimulatory effect on the kinases and [3H]thymidine uptake. ET-1, ET-3, and S6c had approximately the same potency to activate p42 MAPK, MAPKK gel retardation, and [3H]thymidine uptake in ETB-transfected cells. These data suggest that 1) ET isopeptides, through either ETA or ETB receptors, induce the MAPK cascade as well as cell proliferation; and 2) the different potencies of ET isopeptides for activation of the MAPK cascade and induction of cell growth are mainly due to their different affinities toward ETA and ETB.

scholarly journals The vaccinia virus-stimulated mitogen-activated protein kinase (MAPK) pathway is required for virus multiplication

2004 ◽  
Vol 381 (2) ◽  
pp. 437-446 ◽  
Author(s):  
Anderson A. ANDRADE ◽  
Patrícia N. G. SILVA ◽  
Anna C. T. C. PEREIRA ◽  
Lirlândia P. de SOUSA ◽  
Paulo C. P. FERREIRA ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Tyrosine Kinase ◽  
Vaccinia Virus ◽  
Mapk Pathway ◽  
Decisive Role ◽  
Virus Multiplication ◽  
Mitogen Activated Protein Kinase ◽  
Actin Dynamics ◽  
Kinase Activation ◽  
Mitogen Activated Protein

Early events play a decisive role in virus multiplication. We have shown previously that activation of MAPK/ERK1/2 (mitogen-activated protein kinase/extracellular-signal-regulated kinase 1/2) and protein kinase A are pivotal for vaccinia virus (VV) multiplication [de Magalhães, Andrade, Silva, Sousa, Ropert, Ferreira, Kroon, Gazzinelli and Bonjardim (2001) J. Biol. Chem. 276, 38353–38360]. In the present study, we show that VV infection provoked a sustained activation of both ERK1/2 and RSK2 (ribosomal S6 kinase 2). Our results also provide evidence that this pattern of kinase activation depends on virus multiplication and ongoing protein synthesis and is maintained independently of virus DNA synthesis. It is noteworthy that the VGF (VV growth factor), although involved, is not essential for prolonged ERK1/2 activation. Furthermore, our findings suggest that the VV-stimulated ERK1/2 activation also seems to require actin dynamics, microtubule polymerization and tyrosine kinase phosphorylation. The VV-stimulated pathway MEK/ERK1/2/RSK2 (where MEK stands for MAPK/ERK kinase) leads to phosphorylation of the ternary complex factor Elk-1 and expression of the early growth response (egr-1) gene, which kinetically paralleled the kinase activation. The recruitment of this pathway is biologically relevant, since its disruption caused a profound effect on viral thymidine kinase gene expression, viral DNA replication and VV multiplication. This pattern of sustained kinase activation after VV infection is unique. In addition, by connecting upstream signals generated at the cytoskeleton and by tyrosine kinase, the MEK/ERK1/2/RSK2 cascade seems to play a decisive role not only at early stages of the infection, i.e. post-penetration, but is also crucial to define the fate of virus progeny.

scholarly journals APPL1 mediates adiponectin-stimulated p38 MAPK activation by scaffolding the TAK1-MKK3-p38 MAPK pathway

2011 ◽  
Vol 300 (1) ◽  
pp. E103-E110 ◽  
Author(s):  
Xiaoban Xin ◽  
Lijun Zhou ◽  
Caleb M. Reyes ◽  
Feng Liu ◽  
Lily Q. Dong
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Mapk Pathway ◽  
Adaptor Protein ◽  
Mapk Signaling ◽  
Mitogen Activated Protein Kinase ◽  
Scaffolding Protein ◽  
Mapk Activation ◽  
P38 Mapk Pathway ◽  
Mitogen Activated Protein

The adaptor protein APPL1 mediates the stimulatory effect of adiponectin on p38 mitogen-activated protein kinase (MAPK) signaling, yet the underlying mechanism remains unclear. Here we show that, in C2C12 cells, overexpression or suppression of APPL1 enhanced or suppressed, respectively, adiponectin-stimulated p38 MAPK upstream kinase cascade, consisting of transforming growth factor-β-activated kinase 1 (TAK1) and mitogen-activated protein kinase kinase 3 (MKK3). In vitro affinity binding and coimmunoprecipitation experiments revealed that TAK1 and MKK3 bind to different regions of APPL1, suggesting that APPL1 functions as a scaffolding protein to facilitate adiponectin-stimulated p38 MAPK activation. Interestingly, suppressing APPL1 had no effect on TNFα-stimulated p38 MAPK phosphorylation in C2C12 myotubes, indicating that the stimulatory effect of APPL1 on p38 MAPK activation is selective. Taken together, our study demonstrated that the TAK1-MKK3 cascade mediates adiponectin signaling and uncovers a scaffolding role of APPL1 in regulating the TAK1-MKK3-p38 MAPK pathway, specifically in response to adiponectin stimulation.

scholarly journals Both Binding and Activation of p38 Mitogen-Activated Protein Kinase (MAPK) Play Essential Roles in Regulation of the Nucleocytoplasmic Distribution of MAPK-Activated Protein Kinase 5 by Cellular Stress

2002 ◽  
Vol 22 (20) ◽  
pp. 6931-6945 ◽  
Author(s):  
Ole Morten Seternes ◽  
Bjarne Johansen ◽  
Beate Hegge ◽  
Mona Johannessen ◽  
Stephen M. Keyse ◽  
...  
Keyword(s):  
Protein Kinase ◽  
P38 Mapk ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Mapk Pathway ◽  
Nuclear Export Signal ◽  
Mitogen Activated Protein Kinase ◽  
Protein Fusion ◽  
Translational Machinery ◽  
Mitogen Activated Protein

ABSTRACT The p38 mitogen-activated protein kinase (MAPK) pathway is an important mediator of cellular responses to environmental stress. Targets of p38 include transcription factors, components of the translational machinery, and downstream serine/threonine kinases, including MAPK-activated protein kinase 5 (MK5). Here we have used enhanced green fluorescent protein fusion proteins to analyze the subcellular localization of MK5. Although this protein is predominantly nuclear in unstimulated cells, MK5 shuttles between the nucleus and the cytoplasm. Furthermore, we have shown that the C-terminal domain of MK5 contains both a functional nuclear localization signal (NLS) and a leucine-rich nuclear export signal (NES), indicating that the subcellular distribution of this kinase reflects the relative activities of these two signals. In support of this, we have shown that stress-induced activation of the p38 MAPK stimulates the chromosomal region maintenance 1 protein-dependent nuclear export of MK5. This is regulated by both binding of p38 MAPK to MK5, which masks the functional NLS, and stress-induced phosphorylation of MK5 by p38 MAPK, which either activates or unmasks the NES. These properties may define the ability of MK5 to differentially phosphorylate both nuclear and cytoplasmic targets or alternatively reflect a mechanism whereby signals initiated by activation of MK5 in the nucleus may be transmitted to the cytoplasm.

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